Journal articles on the topic 'AR Chemy app'

<strong><em>Abstract</em></strong> <em>The more modern and sophisticated technology of today are undergoing constant transformation. Every area of our life, including education, has changed as a result of contemporary computing technologies. Digital tools are used to enhance the traditional teaching method. Additionally, a lot of software with varied functions is being used. A single one of them is augmented reality (AR). AR gives kids access to real-world experiences. A knowledge-based society, where knowledge is a tremendous power, economy, and potency of an individual and the benefit of a country, has benefited greatly from the advances in science and technology and their application. Its greatness and expansion both have a magnificent detonation. To access and effectively use this rapidly developing technology, we require innovative knowledge. It necessitates total access to and control over the knowledge acquisition process. With the aid of information and communication technological science, it is possible. Over the past ten years, augmented reality (AR) interfaces have proliferated, with an increase in user-based trials. Chemistry note cards have long been a mainstay for remembering new concepts; with AR, they now offer greater content for learning. An open-source computer tracking framework called ARToolkit is used to build powerful augmented reality apps. A digital content repository with the construction of coursework, learning games and simulations, augmented reality, and virtual reality will be developed with efficiency and perfection, according to the National Education Policy (NEP, 2020). The current status, various device types, hardware, software, applications, and content for augmented reality are discussed in this paper.</em>

Introduction Bruton's tyrosine kinase inhibitors (BTKi) have improved 1 st line therapy efficacy in elderly (Wang NEJM 2022) and young, fit, MCL-the latter with intermittent dosing during RCHOP in an alternating RCHOP/cytarabine-based regimen (Dreyling Blood 2022). However direct combination of BTKi &amp; intensive chemo is toxic in DLBCL studies. (Kuruvilla Haematol Oncol 2017). The highly selective BTKi, acalabrutinib (A), has proven efficacy and tolerability in relapsed MCL (Wang Lancet 2018). RDHAOx chemotherapy (rituximab, dexamethasone, cytarabine, oxaliplatin) prior to ASCT provides a complete response (CR) rate of 77% and favourable toxicity compared to many induction regimens (Le Gouill Blood 2017). Furthermore, Obinutuzumab-DHAP yields undetectable minimum residual disease (MRD) in 85% of young MCL patients. (Le Gouill Haematol Oncol 2019). Here we report the primary endpoint from the Australasian Leukaemia &amp; Lymphoma Group phase 2 ‘WAMM’ trial exploring a ‘sandwich’ model of an acalabrutinib-rituximab (AR) ‘window’ before RDHAOx +/- ASCT, followed by fixed-duration AR-maintenance to improve therapy response and minimise additional toxicity. Methods NHL33'WAMM' (ACTRN12619000990123) is a multicentre single-arm phase 2 trial. Key eligibility included: age 18-70 years, untreated histologically-proven stage II-IV MCL, ECOG&amp;lt;2; no contraindications to ASCT or BTKi. Pts received 2 cycles of AR (AR window); ‘A’ dose; 100mg BD PO, ‘R’ dose; 375mg/m 2 IV (day 1) every 4 weeks, followed by RDHAOx x4 cycles. Those with an objective response (CR or partial response-PR) underwent BEAM ASCT (carmustine, etoposide, cytarabine, melphalan) then AR maintenance (A; 1yr continuous &amp; R; 3-monthly x 8 cycles). Those who did not undergo BEAM ASCT were able to remain on study and proceed to maintenance AR in the event of ongoing response. Co-primary endpoints (EP) were safety; defined by lack of prohibitive toxicity causing treatment cessation AND PET-determined complete response (CR) rate after AR+RDHAOx. Secondary EP include overall response rates (ORR), toxicity, overall survival (OS) &amp; progression-free survival (PFS), MRD negativity rates and quality of life. Only Grade 3+ toxicity related to acalabrutinib was collected during the ASCT period. Baseline whole exome sequencing was performed to identify mutations, MRD analysis was done by LymphoTrack® Dx and MRD Assay platform (Invivoscribe, Inc) using Illumina® MiSeq. This study was the first Australian blood cancer trial to use telehealth and a ‘hub-and-spoke’ transplant model. Results 44 pts were enrolled from Sept 2020 to Apr 2022 (43 evaluable for the primary endpoint). Baseline characteristics were typical of a young MCL cohort: median age 59 years (Interquartile range 54-64), 77% were male, ECOG was 0-1 in 98%, 84% had stage IV, lactate dehydrogenase was elevated in 35%, Ki-67 &amp;gt;30% in 66% and blastoid/pleomorphic histology 9%. TP53 by NGS will be reported at presentation. CR rate post AR+RDHAOx induction was 88%; (95%CI 72-95) with ORR 95% and no prohibitive toxicity. AR window ORR was 93% (CR 57%). MRD negativity was achieved in 18% (7/38) post AR window, and 94% (34/36) post RDHAOx. 43 patients remained evaluable for response after R-DHAOX and pre-maintenance with 35 (80%) who underwent ASCT and 38 (86%) who commenced AR maintenance; 7 remain on maintenance. Response in specific subgroups will be reported at the meeting. At data lock, 35 pts (81%) experienced ≥1 G3+ adverse events during induction or maintenance phases, most common were neutropenia (58% of pts), febrile neutropenia (27%), thrombocytopenia (25%), diarrhea (14%). No G5 treatment-related events have occurred to date. There have been 22 SAEs, most common were COVID (3), febrile neutropenia (2) and fever (2). 5 deaths have been reported: 4 in pts with progressive disease and 1 COVID pneumonitis during AR maintenance. After a median follow up of 22 months (range 17-28m), the 2-year OS was 89%. Analysis of survival, quality of life and biomarkers will be reported with more mature follow up. Conclusion AR delivered in a sandwich approach is active and safe. An AR window yields a high ORR and compared to historical studies, improves post-chemo induction CR rates and MRD negativity. A telehealth model allowed rapid recruitment in a rare cancer.

Appropriate trafficking of the β1-adrenergic receptor (β1-AR) after agonist-promoted internalization is crucial for the resensitization of its signaling pathway. Efficient recycling of the β1-AR required the binding of the protein kinase A anchoring protein-79 (AKAP79) to the carboxyl terminus of the β1-AR (Gardner, L. A., Tavalin, S. A., Goehring, A., Scott, J. D., and Bahouth, S. W. (2006) J. Biol. Chem. 281, 33537-33553). In this study we show that AKAP79 forms a complex with the type 1 PDZ-binding sequence (ESKV) at the extreme carboxyl terminus of the β1-AR, which is mediated by the membrane-associated guanylate kinase (MAGUK) protein SAP97. Thus, the PDZ and its associated SAP97-AKAP79 complex are involved in targeting the cyclic AMP-dependent protein kinase (PKA) to the β1-AR. The PDZ and its scaffold were required for efficient recycling of the β1-AR and for PKA-mediated phosphorylation of the β1-AR at Ser312. Overexpression of the catalytic subunit of PKA or mutagenesis of Ser312 to the phosphoserine mimic aspartic acid both rescued the recycling of the trafficking-defective β1-ARΔ PDZ mutant. Thus, trafficking signals transmitted from the PDZ-associated scaffold in the carboxyl terminus of the β1-AR to Ser312 in the 3rd intracellular loop (3rd IC) were paramount in setting the trafficking itinerary of the β1-AR. The data presented here show that a novel β1-adrenergic receptosome is organized at the β1-AR PDZ to generate a scaffold essential for trafficking and networking of the β1-AR.

134 Background: Androgen receptor (AR) signaling is the principal driver of PC at all stages. CC-94676 (BMS-986365) is a heterobifunctional, first-in-class, orally bioavailable AR LDD designed to induce rapid, sustained, and highly selective AR degradation in pts who progressed on standard of care therapies. We report initial results from an open-label, multicenter study, NCT04428788, evaluating CC-94676 in pts with progressive mCRPC. Methods: Pts with mCRPC who progressed on androgen deprivation therapy, ≥ 1 second generation hormonal therapy (eg, enzalutamide [enza], abiraterone [abi], darolutamide, and apalutamide) and taxane chemotherapy (chemo) (unless refused or not indicated) were enrolled to evaluate the safety, tolerability, PK/PD, and preliminary efficacy of CC-94676. Escalation doses evaluated were 100–1200 mg QD and 600–900 mg BID; expansion doses were 600 mg QD and 400–900 mg BID. Results: As of Aug 21, 2023, 95 pts received CC-94676 (median age 71 yrs) with a median of 5 (range 2–12) prior therapies, including enza (80%), abi (72%), both enza &amp; abi (56%), and chemo (56%) (docetaxel 55%; cabazitaxel 20%). There were no ≥ Grade (G) 4 treatment-related adverse events (TRAEs) or discontinuations due to TRAEs. Of 27 pts treated in escalation, treatment (Tx) was well tolerated; 2 pts had non-serious G3 TRAEs at doses ≥ 800 mg QD, which were manageable with dose modifications. One dose-limiting toxicity of asymptomatic QTc prolongation was observed at 900 mg BID and resolved with dose interruption. The maximum tolerated dose was not reached. Of 68 pts treated in expansion, the most common TRAEs were dose-dependent: QTc prolongation (asymptomatic) (43%; G3: 9%), bradycardia (31%; ≥G3: none), and fatigue (24%; ≥G3: none). G3 TRAEs were manageable with dose modifications. The rate of pts with confirmed PSA reductions ≥ 30% (PSA30) increased dose-dependently from 400 to 900 mg BID. 23/68 (34%) pts across all dose levels achieved ≥ PSA30. At 900 mg BID, 11/20 (55%) pts showed PSA30; 7/20 (35%) and 2/20 (10%) had confirmed PSA declines of ≥50% and ≥90%, respectively. PSA responses and radiographic tumor shrinkage occurred across all dose levels, including in pts with AR wildtype (WT) , amplifications, and mutations (by cfDNA), and in heavily pretreated pts who progressed on abi, enza, and chemo. At 900 mg BID, the median duration of Tx was 182 days (range 21-448) and 9/20 (45%) pts remained free of radiographic progression with treatment ongoing at 6 months. Conclusions: CC-94676 is well tolerated with a manageable safety profile. CC-94676 shows promising and prolonged clinical activity in heavily pretreated mCRPC pts who progressed on abi, enza, and chemo with activity seen in pts with tumors expressing WT and mutant ARs. Selection of the recommended phase 2 dose is ongoing. Clinical trial information: NCT04428788 .

252 Background: Liquid biopsy assays in mCRPC are utilized for treatment eligibility of PARP inhibitors (HRR) and Pembrolizumab (MSI High). These ctDNA targeted gene panels provide translational data beyond gene mutations. Tumor Fraction (TF), the percentage of cfDNA from tumor, is a promising prognostic efficacy biomarker in pre-chemo mCRPC, and a potential predictive biomarker to post-chemo PSMA radioligand therapy. Methods: We analyzed ctDNA run with Illumina-TSO500 from the CA209-7DX Ph3 mCRPC trial (Nivo+chemo vs. Chemo in chemo-naïve mCRPC) to determine: TF at baseline or changes at Cycle6 Day1 (C6D1). TF at each timepoint was determined through the maximum somatic allelic frequency. Results: We present a combined analysis of treatment arms as both showed similar association between clinical outcomes and TF levels as well as clearance. Out of 1030 ITT patients TF data was available for 527 (51%) patients. Patients in the highest TF tertile had TF of ≥4.6% and TF&lt;0.3% for the lowest tertile. Patients in the lowest TF tertile had 10 months longer overall survival (OS) to the highest TF tertile (p&lt;0.001). Patients in the highest TF tertile demonstrated higher hazard ratio for OS (HR 3.42,[2.34-4.98] p&lt;0.001) and rPFS (HR 1.95 [1.47-2.59], p&lt;0001) over lowest tertile. This differential was maintained across arms and indicates that TF is a strong prognostic biomarker. High TF outperformed common stratification factors (prior exposure to chemo in CSPC or novel ARPIs) for poor prognostication in a multivariate analysis. TF high patients have higher rates of AR-Amp/AR-LBD mutations (72.6%) than TF low (28.8%). Patients with TF clearance, defined as TF &lt; 1% at C6D1 had 8 months mOS advantage over patients with a TF &gt;1% at baseline and on-treatment. TF clearance strongly correlated with OS (HR=0.42 [0.28-0.62], p&lt;0.001), rPFS benefit (HR=0.24 [0.17-0.34], p&lt;0.001) and PSA response (28% vs. 62%) regardless of treatment arm. Conclusions: This data advocates for plasma TF as a robust prognostic biomarker and as a potential stratification opportunity in mCRPC clinical trials. TF clearance may act as an early indicator of treatment response and hence can be an important monitoring tool for mCRPC patient management. Clinical trial information: NCT04100018 .

Abstract. Winter fog and severe aerosol loading in the boundary layer over northern India, particularly in the Indo-Gangetic Plain (IGP), disrupt the daily lives of millions of people in the region. To better understand the role of aerosol–radiation (AR) feedback on the occurrence, spatial extent, and persistence of winter fog, as well as the associated aqueous chemistry in fog in the IGP, several model simulations have been performed using the Weather Research and Forecasting model coupled with Chemistry (WRF-Chem). While WRF-Chem was able to represent the fog formation for the 23–24 December 2017 fog event over the central IGP in comparison to station and satellite observations, the model underestimated PM2.5 concentrations compared to the Central Pollution Control Board (CPCB) of India monitoring network. While evaluating aerosol composition for fog events in the IGP, we found that the WRF-Chem aerosol composition was quite different from measurements obtained during the Winter Fog Experiment (WiFEX) in Delhi, with secondary aerosols, particularly the chloride aerosol fraction, being strongly underpredicted (∼ 66.6 %). Missing emission sources (e.g., industry and residential burning of cow dung and trash) and aerosol and chemistry processes need to be investigated to improve model–observation agreement. By investigating a fog event on 23–24 December 2017 over the central IGP, we found that the aerosol–radiation feedback weakens turbulence, lowers the boundary layer height, and increases PM2.5 concentrations and relative humidity (RH) within the boundary layer. Factors affecting the feedback include loss of aerosols through deposition of cloud droplets and internal mixing of absorbing and scattering aerosols. Aqueous-phase chemistry increases the PM2.5 concentrations, which subsequently affect the aerosol–radiation feedback by both increased mass concentrations and aerosol sizes. With aerosol–radiation interaction and aqueous-phase chemistry, fog formation began 1–2 h earlier and caused a longer fog duration than when these processes were not included in the WRF-Chem simulation. The increase in RH in both experiments was found to be important for fog formation as it promoted the growth of aerosol size through water uptake, increasing the fog water content over the IGP. The results from this study suggest that the aerosol–radiation feedback and secondary aerosol formation play an important role in the air quality and the intensity and lifetime of fog over the IGP, yet other feedbacks, such as aerosol–cloud interactions, need to be quantified.

Abstract Metastatic prostate cancer is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and are therefore sensitive to androgen deprivation therapies. However, eventual resistance to standard medical castration and secondary chemotherapies including next-generation AR-targeting agents like abiraterone or enzalutamide and taxanes (Docetaxel or cabazitaxel) is nearly universal. Further, neuroendocrine PCa or NEPC, a poorly differentiated aggressive variant of PCa that lacks AR expression, and the presence of cancer stem-like cells with self-renewal and differentiation (epithelial to mesenchymal trans-differentiation or EMT) capacities significantly contribute to the development of clinically most aggressive and lethal variants of PCa. Endogenous Ras/ERK/MAPK signaling is known to drive prostate tumor formation, cancer progression, cell migration, and the acquisition of mesenchymal phenotype (EMT) in metastatic prostate cancers. Ras family members are responsible for modulating the function of most of the receptors upregulated in advanced PCa, including AR, so that AR can be functional even in the absence of physiologic levels of androgen. Previous studies have reported that the NSAID, sulindac, inhibits Ras-induced transformation and directly binds Ras. In this study, we show that a non-COX inhibitory sulindac derivative, ADT-007, is a potent drug candidate against lethal PCa. First, we performed single-cell RNA sequencing as a biomarker-based drug screen and showed that chemo-resistant, drug-tolerant, stem-cell-like PCa cell clusters had high expression of Ras genes, indicating that the Ras inhibitor, ADT-007, may be effective in targeting these aggressive subclones. Next, using several cell-based assays on a large panel of lethal PCa cell lines representing metastatic castration-resistant PCa (mCRPC), NEPC as well as African American ancestry, we showed that ADT-007 is highly effective as a single-agent and in combination with taxanes. Combination index values were &amp;lt;0.7, and BLISS scores were &amp;gt;10, indicating very high synergy. Finally, using pre-vs-post-treatment bulk RNAseq, we demonstrated that ADT-007 is indeed effective in downregulating KRas and NRas in a dose-dependent manner. In addition, ingenuity pathway analysis (IPA) predicted significant downregulation of cell cycle, DNA replication, CCND1, AR, and the lncRNA ELDR, and upregulation of autophagy and interferon pathway genes, and the microRNA miR-96. Finally, using PCa patient datasets, we showed that ADT-007 is potentially capable of reversing the expression of several genes associated with biochemical recurrence. Thus, our results show that ADT-007 is a novel drug candidate that circumvents subclonal aggressiveness, drug resistance, and stemness in lethal PCa through simultaneous inhibition of multiple oncogenic factors/pathways. Citation Format: Taraswi Mitra Ghosh, Adam B. Keeton, Xi Chen, Salsabil Ahmed, Razan S. Waliagha, Gary A. Piazza, Amit K. Mitra. Validation of a novel pan-Ras inhibitor, ADT-007, against lethal variants of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1638.

237 Background: AA/P is an approved treatment for mCRPC but there are no known predictive markers of response or resistance. We conducted a prospective trial to evaluate if Androgen Receptor (AR) &amp; AR-variant (ARV) expression in tissue metastases can predict resistance to AA/P. Methods: mCRPC stage patients (pts) initiating pre-chemo AA/P underwent metastatic site biopsies prior to (pre-AA/P) and after 12 weeks of treatment. Composite progression at 12 weeks, (primary endpoint) was evaluated with PSA, RECIST, bone scan and symptoms (per PCWG2). mRNA expressions of pre-AA/P ARFL, ARV3, ARV7, ARV9, ARV23, ARV45, four cell cycle division genes, Chromogranin-A (CHGA) together with PSA/testosterone levels, Gleason Score (GS) at initial diagnosis; high versus low volume disease; time from starting hormone therapy to mCRPC stage and serum CHGA levels were evaluated using a logistic regression model for predicting resistance at 12 weeks of therapy. A final multivariate model fitted only those factors thought to be clinically relevant or with an entry threshold of p ≤ 0.3 in univariate analysis. Results: Between 6/2013 &amp; 3/2015, 82 pts were enrolled of which 52 had complete mRNA expression &amp; disease assessment data at the12-week time point for analysis. Median age of the cohort was 72.5 yrs (IQR: 68.5-78); median pre-AA/P PSA was 18 ng/ml (IQR: 8.1- 46.6); GS distribution at initial diagnosis for GS 2-6; 7; 8-10 was 11; 14; 27 respectively. Progression was observed in 21/52 pts after 12 weeks. At the univariate level elevated pre-AA/P expression of ARV3 (p = 0.08), ARV7 (p = 0.26), ARV9 (p = 0.04), and cell division cycle gene CDC45 (p = 0.19) along with GS at diagnosis (p = 0.29) met the threshold for inclusion into multivariate analysis. Elevated expression of pre-therapy ARV9 in metastases alone was associated with progression at 12 weeks (OR: 3.9; CI 1.07 – 14.16; C-Index: 0.63). The 12-week biopsy of pts with progression had increased ARV9 mRNA expression compared to pts responding at 12 weeks (p = 0.14). Conclusions: Increased ARV9 mRNA expression in metastases is associated with early resistance to AA/P. This observation will need further validation in comparable datasets. Clinical trial information: NCT #0195364.

SummaryPlatelet activation results in shape change, aggregation, generation of thromboxane A2, and release of granule contents. We have recently demonstrated that secreted ADP is essential for thromboxane A2-induced platelet aggregation (J. Biol. Chem. 274: 29108-29114, 1999). The aim of this study was to investigate the role of secreted ADP interacting at P2 receptor subtypes in platelet secretion. Platelet secretion induced by the thromboxane A2 mimetic U46619 was unaffected by adenosine-3’phosphate-5’-phosphate, a P2Y1 receptor selective antagonist. However, AR-C66096, a selective antagonist of the P2T AC receptor, inhibited U46619-induced platelet secretion, indicating an important role for Gi signaling in platelet secretion. Selective activation of either the P2T AC receptor or the α2A adrenergic receptor did not cause platelet secretion, but potentiated U46619-induced platelet secretion. SC57101, a fibrinogen receptor antagonist, failed to inhibit platelet secretion, demonstrating that outside-in signaling was not required for platelet secretion. Since Gi signaling results in reduction of basal cAMP levels through inhibition of adenylyl cyclase, we investigated whether this is the signaling event that potentiates platelet secretion. SQ22536 or dideoxyadenosine, inhibitors of adenylyl cyclase, failed to potentiate U46619-induced primary platelet secretion, indicating that reduction in cAMP levels does not directly contribute to platelet secretion. Wortmannin, a selective inhibitor of PI-3 kinase, minimally inhibited U46619-induced platelet secretion when it was solely mediated by Gq, but dramatically ablated the potentiation of Gi signaling. We conclude that signaling through the P2TAC receptor by secreted ADP causes positive feedback on platelet secretion through a PI-3 kinase pathway.

Triple-negative breast cancer (TNBC), characterized by the absence or low expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2), is the most aggressive subtype of breast cancer. TNBC accounts for about 15% of breast cancer cases in the U.S., and is known for high relapse rates and poor overall survival (OS). Chemo-resistant TNBC is a genetically diverse, highly heterogeneous, and rapidly evolving disease that challenges our ability to individualize treatment for incomplete responders and relapsed patients. Currently, the frontline standard chemotherapy, composed of anthracyclines, alkylating agents, and taxanes, is commonly used to treat high-risk and locally advanced TNBC. Several FDA-approved drugs that target programmed cell death protein-1 (Keytruda) and programmed death ligand-1 (Tecentriq), poly ADP-ribose polymerase (PARP), and/or antibody drug conjugates (Trodelvy) have shown promise in improving clinical outcomes for a subset of TNBC. These inhibitors that target key genetic mutations and specific molecular signaling pathways that drive malignant tumor growth have been used as single agents and/or in combination with standard chemotherapy regimens. Here, we review the current TNBC treatment options, unmet clinical needs, and actionable drug targets, including epidermal growth factor (EGFR), vascular endothelial growth factor (VEGF), androgen receptor (AR), estrogen receptor beta (ERβ), phosphoinositide-3 kinase (PI3K), mammalian target of rapamycin (mTOR), and protein kinase B (PKB or AKT) activation in TNBC. Supported by strong evidence in developmental, evolutionary, and cancer biology, we propose that the K-RAS/SIAH pathway activation is a major tumor driver, and SIAH is a new drug target, a therapy-responsive prognostic biomarker, and a major tumor vulnerability in TNBC. Since persistent K-RAS/SIAH/EGFR pathway activation endows TNBC tumor cells with chemo-resistance, aggressive dissemination, and early relapse, we hope to design an anti-SIAH-centered anti-K-RAS/EGFR targeted therapy as a novel therapeutic strategy to control and eradicate incurable TNBC in the future.

Abstract Background: Preclinical models of prostate adenocarcinoma (PAC) are challenging to develop and maintain. We previously reported (AACR2022/ENA2023) the successful establishment and characterization of four parental and three conditioned prostate adenocarcinoma models in intact immunodeficient male mice. Using this experience, we have expanded our platform with six additional PAC models designated ST6084, ST6838, ST7482, ST7695, STF1159 and STM344. These models were characterized for receptor expression, genomic alterations, and in vivo drug sensitivity to relevant therapies. Methods: XPDX models representing prostate cancer were established from primary (ST6084, ST6838, ST7695, STF1159) or metastatic (ST7482, STM344) samples implanted into intact CB-17 SCID mice. Resulting models were passaged serially and further developed in both intact and castrated mice until growth stabilization. Resulting models were characterized using genomic analysis, including WES and RNAseq, receptor expression, and in vivo drug sensitivity studies. For in vivo studies, docetaxel, abiraterone and enzalutamide were evaluated at standard treatment regimens. Study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C&amp;lt;0%) versus Day 0 tumor volume was also reported. Results: Most models lacked AR or PSMA staining but ST6838 and STM344 were positive for AR-V7. Genomic characterization identified KDM6A loss in ST7482, a model from a patient post-apalutamide. An ATM aberration was present in both ST7482 and STM344, the latter derived from a patient post-chemo and targeted therapies including a five-month response on an ATR inhibitor. No tested models were sensitive to abiraterone or enzalutamide (%T/C&amp;gt;20%) and only ST7482 was sensitive to docetaxel. Conclusion: We have established and characterized an expanded panel of PAC XPDX models. These models can be utilized as a valuable tool in better understanding prostate cancer and in developing novel therapies for drug-resistant patients. Citation Format: Johnnie Flores, Alyssa Simonson, Anna Stackpole, Jennifer Garcia, Shaquille Johnson, Pedro Cesenes, Mia Lopez, Peter Forofontov, Lisa Gonzales, Garrett Mercer, Amarissa Rojas, Nehal Lakhani, Emiliano Calvo, Andrew Cunningham, Michael J. Wick. Establishment and characterization of prostate adenocarcinoma XPDX models from naïve and pretreated patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 43.

185 Background: Targeted radionucleotide therapy (TRT) has become a standard of care. α-emitters have a higher energy transfer over a shorter range than β-emitters. PSMA-targeting antibodies have different biodistribution than small molecules and may improve intracellular retention based on pre-clinical models. Here, we present mature follow up of a phase 1 dose-escalation trial of 225Ac-J591 plus 177Lu-PSMA-I&amp;T (aka PNT2002). Methods: Inclusion criteria:progressive mCRPC with ≥1 prior AR pathway inhibitor (ARPI), prior chemo (or unfit/refused), and with ≥1 lesion on PSMA PET where SUVmax &gt;liver. 177Lu-PSMA-I&amp;T (6.8 GBq) and 225Ac-J591 (30, 35, or 40 KBq/kg) given up to 2 doses 8 weeks apart. Primary outcome was dose-limiting limiting toxicity and recommended phase 2 dose (RP2D). Preliminary efficacy outcomes examined were overall survival (OS), progression-free survival (PFS), PSA response, and circulating tumor cell (CTC) changes. Results: 18 patients (6 at each dose level) with median age 70, median PSA of 54.4 (2.43-9614). Previous therapies: 11 (61%) with &gt;1 ARPI, 12 (67%) chemo, 5 (28%) sip-T, 3 (17%) radium-223. Baseline CTCs: 15 detectable, 9 unfavorable. The median SUVmax of the most avid lesion on PSMA-PET was 31.4 (95% CI 11-82.7). Metastatic sites: 13 bone, 9 lymph node, 4 visceral. 8 (44%) were Halabi high risk. Treatment emergent adverse events (AEs) included neutropenia in 3 patients (17%), all Grade &lt;2; thrombocytopenia occurred in 12 (67%) (3 GR 3); anemia in 10 (56%, 3 GR 3); 12 (67%) xerostomia (one Gr2); one (6%) with acute renal failure. Other AEs: 10 (56%) pain flare (1 Gr3 in pt with cord compression), 11 (61%) nausea (all Gr 1), 9 (50%) fatigue (all Gr 1). The RP2D of 225Ac-J591 was 35 KGBq/kg. 17 (94%) patients experienced a decline in PSA levels, with 11 out of 17 (64%) achieving PSA50 response. 4/5 patients (80%) converted from unfavorable to favorable CTC count, 4/8 (50%) from detectable to undetectable, 1 of 2 (50%) remained undetectable. Median biochemical PFS was 7.3 months (95% CI 2.7-15.8), and median OS was 29.8 mo (95% CI 7.4-NR), with 10 patients still alive at time of submission. 5 were progression free at one year, including 3 of 6 treated at RP2D. With longer follow up, no new safety signals were identified. Conclusions: The combination of PSMA-targeted alpha (via antibody) plus beta (via small molecule) was feasible. High grade AEs were rare and no new safety signals emerged with longer term follow up. Nearly all patients had PSA decline, and 5 had durable disease control off therapy. Clinical trial information: NCT04886986 .

156 Background: Whether EGFR is a critical target in met TNBC is unknown. Here we report the clinical history &amp; tumor molecular alterations in a patient with refractory metTNBC who had an ExRx to pac/cis. Methods: Following IRB-approved informed consent, targeted NGS (Foundation Medicine) and WGS (TGEN) was performed on the pt’s FFPE primary TNBC and 2 recurrent lymph nodes to characterize all classes of genomic alterations in cancer-related genes. RPPA was performed at a CLIA-certified laboratory (Theranostics Health) and George Mason Univ where immunostaining was directed against HER1/2/3 pathway and other proteins. Results: At age 58 in 2006, pt had T1c 1+ node TNBC treated with FAC/T. Between 2008 and 2011 she had 4 chemotherapy-refractory recurrences in axilla, supraclavicular (SC), internal mammary (IM) LNs treated unsuccessfully with surgery, radiation and multiple cytotoxic agents including carboplatin. In 2011, following SC LN biopsy, she was treated with BEZ, a PI3K/mTOR, ATM, ATR, DNA-PKcs inhibitor, had a 3 mo response, followed by rapidly enlarging progressive disease (PD) in IM LNs pushing sternum anteriorly. She was treated with pac/cis and had an ongoing complete response (CR) of 2.5+ yrs. NGS of 2011 SC LN (pre-BEZ) &amp; 2012 IM LN (post-BEZ): TP53 &amp; BRCA2 (somatic 15% mutant allele freq) mutations, FOXM1 amplification; SMARCA4 (BRG1) deletion RPPA (GMU) 2011 SC LN (Pre-BEZ): 3+ EGFR; 2+ p-EGFR, p-AKT, p-MEK1/2, p-mTOR RPPA (Theranostics) 2012 IM LN (post-BEZ): 3+ p-MEK1/2 (EGFR &amp; p-EGFR 0) (AR-). Conclusions: Strong EGFR signaling associated with chemo-resistant metTNBC in 2011 SC LN was not present in post-BEZ rapid PD in IM LN which then had durable CR with pac/cis. BEZ inhibits DSB repair, sensitizing cancers to DNA damaging agents (Gil del Alcazar, Clin Ca Res 20:1235, 2014). Progression of p-AKT-activated TNBC following response to inhibitors of PI3K &amp; DNA repair shows DSB repair-deficiency and MAPK activation (Juvekar, Cancer Dis 2:1048, 2012). A prospective trial of BEZ followed at PD by pac/cis in metTNBC is warranted.

45 Background: We performed the 1st dose-escalation study of PSMA-targeted radionuclide therapy with 177Lu-PSMA-617. Using dose-fractionation, we intended to deliver a dose-intense regimen designed to minimize radioresistance due to repopulation. Radionuclide therapy may be able to treat symptoms due to tumor and therefore may be associated with improvement in PRO. Methods: Inclusion: progressive mCRPC following potent AR-pathway inhibitor (ARPI, e.g. abi/enza) and taxane (or unfit/refuse chemo) without limit of # prior therapies, adequate organ function, ECOG performance status 0-2, without preselection for PSMA expression. Treatment was a single cycle of fractionated dose 177Lu-PSMA-617 on D1 and D15 (7.4 to 22 GBq in phase 1; 22.2 GBq in phase 2). PRO tools included FACT-P and BPI-SF at baseline and follow up. Results: 44 men with median age 69 (range 55-91), median PSA 182.97 (range 0.89-5541) were treated. 93% with bone, 45% nodal, 18% lung, 9% liver, 9% other visceral metastases. 55% with at least 1 prior chemo regimen, 52% &gt;2 prior ARPI, 27% with Ra223, 30% sip-T, 5% 177Lu-J591. 59.1% with &gt;50% PSA decline (66.7% at 22.2 GBq, n=21), median overall survival 16 months (95% CI 11-NR). High grade (Gr) toxicity was rare with 6.8% Gr 3 anemia and 2.3% Gr 3 platelets. Gr 1/2 treatment-emergent AE’s include 81.8% with pain flare, 61.4% xerostomia, 29.5% fatigue, 25% platelets, 25% anemia, 25% pain, 15.5% nausea. FACT-P scores tended to improve in all categories by D22 (1 week later), with overall FACT-P scores improving by mean of 8.9 points (p=0.07) at D22 and remaining improved at 12 wks. All BPI scores also improved, with BPI overall severity score improving by mean of 3.0 at D22 (p=0.008) and remained better than baseline at 12 wks. There was no clear association with any AE and PRO changes, but those with PSA decline tended to have improved pain scores (p=0.1). Conclusions: A single cycle of up to 22.2 GBq of 177Lu-PSMA-617 is safe with fractionated (D1 &amp; D15) dosing, with encouraging early efficacy signals in a population unselected for PSMA expression and improved quality of life and pain scores by validated PRO instruments. Clinical trial information: NCT03042468.

5018 Background: PSMA may be targeted by antibodies (mAb) or small molecules (SML), with different kinetics and biodistribution. mAb with longer circulation time and marrow exposure, but decreased access to luminal PSMA expression (e.g. salivary glands, intestine, kidney). SML diffuse to all sites of PSMA expression and then excreted. Alpha radionuclides emit more energy over shorter range vs beta. Pre-clinical data combining mAb and SML supports synergy, with enhanced uptake and retention of SML. Here, we present phase I dose-escalation results of a phase I/II trial investigating combo 225Ac-J591 with 177Lu-PSMA-I&amp;T (aka PNT2002). Methods: Eligibility criteria include progressive mCRPC, ≥1 prior AR pathway inhibitor (ARPI), prior chemo (or unfit/refuse), ≥1 lesion with SUVmax &gt;liver. Doses: 177Lu-PSMA-I&amp;T (6.8 GBq); 225Ac-J591 (30, 35, or 40 KBq/kg), up to 2 doses of combo TRT 8 weeks (wks) apart with 177Lu SPECT following each dose. Primary objectives: dose-limiting toxicity (DLT) and recommended phase II dose; phase II will test the proportion of patients (pts) obtaining &gt;50% PSA decline (PSA50). DLT defined as G4 myelosuppression lasting &gt;1 wk, Gr&gt;2 non-hematologic adverse event (AE), any Gr attributed AE delaying therapy &gt;3 wks. Additional endpoints include progression-free and overall survival, radiographic response rate, safety, circulating tumor cell (CTC) changes, pt reported outcomes, PSMA imaging, blood/tissue correlatives. Results: 18 pts treated (6 at each dose level); 3 did not receive the 2nd dose due to progressive disease or withdrawal. Median age 70 (range 54-86), PSA 54.4 (2.43-9614). 13 (72%) with bone, 9 (50%) lymph node, 2 (11%) liver, 2 (11%) lung mets. 10 (56%) pts CALGB risk category high, 7 (39%) intermediate, 1 low. Previous therapies: 11 (61%) with &gt;1 ARPI, 12 (67%) chemo, 5 (28%) sip-T, 3 (17%) radium-223. 2 of 6 pts with DLT at 40 KBq/Kg (Gr 2 or 3 thrombocytopenia delaying cycle 2 by &gt;3 wk); no DLT observed in other cohorts. As submission, 7 pts (39%) remain on study, including 1 without progression at 16 months and another with undetectable PSA at 10 months. With follow up ongoing, 17 (94%) with PSA decline, 9 (50%) with PSA50. Of those with paired CTC counts, 4 of 5 (80%) converted from unfavorable to favorable CTC count, 4 of 8 (50%) from detectable to undetectable, 1 of 2 (50%) remained undetectable. AEs include 3 (17%) neutropenia (all Gr &lt;2), 12 (67%) thrombocytopenia (3 Gr 3), 10 (56%) anemia (3 Gr 3), 10 (56%) pain flare (1 Gr3 in pt with cord compression), 12 (67%) xerostomia (one Gr2), 11 (61%) nausea (all Gr 1), 9 (50%) fatigue (all Gr 1). Conclusions: The combination of dual-PSMA targeting with mAb + SML and alpha + beta is feasible with follow up ongoing. Efficacy will formally be tested in the upcoming phase II portion of the study with 225Ac-J591 at 35 KBq/Kg and 177Lu-PSMA I&amp;T at 6.8 GBq. Clinical trial information: NCT04886986 .

Abstract Background: PSMA-based targeted radionuclide therapy is now a standard of care for mCRPC since approval of 177Lu-PSMA-617. Use of antibodies (e.g., J591) to target PSMA with higher potency radionuclides (e.g., 225Ac) impacts kinetics, biodistribution, clinical efficacy, and toxicities. In our first-in-human ph I dose-escalation study of single dose 225Ac-J591 in patients with mCRPC, no MTD was reached (max dose 93.3 KBq/kg). Following this, we developed ph I/II parallel dose-escalation studies of fractionated (D1, D15) single cycle and multiple (q6w) dose regimens. Here we present safety data from the initial fractionated study in predominantly 177Lu-naive. Methods: Eligible patients had adequate organ function, ECOG performance status 0-2, and progressive mCRPC following potent AR pathway inhibitor (ARPI) and chemo (or unfit/refusing). Prior 177Lu-PSMA was allowed until an amendment developed a separate study for post-177Lu-PSMA. Baseline 68Ga-PSMA PET scans were performed, but not used to determine eligibility. A 3+3 dose-escalation design was used. Phase I primary objective: determination of dose-limiting toxicity (DLT) and recommended phase II dose (RP2D). DLT was defined as within 8 weeks of first dose: neutropenia (Gr 4 or febrile neutropenia), thrombocytopenia (TCP) (Gr 4, or Gr 3 with clinically significant bleeding), any Gr &amp;gt;2 non-hematologic toxicity deemed to be at least possibly related to 225Ac-J591, or any attributable toxicity precluding or delaying the second dose by &amp;gt;2 weeks. Secondary/exploratory objectives: efficacy measures (e.g., PSA decline, radiographic RR, biochemical/radiographic PFS, OS, CTCs, patient reported outcomes), safety (CTCAE v5), and correlatives (plasma and tissue genomics, PSMA imaging). Results: 24 patients were enrolled in phase I. Median age 73.5 (57-91), PSA 25.78 (3.39-2133.41); 53% (n=13) &amp;gt;1 prior ARPI, 58% (n=14) taxane chemo, 8% (n=2) anti-PSMA therapy, 12.5% (n=3) prior 223Ra. CALGB prognostic groups: Good 4 (16%), Intermediate 8 (33%), Poor 12 (50%). No DLTs were observed in Cohort 1 (n=3) or 2 (n=6). In C3, 2/6 subjects experienced DLTs (Gr 3 weakness, Gr 2 TCP with &amp;gt;2 week delay in second fraction). 8 patients were enrolled in an intermediate dose cohort (2.5) with 1 DLT (Gr 4 TCP). Two patients withdrew before the second dose (intercurrent illness; interruption of 225Ac supply). Most common low gr non-hematologic treatment emergent AEs: fatigue (95%), xerostomia (69%), and nausea (57%). Among evaluable patients for PSA change (n=22), 21 (95%) experienced PSA decline with 14 (67%) with decline of 50% and 6 (37%) with decline of 90%. 13/21 patients had CTCs samples collected at baseline and 12 wks; 5 were unfavorable at baseline (≥5/7.5 mL); 10/13 (77%) remained favorable or converted from unfavorable to favorable; 6/12 (50%) had 50% decline in CTC count; and 5/13 (38%) converted from detectable to undetectable. Conclusions: A single fractionated cycle of 225Ac-J591 was delivered with few high grade AEs and with evidence of preliminary efficacy by PSA and CTC changes across all dose levels. Citation Format: Jones T. Nauseef, Michael Sun, Charlene Thomas, Mahelia Bissassar, Escarleth Fernandez, Zachary Davidson, Amie Patel, Angela Tan, Tessa A. Chamberlain, Kara Earle, Rebecca Wunder, Sabrina Guervil, Sandra H. Castellanos, Judith Stangl-Kremser, Peter S. Gregos, Joseph R. Osborne, Karla V. Ballman, Ana M. Molina, Cora N. Sternberg, David M. Nanus, Neil H. Bander, Scott T. Tagawa. Phase I dose-escalation study of fractionated dose 225Ac J591 for metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT014.

Abstract Objectives Wheat bran (WB) is a rich source of dietary fiber and phytochemicals with health-promoting properties. However, the active components especially the interaction between different components in whole grain (WG) wheat have not been fully explored. This study aimed to investigate whether WB phytochemicals, alkylresorcinols (ARs), and the active intestinal microbial metabolite of fiber, butyrate, could synergistically suppress human colon cancer cells. Methods Cell viability was determined by MTT assay in HCT-116 and HT-29 cells after the treatments of the combination of ARs, C21 and C19, and NaB, respectively. Apoptosis was determined by Cell Death ELISA Kit. The further mechanism was investigated by western blot associated with apoptosis, autophagy, and ER stress pathways. The C21 levels in gastrointestinal tract were measured by HPLC in mice treated with human-relevant doses of C21. Isobologram analysis was employed for the determination of synergistic or additive anticancer effects of the co-treatments of AR and NaB. Results The combination of C21 or C19 and butyrate synergistically inhibited the growth of colon cancer cells and induced apoptosis. Further mechanistic studies demonstrated that the co-treatment of C21 and butyrate induced significant upregulations in cleaved Poly(ADP-ribose) polymerase (PARP), cleaved caspase 3, p53 upregulated modulator of apoptosis (PUMA), cytochrome C, lipid-conjugated membrane-bound form of microtubule-associated protein 1A/1B-light chain 3 (LC3-II), and C/EBP homologous protein (CHOP) expressions. Notably, the C21 concentrations in the large intestinal tract of mice treated with human-relevant doses of C21, were from 0.86 to 1.78 μmol/g, suggesting the C21 doses used in vitro may be achievable after daily WG wheat intake. Conclusions We demonstrated for the first time that phytochemical component ARs and the microbial metabolites of WB fiber exhibit synergistic anticancer effects in human colon cancer cells, which may be associated with the induction of apoptosis, autophagy, and ER stress pathways. The present study provides novel insights into the understanding of the chemo-preventive effects of WG wheat against CRC. However, the mechanism of the synergistic anticancer effect of phytochemicals and fiber is complicated and still needs to be further investigated in vivo. Funding Sources USDA NIFA R01.

Abstract Background: No effective tool is available to predict response to neoadjuvant chemotherapy (NACT). Aim: to enable an accurate prediction of the treatment response for BC patients, with the aid of machine learning based analysis of tumour histopathological, molecular &amp; imaging features before NACT. Methods: The baseline biopsies were immunohistochemistry (IHC) stained for ER, PR, HER2, Ki67 &amp; SPAG5 and genomically analysed. To build up a genomic profiler &amp; multiplex analysis, a tailored mRNA code set of 450 genes was designed using NanoString nCounter Flex Analysis System. In addition, samples have been analysed for currently used prognostic &amp; predictive genomic panels including: Oncotype DX [Genomic Health], MammaPrint [Agendia], Prosigna [Nanostring Technologies] &amp; MD Anderson Genomic Chemo Sensitivity Predictor. The imaging features were extracted from MRI using radiomics measurements and deep learning methods. Our predictive models were trained in a local cohort of 400 BC (300 HER2 negative (HER2-) received 8 cycles of anthracycline &amp; Taxane &amp; 100 HER2 positive (HER2+) received Trastuzumab, anthracycline &amp;Taxane) &amp; was validated in 150 patients from local clinical trial patients. I-SPY-1 clinal trial genomic &amp; imaging data were used as an external validation. The pathological complete response (pCR) defined as absence of tumor cells in both breast &amp; lymph node was used as endpoint. Results: Compared to HER- BC, HER2+ BC had higher expression of GRB7, ERBB2, SPAG5, FGRF4 &amp; CDC6. While HER2 negative BC showed higher level of CCND1, CDK6, FOXM1, FOXC1, MAI, MYC, ANP32E, SFRP1, ACTR3B, RAD51AP1, MCM3, LIN9, CDCA7 &amp; PMAIP1 (Table 1). In both HER2 positive and negative BC; the reduction of &amp;gt;70% in tumour volume (OR (95% CI)=1.72 (1.35-2.7, p&amp;lt;0.0001) &amp; 1.87 (1.44-2.42, p&amp;lt;0.0001);respectively), high mitotic count (OR (95% CI)=2.6 (1.73-3.91), p&amp;lt;0.0001) &amp; 1.98 (1.49-2.65), p&amp;lt;0.000);respectively), ER- (OR (95% CI)=0.51 (0.41-0.64); p&amp;lt;0.0001 &amp; 0.53 (0.42-0.68; p&amp;lt;0.0001; respectively, IHC), PR- (OR (95% CI)=0.67 (0.57-0.80: p&amp;lt;0.0001 &amp; 0.62 (0.51-0.77; p&amp;lt;0.0001; respectively, IHC), SPAG5+ (OR (95% CI)=2.36 ( 1.77-3.15; p&amp;lt;0.0001) &amp; 2.15 (1.51-3.06); p=&amp;lt;0.0001); respectively, IHC) and over-expressions of ERBB2 (OR (95% CI)=3.51 (2.26-5.45) &amp; 3.32 (2.15-5.10) ; ps&amp;lt;0.0001, respectively), EGFR (OR (95% CI)=1.43. (1.01-2.03; p=0.004) &amp; 1.48 (1.16-1.89); p=0.002; respectively), GRB7 (OR (95% CI)= 3.78 (2.52-5.64) &amp; 3.56 (2.39-5.29); respectively, ps&amp;lt;0.0001), and CDC6 (OR (95% CI)=2.19 1.65-2.90; p=0.0002 &amp; 2.1 (1.60-2.77); p=0.0005; respectively) were associated with higher pCR after receiving NACT. In HER2+ BC, the differential expression of DLC1, EBB4, RUNDC1, ARHGEF9 &amp; PIK3CD were associated with higher response to Trastuzumab (ps&amp;lt;0.01).In HER2- BC: the intensity of histogram skewness in MRI (p=0.03) and low expression of CXXC5, AR, TGFB3, TACC3, TUBA4A, AGR2, ESR1, TP73 and BAG1 were associated with high response to chemotherapy (ps&amp;lt;0.01). Integrated models were developed for predicting response to NACT of HER2- BC (AUC (95% CI)= 0.83 (0.76-0.91; p&amp;lt;0.0001) &amp; to chemotherapy plus Trastuzumab in HER2+ BC (AUC (95% CI) = 0.79 (0.67-0.91; p&amp;lt;0.0001). Conclusions: A predictive model was developed for HER2+ and another for HER2- BC with &amp;gt;= 80% accuracy prediction of pCR. AI &amp; multiplex technology could enable robust biomarker discovery. ORLower 95% CIUpper 95% CIAdjusted pGRB7-mRNA9.986.7714.706.76E-24ERBB2-mRNA7.765.3011.401.12E-20FOXC1-mRNA0.100.060.166.44E-19MIA-mRNA0.180.110.282.63E-12ANP32E-mRNA0.500.410.622.30E-09MYC-mRNA0.520.410.678.82E-07SFRP1-mRNA0.310.190.491.65E-06CD99-mRNA1.481.261.732.73E-06SPAG5-mRNA1.891.452.474.68E-06PMAIP1-mRNA0.510.390.685.03E-06CDK6-mRNA0.520.390.699.09E-06FGFR4-mRNA2.621.733.979.21E-06CDCA7-mRNA0.400.270.591.13E-05LIN9-mRNA0.610.490.761.25E-05CDC6-mRNA1.941.452.591.44E-05MCM3-mRNA0.660.550.801.55E-05RAD51AP1-mRNA0.570.450.731.58E-05CCND1-mRNA0.450.310.641.93E-05FOXM1-mRNA0.510.380.681.97E-05 Citation Format: Tarek Ma Abdel-Fatah, Graham Ball, Xin Chen, Dalia Mehaisi, Elisabetta Giannotti, Dorothee Auer, Jayakumar Vadakekolathu, Ruizhe Li, Graham Pockley, Stephen Chan. Utilising artificial intelligence (AI) for analysing multiplex genomic and magnetic resonance imaging (MRI) data to develop multimodality predictive system for personalised neoadjuvant treatment of breast cancer (BC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-19.

142 Background: Robust biomarkers for personalization of NHT treatment decisions remains an unmet need. Most assessments of candidate biomarkers to predict NHT resistance have been conducted in clinical trials or academic centers, meriting additional validation in diverse community settings. We sought to correlate real-world outcomes on NHT with comprehensive genomic profiling (CGP)-reported alterations, hypothesizing that AR amplification ( ARamp) and deleterious genomic alterations (GAs) in BRCA2, PTEN, RB1, TP53 would correlate with worse outcomes on NHT. Methods: Following a prespecified analysis plan, pts were selected from Flatiron Health (FH)-Foundation Medicine (FMI) clinico-genomic database (CGDB), a nationwide deidentified electronic health record database linked to FMI CGP. Inclusion criteria: mCRPC diagnosis, treatment in FH network and CGP result between 1/1/11-3/30/20 where tissue collected prior to initiation of first line (1L) or second (2L) NHT (within 180 days for ARamp +/- comparison). A priori power analyses were conducted. Adjusted hazard ratios (aHR) from multivariable Cox proportional hazard models were utilized for TTD and OS comparisons from start of NHT including: GA groups, adjusted for age, line number, practice type, and left truncation. Results: Among 1626 evaluable pts, 397 received 1L (n = 287; 72%) or 2L (n = 110; 28%) NHT with majority treated in community setting (n = 297; 75%). Abiraterone (n = 242; 61%) and enzalutamide (n = 145; 39%) were most common NHTs. Incidence: ARamp (15%) and deleterious GA in TP53 (45%), PTEN (28%), RB1 (3%), and BRCA2 (8%). Cohort was strongly powered to assess TP53 &amp; PTEN, moderately for ARamp &amp; BRCA2, weakly for RB1. As hypothesized, ARamp correlated with worse TTD (aHR: 3.37 [1.26-9.0]) and OS (aHR: 4.92 [1.47-16.5]). BRCA2 GA correlated with improved OS (aHR: 0.41 [0.21-0.81]), but no differences in TTD (aHR: 1.25 [0.82-1.9]). RB1 GA had trends for worse OS (aHR: 2.0 [0.93-4.28]) and worse TTD (aHR – 1.41 [0.72-2.8]). TP53 GA had worse OS (aHR: 1.47 [1.1-2.0]), but no difference in TTD (aHR: 1.08 [0.85-1.4]), and PTEN GA did not correlate with TTD (aHR: 0.94 [0.72-1.2]) or OS (aHR: 1.01 [0.74-1.38]). Conclusions: ARamp is associated with worse TTD and OS in mCRPC pts treated with NHT in real-world, mostly community practice, consistent with prior academic center studies and trials data. Surprisingly, BRCA2 GA correlated with improved OS but not TTD. RB1 GA were directionally consistent with prior studies but underpowered. This study supports using CGP to inform decisions for escalation of non-NHT treatment use in conjunction with patient goals and predictive CGP biomarkers for other drugs. Additional biomarkers, multivariable models and, NHT vs taxane chemo predictive assessments will be reported at symposium.

Introduction Oxide-type all-solid-state Na batteries (ASSBs) are attracted attention owing to their high safety, resource abundant and intrinsic chemical stability, toward electric storage system for renewable energies, such as photovoltaics and wind generations, using electrochemical devices in urban locations. Although the ASSBs shows low output performances and high internal resistances due to their low sinter density in general, the improvement of these properties have been strenuously studied by using the spark-plasma-sintering (SPS) and sintering additives of low melting point material such as B2O3. However, charge-discharge process is complex reactions extremely, which includes redox reactions of electrode, ionic transport of solid electrolyte at macro / micro-scales every component material. Therefore, to perspective understanding of charge-discharge reactions in the ASSBs, the construction of analysis technique is strongly desired by combination of evaluation methods with suitable selection for each scale. Herein, the multi-scale analysis was demonstrated by using Operando scanning electron microscopy / energy dispersive X-ray spectroscopy (SEM / EDS) (µm-scale) and Raman spectroscopy (atomic-order) for observing changes of Na concentration, bonding states during charge-discharge processes, respectively, in addition evaluation of elemental distributions through the time-of-fright secondary ion mass spectrometry (TOF-SIMS) (nm-scale). Experimental The ASSBs were fabricated by multi-step-sintering method using SPS1 from Na3V2(PO4)3 (NVP) as positive (PE) and negative electrode (NE) active materials, Na3Zr2Si2PO12 (NZSP) as solid electrolyte (SE), B2O3 as adhesive between PE/SE and SE/NE layers. The electrode composition were NVP : NZSP : carbon = 25 : 60 : 15 by weight ratio. The sintered ASSB was physically split for arbitrary size, and then obtained piece loaded into a sample holder of Ar-ion milling system in Ar atmosphere to prepare smooth surface of cross-sectional direction. After the Ar-ion milled sample was transferred to holder enabled to apply voltage, Operando SEM-EDS was performed by cyclic voltammetry (CV) with 0.2 mV s-1 of scan rate, 0.5 - 2.5 V of voltage range at room temperature, and acquired elemental mapping images every 0.5 V for 3 min of recording time. As evaluation at 2, 3 cycle, Operando Raman spectroscopy using the same sample was performed by CV with same conditions at room temperature (at 2 cycle) and 60oC (at 3 cycle), using electrochemical measurement cell having observation window of quartz glass. After 3 cycles, the TOF-SIMS mappings were acquired from all area of the cross-sectional ASSB by using plus mode. Results &amp; Discussion From the Operando SEM / EDS, the distributions of Na element clear changed in PE and NE layers under charge-discharge processes, the 2D-dimensional profile on Na counts normalized by P, which is estimated to be not contribute electrochemical reactions, is shown Figure 1 (a). The Na concentration were confirmed decrease / increase trends at charge process, even though there were decreased / increased at discharge process in the PE and NE layers, respectively. These behaviors of concentration change mean to Na transport between the PE and NE layers with deintercalation and intercalation reactions of NVP during charge-discharge processes. On other hand, Figure 1 (b), (c) shows the Raman spectra changes for PE and NE layers at 2 cycle. Although the D- (1380 cm-1) and G- (1520 cm-1) bands related to carbon material did not change, the integrated intensities at about 780 cm-1 clear increased / decreased at PE layer in charge-discharge processes, respectively. These peaks were assigned to PO4 bonding of NVP, and suggested to reversible structural change owing to intercalation and deintercalation reactions of Na. In addition, the calculation result of Na pathway in the NVP was reported by migration between PO4 tetrahedral along the x direction with low activation energy, and should affect bonding states owing to change of backbone structure during charge-discharge processes.2 Therefore, the peak of PO4 bonding is considered as estimation index on the state-of-charge with electrode redox reactions in ASSBs. In other words, intercalation / deintercalation of Na affects not only structural change of NVP, but also concentration change in all areas of PE and NE layers and these reactions could be directly observed by combination of Operando SEM / EDS and Ram.an spectroscopy. In this presentation, we will also report the results of elemental distributions after 3 cycles by acquired from the TOF-SIMS analysis and correlationship between each evaluation technique for ASSB. D. Kutsuzawa, et al., ACS Appl. Energy Mater., 5, 4025–4028 (2022) W. Song et al., J. Mater. Chem. A, 2, 5358 (2014). Figure 1

Most active electrocatalysts include platinum-group metals (PGMs) and their alloys for electrochemical water splitting, but they are strongly limited by material cost and scarcity. [1] On the other hand, transition metal nitrides overcome this bottleneck and show high electrical conductivity, making them catalytically active for water electrolysis. The high conductivity is based on the incorporation of nitrogen atoms into the unit cell of metal, leading to their d-band contraction with electron density enhancement below Fermi level. [2] For instance, metallic nickel nitride (Ni3N) has been widely reported as a promising electrocatalyst material to accelerate the oxygen evolution reaction (OER) by in-situ forming a nickel (oxy)hydroxide [Ni(OH)2 and NiOOH] layer. [3] The Ni3N materials can be prepared by single step soft-urea route to obtain pure crystal phase [4] or a composite with carbon [5], which rises the following two research questions: 1. What are the optimal synthesis parameters of the soft-urea route to control the structure of Ni3N? 2. What is the relationship between structure and OER activity for Ni3N materials? In this work, we used the soft-urea route to prepare Ni3N materials by varying the ratios of NiCl2, urea and ethanol as precursor chemicals. The precursors were made into a gel by magnetic stirring having metal:urea ratio of 1:5, and then heated up to 400 ºC in a tubular furnace with constant flow of argon. The as-prepared Ni3N were then characterized by X-ray diffraction (XRD), elemental analysis (CHN), transmission electron microscopy (TEM) equipped with an energy-dispersive X-ray spectroscopy (EDX) and X-ray absorption spectroscopy (XAS). Based on the quantitative Rietveld refinement, the formation of pristine Ni3N materials is strongly controlled by the annealing temperature. In addition to the Ni3N phase, we observed the formation of the fcc Ni phase (0 – 10 wt.%) as a minor phase. The TEM images show individual particles of below 100 nm in size. Rotating disc electrode (RDE) technique was employed to establish the electrochemical OER activity of Ni3N materials by sweeping the potential from 1.3 to 1.8 VRHE at 10 mV s-1 in Ar-saturated 0.1 M KOH. Polycrystalline Ni and Ni particles are taken as reference materials. We observed an improvement of the OER activity as the overpotential at 10 mA cm-2 (normalized by the geometric surface area) reduced from 379 mV (poly-Ni) to 358 mV (Ni3N). Furthermore, in-situ XAS studies were performed to better understand the nature of the redox reactions between Ni species and the in-situ formation of nickel (oxy)hydroxide [Ni(OH)2 and NiOOH] layers before and during the OER as a function of the applied potential. Based on the in-situ XANES data, we correlated the structure of Ni3N/fcc-Ni/carbon composite with observed OER activity under alkaline conditions. To summarize, we were able to prepare a composite of Ni3N/fcc-Ni/carbon material with improved OER activity via soft-urea route. The in-situ formation of the layered nickel (oxy)hydroxide [Ni(OH)2 and NiOOH] layers as the catalytically active OER species were probed by in-situ XANES data. References: [1] M. Tahir, et.al., Nano Energy. 37 (2017) 136–157. DOI:10.1016/j.nanoen.2017.05.022. [2] S. Wirth et al., Appl. Catal. B Environ. 126 (2012) 225–230. DOI: 10.1016/j.apcatb.2012.07.023. [3] M. Shalom, et al., J.Mater.Chem.A, 3 (2015) 8171–8177. DOI: 10.1021/cm503258z [4] Mohammad Mazloum-Ardakani et al., Materials Science &amp; Engineering B 229 (2018) 201–205. DOI: 10.1016/j.mseb.2017.12.038. [5] Clavel et al., Chem. Eur. J. 20 (2014) 9018 – 9023, https://doi.org/10.1002/chem.201400398

Abstract Background: PSMA is overexpressed by the majority of PC and may be targeted by both antibodies (mAb) and small molecule ligands (SML), each with differing PSMA binding sites, kinetics, and biodistributions. mAbs have long circulating times (exposing organs such a bone marrow) but are too large to target normal tissue luminal PSMA expression in salivary glands, small bowel, and kidney; SMLs rapidly diffuse to all PSMA+ sites and are excreted by the kidneys within hours. Alpha emitters have high potency over a short range whereas beta emitters have lower energy over a significantly longer range. We hypothesized that combining mAb and SML targeting plus combining alpha and beta emitters offered complementary benefits and would be safe and effective. Methods: A series of cell line and xenograft studies were performed to test mAb vs SML vs combo binding, uptake, and efficacy. A phase I/II clinical trial was initiated, enrolling patients with progressive, PSMA+ (by PSMA PET) mCRPC following potent AR pathway inhibition and chemotherapy (or chemo ineligible/refused)[NCT04886986]. The primary endpoint of phase I is assessment of dose-limiting toxicity (DLT) with secondary endpoints of response, progression-free and overall survival, genomic and imaging correlatives, and patient-reported outcomes. Results: Binding studies demonstrated that mAb plus SML binding was additive rather than competitive in LNCaP and CWR22Rv1 cell lines. Uptake of 177Lu-mAb plus -SML in LNCaP, CWR22rv1, and PC3/PSMA xenograft models demonstrated synergism, with 44-65% greater tumor radioactivity in combination than the sum of the individual agents. Intracellular tracking studies indicate that the mAb re-directs SML to lysosomes from recycling endosomes, maintains retention of SML in tumor cells, and, therefore, prolongs intra-tumoral radioactivity exposure. Survival was prolonged in animals receiving 225Ac-mAb plus 177Lu-SML vs either drug alone (or control). Nine men with median age 68 (range 55-87), PSA 140 (2.4-9614) have been enrolled in both of 2 planned dose-escalation cohorts. 89% with bone, 44% lymph node, 22% liver, 22% lung metastases; 88% with detectable CTC count (75% unfavorable). All had at least 1 metastatic lesion with PSMA PET SUVmax &amp;gt; liver SUVmean, with SUVmax of the single hottest lesion ranging from 11.6-69.9. For the primary endpoint, 0 of 3 with DLT in cohort 1, and with follow up ongoing, no DLT has occurred to date in 6 patients in cohort 2. Of evaluable patients, 5 of 6 with PSA decline (8-97% decline). Conclusions: Targeting PSMA with the combination of mAb and SML leads to synergistic radioactivity in preclinical studies. The combination on mAb and SML radiolabeled with alpha and beta emitters appears safe with short-term follow up with phase 2 enrollment planned. Citation Format: Scott T. Tagawa, Edward Fung, Muhammad O. Niaz, Mahelia Bissassar, Sharon Singh, Amie Patel, Angela Tan, Juana Martinez Zuloaga, Sandra Huicochea Castellanos, Jones T. Nauseef, Ana Molina, Cora Sternberg, David M. Nanus, Joseph Osborne, Neil H. Bander. Results of combined targeting of prostate-specific membrane antigen (PSMA) with alpha-radiolabeled antibody 225Ac-J591 and beta-radiolabeled ligand 177Lu-PSMA I&amp;T: preclinical and initial phase 1 clinical data in patients with metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT143.

Abstract Background: Clear cell ovarian carcinoma (CCOC) is characterized by a distinct histologic and molecular profile, and associated with very poor responses to standard treatment consisting of surgery and carboplatin:taxol chemotherapy. CCOC is chemo-resistant at the time of diagnosis and response to chemotherapy in the recurrent setting is less than 10%. Literature suggests a potential role for immune checkpoint inhibitors (ICI). However, only a subgroup of patients (20%) responds to ICI and little is known about the mechanisms of response and resistance to therapy. We postulate that a better understanding of CCOC tumor microenvironment (TME) could help predict patients’ response to ICI. Objective: The aim of this study is to investigate the relationship between TME immune infiltrate and clinical/outcome in 22 CCOC cases and identify subsets of CCOC who may benefit from immunotherapy. Material &amp; Methods: We characterized the immune landscape of 11 early and 11 advanced CCOC through a multiplex IHC Discovery Platform. Spatial single-cell proteomics analyses (cyclic-IF) and spatially-resolved RNAseq in 10 CCOC cases using an OC tissue microarray (TMA) were performed. Results were corelated with clinical and treatment outcome. Results: The percent of CD8, CD4, CD20 B cells Tregs, PD1, PDL1, monocytes and M2 monocytes and myeloid cells was significantly higher in advanced than early-stage cancers. Recurrent cancers were more immunosuppressive than cases with no recurrence. Tumor infiltrate was dense in 4 cases. Four patients with early-stage disease had a high number of CD8 naïve cells and experienced no recurrence. TME analysis of single case of advanced CCOC (before and after chemotherapy), revealed that hot tumor changed to cold tumor, suggesting the resistance to treatment. Cyclic IF analyses identified 3 tumor phenotype groups in a subset of 10 CCOC present on the OC TMA. The majority of CCOC had high expression of CCNE and high PI3K-AKT-mTor activity, low expression of hormone receptors (AR, ERa, PRg) and low cell cycle activity. Some tumors were also high for HER2. The stromal compartment was enriched in collagen VI, aSMA and PDGFR. The immune monitoring of several of those samples also revealed an immunosuppressive microenvironment, including the presence of M2 macrophages and expression of immune checkpoint proteins PD-L1 and B7-H4. Conclusion: Our study shows that various phenotypes of CCOC are defined by the cancer cell profiles and TME content. Importantly, a strong immunosuppressive microenvironment was detected in many samples, suggesting a potential response to ICI. Furthermore, we detected the expression of several therapeutic targets (MAPK, CCNE, PI3K-AKT-mTOR, HSP90, HER2), including oncogenic signaling pathways (RTK, MAPK). Different tumor phenotypes identified across our CCOC samples suggest that clear cell carcinoma could be subclassified into subtypes that should be treated differently. Citation Format: Tanja Pejovic, Sonali Joshi, Shawn Campbell, Dhanir Tailor, Joanna Pucilowska, Benjamin Tate, Pierre-Valérien Abate, Korina Mouzakitis, Marilyne Labrie, Elizabeth Munro, Jenna Emerson, Sanjay V. Malhotra. Study of tumor microenvironment of ovarian clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 79.

Abstract Introduction Acute lymphoblastic leukemia (ALL) is a clonal hematopoietic disorder that originates from B or T lymphoid progenitors and has well validated prognostic and predictive factors that influence outcomes. One of the strongest prognostic factors is the detection of minimal residual disease (MRD) which measures residual cell population after treatment when a morphologic complete response has been achieved. MRD positivity is associated with a higher risk of relapse and poor response to chemo or radiotherapy Objectives The aim of the study was to assess the prognostic impact of post-induction MRD status in a cohort of ALL Colombian patients in terms of relapse-free survival (RFS) and overall survival (OS). Methods This is a retrospective observational study conducted at a Colombian university hospital and included a cohort of ALL patients diagnosed between 2013 and 2020 treatment according to protocol PETHEMA (Spanish Program for Hematology Treatments). MRD was measured with 8-color flow cytometry evaluate on bone marrow. MRD status was classified as negative MRD (NMRD) or positive MRD (PMRD) based on a sensitivity threshold of &amp;lt;0,01% or ≥0,01% leukemic cells, respectively, following to international guides. Demographic and clinical characteristics were analyzed using descriptive statistics. Kaplan-Meier method was used to assess overall RFS and OS. Results A total 128 patients were included. The median age at diagnosis was 34 years (range 0-89 years), 54% were men, 26% were overweight, 22% obese, 6.2% had type 2 DM (T2DM), and most had a ECOG PS of £2 (94%). Most patients (80.5%) had high risk according PETHEMA, B-ALL and were classified as ALL-2 (58%) according to FAB classification with Pre-B cell ALL being the most common phenotype (54.7%). Ph+ ALL was diagnosed in 12% of patients. Most used treatments protocols were PETHEMA-AR and PETHEMA-RI in 43.8% and 11.6% of patients, respectively. Post-induction MRD measurement was available in 98 patients, 36 (36.7%) had NMRD and 62 (63.3%) PMRD. From the 36 patients with NMRD, eight patients (22.2%) received Allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT): two of them, were transplanted in first complete remission, one because of high risk and the other one for BCR-ABL positivity. The other six patients received alloHSCT in second remission and all of them relapsed after late consolidations. Finally, alloHSCT was done in 28 patients with PMRD (45.2%). The 12-month OS for patients with NMRD was 68.7% (95%CI 50.5-81.2) compared to 63.7% (95%CI 50.3-74.4) in the ones with PMRD, p=0.375. 12-month RFS was 83.3% (95%CI 61.5-93.4) in patients with NMRD and 90.0% (95%CI 72.1-96.7) in patients with PMRD, p=0.436. (Figure 1). OS was significantly higher for the PMRD patients who underwent AlloHSCT 96.4% (95%CI 77.2-99.4) versus not underwent 36.8% (95%CI 20.6-53.2), HR: 0.39 (95%CI 0.005-0.29) p=0.002 (Figure 2). Conclusion MRD assessment is a strong prognostic in ALL, however it was not associated with significant differences in RFS or OS in this single institution cohort of Colombian patients. Patients with PMRD taken to AlloHSCT had superior OS compared to NMRD that underwent transplant. A small sample size and short follow-up could explain our results. Larger cohorts with extended follow up and with different MRD methods are needed to better understand the role of MRD assessment in minority ALL populations, such as Colombian patients. Figure 1 Figure 1. Disclosures Peña: Amgen: Research Funding. Salazar: Amgen: Research Funding. Sandoval-Sus: BMS: Other: Advisory Board, Speakers Bureau; SeaGen, Janssen, MassiveBio, TG: Other: Advisory Board. Sossa: Amgen: Research Funding.

Размерные эффекты существенно меняют состояние и физико-химические свойства дисперсных систем. Особенности химических процессов, протекающих в малых (нано-, пико-, фемтолитровых) объемах, важны для технологий получения уникальных материалов. Целью работы явилось экспериментальное подтверждение размерных эффектов при химических процессах в малых объёмах и их интерпретация на основе представлений химической термодинамики.Объектом исследования были реакции органического синтеза, проводимые в ансамблях сидячих капель водных растворов органических соединений, с участием газовой среды. Для наблюдения использовались методы оптической микроскопии с цифровой обработкой изображений. Эксперименты однозначно демонстрируют влияние геометрических параметров (радиус, краевой угол) на кинетику фазовых и химических превращений в полидисперсных ансамблях сидячих капель органических и водно-органических смесей, взаимодействующих с летучими реагентами в газовой среде. 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Amorphous thin film materials in the LiPON(1) or LiSiPON(2) systems have been prepared for the first time in the early 1990s using magnetron sputtering. Since then, LiPON materials have been used as state-of-the-art solid electrolytes into all-solid-state thin film micro-batteries thanks to their outstanding properties (electrochemical stability vs Li°, wide electrochemical stability window of ~[4-0] V vs. Li+/Li0, isotropic and homogeneous medium, beneficial mechanical properties(3), very low electronic conductivity). The ionic conductivity of LiPON prepared from Li3PO4 targets remains rather moderate, reaching a maximum of 3.10-6 S.cm-1 at room temperature. However, it has recently been demonstrated that the introduction of SiO2 as a second glass former can increase this value up to 2.10-5 S.cm-1, and also that the ionic conductivity of these compounds does not increase steadily with their Li content.(4)This highlights the convoluted effects of mixed formers(5) (Si, P, B...), mixed anions(6) (O, N, S...) and Li concentration on the ionic conductivity. Nevertheless, the latter are usually observed on limited series of compositions, and barely not studied on large sets of samples, which can limit the understanding of composition-structure-conductivity relationships. In this context the aim of our work was to build a High Throughput Screening (HTS) approach to explore the LixSiyPzOuNw system as a case study (Fig.1). Indeed, HTS approaches aim to accelerate material breakthrough discovery, and to understand beyond the material mechanisms. In material science, the goal is to accelerate the discovery of new keys organic, inorganic or composite materials. It may also bring beyond ‘classical’ iterative method regarding the study of complex systems (ternary, quaternary...)(7). To do so, wide ranges of material libraries are synthesized, prior to be tested through an automated characterization workflow. Our specific approach starts with the preparation of material libraries by combinatorial synthesis using magnetron co-sputtering with tilted targets, then goes through automated and fast characterizations targeting specific properties, relevant to its application as ionic conductor. Deposition of thin films displaying composition and thickness gradients at the surface of a 4’’ silicon substrate is achieved by co-sputtering three target materials in a pure Ar or N2 atmosphere. Deposition through a shadow mask allows to discretize the continuum and to prepare a library of 76 separate LixSiyPzOuNw samples in one experiment. Then, by tuning the sputtering parameters (i.e discharge gas, gas pressure, incident power, target tilt, target-to-substrate distance,...) different compositional domains can be explored. Relevant automated characterization techniques are then applied to the material library spread on the 4’’ substrate. For the assessment of these amorphous ionic conducting films, dimensional (thickness), physical-chemical (composition &amp; local structure) and functional properties (ionic/electronic conductivities) are studied.In this sequence, determining the composition of the thin film is a real challenge due to a number of requirements :(i) light elements analysis, especially lithium; (ii) need of localized analysis (mapping) on few mm² over large substrate (4” wafer); (iii) scarce material quantity available (~ μg) and finally (iv) fast analysis. Over lab-scale techniques available, only Laser Induced Breakdown Spectroscopy technique (LIBS) seems to fulfil all these requirements. To this purpose, LIBS is developed as a HTS mapping technique for chemical analysis. A sample calibration approach is implemented through coupling with robust chemical analysis techniques (RBS, NRA, ICP-OES, SEM-EDS). In short, this specific HTS procedure will be discussed and results about composition-structure-conductivity relationships in the LiSiPON system will be presented (Fig.2). References J. B. Bates et al., J. Power Sources. 43, 103–110 (1993). N. J. Dudney, J. B. Bates, J. D. Robertson, J. Vac. Sci. Technol. A. 11, 377–389 (1993). A. S. Westover et al., Chem. Mater. 35, 2730–2739 (2023). T. Famprikis, J. Galipaud, O. Clemens, B. Pecquenard, F. Le Cras, ACS Appl. Energy Mater. 2, 4782–4791 (2019). Y. Su et al., Phys. Status Solidi B. 254, 1600088 (2017). N. Mascaraque, J. L. G. Fierro, A. Durán, F. Muñoz, Solid State Ion. 233, 73–79 (2013). E. J. Amis, X.-D. Xiang, J.-C. Zhao, MRS Bull. 27, 295–300 (2002). Figure 1

For widespread utilization of fuel cell system in the future, we have focused on non-precious metal electrocatalyst against high cost of system. Especially, we have studied and reported group 4 and 5 metal oxide-based electrocatalyst as non-platinum catalysts for the oxygen reduction reaction (ORR) because of low-cost, abundant reserves, and high stability in acidic electrolytes [1-2]. We have found excellent ORR activity of Zr oxide-based electrocatalysts prepared from pyrazinecarboxylic acid as a precursor [3-4]. In the case of Ti oxide-based electrocatalysts, the sample prepared under low partial pressure of oxygen with the heat treatment shows high ORR activity when it uses nitrogen-containing porphyrin cyclic organic Ti complexes (TiOTPPz) with multi-walled carbon nanotubes (MWCNTs) as a precursor. However, the heat treatment conditions have not widely investigated yet [5]. In this study, we have investigated to improve ORR activity of Ti oxide-based electrocatalyst prepared from TiOTPPz by varying the oxygen concentration during heat treatment. Oxytitanium tetrapyrazinoporphyrazine (TiOTPPz, [TiOC24H8N16]), a porphyrin cyclic nitrogen-containing organic Ti complex, was used as a starting material. 0.39 g of TiOTPPz and 0.26 g of MWCNT were mixed and treated in a dry ball mill for 1 h to obtain the precursor. The precursor was heated up to 900 oC in an Ar atmosphere and then annealed at 900 oC for 3 h in a low-oxygen atmosphere to obtain a catalyst with precipitated carbon and Ti oxide dispersed on the MWCNTs. The conditions of the hypoxic atmosphere were 2 %H2 + n %O2 / inert gas, with n = 0.05, 0.1, 0.5, 1.0, and 2.0. Catalysts are denoted by TiCNO_(O2 concentration during heat treatment). The catalyst powder was dispersed into 1-propanol with Nafion solution to prepare a catalyst ink. The ink was dropped on a glassy carbon rod, and dried for an hour to use as a working electrode in electrochemical measurement. Electrochemical measurements were performed in 0.5 mol dm-3 H2SO4 at 30 oC with a conventional 3-electrode cell. A reversible hydrogen electrode (RHE) and a glassy carbon plate were used as used as a reference and counter electrode, respectively. Slow scan voltammetry (SSV) was performed at a scan rate of 5 mV s-1 from 0.2 V to 1.2 V vs. RHE under O2 and N2. The ORR current (i ORR) was determined by calculating the difference between the current under O2 and N2. Figure 1 shows oxygen reduction activity of Ti oxide-based electrocatalyst. The vertical axis shows the |i ORR@0.8 V|. The |i ORR@0.8 V| was less than 100 mA gcat -1 at O2 concentrations of 0.05 and 0.1 %, and was greatest at 0.5 % at 413 mA gcat -1. The concentration of O2 further decreased with increasing O2 concentration, and was the smallest at 2.0%. It is suggested that the O2 concentration during heat treatment may have an optimum value around 0.5%. We have also analyzed the catalysts by the XRD before electrochemical measurements. According to XRD spectra, the presence of TiC0.3O0.7 was confirmed in all catalysts. In Ti-CNO_0.5 %, the most active catalyst, the diffraction peaks identified TiO2 rutile and TiO2 anatase were observed. In the case of Ti-CNO_1 and 2 %, the peak intensities of TiO2 anatase increased and the ORR activity decreased. This indicates that the degree of Ti oxidation can be controlled by the atmosphere during heat treatment. Acknowledgement: The authors thank for providing TiOTPPz from Dainichiseika Color &amp; Chemicals Mfg. Co., Ltd. Reference [1] A. Ishihara, Y. Ohgi, K. Matsuzawa, S. Mitsushima, and K. Ota, Electrochim. Acta, 55, 8005 (2010). [2] A. Ishihara, S. Tominaka, S. Mitsushima, H. Imai, H. Imai, O. Sugino, and K. Ota, Curr. Opin. Electrochem., 21 , 234 (2020). [3] Y. Takeuchi, K. Matsuzawa, T. Nagai, K. Ikegami, Y. Kuroda, R. Monden and A. Ishihara, Bull. Chem. Soc. Jpn., 96, 175 (2023). [4] Y. Takeuchi, K. Matsuzawa, Y. Matsuoka, K. Watanabe, T. Nagai, R. Monden and A. Ishihara, Electrochemistry, (2023) in press [5] T. Hayashi, A. Ishihara, T. Nagai, M. Arao, H. Imai, Y. Kohno, K. Matsuzawa, S. Mitsushima and K. Ota, Electrochim. Acta, 209, 1 (2016). Figure 1

<strong>ABSTRACT</strong> Within the testis, glucose is essential for spermatogenesis. Melatonin cooperates with insulin in the regulation of glucose metabolism. Since the effects of melatonin on male reproductive system remain largely indefinite, we investigate the effects of melatonin on (GLUT-1), male hormonal level and oxidative state in testicular tissue in metabolic syndrome. Twenty-four rats had been divided randomly into three groups: control, fructose, and fructose plus melatonin. Metabolic syndrome (MS) was induced by fructose rich diet and melatonin was injected at a dose of 5 mg/kg dissolved in 1% ethanol in normal saline. After the end of the 6th-week of experimental period, body weight, testicular weight, and fat accretion were assessed. Serum lipid profile, glucose, insulin levels, insulin resistance, serum testosterone were measured. Rats were sacrificed by cervical decapitation, fresh testis were used; one for preparation of homogenate (GSH &amp; MDA in testicular homogenate) the other testis underwent immunohistochemistry for GLUT-1 receptors. Fructose consumption significantly increased fasting glucose, fat accretion, serum lipids, insulin levels and insulin resistance, successful establishment of the MS model. Serum testosterone was significantly decreased compared to the control group. In addition, testicular MDA significantly increased and testicular GSH significantly decreased compared to the control group. GLUT-1 expression was increased compared to the control group. Melatonin supplementation significantly decreased fasting blood glucose, fat accretion, serum lipids, insulin levels and insulin resistance, compared to fructose group. Serum testosterone was significantly increased compared to the control group. In addition, testicular MDA significantly decreased and testicular GSH significantly increased compared to the control group. GLUT-1 expression was decreased compared to the control group. Conclusion: GLUT-1 expression may be concerned in glucose metabolism of testicular tissue in fructose induced MS. Melatonin protective effect may be linked to its antioxidant &amp; lipid lowering effect with increased GLUT-1 expression. <strong>Key Words:</strong> Melatonin; fructose; metabolic syndrome; insulin resistance; GLUT-1<em>.</em> <strong>REFERENCES</strong> Srikanthan K, Feyh A, Visweshwar H, Shapiro JI, Sodhi K. Systematic Review of Metabolic Syndrome Biomarkers: A Panel for Early Detection, Management, and Risk Stratification in the West Virginian Population. Int J Med Sci. 2016 Jan 1;13(1):25-38. Malik S, Wong ND, Franklin SS, et al. Impact of the metabolic syndrome on mortality from coronary heart disease, cardiovascular disease, and all causes in United States adults. 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Melatonin improves glucose homeostasis and endothelial vascular function in high-fat diet-fed insulin-resistant mice. Endocrinology 2009, 150, 5311&ndash;5317. Vanecek J. Inhibitory effect of melatonin on GnRH-induced LH release. Rev Reprod 1999, 4:67&ndash;72. Reiter RJ, Rosales-Corral SA, Manchester LC, Tan DX Peripheral reproductive organ health and melatonin: ready for prime time. Int J Mol Sci 2013a; 14:7231-7272. Rato L, Alves MG, Dias TR, Lopes G, Cavaco JE, Socorro S, Oliveira PF High-energy diets may induce a pre-diabetic state altering testicular glycolytic metabolic profile and male reproductive parameters. Andrology 2013; 1:495-504. Oliveira PF, Alves MG, Rato L, Laurentino S, Silva J, Sa R, Barros A, Sousa M, Carvalho RA, Cavaco JE, et al. Effect of insulin deprivation on metabolism and metabolism-associated gene transcript levels of in vitro cultured human Sertoli cells. Biochim Biophys Acta 2012;1820:84-89 Kokk K, Ver&auml;j&auml;nkorva E, Wu XK, Tapfer H, P&otilde;ldoja E, P&ouml;ll&auml;nen P.:Immunohistochemical detection of glucose transporters class I subfamily in the mouse, rat and human testis. Medicina (Kaunas). 2004; 40(2):156-60. Chen Y, Nagpal ML and Lin T Expression and regulation of glucose transporter 8 in rat Leydig cells Journal of Endocrinology 2003; 179, 63&ndash;72. Go&acute;mez O., Romero A., Terrado J and Mesonero JE GLUT8 expression in mouse testis&nbsp;&nbsp;&nbsp; Reproduction 2006; 131 63&ndash;70. Scheepers A, Joost HG, Schurmann A. The glucose transporter families SGLT and GLUT: molecular basis of normal and aberrant function. JPEN J Parenter EnteralNutr 2004; 28: 364&ndash;71 Ding GL, Liu Y, Liu ME, Pan JX, Guo MX, Sheng JZ, Huang HF1.: The effects of diabetes on male fertility and epigenetic regulation during spermatogenesis.Asian J Androl. 2015; 17(6):948-53. 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Hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days. Equal amounts of proteins from urinary bladder or liver extracts of control and arsenic-treated hamsters were labeled with Cy3 and Cy5 dyes, respectively. After differential in gel electrophoresis and analysis by the DeCyder software, several protein spots were found to be down-regulated and several were up regulated. Our experiments indicated that in the bladder tissues of hamsters exposed to arsenite, transgelin was down-regulated and GST-pi was up-regulated. The loss of transgelin expression has been reported to be an important early event in tumor progression and a diagnostic marker for cancer development [29-32]. Down-regulation of transgelin expression may be associated with the carcinogenicity of inorganic arsenic in the urinary bladder. In the liver of arsenite-treated hamsters, ornithine aminotransferase was up-regulated, and senescence marker protein 30 and fatty acid binding protein were down-regulated. The volume ratio changes of these proteins in the bladder and liver of hamsters exposed to arsenite were significantly different than that of control hamsters.&#x0D; Introduction Chronic exposure to inorganic arsenic can cause cancer of the skin, lungs, urinary bladder, kidneys, and liver [1-6]. The molecular mechanisms of the carcinogenicity and toxicity of inorganic arsenic are not well understood [7-9). Humans chronically exposed to inorganic arsenic excrete MMA(V), DMA(V) and the more toxic +3 oxidation state arsenic biotransformants MMA(III) and DMA (III) in their urine [10, 11], which are carcinogen [12]· After injection of mice with sodium arsenate, the highest concentrations of the very toxic MMA(III) and DMA(III) were in the kidneys and urinary bladder tissue, respectively, as shown by experiments of Chowdhury et al [13].&#x0D; Many mechanisms of arsenic toxicity and carcinogenicity have been suggested [1, 7, 14] including chromosome abnormalities [15], oxidative stress [16, 17], altered growth factors [18], cell proliferation [19], altered DNA repair [20], altered DNA methylation patterns [21], inhibition of several key enzymes [22], gene amplification [23] etc. Some of these mechanisms result in alterations in protein expression.&#x0D; Methods for analyzing multiple proteins have advanced greatly in the last several years. In particularly, mass spectrometry (MS) and tandem MS (MS/MS) are used to analyze peptides following protein isolation using two-dimensional (2-D) gel electrophoresis and proteolytic digestion [24]. In the present study, Differential In Gel Electrophoresis (DIGE) coupled with Mass Spectrometry (MS) has been used to study some of the proteomic changes in the urinary bladder and liver of hamsters exposed to sodium arsenite in their drinking water. Our results indicated that transgelin was down-regulated and GST-pi was up-regulated in the bladder tissues. In the liver tissues ornithine aminotransferase was up-regulated, and senescence marker protein 30, and fatty acid binding protein were down-regulated.&#x0D; Materials and Methods &#x0D; &#x0D; Chemicals &#x0D; &#x0D; Tris, Urea, IPG strips, IPG buffer, CHAPS, Dry Strip Cover Fluid, Bind Silane, lodoacetamide, Cy3 and Cy5 were from GE Healthcare (formally known as Amersham Biosciences, Uppsala, Sweden). Thiourea, glycerol, SDS, DTT, and APS were from Sigma-Aldrich (St. Louis, MO, USA). Glycine was from USB (Cleveland, OH, USA). Acrylamide Bis 40% was from Bio-Rad (Hercules, CA, USA). All other chemicals and biochemicals used were of analytical grade. All solutions were made with Milli-Q water.&#x0D; &#x0D; Animals &#x0D; &#x0D; Male hamsters (Golden Syrian), 4 weeks of age, were purchased from Harlan Sprague Dawley, USA. Upon arrival, hamsters were acclimated in the University of Arizona animal care facility for at least 1 week and maintained in an environmentally controlled animal facility operating on a 12-h dark/12-h light cycle and at 22-24°C. They were provided with Teklad (Indianapolis, IN) 4% Mouse/Rat Diet # 7001 and water, ad libitum, throughout the acclimation and experimentation periods.&#x0D; &#x0D; Sample preparation and labelling &#x0D; &#x0D; Hamsters were exposed to sodium arsenite (173 mg) in drinking water for 6 days and the control hamsters were given tap water. On the 6th day hamsters were decapitated rapidly by guillotine. Urinary bladder tissues and liver were removed, blotted on tissue papers (Kimtech Science, Precision Wipes), and weighed. Hamster urinary bladder or liver tissues were homogenized in lysis buffer (30mMTris, 2M thiourea, 7M urea, and 4% w/w CHAPS adjusted to pH 8.5 with dilute HCI), at 4°C using a glass homogenizer and a Teflon coated steel pestle; transferred to a 5 ml acid-washed polypropylene tube, placed on ice and sonicated 3 times for 15 seconds. The sonicate was centrifuged at 12,000 rpm for 10 minutes at 4°C. Small aliquots of the supernatants were stored at -80°C until use (generally within one week). Protein concentration was determined by the method of Bradford [25] using bovine serum albumin as a standard. Fifty micrograms of lysate protein was labeled with 400 pmol of Cy3 Dye (for control homogenate sample) and Cy5 Dye (for arsenic-treated urinary bladder or liver homogenate sample). The samples containing proteins and dyes were incubated for 30 min on ice in the dark. To stop the labeling reaction, 1uL of 10 mM lysine was added followed by incubation for 10 min on ice in the dark. To each of the appropriate dye-labeled protein samples, an additional 200 ug of urinary bladderor liver unlabeled protein from control hamster sample or arsenic-treated hamster sample was added to the appropriate sample. Differentially labeled samples were combined into a single Microfuge tube (total protein 500 ug); protein was mixed with an equal volume of 2x sample buffer [2M thiourea, 7M urea, pH 3-10 pharmalyte for isoelectric focusing 2% (v/v), DTT 2% (w/v), CHAPS 4% (w/v)]; and was incubated on ice in the dark for 10 min. The combined samples containing 500 ug of total protein were mixed with rehydration buffer [CHAPS 4% (w/v), 8M urea, 13mM DTT, IPG buffer (3-10) 1% (v/v) and trace amount of bromophenol blue]. The 450 ul sample containing rehydration buffer was slowly pipetted into the slot of the ImmobilinedryStripReswelling Tray and any large bubbles were removed. The IPG strip (linear pH 3-10, 24 cm) was placed (gel side down) into the slot, covered with drystrip cover fluid (Fig. 1), and the lid of the Reswelling Tray was closed. The ImmobillineDryStrip was allowed to rehydrate at room temperature for 24 hours.&#x0D; &#x0D; First dimension Isoelectric focusing (IEF) &#x0D; &#x0D; The labeled sample was loaded using the cup loading method on universal strip holder. IEF was then carried out on EttanIPGphor II using multistep protocol (6 hr @ 500 V, 6 hr @ 1000 V, 8 hr @ 8000 V). The focused IPG strip was equilibrated in two steps (reduction and alkylation) by equilibrating the strip for 10 min first in 10 ml of 50mM Tris (pH 8.8), 6M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 0.5% (w/v) DTT, followed by another 10 min in 10 ml of 50mM Tris (pH 8.8), 6M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 4.5% (w/v) iodoacetamide to prepare it for the second dimension electrophoresis.&#x0D; &#x0D; Second dimension SDS-PAGE &#x0D; &#x0D; The equilibrated IPG strip was used for protein separation by 2D-gel electrophoresis (DIGE). The strip was sealed at the top of the acrylamide gel for the second dimension (vertical) (12.5% polyacrylamide gel, 20x25 cm x 1.5 mm) with 0.5% (w/v) agarose in SDS running buffer [25 mMTris, 192 mM Glycine, and 0.1% (w/v) SDS]. Electrophoresis was performed in an Ettan DALT six electrophoresis unit (Amersham Biosciences) at 1.5 watts per gel, until the tracking dye reached the anodic end of the gel.&#x0D; &#x0D; Image analysis and post-staining &#x0D; &#x0D; The gel then was imaged directly between glass plates on the Typhoon 9410 variable mode imager (Sunnyvale, CA, USA) using optimal excitation/emission wavelength for each DIGE fluor: Cy3 (532/580 nm) and Cy5 (633/670 nm). The DIGE images were previewed and checked with Image Quant software (GE Healthcare) where all the two separate gel images could be viewed as a single gel image. DeCyde v.5.02 was used to analyze the DIGE images as described in the Ettan DIGE User Manual (GE Healthcare). The appropriate up-/down regulated spots were filtered based on an average volume ratio of ± over 1.2 fold. After image acquisition, the gel was fixed overnight in a solution containing 40% ethanol and 10% acetic acid. The fixed gel was stained with SyproRuby (BioRad) according to the manufacturer protocol (Bio-Rad Labs., 2000 Alfred Nobel Drive, Hercules, CA 94547).&#x0D; &#x0D; Identification of proteins by MS &#x0D; &#x0D; Protein spot picking and digestion &#x0D; &#x0D; &#x0D; &#x0D; Sypro Ruby stained gels were imaged using an Investigator ProPic and HT Analyzer software, both from Genomic Solutions (Ann Arbor, MI). Protein spots of interest that matched those imaged using the DIGE Cy3/Cy5 labels were picked robotically, digested using trypsin as described previously [24] and saved for mass spectrometry identification.&#x0D; &#x0D; Liquid chromatography (LC)- MS/MS analysis &#x0D; &#x0D; LC-MS/MS analyses were carried out using a 3D quadrupole ion trap massspectrometer (ThermoFinnigan LCQ DECA XP PLUS; ThermoFinnigan, San Jose, CA) equipped with a Michrom Paradigm MS4 HPLC (MichromBiosources, Auburn, CA) and a nanospray source, or with a linear quadrupole ion trap mass spectrometer (ThermoFinnigan LTQ), also equipped with a Michrom MS4 HPLC and a nanospray source. Peptides were eluted from a 15 cm pulled tip capillary column (100 um I.D. x 360 um O.D.; 3-5 um tip opening) packed with 7 cm Vydac C18 (Vydac, Hesperia, CA) material (5 µm, 300 Å pore size), using a gradient of 0-65% solvent B (98% methanol/2% water/0.5% formic acid/0.01% triflouroacetic acid) over a 60 min period at a flow rate of 350 nL/min. The ESI positive mode spray voltage was set at 1.6 kV, and the capillary temperature was set at 200°C. Dependent data scanning was performed by the Xcalibur v 1.3 software on the LCQ DECA XP+ or v 1.4 on the LTQ [27], with a default charge of 2, an isolation width of 1.5 amu, an activation amplitude of 35%, activation time of 50 msec, and a minimal signal of 10,000 ion counts (100 ion counts on the LTQ). Global dependent data settings were as follows: reject mass width of 1.5 amu, dynamic exclusion enabled, exclusion mass width of 1.5 amu, repeat count of 1, repeat duration of a min, and exclusion duration of 5 min. Scan event series were included one full scan with mass range of 350-2000 Da, followed by 3 dependent MS/MS scans of the most intense ion.&#x0D; &#x0D; Database searching &#x0D; &#x0D; Tandem MS spectra of peptides were analyzed with Turbo SEQUEST, version 3.1 (ThermoFinnigan), a program that allows the correlation of experimental tandem MS data with theoretical spectra generated from known protein sequences. All spectra were searched against the latest version of the non redundant protein database from the National Center for Biotechnology Information (NCBI 2006; at that time, the database contained 3,783,042 entries).&#x0D; &#x0D; Statistical analysis &#x0D; &#x0D; The means and standard error were calculated. The Student's t-test was used to analyze the significance of the difference between the control and arsenite exposed hamsters. P values less than 0.05 were considered significant. The reproducibility was confirmed in separate experiments.&#x0D; Results &#x0D; &#x0D; Analysis of proteins expression &#x0D; &#x0D; After DIGE (Fig. 1), the gel was scanned by a Typhoon Scanner and the relative amount of protein from sample 1 (treated hamster) as compared to sample 2 (control hamster) was determined (Figs. 2, 3). A green spot indicates that the amount of protein from sodium arsenite-treated hamster sample was less than that of the control sample. A red spot indicates that the amount of protein from the sodium arsenite-treated hamster sample was greater than that of the control sample. A yellow spot indicates sodium arsenite-treated hamster and control hamster each had the same amount of that protein.&#x0D; Several protein spots were up-regulated (red) or down-regulated (green) in the urinary bladder samples of hamsters exposed to sodium arsenite (173 mg As/L) for 6 days as compared with the urinary bladder of controls (Fig. 2).&#x0D; In the case of liver, several protein spots were also over-expressed (red) or under-expressed (green) for hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days (Fig. 3).&#x0D; The urinary bladder samples were collected from the first and second experiments in which hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days and the controls were given tap water. The urinary bladder samples from the 1st and 2nd experiments were run 5 times in DIGE gels on different days. The protein expression is shown in Figure 2 and Table 1.&#x0D; The liver samples from the 1st and 2nd experiments were also run 3 times in DIGE gels on different days. The proteins expression were shown in Figure 3 and Table 2. The volume ratio changed of the protein spots in the urinary bladder and liver of hamsters exposed to arsenite were significantly differences than that of the control hamsters (Table 1 and 2).&#x0D; &#x0D; Protein spots identified by LC-MS/MS&#x0D; &#x0D; Bladder &#x0D; &#x0D; &#x0D; &#x0D; The spots of interest were removed from the gel, digested, and their identities were determined by LC-MS/MS (Fig. 2 and Table 1). The spots 1, 2, &amp; 3 from the gel were analyzed and were repeated for the confirmation of the results (experiments; 173 mg As/L). The proteins for the spots 1, 2, and 3 were identified as transgelin, transgelin, and glutathione S-transferase Pi, respectively (Fig. 2).&#x0D; &#x0D; Liver &#x0D; &#x0D; We also identified some of the proteins in the liver samples of hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days (Fig. 3). The spots 4, 5, &amp; 6 from the gels were analyzed and were repeated for the confirmation of the results. The proteins for the spots 4, 5, and 6 were identified as ornithine aminotransferase, senescence marker protein 30, and fatty acid binding protein, respectively (Fig. 3)&#x0D; Discussion The identification and functional assignment of proteins is helpful for understanding the molecular events involved in disease. Weexposed hamsters to sodium arsenite in drinking water. Controls were given tap water. DIGE coupled with LC-MS/MS was then used to study the proteomic change in arsenite-exposed hamsters. After electrophoresis DeCyder software indicated that several protein spots were down-regulated (green) and several were up-regulated (red).&#x0D; Our overall results as to changes and functions of the proteins we have studied are summarized in Table 3.&#x0D; &#x0D; Bladder &#x0D; &#x0D; In the case of the urinary bladder tissue of hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days, transgelin was down-regulated and GST-pi was up-regulated. This is the first evidence that transgelin is down-regulated in the bladders of animals exposed to sodium arsenite.&#x0D; Transgelin, which is identical to SM22 or WS3-10, is an actin cross linking/gelling protein found in fibroblasts and smooth muscle [28, 29]. It has been suggested that the loss of transgelin expression may be an important early event in tumor progression and a diagnostic marker for cancer development [30-33]. It may function as a tumor suppressor via inhibition of ARA54 (co-regulator of androgen receptor)-enhanced AR (androgen receptor) function. Loss of transgelin and its suppressor function in prostate cancer might contribute to the progression of prostate cancer [30]. Down-regulation of transgelin occurs in the urinary bladders of rats having bladder outlet obstruction [32]. Ras-dependent and Ras-independent mechanisms can cause the down regulation of transgelin in human breast and colon carcinoma cell lines and patient-derived tumorsamples [33]. Transgelin plays a role in contractility, possibly by affecting the actin content of filaments [34]. In our experiments loss of transgelin expression may be associated or preliminary to bladder cancer due to arsenic exposure. Arsenite is a carcinogen [1].&#x0D; In our experiments, LC-MS/MS analysis showed that two spots (1 and 2) represent transgelin (Fig. 2 and Table 1). In human colonic neoplasms there is a loss of transgelin expression and the appearance of transgelin isoforms (31).&#x0D; GST-pi protein was up-regulated in the bladders of the hamsters exposed to sodium arsenite. GSTs are a large family of multifunctional enzymes involved in the phase II detoxification of foreign compounds [35]. The most abundant GSTS are the classes alpha, mu, and pi classes [36]. They participate in protection against oxidative stress [37]. GST-omega has arsenic reductase activity [38].&#x0D; Over-expression of GST-pi has been found in colon cancer tissues [39]. Strong expression of GST-pi also has been found in gastric cancer [40], malignant melanoma [41], lung cancer [42], breast cancer [43] and a range of other human tumors [44]. GST-pi has been up-regulated in transitional cell carcinoma of human urinary bladder [45]. Up-regulation of glutathione – related genes and enzyme activities has been found in cultured human cells by sub lethal concentration of inorganic arsenic [46].&#x0D; There is evidence that arsenic induces DNA damage via the production of ROS (reactive oxygen species) [47]. GST-pi may be over-expressed in the urinary bladder to protect cells against arsenic-induced oxidative stress.&#x0D; &#x0D; Liver &#x0D; &#x0D; In the livers of hamsters exposed to sodium arsenite, ornithine amino transferase was over-expressed, senescence marker protein 30 was under-expressed, and fatty acid binding protein was under-expressed. Ornithine amino transferase has been found in the mitochondria of many different mammalian tissues, especially liver, kidney, and small intestine [48]. Ornithine amino transferase knockdown inhuman cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death [49]. It has been suggested that ornithine amino transferase has a role in regulating mitotic cell division and it is required for proper spindle assembly in human cancer cells [49].&#x0D; Senescence marker protein-30 (SMP30) is a unique enzyme that hydrolyzes diisopropylphosphorofluoridate. SMP30, which is expressed mostly in the liver, protects cells against various injuries by stimulating membrane calcium-pump activity [50]. SMP30 acts to protect cells from apoptosis [51]. In addition it protects the liver from toxic agents [52]. The livers of SMP30 knockout mice accumulate phosphatidylethanolamine, cardiolipin, phosphatidyl-choline, phosphatidylserine, and sphingomyelin [53].&#x0D; Liver fatty acid binding protein (L-FABP) also was down- regulated. Decreased liver fatty acid-binding capacity and altered liver lipid distribution hasbeen reported in mice lacking the L-FABP gene [54]. High levels of saturated, branched-chain fatty acids are deleterious to cells and animals, resulting in lipid accumulation and cytotoxicity. The expression of fatty acid binding proteins (including L-FABP) protected cells against branched-chain saturated fatty acid toxicity [55].&#x0D; Limitations: we preferred to study the pronounced spots seen in DIGE gels. Other spots were visible but not as pronounced. Because of limited funds, we did not identify these others protein spots.&#x0D; In conclusion, urinary bladders of hamsters exposed to sodium arsenite had a decrease in the expression of transgelin and an increase in the expression of GST-pi protein. Under-expression of transgelin has been found in various cancer systems and may be associated with arsenic carcinogenicity [30-33). Inorganic arsenic exposure has resulted in bladder cancer as has been reported in the past [1]. Over-expression of GST-pi may protect cells against oxidative stress caused by arsenite. In the liver OAT was up regulated and SMP-30 and FABP were down regulated. These proteomic results may be of help to investigators studying arsenic carcinogenicity.&#x0D; The Superfund Basic Research Program NIEHS Grant Number ES 04940 from the National Institute of Environmental Health Sciences supported this work. Additional support for the mass spectrometry analyses was provided by grants from NIWHS ES06694, NCI CA023074 and the BIOS Institute of the University of Arizona. &#x0D; Acknowledgement &#x0D; The Author wants to dedicate this paper to the memory of his former supervisor Dr. H. VaskenAposhian who passed away in September 6, 2019. He was an emeritus professor of the Department of Molecular and Cellular Biology at the University of Arizona. 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This development research is motivated by the low learning outcomes of students and the lack of motivation students in learning chemistry, especially on solubility and solubility result at SMAN 1 Mandor. This study aims to produce worksheets based on chemo-edutainment student on solubility and solubility result. This type of research is the development of the ADDIE Research and Development (R&amp;D) model. The instruments used were validation sheets, questionnaire sheet for teacher and student responses and test sheets for pretest and posttest questions. The analysis shows the validity criteria obtaine by the validator 1.0 value with a very valid category. The results of the questionaire respons of primary field trial students obtained an average percentage value of 88.57 %. The efective criteria for chemo-edutainment based worksheets were seen from the pretest and posttest results of students calculated using N-gain formula, the value obtained in the main field trial is 0.85 (very high). Based on these results, it can be conculuded that the development of chemo-edutainment based student worksheets is feasible to be used as teaching material on solubility material and solubility results in class XI IPA in SMAN 1 Mandor.

Gas-phase dissociation of fluorine ( 1 Ʃ + g ) molecules in an agron bath at 3000 K was studied by using the 3D Monte Carlo classical trajectory (3DMCCT) method. To assess the importance of the potential energy surface (PES) in such calculations, three surfaces, with a fixed, experimentally determined F 2 dissociation energy, were constructed. These surfaces span the existing experimental uncertainties in the shape of the F 2 potential. The first potential was the widest and softest; in the second potential the anharmonicity was minimized. The intermediate potential was constructed to ‘localize’ anharmonicity in the energy range in which the collisions are most reactive. The remaining parameters for each PES were estimated from the best available data on interatomic potentials. By using the single uniform ensemble (SUE) method (Kutz, H. D. &amp; Burns, G. J. chem. Phys . 72, 3652-3657 (1980)), large ensembles of trajectories (LET) were generated for the PES. Two such ensembles consisted of 30000 trajectories each and the third of 26200. It was found that the computed one-way-flux equilibrium rate coefficients (Widom, B. Science 148, 1555-1560 (1965)) depend in a systematic way upon the anharmonicity of the potential, with the most anharmonic potential yielding the largest rate coefficient. Steady-state reaction-rate constants, which correspond to experimentally observable rate constants, were calculated by the SUE method. It was determined that this method yields (for a given trajectory ensemble, PES and translational temperature) a unique steady-state rate constant, independent of the initial, arbitrarily chosen, state (Tolman, R. C. The principles of statistical mechanics , p. 17. Oxford University Press (1938)) of the LET, and consequently independent of the corresponding initial value of the reaction rate coefficient. For each initial state of the LET, the development of the steady-state rate constant from the equilibrium rate coefficient was smooth, monotonic, and consistent with the detailed properties of the PES. It was found that, although the increased anharmonicity of the F 2 potential enhanced the equilibrium rate coefficients, it also enhanced the non-equilibrium effects. As a result, the steady-state rate constants were found to be insensitive to the variation of the PES. Thus, the differences among the steady-state rate constants for the three potentials were of the order of their standard errors, which was about 15% or less. On the other hand, the calculated rate constants exceeded the experimental rate constant by a factor of five to six. Because within the limitations of classical mechanics the calculations were ab initio , it was tentatively concluded that the discrepancy of five to six is due to the use of classical mechanics rather than details of the PES structure.

The beta 1 and beta 2 adrenoceptors (β1 and β2 AR) are GPCRs for the catecholamines norepinephrine and epinephrine. Both of these receptors couple to Gsα. Their activation causes cAMP elevation, which in turn controls cellular signaling through its downstream effectors PKA and the cAMP guanine nucleotide exchange factors (GEFs) Epac1 and Epac2. The β2AR has also been shown to signal via engagement of β‐arrestin, which acts as a scaffold for a variety of signaling proteins, including the MAP kinase ERK. We have recently characterized a neuronal and endocrine‐specific cAMP sensor, a GEF related to Epac 1 and 2, and to the previously characterized non‐cAMP‐activated GEF PDZ‐GEF1, which we have named NCS (neuritogenic cyclic AMP sensor)‐Rapgef2 (a protein product of the Rapgef2 gene). This sensor mediates activation of ERK leading to neuritogenesis in the PC12 and NS‐1 neuroendocrine cell lines (Emery et al., Sci. Signal. 6, ra51, 2013; Emery et al., J. Biol. Chem. 289: 10125, 2014). We have created NS‐1 cell lines stably expressing either β1AR or β2AR, and examined signaling to each of the three cAMP sensors present in these cells following treatment with isoproterenol. In β1AR‐expressing cells, agonist treatment caused activation of all measurable cAMP‐dependent signaling pathways in these cells: Epac2/p38‐dependent growth arrest; PKA‐dependent CREB phosphorylation; and NCS‐Rapgef2/ERK‐dependent neuritogenesis. In contrast, agonist stimulation of β2AR‐expressing cells caused isoproterenol‐initiated Epac2/p38‐dependent growth arrest and PKA‐dependent CREB phosphorylation, but did not couple to NCS‐Rapgef2/ERK‐dependent neuritogenesis. To compare the desensitization profiles of the two receptors, a biosensor that allowed for continuous real‐time cyclic AMP measurements was co‐expressed in each cell line (Emery et al., Peptides, 79: 39, 2016). In β2AR‐expressing cells, the maximal effect of isoproterenol on cAMP was observed after 10 minutes of treatment and decreased rapidly thereafter. In contrast, isoproterenol‐dependent β1AR activation caused persistent cAMP elevation, observed at approximately maximal levels at least 40 minutes following agonist addition. Unlike the mode of ERK phosphorylation observed following β1AR activation (NCS‐Rapgef2‐dependent), ERK activation elicited by β2AR (NCS‐Rapgef2‐independent) most likely occurs in a cellular compartment restricted from transcriptional regulation, which is required for neuritogenesis in this cell type (Ravni et al., Mol. Pharmacol. 73: 1688, 2008). We conclude that there is an inverse relationship between adrenergic receptor desensitization, and engagement of NCS‐Rapgef2 of sufficient duration to support the sustained activation of ERK necessary to promote neuritogenesis in NS‐1 cells. We notice the same inverse relationship in receptors for dopamine (D1) and adenosine (A2A), as well as the neuropeptides PACAP, VIP, and GLP‐1 (PAC1, VPAC1, VPAC2, and GLP‐1R).Support or Funding InformationThis work was supported by NIMH Intramural Research Program Project ZIAMH002386 and by a 2014 NARSAD Young Investigator Grant to A.C.E. from the Brain and Behavior Research Foundation (Grant 21356).

Authors: Jinquan Yu ### Abstract The most common bond in many organic compounds is the C–H bond. Hence, it is a great challenge to selectively cleave a particular C–H bond in the presence of multiple ones. One of most widely used approach to this problem is the use of -chelating directing groups (1). However, the insertion of the transition metal is strictly restricted to the ortho-C–H bond through a six- or seven-membered cyclic pre-transition state (TS). Although many strategies have been developed to selectively functionalize meta- and para-C–H bonds (2–4), this newly developed template approach overcomes the intrinsic steric and electronic bias of the substrates, and allows for the activation of remote C–H bonds. ### Introduction The most common bond in many organic compounds is the C–H bond. Hence, it is a great challenge to selectively cleave a particular C–H bond in the presence of multiple ones. One of most widely used approach to this problem is the use of -chelating directing groups (1). However, the insertion of the transition metal is strictly restricted to the ortho-C–H bond through a six- or seven-membered cyclic pre-transition state (TS). Although many strategies have been developed to selectively functionalize meta- and para-C–H bonds (2–4), this newly developed template approach overcomes the intrinsic steric and electronic bias of the substrates, and allows for the activation of remote C–H bonds. ### Reagents 1. Palladium pivalate (Sigma-Aldrich, cat. no. 721611) - Palladium(II) acetate (Sigma-Aldrich, cat. no. 205869) - Ethyl acrylate, contains 10-20 ppm MEHQ as inhibitor (Sigma-Aldrich, cat. no. E9706) - Silver pivalate (made from silver nitrate and pivalic acid) - Silver acetate (Sigma-Aldrich, cat. no. 85140) - Ac-Gly-OH (Novabiochem cat. no. 04-12-8006) - 1,2-Dichloroethane, anhydrous grade (Sigma-Aldrich, cat. no. 284505) - 1,1,1,3,3,3-Hexafluoro-2-propanol (Oakwood Products, cat. no. 003409) - Celite® 545 coarse (EMD Chemical, cat. no. CX0574-3) - 2 M Hydrochloric acid (prepared from concentrated HCl, EMD Chemicals, ACS grade, cat. no. HX0603-75) - Diethyl ether, (Fisher Chemical, Anhydrous; BHT Stabilized/Certified ACS; cat. no. E13820) - Ethyl acetate, (EMD Chemical, Reagent A.C.S, cat. no. CX0240-3) - Hexanes, (Avantor Performance Materials, AR A.C.S grade, cat. no. MK518922 ) - Thin-layer chromatography plates on glass backing, silica gel 60 F254 (Merck) - Potassium permanganate thin-layer chromatography visualizing stain - Preparative TLC plates, 500 μm with fluorescent indicator (Sigma-Aldrich, cat. no. Z513032) ### Equipment 1. Magnetic hotplate stirrer (IKA® RCT Basic or Corning PC420D) - Digital temperature probe - Oil bath (Silicone oil from Alfa Aesar, cat. no. A12728-0E) - Pressure vessels, heavy wall, with a Teflon bushing, 15 mL or 30 ml (Chemglass, cat. no. CG-1880-01 or CG-1880-02) - Disposable syringes (Norm-Ject cat. no. 53548-000) - Disposable needles (BD Presicion needle, cat. no. 305196) - Teflon-coated magnetic stirrer bar (various brand?) - Balloon fitted to disposable 2.5 mL syringe barrel - Büchner filter funnels with inner joints and coarse frit, 15mL - Rotary evaporator (Heidolph) - Pyrex chromatographic column (approx. diameter 3 cm) - NMR tubes ### Procedure **Toluene derivatives** 1. On the same piece of weighing paper, weigh all the solids. Weigh the substrate (0.10 mmol) first, followed by Pd(OPiv)2 (3.0 mg, 0.010 mmol, 0.10 equiv.) and AgOPiv (62.7 mg, 0.30 mmol, 3.0 equiv.). The solids were carefully transferred into the bottom of a 15 mL pressure vessel equipped with a Teflon-coated magnetic stirrer bar. - Add ethyl acrylate (16.5 µL, 0.15 mmol, 1.5 equiv.) to the solid mixture. - Wash down the solids on the sides of wall with 1,2-dichloroethane (1.0 mL). - Cap the tube and submerge it into a pre-heated 90 °C (controlled by a digital temperature probe) oil bath. - Cover the tube and oil bath with aluminum foil and leave the reaction stirring for a total of 42–48 hours. - Lift vessel out of the oil bath and submerge it into ice bath for 10 minutes. - Filter the reaction mixture through a short pad of Celite® into a scintillation vial and wash the tube and Celite® pad three times with 2 mL of diethyl ether. Evaporate the solvent to dryness using a rotary evaporator. - Purify the desired product by preparative silica gel thin-layer chromatography eluting with hexane:ethyl acetate to yield the desired olefinated product. **Hydrocinnamic acid derivatives** 1. On the same piece of weighing paper, weigh all the solids sequentially as followed: Pd(OAc)2 (2.3 mg, 0.010 mmol, 10 mol%), Ac-Gly-OH (2.4 mg, 0.020 mmol, 20 mol%), AgOAc (50 mg, 0.30 mmol, 3.0 equiv.) and the substrate (0.10 mmol). The solids were carefully transferred into the bottom of a 35 mL pressure vessel pre-equipped with a Teflon-coated magnetic stirrer bar. - HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol) (0.60 mL) was added to the mixture to wash down the solids on the sides of wall, followed by ethyl acrylate (22 µL, 2.0 equiv.) and then another 0.60 mL of HFIP. - Cap the tube tightly and submerge it into a pre-heated 90 °C (controlled by a digital temperature probe) oil bath. - Leave the reaction stirring for 24 hours. - Lift vessel out of the oil bath and filtrate the reaction mixture through a short pad of Celite® into a 50 ml round bottom flask after it cools down. - Evaporate the solvent to dryness using a rotary evaporator. - Isolate the desired product by preparative silica gel thin-layer chromatography eluting with hexane:ethyl acetate to yield the desired olefinated product. ### Timing - Toluene derivatives: 42 hours (electron-donating substituents); 48 hours (electron-withdrawing substituents) - Hydrocinnamic acid derivatives: 24 hours ### Troubleshooting - Toluene derivatives - Poor separation of the major meta-isomer from the minor isomers: Preparative thin-layer chromatography is usually the first choice to achieve better separation. Repetitive running of the thin-layer chromatography using less polar eluent is recommended to achieve good resolution. - Hydrocinnamic acid derivatives - Problem: Poor separation of mono-olefinated meta-isomer from the di-olefinated meta-isomer (when applicable), and poor separation of the major meta-isomer from the trace minor isomers for some substrates. - Solutions: Especially for those substrates with di-olefinated product, preparative thin-layer chromatography is usually employed to achieve better separation. Repetitive developing of the thin-layer chromatography (3 to 5 times) using relative less polar eluent is recommended. ### Anticipated Results Typical isolated yield of toluene derivatives should be 46–98% depending on the substituents on the aromatic ring and olefins used. Typical yield of hydrocinnamic acid derivatives should be 67–93% depending on the substituents on the aromatic ring. Longer reaction time than 24 hours may lead to more di-olefinated products; however, shorter reaction time could cause incomplete reaction. ### References 1. Engle, K. M., Mei, T.-S., Wasa, M. &amp; Yu, J.-Q. Weak coordination as a powerful means for developing broadly useful C–H functionalization reactions. *Acc. Chem. Res*., DOI:10.1021/ar200185g (2011). - Zhang, Y.-H., Shi, B.-F. &amp; Yu, J.-Q. Pd(II)-catalyzed olefination of electron-deficient arenes using 2,6-dialkylpyridine ligands. *J. Am. Chem. Soc*. 131, 5072–5074 (2009). - Wang, X., Leow, D. &amp; Yu, J.-Q. Pd(II)-catalyzed para-selective C–H arylation of monosubstituted arenes. *J. Am. Chem. Soc*. 133, 13864–13867 (2011). - Ye, M., Gao, G.-L. &amp; Yu, J.-Q. Ligand-promoted C-3 selective C–H olefination of pyridines with Pd catalysts. *J. Am. Chem. Soc*. 133, 6964–6967 (2011). - Leow, D., Li, G., Mei, T.-S. &amp; Yu, J.-Q. Activation of Remote meta-C–H Bonds Assisted by an End-on Template. *Nature*, in press (2012). ### Associated Publications **Activation of remote meta-C–H bonds assisted by an end-on template**. Dasheng Leow, Gang Li, Tian-Sheng Mei, and Jin-Quan Yu. *Nature* 486 (7404) 518 - 522 27/06/2012 [doi:10.1038/nature11158](http://dx.doi.org/10.1038/nature11158) ### Author information **Jinquan Yu**, Yu Lab, The Scripps Research Institute Correspondence to: Jinquan Yu (yu200@scripps.edu) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2390) (2012) doi:10.1038/protex.2012.018. Originally published online 29 June 2012*.

Abstract STUDY QUESTION What is the influence of age and chemotherapy regimen on the longitudinal blood anti-Müllerian hormone (AMH) variations in a large series of adolescents and young adult (AYA) (15–24 years old) and non-AYA (25–35 years old) lymphoma patients? SUMMARY ANSWER In case of alkylating regimen treatment, there was a deep and sustained follicular depletion in AYA as well as non-AYA patients; however in both groups, the ovarian toxicity was extremely low in cases of non-alkylating treatments. WHAT IS KNOWN ALREADY AMH is now well-recognised to be a real-time indicator of ovarian follicular depletion and recovery in women treated by chemotherapy. Its longitudinal variations may discriminate between highly and minimally toxic protocols regarding ovarian function. It has been shown, in different cancer types, that age, type of chemotherapy regimen and pre-treatment AMH levels are the main predictors of ovarian recovery. Large studies on longitudinal AMH variations under chemotherapy in lymphoma patients are few but can provide the opportunity to assess the degree of follicle loss at a young age. STUDY DESIGN, SIZE, DURATION This prospective cohort study was conducted in the Fertility Observatory of the Lille University Hospital. Data were collected between 2007 and 2016. Non-Hodgkin or Hodgkin lymphoma patients (n = 122) between 15 and 35 years old were prospectively recruited before commencing chemotherapy. Patients were treated either by a non-alkylating protocol (ABVD group; n = 67) or by an alkylating regimen (alkylating group; n = 55). PARTICIPANTS/MATERIALS, SETTING, METHODS Serial AMH measurements were performed at baseline (AMH0), 15 days after the start of chemotherapy (AMH1), 15 days before the last chemotherapy cycle (AMH2), and at time 3, 6, 9, 12, 18 and 24 months from the end of chemotherapy. The whole study population was divided into two groups according to age: AYA (15–24; n = 65) and non-AYA (25–35; n = 57). All patients received a once monthly GnRH agonist injection during the whole treatment period. A linear mixed model was used to account for the repeated measures of single patients. MAIN RESULTS AND THE ROLE OF CHANCE At baseline, non-AYA patients had higher BMI and lower AMH levels than AYA patients. All AYA and non-AYA patients having received ABVD protocols had regular cycles at 12 months of follow-up. In case of alkylating regimens, amenorrhoea was more frequent in non-AYA patients than in AYA patients at 12 months (37% vs 4%, P = 0.011) and at 24 months (24% vs 4%, P = 0.045). We distinguished a similar depletion phase from AMH0 to AMH2 between ABVD and alkylating groups but significantly different recovery phases from AMH2 to AMH + 24 months. AMH recovery was fast and complete in case of ABVD protocols whatever the age: AMH reached pre-treatment values as soon as the 6th month of follow-up in the AYA group (mean (95% CI) in log AMH M0 vs M6: 3.07 (2.86 to 3.27) vs 3.05 (2.78 to 3.31), P = 1.00) and in the non-AYA group (mean (95% CI) in log AMH M0 vs M6: 2.73 (2.40 to 3.05) vs 2.47 (2.21 to 2.74), P = 1.00). In contrast, no patients from the alkylating group returned to pre-treatment AMH values whatever the age of patients (AYA or non-AYA). Moreover, none of the AMH values post-chemotherapy in the non-AYA group were significantly different from AMH2. Conversely in the AYA group, AMH levels from 6 months (mean (95% CI) in log AMH: 1.79 (1.47 to 2.11), P &amp;lt; 0.001) to 24 months (mean (95% CI) in log AMH: 2.16 (1.80 to 2.52), P ≤ 0.001) were significantly higher than AMH2 (mean (95% CI) in log AMH: 1.13 (0.89 to 1.38)). Considering the whole study population (AYA and non-AYA), pre-treatment AMH levels influenced the pattern of the AMH variation both in alkylating and ABVD protocols (interaction P-value = 0.005 and 0.043, respectively). Likewise, age was significantly associated with the pattern of the recovery phase but only in the alkylating group (interaction P-value =0.001). BMI had no influence on the AMH recovery phase whatever the protocol (interaction P-value = 0.98 in alkylating group, 0.72 in ABVD group). LIMITATIONS, REASONS FOR CAUTION There was a large disparity in subtypes of protocols in the alkylating group. The average duration of chemotherapy for patients treated with alkylating protocols was longer than that for patients treated with ABVD. WIDER IMPLICATIONS OF THE FINDINGS These results make it possible to develop strategies for fertility preservation according to age and type of protocol in a large series of young lymphoma patients. In addition, it was confirmed that young age does not protect against ovarian damage caused by alkylating agents. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by Agence Régionale de Santé Hauts de France and Agence Onco Hauts-de-France who provided finances for AMH dosages (n° DOS/SDES/AR/FIR/2019/282). There are no competing interests. TRIAL REGISTRATION NUMBER DC-2008-642 and CNIL DEC2015-112.

Authors: Afzal Hussain, Iqbal Hussain, Mohamed F. Al-Ajmi &amp; Imran Ali ### Abstract Small peptides (di-, tri-, tetra- penta- hexa etc. and peptides) control many chemical and biological processes. The biological importance of stereomers of peptides is of great value. The stereo-separations of peptides are gaining importance in biological and medicinal sciences and pharmaceutical industries. There is a great need of experimental protocols of stereo-separations of peptides. The various chiral selector used were polysaccharides, cyclodextrins, Pirkle’s types, macrocyclic antibiotics, crown ethers, ligand exchange, etc. The attempts have been made to develop stereo-separations protocols for peptides using capillary electrophoretic and chromatographic techniques. In addition to these, the optimization strategies of stereo-separations were also discussed in the details. The efforts are also made to discuss the future perspectives of peptides stereo-separations. ### Introduction Its 21st century scientists are attempting to provide the best lives to the society. Medication is one of the most important aspects in our lives. Chirality in the drugs is a complex phenomenon creating confusion in medications. The demand of chiral drugs is increasing constantly due to different pahramaceutical activities of drugs enantiomers. One enantiomers may be active while the other inactive, toxic or ballast; leading to various side effects and problems [1]. It is because of chiral nature of our biological systems. Mostly biological reactions are stereo-selective because of different enantioselective distribution rates, metabolisms, excretion and clearances of enantiomers. Due to these facts, scientists, clinicians, industrialists, academicians and government authorities are asking data for optically active drugs and other biological important molecules. US FDA, Health Canada, European Committee for Proprietary Medicinal Products and Pharmaceutical and Medical Devices Agencies of Japan, have banned the marketing of all racemic drugs [2-4]. Small peptides (monomers n &lt; 6) are of great importance because of contribution in various biological processes. The biological activities of small peptides include protein synthesis, fertility, neurotransmission, inflammation process, pathogenic microorganisms activities and other functions of human beings. These functions made peptide vital molecules in drug development and health care [5-7]. Besides, these peptides are also being used as biological markers in the biological systems [8]. The small peptides are also considered as important molecules in food and nutrition industries. For examples, aspartame, carnosine, etc. are being prepared at industrial scale [7]. It is important to mention here that biological functions of peptides are stereoselective; especially related to enzymatic reactions. In view of these facts, stereo-separation of small peptides is very important in drugs development and health care. Stereo-separation of peptides may be achieved by capillary electrophoresis and chromatography. Literature has many papers on stereo-separation of peptides [9-12]. Recently, Ali et al. [13] reviewed stereo-separations of small peptides by capillary electrophoresis and chromatography techniques. It was observed that all these papers contain sufficient information on stereo-separations of peptides but no one describes the experimental procedures, methods development and optimization strategies in details, which are urgently required at laboratory level globally. Therefore, the attempts have been made to describe a protocol for stereo-separations of peptides by capillary electrophoresis and chromatography. The present article describes the state-of-the-art of stereo-separations of peptides using capillary electrophoresis, chromatography. The efforts have been made to discuss optimization strategies and future perspectives of stereo-separations of peptides. ### Materials 1. All solvents and reagents should be of HPLC and AR grades - Optically active pure and racemic peptides standards - Deionised water - Acetonitrile - Methanol - Reagents for the preparation of phosphate, acetate and borate buffers. - Required chiral selectors - Acids and bases for pH adjustment ### Equipment 1. Capillary Electrophoresis Instrument - Personal computer (PC) for data acquisition - Fused silica capillaries (~ 50 cm effective length with 50 or 75 μm inner diameter) - Special capillary cutting blade - pH meter - UV–Vis. Spectrometer - Degasification unit - Filtration unit - Micro balance ### Procedure 1. Prepare stock solutions of peptides (optically active pure and racemic) in water (0.1 mg/mL). - Prepare required BGE and dissolve suitable and appropriate amount of chiral selector in it. - Protocols given in Figure 2 should be used before selecting and preparing BGE. - Filter through 0.45 μm membrane and degas by sonication. - Rinse the capillary for 5 min. with 0.5 M NaOH followed by 10 min with deionize water. - Fill BGE in CE reservoirs and dip the ends of capillary into them. - Rinse capillary for 5 min with BGE. - Inject racemic peptides sample solutions. - Apply appropriate potential and run CE instrument. - Among the measurements, rinse capillary for 2 min with BGE from time to time. - Optimize the stereo-separations. - Identify the resolved enantiomers by running standard pure optically active stereomers. - Wash capillary by deionised water before stopping HPLC instrument. - Calculate capillary electrophoretic parameters using standard equations [14]. - Determine the qualitative and quantitative stereo-separations. ### Timing - Rinse the capillary for 5 min. with 0.5 M NaOH followed by 10 min with deionize water. - Rinse capillary for 5 min with BGE - Among the measurements, rinse capillary for 2 min with BGE from time to time. ### Troubleshooting 1. CE is gaining importance in stereo-separations of peptides. - CE instrument has two injection modes i.e. electrokinetic and pressure injection modes. - pH meter should be calibrated using pH 4.0 and pH 10.0 standards. - First filter about 10 mL of deionized water in order to remove any impurities from filtration unit. - pH of electrolyte is a crucial parameter and should be adjusted as per the requirements. - The addition of organic modifiers improves the stereo-separations. - Generally, organic modifiers are toxic to health. - Care should be taken to avoid skin contact, inhalation and swallowing. - Organic modifiers should be handled with cautions using gloves, glasses, etc. - These organic modifiers should be stored in cool, dry and well ventilated places. - Both ends of capillary should be sealed by heating; if instrument is kept for long time. ### Anticipated Results Stereo-separation of small peptides is a growing research area since early 1990s. The metabolism of D-amino acid containing peptides stimulated stereo-separations of peptides The stereo-separations methods are gaining advancement with respect to time. For example, switching from indirect methods to direct stereo-separations and a gradual replacement of protocols requiring derivatization of samples to the analysis of underivatized peptides are the advancements. The cyclodextrines and macrocyclic antibiotics are the most commonly used chiral selectors. However, in recent time polysaccharides and other chiral selectors have been used. During last few years, hyphenation of CE and HPLC with MS detectors, 2D-LC [57] and the development of miniaturized analytical devices are other developments [58]. Reducing analysis time and complication of analyzed samples have economic impact [59]. Fast speed UPLC instrument has not been used in chiral chromatograph1 of peptides. But UPLC is used for stereo-separations of derivatized amino acids [60]. Therefore, it is expected that UPLC may acquire a good position in stereoseparations of peptide. The stereo-separation of peptides diastereomers is important in physiological researches and industries. This is due to the fact that multi-components, multistereoisomer mixtures are found in the food and pharmaceutical industries. Such situation will expand industries with respect to enantiopure peptides. Keeping these facts into consideration, the stereo-separations of peptides at preparative scale is the requirement of today. Literature survey indicates only one report at preparative scale [61]. Therefore, there is a great need for stereo-separation of peptides at preparative scale. Really, optically active pure di- and tri-peptides can be obtained by chiral synthetic methods but not larger peptides. This is a niche for preparative chromatographic methods. 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Chromatogr*. 414, 313-322 (1987). ### Acknowledgements The author would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University, Riyadh, Saudi Arabia for its funding this research group No. RGP-150. **Figure 1: Diastereomers of N-(α-Aspartyl)-phenylalanine. LL-aspartame is sweet**.  **Figure 2: Protocol for development and optimization of CE conditions for chiral resolution**.  **Figure 3: Protocol for development and optimization of mobile phases on polysaccharides CSPs under normal phase mode.**  **Figure 4: Protocol for development and optimization of mobile phases on polysaccharides CSPs under reversed phase mode.**  **Figure 5: Protocol for development and optimization of normal mobile phases on CDs based CSPs under normal phase mode.**  **Figure 6: The Protocol for development and optimization of normal mobile phases on CDs based CSPs under reversed phase mode.**  **Figure 7: The protocol for the development and optimization of mobile phases on CDs based CSPs under polar organic phase mode.**  ### Author Information Afzal Hussain, College of Pharmacy, King Saud University Iqbal Hussain &amp; Mohamed F. Al-Ajmi, Unaffiliated Imran Ali, Department of Chemistry, Jamia Millia Islamia, New Delhi, India Correspondence to: Imran Ali ([drimran_ali@yahoo.com, drimran.chiral@gmail.com](drimran_ali@yahoo.com, drimran.chiral@gmail.com)) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/3455). Originally published online 31 October 2014*.

Introdução: A Odontologia do Esporte vem ganhando espaço e se tornando mais conhecida; mostrando que assim como a medicina, a nutrição e a fisioterapia, ela também possui sua importância no desempenho do atleta. Objetivo: Realizar uma revisão de literatura fazendo uma reflexão sobre a relação do desempenho do atleta com a manutenção da saúde bucal e o papel do cirurgião dentista nesse cenário. Material e Método: A pesquisa bibliográfica foi realizada utilizando as bases de dados Scielo, Lilacs, Pubmed e Teses USP. Resultados: A presente revisão aborda os tópicos: Odontologia do esporte; seu reconhecimento com especialidade e áreas de atuação; traumatismos; protetores bucais e faciais; desordens odontológicas com impacto na prática do esporte; doping e Odontologia. Conclusão: É necessária maior divulgação e conscientização sobre a importância da especialidade de Odontologia do esporte, do uso de protetores bucais personalizados e do quanto que um foco infeccioso e/ou oclusão deficiente podem influenciar no desempenho do esportista.Descritores: Protetores Bucais; Traumatismos Dentários; Traumatismos em Atletas.ReferênciasPadilha C, Namba EL, Coto NP. Qual o papel dos protetores bucais na redução da prevalência e da gravidade da concussão cerebral em esportes? Rev cir traumatol buco-maxilo-fac. 2014;14(3):73-8.Vanz MP, Gehlen GLA, Rovani G, Conto F, Flores ME. Alteração do desempenho esportivo associado a causas bucais. In: Linden MSS, Carli JP, Magro ML, Trentin MS, Silva SO, organizadores. Odonto Science: 53 Anos FOUPF. São José dos Pinhas: Editora Plena; 2014. p.77-81.Bastos RS, Vieira EMM, Simões CAD, Sales Peres SHC, Caldana ML, Lauris JRP et al. Odontologia desportiva: proposta de um protocolo de atenção à saúde bucal do atleta. RGO. 2013;61(Suppl 1):461-68.Reinhel AF, Scherma AP, Peralta FS, Palma ICR. Saúde bucal e performance física de atletas. ClipeOdonto UNITAU 2013;7(1):45-56.Sequeira E. Odontologia Desportiva: O Esporte e a Saúde Bucal. 2005. Disponível em:&lt;http://www. saudetotal.com.br/artigos/saudebucal/odontodesp ortiva.asp&gt; Acesso em: 26 de abril 2016.Dias RB, Silva CMF, Gennari MG, Coto NP. Problemas Odontológicos X Rendimento Desportivo. Rev odontol Univ St Amaro. 2005;10(2):28-31.Moura APF. Odontologia desportiva e o desempenho dos atletas. 2004. Disponível em: &lt;http://www.explorevale.com.br/2umatutino/materias/higiene_ odontologia_esportiva.pdf &gt; Acesso em: 10 de maio de 2016.Rosa AF, Costa SB, Silva PRS, Roxo CDMN, Machado GS, Teixeira AAA, et al. Estudo descritivo de alterações odontológicas verificadas em 400 jogadores de futebol. Rev Bras Med Esporte. 1999; 5(2):55-8.Lemos LFC, Oliveira RS. Odontologia desportiva. Uma breve revisão sobre essa nova tendência no esporte. Rev. Digital Educ Fis Deportes.2007; 12:1-5.Santos V. Odontologia do Esporte. Odonto Magazine 2013; 3:18-20.Yavich LG. Jornada Odontológica Interdisciplinar: Patologias da ATM e a sua conexão com a saúde integral do ser humano. 2016. Disponível em: &lt;https://lidiayavi ch.com/tag/sintomatologia/&gt; Acesso em: 10 de abril de 2017.Di Leone CCL, Barros IRCN, Salles AG, Antunes LAA, Antunes LS. O uso do protetor bucal nas artes marciais: consciência e atitude. Rev Bras Med Esporte. 2014;20(6):451-55.Sizo SR, Silva ES, Rocha MPC, Klavtav EB. Avaliação do conhecimento em odontologia e educação física acerca dos protetores bucais. Rev Bras Med Esporte. 2009;15(4):282-86.Souza JGS, Soares LA, Souza TCS, Pereira AR, Souza AGS. Traumatismos faciais decorrentes da prática esportiva. Rev bras cir cabeça pescoço. 2013;42(1):53-7.Jerolimov V. Temporomandibular injuries and disorders in sport. Medical Sciences 2010;34:149-65.Fonseca R. Trauma bucomaxilofacial. 4ª. ed. Rio de Janeiro: Elsevier; 2015.Costa SS. Odontologia desportiva na luta de reconhecimento. Rev Odontol Univ Cidade de São Paulo. 2009;21(2):162-68.Lopes LBPM, Ferreira JF. Dental trauma in contact sports. RGO. 2017;65(3):237-42.Coto NP, Meira JB, Brito e Dias R, Driemeier L, de Oliveira Roveri G, Noritomi PY. Assessment of nose protector for sport activities: finite element analysis. Dent Traumatol. 2012;28(2):108-13.Mcintosh L, Cordell JM, Johnson AJW. Impact of bone geometry on effective properties of bone scaffolds. Acta Biomater 2009;5(2):680-92. Westerman B, Stringfellow PM, Eccleston JA, Harbrow DJ. Effect of ethylene vinyl acetate (EVA) closed cell foam on trasmitted forces in mouthguard material. Br J Sports Med. 2002;36(3):205-8.Goiato MC, Santos DM, Moreno A, Haddad MF, Pesqueira A A, Turcio KHL, Dekon SFC et al. Use of facial protection to prevent reinjury during sports practice. J Craniofacial Surg. 2012;23(4):1201-2.Anjos DSC, Sá FC, Azevedo EN, Revoredo ECV, Galembeck A. Blendas de silicone-acrilato para próteses faciais. Anais - Natal: Sociedade Latina Americana de Biomateriais, Orgãos Artificias e Engenharia de Tecidos, 2012.Antunez MEM, Reis YB. O binômio esporte – odontologia. Rev Adolesc Saúde. 2010;7(1):37-9.Nishimura CM, Gimenez SRML. Perfil da fala do respirador oral. Rev CEFAC. 2010;12(3):36- 9.Leite JVM, Feitosa GM, Souza FASP, Pedrosa JCP, Antunes LL, Bezerra R. Odontologia Desportiva x performance física. 2007. Disponível em: &lt;http://worldsoccerone.blogspot.com.br/ search/label/Odontologia%20Desporti va&gt; Acesso em: 26 de abril de 2016.Souza LA, Elmadjian TR, Dias RB, Coto NP. Prevalence of malocclusions in the 13-20 year old categories of football athletes. Braz Oral Res. 2011;25(1):19-22.Ashley P, Di Iorio A, Cole E, Tanday A, Needleman I. Oral health of elite athletes and association with performance: a systematic review. Br J Sports Med. 2015;49(1):14-9.Needleman I, Ashley P, Meehan L, Petrie A, Weiler R, McNally S et al. Poor oral health including active caries in 187 UK professional male football players: clinical dental examination performed by dentists. Br J Sports Med. 2016;50(1):41-4.Buischi Y. Bebidas e alimentos que causam erosão dentária. 2004. Disponível em: &lt;http://odontologia.com.br.asp/?id=366idesp=1&amp;ler=s&gt; 22 mar 2004.Ranalli D. Adolescent athletes: perspectives for dental practitioners. Northwest Dentistry. 2007:15-20.Mello AB, Flório FM. Odontologia do esporte: como atuar em equipe na prescrição segura de medicamentos? FIEP Bulletin 2010; 80, Special Edition, Article II, disponível em http://www.fiepbulletin.net.Rose EH, NETO FRA, Levy R. Informações sobre o uso de medicamentos no esporte. 2006. Disponível em: http://www.cbh.org.br/arquivos/ Informacoes%20sobre%20o%20uso%20de%20medicamentos%20no%20 Esporte%20%20COB%20Cavaleiros%20Amazonas.pdf &gt; Acesso em: 10 de abril de 2017.Anizan S, Huestis MA. The potential role of oral fluid in antidoping testing. 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Authors: Massmiliano Cavallini, Denis Gentili, Pierpaolo Greco, Francesco Valle &amp; Fabio Biscarini ### Abstract This protocol provides the instructions for designing and fabricating stamping tools with features ranging from nanometer to micrometer scale, including the fabrication using commercial tools such as compact disks or digital video disks. In particular the reported procedures are oriented towards the tools fabrication for lithographically controlled wetting and soft lithography. Because the versatility of these methods that has almost no restrictions concerning the materials used for the stamps, a wide range of methods are provided in this protocol including photolithography, electron beam lithography, replica molding, laser engraving and nanoimprinting. ### Introduction **GENERAL INFORMATION** **PHOTOLITHOGRAPHY** Presently the most common method to fabricate rigid stamps (usually in silicon) and masters required for replica molding of the soft stamps is photolithography (see the protocol in ref.(1)). This process, widely used for microfabrication, is based on the selective removing of parts of a film upon the exposition to light (usually UV). This is achieved thanks to some materials known as photoresists, that are light-sensitive, meaning that, according to their composition, they become soluble or insoluble to their developing solution upon illumination. The first family is called positive photoresist and the second negative. The light is sent through a photomask that shadows the parts of the film that, according to the kind of resist used, must be removed or preserved. Different kind of masks can be used but their common feature is the ability to shadow as much as possible some parts of the photoresist below (contrasting power), the most used are transparent foils with pattern drawn by an ink non-transparent to UV-light or highly reflecting foils (usually metallic) where some portion have been removed by laser engraving. The current limits of photolithography is approximately 250 nm and the minimum feature size is 100 nm (2). These values are important on the one hand to define the ranges of fabrication of soft stamps for LCW but on the other hand for the forthcoming comparison with the feature sizes and resolution that LCW can achieve. The masks are usually drawn using CAD software and then transferred either by common printers on transparent foils, where the marks will represent the shadowed areas, or by laser engraving of highly reflective metallic sheets where the removed part will corresponding to the illuminated path. The photoresist film, upon illumination is then developed in the appropriate solution leaving on the surface only the features that will be used as master for replica molding or directly as stamp. **ELECTRON BEAM LITHOGRAPHY** When the resolution limits required by the process are below those of photolithography (diffraction limit), electron beam lithography (EBL) is usually chosen for fabricating the masters. This technique is in fact able to achieve resolution of 20-30 nm in lateral size (see the protocol in ref.(1)), because it make use of an electron beam to locally alter the chemical properties of a material (resist) that will be then removed in a development step. Beside the illustrated resolution advantage one must remind that EBL is an expensive technology and it is a serial technique, thus it is much slower than photolithography. **REPLICA MOLDING** Replica molding (RM) is the most common way of fabricating the elastomeric stamp for soft lithography and LCW and it is one of the most important tools for these techniques. RM is based on the reticulation of the elastomeric precursors onto the master that is then removed, upon curing, by peeling it off. It is worth mentioning that the LCW is not limited to the elastomeric materials as other soft-lithographic methods and it makes wide use of rigid and metallic materials as it is shown other sections of this protocol. Nevertheless replica molding of the elastomeric materials still plays a crucial role because, making use of well known materials such as Polydimethylsiloxane (PDMS), it allows tuning the surface properties of the stamp by liquid or plasma chemical treatments. Elastomeric stamps made of PDMS are deformed easily under the effect of capillary adhesion. The attractive force, exerted by the solution trapped between the substrate and the stamp, may involve sagging of the PDMS stamp and poor patterning in the region where the PDMS displaced the solution. The capillary force may be estimated according to De Gennes,(3) and needs to be considered during the design of the spacer and the dimensioning of the full PDMS replica. In this case, an effective parameter is the aspect ratio AR, defined as the ratio of width over thickness of the window delimited by the spacer. If we put PDMS Young modulus E ≈ 460 kPa, contact angle of solution θ = π/6, and surface tension γ = 0.02 N/m (organic solvents), we derived the critical value for AR that will induce sagging for various distances between the substrate and the PDMS replica, by finite element calculation (Figure 1). The empirical relationship can be summarized with a linear abacus for fast design, providing that the overall dimension of the stamp is exceeding the window defined by the spacer. **NANOIMPRINT LITHOGRAPHY** Nanoimprint lithography (NIL) is another important technique useful to fabricate tools (i.e. master for RM and stamps) for LCW. NIL consists of a physical (morphological) deformation of a thermoplastic material in a temperature and pressure controlled printing process. A silicon stamp fabricated by a conventional lithography such as EBL or photolithography is usually employed. In NIL the thermoplastic material (usually a polymer) is deformed by pressing the stamp into the polymer at a temperature above the polymer’s glass-transition temperature. The polymer is then cooled down below the glass-transition temperature and the stamp is removed. **VACUUM SUBLIMATION OF METALS** One of the advantages of LCW is the versatility with respect to the material of the stamp used for patterning. In this frame, a role is played also by metallic stamps, some of them (gold for instance) can be functionalized using thiol based Self Assembled Monolayers (SAM) to tune their surface properties. These stamps are usually fabricated by vacuum sublimation of a thin metallic layer on a master previously realized by one of the above mentioned techniques. For some substrates such as silicon or mica an adhesive layer (usually made of chromium or titanium) has to be previously evaporated on the underlying material to favour the stability of the metallic film. **COMMERCIAL AVAILABLE MASTER/STAMPS** Commercial metallic grids commonly used for Electron Microscopy experiments have proven to be very suitable for as a stamp for LCW (See Fig 2). When the grid comes as a large sheet that must be cut, it is crucial to cut it with very sharp scissors to avoid large deformations of the areas near the cut. For the most kind of patterning inexpensive masters such as Compact Disc (CD) Digital versatile disk (DVD) or diffraction gratings are commercially available. This kind of devices are made of: Blank CD, parallel stripes 1.5 µm pitch, 500 nm width and 220 nm depth. Blank DVD, parallel stripes with 750 µm pitch, 300 nm width and 110 nm depth. Written disks contain a pattern o doth and line with the same size containing an information in digital (binary) code. Figure 3 shows typical AFM images CD and DVD masters. ### Reagents 1. Photoresists SU-8 - SU-8 developer 1-Methoxy-2-propanol acetate - PolyMethylMethacrylate (PMMA) (Allresist, AR-P679.01) - Sylgard 184 silicone elastomer base and Sylgard 184 elastomer curing agent (Dow Corning). CRITICAL Sylgard 184 is a thermal curable elastomer, which is provided as a two-component kit consisting of the base and the curing agent. The standard ratio between base and the curing agent 1:10 small variation led to adjust the softness of cured elastomer (higher value of 10% led more rigid stamp). Use glove powder-free for these operations. - Ethanol (Sigma-Aldrich, cat. no. 459836) - Hydrogen peroxide (Sigma-Aldrich, cat. no. 216763) - Sulfuric acid (Sigma-Aldrich, cat. no. 320501) - Masks (supplier Toppan inc.) **REAGENT SETUP** - Piranha solution 3:1 mixture of concentrated H2SO4 and H2O2 (30% vol/vol). Add very slowly and mix with a glass rod one volume of H2O2 to three volumes of H2SO4 in a clean beaker whose volume is at least 10 times the volume of the final solution. - CRITICAL Piranha solution must be prepared fresh and cooled to room temperature before use. - ! CAUTION Keep attention because there are exothermic processes involved when you add the hydrogen peroxide to the sulphuric acid. Piranha solution can reacts violently with organic compounds, is very aggressive to skin, and should be handled with care. It is important to work in a fume hood and wear personal protective clothing (e.g., nitrile or latex gloves, lab coat, safety glasses) when handling solutions and keep away from organic chemicals ### Equipment 1. IR fiber laser marker (for metallic photomask engraving e.g., LaserPoint, Marko 10P) - Laser marker IR 1064 nm - Spin coater (for the application of thin films of resists, e.g., Laurell ws-650-6NNP/Lite) - UV mask aligner (for photolithography, e.g., Karl Suss, Mask Aligner MJB4) - Electron-beam writer (for e-beam lithography, e.g., SEM-FEG Hitachi S4000 with Nabity NPGS e-beam source) - Vacuum line (for removal of bubbles) - Hotplates (for baking resist films and surface cleaning, e.g., MR Hei-Standard, Heidolph) - Nitrogen gas line (for drying stamps and substrates) - Precision hydraulic press (for imprint semirigid stamps, e.g, PW20, P/O Weber) - Ultrasonic cleaner (for surface cleaning, e.g., Elmasonic S30H) - System for metal vacuum sublimation (for preparing supported thin films of gold or, e.g., EDUARS System) - Optical microscope (for characterizing patterns on masters and stamps at the microscale, e.g., Nikon Eclipse 80i) - Scanning electron microscope (SEM, for characterizing patterns on masters and stamps at both micro- and nanoscale, e.g., SEM-FEG Hitachi S4000) - Atomic force microscope (AFM, for characterizing patterns on masters and stamps at both micro- and nanoscale, e.g., NT-MDT NTEGRA). ### Procedure Photolithography: fabrication of masters and rigid stamps - TIMING 1-3 days Follow protocols in refs.(1),(4),(5) or: 1. Design the masks by CAD or other vectorial graphic software. - CRITICAL STEP Define properly the distances and geometry of the stamp to prevent sagging using soft stamps. - Photomask fabrication: - A. for low-resolution mask (feature size &gt;20 µm), print by a high-resolution printer the features on transparent plastic sheet. - B. for high-resolution mask (feature size 10 µm &lt; Size &lt; 20 µm), print by laser marker to engrave metallic foils. - CRITICAL STEP: It is important for the metallic layer to be enough thick to shadow the UV light during the photolithography illumination. It is also crucial that material supporting the the thin metallic layer is homogeneouly transparent to the IR laser to avoid artifactual shadowings. - C. a commercial chrome photomask (photomask with micrometric feature can be fabricated in 1-2 week by supplier) - Clean accurately the substrate supporting the photoresist layer: using a silicon wafer 30 minutes in Piranha solution. Then rinse with ultrapure water and dry with clean gas flow. If it has not to be used immediately, store the substrate in ultrapure water. - Fix the silicon on the spin coater, spread the photoresist and start the chosen program. For SU-8 a two steps program with a first step of 5 seconds at 300 rpm followed by a second one of 50 seconds at 1000 rpm is suitable for a 20 μm layer (for 10 μm double the rpm reaching 2000). - Heat sample to remove the solvent (3 min. at 65°C followed by 7 min. at 95°C) before the exposition - Expose the photoresist film to the UV light using the Mask Aligner, chose times between 6 s for a 40 μm layer and 1 sec. for 1 μm. - Remove the sample from the Mask Aligner and place on the heater for 2 min. at 65°C followed by 4 min. at 95°C. - Develop the sample using the following two solutions: SU-8 developer and isopropanol; the former remove the material and the latter stop the process. The time within the developer can range between 30 s and 3 minutes according to the thickness. - CRITICAL The substrate holding the photoresist layer must be extremely clean and, during the exposition, the photomask has to adhere well to the photoresist. - PAUSE POINT The photolithographic fabricated master can also be used both directly of through replica molding of elastomeric stamps for LCW. Electron beam lithography: fabrication of masters and rigid - TIMING 1-2 days Follow protocol as in refs.(1),(5) or 1. Spin coat PMMA, 5000rpm for 5 min, on silicon previously cleaned as described for photolithography. - Bake the sample on a hot plate at 180 °C for 60 s to flatten the film, get rid of residual solvent, and enhance the adhesion between PMMA and the substrate. - Insert the PMMA film in the Scanning Electron Microscope chamber and create the vacuum. - Start writing the pattern with the electron beam using respectively Vacc=30kV and IEM=10μA to generate a pattern of holes in a PMMA film and Vacc=30kV and IEM=20μA to generate a pattern of reliefs in the same film. - Optimise the focus gradually until reaching the highest magnification and measure the current in a hole of the Faraday Cup; for a pattern of holes in PMMA IFC ∼0.6 nA, for reliefs IFC ∼4 nA. - Chose the dose (this will fix the time you need for patterning the chosen area); for holes in PMMA the Doses must be 500-5000 μC/cm2 for reliefs 5000-50000 μC/cm2. - Remove the sample from the SEM chamber and develop the film: - A. for a pattern of holes in PMMA use a solution 1:3 methyl isobutylketone:isopropanol (MIBK:IPA) at 20°C for 60 s, then rinse in pure IPA for 60 s, and finally dry with nitrogen gas. - B. for a pattern of reliefs leave the sample for 10-20 s in acetone and then rinse in pure water for 60 s and dry with nitrogen gas. - PAUSE POINT The EBL fabricated master can also be used both directly of through replica molding of elastomeric stamps for LCW. Replica molding of a master: fabrication of soft stamps - TIMING 6-12 h Follow protocols in refs.(1),(6) or: 1. Pipette inside a cup 10 parts of Sylgard 184 base and 1 part of the curing agent (w/w). - Use a spatula or a spoon to carefully mix the base and curing agent for at least 3 min. - CRITICAL STEP Ensure that the two components are perfectly mixed. Incomplete mixing may affect the curing behaviour, homogeneity and mechanical properties of the resulting stamp. - Place the master on a Petri dish and put on top of it a thickness mask to define size and shape of the stamp (e.g. a metallic washer). - CRITICAL STEP use clean tweezers to manipulate the master avoid touch any feature present on top of the master. Plastic tweezers are preferred for these operations. - Pipette the fluid mixture to the desired thickness inside the washer; remove the bubbles by placing the Petri dish in a box (e.g. a desiccator) and connect to the vacuum line of the fume hood for 15 min. - CRITICAL STEP It is important to complete this step after pouring the mixed PDMS onto the master so that no bubbles will be trapped at the interface between the master and PDMS. - Turn off the vacuum and vent the box. - Curing: Place the Petri dish containing both the master and the PDMS/curing mixture 48 h in the desiccator at room temperature (20°C) or, for a faster preparation, in an oven set to 70 °C and cure it for 6 h. - Peel-off the PDMS stamp and the washer from the master. Use a finger or a spatula to remove the PDMS stamp from the washer. Nanoimprinting: fabrication of semi-rigid stamps - TIMING 6-12 h 1. Nanoimprinting of polycarbonate: Insert a foil &gt;500 µm thick of polycarbonate in between the plate of a press. A thickness ranging between 0.5 mm and 1mm are ideal for an easy manipulation by tweezers normally the flat side of commercial CD or DVD can work properly (Note the unpatterned side of CD or DVD can properly work). - Place the master on top of the polycarbonate foil with the features in contact with the polymer. - Put the plates of the press in contact, sandwich the polycarbonate and master and apply a low pressure &lt;5 bar. By this operation the master will be conformal with the polycarbonate. - Heat the plates at 130°C; wait the thermalization of the system for 20 min. - Apply a pressure of 75 bar for 1 min. - Deprint the plates. - Remove the master from the polycarbonate foil with the help of a spatula ! CAUTION be sure that the temperature reach above room temperature). - CRITICAL STEP pay attention to the cleaning of the environment and in particular the interface between the master and the foil, usually for sub-micrometric resolution you can work under laminar wood, in the case of &lt;200 nm feature the use of a clean room is desirable furthermore the stamp surface must be functionalised by anti-adhesion SAM as step 9-12 of the protocol. Vacuum sublimation of metals: stamp metallization - TIMING 6-12 h Stamp metallization is necessary only if you use aggressive solvents, it must be done using vacuum system, which is commercially apparatus. Metallization system it is usually available in common preparation facilities. See also protocol in ref.4 1. Fix the stamp on a sample holder with the features exposed. - Put the system in vacuum with pressure&lt;10-6 bar - Evaporate &gt;50 nm of Au taking the crucible at a temperature suitable for a rate &lt;1 nm/min. (thickness ranging between 50 and 100 nm offer adequate protection of the polymer without alter the stamp features) the sample must kept at room temperature. Monitor the growth rate by the quartz microbalance (a quartz microbalance tool is usually present in each commercial metallization system). - CRITICAL POINT the metallization take all polymers impermeable to the solvent, thus in some case it changes the result of your patterning. Metallic grids and masks fabrication - TIMING 1-2 days 1. Design the masks by CAD or other vectorial graphic software. - Take a metallic foil thick enough to be self standing, aluminium is the most suitable and easier to find as a commercial product, and let it adhere well and uniformly onto a glass support or onto any other material that is flat and transparent to IR light. - Place the metal+support onto the scanner of a IR marker and start writing (Note: the laser intensity and the focus of the beam will define the size of the feature where metal is removed). - CRITICAL POINT Tuning the laser intensity one has to pay attention to avoid the formation of rims of metal on the border of the engraved areas and to tune the speed of the laser to have the borders of the features as much as possible uniform. - Detach the metallic foil from the support and use this mask for PRINTING. Metallized stamp from blank CD - TIMING 20 min 1. Rinse the label layer of the blank CD with ethanol and dry in a stream of nitrogen for 60 s. - Rinse the solid substrate (e.g., glass, silicon wafer) with ethanol and dry in a stream of nitrogen for 30 s. - Stick a double-sided tape on the solid substrate and use a razor blade to remove the tape in excess. - CRITICAL STEP it is important to use a double-sided tape compatible with the solvent used in the next steps. - Stick the substrate on the label layer of the blank CD using the second adhesive layer of the tape and press down with hand to achieve a homogeneous contact. Use a razor blade to cut the label layer of the blank CD along the edges of the substrate. - Peel off the substrate with the metal layer of the blank CD carefully with the aid of a flat head tweezers. - CRITICAL STEP it is important to peel off the substrate slowly to prevent the breaking of the metal layer. - Rinse the metallic side of the blank CD with ethanol to remove the organic dye and dry in a stream of nitrogen for 30 s. Soft stamp from blank CD - TIMING 8 h 1. Peel-off the label (and metallic) layer of the CD from its polycarbonate layer using an adhesive tape. - CRITICAL STEP To facilitate this step, make a little scratch on the border of the label layer with a razor blade. - Rinse the polycarbonate layer of the blank CD with ethanol to remove the organic dye and dry in a stream of nitrogen for 60 s. - Follow fabrication of soft stamp using the polycarbonate side of the CD as master. (Note to achieve stamps with a thickness in the range of 0.3-0.5 cm use 25 g of mixture for single CD). - CRITICAL STEP It is important to peel off the substrate slowly to prevent the breaking of the metal layer. Metallized stamp from Digital Video Disc (DVD) - TIMING 10 min 1. Separate the two polycarbonate layers of the DVD using spatula and robust tweezers.  CRITICAL STEP It is important to separate the substrates slowly to prevent the breaking of the metal layer. 2. Rinse the metallic layer of the blank DVD with ethanol to remove the organic dye and dry in a stream of nitrogen for 60 s. 3. Cut the metallic layer supported on polycarbonate with a razor blade into stamps with the desired dimensions. Soft stamp from diffraction grating - TIMING 8 h 1. Rinse the diffraction grating with the proper solvent and dry in a stream of nitrogen for 60 s. - CRITICAL STEP It is important to use a solvent that does not damage the diffraction grating. - Follow fabrication of soft stamp using the diffraction grating as master. ### References 1. Qin, D., Xia, Y.N. &amp; Whitesides, G.M. Soft lithography for micro- and nanoscale patterning. *Nat. Protoc*. 5, 491-502 (2010). - Xia, Y.N. &amp; Whitesides, G.M. Soft lithography. *Angew. Chem*., Int. Ed. 37, 551-575 (1998). - de Gennes, P.-G., Brochard-Wyart, F. &amp; Quere, *D. Capillarity and Wetting Phenomena: Drops, Bubbles, Pearls, Waves*. (Springer, 2004). - Kim, P., Adorno-Martinez, W.E., Khan, M. &amp; Aizenberg, J. Enriching libraries of high-aspect-ratio micro- or nanostructures by rapid, low-cost, benchtop nanofabrication. *Nat. Protoc*. 7, 311-327 (2012). - Nair, P.M., Salaita, K., Petit, R.S. &amp; Groves, J.T. Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling. *Nat. Protoc*. 6, 523-539 (2011). - Nelson, C.M., Inman, J.L. &amp; Bissell, M.J. Three-dimensional lithographically defined organotypic tissue arrays for quantitative analysis of morphogenesis and neoplastic progression. *Nature Protocols* 3, 674-678 (2008). ### Acknowledgements The work was supported by the project ESF-EURYI DYMOT, ESF-EuroBioSAS ICS, EU Collaborative Project HYSENS FP7-NMP3-SL-2011-263091 and national project PRIN prot. 2009N9N8RX_003. ### Figures **Figure 1: Finite element calculation of stamp deformation** [Download Figure 1](http://www.nature.com/protocolexchange/system/uploads/2158/original/1.tif?1338298551) *a) Linear dependence of critical aspect ratio (sagging) vs spacer thickness in LCW, for plasma treated PDMS. For thin replicas sagging occurs also at larger spacer thicknesses, and the dependence is not linear b) Deformation of elastomeric stamp as output from finite element calculation, showing sagging due to capillary adhesive forces*. **Figure 2: Commercial grids** [Download Figure 2](http://www.nature.com/protocolexchange/system/uploads/2159/original/2.tif?1338299266) **Figure 3: Commercial tools CD and DVD**. [Download Figure 3](http://www.nature.com/protocolexchange/system/uploads/2160/original/Fig_3.tif?1338299361) *Scheme of the CD-ROM and DVD structures with the corresponding Atomic Force Microscopy images*. **Associated Publications** **Micro- and nanopatterning by lithographically controlled wetting**. Massimiliano Cavallini, Denis Gentili, Pierpaolo Greco, Francesco Valle, and Fabio Biscarini. *Nature Protocols* 7 (9) 1668 - 1676 doi:10.1038/nprot.2012.094 ### Author information **Massmiliano Cavallini**, Consiglio Nazionale delle Ricerche (CNR) - Istituto per lo Studio dei Materiali Nanostrutturati (ISMN), Via P. Gobetti 101, 40129 Bologna, Italy. **Denis Gentili, Francesco Valle &amp; Fabio Biscarini**, CNR -ISMN Bologna **Pierpaolo Greco**, Scriba Nanotecnologie S.r.l Correspondence to: Massmiliano Cavallini (m.cavallini@bo.ismn.cnr.it) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2401) (2012) doi:10.1038/protex.2012.029. Originally published online 20 August 2012*.

Follicular lymphoma (FL) is the most common type of indolent lymphoma in the Western world, accounting for approximately 30% of lymphoma cases. FL is known for its recurrent nature, necessitating diverse treatment options. The introduction of rituximab, an anti-CD20 antibody, has greatly improved FL outcomes and paved the way for targeted therapies. In this review, we thoroughly explore the structure, mechanism of action, clinical outcomes, and side effects of currently approved monoclonal antibodies (mAb) for FL. Furthermore, we provide insights into ongoing clinical trials and emerging monoclonal antibodies that hold promise for the future of FL treatment. A comprehensive literature search was conducted using various medical databases, including ASH and ASCO publications, as well as PubMed. The clinicaltrials.gov website was used to compile a list of investigational monoclonal antibodies from ongoing clinical trials. The future of antibody-based therapy for follicular lymphoma shows great promise, with a focus on enhancing antibody efficacy, prioritizing optimized combination therapies to address treatment resistance, and evaluating bispecific antibodies as first-line therapies, all while carefully balancing risks and benefits and sequencing treatments appropriately for better disease management. These directions have the potential to establish antibodies as a central component of follicular lymphoma treatment. Article Details How to Cite MOUSTAFA, Muhamad Alhaj. A Comprehensive Review of Monoclonal Antibodies for the Treatment of Follicular Lymphoma Including Both Approved and Investigational Options. Medical Research Archives, [S.l.], v. 11, n. 11, nov. 2023. ISSN 2375-1924. Available at: &lt;https://esmed.org/MRA/mra/article/view/4745&gt;. Date accessed: 02 dec. 2023. doi: https://doi.org/10.18103/mra.v11i11.4745. 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Authors: David Blum, Samuel LaBarge, The Reproducibility Project: Cancer Biology†* ### Abstract The [Reproducibility Project: Cancer Biology](https://osf.io/e81xl/wiki/home/) seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012. This Registered Report describes the proposed replication plan of key experiments from “Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion” by Straussman and colleagues, published in Nature in 2012 (Straussman et al., 2012).The key experiments being replicated in this study are from Figures 2A, C and D (and Supplemental Figure 11) and Figure 4C (and Supplemental Figure 19) (Straussman et al., 2012). Figure 2 demonstrates resistance to drug sensitivity conferred by co-culture with some stromal cell lines, and identifies the secreted factor responsible as HGF. In Figure 4, Straussman and colleagues show that blocking the HGF receptor MET abrogates HGF’s rescue of drug sensitivity. The Reproducibility Project: Cancer Biology is a collaboration between the [Center for Open Science](http://centerforopenscience.org/) and [Science Exchange](https://www.scienceexchange.com/), and the results of the replications will be published by eLife. ### Introduction Resistance to oncoprotein-targeted chemotherapy is a common occurrence during cancer treatment and identifying the mechanisms of resistance is important in improving treatment options. Specifically, BRAF-mutant melanomas, which show an initial response to RAF inhibitors, usually become resistant to the therapy (Nickoloff and Vande Woude, 2012). The identification of stroma-mediated resistance in BRAF-mutant melanomas, through the secretion of hepatocyte growth factor (HGF), therefore indicates a potential therapeutic strategy through combination treatment of RAF inhibitors and inhibition of the HGF activated pathway (Straussman et al., 2012). This report is the first to identify paracrine HGF as a potential mechanism for the development of drug resistance (Ghiso and Giordano, 2013; Glaire et al., 2012). In Figure 2A of their paper, Straussman and colleagues tested the effect of fibroblast-conditioned medium on the proliferation of BRAF-mutant melanoma cells grown in the presence of the BRAF inhibitor PLX4720. Using a cell proliferation assay, they reported that fibroblast-conditioned medium rescued BRAF-mutant melanoma cells from PLX4720 sensitivity, which indicated that a secreted factor was involved. This was a key finding demonstrating that the stromal environment of the tumor cells could mediate their response to drug treatment. This experiment will be replicated in Protocol 3. Straussman and colleagues went on to identify the secreted factor responsible for acquired drug resistance as HGF. In Figure 2C, they demonstrate that treating melanoma cell lines with PLX4720 in combination with increasing concentrations of exogenous HGF increased proliferation as compared to treatment with drug alone. This finding showed a similar effect to treatment with conditioned media from stromal cells that secrete HGF (see Figure 2A) and supports the hypothesis that HGF is the growth factor responsible for rescuing melanoma cells from drug sensitivity. This experiment will be replicated in Protocol 4. Straussman and colleagues demonstrated that the HGF-mediated rescue of melanoma cells from drug sensitivity was mediated through HGF’s cognate receptor tyrosine kinase MET by treating melanoma cell lines co-cultured with stromal cell lines in the presence of PLX4720 with the MET inhibitor crizotinib, as shown in Figure 2D and Supplemental Figure 11. Treatment with crizotinib reduced the increase in proliferation due to co-culture with an HGF-secreting stromal cell line. This experiment provided further support for the hypothesis that HGF was responsible for rescue from drug sensitivity, and also provided evidence that that rescue was MET dependent. This experiment is replicated in Protocol 5. Lastly, Straussman and colleagues reported sustained activation of both ERK and AKT in HGF-treated melanoma cells during BRAF inhibition, and to a lesser extent MEK inhibition, as shown in Figure 4C and Supplemental Figure 19 by Western blot. This confirms activation of pro-survival pathways in response to HGF treatment even in the presence of PLX4720. These experiments are replicated in Protocol 6. To date, a direct replication has been reported; Lezcano and colleagues (Lezcano et al., 2014) published a replication of Figure 3 of Straussman et al Nature 2013 wherein Straussman and colleagues evaluated HGF expression in patient-derived primary melanoma samples and observed a negative correlation between expression of HGF and response to therapy (Straussman et al., 2012). While Lezcano and colleagues’ replication also detected the presence of HGF in human melanoma tumor cells and stromal cells with increased expression at disease progression, they did not identify a statistically significant correlation between HGF expression and clinical outcome (Lezcano et al., 2014). While both of the studies come to different conclusions about the association of stromal HGF and clinical outcome, the 95% confidence intervals of the standardized measure of the effect (cohen’s d) for each study substantially overlap. A study published around the same time as the work of Straussman and colleagues supports the negative association between HGF and clinical response to RAF inhibitor treatments through an analysis of HGF levels in patient plasma samples (Wilson et al., 2012). In other systems, additional labs have observed a similar role for HGF in acquired drug resistance. Caenepeel and colleagues reported that HGF rescued melanoma cell lines, notably SK-MEL-5, from BRAF or MEK inhibition using vemurafenib (an analogue of PLX4720) or PD0325901, respectively, and the rescue was attenuated by MET inhibition (Caenepeel et al., 2013). Nakagawa and colleagues observed that tumor-secreted (not stromal secreted) HGF could induce resistance to the VEGFR inhibitor lenvatinib, and that this resistance could be overcome by co-treatment with golvatinib, a MET inhibitor (Nakagawa et al., 2014). Etnyre and colleagues reported that c-MET and BRAF inhibitors had synergistic inhibitory effects when exposed in combination to melanoma cell lines (Etnyre et al., 2013). Casbas-Hernandez and colleagues co-cultured MCF10 cells with immortalized mammoplasty-derived fibroblasts, and observed a correlation between the levels of fibroblast-secreted HGF and the differentiation of the MCF10 cells towards a ductal carcinoma phenotype. They also observed a correlation between HGF expression and the more invasive basal-like tumors as opposed to the less invasive luminal tumors (Casbas-Hernandez et al., 2013). HGF is also being evaluated as a potential biomarker to indicate potential treatment choices (Penuel et al., 2013; Xie et al., 2013). ### Materials and Methods Unless otherwise noted, all protocol information was derived from the original paper, references from the original paper, or information obtained directly from the authors. **Protocol 1: Determining the range of detection of the replicating lab’s plate reader** This is a general protocol that determines the range of detection of the plate reader. Because the plate reader in use by the replicating lab is different than the plate reader used in the original study, we are determining what the range of detection is for the replicating lab’s plate reader. Sampling: - SK-MEL-5 - 8,000 cells/well x 4 replicates - 4,000 cells/well x 4 replicates - 2,000 cells/well x 4 replicates - 1,000 cells/well x 4 replicates - 500 cells/well x 4 replicates -250 cells/well x 4 replicates - 125 cells/well x 4 replicates - 62.5 cells/well x 4 replicates - 31.25 cells/well x 4 replicates - The experiment is done a total of once. Materials and Reagents: - Reagents that are different from ones originally used are noted with an asterisk (*).  Procedure: 1. Seed 4 wells of a 384-well black plate with 8,000 cells/well all the way to 31.25 cells/well (serial 1:2 dilutions) with pLex-TRC206 SK-MEL-5 cells in 60 µl per well using phenol-red free medium using an automated workstation. Note: All cells will be sent for mycoplasma testing and STR profiling. Note: Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or puromycin (0.5-2 µg/ml), however do not grow cells under antibiotic selection on a regular basis. - a. Total wells seeded = 36 - b. Medium for assay: phenol-red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut. - c. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment. - The next day after seeding, read GFP fluorescence (Synergy HT Microplate Reader). - Subtract the average reading from media-only wells from the wells with cells. Deliverables: - Data to be collected: - Raw GFP fluorescence readings. - Graph of GFP fluorescence readings vs cell number. Confirmatory analysis plan: - Statistical Analysis: - Coefficient of determination of data values. Known differences from the original study: - Synergy HT Microplate Reader used instead of Molecular Devices SpectraMax M5e Microplate Reader – both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for sensitivity of detection (Protocol 1) and to determine if the gradient is similar to the original study (≤ 5%) (Protocol 2). Provisions for quality control: This protocol will ensure that the replicating lab’s plate reader is comparable to the original lab’s plate reader. - A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. **Protocol 2: Determining the detection variability of the replicating lab’s plate reader** This is a general protocol that determines the variability in detection of the plate reader. Because the plate reader in use by the replicating lab is different than the plate reader used in the original study, we are determining what the variability of detection is for the replicating lab’s plate reader. Sampling: - SK-MEL-5: - 2,000 cells/well x 384 replicates - Experiment will be done a total of once. Materials and Reagents: - Reagents that are different from ones originally used are noted with an asterisk (*)  Procedure: 1. Seed all wells of a 384-well black plate with 2,000 pLex-TRC206 SK-MEL-5 cells (provided by authors) in 60 µl per well using phenol-red free medium using an automated workstation. - Note: All cells will be sent for mycoplasma testing and STR profiling. - Note: Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. - a. Medium for assay: phenol-red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut. - b. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment. - The next day after seeding, read GFP fluorescence (Synergy HT Microplate Reader). - a. Subtract the average reading from media-only wells from the wells with cells. Deliverables: - Data to be collected: - Raw GFP fluorescence readings. - Difference of each individual well and the average reading across the plate. Confirmatory analysis plan: - Statistical Analysis: - Standard deviation of data values. Known differences from the original study: - Synergy HT Microplate Reader used instead of Molecular Devices SpectraMax M5e Microplate Reader – both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for sensitivity of detection (Protocol 1) and to determine if the gradient is similar to the original study (≤ 5%) (Protocol 2). Provisions for quality control: This protocol will ensure that the replicating lab’s plate reader is comparable to the original lab’s plate reader. - A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. **Protocol 3: Co-culture proliferation assay** This protocol outlines how to culture melanoma cell lines with conditioned medium from three stromal cell lines with or without the RAF inhibitor PLX4720 to analyze cell proliferation rates, as is described in Figure 2A. Sampling: - Experiment to be repeated a total of 4 times for a minimum power of 81%. - See Power Calculations section for details - Each experiment has six conditions to be run in quadruplicate per experiment: - SK-MEL-5 untreated control [additional control] - SK-MEL-5 vehicle (DMSO) control - SK-MEL-5 treated with 2 µM PLX4720 and with unconditioned medium - SK-MEL-5 treated with 2 µM PLX4720 and with conditioned medium from CCD-1090Sk cells that do not secrete HGF - SK-MEL-5 treated with 2 µM PLX4720 and with conditioned medium from PC60163A1 cells that do secrete HGF - SK-MEL-5 treated with 2 µM PLX4720 and with conditioned medium from LL 86 cells that do secrete HGF Materials and Reagents: - Reagents that are different from ones originally used are noted with an asterisk (*)  Procedure: 1. Prepare Pre-Conditioned Medium (PCM); fresh PCM must be prepared the same day it is used in the treatment of SK-MEL-5 cells; this step is repeated three times to ensure fresh PCM is available on the needed day: - a. Three days before the PCM is needed, seed 3 x 10 cm tissue culture plates with 0.5x10e6 LL 86 cells each, 3 x 10 cm tissue culture plates with 1x10e6 PC60163A1 cells each, and 3 x 10 cm tissue culture plates with 2x10e6 CCD-1090Sk cells each (9 plates total) in 10 ml of phenol-red free medium each and grow for 3 days. - b. 3 days after seeding, collect medium from each cell line using the plate closest to 80-90% confluent. - i. 75-95% confluency can be used. - c. Filter through 0.45 µm syringe filter with a 10 ml syringe and dilute filtered PCM 1:1 in fresh phenol-red free medium. Total volume = 20 ml. - i. Use same day. - ii. Do not dilute for day 0 of treatment (these wells will already have 20 µl of media in them). - On day 0, seed 120 wells of a 384-well black plate with 1,900 pLex-TRC206 SK-MEL-5 cells in 20 µl per well using phenol-red free medium using an automated workstation. - Note: - All cells will be sent for mycoplasma testing and STR profiling. - Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. - Do not exceed a rate of 5-10 µl/sec and do not let the tip end closer than 1mm to the well bottom. - a. Fill wells with 50 µl/well of media in at least 2 rows and 2 columns around wells that are being included in the experiment. - i. Medium for assay: phenol-red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut. - b. To wells in step a, add 20 µl of fresh undiluted PCM from appropriate stromal cells generated as described in step 1 (see Sampling section for Cohorts) or phenol-red free medium alone (Cohort 1). - On day 1 after seeding, read GFP fluorescence (Synergy HT Microplate Reader). - a. Subtract the average reading from media-only wells from the wells with cells. - After reading GFP fluorescence, refresh media and add drug using an automated workstation. - a. Change medium for each cohort to 40 µl fresh diluted PCM from appropriate stromal cell lines generated as described in step 1 or phenol-red free medium alone. - b. Within each cohort, add 10 µl of 5X PLX4720, DMSO dilution or 10 µl phenol-red free medium to each appropriate well to bring the final volume per well up to 50 µl. - i. 5X PLX4720: Make up stocks of 10mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 10µM PLX4720. This is a 5X stock. - ii. DMSO dilution: Dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO wells. - __1. These dilutions in media prevent toxicity from excess DMSO. - On day 4 after seeding, read GFP fluorescence. - a. Subtract the average reading from media-only wells from the wells with cells. - After reading GFP fluorescence, change medium in appropriate wells to 40 µl fresh diluted PCM from appropriate stromal cell lines generated as described in step 1 or phenol-red free medium alone using an automated workstation. - a. Add 10 µl of 5X PLX4720, DMSO dilution or 10 µl phenol-red free medium to each appropriate well to bring the final volume per well up to 50 µl. - i. 5X PLX4720: Make up stocks of 10 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 10 µM PLX4720. This is a 5X stock. - ii. DMSO dilution: Dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO wells. - On day 7 after seeding, read GFP fluorescence and document bright-field and GFP images (BD, Pathway 435 Bioimager). - a. Subtract the average reading from media-only wells from the wells with cells. - Data analysis: - a. Remove background fluorescence by subtracting the average reading from media-only wells from the wells with cells for each plate reading. - b. Subtract the readings of day 1 from the other plates (day 4 and day 7) for the same wells. - c. Average the quadruplicates. - d. Calculate the effect of PLX4720 in the presence or absence of conditioned media by normalizing the number of cells after 7 days of treatment (as measured by GFP fluorescence) to the number of cells present in the SK-MEL-5 vehicle control condition. - Repeat experiment independently three additional times. Deliverables: - Data to be collected: - Raw GFP fluorescence readings from days 1, 4 and 7. - Normalized fluorescence proliferation data. - Fluorescent and bright field micrographs of cells from day 7. - Bar chart of relative proliferation as a % of untreated control for all conditions. (Use data from Day 7 - Day 1 background) (Compare to Figure 2A) - A semi-logarithmic graph of proliferation (log) vs time (linear) over 3 time points after seeding. Confirmatory Analysis Plan: - Statistical Analysis of the Replication Data: - One-way ANOVA comparing the proliferation of PLX4720-treated cells cultured with unconditioned medium, CCD-1090Sk conditioned medium, LL 86 conditioned medium, or PC60163A1 conditioned medium. - Planned comparisons with the Bonferroni correction: - unconditioned medium to PC60163A1 conditioned medium - unconditioned medium to LL 86 conditioned medium - CCD-1090Sk to PC60163A1 conditioned medium - CCD-1090Sk to LL 86 conditioned medium - Meta-analysis of original and replication attempt effect sizes: - Compare the effect sizes of the original data to the replication data and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot. Known differences from original study: - The replication will only use one of the three melanoma cell lines used by the original authors, the SK-MEL-5 cell line. The replication will exclude SK-MEL-28 and G-361 cells. - The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study. - A Synergy HT Microplate Reader will be used instead of a Molecular Devices SpectraMax M5e Microplate Reader – both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for range of detection (Protocol 1) and detection variability (Protocol 2) - A BD Pathway 435 Bioimager used instead of a Zeiss Axio Observer.Z1 – both are fluorescence microscopes with high-throughput screening capabilities. - The replicating lab does not have a ViCell XR cell viability counter, and thus will seed a larger number of cells per well (1,900 instead of 1,700 cells/well). Provisions for quality control: All data obtained from the experiment - raw data, data analysis, control data and quality control data - will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/). - A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. **Protocol 4: Recombinant HGF proliferation assay** This protocol assesses changes in proliferation when melanoma cells are treated with the RAF inhibitor PLK4720 with or without HGF, as is described in Figure 2C. The cells are also treated with a MEK inhibitor, PD184352. Sampling: - Experiment to be repeated a total of 3 times for a final power of 99%. - See Power Calculations section for details - Each experiment has 12 conditions to be done in quadruplicate per experiment: - SK-MEL-5 untreated control [additional control] - SK-MEL-5 vehicle (DMSO) control - SK-MEL-5 2 µM PLX4720 + 0 ng/ml HGF - SK-MEL-5 2 µM PLX4720 + 6.25 ng/ml HGF - SK-MEL-5 2 µM PLX4720 + 12.5 ng/ml HGF - SK-MEL-5 2 µM PLX4720 + 25 ng/ml HGF - SK-MEL-5 2 µM PLX4720 + 50 ng/ml HGF - SK-MEL-5 1 µM PD184352 + 0 ng/ml HGF - SK-MEL-5 1 µM PD184352 + 6.25 ng/ml HGF - SK-MEL-5 1 µM PD184352 + 12.5 ng/ml HGF - SK-MEL-5 1 µM PD184352 + 25 ng/ml HGF - SK-MEL-5 1 µM PD184352 + 50 ng/ml HGF Materials and Reagents: - Reagents that are different from the ones originally used are noted with a *.  Procedure: 1. On day 0, seed 48 wells of a 384 well clear-bottom plate with 2,800 pLex-TRC206 SK-MEL-5 cells in 40 µl of phenol-red free medium each using an automated workstation. - Note: - All cells will be sent for mycoplasma testing and STR profiling. - Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. - Do not exceed a rate of 5-10 µl/sec and do not let the tip end closer than 1 mm to the well bottom. - a. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment. - b. Medium of all cell lines for assay: phenol-red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut. - On day 1 after seeding, read GFP fluorescence (Synergy HT Microplate Reader). - a. Subtract the average reading from media-only wells from the wells with cells. - After reading GFP fluorescence, add to the appropriate wells; 10 µl 6X HGF or phenol-red free medium alone. Then add to the appropriate wells the following; 10 µl 6X PLX4720, 10 µl 6X PD184352, 10 µl DMSO dilution, or 10 µl phenol-red free medium alone. - a. 6X HGF: Make up stocks of 100 ug/ml HGF, then dilute accordingly to make 6X working concentrations of each required HGF dilution. - b. 6X PLX4720: Make up stocks of 12 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 12 uM PLX4720 for use at 6X for the assay to avoid excessive DMSO toxicity. - c. 6X PD184352: Make up stocks of 6 mM PD184352 in DMSO, then dilute 1:1000 in media to make up 6 uM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. - d. DMSO dilution: Dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO dilution wells. a. These media dilutions are to avoid toxicity from excessive DMSO. - On day 4 after seeding, read GFP fluorescence. - a. Subtract the average reading from media-only wells from the wells with cells. - After reading GFP fluorescence, change medium in all wells to 40 µl fresh phenol-red free medium using an automated workstation. Then add to the appropriate wells; 10 µl 6X HGF or phenol-red free medium alone. Then add to the appropriate wells the following; 10 µl 6X PLX4720, 10 µl 6X PD184352, 10 µl DMSO dilution, or 10 µl phenol-red free medium alone. - a. 6X HGF: Make up stocks of 100 ug/ml HGF, then dilute accordingly to make 6X working concentrations of each required HGF dilution. - b. 6X PLX4720: Make up stocks of 12 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 12 uM PLX4720 for use at 6X for the assay to avoid excessive DMSO toxicity. - c. 6X PD184352: Make up stocks of 6 mM PD184352 in DMSO, then dilute 1:1000 in media to make up 6 uM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. - d. DMSO dilution: Dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO dilution wells. - a. These media dilutions are to avoid toxicity from excessive DMSO. - On day 7 after seeding, read GFP fluorescence and document bright-field and GFP images (BD, Pathway 435 Bioimager). - a. Subtract the average reading from media-only wells from the wells with cells. - Data analysis: - a. Remove background fluorescence by subtracting the average reading from media-only wells from the wells with cells for each plate reading. - b. Subtract the readings of day 1 from the other plates (day 4 and day 7) for the same wells. - c. Average the quadruplicates. - d. Calculate the effect of PLX4720 and PD184352 in the presence or absence of HGF by normalizing the number of cells after 7 days of treatment (as measured by GFP fluorescence) to the number of cells present in the SK-MEL-5 vehicle control condition. - Repeat experiment independently two additional times. Deliverables: - Data to be collected: - Raw GFP fluorescence readings from days 1, 4 and 7. - Normalized fluorescence proliferation data. - Fluorescent and bright field micrographs of cells from day 7. - Bar chart of relative proliferation as a % of untreated control for all conditions. (Use data from Day 7 - Day 1 background) (Compare to Figure 2C) - A semi-logarithmic graph of proliferation (log) vs time (linear) over 3 time points after seeding. Confirmatory Analysis Plan: - Statistical Analysis: - Compare the proliferation rate of PLX4720 treated cells treated with 0, 6.25, 12.5, 25, or 50 ng/ml HGF. Also compare each HGF cohort to the proliferation rate of vehicle treated and untreated cells. - One-way ANOVA - Compare the proliferation rate of PD184352 treated cells treated with 0, 6.25, 12.5, 25, or 50 ng/ml HGF. Also compare each HGF cohort to the proliferation rate of vehicle treated and untreated cells. - One-way ANOVA - Meta-analysis of original and replication attempt effect sizes: - Compare the effect sizes of the original data to the replication data use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot. Known differences from original study: - The replication will only use one of the three melanoma cell lines used by the original authors, the SK-MEL-5 cell line. The replication will exclude SK-MEL-28 and G-361 cells. - The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study. - A Synergy HT Microplate Reader used instead of a Molecular Devices SpectraMax M5e Microplate Reader – both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for range of detection (Protocol 1) and detection variability (Protocol 2) - A BD Pathway 435 Bioimager used instead of a Zeiss Axio Observer.Z1 – both are fluorescence microscopes with high-throughput screening capabilities. - The replicating lab does not have a ViCell XR cell viability counter, and thus will seed a larger number of cells per well (2,800 instead of 2,500 cells/well). Provisions for quality control: All data obtained from the experiment - raw data, data analysis, control data and quality control data - will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/). - A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. **Protocol 5: Inhibitor proliferation assay** This experiment confirms that the rescue from drug sensitivity is due to HGF signaling by co-treating cells with crizotinib, an inhibitor of MET, the receptor tyrosine kinase for HGF, as seen in Figure 2D and Supplemental Figure 11. Sampling: - Run the experiment six times in total for a minimum power of 80%. - See Power Calculations section for details - Each experiment has 10 cohorts: - Each cohort consists of - SK-MEL-5 cells alone - SK-MEL-5 co-cultured with LL86 cells - SK-MEL-5 co-cultured with CCD-1090Sk cells - __Each condition will be run in quadruplicate. - The cohorts are treated with the following drugs: - Cohort 1: no drug treatment [additional control] - Cohort 2: Treated with vehicle (DMSO) control - Cohort 3: Treated with 0.2 µM crizotinib and vehicle - Cohort 4: Treated with 0.2 µM PHA-665752 and vehicle [additional control] - Cohort 5: Treated with 2 µM PLX4720 and vehicle - Cohort 6: Treated with 2 µM PLX4720 and 0.2 µM crizotinib - Cohort 7: Treated with 2 µM PLX4720 and 0.2 µM PHA-665752 [additional] - Cohort 8: Treated with 1 µM PD184352 and vehicle - Cohort 9: Treated with 1 µM PD184352 and 0.2 µM crizotinib - Cohort 10: Treated with 1 µM PD184352 and 0.2 µM PHA-665752 [additional control] Materials and Reagents: - Reagents that are different from ones originally used are noted with an asterisk (*)  Procedure: 1. On day 0, seed 40 wells of a 384 well clear-bottom plate with 1,900 LL86 stromal cells in 20 µl phenol-red free media, seed 40 wells with 1,900 CCD-1090Sk stromal cells in 20 µl media, and seed 40 wells with phenol-red free medium alone using an automated workstation. - Note: - All cells will be sent for mycoplasma testing and STR profiling. - Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. - Do not exceed a rate of 5-10 µl/sec and do not let the tip end closer than 1 mm to the well bottom - a. Total wells seeded: 120 - b. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment. - c. Medium of all cell lines for assay: phenol-red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut. - In wells from Step 1, seed 1,900 pLex-TRC206 SK-MEL-5 cells in 20 µl phenol-red free medium per well using an automated workstation. - On day 1 after seeding, read GFP fluorescence (Synergy HT Microplate Reader). a. Subtract the average reading from media-only wells from the wells with cells. - Add appropriate drugs to each well (final volume = 60 µl). - a. Formulation of drug stock solutions: - i. 6X PLX4720: Make up stocks of 12 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 12 µM PLX4720 for use at 6X for the assay to avoid excessive DMSO toxicity. - ii. 6X PD184352: Make up stocks of 6 mM PD184352 in DMSO, then dilute 1:1000 in media to make up 6 µM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. - iii. 6X crizotinib: Make up stocks of 1.2 mM crizotinib in DMSO, then dilute 1:1000 in media to make up 1.2 µM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. - iv. 6X PHA-665752: Make up stocks of 1.2 mM PHA-665752 in DMSO, then dilute 1:1000 in media to make up 1.2 µM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. - v. DMSO dilution: Dilute DMSO 1:1000 in medium to avoid excessive DMSO toxicity. - b. Cohort 1: Add 20 µl phenol-red free medium - c. Cohort 2: Add 10 µl DMSO dilution and 10 µl medium - d. Cohort 3: Add 10 µl 6X crizotinib and 10 µl medium - e. Cohort 4: Add 10 µl 6X PHA-665752 and 10 µl medium - f. Cohort 5: Add 10 µl 6X PLX4720 and 10 µl medium - g. Cohort 6: Add 10 µl 6X PLX4720 and 10 µl 6X crizotinib - h. Cohort 7: Add 10 µl 6X PLX4720 and 10 µl 6X PHA-665752 - i. Cohort 8: Add 10 µl 6X PD184352 and 10 µl medium - j. Cohort 9: Add 10 µl 6X PD184352 and 10 µl 6X crizotinib - k. Cohort 10: Add 10 µl 6X PD184352 and 10 µl 6X PHA-665752 - On day 4 after seeding, read GFP fluorescence. - a. Subtract the average reading from media-only wells from the wells with cells. - Change medium in relevant wells to 40 µl fresh media, then add appropriate drugs as per Step 4 using an automated workstation. - On day 7 after seeding, read GFP fluorescence and document bright-field and GFP images (BD, Pathway 435 Bioimager). - a. Subtract the average reading from media-only wells from the wells with cells. - Data analysis: - a. Remove background fluorescence by subtracting the average reading from media-only wells from the wells with cells for each plate reading. - b. Subtract the readings of day 1 from the other plates (day 4 and day 7) for the same wells. - c. Average the quadruplicates. - d. Calculate the effect of PLX4720, PD184352, Crizotinib, PHA-665752, PLX4720 + Crizotinib, PLX4720 + PHA665752, PD184352 + Crizotinib, PD184352 + PHA665752, DMSO, or untreated in the presence or absence of stromal cells by normalizing the number of cells after 7 days of treatment (as measured by GFP fluoresence) to the number of cells present in the vehicle control treated SK-MEL-5 cells alone condition. - Repeat experiment independently five additional times. Deliverables - Data to be collected: - Raw GFP fluorescence readings from days 1, 4 and 7. - Normalized fluorescence proliferation data. - Fluorescent and bright field micrographs of cells from day 7. - Bar chart of relative proliferation as a % of untreated control for all conditions. (Use data from Day 7 - Day 1 background) (Compare to Figure SF11) - A semi-logarithmic graph of proliferation (log) vs time (linear) over 3 time points after seeding. Confirmatory analysis plan: - Statistical Analysis of replication data: - Three-way ANOVA comparing the proliferation of vehicle-treated, PLX4720-treated, or PD184352-treated cells also treated with vehicle, crizotinib, or PHA-665752 cultured with or without stromal cells followed by: - Two-way ANOVA comparing the proliferation of vehicle-treated cells treated with vehicle, crizotinib, or PHA-665752 cultured with or without stromal cells. - Two-way ANOVA comparing the proliferation of PLX4720-treated cells treated with vehicle, crizotinib, or PHA-665752 cultured with or without stromal cells. - Planned comparisons with the Bonferroni correction: - Vehicle-treated LL 86 cells compared to vehicle-treated no stromal cells - Vehicle-treated LL 86 cells compared to vehicle-treated CCD-1090Sk cells - Vehicle-treated LL 86 cells compared to crizotinib-treated LL 86 cells - Vehicle-treated LL 86 cells compared to PHA-665752-treated LL 86 cells - Two-way ANOVA comparing the proliferation of PD184352-treated cells treated with vehicle, crizotinib, or PHA-665752 cultured with or without stromal cells. - Planned comparisons with the Bonferroni correction: -__Vehicle-treated LL 86 cells compared to crizotinib-treated LL 86 cells -__Vehicle-treated LL 86 cells compared to PHA-665752-treated LL 86 cells - Meta-analysis of original and replication attempt effect sizes: - Compare the effect sizes of the original data to the replication data use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot. Known differences from original study: - Supplemental Figure 11 tests co-culture of SK-MEL-5 cells with 9 stromal cell lines. We have chosen LL86 cells, which showed the largest rescue of proliferation, and CCD-1090Sk cells, which showed the least rescue. - Additional controls added by the replication team: - Treatment with PHA-665752 - In addition to inhibiting MET, crizotinib also targets ALK, ROS1 and RON. In order to confirm that the effects of crizotinib are due to targeting of MET, we will also use a more selective MET inhibitor, PHA-665752 (Cui, 2014; Parikh et al., 2014). - The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study. - A Synergy HT Microplate Reader used instead of a Molecular Devices SpectraMax M5e Microplate Reader - Both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for range of detection (Protocol 1) and detection variability (Protocol 2) - A BD Pathway 435 Bioimager used instead of a Zeiss Axio Observer.Z1 - Both are fluorescence microscopes with high-throughput screening capabilities. - The replicating lab does not have a ViCell XR cell viability counter, and thus will seed a larger number of cells per well (1,900 instead of 1,700 cells/well). Provisions for quality control: All data obtained from the experiment - raw data, data analysis, control data and quality control data - will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/). - A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. **Protocol 6: Inhibitor western blot assay of ERK and AKT signaling** This experiment assesses the protein levels of various activated downstream pathway signaling component proteins in the presence or absence of HGF and drugs, as seen in Figure 4C and Supplemental Figure 19. Sampling: - Repeat the experiment six times in total for a minimum power of 85%. - See Power Calculations section for details - Each experiment contains seven conditions: - SK-MEL-5 cells treated with: - untreated [additional control] - vehicle (DMSO) control - 2 µM PD184352 - 2 µM PLX4720 - 25 ng/ml HGF + vehicle - 25 ng/ml HGF + 2µM PD184352 - 25 ng/ml HGF + 2µM PLX4720 Materials and Reagents: - Reagents that are different from ones originally used are noted with an asterisk (*)   Procedure: 1. On day 0, plate 5x10e5 pLex-TRC206 SK-MEL-5 cells in 2 ml media per well for a total of 7 wells across 2 x 6-well plates. - Note: - All cells will be sent for mycoplasma testing and STR profiling. - Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. - a. Medium of all cell lines for assay: DMEM supplemented with 10% FBS and 1X Pen-Strep. - On day 1 add the appropriate additives to each well. - a. Formulation of stock solutions: Note: These dilutions are to avoid toxicity from excessive DMSO. - i. 1000X HGF: Make a stock of 25 µg/ml HGF. - ii. 1000X PLX4720: Make a stock of 20 mM PLX4720 in DMSO, then dilute 1:10 in media to make a 2 mM PLX4720 working solution. - iii. 1000X PD184352: Make a stock of 20 mM PD184352 in DMSO, then dilute 1:10 in media to make a 2 mM working solution. - iv. DMSO dilution: Dilute DMSO 1:10 in medium. - b. For media only: add 2 µl media - c. For DMSO: Add 2 µl DMSO dilution - d. For 2 µM PD184352: Add 2 µl 1000X PD184352 - e. For 2 µM PLX4720: Add 2 µl 1000X PLX4720 - f. For 25 ng/ml HGF + DMSO: Add 2 µl 1000X HGF and 2 µl DMSO dilution - g. For 25 ng/ml HGF + 2 µM PD184352: Add 2 µl 1000X HGF and 2 µl 100X PD184352 - h. For 25 ng/ml HGF + 2 µM PLX4720: Add 2 µl 1000X HGF and 2 µl 100X PLX4720. - 24 hr after drug treatment, prepare cells for lysis. - a. Quickly wash cells with ice-cold PBS and remove excess PBS. - b. Add 0.5 ml or less of ice-cold lysis buffer to wells on ice. - i. Lysis Buffer: 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1mg/ml NaF and one pellet per 10 ml each of PhosSTOP Phosphatase Inhibitor and Complete Mini Protease Inhibitor. - c. Scrape cells off dish with cell scraper. - d. Collect cells in a 1.5 ml centrifuge tube on ice. - e. Incubate on ice for 30 min with periodic vortexing. - f. Spin down at 4˚C and remove supernatant into separate tube. - Determine protein concentration by using the DC Protein Assay Kit II following manufacturer’s instructions. - Mix 50 µg total cell lysate with NuPAGE Sample Reducing Agent and run on two 4-12% Bis-Tris gels with a protein molecular weight marker at 120 V. - Transfer onto membrane using replicating lab’s transfer protocol. - After the transfer, stain the membrane with Ponceau to visualize transferred protein. Image membrane, then wash out the Ponceau stain. [additional quality control step] - Wet membrane with PBS for 5 min, then block membranes in Odyssey blocking buffer (LI-COR, 927-40000) following manufacturer’s instructions. - Probe membrane with the following primary antibodies diluted in Odyssey blocking buffer at 4°C with gentle shaking, overnight. - a. mouse anti-c-Met (Cell Signaling, 3148); 1:1000; 145kDa - b. rabbit anti-pMet Tyr 1349 (Cell Signaling, 3133); 1:1000; 145kDa - c. mouse anti-AKT (Cell Signaling, 2920); 1:2000; 60kDa - d. rabbit anti-pAKT (Cell Signaling, 4060); 1:2000; 60kDa - e. mouse anti-MEK (Cell Signaling, 4694); 1:1000; 45kDa - f. rabbit anti-pMEK (Cell Signaling, 9154); 1:1000; 45kDa - g. mouse anti-ERK (Santa Cruz, 135900); 1:200; 44,42kDa - h. rabbit anti-pERK (Cell Signaling, 4370); 1:2000; 44,42kDa - i. rabbit anti-GAPDH (Cell Signaling, 2118); 1:1000; 37kDa - i. Loading control - j. Note: multiple gels will need to be run to probe for this many proteins. Do not strip between probing with different phospho antibodies, just wash membrane well (4 x 10 min PBS-T) and then add next antibody. Suggest grouping as follows: - i. Gel 1: Probe pAKT [rabbit 60kDa], then pMEK [rabbit 45kDa], then AKT [mouse 60kDa], then MEK [mouse 45kDa], then GAPDH [rabbit 37kDa]. - ii. Gel 2: Probe pMet Tyr 1349 [rabbit 145kDa], then pERK [rabbit 44,42kDa], then c-Met [mouse 145kDa], then ERK [mouse 44,42kDa], then GAPDH [rabbit 37kDa]. - Wash membranes in PBS + 0.1% Tween 20 4 x 5 min. - Detect primary antibodies with anti-rabbit or anti-mouse IRDye secondary antibodies (LICOR) diluted in Odyssey blocking buffer for 30-60 min protected from light following manufacturer’s instructions. - Wash membranes in PBS + 0.1% Tween 20 4 x 5 min. - Rinse membrane with PBS to remove residual Tween 20. - Detect near infrared fluoresence with the Odyssey Infrared Imaging System. - Quantify signal intensity with Odyssey Application Software. - a. For each antibody subtract background intensity from values and then divide by the GAPDH loading control. - b. Calculate the effect of PLX4720, PD184352, or vehicle in the presence or absence of HGF by normalizing the band intensities (after background and loading correction) to the band intensity of the SK-MEL-5 vehicle control condition. - Repeat experiment independently five additional times. Deliverables: - Data to be collected: - Odyssey images of probed membranes (full images with ladder) - Raw and quantifed signal intensities normalized for GAPDH loading and total pan-protein levels. - Bar graphs of normalized mean signal intensities (Compare to Figure S19) Confirmatory Analysis plan: - Statistical Analysis of replication data: - Two-way ANOVA comparing the relative phopho-AKT band intensities of cells treated with vehicle, PLX4720, or PD184352 in the presence or absence of HGF. - Planned comparisons with the Bonferroni correction: - PLX4720-treated cells in the absence of HGF compared to PLX4720-treated cells in the presence of HGF - Two-way ANOVA comparing the relative phopho-ERK band intensities of cells treated with vehicle, PLX4720, or PD184352 in the presence or absence of HGF. - Planned comparisons with the Bonferroni correction: - PLX4720-treated cells in the absence of HGF compared to PLX4720-treated cells in the presence of HGF - Two-way ANOVA comparing the relative phopho-MET (Tyr1349) band intensities of cells treated with vehicle, PLX4720, or PD184352 in the presence or absence of HGF. - Planned comparisons with the Bonferroni correction: - Cells treated in the absence of HGF and treated with vehicle, PLX4720, or PD184352 compared to cells treated in the presence of HGF and treated with vehicle, PLX4720, or PD184352 - Meta-analysis of original and replication attempt effect sizes: - Compare the effect sizes of the original data to the replication data and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot. Known differences from original study: - Provider lab transfer protocol used instead of iBlot Gel Transfer Device (Invitrogen, IB1001) using Program 4 – both are capable of transferring protein efficiently, and to determine completeness of the transfer, the gel may be stained (Step 8). - The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study. - The replication will not include the pMet Tyr1234/5, RAF1, and pRAF1 antibodies included in the original study. Provisions for quality control: All data obtained from the experiment - raw data, data analysis, control data and quality control data - will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/). - A lab from the Science Exchange network with extensive experience in conducting cell viability assays and performing Western blots will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. ### Power Calculations All calculations are determined in order to reach at least 80% power. **Protocol 1** No power calculations required. **Protocol 2** No power calculations required. **Protocol 3** Summary of original data: Note: Original data values were shared by authors.  - Standard deviation was calculated using formula SD = SEM*(SQRT n) Test family - ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with Graphpad Prism 6.0 - Partial η2 calculated from (Lakens, 2013) Power calculations for replication:  Test family - two tailed t-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.0125. - Calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power Calculations for replication:  **Protocol 4** Summary of original data: Note: Original data values were shared by authors.  Test family: - ANOVA: Fixed effects, omnibus, one way, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with Graphpad Prism 6.0 - Partial η2 calculated from (Lakens, 2013)  Summary of original data: Note: Original data values were shared by authors.  Test family: - ANOVA: Fixed effects, omnibus, one way, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with Graphpad Prism 6.0 - Partial η2 calculated from (Lakens, 2013) Power Calculations for replication:  **Protocol 5** Summary of original data: Note: numbers were shared by original authors.  Test family: - 3-way ANOVA Between subjects: Fixed effects, special, main effects and interactions, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with R software 3.1.1 (R Core Team, 2014) - Partial η2 calculated from (Lakens, 2013) Power Calculations for replication:  Test family: - 2-way ANOVA Between subjects: Fixed effects, special, main effects and interactions, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with Graphpad Prism 6.0 - Partial η2 calculated from (Lakens, 2013) Power Calculations for replication (BRAF/MEK inhibitor):  Test family: - two tailed *t*-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.0125. - Power calculations were performed for effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power Calculations for replication (PLX4720 group):  Test family: - two tailed *t*-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.025. - Power calculations were performed for effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power Calculations for replication (PD184352 group):  **Protocol 6** Summary of original data: Note: numbers were estimated from bar chart in Supplemental Figure S19.  Test family: - 2-way ANOVA Between subjects: Fixed effects, special, main effects and interactions, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with Graphpad Prism 6.0 - Partial η2 calculated from (Lakens, 2013) Power Calculations for replication:  Test family - two tailed *t*-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.05. - Note: Calculations were performed for effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power calculations for replication (pAKT group):  Note: HGF/PD184352 compared to vehicle/PD184352 is not included as the number of needed samples is too large Test family - two tailed *t*-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.05. - Note: Calculations were performed for effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power calculations for replication (pERK group):  Note: HGF/PD184352 compared to vehicle/PD184352 is not included as the number of needed samples is too large. Test family - two tailed *t*-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.05. - Note: Calculations were performed for effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power calculations for replication (pMET(Tyr1349) group):  ### Acknowledgements The Reproducibility Project: Cancer Biology core team would like to thank the original authors, in particular Ravid Straussman and Michal Barzily-Rokni, for generously sharing critical information and reagents to ensure the fidelity and quality of this replication attempt. We would also like to thank the following companies for generously donating reagents to the Reproducbility Project: Cancer Biology; BioLegend, Charles River Laboratories, Corning Incorporated, DDC Medical, EMD Millipore, Harlan Laboratories, LI-COR Biosciences, Mirus Bio, Novus Biologicals, and Sigma-Aldrich. ### References 1. Caenepeel, S, Cajulis, E, Kendall, R, Coxon, A, and Hughes, P. Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines. In: *Proceedings of the 104th Annual Meeting of the American Association for Cancer Research*, Washington, DC. 2013. Cancer Res 73: Abstract. doi: 10.1158/1538-7445.AM2013-3405. - Casbas-Hernandez, P, Arcy, MD, Roman-Perez, E, Brauer, HA, McNaughton, K, Miller, SM, Chhetri, RK, Oldenburg, AL, Fleming, JM, Amos, KD, Makowski, L, and Troester, MA. 2013. Role of HGF in epithelial-stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ. *Breast Cancer Research* 15:R82. doi: 10.1186/bcr3476. - Cui, JJ. 2014. Targeting Receptor Tyrosine Kinase MET in Cancer: Small Molecule Inhibitors and Clinical Progress. *J. Med. Chem*. 57:4427-53. doi: 10.1021/jm401427c. - Faul, F, Erdfelder, E, Lang, AG, and Buchner, A. 2007. G* Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. *Behavior research methods* 39:175-91. - Lezcano, C, Lee, C-W, Larson, AR, Menzies, AM, Kefford, RF, Thompson, JF, Mihm, MC, Ogino, S, Long, GV, Scolyer, RA, and Murphy, GF. 2014. 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Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. *Nature* 487:505-09. doi: 10.1038/nature11249. - Xie, Q, Su, Y, Dykema, K, Johnson, J, Koeman, J, De Giorgi, V, Huang, A, Schlegel, R, Essenburg, C, Kang, L, Iwaya, K, Seki, S, Khoo, SK, Zhang, B, Buonaguro, F, Marincola, FM, Furge, K, Vande Woude, GF, and Shinomiya, N. 2013. Overexpression of HGF Promotes HBV-Induced Hepatocellular Carcinoma Progression and Is an Effective Indicator for Met-Targeting Therapy. *Genes &amp; Cancer* 4:247-60. doi: 10.1177/1947601913501075. ### Author Information † The RP:CB core team consists of Elizabeth Iorns (Science Exchange, Palo Alto, California), William Gunn (Mendeley, London, United Kingdom), Fraser Tan (Science Exchange, Palo Alto, California), Joelle Lomax (Science Exchange, Palo Alto, California), and Timothy Errington (Center for Open Science, Charlottesville, Virginia). David Blum, Bioexpression and Fermentation Facility, University of Georgia, Athens, Georgia Samuel LaBarge, City of Hope, Duarte, California Correspondence to Fraser Tan, fraser@scienceexchange.com. Competing Interests: We disclose that EI, FT, and JL are employed by and hold shares in Science Exchange Inc. The experiments presented in this manuscript will be conducted by EG at the Monoclonal Antibody Core Facility, which is a Science Exchange lab. No other authors disclose conflicts of interest related to this manuscript. Funding: The Reproducibility Project: Cancer Biology is funded by the Laura and John Arnold Foundation, provided to the Center for Open Science in collaboration with Science Exchange. The funder had no role in study design or the decision to submit the work for publication.