Journal articles on the topic 'SynNotch'

Abstract BACKGROUND: The development of effective chimeric antigen receptor (CAR) T-cell therapies for malignant pediatric brain tumors remains a challenge due to multiple barriers, including antigenic heterogeneity, on-target off-tumor toxicities, and T cell exhaustion. We have adopted a novel synthetic Notch “synNotch” receptor system and developed innovative T-cell circuits that recognize tumor cells based on the “prime-and-kill” strategy. METHODS: We created a novel synNotch-CAR circuit in which the brain/glioma-specific antigen Brevican (BCAN) primes the T cells to induce expression of a CAR that recognizes interleukin-13 receptor α2 (IL-13Rα2) and ephrin type A receptor (EphA2), thereby eradicating glioma cells expressing either antigen. Immunocompromised mice bearing the SF8628 DIPG cell line in the frontal lobe or brain stem received a single intravenous (IV) infusion of synNotch CAR T-cells (2.5 x 106 each of CD4+ and CD8+ T cells) on day 6 following the tumor inoculation. Mice were monitored for toxicity and tumor growth. RESULTS: Following this synNotch CAR T-cell dose, although tumors in the brainstem did not regress, 3 of the 5 mice with frontal lobe tumors demonstrated complete and sustained remission. Our histological analyses revealed primed CAR T-cells both within and surrounding the tumor in both settings. By flow cytometry, we confirmed the CAR T-cells in the CNS were primed and mostly did not express an exhaustive phenotype. In the spleen, we also found the CAR T-cells in a more naïve and central memory state. CONCLUSIONS: Our work so far has demonstrated that synNotch-CAR T-cells are able to traffic to the tumor microenvironment even in the brainstem, are primed to express the CAR, and most do not express an exhaustive phenotype. Future work will include CAR T-cell dose optimization, continued assessments of the tumor microenvironment, and investigating for antigen escape.

Synthetic receptor biology and genome editing are emerging techniques, both of which are currently beginning to be used in preclinical and clinical applications. We were interested in whether a combination of these techniques approaches would allow for the generation of a novel type of reporter cell that would recognize transient cellular events through specifically designed synthetic receptors and would permanently store information about these events via associated gene editing. Reporting cells could be used in the future to detect alterations in the cellular microenvironment, including degenerative processes or malignant transformation into cancer cells. Here, we explored synthetic Notch (synNotch) receptors expressed in human embryonic kidney cells to investigate the efficacy of antigen recognition events in a time- and dose-dependent manner. First, we evaluated the most suitable conditions for synNotch expression based on dsRed-Express fluorophore expression. Then, we used a synNotch receptor coupled to transcriptional activators to induce the expression of a Cas9 nuclease targeted to a specific genomic DNA site. Our data demonstrate that recognition of various specific antigens via synNotch receptors robustly induced Cas9 expression and resulted in an indel formation frequency of 34.5%–45.5% at the targeted CXCR4 locus. These results provide proof of concept that reporter cells can be designed to recognize a given event and to store transient information permanently in their genomes.

Abstract Heterogeneous expression of target antigens allows tumor escape from chimeric antigen receptor-transduced T-cell (CART) therapy targeting a single antigen. While epidermal growth factor receptor (EGFR)vIII represents a glioblastoma (GBM)-specific antigen, its expression is heterogeneous within the tumor. On the other hand, most other antigens expressed more uniformly in GBMs are non-mutated, glioma-associated antigens (GAAs), such as EphA2. Although these GAAs are not expressed in the normal brain, they are expressed at low levels in other normal organs. As a way to safely target GAAs in the tumor without attacking normal cells expressing the same GAAs outside of the brain, we adapted a novel synthetic Notch (synNotch) receptor system, and established a “prime and kill” sequential two-receptor CAR circuit: the first is a transcriptional CAR against EGFRvIII, and the second is a CAR against a GAA (e.g. EphA2). When the first CAR binds to EGFRvIII, it induces the expression of the second CAR against GAA. While the first CAR does not trigger the cytotoxic function itself, the second CAR mediates the cytotoxicity upon recognition of the target GAA. We validated this system in vitro using the mixture of U87 GBM cells and those transduced with EGFRvIII. SynNotch CART effectively lysed both EGFRvIII+ and EGFRvIII-negative U87 cells when they are mixed, but never lysed EGFRvIII-negative cells in the absence of EGFRvIII+ cells. SynNotch CART cells exhibited precise local effect in vivo, sparing EGFRvIII-negative subcutaneous tumor while efficiently clearing intracranial EGFRvIII heterogeneous tumor. In the orthotopic GBM6 model, which is a patient-derived xenograft with heterogeneous expression of EGFRvIII, intravenous infusion of synNotch CART resulted in long-term (over 50 days) survival and eradication of the heterogeneous tumor in 6 of 6 mice, while the conventional anti-EGFRvIII CART failed to do so. These data provide a strong basis for developing synNotch CART therapy for GBM.

Neuronal apoptosis is an early and critical pathological hallmark of many chronic neurodegenerative diseases, often occurring silently long before the appearance of overt clinical symptoms. In this study, we engineered astrocytes utilizing a dual-biomarker recognition synNotch system (dual-synNotch). This system is designed to specifically identify neuronal apoptosis through the ‘AND Gate’ activation mechanism, which is triggered by the simultaneous sensing of the apoptotic signal phosphatidylserine (PS) and the neuronal signal ganglioside Gt1b. Upon detection of these neuronal apoptotic signals, the synNotch receptors are activated, inducing the expression of two key molecules: secreted Gaussia luciferase (GLuc), a highly detectable reporter that can cross the blood–brain barrier (BBB), and brain-derived neurotrophic factor (BDNF), a neuroprotective molecule that promotes neuronal survival by inhibiting apoptosis and enhancing memory and cognitive function. This engineered system effectively converts and amplifies early, imperceptible neuronal apoptotic signals into detectable outputs, enabling convenient in vitro monitoring and diagnosis. Therefore, it represents a promising strategy for the early detection and intervention of neurodegenerative diseases associated with neuronal apoptosis.

Treatment of solid cancers with chimeric antigen receptor (CAR) T cells is plagued by the lack of ideal target antigens that are both absolutely tumor specific and homogeneously expressed. We show that multi-antigen prime-and-kill recognition circuits provide flexibility and precision to overcome these challenges in the context of glioblastoma. A synNotch receptor that recognizes a specific priming antigen, such as the heterogeneous but tumor-specific glioblastoma neoantigen epidermal growth factor receptor splice variant III (EGFRvIII) or the central nervous system (CNS) tissue-specific antigen myelin oligodendrocyte glycoprotein (MOG), can be used to locally induce expression of a CAR. This enables thorough but controlled tumor cell killing by targeting antigens that are homogeneous but not absolutely tumor specific. Moreover, synNotch-regulated CAR expression averts tonic signaling and exhaustion, maintaining a higher fraction of the T cells in a naïve/stem cell memory state. In immunodeficient mice bearing intracerebral patient-derived xenografts (PDXs) with heterogeneous expression of EGFRvIII, a single intravenous infusion of EGFRvIII synNotch-CAR T cells demonstrated higher antitumor efficacy and T cell durability than conventional constitutively expressed CAR T cells, without off-tumor killing. T cells transduced with a synNotch-CAR circuit primed by the CNS-specific antigen MOG also exhibited precise and potent control of intracerebral PDX without evidence of priming outside of the brain. In summary, by using circuits that integrate recognition of multiple imperfect but complementary antigens, we improve the specificity, completeness, and persistence of T cells directed against glioblastoma, providing a general recognition strategy applicable to other solid tumors.

Abstract One of the key challenges in the development of CAR-T therapy is the absence of target antigens that are tumor-specific and have homogenous expression. To safely target glioblastoma-associated antigens (GAAs) in the tumor without attacking normal tissue expressing the same GAAs outside of the brain, we adapted a novel synthetic Notch (synNotch) receptor system and established a “prime and kill” sequential two-receptor CAR circuit. We used glioblastoma- (GBM) specific neoantigen EGFRvIII as a priming signal for synNotch receptor and have reported robust antitumor response in the mice bearing intracerebral PDX tumor with heterogenous EGFRvIII expression. However, less than 20% of adult GBM cases express EGFRvIII. Furthermore, EGFRvIII expression can diminish over time even after its detection in the primary GBM, and the EGFRvIII-synNotch primed CAR T cells may deplete EGFRvIII-expressing GBM cells via their cytotoxic effects, thereby losing the priming signal. To overcome these inherent challenges of the EGFRvIII-priming strategy, we used CNS tissue-specific antigens as priming signal to drive localized expression of the CAR against EphA2 and IL-13Rα2 and bypassed the need for tumor-specific antigen. We have found BCAN (also known as Brevican) as the most promising priming antigen among several CNS-specific cell surface proteins that we evaluated. When mice bearing intracerebral GBM6 PDX received a single IV infusion of T cells engineered with the α-BCANsynNotch- > α-EphA2/IL-13Rα2 CAR (B-SYNC) circuit, all mice (10/10) demonstrated complete regression of the tumor. Furthermore, these B-SYNC T cells were significantly more efficacious than constitutively expressed EphA2/IL-13Rα2 CAR T cells, associated with superior persistence and less exhausted phenotype. Thus, the B-SYNC approach represents a conceptual novelty of CNS tissue-specific CAR activation. This also enhances the translational significance of synNotch-CAR T cells by widely extending the eligibility to EGFRvIII-negative glioma patients. We will develop and conduct a phase I study to evaluate our hypothesis.

Abstract HER2 amplification occurs in about 5% of colorectal cancers (CRC) and is only partially associated with clinical response to combined HER2/EGFR targeted treatment. An alternative approach, based on Adoptive Cell Therapy (ACT) using T cells engineered with anti-HER2 Chimeric Antigen Receptor (CAR), proved toxic due to "on-target off-tumor" activity. Therefore, we developed a combinatorial ACT strategy to safely target HER2 amplification in CRC using a synNotch-based artificial regulatory network. A synthetic Notch receptor was employed in which the extracellular domain is an anti-HER2 scFv and the intracellular domain contains the GAL4VP64 artificial transcription factor. Engagement of the HER2-synNotch by overexpressed HER2 on the surface of target cells induces GAL4VP64 cleavage and translocation to the nucleus, where it drives expression of a CAR under a GAL4-responsive promoter. In this way, only cells co-expressing both HER2 and the CAR target are killed. Carcinoembryonic antigen (CEA) was selected as a colon-specific CAR target: CEA expression is indeed restricted to the digestive tract and is increased in CRC. The natural killer cell line NK-92 was chosen as effector to be engineered. NK-92 cells transduced with HER2-synNotch and inducible CEA-CAR lentiviral vectors were subsequently sorted in the OFF and ON state to select those with the higher CAR induction after synNotch engagement. Subsequently, cloning of sorted cells led to identification of an optimally responsive clone (clone 5F), in terms of specificity and amplitude of CAR induction. The 5F clone demonstrated significant activity in vitro and in vivo specifically against HER2amp CRC models, with no effects on cells with physiological HER2 levels. In vitro, the 5F clone displayed selective cytotoxicity against HER2amp/CEA+ CRC cells, with minimal killing activity against HER2amp/CEA- cells, or HER2norm/CEA+ cells. Additional assays on 3D organoids highlighted better recruitment and infiltration by the 5F clone respect to NK-92 WT cells, only in HER2amp models. In vivo, NK-92.5F significantly impaired tumor growth in two different HER2amp CRC models, with no effect on HER2norm CRC cells. To further improve survival, tumor penetration and in vivo efficacy of the NK-92.5F clone, a more complex system was built, in which HER2 synNotch engagement drives expression of both the CEA-CAR and IL2. 5F-IL2 cells displayed further increased cytotoxicity in vitro. In vivo, 5F-IL2 cells drastically increased survival of mice carrying HER2amp CRC xenografts with respect to the parental 5F clone. The observed selective therapeutic efficacy both in vitro and in vivo of this innovative HER2 synNotch/CEA-CAR system, and its future evolutions, opens perspectives for possible clinical applications in cases of HER2amp CRC resistant or partially responsive to HER2/EGFR blockade. Citation Format: Marco Cortese, Erica Torchiaro, Alice D'Andrea, Consalvo Petti, Federica Invrea, Letizia Franco, Chiara Donini, Valeria Leuci, Simonetta M. Leto, Valentina Vurchio, Francesca Cottino, Claudio Isella, Sabrina Arena, Elisa Vigna, Andrea Bertotti, Livio Trusolino, Dario Sangiolo, Enzo Medico. Preclinical efficacy of a synthetic Notch-based combinatorial immunotherapy against colorectal cancer with HER2 amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3998.

Abstract Adoptive immunotherapy based on chimeric antigen receptor (CAR)-T cells has led to successful treatment of some hematological malignancies, but it remains extremely challenging for solid tumors, mostly because of “on-target off-tumor” toxicity, as observed in the case of anti-HER2 CAR-T treatment of colorectal cancer (CRC) with HER2 amplification. To enable adoptive immunotherapy against HER2-amplified CRC, we therefore considered a combinatorial strategy based on the synNotch-based artificial regulatory network. A synthetic Notch receptor was employed in which the extracellular domain is an anti-HER2 scFv and the intracellular domain contains the GAL4-VP64 artificial transcription factor. Engagement of the anti-HER2 domain by target cells drives GAL4-VP64 cleavage and translocation to the nucleus, where it drives expression of a CAR under a GAL4-responsive promoter. In this way, only cells co-expressing both HER2 and the CAR target are killed. As a CRC-specific CAR target we selected CEA, product of the CEACAM5 gene. CEA expression is restricted to the digestive tract and is increased in cancer. For the generation of HER2-synNotch CEA-CAR effectors, we chose the natural killer cell line NK-92 and transduced it with two lentiviral vectors, encoding respectively the HER2 synNotch and the second-generation anti-CEA CAR with CD28 costimulatory domain. Transduced cells were repeatedly sorted in the ON and OFF state to select those with the best CAR induction after synNotch engagement; cloning of sorted cells led to identification of an optimally responsive clone (clone 5F), showing no basal CEA-CAR expression and massive induction in the presence of HER2-overexpressing cancer cells. In vitro, the 5F clone displayed selective cytotoxicity against HER2++/CEA++ CRC cells, with minimal killing activity against HER2++/CEA- breast cancer cells, or against CRC cells expressing CEA but without HER2 amplification. In vitro 3D models highlighted better recruitment and infiltration by NK-92 clone 5F respect to NK-92 WT cells, only of HER2++ organoids. In vivo, the clone 5F significantly impaired tumor growth in two different HER2++ CRC models. The observed selective efficacy both in vitro and in vivo of the HER2-synNotch/CEA-CAR approach opens a perspective for possible clinical applications in cases of HER2-amplified CRC displaying primary or secondary resistance to HER2/EGFR blockade. Citation Format: Marco Cortese, Erica Torchiaro, Federica Invrea, Consalvo Petti, Alice D'Andrea, Sabrina Arena, Enzo Medico. Design, characterization and preclinical validation of a combinatorial CAR-based immunotherapy against colorectal cancer with HER2 amplification [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 LB091.

Abstract BACKGROUND Diffuse midline glioma (DMG), including Diffuse intrinsic pontine glioma (DIPG), is an aggressive brain tumor in children with limited treatment options. Recent developments of phase 1 clinical trials have shown early promise for chimeric antigen receptor (CAR) T cells in patients with DMG/DIPG. However, several barriers such as the absence of tumor-specific antigens, restricted trafficking to the tumor site, and poor persistence hinder the full therapeutic potential of CAR T cell therapy in DMG/DIPG. METHODS To safely target the glioma-associated antigens (GAAs) without attacking nomal tissues expressing the same antigens, we adopted a novel synthetic Notch (synNotch) receptor system and engineered T cell circuits employing a “prime-and-kill” strategy. In this system, a synNotch receptor recognizing the priming antigen, Brevican (BCAN), exclusively expressed on cells in the central nervous system (CNS) but not on non-CNS cells, locally induces the expression of a tandem CAR against GAAs, ephrin type A receptor (EphA2) and interleukin-13 receptor a2 (IL13Ra2), there by homogenously eliminating DMG/DIPG cells. RESULTS The α-BCAN synNotch⋄α-EphA2/IL-13Rα2 CAR (B-SYNC) T cells efficiently killed DMG/DIPG (BT-245, SF8628, SU-DIPGXIII, and SU-DIPGXVII) cells in vitro in the presence of priming cells (K562 cells expressing BCAN). Moreover, a single intravenous infusion of B-SYNC T cells significantly (P< 0.001) prolonged the survival of immunodeficient mice bearing aggressive, orthotopic SF8628 DIPG xenografts and completely eradicated the tumor in more than 50% (4/7) of mice. Strikingly, we observed excellent homing, priming, activation, and persistence of B-SYNC T cells in the brain stem of mice bearing SF8628 xenografts. In contrast, constitutively active α-EphA2/IL-13Rα2 CAR T cells or untransduced T cells demonstrated a failure to migrate to the brain stem and improve the survival of mice. CONCLUSIONS Collectively, our findings strongly support the development of clinical trials evaluating the efficacy of B-SYNC T cells in DMG/DIPG patients.

Natural killer (NK) cells are crucial components of innate immunity, known for their potent tumor surveillance abilities. Chimeric antigen receptors (CARs) have shown promise in cancer targeting, but optimizing CAR designs for NK cell functionality remains challenging. CAR-NK cells have gained attention for their potential to reduce side effects and enable scalable production in cancer immunotherapy. This study aimed to enhance NK cell anti-tumor activity by incorporating PD1-synthetic Notch (synNotch) receptors. A chimeric receptor was designed using UniProt database sequences, and 3D structure models were generated for optimization. Lentiviral transduction was used to introduce PD1-Syn receptors into NK cells. The expression of PD1-Syn receptors on NK cell surfaces was assessed. Engineered NK cells were co-cultured with PDL1+ breast cancer cells to evaluate their cytotoxic activity and ability to produce interleukin-12 (IL-12) and interferon-gamma (IFNγ) upon interaction with the target cells. This study successfully expressed the PD1-Syn receptors on NK cells. CAR-NK cells secreted IL-12 and exhibited target-dependent IFNγ production when engaging PDL1+ cells. Their cytotoxic activity was significantly enhanced in a target-dependent manner. This study demonstrates the potential of synNotch receptor-engineered NK cells in enhancing anti-tumor responses, especially in breast cancer cases with high PDL1 expression.

BackgroundSolid tumors such as GBM are particularly difficult to treat, being largely resistant to traditional treatments, fueling interest in alternative treatment approaches, including cell-based immunotherapy. Natural killer (NK) cells have emerged as promising effectors to target GBM through genetic modifications and ex vivo manipulation. However, immunosuppressive conditions within the tumor microenvironment (TME) further complicate NK cell-based treatments. Specifically, within the TME tumor cells release of high levels of ATP extracellularly. While intracellular ATP is necessary for cell metabolism, extracellular ATP is converted into adenosine (ADO) by ectonucleotidases CD39 and CD73, both overexpressed on GBM.1 Extracellular ADO induces immunometabolic suppression of NK cells through binding with A2A adenosine receptors (A2ARs) on NK cells, suppressing cytokine secretion, proliferation, and other functional activities. 2–4 Adding to the suppression of NK cells is the interaction between CD155, expressed highly on GBM and other solid tumors, and T cell immunoreceptor with Ig and ITIM domains (TIGIT) expressed on NK cells. This interaction signals inhibition of NK cell cytolytic function, allowing for cancer cell immune-evasion.5,6MethodsTo restore impaired NK cell anti-tumor activity, we have engineered NK cells to concomitantly target CD155 and CD73-induced immunosuppression on GBM using a tumor-responsive genetic construct. The construct is capable of blocking the immunosuppressive CD155/TIGIT interaction, and, upon binding, release a CD73-blocking scFv to inhibit the accumulation of extracellular ADO and mitigate immunosuppression of NK cells. Such localized response enhances specificity and reduces off-target effects of NK-based targeting.ResultsPrimary NK cells were successfully electroporated to express our synthetic TIGIT-synNotch construct, as evidenced by increased expression levels of TIGIT (% and MFI) (figure 1). To evaluate the functionality of engineered NK cells against GBM targets, we tested the cytotoxicity of our engineered NK cells against a primary, patient-derived GBM cell line, GBM43. Overall, cytolytic function of engineered NK cells against GBM was significantly higher than that of non-engineered NK cells, with or without CD73 (10 ug/mL) and TIGIT (50 ug/mL) antibodies, for E:T ratios of 5:1 and 10:1 (figure 2), demonstrating the functional efficacy of our genetic construct. Further, engineered NK cells (T-PNK) expressed significantly higher levels of CD107a in response to GBM43 stimulation than non-engineered PNK at E:T ratios 2.5:1 and 10:1 (figure 3).Abstract 123 Figure 1TIGIT-synNotch gene expressionGene expression (left: %, right, MFI) of electroporated NK cells engineered with anti-CD73 and TIGIT blocking mRNAAbstract 123 Figure 2Engineered NK cell cytotoxicityCytotoxicity of NK cells against GBM43 cells at E:T ratios of 2.5:1, 5:1, and 10:1. NK cells were either un-transfected (with and without CD73 and TIGIT mAbs), transfected with the TIGIT-synNotch construct, or transfected with the TIGIT-synNotch and CD73 genetic constructsAbstract 123 Figure 3Engineered NK cell degranulationCD107a expression measured on transfected and non-transfected NK cells stimulated with GBM43 at E:T ratios of 2.5:1, 5:1, and 10:1ConclusionsOverall, we have shown that co-targeting CD155 and CD73 in a localized, responsive manner can dampen immunosuppression and significantly enhance the killing potential of engineered NK cells against aggressive patient-derived GBM tumors.ReferencesChambers AM, et al. Adenosinergic Signaling Alters Natural Killer Cell Functional Responses. Front. Immunol 2018;9:2533.Chambers AM, Lupo KB & Matosevic S. Tumor microenvironment-induced immunometabolic reprogramming of natural killer cells. Front Immunol 2018;9:2517.Chambers AM, et al. Adenosinergic signaling alters natural killer cell functional responses. Front. Immunol 2018;9:2533.Wang, J., Lupo, K. B., Chambers, A. M. & Matosevic, S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. J. immunotherapy cancer 2018;6:136.Zhang B, et al. Immunoreceptor TIGIT inhibits the cytotoxicity of human cytokine-induced killer cells by interacting with CD155. Cancer Immunol Immunother 2016;65:305–314.Lupo KB & Matosevic S. CD155 immunoregulation as a target for natural killer cell immunotherapy in glioblastoma. J Hematol Oncol 2020;13:76.

Abstract Background Multiple myeloma (MM) remains an incurable malignancy, with most patients relapsing and dying from the disease. Anti-myeloma drugs, such as proteasome inhibitors (PIs) bortezomib and carfilzomib (CFZ), have considerably improved prognosis in myeloma. Despite these advances, disease heterogeneity, early relapse and treatment resistance still pose major challenges in MM treatment. Understanding the mechanisms that mediate PI resistance provide a key to targeting both, PI-resistant minimal residual disease that drives relapsed MM after prolonged PI-containing frontline therapy, as well as PI-refractory, aggressive advanced MM. While key mechanisms of the in vitro-generated PI resistance in MM have been revealed in cell line models, we lack understanding of PI resistance in vivo, where in particular clonal heterogeneity and the tumor microenvironment (TME) within the bone marrow (BM) add additional levels of complexity. Therefore, the aim of our study was to analyze the molecular landscape and changes occurring during MM progression under CFZ treatment in vivo and to identify key molecular processes contributing to CFZ-resistance of MM cells in the presence of stromal cells in vitro, to ultimately identify new molecular pathways and develop innovative treatment strategies in PI-resistant MM. Methods The NSG mice intrafemorally engrafted with human RPMI-8226 cells were either untreated or treated long-term with 4 mg/kg CFZ (intravenously) until they became drug resistant. At this point, CFZ naïve and CFZ-resistant cells were isolated and processed for single-cell RNA sequencing (scRNA-seq, 10x Genomics) with the aim to characterize a transcriptional CFZ-resistance signature in refractory cells. To investigate the role of the TME as well as the importance of cell-cell interactions in CFZ-resistance in vitro, we performed two independent genome-wide CRISPR/Cas9 library screenings. In the first one, Brunello library transduced RPMI-8226 cells were co-cultured with human stromal cells (HS5) and treated with CFZ to identify CFZ sensitivity/resistance candidate genes. In the second experiment, Brunello library and synthetic Notch (synNotch) receptor transduced HS5 cells were co-cultured with synNotch ligand transduced RPMI-8226 cells to identify genes that are essential for establishing cell-cell contacts between stromal and MM cells. Subsequent functional analysis of the highest-ranking CFZ sensitivity/resistance candidates in the RPMI-8226+HS5 co-culture included shRNA-silencing, single-gene knockouts, viability assays, cell cycle analysis and protein synthesis analysis using the SUnSET assay. Results ScRNA-seq analysis of CFZ-refractory RPMI-8226 cells growing in the BM of NSG mice showed a different transcriptional landscape, compared to CFZ-naïve cells isolated from the BM of untreated mice. The unsupervised clustering analysis, using UMAP, revealed that cells exposed to CFZ show distinct populations with a strong increase in the OXPHOS and protein folding capacity as well as down-regulation of several genes involved in proliferation and apoptosis, when compared to naïve cells. The CRISPR/Cas9 library screening where RPMI-8226 cells were co-cultured with HS5 cells and exposed to CFZ revealed several CFZ sensitivity candidates at the cut-off of false discovery rate (FDR) < 0.01 and fold change above 1.5-fold. Those genes are involved in cytokine signaling, cell growth, invasion, metastasis and quality control of translational elongation. At the same time, the CRISPR/Cas9 library screening, where synNotch receptor transduced HS5 cells were co-cultured with synNotch ligand transduced RPMI-8226 cells revealed gene candidates at the cut-off of FDR < 0.01 and fold change greater than 1.5-fold, which mediate stronger or weaker cell-cell interaction. Those genes are particularly involved in cytokine signaling and mitochondrial metabolism. Conclusion In conclusion, MM cells that acquired CFZ-resistance upon cell-cell contact with certain cell types within the TME, such as stromal cells, differ significantly from CFZ-naïve cells. CFZ-resistance, caused by cell-cell contact with stromal cells, is presumably mediated via decreased proliferative as well as protein synthesis capacity of MM cells. Therefore, stimulation of MM cells to proliferate and synthesize more proteins may be a key to targeting CFZ-resistance in vivo. Disclosures No relevant conflicts of interest to declare.

BackgroundNatural killer (NK) cells have emerged as promising effectors to target GBM and other solid tumors through genetic modifications and ex vivo manipulation. However, immunosuppressive conditions within the tumor microenvironment (TME) and interactions between NK cell activating and inhibitory receptors further complicate NK cell-based treatments. In particular, the T cell immunoreceptor with Ig and ITIM domains (TIGIT) is expressed on NK cells and interacts with CD155 to induce immunosuppression of NK cell cytolytic functions.1 2 Although CD155 also binds with activating receptors DNAM-1 and CD96 on NK cells, spurring NK cell activity, TIGIT has predominantly been reported as having an inhibitory effect on NK cells.3–5 Further, tumor cells release of high levels of ATP extracellularly. While intracellular ATP is necessary for cell metabolism, extracellular ATP is converted into adenosine (ADO) by ectonucleotidases CD39 and CD73, both overexpressed on GBM and other solid tumors.6 Extracellular ADO induces immunometabolic suppression of NK cells through binding with A2A adenosine receptors (A2ARs) on NK cells, suppressing cytokine secretion, proliferation, and other functional activities.7–9 We found that TIGIT and CD73 are effective combination targets in GBM for both primary and iPSC-derived NK cells.MethodsIn order to effectively target immunometabolic reprogramming induced by CD73-produced adenosine and the immunosuppressive TIGIT-CD155 axis, we have engineered NK cells to concomitantly target CD155 and CD73-induced immunosuppression on GBM using a tumor-responsive genetic construct based on the synNotch signaling system. The construct is capable of blocking the immunosuppressive CD155/TIGIT interaction, and, upon binding, release a CD73-blocking scFv to inhibit the accumulation of extracellular ADO and mitigate immunosuppression of NK cells. Such localized response enhances specificity and reduces off-target effects of NK-based targeting.ResultsPrimary NK cells and iPSC-derived NK cells were successfully engineered to express the synthetic TIGIT-synNotch construct, measured through expression of TIGIT. To evaluate the functionality of engineered NK cells against GBM targets, we tested the cytotoxicity of our engineered NK cells against a primary, patient-derived GBM cell line, GBM43. Overall, cytolytic function of engineered NK cells against GBM was significantly higher than that of non-engineered NK cells, with or without CD73 (10 ug/mL) and TIGIT (50 ug/mL) antibodies, for E:T ratios of 5:1 and 10:1, demonstrating the functional efficacy of our genetic construct.ConclusionsOverall, we have shown that co-targeting CD155 and CD73 in a localized, responsive manner can dampen immunosuppression and significantly enhance the killing potential of engineered NK cells against aggressive patient-derived GBM tumors.ReferencesZhang B, et al. Immunoreceptor TIGIT inhibits the cytotoxicity of human cytokine-induced killer cells by interacting with CD155. Cancer Immunol Immunother 2016;65:305–314.Lupo KB & Matosevic S. CD155 immunoregulation as a target for natural killer cell immunotherapy in glioblastoma. J Hematol Oncol 2020;13:76.Hung AL, et al. TIGIT and PD-1 dual checkpoint blockade enhances antitumor immunity and survival in GBM. OncoImmunology 2018; e1466769. doi:10.1080/2162402X.2018.1466769.Mahnke K & Enk, AH. TIGIT-CD155 Interactions in Melanoma: A Novel Co-Inhibitory Pathway with Potential for Clinical Intervention. Journal of Investigative Dermatology 2016; 136, 9–11.Stanietsky N, et al. Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR: Innate immunity. Eur J Immunol 2013; 43:2138–2150.Chambers AM, et al. Adenosinergic Signaling Alters Natural Killer Cell Functional Responses. Front Immunol 2018;9:2533.Chambers AM, Lupo KB & Matosevic S. Tumor Microenvironment-Induced Immunometabolic Reprogramming of Natural Killer Cells. Front Immunol 2018;9:2517.Chambers AM. et al. Adenosinergic Signaling Alters Natural Killer Cell Functional Responses. Front Immunol 2018;9:2533.Wang J, Lupo KB, Chambers AM & Matosevic S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. J Immunotherapy Cancer 2018;6:136.Ethics ApprovalPrimary human NK cells were obtained from healthy adult donors approved under Purdue University’s Institutional Review Board (IRB) (IRB-approved protocol #1804020540). Donors gave written informed consent prior to taking part in the study.

Este trabajo se inscribe en el marco de los planteamientos clásicos y contemporáneos de la sociología de los sentidos, así como el interés por la relación entre la ciudad y los olores. Las investigaciones sobre el olor han acudido a diversas referencias literarias (Howes, Classen y Synnott, 1994, p. 4; Synnott, 2003, p. 446; Drobnick, 2006; Porteous, 2006; Waskul, Vannini y Wilson, 2009; Solander, 2010; Verbeek y Campen, 2013; Low, 2013, p. 292; Rindisbacher, 2015; Jaquet; 2016, p. 168) como referentes significativos que permiten enunciar algunas problematizaciones asociadas a la investigación sociohistórica y cultural sobre los olores. Es notable la referencia a la obra de escritores varones, mientras que la alusión a escritoras está subrepresentada. El objetivo de este artículo es plantear cómo se representan (Lahire, 2006; Becker, 2015) los olores de la ciudad, en dos escritoras contemporáneas mexicanas: Guadalupe Nettel (1973) y Ana Clavel (1961). Específicamente, me interesa mostrar cómo se narran ciertos significados atribuidos a experiencias olfativas desagradables en la ciudad, desde dos narrativas que recurren constantemente a las diferencias genéricas. La intención es visibilizar otras voces con rendimiento heurístico para la investigación sociológica de los olores, el género y su relación con entidades humanas y no humanas en las grandes urbes.

AbstractWe describe an investigation of the science potential of the filled Mills cross orbiting, imaging, optical interferometer proposed by Synnott et al. and the constraints that science places on the design of the instrument. Imaging simulations were performed with this design in order to determine its capabilities in the area of spatial resolution, dynamic range, and sensitivity. In addition, specific astronomical objects of high interest were chosen as candidate program sources to determine what science was desired for these objects, what spatial resolution, etc. was needed to perform that science, and whether or not this interferometer design could do the science.

Résumé Cet article propose une analyse critique de la pratique du défrisage chez les femmes et les hommes au Cameroun et en France. L’enquête de terrain menée entre 2011 et 2014 en France et au Cameroun auprès d’une centaine de femmes et d’hommes démontre d’une part la mixité de cette pratique, d’autre part des différences dans son traitement et son appréciation en fonction du sexe. Enfin, les résultats révèlent une inégalité en défaveur des femmes et confortent la théorie des contraires (Synnott, 1987) qui opposent notamment pilosités masculine et féminine. Le poids du stigmate « cheveu crépu » pèse plus lourdement sur elles que les hommes en raison même de leur sexe.

Nesse trabalho procuramos situar as relações entre a antropologia dos sentidos e a etnografia sensorial observando as dissonâncias, assonâncias e ressonâncias presentes em seus escopos teórico-metodológicas. Apresentamos algumas perspectivas e fontes contemporâneas desta relação, notadamente as reflexões propostas a partir da antropologia interpretativa de Geertz (1989), aquelas dos autores do “Seminário de Santa Fé” (1984), da antropologia dos sentidos proposta por Howes; Synnott; Classen, (1994) e da antropologia modal de Laplantine (2017). Nossa discussão também está ancorada na contribuição de Ingold (2000; 1008; 2011; 2012) e de Pink (2006; 2008; 2011; 2017). Desta maneira, por meio da síntese que esses autores fazem do debate a respeito da natureza reflexiva da etnografia.

Tujuan dari penelitian ini adalah untuk menunjukkan cara rambut perempuan dikonstruksi oleh mitos kecantikan dalam novel metropop Hair-quake (2014) karya Mariskova. Metode yang digunakan dalam penelitian ini adalah deskriptif kualitatif, dengan pendekatan kritik sastra feminis. Data berupa kata, frasa, dan kalimat dikumpulkan melalui pembacaan tertutup, dengan teknik dokumentasi. Data kemudian diinterpretasi dan dianalisis dengan landasan teoretis yang relevan. Dalam penelitian ini, gagasan yang diapropriasi adalah teori-teori dari Synnott, Manning, dan Wolf. Hasil penelitian menunjukkan bahwa rambut sebagai simbol identitas perempuan di satu sisi bersifat fisik dan privat, namun di sisi lain bersifat publik. Keberadaannya terus didefinisikan oleh tren yang dikonstruksi dan disosialisasikan media dan pihak dominan. Mitos kecantikan dalam novel Hair-quake beroperasi pada wilayah pekerjaan, kultur, dan seksualitas perempuan. Protagonis sebagai perempuan dewasa, lajang, cerdas, dan mandiri, masih memaknai tubuhnya melalui negosiasi dengan nilai-nilai yang ditawarkan pihak orang lain.

Flower seed threshing and cleaning are challenging because many flowers have tiny seed, e.g., the 1000-seed weight of Begonia is 0.01 g, and others have odd-shaped seed, e.g., Tagetes has pappus-bearing seed and Fibigia has winged seed. There is a lack of information on the threshing and cleaning of flower seeds. At the Ornamental Plant Germplasm Center, a small-plot grain belt thresher was modified by disengaging its winnower and a special chute installed to collect the threshed seed and chaff together for cleaning. A custom-made threshing board is used for small samples. The seed with chaff is passed through screen with mesh size that allows all the seed to pass through so that the big pieces of chaff are retained and separated, i.e., scalping. Accurate selection of the next scalping screen (SS) is critical so that the mesh size is just right for at least 95% of the seed to pass through to remove all the chaff larger than the seed. The seed is then sieved on a grading screen (GS) of mesh size that retains at least 95% of the seed to remove all the chaff smaller than the seed. A seed blower is used to further separate the remaining chaff and empty seed based on weight and surface area by adjusting the blowing velocity (BV). A vibratory separator (VS) is used for species with round seed, e.g., Antirrhinum. An X-ray machine is used to monitor the cleaning process. The SC, GS, BV, and VT are given for Agastache, Anisodontea, Antirrhinum, Aquilegia, Aster, Astilboides, Begonia, Belamcanda, Bergenia, Cleome, Coreopsis, Dianthus, Eupatorium, Gaillardia, Geranium, Gypsophila, Iris, Lilium, Lysimachia, Myosotis, Nothoscordom, Oenothera, Passiflora, Penstemon, Petunia, Platycodon, Ranunculus, Rudbeckia, Silene, Stokesia, Synnotia, Tagetes, Talinum, Thalictrum, Verbena, Veronica, and Zinnia.

Las contribuciones del presente dossier se han estructurado en tres ejes alrededor de la idea de seducción del olfato. El primero se centra en la cuestión del uso de los olores en la medicina y en la higiene, en particular para combatir la pestilencia y purificar el aire. El segundo eje se dedica a la fascinación por los olores como incentivo creativo e innovador de técnicas o artísticas. El tercero se enfoca en los potentes efectos de los perfumes en el campo de la espiritualidad y la devoción.En los últimos años, los cuerpos han recuperado centralidad en las investigaciones históricas, por lo que la materia sensible de las sociedades del pasado se posicionó como una vía de pesquisa plausible y originó el campo de los estudios sensoriales. El estudio de las sensaciones de los sujetos históricos resulta atrayente y pertinente ya que no son abstracciones intelectuales, sino experiencias vividas, que nos pueden cómo las sociedades han utilizado a los sentidos para ordenar e integrar a sus miembros. Los esquemas sensoriales son analizados de forma holística o individual, de modo que algunos sentidos han recibido más atención que otros. Durante largo tiempo, el olfato fue un sentido olvidado por los historiadores, sea por su sutileza, por la evanescencia de los testimonios o por el desprecio que se le tiene a causa de considerar el olor como un elemento de la vanidad. Al olfato, en efecto, se lo ha colocado en el nivel más bajo de la jerarquía de los sentidos, como lo demostró Anthony Synnott. Pero, desde el incienso de la misa, los rituales funerarios, los olores de la comida, hasta los perfumes del cuerpo, el olor es omnipresente e indispensable para comprender el vínculo social de los integrantes de una cultura.Los olores se perciben a través de filtros culturales y provocan sensaciones que nacen de una construcción social.


 
 
 La Facultad de Ciencias Forestales cumple 30 años de su fundación este año, 2013. La constitución de la prestigiada Facultad de Ciencias Forestales, de la Universidad Autónoma de Nuevo León, se gestó en el año 1980, cuando el entonces Rector, el Dr. Alfredo Piñeyro López le encomendó al Dr. Timothy J. Synnott un estudio de factibilidad de una Facultad en donde se enseñaran las ciencias forestales. Esta inquietud fue presentada, asimismo, al M. en C. Glafiro Alanís Flores y, de manera conjunta, analizaron diversas opciones para definir en qué área geográfica del estado sería conveniente establecer dicha dependencia; se llegó a definir que el sitio más adecuado era la ciudad de Linares, N L, debido a que existían planes para que la propia Universidad se descentralizara hacia ese municipio enclavado al sur del estado. La ubicación de este centro de estudios fue idónea, ya que está en un área próxima a dos tipos de vegetación muy contrastantes: el matorral espinoso tamaulipeco y los bosques templados de la Sierra Madre Oriental, entorno que favorece el desarrollo de investigaciones sobre el aprovechamiento sustentable de los recursos forestales, agroforestales, manejo y conservación de suelos y la diversidad biológica.
 
 
 
 Como resultado del estudio encomendado por el Dr. Alfredo Piñeyro López, el 23 de marzo de 1981 se aprobó la creación del Instituto de Silvicultura y Manejo de Recursos Renovables. Posteriormente, la Universidad adquirió el edificio de la ex-Hacienda de Guadalupe, y lo adecuó para que fuera la sede del naciente Instituto, que fue inaugurado el 24 de septiembre de 1981 por el entonces gobernador del estado Don Alfonso Martínez Domínguez. El grupo de trabajo inicial estaba integrado por el M. C. Glafiro J. Alanís Flores, que fungía como Coordinador, y por el Dr. Burkhard Müller-Using, el Dr. Reinout J. de Hoogh, el Dr. Dietrich Heiseke, el Dr. Rahim Foroughbakach, el Dr. Jaime Flores Lara y el Dr. Franz Wolf.
 
 
 
 
 

The Australian species of the water beetle genus Hydraena Kugelann, 1794, are revised, based on the study of 7,654 specimens. The 29 previously named species are redescribed, and 56 new species are described. The species are placed in 24 species groups. High resolution digital images of all primary types are presented (online version in color), and geographic distributions are mapped. Male genitalia, representative female terminal abdominal segments and representative spermathecae are illustrated. Australian Hydraena are typically found in sandy/gravelly stream margins, often in association with streamside litter; some species are primarily pond dwelling, a few species are humicolous, and one species may be subterranean. The areas of endemicity and species richness coincide quite closely with the Bassian, Torresian, and Timorian biogeographic subregions. Eleven species are shared between the Bassian and Torresian subregions, and twelve are shared between the Torresian and Timorian subregions. Only one species, H. impercepta Zwick, is known to be found in both Australia and Papua New Guinea. One Australian species, H. ambiflagellata, is also known from New Zealand. New species of Hydraena are: H. affirmata (Queensland, Palmerston National Park, Learmouth Creek), H. ambiosina (Queensland, 7 km NE of Tolga), H. antaria (New South Wales, Bruxner Flora Reserve), H. appetita (New South Wales, 14 km W Delagate), H. arcta (Western Australia, Synnot Creek), H. ascensa (Queensland, Rocky Creek, Kennedy Hwy.), H. athertonica (Queensland, Davies Creek), H. australula (Western Australia, Synnot Creek), H. bidefensa (New South Wales, Bruxner Flora Reserve), H. biimpressa (Queensland, 19.5 km ESE Mareeba), H. capacis (New South Wales, Unumgar State Forest, near Grevillia), H. capetribensis (Queensland, Cape Tribulation area), H. converga (Northern Territory, Roderick Creek, Gregory National Park), H. cubista (Western Australia, Mining Camp, Mitchell Plateau), H. cultrata (New South Wales, Bruxner Flora Reserve), H. cunninghamensis (Queensland, Main Range National Park, Cunningham's Gap, Gap Creek), H. darwini (Northern Territory, Darwin), H. deliquesca (Queensland, 5 km E Wallaman Falls), H. disparamera (Queensland, Cape Hillsborough), H. dorrigoensis (New South Wales, Dorrigo National Park, Rosewood Creek, upstream from Coachwood Falls), H. ferethula (Northern Territory, Cooper Creek, 19 km E by S of Mt. Borradaile), H. finniganensis (Queensland, Gap Creek, 5 km ESE Mt. Finnigan), H. forticollis (Western Australia, 4 km W of King Cascade), H. fundaequalis (Victoria, Simpson Creek, 12 km SW Orbost), H. fundata (Queensland, Hann Tableland, 13 km WNW Mareeba), H. hypipamee (Queensland, Mt. Hypipamee National Park, 14 km SW Malanda), H. inancala (Queensland, Girraween National Park, Bald Rock Creek at "Under-ground Creek"), H. innuda (Western Australia, Mitchell Plateau, 16 mi. N Amax Camp), H. intraangulata (Queensland, Leo Creek Mine, McIlwrath Range, E of Coen), H. invicta (New South Wales, Sydney), H. kakadu (Northern Territory, Kakadu National Park, Gubara), H. larsoni (Queensland, Windsor Tablelands), H. latisoror (Queensland, Lamington National Park, stream at head of Moran's Falls), H. luminicollis (Queensland, Lamington National Park, stream at head of Moran's Falls), H. metzeni (Queensland, 15 km NE Mareeba), H. millerorum (Victoria, Traralgon Creek, 0.2 km N 'Hogg Bridge', 5.0 km NNW Balook), H. miniretia (Queensland, Mt. Hypipamee National Park, 14 km SW Malanda), H. mitchellensis (Western Australia, 4 km SbyW Mining Camp, Mitchell Plateau), H. monteithi (Queensland, Thornton Peak, 11 km NE Daintree), H. parciplumea (Northern Territory, McArthur River, 80 km SW of Borroloola), H. porchi (Victoria, Kangaroo Creek on Springhill Rd., 5.8 km E Glenlyon), H. pugillista (Queensland, 7 km N Mt. Spurgeon), H. queenslandica (Queensland, Laceys Creek, 10 km SE El Arish), H. reticuloides (Queensland, 3 km ENE of Mt. Tozer), H. reticulositis (Western Australia, Mining Camp, Mitchell Plateau), H. revelovela (Northern Territory, Kakadu National Park, GungurulLookout), H. spinissima (Queensland, Main Range National Park, Cunningham's Gap, Gap Creek), H. storeyi (Queensland, Cow Bay, N of Daintree River), H. tenuisella (Queensland, 3 km W of Batavia Downs), H. tenuisoror (Australian Capital Territory, Wombat Creek, 6 km NE of Piccadilly Circus), H. textila (Queensland, Laceys Creek, 10 km SE El Arish), H. tridisca (Queensland, Mt. Hemmant), H. triloba (Queensland, Mulgrave River, Goldsborough Road Crossing), H. wattsi (Northern Territory, Holmes Jungle, 11 km NE by E of Darwin), H. weiri (Western Australia, 14 km SbyE Kalumburu Mission), H. zwicki (Queensland, Clacherty Road, via Julatten).

Li anodes represent a theoretical 10-fold upgrade over the capacity of graphite at comparable potential, and as such are key anode candidates for next-generation high energy density (> 1000 Wh/L) batteries. However, Li still falls below the 99.95-99.97% CE required for long-life cycling(1) and displays rate capability an order-of-magnitude lower than necessary for fast-charging (>2 C).(2) Interestingly, intrinsic Li0/Li+ redox has been reported to be more facile (j 0 > 10 mA/cm2)(3) than graphite intercalation (j 0 = 1-3 mA/cm2)(4). In spite of this, the chemical exchange of Li+ through the native solid electrolyte interphase (SEI) on Li is typically slow (0.5-3 mA/cm2),(5) and as such it can bottleneck Li+/Li0 redox(3) and increase charge-transfer resistance.(6) The SEI thus manifests itself by substantially decreasing the exchange current j 0 measured on Li down from its intrinsic kinetic value.(7) While multiple recent studies see j 0 as a relevant property in determining Li reversibility,(1) measuring j 0 in the presence of an SEI is not straightforward. Consequently, current literature presents widely varying numerical values of j 0 in the presence of an SEI, making it challenging to discern the relationship between j 0 and CE. To bridge this gap, Li+ exchange at the Li anode is here systematically quantified using cyclic voltammetry (CV) at slow scan rates and electrochemical impedance spectroscopy (EIS), both of which allow an SEI to develop natively. To avoid ambiguity with the intrinsic Li0/Li+ redox exchange current j 0, exchange rates are here interpreted in the framework of a “pseudo”-exchange current, j 0 p, that represents the total rate of Li+ exchange on the electrode. j 0 p was measured across a selection of historically-relevant and modern electrolytes, spanning low (78.0%) to high (99.3%) CE. In both methodologies, a strong dependence of j 0 p on electrolyte chemistry was identified. These differences reflect a strong correlation between CE and j 0 p, with electrolytes that display higher j 0 p typically also displaying higher CE. Upon cycling, a dynamic behavior of Li+ exchange on both Cu and Li were observed, with j 0 p typically increasing through cycling, attributed to morphological changes induced by non-uniform plating/stripping inherent to Li electrochemistry.(8) We will discuss the implications of this dynamic behavior on both the formation cycle on Cu, as well as how j 0 p changes report on SEI evolution during cycling. Finally, it was found that cycling Li with current densities j beyond j 0 p leads to substantial capacity loss and low CE, whereas electrolytes that can sustain high j 0 p are insensitive to j. Altogether, our results indicate that Li+ exchange plays a dominant role in determining the rate capability and CE of Li anodes, with high-j 0 p electrolytes displaying higher CE and better rate capability than their low-j 0 p counterparts. G. M. Hobold, J. Lopez, R. Guo, N. Minafra, A. Banerjee, Y. Shirley Meng, Y. Shao-Horn and B. M. Gallant, Nature Energy, 6, 951 (2021). P. Albertus, S. Babinec, S. Litzelman and A. Newman, Nature Energy, 3, 16 (2018). D. T. Boyle, X. Kong, A. Pei, P. E. Rudnicki, F. Shi, W. Huang, Z. Bao, J. Qin and Y. Cui, ACS Energy Letters, 5, 701 (2020). Y.-C. Chang, J.-H. Jong and G. T.-K. Fey, Journal of The Electrochemical Society, 147, 2033 (2000). A. B. Gunnarsdóttir, S. Vema, S. Menkin, L. E. Marbella and C. P. Grey, Journal of Materials Chemistry A, 8, 14975 (2020). A. Zaban, E. Zinigrad and D. Aurbach, The Journal of Physical Chemistry, 100, 3089 (1996). J. N. Butler, D. R. Cogley and J. C. Synnott, Journal of Physical Chemistry, 73, 4026 (1969). J. Z. Lee, T. A. Wynn, M. A. Schroeder, J. Alvarado, X. Wang, K. Xu and Y. S. Meng, ACS Energy Letters, 4, 489 (2019).

Aria, M., & Cuccurullo, C. (2017). bibliometrix: An R-tool for comprehensive science mapping analysis. Journal of Informetrics, 11(4), 959–975. https://doi.org/10.1016/j.joi.2017.08.007 Arno, A., Elliott, J., Wallace, B., Turner, T., & Thomas, J. (2021). The views of health guideline developers on the use of automation in health evidence synthesis. Systematic Reviews, 10(1), 16. https://doi.org/10.1186/s13643-020-01569-2 Ashiq, M., & Warraich, N. F. (2022). A systematized review on data librarianship literature: Current services, challenges, skills, and motivational factors. Journal of Librarianship and Information Science, 55(2), 414–433. https://doi.org/10.1177/09610006221083675 Beller, E., Clark, J., Tsafnat, G., Adams, C., Diehl, H., Lund, H., Ouzzani, M., Thayer, K., Thomas, J., Turner, T., Xia, J., Robinson, K., & Glasziou, P. (2018). Making progress with the automation of systematic reviews: Principles of the International Collaboration for the Automation of Systematic Reviews (ICASR). Systematic Reviews, 7(1), 77. https://doi.org/10.1186/s13643-018-0740-7 Brierley, L., Nanni, F., Polka, J. K., Dey, G., Pálfy, M., Fraser, N., & Coates, J. A. (2022). Tracking changes between preprint posting and journal publication during a pandemic. PLOS Biology, 20(2), e3001285. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8806067/ Briscoe, S., Abbott, R., & Melendez‐Torres, G. J. (2022). Expert searchers identified time, team, technology and tension as challenges when carrying out supplementary searches for systematic reviews: A thematic network analysis. Health Information & Libraries Journal. Advance online publication. https://doi.org/10.1111/hir.12468 Brody, S., Loree, S., Sampson, M., Mensinkai, S., Coffman, J., Mueller, M., Askin, N., Hamill, C., Wilson, E., McAteer, M. B., & Staines, H. (2023). Searching for evidence in public health emergencies: A white paper of best practices. Journal of the Medical Library Association, 111(1), 566–578. https://doi.org/10.5195/jmla.2023.1530 Butcher, R., Sampson, M., Couban, R. J., Malin, J. E., Loree, S., & Brody, S. (2022). The currency and completeness of specialized databases of COVID-19 publications. Journal of Clinical Epidemiology, 147, 52–59. https://doi.org/10.1016/j.jclinepi.2022.03.006 CABI Digital Library. (2023). searchRxiv. Retrieved October 12, 2023, from https://www.cabidigitallibrary.org/journal/searchrxiv Callaway, J. (2021). The Librarian Reserve Corps: An emergency response. Medical Reference Services Quarterly, 40(1), 90–102. https://doi.org/10.1080/02763869.2021.1873627 Chappell, M., Edwards, M., Watkins, D., Marshall, C., & Graziadio, S. (2023). Machine learning for accelerating screening in evidence reviews. Cochrane Evidence Synthesis and Methods, 1(5), e12021. https://doi.org/10.1002/cesm.12021 Chen, Y. Y., Bullard, J., & Giustini, D. (2023). Automated indexing using NLM's Medical Text Indexer (MTI) compared to human indexing in Medline: A pilot study. Journal of the Medical Library Association, 111(3), 684–695. https://doi.org/10.5195/jmla.2023.1588 Clyne, B., Walsh, K. A., O'Murchu, E., Sharp, M. K., Comber, L., O’ Brien, K. K., Smith, S. M., Harrington, P., O'Neill, M., Teljeur, C., & Ryan, M. (2021). Using preprints in evidence synthesis: Commentary on experience during the COVID-19 pandemic. Journal of Clinical Epidemiology, 138, 203–210. https://doi.org/10.1016/j.jclinepi.2021.05.010 Cooper, C., Booth, A., Husk, K., Lovell, R., Frost, J., Schauberger, U., Britten, N., & Garside, R. (2022). A Tailored Approach: A model for literature searching in complex systematic reviews. Journal of Information Science. Advance online publication. https://doi.org/10.1177/01655515221114452 De Brún, C. (2022, June 1–3). Knowledge makes the world go round: Librarians working together to fight the COVID infodemic [Poster session]. European Association for Health Information and Libraries, Rotterdam, Netherlands. de Kock, S., Stirk, L., Ross, J., Duffy, S., Noake, C., & Misso, K. (2020). Systematic review search methods evaluated using the Preferred Reporting of Items for Systematic Reviews and Meta-Analyses and the Risk of Bias in Systematic reviews tool. International Journal of Technology Assessment in Health Care, 37(1), E18. https://doi.org/10.1017/S0266462320002135 EBI-SIG. (2023). Library of Search Strategy Resources. Retrieved October 12, 2023, from https://sites.google.com/view/searchresourceslib/home El Mikati, I. K., Khabsa, J., Harb, T., Khamis, M., Agarwal, A., Pardo-Hernandez, H., Farran, S., Khamis, A. M., El Zein, O., El-Khoury, R., Schünemann, H. J., & Akl, E. A. (2022). A Framework for the development of living practice guidelines in health care. Annals of Internal Medicine, 175(8), 1154-1160. https://doi.org/10.7326/M22-0514 Elliott, J. H., Synnot, A., Turner, T., Simmonds, M., Akl, E. A., McDonald, S., Salanti, G., Meerpohl, J., MacLehose, H., Hilton, J., Tovey, D., Shemilt, I., & Thomas, J. (2017). Living systematic review: 1. Introduction—the why, what, when, and how. Journal of Clinical Epidemiology, 91, 23–30. https://doi.org/10.1016/j.jclinepi.2017.08.010 Foster, M. J., & Jewell, S. T. (2022). Piecing together systematic reviews and other evidence syntheses. Rowman & Littlefield. Greenhalgh, T., Fisman, D., Cane, D. J., Oliver, M., & Macintyre, C. R. (2022). Adapt or die: How the pandemic made the shift from EBM to EBM+ more urgent. BMJ Evidence-Based Medicine, 27(5), 253–260. https://doi.org/10.1136/bmjebm-2022-111952 Greenhalgh, T., & Peacock, R. (2005). Effectiveness and efficiency of search methods in systematic reviews of complex evidence: Audit of primary sources. BMJ, 331(7524), 1064–1065. https://doi.org/10.1136/bmj.38636.593461.68 Johnson, E. E., O’Keefe, H., Sutton, A., & Marshall, C. (2022). The Systematic Review Toolbox: Keeping up to date with tools to support evidence synthesis. Systematic Reviews, 11(1), 258. https://doi.org/10.1186/s13643-022-02122-z Khalil, H., Tamara, L., Rada, G., & Akl, E. A. (2021). Challenges of evidence synthesis during the 2020 COVID pandemic: A scoping review. Journal of Clinical Epidemiology, 142, 10–18. https://doi.org/10.1016/J.JCLINEPI.2021.10.017 Kirkham, J. J., Penfold, N. C., Murphy, F., Boutron, I., Ioannidis, J. P., Polka, J., & Moher, D. (2020). Systematic examination of preprint platforms for use in the medical and biomedical sciences setting. BMJ Open, 10(12), e041849. https://doi.org/10.1136/bmjopen-2020-041849 Levay, P., & Craven, J. (2019). Conclusion: Where do we go from here? In P. Levay, & J. Craven (Eds.), Systematic searching: Practical ideas for improving results (pp. 289–292). Facet Publishing. Levay, P., Heath, A., & Tuvey, D. (2022a). Efficient searching for NICE public health guidelines: Would using fewer sources still find the evidence? Research Synthesis Methods, 13(6), 760–789. https://doi.org/10.1002/jrsm.1577 Levay, P., Walsh, N., & Foster, L. (2022b). The National Institute for Health and Care Excellence information specialist development pathway: Developing the skills, knowledge and confidence to quality assure search strategies. Health Information and Libraries Journal, 39(4), 392–399. https://doi.org/10.1111/hir.12460 MacFarlane, A., Russell-Rose, T., & Shokraneh, F. (2022). Search strategy formulation for systematic reviews: Issues, challenges and opportunities. Intelligent Systems with Applications, 15, 200091. https://doi.org/10.1016/j.iswa.2022.200091 McDonald, S., Sharp, S., Morgan, R. L., Murad, M. H., & Fraile Navarro, D. (2023). Methods for living guidelines: Early guidance based on practical experience. Paper 4: Search methods and approaches for living guidelines. Journal of Clinical Epidemiology, 155, 108–117. https://doi.org/10.1016/j.jclinepi.2022.12.023 Metzendorf, M., & Featherstone, R. M. (2021). Evaluation of the comprehensiveness, accuracy and currency of the Cochrane COVID-19 Study Register for supporting rapid evidence synthesis production. Research Synthesis Methods, 12(5), 607–617. https://doi.org/10.1002/jrsm.1501 Metzendorf, M., Weibel, S., Reis, S., & McDonald, S. (2022). Pragmatic and open science-based solution to a current problem in the reporting of living systematic reviews. BMJ Evidence-Based Medicine, 8(4), 267–272. https://doi.org/10.1136/bmjebm-2022-112019 Naicker, R. (2022). Critically appraising for antiracism. Education for Information, 38(4), 291–308. https://doi.org/10.3233/EFI-220052 National Library of Medicine. (2022). Indexing FAQs. Retrieved October 12, 2023, from https://support.nlm.nih.gov/knowledgebase/article/KA-05326/en-us O’Mara-Eves, A., Thomas, J., McNaught, J., Miwa, M., & Ananiadou, S. (2015). Using text mining for study identification in systematic reviews: A systematic review of current approaches. Systematic Reviews, 4(1), 5. https://doi.org/10.1186/2046-4053-4-5 Pierre, O., Riveros, C., Charpy, S., & Boutron, I. (2021). Secondary electronic sources demonstrated very good sensitivity for identifying studies evaluating interventions for COVID-19. Journal of Clinical Epidemiology, 141, 46–53. https://doi.org/10.1016/j.jclinepi.2021.09.022 Qureshi, R., Shaughnessy, D., Gill, K. A. R., Robinson, K. A., Li, T., & Agai, E. (2023). Are ChatGPT and large language models “the answer” to bringing us closer to systematic review automation? Systematic Reviews, 12(1), 72. https://doi.org/10.1186/s13643-023-02243-z Ramirez, D., Foster, M. J., Kogut, A., & Xiao, D. (2022). Adherence to systematic review standards: Impact of librarian involvement in Campbell Collaboration's education reviews. The Journal of Academic Librarianship, 48(5), 102567. https://doi.org/10.1016/j.acalib.2022.102567 Rethlefsen, M. L., Kirtley, S., Waffenschmidt, S., Ayala, A. P., Moher, D., Page, M. J., & Koffel, J. B. (2021). PRISMA-S: An extension to the PRISMA statement for reporting literature searches in systematic reviews. Systematic Reviews, 10(1), 39. https://doi.org/10.1186/s13643-020-01542-z Rosonovski, S., Levchenko, M., Ide‐Smith, M., Faulk, L., Harrison, M., & McEntyre, J. (2023). Searching and evaluating publications and preprints using Europe PMC. Current Protocols, 3(3), e694. https://doi.org/10.1002/cpz1.694 Sampson, M. (2019). Communication for information specialists. In P. Levay, & J. Craven (Eds.), Systematic searching: Practical ideas for improving results (pp. 249–268). Facet Publishing. Shemilt, I., Noel-Storr, A., Thomas, J., Featherstone, R., & Mavergames, C. (2022). Machine learning reduced workload for the Cochrane COVID-19 Study Register: Development and evaluation of the Cochrane COVID-19 Study Classifier. Systematic Reviews, 11(1), 15. https://doi.org/10.1186/s13643-021-01880-6 Shokraneh, F., & Adams, C. E. (2019). Study-based registers reduce waste in systematic reviewing: Discussion and case report. Systematic Reviews, 8(1), 129. https://doi.org/10.1186/s13643-019-1035-3 Stansfield, C., Stokes, G., & Thomas, J. (2022). Applying machine classifiers to update searches: Analysis from two case studies. Research Synthesis Methods, 13(1), 121–133. https://doi.org/10.1002/jrsm.1537 Svarre, T., & Russell-Rose, T. (2022). Think outside the search box: A comparative study of visual and form-based query builders. Journal of Information Science. Advance online publication. https://doi.org/10.1177/01655515221138536 Thomas, J., McDonald, S., Noel-Storr, A., Shemilt, I., Elliott, J., Mavergames, C., & Marshall, I. J. (2021). Machine learning reduced workload with minimal risk of missing studies: Development and evaluation of a randomized controlled trial classifier for Cochrane Reviews. Journal of Clinical Epidemiology, 133, 140–151. https://doi.org/10.1016/j.jclinepi.2020.11.003 Townsend, W., Anderson, P., Haines, K., Hansen, S., James, L., MacEachern, M., Rana, G., Saylor, K. & Saylor, K. (2022). Addressing antiquated, non-standard, exclusionary, and potentially offensive terms in evidence syntheses and systematic searches. Retrieved October 12, 2023, from https://doi.org/10.7302/6408 van Noorden, R. (2023). ChatGPT-like AIs are coming to major science search engines. Nature, 620(7973), 258. https://doi.org/10.1016/j.jclinepi.2022.12.023 Verdugo-Paiva, F., Vergara, C., Ávila, C., Castro-Guevara, J., Cid, J., Contreras, V., Jara, I., Jiménez, V., Lee, M. H., Muñoz, M., Rojas-Gómez, A. M., Rosón-Rodríguez, P., Serrano-Arévalo, K., Silva-Ruz, I., Vásquez-Laval, J., Zambrano-Achig, P., Zavadzki, G., & Rada, G. (2022). COVID-19 Living Overview of Evidence repository is highly comprehensive and can be used as a single source for COVID-19 studies. Journal of Clinical Epidemiology, 149, 195–202. https://doi.org/10.1016/j.jclinepi.2022.05.001 Waffenschmidt, S., & Hausner, E. (2019). Collaborative working to improve searching. In P. Levay, & J. Craven (Eds.), Systematic searching: Practical ideas for improving results (pp. 229–248). Facet Publishing. Wang, S., Scells, H., Koopman, B., & Zuccon, G. (2023). Can ChatGPT write a good Boolean query for systematic review literature search? SIGIR '23: Proceedings of the 46th International ACM SIGIR Conference on Research and Development in Information Retrieval, Taipei. 1426–1436. https://doi.org/10.1145/3539618.3591703

AbstractObjectivessynNotch receptors can get different inputs about the outside environment into cells; however, synNotch system doesn’t work for soluble ligands. This study aims to explore the activation efficiency of different ligand carriers on synNotch system.MethodsSynNotch-based contact-dependent activation system was constructed in 293T cells (293T-synNotch), then the synNotch receptor ligands eGFP proteins were provided by three different carriers: 400 nm Ni magnetic beads (Mag Ni-eGFP), 300 nm carboxyl modified Fe3O4 magnetic beads (Mag COOH-deGFP), and 4T1 cell (4T1-mGFP). Three eGFP carriers were incubated with 293T-synNotch, the activation efficiencies of these three eGFP carriers were evaluated by fluorescence microscopy.ResultsThe 293T-synNotch cell activated by Mag Ni-eGFP shows no observable red fluorescence; when Mag COOH-deGFP are incubated with 293T-synNotch, about 50‰ cells with red fluorescence appeared, also eGFP proteins have not dropped off from the Mag COOH-deGFP beads; 4T1-mGFP cells show the highest activation efficiency, about 21% synNotch cells are activated, when 4T1-mGFP and 293T-synNotch are incubated at high cell density.ConclusionThe 4T1-mGFP cells are the most effective ligands for synNotch receptor activation.

The synthetic Notch receptor has emerged as a potent tool for precisely modulating cellular functions. It constitutes a receptor system rooted in the Notch signaling pathway. SynNotch receptors, coupled with downstream transcription programs, hold promise for organoid and 3D tissue construction. Additionally, it enables the tracking and visualization of intercellular communication. Moreover, engineering SynNotch cells to carry specific receptors markedly enhances the efficacy and safety of immunotherapy. This review delineates the subdomains and tunable mechanisms of SynNotch, summarizing four core modes of combinatorial multiplexing potentially pivotal for regulating SynNotch cell functions. Furthermore, this review summarizes the multifaceted applications, advantages, and limitations of SynNotch, offering fresh insights into its future biomedical utilization.

AbstractSynthetic Notch (synNotch) receptors are genetically encoded, modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Although some materials have been modified to present synNotch ligands with coarse spatial control, applications in tissue engineering generally require extracellular matrix (ECM)-derived scaffolds and/or finer spatial positioning of multiple ligands. Thus, we develop here a suite of materials that activate synNotch receptors for generalizable engineering of material-to-cell signaling. We genetically and chemically fuse functional synNotch ligands to ECM proteins and ECM-derived materials. We also generate tissues with microscale precision over four distinct reporter phenotypes by culturing cells with two orthogonal synNotch programs on surfaces microcontact-printed with two synNotch ligands. Finally, we showcase applications in tissue engineering by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined micropatterns. These technologies provide avenues for spatially controlling cellular phenotypes in mammalian tissues.

Abstract Muscle-invasive bladder cancer (MIBC), a highly heterogeneous disease, shows genomic instability and a high mutation rate, making it difficult to treat. Recent studies revealed that cancer stem cells (CSCs) play a critical role in MIBC frequent recurrence and high morbidity. Previous research has shown that Cyclooxygenases-2 (COX-2) is particularly highly expressed in bladder cancer cells. In recent years, the development of oncolytic adenoviruses and their use in clinical trials have gained increased attention. In this study, we composed a conditionally replicative adenovirus vector (CRAd-synNotch) that carries the COX-2 promotor driving adenoviral E1 gene, the synNotch receptor therapeutic gene, and the Ad5/35 fiber gene. Activation of the COX-2 promoter gene causes replication only within COX-2 expressing cancer cells, thereby leading to tumor oncolysis. Also, CD44 and HIF signals contribute to cancer stemness and maintaining CSCs in bladder cancer, and the transduced synNotch receptor inhibits both CD44 and HIF signals simultaneously. We performed an in vivo study using a mouse xenograft model of T24 human MIBC cells and confirmed the significant antitumor activity of CRAd-synNotch. Our findings in this study warrant the further development of CRAd-synNotch for treating patients with MIBC.

Cancer immunotherapy has been transformed by chimeric antigen receptor (CAR) T-cell treatment, which has shown groundbreaking results in hematological malignancies. However, its application in solid tumors remains a formidable challenge due to immune evasion, tumor heterogeneity, and safety concerns arising from off-target effects. A long-standing effort in this field has been the development of synthetic receptors to create new signaling pathways and rewire immune cells for the specific targeting of cancer cells, particularly in cell-based immunotherapy. This field has undergone a paradigm shift with the introduction of synthetic Notch (synNotch) receptors, which offer a highly versatile signaling platform modeled after natural receptor-ligand interactions. By functioning as molecular logic gates, synNotch receptors enable precise, multi-antigen regulation of T-cell activation, paving the way for enhanced specificity and control. This review explores the revolutionary integration of synNotch systems with CAR T-cell therapy, emphasizing cutting-edge strategies to overcome the inherent limitations of traditional approaches. We delve into the mechanisms of synNotch receptor design, focusing on their ability to discriminate between cancerous and normal cells through spatiotemporally controlled gene expression. Additionally, we highlight recent advancements to improve therapeutic efficacy, safety, and adaptability in treating solid tumors. This study highlights the potential of synNotch-based CAR-T cells to transform the field of targeted cancer therapy by resolving present challenges and shedding light on potential future paths.

ABSTRACT The application of immunotherapeutic strategies, such as chimeric antigen receptor-T cells and broadly neutralizing antibodies (bNAbs), for the treatment of human immunodeficiency virus (HIV) infection is hindered by the latent reservoirs and viral escape. Achieving long-term control of viral load in the absence of antiretroviral therapy requires a combination approach utilizing these immunotherapeutic strategies. For this purpose, we developed novel anti-HIV-1 synthetic Notch (synNotch) receptor-T cells, termed CD4-17b-VN, which express both a bNAb (VRC01) and a bispecific T cell-engaging protein (N6-αCD3) with antigenic control. The synNotch receptor-expressing cells can sense the viral antigen presented on both HIV-1 particles and the surface of target cells. A human T cell line equipped with the CD4-17b-VN circuit could effectively control VRC01 and N6-αCD3 secretion upon sensitization, suppress the infection of diverse subtypes of HIV-1 strains, and mediate specific bypass cytotoxic activity against infected and latency-reactivated cells. Additionally, CD4-17b-VN CD8 + T cells exhibited long-lasting suppression of infected cells and stronger killing effect on latency-reactivated cells in vitro . Importantly, we demonstrated that the synNotch receptor did not increase susceptibility to HIV-1 infection in the engineered cells. Our study validates the concept of a synNotch platform-based T cell therapeutic approach that can deliver broadly therapeutic antibodies in an HIV-1 antigen-controlled manner, which may have important implications for the functional cure of AIDS. IMPORTANCE Adoptive transfer of effector T cells modified with a chimeric antigen receptor has been proposed as an applicable approach to treat human immunodeficiency virus (HIV) infection. The synNotch receptor (SNR) system serves as a versatile tool, enabling customized programming of input and output functions in mammalian cells. Herein, we report a novel synNotch platform-based approach for T cell engineering targeting both cell-free particles and infected cells by coupling antibody neutralization with cytotoxicity. Our findings demonstrate that the engineered CD4-17b SNR enables controllable production of functional anti-HIV-1 broadly neutralizing antibody and bispecific T cell-engaging protein upon recognition of the viral particle and cell surface antigens by the bifunctional synNotch-T cells. Human primary CD8 + T cells equipped with the bifunctional synNotch circuit CD4-17b-VN can effectively suppress long-term viral replication and reduce latency-reactivated cells in vitro , without the undesired risk of being infected by the virus, suggesting their potential candidacy for AIDS therapy with prospects for future clinical applications.

Introduction: Cellular immunotherapy has greatly improved cancer treatment in recent years. For instance, chimeric antigen receptor (CAR) T cell therapy has been proven highly effective in treating hematological malignancies, and many CAR cell designs are being explored for solid tumors. However, many questions remain why responses differ across patients and some tumor types are resistant. Improved and relatively inexpensive ways to monitor these cells could provide some answers. Clinically, blood tests are regularly used to monitor these therapies, but blood signals often do not reflect the activity of immune cells within the tumor(s). Here, using the synthetic Notch (synNotch) receptor that tethers antigen binding to customized transgene expression, we linked intratumoral immune-cancer cell communication to a simple secreted reporter blood test. Specifically, we engineered immune cells with a CD19-targeted synNotch receptor and demonstrated that binding to CD19 on cancer cells in vivo resulted in the production of secreted embryonic alkaline phosphatase (SEAP) at levels that are readily detected in the blood.Methods and Results: Jurkat T cells were engineered via sequential lentiviral transduction of two components: an anti-CD19 synNotch receptor and a synNotch response element encoding SEAP. Co-culture of engineered cells with CD19+, but not CD19−, Nalm6 cells, resulted in significantly elevated SEAP in media. Nod-scid-gamma (NSG) mice were subcutaneously injected with either CD19+ or CD19− Nalm6 cells. Intratumoral injection of engineered T cells (1x107) resulted in significantly elevated blood SEAP activity in mice bearing CD19+ tumors (n = 7), but not CD19− tumors (n = 5).Discussion: Our synNotch reporter system allows for the monitoring of antigen-dependent intratumoral immune-cancer cell interactions through a simple and convenient blood test. Continued development of this system for different target antigens of interest should provide a broadly applicable platform for improved monitoring of many cell-based immunotherapies during their initial development and clinical translation, ultimately improving our understanding of design considerations and patient-specific responses.