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1
Shi, Ying, Yan-ran Wu, Jian-qiang Yu, Wan-nian Zhang, and Chun-lin Zhuang. "DNA-encoded libraries (DELs): a review of on-DNA chemistries and their output." RSC Advances 11, no. 4 (2021): 2359–76. http://dx.doi.org/10.1039/d0ra09889b.
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We summarize a series of novel DNA-compatible chemistry reactions for DNA-encoded chemical library (DEL) building blocks and analyse the druggability of screened hit molecules via DELs in the past five years.
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Fan, Zhoulong, Shuai Zhao, Tao Liu, Peng-Xiang Shen, Zi-Ning Cui, Zhe Zhuang, Qian Shao, et al. "Merging C(sp3)–H activation with DNA-encoding." Chemical Science 11, no. 45 (2020): 12282–88. http://dx.doi.org/10.1039/d0sc03935g.
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Tran-Hoang, Nam, and Thomas Kodadek. "Solid-Phase Synthesis of β-Amino Ketones Via DNA-Compatible Organocatalytic Mannich Reactions." ACS Combinatorial Science 20, no. 2 (January 24, 2018): 55–60. http://dx.doi.org/10.1021/acscombsci.7b00151.
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Shu, Keitou, and Thomas Kodadek. "Solid-Phase Synthesis of β-Hydroxy Ketones Via DNA-Compatible Organocatalytic Aldol Reactions." ACS Combinatorial Science 20, no. 5 (March 26, 2018): 277–81. http://dx.doi.org/10.1021/acscombsci.8b00001.
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Favalli, Nicholas, Gabriele Bassi, Tania Zanetti, Jörg Scheuermann, and Dario Neri. "Screening of Three Transition Metal‐Mediated Reactions Compatible with DNA‐Encoded Chemical Libraries." Helvetica Chimica Acta 102, no. 4 (April 2019): e1900033. http://dx.doi.org/10.1002/hlca.201900033.
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Liu, Wentao, Wei Huang, Qian Lin, Mei-Hsuan Tsai, Rui Zhang, Lijun Fan, Jack D. Scott, Guansai Liu, and Jinqiao Wan. "Development of DNA-compatible hydroxycarbonylation reactions using chloroform as a source of carbon monoxide." Bioorganic & Medicinal Chemistry 38 (May 2021): 116118. http://dx.doi.org/10.1016/j.bmc.2021.116118.
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Wang, Jie, Helena Lundberg, Shota Asai, Pedro Martín-Acosta, Jason S. Chen, Stephen Brown, William Farrell, et al. "Kinetically guided radical-based synthesis of C(sp3)−C(sp3) linkages on DNA." Proceedings of the National Academy of Sciences 115, no. 28 (June 26, 2018): E6404—E6410. http://dx.doi.org/10.1073/pnas.1806900115.
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DNA-encoded libraries (DEL)-based discovery platforms have recently been widely adopted in the pharmaceutical industry, mainly due to their powerful diversity and incredible number of molecules. In the two decades since their disclosure, great strides have been made to expand the toolbox of reaction modes that are compatible with the idiosyncratic aqueous, dilute, and DNA-sensitive parameters of this system. However, construction of highly important C(sp3)−C(sp3) linkages on DNA through cross-coupling remains unexplored. In this article, we describe a systematic approach to translating standard organic reactions to a DEL setting through the tactical combination of kinetic analysis and empirical screening with information captured from data mining. To exemplify this model, implementation of the Giese addition to forge high value C–C bonds on DNA was studied, which represents a radical-based synthesis in DEL.
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Kundu, Nandini, Brian E. Young, and Jonathan T. Sczepanski. "Kinetics of heterochiral strand displacement from PNA–DNA heteroduplexes." Nucleic Acids Research 49, no. 11 (June 14, 2021): 6114–27. http://dx.doi.org/10.1093/nar/gkab499.
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Abstract Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as ‘heterochiral’ strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction – strand displacement from PNA–DNA heteroduplexes – remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA–DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA–DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.
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Qu, Yi, Huanan Wen, Rui Ge, Yanfen Xu, Hong Gao, Xiaodong Shi, Jiangong Wang, et al. "Copper-Mediated DNA-Compatible One-Pot Click Reactions of Alkynes with Aryl Borates and TMS-N3." Organic Letters 22, no. 11 (May 8, 2020): 4146–50. http://dx.doi.org/10.1021/acs.orglett.0c01219.
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Whang, I., J. Lee, and M. Jayaram. "Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination." Molecular and Cellular Biology 14, no. 11 (November 1994): 7492–98. http://dx.doi.org/10.1128/mcb.14.11.7492.
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A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.
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Whang, I., J. Lee, and M. Jayaram. "Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination." Molecular and Cellular Biology 14, no. 11 (November 1994): 7492–98. http://dx.doi.org/10.1128/mcb.14.11.7492-7498.1994.
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A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.
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Favalli, Nicholas, Gabriele Bassi, Davide Bianchi, Jörg Scheuermann, and Dario Neri. "Large screening of DNA-compatible reaction conditions for Suzuki and Sonogashira cross-coupling reactions and for reverse amide bond formation." Bioorganic & Medicinal Chemistry 41 (July 2021): 116206. http://dx.doi.org/10.1016/j.bmc.2021.116206.
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Haegi, Anita, Valentina Catalano, Laura Luongo, Salvatore Vitale, Michele Scotton, Nadia Ficcadenti, and Alessandra Belisario. "A Newly Developed Real-Time PCR Assay for Detection and Quantification of Fusarium oxysporum and Its Use in Compatible and Incompatible Interactions with Grafted Melon Genotypes." Phytopathology® 103, no. 8 (August 2013): 802–10. http://dx.doi.org/10.1094/phyto-11-12-0293-r.
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A reliable and species-specific real-time quantitative polymerase chain reaction (qPCR) assay was developed for detection of the complex soilborne anamorphic fungus Fusarium oxysporum. The new primer pair, designed on the translation elongation factor 1-α gene with an amplicon of 142 bp, was highly specific to F. oxysporum without cross reactions with other Fusarium spp. The protocol was applied to grafted melon plants for the detection and quantification of F. oxysporum f. sp. melonis, a devastating pathogen of this cucurbit. Grafting technologies are widely used in melon to confer resistance against new virulent races of F. oxysporum f. sp. melonis, while maintaining the properties of valuable commercial varieties. However, the effects on the vascular pathogen colonization have not been fully investigated. Analyses were performed on ‘Charentais-T’ (susceptible) and ‘Nad-1’ (resistant) melon cultivars, both used either as rootstock and scion, and inoculated with F. oxysporum f. sp. melonis race 1 and race 1,2. Pathogen development was compared using qPCR and isolations from stem tissues. Early asymptomatic melon infections were detected with a quantification limit of 1 pg of fungal DNA. The qPCR protocol clearly showed that fungal development was highly affected by host–pathogen interaction (compatible or incompatible) and time (days postinoculation). The principal significant effect (P ≤ 0.01) on fungal development was due to the melon genotype used as rootstock, and this effect had a significant interaction with time and F. oxysporum f. sp. melonis race. In particular, the amount of race 1,2 DNA was significantly higher compared with that estimated for race 1 in the incompatible interaction at 18 days postinoculation. The two fungal races were always present in both the rootstock and scion of grafted plants in either the compatible or incompatible interaction.
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Dawadi, Surendra, Nicholas Simmons, Gabriella Miklossy, Kurt M. Bohren, John C. Faver, Melek Nihan Ucisik, Pranavanand Nyshadham, Zhifeng Yu, and Martin M. Matzuk. "Discovery of potent thrombin inhibitors from a protease-focused DNA-encoded chemical library." Proceedings of the National Academy of Sciences 117, no. 29 (July 8, 2020): 16782–89. http://dx.doi.org/10.1073/pnas.2005447117.
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DNA-encoded chemical libraries are collections of compounds individually coupled to unique DNA tags serving as amplifiable identification barcodes. By bridging split-and-pool combinatorial synthesis with the ligation of unique encoding DNA oligomers, million- to billion-member libraries can be synthesized for use in hundreds of healthcare target screens. Although structural diversity and desirable molecular property ranges generally guide DNA-encoded chemical library design, recent reports have highlighted the utility of focused DNA-encoded chemical libraries that are structurally biased for a class of protein targets. Herein, a protease-focused DNA-encoded chemical library was designed that utilizes chemotypes known to engage conserved catalytic protease residues. The three-cycle library features functional moieties such as guanidine, which interacts strongly with aspartate of the protease catalytic triad, as well as mild electrophiles such as sulfonamide, urea, and carbamate. We developed a DNA-compatible method for guanidinylation of amines and reduction of nitriles. Employing these optimized reactions, we constructed a 9.8-million-membered DNA-encoded chemical library. Affinity selection of the library with thrombin, a common protease, revealed a number of enriched features which ultimately led to the discovery of a 1 nM inhibitor of thrombin. Thus, structurally focused DNA-encoded chemical libraries have tremendous potential to find clinically useful high-affinity hits for the rapid discovery of drugs for targets (e.g., proteases) with essential functions in infectious diseases (e.g., severe acute respiratory syndrome coronavirus 2) and relevant healthcare conditions (e.g., male contraception).
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Tsuji, Gakushi, Satoshi Fujii, Takeshi Sunami, and Tetsuya Yomo. "Sustainable proliferation of liposomes compatible with inner RNA replication." Proceedings of the National Academy of Sciences 113, no. 3 (December 28, 2015): 590–95. http://dx.doi.org/10.1073/pnas.1516893113.
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Although challenging, the construction of a life-like compartment via a bottom–up approach can increase our understanding of life and protocells. The sustainable replication of genome information and the proliferation of phospholipid vesicles are requisites for reconstituting cell growth. However, although the replication of DNA or RNA has been developed in phospholipid vesicles, the sustainable proliferation of phospholipid vesicles has remained difficult to achieve. Here, we demonstrate the sustainable proliferation of liposomes that replicate RNA within them. Nutrients for RNA replication and membranes for liposome proliferation were combined by using a modified freeze–thaw technique. These liposomes showed fusion and fission compatible with RNA replication and distribution to daughter liposomes. The RNAs in daughter liposomes were repeatedly used as templates in the next RNA replication and were distributed to granddaughter liposomes. Liposome proliferation was achieved by 10 cycles of iterative culture operation. Therefore, we propose the use of culturable liposomes as an advanced protocell model with the implication that the concurrent supplement of both the membrane material and the nutrients of inner reactions might have enabled protocells to grow sustainably.
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Surrette, Christine, David Shoudy, Alex Corwin, Wei Gao, Maria I. Zavodszky, Stanislav L. Karsten, Todd Miller, et al. "Microfluidic Tissue Mesodissection in Molecular Cancer Diagnostics." SLAS TECHNOLOGY: Translating Life Sciences Innovation 22, no. 4 (November 19, 2016): 425–30. http://dx.doi.org/10.1177/2211068216680208.
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We present a mesodissection platform that retains the advantages of laser-based dissection instrumentation with the speed and ease of manual dissection. Tissue dissection in clinical laboratories is often performed by manually scraping a physician-selected region from standard glass slide mounts. In this manner, costs associated with dissection remain low, but spatial resolution is compromised. In contrast, laser microdissection methods maintain spatial resolution that matches the requirements for analysis of important tissue heterogeneity but remains costly and labor intensive. We demonstrate a microfluidic tool for rapid extraction of histological regions of interest from formalin-fixed paraffin-embedded tissue, which uses a simple and automated method that is compatible with most downstream enzymatic reactions, including protocols used for next-generation DNA sequencing.
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Szymanski, Irma O., and Sharyn L. Orton. "Since the Process of Transfusion-Induced Alloimmunization Is Not Harmless, Can We Mitigate It?" Blood 124, no. 21 (December 6, 2014): 5101. http://dx.doi.org/10.1182/blood.v124.21.5101.5101.
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Abstract Transfusion protocols have remained unchanged for decades. Accordingly, it is necessary to avoid transfusion reactions by giving patients ABO- and irregular antibody-compatible red blood cells (RBC). Prevention of immunization to the Rh antigen D is also required since anti-D may cause a serious hemolytic disease of fetus and newborn. Prevention of transfusion-induced immunization to other blood group antigens is neither required nor feasible for the general transfused population. We consider the process of alloimmunization to be harmful due to the following untoward effects: 1) Destruction of transfused RBC negates transfusion's intended therapeutic benefits and may cause transfusion reactions. 2) Some alloantibodies become undetectable after a relatively short period (e.g., anti-Jka) so that in the future such seemingly compatible transfusions cause severe, sometimes fatal delayed transfusion reactions. 3) RBC autoimmunity may follow alloimmunization and cause hemolytic anemia in the recipient. To take the first step toward preventing transfusion-induced alloimmunization we calculated, using our antibody database, the immunogenicity of RBC antigens in a predominantly white female and male populations according to the methods of *Giblett (Transfusion 1961;1(4): 233) and **Tormey and Stack. (Blood 2009;114: 4279). The latter method corrects immunogenicities for antibody evanescence. The Table below shows that the three highly immunogenic antigens against which clinically significant antibodies were frequently formed were E, Jka and K, both in females and males. Immunogenicity of Rh C antigen was not calculated, since anti-C occurs most often with anti-D. Figure 1 Figure 1. In summary, we suggest a first step toward improving immunological safety of RBC transfusions by preventing immunization to E, Jka and K antigens. Automated typing or DNA analysis would facilitate mass typing of both donors' and recipients' RBC antigens. Disclosures No relevant conflicts of interest to declare.
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Rooney, Sean, Frederick W. Alt, David Lombard, Scott Whitlow, Mark Eckersdorff, James Fleming, Sebastian Fugmann, David O. Ferguson, David G. Schatz, and JoAnn Sekiguchi. "Defective DNA Repair and Increased Genomic Instability in Artemis-deficient Murine Cells." Journal of Experimental Medicine 197, no. 5 (March 3, 2003): 553–65. http://dx.doi.org/10.1084/jem.20021891.
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In developing lymphocytes, the recombination activating gene endonuclease cleaves DNA between V, D, or J coding and recombination signal (RS) sequences to form hairpin coding and blunt RS ends, which are fused to form coding and RS joins. Nonhomologous end joining (NHEJ) factors repair DNA double strand breaks including those induced during VDJ recombination. Human radiosensitive severe combined immunodeficiency results from lack of Artemis function, an NHEJ factor with in vitro endonuclease/exonuclease activities. We inactivated Artemis in murine embryonic stem (ES) cells by targeted mutation. Artemis deficiency results in impaired VDJ coding, but not RS, end joining. In addition, Artemis-deficient ES cells are sensitive to a radiomimetic drug, but less sensitive to ionizing radiation. VDJ coding joins from Artemis-deficient ES cells, which surprisingly are distinct from the highly deleted joins consistently obtained from DNA-dependent protein kinase catalytic subunit–deficient ES cells, frequently lack deletions and often display large junctional palindromes, consistent with a hairpin coding end opening defect. Strikingly, Artemis-deficient ES cells have increased chromosomal instability including telomeric fusions. Thus, Artemis appears to be required for a subset of NHEJ reactions that require end processing. Moreover, Artemis functions as a genomic caretaker, most notably in prevention of translocations and telomeric fusions. As Artemis deficiency is compatible with human life, Artemis may also suppress genomic instability in humans.
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Parham, Nicholas J., François J. Picard, Régis Peytavi, Martin Gagnon, Grégoire Seyrig, Pier-Ann Gagné, Maurice Boissinot, and Michel G. Bergeron. "Specific Magnetic Bead–Based Capture of Genomic DNA from Clinical Samples: Application to the Detection of Group B Streptococci in Vaginal/Anal Swabs." Clinical Chemistry 53, no. 9 (September 1, 2007): 1570–76. http://dx.doi.org/10.1373/clinchem.2007.091389.
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Abstract Background: Group B streptococci (GBS) are a leading cause of sepsis and meningitis in newborns. We previously developed a rapid diagnostic system for GBS detection from vaginal/anal samples obtained from pregnant women during delivery. To facilitate the adaptation of this method for point-of-care testing, we have developed a specific and efficient GBS DNA capture method that is compatible with both PCR and nonamplification detection technologies. Methods: Superparamagnetic beads were functionalized with oligonucleotide capture probes of different lengths and used to capture GBS genomic DNA (gDNA). A rapid extraction procedure was used to provide DNA from GBS cultures or vaginal/anal samples with added GBS. Hybridization reactions consisting of functionalized beads and target DNA in 30 μL of hybridization buffer were performed for 1 h at room temperature, followed by washing and resuspension in water. Captured DNA was then detected using quantitative PCR. Results: A 25-mer capture probe allowed detection of 1000 genome copies of purified GBS DNA. The ability to detect GBS was improved by use of a 50-mer (100 copies) and a 70-mer capture probe (10 copies). Detection of approximately 1250 CFU/mL was achieved for diluted GBS broth culture and for vaginal/anal swab samples with added GBS. Conclusion: Oligonucleotide-functionalized superparamagnetic microbeads efficiently capture GBS gDNA from both bacterial cultures and vaginal/anal samples with added GBS. Efficiency of gDNA capture increases with oligonucleotide length. This technology could be combined with sample preparation and detection technologies in a microfluidic system to allow point-of-care testing for GBS.
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Matsumura, Ichiro. "Methylase-assisted subcloning for high throughput BioBrick assembly." PeerJ 8 (September 11, 2020): e9841. http://dx.doi.org/10.7717/peerj.9841.
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The BioBrick standard makes possible iterated pairwise assembly of cloned parts without any depletion of unique restriction sites. Every part that conforms to the standard is compatible with every other part, thereby fostering a worldwide user community. The assembly methods, however, are labor intensive or inefficient compared to some newer ones so the standard may be falling out of favor. An easier way to assemble BioBricks is described herein. Plasmids encoding BioBrick parts are purified from Escherichia coli cells that express a foreign site-specific DNA methyltransferase, so that each is subsequently protected in vitro from the activity of a particular restriction endonuclease. Each plasmid is double-digested and all resulting restriction fragments are ligated together without gel purification. The ligation products are subsequently double-digested with another pair of restriction endonucleases so only the desired insert-recipient vector construct retains the capacity to transform E. coli. This 4R/2M BioBrick assembly protocol is more efficient and accurate than established workflows including 3A assembly. It is also much easier than gel purification to miniaturize, automate and perform more assembly reactions in parallel. As such, it should streamline DNA assembly for the existing community of BioBrick users, and possibly encourage others to join.
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Brenier-Pinchart, Marie-Pierre, Emmanuelle Varlet-Marie, Florence Robert-Gangneux, Denis Filisetti, Juliette Guitard, Yvon Sterkers, Hélène Yera, Hervé Pelloux, and Patrick Bastien. "Impact of pre-analytic step duration on molecular diagnosis of toxoplasmosis for five types of biological samples." PLOS ONE 16, no. 2 (February 17, 2021): e0246802. http://dx.doi.org/10.1371/journal.pone.0246802.
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Introduction Toxoplasma-PCR is essential to diagnose ocular, cerebral, disseminated and congenital toxoplasmosis. This multicenter study evaluated the impact of sample storage duration at +4°C on PCR assay performances in order to propose guidelines for the storage of samples during shipment or/and before PCR. Materials and methods Five matrices, amniotic (AF), cerebrospinal (CSF), and bronchoalveolar lavage fluids (BALF), whole blood (WB) and buffy coat (BC), were artificially spiked with different amounts of Toxoplasma gondii (20, 100, 500 tachyzoites per mL of sample) or with previously infected THP1 cells. DNA extractions were performed at day 0 and after 2, 4 and 7 days of storage at +4°C. Each extract was amplified at least twice by real-time PCR. Results A total of 252 spiked samples was studied. No increase of crossing point was observed and all samples were positive for AF, BALF, BC and infected THP1-spiked WB after up to 7 days at 4°C. For CSF spiked with 20 parasites/mL, only 50% of PCR reactions were positive at D7 (p<0.05). For WB spiked with type II parasites, all reactions remained positive at D7 but amplifications were significantly delayed from D2; and for WB spiked with RH strain, the proportion of positive reactions decreased at D7. Conclusion The storage of clinical samples at +4°C is compatible with the molecular detection of T. gondii parasites. Provided that PCR assays are performed in duplicate, storage of samples is possible up to 7 days. However, from the fifth day onwards, and for samples susceptible to contain low parasitic loads, we recommend to perform the PCR in multiplicate.
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Mokany, Elisa, Yee Lee Tan, Simon M. Bone, Caroline J. Fuery, and Alison V. Todd. "MNAzyme qPCR with Superior Multiplexing Capacity." Clinical Chemistry 59, no. 2 (February 1, 2013): 419–26. http://dx.doi.org/10.1373/clinchem.2012.192930.
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BACKGROUND MNAzymes (nucleic acid enzymes formed from multiple partial enzymes) can be linked to PCR to provide a highly specific method for target detection and quantification. We investigated the feasibility of multiplexing MNAzyme quantitative PCR (qPCR) methods. METHODS We combined MNAzyme components with PCR primers and standard qPCR reagents to perform MNAzyme qPCR and reverse-transcription qPCR (RT-qPCR) assays with a set of universal reporter probes. Assays were performed on single targets and in multiplex formats that combined up to 5 different targets in a single reaction. RESULTS A comparison of 3 targets amplified in single and triplex formats showed no significant differences with respect to detection limit or amplification efficiency. Likewise, we successfully converted single-target assays for 11 transcripts of interest to triplex assays containing 2 reference transcripts without having to optimize or modify the conditions. A quintuplex RT-qPCR that simultaneously quantified 5 transcripts with 5 universal probes produced high amplification efficiencies and r2 values for all transcripts. Despite the large numbers of oligonucleotides in the reactions, we observed no false-positive signals, owing to the requirement of 4 target-specific binding events to produce a signal. A quadruplex assay that combined MNAzymes with methylation-specific PCR to measure epigenetic biomarkers of prostate cancer was capable of detecting a single methylated DNA allele in a BACKGROUND of 1000–10 000 unmethylated alleles. The MNAzyme qPCR was compatible with a rapid-cycling protocol. CONCLUSIONS MNAzymes offer a flexible and unique approach to qPCR that is specific, sensitive, and easily multiplexed. The universal nature of MNAzyme reporter probes removes the need for target-specific probes, thereby making the development of new assays easier and cheaper.
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Kohn, L. M. "The clonal dynamic in wild and agricultural plant–pathogen populations." Canadian Journal of Botany 73, S1 (December 31, 1995): 1231–40. http://dx.doi.org/10.1139/b95-383.
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The stability or change in clone frequencies during the disease cycle and from year to year is what I term the clonal dynamic. Among pathogenic fungi, the prevalence of efficient asexual reproduction affords the opportunity for invasive, epidemic, clonal colonization and spread. Clonality is probably most extreme in monoculture, although it could be expected to be important in wild plants and in transfers of adaptive pathogenic genotypes between wild and cultivated plants. The clonal dynamic was studied in Sclerotinia sclerotiorum in two experiments, one on four Canadian field populations of cultivated canola and the other on two Norwegian populations of a wild perennial plant, Ranunculus ficaria. Additional samples were made from canola and other crops in Canada and Norway. Four major differences between the agricultural and wild populations were observed. First, in agricultural populations, DNA fingerprint (multilocus haplotype) and mycelial compatibility group were coupled; all individual members of a clone shared a unique fingerprint and all were mycelially compatible. In wild populations, DNA fingerprint and mycelial compatibility group were decoupled. Second, in agricultural populations fingerprint diversity was high, with 594 genotypes recovered from 2747 isolates, but frequently sampled clones were recovered from a wide geographical area repeatedly over a 3-year period; in wild populations fingerprint diversity was low, with 7 genotypes from 300 isolates, and highly localized. Third, in agricultural populations, no evidence of outcrossing and segregation was observed; in the wild populations, some sibling ascospores showed different mycelial compatibility reactions, indicating that crossing had occurred. Last, in agricultural populations, clones were randomly dispersed spatially, probably the result of immigration and mixing of inoculum in air; in the apparently isolated wild populations, strong spatial substructuring was indicated by the distribution of fingerprints, apparently the result of highly localized inbreeding. Clonality was therefore clearly detected in the cultivated plant populations but was difficult to distinguish from inbreeding in the wild populations. Key words: multilocus haplotype, clonality, asexual reproduction, population genetics.
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Zheng, Zhi, Yuling Luo, and Gary McMaster. "Sensitive and Quantitative Measurement of Gene Expression Directly from Peripheral Whole Blood, without RNA Isolation and Target Amplification." Blood 106, no. 11 (November 16, 2005): 4518. http://dx.doi.org/10.1182/blood.v106.11.4518.4518.
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Abstract Peripheral blood gene expression analysis is increasingly used for diagnosis and prognosis of hematological diseases, as well as for surrogate biomarker discovery in a wide range of non-hematological disorders. However, a critical issue concerning the potential clinical application of these research findings relates to the validity and reproducibility of the blood mRNA quantitation results. Current gene expression technologies often depend on multiple steps: 1) blood isolation; 2) RNA purification and 3) subsequent enzymatic reactions, which affect the accuracy and consistency of results. We describe an assay to measure single- and multi-plexed gene expression directly from whole blood without RNA purification and target amplification. The hybridization-based assay uses branched DNA signal amplification technology with a whole blood lysis protocol that preserves the RNA integrity. The assay is sensitive enough to quantitatively measure genes expressed at low levels in a minority of cells from less than 30ul of whole blood. The coefficients of variations are less than 10% and the dynamic range is 3–4 logs for both singleplex and multiplex formats. The assay signals are several times higher than purified RNA from an equivalent amount of blood. Blood proteins, genomic DNA and reticulocyte mRNAs do not interfere with the assay. The assays are compatible with common anticoagulants and Paxgene treated samples. However, we found the Paxgene induced expression of antiapoptotic genes during processing of the whole blood. We used the multiplex assay to evaluate the impact of common blood processing on the expression of a panel of 30 cytokine and apoptosis genes known to be sensitive to ex vivo purtabation. Minimal impact was found with RBC lysis, followed by whole blood RNA extraction, PBMC isolation and Paxgene stabilization. The lowest correlation of expression was found between RNA extracted from whole blood and RNA extracted from same blood treated with Paxgene (R square=0.77). We believe this assay will contribute to fundamental and therapeutic applications where quantitative gene expression and/or throughput are required.
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Riccio, Gennaro, Eduardo Sommella, Nadia Badolati, Emanuela Salviati, Sara Bottone, Pietro Campiglia, Monica Dentice, Gian Tenore, Mariano Stornaiuolo, and Ettore Novellino. "Annurca Apple Polyphenols Protect Murine Hair Follicles from Taxane Induced Dystrophy and Hijacks Polyunsaturated Fatty Acid Metabolism toward β-Oxidation." Nutrients 10, no. 11 (November 20, 2018): 1808. http://dx.doi.org/10.3390/nu10111808.
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Chemotherapy-induced alopecia (CIA) is a common side effect of conventional chemotherapy and represents a major problem in clinical oncology. Even months after the end of chemotherapy, many cancer patients complain of hair loss, a condition that is psychologically difficult to manage. CIA disturbs social and sexual interactions and causes anxiety and depression. Synthetic drugs protecting from CIA and endowed with hair growth stimulatory properties are prescribed with caution by oncologists. Hormones, growth factors, morphogens could unwontedly protect tumour cells or induce cancer cell proliferation and are thus considered incompatible with many chemotherapy regimens. Nutraceuticals, on the contrary, have been shown to be safe and effective treatment options for hair loss. We here show that polyphenols from Malus Pumila Miller cv Annurca are endowed with hair growth promoting activity and can be considered a safe alternative to avoid CIA. In vitro, Annurca Apple Polyphenolic Extract (AAE) protects murine Hair Follicles (HF) from taxanes induced dystrophy. Moreover, in virtue of its mechanism of action, AAE is herein proven to be compatible with chemotherapy regimens. AAE forces HFs to produce ATP using mitochondrial β-oxidation, reducing Pentose Phosphate Pathway (PPP) rate and nucleotides production. As consequence, DNA replication and mitosis are not stimulated, while a pool of free amino acids usually involved in catabolic reactions are spared for keratin production. Moreover, measuring the effect exerted on Poly Unsaturated Fatty Acid (PUFA) metabolism, we prove that AAE promotes hair-growth by increasing the intracellular levels of Prostaglandins F2α (PGF2α) and by hijacking PUFA catabolites toward β-oxidation.
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Spyridonidis, Alexandros, Philipp Faber, Loizos Petrikkos, and Jurgen Finke. "Alloanatigenic Reactions after Hematopoietic Cell Transplantation Induce Genomic Alterations in Epithelial Cells as Shown in Human Studies and in an In Vitro Model." Blood 108, no. 11 (November 16, 2006): 3213. http://dx.doi.org/10.1182/blood.v108.11.3213.3213.
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Abstract Previous studies from our group demonstrated frequent genomic alterations measured by microsatellite instability (MSI) in non-neoplastic epithelial tissues of patients who underwent allogeneic hematopoietic cell transplantation (HCT) (Blood2006;107:3389–3396). These genomic alterations were found only after allogeneic but not after autologous HCT, and therefore we hypothesized that an “allogeneic” effect is substantially involved in the mutation process. We extended our previous analyses by examining 210 bucall swabs obtained from 70 patients between day (d+) 26 and d+3514 after allogeneic HCT for the presence of MSI. MSI analysis was performed by PCR and denaturing capillary electrophoresis at three tetranucleotide (THO-1, SEE33, D14S120) and three mononucleotide microsatellite (ZP3, BAT26, SRY) loci. MSI was found in the buccal smears of 38% allografted patients (median time of occurence 322 days). In a prospective trial, in which patients were followed from time before transplantation until d+365, 5 out of 14 (35%) patients exhibited MSI after transplantation although all of them showed stable microsatellites before transplantation. We are currently pefroming statistical analyses in order to identify which clinical factors influence the presence of MSI and we will present the data in the meeting. To test the hypothesis that an “alloantigenic” effect is responsible for th induction of MSI, we developed a model system in which keratinocyte (HaCaT) cells were transfected with a palsmid vector which carries a G418 (neo) selectable marker and a microsatellite repeat (CA) that places the sequence for Hygromycin Resistance (HygR) out of frame for protein translation. In this reporter system, DNA slippage mutations can restore the HygR reading frame and become detectable by hygromycin treatment as hygromycin resistant (HygR+) colonies. Pools of stably transfected (neo+) HaCaT cells were treated with supernatant (SN) of major histocompatibility complex nonmatched mixed lymphocyte cultures (MLC) and assayed for HygR+ colonies 48h later. Cells transfected with a control, in-frame hygromycin B gene construct (p12) were used as positive controls. Using this system, we found that HaCaT cells aquire hygromycin resistance after treatment with supernatatant from MLC. Treatment of cells with hydrogen hyperoxid which has been shown in a E. Coli system to induce MSI (PNAS1998, 95:12468–12473) generated HygR+ colonies at a >80% lower frequency than the SN-MLC treatment. Control p12 transfected cells were grown with high efficiency in the presence of hygromycin B. In summary, our in vivo data confirm our previous results and provide evidence of genomic alterations after allogeneic HCT and our in vitro data are compatible with the hypothesis that an “alloantigenic” factor is the driving force in producing detectable MSI in the allografted patients. Elucidating the ultimate mechanisms underlying the genomic instability following allogeneic HCT may prove to be of major therapeutic value.
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Petersen, Jesper, Martin Dufva, and Henrik Birgens. "Inexpensive DNA Microarray Application for Screening of Beta-Globin Mutations in Low-Resource Settings." Blood 114, no. 22 (November 20, 2009): 4067. http://dx.doi.org/10.1182/blood.v114.22.4067.4067.
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Abstract Abstract 4067 Poster Board III-1002 Development of public health related products are, in general, driven by clinical usefulness in combination with economic feasibility. Not surprisingly, a high throughput, automated robotic system is not an option in a low-resource setting as exists in the majority of countries with high prevalence of hemoglobinopathies. Diagnostics developed for resource rich areas may not be feasible in such areas but certain parts of these technologies can be transformed into low-cost diagnostics suitable for resource poor settings. Hemoglobinopathies are traditionally presumptively diagnosed using a combination of methods including complete blood count, MCV or MCH, and hemoglobin electrophoresis or HPLC. Recently, sequence specific nucleic acid recognition methodologies have allowed the discovery of more than 200 mutations which cause thalassemia and an even higher number of mutations leading to hemoglobin variants. Importantly, such methods provide a more precise diagnosis and allow for prenatal analysis. Microarrays use specific nucleic acid sequences to detect up to thousands of mutations simultaneously but, due to their cost and requirements of equipment, are principally only suited to resource rich areas. However, pairing microarray technology with alkaline phosphatase colorimetric staining, an established visualization technology, significantly reduces assay cost and initial cost and complexity of required equipment and microarray-based diagnostics can thus become applicable to resource poor settings. This study focused on the development of a highly specific and low-cost method for genotyping beta-thalassemia mutations and hemoglobin variants using colorimetric staining of allele specific DNA microarrays. Target was easily prepared by in vitro incorporation of a small amount of biotin-labeled ribonucleotides after an initial PCR reaction and was immediately ready for subsequent visualization via the well characterized and highly specific biotin-streptavidin interaction. Streptavidin-conjugated alkaline phosphatase coupled to commercially available BCIP/NBT, well established in ELISA and western blots, allowed results visualization. Microarray assays that correctly and accurately identify mutations in multiple nucleic acid sequences (genechips) are difficult to produce as these are usually processed at a single condition. This single condition does not represent the optimum condition for all the individual and necessary reactions required to detect all mutations. This can be circumvented by changing reaction temperature during analysis. This requires multiple readouts during a genotyping cycle and precise thermal control, both of which are not yet compatible with a low-budget application. In a previous paper, we demonstrated a very low cost alternative solution that used buffers with different ionic strength ([Na+]) and a polymeric microfluidic assembly rather than temperature control to optimize microarray conditions. Arrays, including an internal batch quality control, were manufactured on ordinary glass microscope slides coated with a film of agarose. Detection is based on colorimetric grayscale readout from an ordinary flatbed desktop scanner at low resolution (1200 dpi). In this ongoing study, 30 of the most common mutations leading to beta-thalassemia and those responsible for HbC, HbS, HbD-Punjab, HbE and HbO-Arab hemoglobin variants were included. The microarray based technique is currently being compared to molecular diagnosis via sequencing. To date, a 100% concordance has been obtained. The study also continues to optimize probes for mutagenic sites in order to increase the number of analysis sites and confidence in genotype calls. In conclusion, combining a polymeric device easily manufacturable by high volume methods such as injection molding, microarrays printed on ordinary glass slides, and detection based on colorimetric staining a low-budget analysis system for genetic variations was obtained. Start-up and assay costs were roughly $2000 and $5 US, respectively. As such the method provides interesting and new diagnostic possibilities for those third world countries where beta-thalassemia and other hemoglobinopathies are highly prevalent and where the socioeconomic conditions exclude introduction of expensive methods for molecular diagnostics. Disclosures: No relevant conflicts of interest to declare.
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Martinez, Aline Amorim, Alessandra Midori Kuniyoshi, Mihoko Yamamoto, Maria de Lourdes Chauffaille, André Luiz Vettore, Akemi Kuroda Chiba, Eliza Yuriko Sugano Kimura, Aline Akemi Nagado, and José Orlando Bordin. "Loss of red cell ABH Antigens and Hypermethylation of the ABO Gene Promoter region is frequent in Myeloid Malignancies." Blood 114, no. 22 (November 20, 2009): 3151. http://dx.doi.org/10.1182/blood.v114.22.3151.3151.
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Abstract Abstract 3151 Poster Board III-88 Background Loss of red blood cell (RBC) antigens may occur in solid tumors and in hematological diseases; however the mechanisms involved in these changes are not fully understood. Aberrant DNA hypermethylation is thought to be involved in acute myeloid leukemia (AML) as well as in myelodysplastic syndromes (MDS), and the methylation of cytosines residues in the dinucleotide CpG may account for the expression patterns of the ABO genes. In this study we investigated loss of RBC ABH antigens and the possible role of DNA methylation in the ABO promoter gene in patients with myeloid diseases. Methods Forty-three patients were included in our study (MDS=16, chronic myeloid leukemia (CML)=12, AML=4, polycythemia vera (PV)=3, essential thrombocythemia (ET)=3, chronic myelomonocytic leukemia (CMML)=3, myelofibrosis (MF)=1 and chronic neutrophilic leukemia (CNL)=1). Forty-one patients were evaluated according to their ABO group using serological immunohematological tests (Diamed Inc., Brazil). ABH secreted antigens were investigated in saliva and a PCR-based ABO genotyping using restriction enzyme digestion (Alu and Kpn) was performed to confirm the ABO blood type. In addition, the expression of the A, B and H antigens was analyzed by flow cytometry in 26 patients (median age=63 yrs; 6M/17F) and 81 healthy controls (median age=33 yrs; 38M/43F). Methylation of CpG islands was investigated in 45 bone marrow samples using methylation specific PCR (MSP) technique with methylated and unmethylated primer sets for region from -200 to +26 sequence of the ABO gene. Results The investigation of the secreted antigens in saliva and the genotyping studies confirmed the results of the ABO serological tests in 40 patients, but we found that the RBCs of one patient were not agglutinated by anti-B while his genotype result was compatible with BO indicating loss of the B antigen in his RBCs. Overall, loss of A, B or H antigen was detected by flow cytometry in 11/26 (42%) patients. Hypermethylation of the ABO promoter gene was detected in 51% (23/45) of the analyzed bone marrow samples. Among patients with hypermethylation in the ABO promoter gene, 44% had loss of A or B antigens confirmed by serological or flow cytometric tests compared with 28% in the group of patients with unmethylated ABO promoter gene (p=0.632). Conclusions Epigenetic changes including methylation of cytosine residues are recognized as major contributors to gene silencing, disease progression and worse outcome in several cancer patients. Our data showed that the hypermethylation of the ABO gene is frequent in patients with myeloid malignancies corresponding to the pathogenesis already described for AML and MDS. However, not all patients showing loss of RBCs antigens had ABO methylation evidence, suggesting that other mechanisms may take place. Patients with myeloid malignancies often need blood transfusion support and loss of RBCs antigens can lead to changing in ABO blood group increasing the risk of serious blood transfusion reactions. The relatively high rate (42%) of loss of ABH antigens found in this study demonstrated that these patients have to be carefully managed to avoid such severe transfusion reactions. (Grants supported by CNPq, 478814/2006-2). Disclosures No relevant conflicts of interest to declare.
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François, Celia, Celia Martinez, Clement Faye, Nathalie Pansu, Catherine Dunyach-Remy, Laurent Garrelly, Benoit Roig, and Axelle Cadiere. "The Utilization of Linear Polylysine Coupled with Mechanic Forces to Extract Microbial DNA from Different Matrices." Microorganisms 8, no. 12 (November 30, 2020): 1901. http://dx.doi.org/10.3390/microorganisms8121901.
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Molecular approaches are powerful tools that are used for medical or environmental diagnoses. However, the main limitations of such a tools are that they extract low levels of DNA and they do not remove the inhibitors of polymerase chain reaction (PCR). Although the use of polycation to complex and purify DNA has been described in the literature, elution often requires a high ionic strength or pH levels not compatible with molecular analyses. In this paper, we described a new process that is based on the complexation of DNA with linear polylysine, followed by capturing the complex by a cation exchange resin. The originality of the process consisted of using mechanic force to elute DNA from the complex. The extraction method showed several advantages when compared to existing methods, such as being compatible with pH levels that range from 5 to 11, as well as high levels of DNA recovery and elimination of PCR inhibitors from complex samples. This method was successfully applied to different types of samples, such as environmental samples, beverage samples, and medical samples. Furthermore, it was proven to be a good solution for removing PCR inhibitors and assuring good DNA recovery yield.
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Barouqa, Mohammad, Kenji Ikemura, Henny Billett, Margarita Kushnir, Kateryna Fedorov, David Yin, and Morayma Reyes Gil. "Neutrophilic Extracellular Traps (NETs); A Subset of Smudge Cells Identifiable by Peripheral Smear Autoanalyzers in the Rising Era of Artificial Intelligence." American Journal of Clinical Pathology 154, Supplement_1 (October 2020): S10—S11. http://dx.doi.org/10.1093/ajcp/aqaa137.018.
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Abstract Background Digitized microscopy such as CellaVision® technology has revolutionized the laboratory. Smudge cells, also called basket cells, are usually seen in lymphoproliferative disorders representing remnants from degenerated lymphocytes (DLs). CellaVision® classifies DLs and web-like remnants as smudge cells. The morphology of the web-like remnants is compatible with Neutrophil Extracellular Traps (NETs) where extracellular decondensed DNA chromatin network is formed as one of several neutrophilic reactions to stress. Currently, we lack clinical tests that reliably identify and quantify NETs. Aims To develop an in-vitro model for NETs formation in blood, create a library of their morphological changes at different maturation stages; correlate their presence to infections in absence of leukocytosis and develop an artificial intelligence platform (AI-Heme-1) for their detection. Methods A library was built to develop AI-Heme-1 where NETs were induced with classic triggers (phorbol-myristate-acetate, lipopolysaccharide and ionomycin) in EDTA whole blood from normal subjects. Smears were prepared at 30 minutes intervals for 24 hours to identify NETs by Immunofluorescence and immunohistochemistry. WBC differentials were performed by CellaVision® to capture different stages of NETs. AI-Heme-1 was modified from Python online convolutional neural network. For the clinical correlation, smears with >20% smudge cells were classified morphologically as NETs vs. DLs compared to a control group, < 5% smudge cells. We used morphologic characteristics, immunohistochemistry, immunofluorescence and flow cytometry to differentiate NETs from DLs. Medical chart review performed by blinded investigators, included patient demographics, CBC and presence of microbial infection occurring < 1 week of sample collection. Statistical analyses included two sided t-test and chi square. Results The classical triggers for Netosis showed consistent morphological changes following a canonical order: vacuolation, nuclear decondensation, degranulation and chromatin ejection. These cell remnants were positive for citrullinated histones, myeloperoxidase, leukocyte alkaline phosphatase and neutrophil elastase by immunofluorescence. On Wright Giemsa stain, web-like remnants resembling NETs stained for SytoxGreen. On flow cytometry, NETs were large with extracellular DNA and MPO. For the clinical study group of >20% smudge cells, 88 were morphologically designated as NETs, 8 as DL vs. 59 as control group. A random sampling from >20% smudge cells showed cases with NET subclassification stained strongly with myeloperoxidase, neutrophil elastase and SytoxGreen while DLs were negative. Comparing patients with >20% smudge and NET sub-classification to <5% smudge cells, the formers had higher incidence of bacterial and viral infections (p=0.009/0.005 and p=0.008/0.007). Conclusions Our study was able to identify NETs on peripheral smears performed by a routine Hematology Autoanalyzer using a reliable set of morphologic characteristics, immunohistochemical stains and flow cytometry. It supports data that associate NETs with infections in the absence of leukocytosis. AI-Heme-1 was able to identify NETs on blood smears. This approach can provide a rapid, early and accurate tool to screen patients with infections.
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Woodhall, J. W., B. Lutomirska, J. C. Peters, and P. S. Wharton. "Rhizoctonia solani Anastomosis Group 3 is Predominant in Potato Tubers in Poland." Plant Disease 97, no. 9 (September 2013): 1245. http://dx.doi.org/10.1094/pdis-05-12-0482-pdn.
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Rhizoctonia solani is a species complex of 13 related but genetically distinct anastomosis groups (AGs). In potato, R. solani can infect the stems, stolons, and roots, resulting in quantitative losses. It can also cause qualitative losses through blemishes occurring on progeny tubers, such as black scurf and elephant hide (corky cracking). Knowledge of the AG in local populations is important because they differ in host range, fungicide sensitivity, and disease severity (2). To determine the AGs present in Poland, 54 tuber samples displaying typical R. solani symptoms were taken from six different fields in 2011. The fields were representative of five different administrative regions of Poland and from at least 10 different varieties. Rhizoctonia was isolated from tubers by placing symptomatic material on to tap water agar amended with streptomycin and penicillin and after 2 to 3 days Rhizoctonia colonies were identified and hyphal tips of these transferred to potato dextrose agar. Rhizoctonia was successfully isolated from 48 tubers displaying black scurf and two tubers displaying elephant hide symptoms. DNA was extracted from Rhizoctonia cultures using a Wizard Food kit (Promega) and the AG was determined using specific real-time PCR assays (1). All Rhizoctonia isolates were determined to be AG3 and this was confirmed for 10 selected isolates by observing hyphal fusion with a known AG3 tester isolate (Rs08) as described previously (3). Pairings were also conducted amongst the 10 Polish isolates, C2 reactions were typically observed indicating numerous vegetative compatible groups are present. This study shows that AG3 is likely to be the predominant AG in potato tubers in Poland. This is similar to other studies in Europe, which have all determined that AG3 accounts for at least 92% of isolates from potato (2,3). AG2-1, 4, and 5 have also been found in tubers worldwide and climate and certain crop rotations can influence the presence of these other AGs in potato tubers (2). However, climate and crop rotations in Poland are similar to other parts of Europe so the predominance of AG3 is expected. AG3 was also isolated from elephant hide symptoms; however, it was more frequently isolated from sclerotia. The ability of AG3 to prolifically produce sclerotia and thereby survive on seed tubers may explain its predominance in potato crops (4). Therefore, studies focusing on the management of Rhizoctonia potato disease in Poland should consider AG3 in the first instance. References: (1) G. E. Budge et al. Plant Pathol. 58:1071, 2009. (2) L. Tsror. J. Phytopathol. 158:649, 2010. (3) J. W. Woodhall et al. Plant Pathol. 56:286, 2007. (4) J. W. Woodhall et al. Plant Pathol. 57:5, 2008.
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Shtenberg, Giorgi, Naama Massad-Ivanir, Oren Moscovitz, Sinem Engin, Michal Sharon, Ljiljana Fruk, and Ester Segal. "Biosensor based on DNA directed immobilization of enzymes onto optically sensitive porous Si." MRS Proceedings 1569 (2013): 195–200. http://dx.doi.org/10.1557/opl.2013.806.
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ABSTRACTOptical biosensor for monitoring proteolytic activity is constructed by DNA-directed immobilization of enzymes onto porous Silicon nanostructures. This sensor configuration allows both protease recycling and easy surface regeneration for subsequent biosensing analysis by means of mild dehybridization conditions. We demonstrate real-time analysis of minute quantities of proteases paving the way for substrate profiling and the identification of cleavage sites. The biosensor is compatible with common proteomic methods and allows for a successful downstream mass spectrometry analysis of the reaction products.
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Hu, Hezhi, Jingmeng Cheng, Chunyang Wei, Shanshan Li, Chengzhuang Yu, Xiaoshuai Meng, and Junwei Li. "Pre-Degassed Microfluidic Chamber-Based Digital PCR Device for Meat Authentication Applications." Micromachines 12, no. 6 (June 14, 2021): 694. http://dx.doi.org/10.3390/mi12060694.
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Droplet digital polymerase chain reaction (ddPCR) suffers from the need for specific equipment and skilled personnel; thus, we here present a chamber-based digital PCR microfluidic device that is compatible with fluorescence image read-out systems and removes bubbles by a pre-degassed microfluidic device that consists of a pilot channel and micro chamber arrays. Digitalized PCR reagents are introduced into micro chambers, and thermocycles are taken to perform a DNA amplification process. Then, fluorescence images of a micro chamber array are read out and analyzed to obtain the total number of positive chambers. Thereby, the copy numbers of target DNA are calculated for quantitative detections. As a validation, this device is evaluated by the application of meat authentication. We performed dPCR tests using DNA templates extracted from a pure mutton DNA template with different dilutions. Then, the dPCR chip was used to identify the meat authentication using mutton–chicken mixtures with different mass ratios, showing its performance in real biotechnical applications.
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Yang, Kun, Giovanni Stracquadanio, Jingchuan Luo, Jef D. Boeke, and Joel S. Bader. "BioPartsBuilder: a synthetic biology tool for combinatorial assembly of biological parts." Bioinformatics 32, no. 6 (November 14, 2015): 937–39. http://dx.doi.org/10.1093/bioinformatics/btv664.
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Abstract Summary: Combinatorial assembly of DNA elements is an efficient method for building large-scale synthetic pathways from standardized, reusable components. These methods are particularly useful because they enable assembly of multiple DNA fragments in one reaction, at the cost of requiring that each fragment satisfies design constraints. We developed BioPartsBuilder as a biologist-friendly web tool to design biological parts that are compatible with DNA combinatorial assembly methods, such as Golden Gate and related methods. It retrieves biological sequences, enforces compliance with assembly design standards and provides a fabrication plan for each fragment. Availability and implementation: BioPartsBuilder is accessible at http://public.biopartsbuilder.org and an Amazon Web Services image is available from the AWS Market Place (AMI ID: ami-508acf38). Source code is released under the MIT license, and available for download at https://github.com/baderzone/biopartsbuilder. Contact: joel.bader@jhu.edu Supplementary information: Supplementary data are available at Bioinformatics online.
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Schroeder, Max R., and Vladimir Loparev. "Rapid Inactivation of Non-Endospore-Forming Bacterial Pathogens by Heat Stabilization is Compatible with Downstream Next-Generation Sequencing." Applied Biosafety 24, no. 3 (July 15, 2019): 129–33. http://dx.doi.org/10.1177/1535676019861261.
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Introduction: Heat stabilization treatment preserves the in vivo state of biological samples by rapidly inactivating enzymes that cause degradation of proteins and nucleic acids. Historically, proteomics studies used this technique as an alternative to chemical fixation. More recently, microbiologists discovered that heat stabilization treatment rapidly inactivates pathogens present in tissue samples and preserves deoxyribonucleic acid (DNA) in the tissue. However, these recent studies did not investigate the inactivation of high-density bacterial suspensions and the quality of bacterial DNA. Methods and Results: High-density suspensions of Escherichia coli (>109 cfu/mL) were completely inactivated by heat stabilization treatment using the Denator Stabilizor T1 instrument at 72°C and 95°C for 45 seconds. Using the heat stabilization instrument, a panel of 30 species, 20 Gram-negative and 10 non-endospore-forming Gram-positive species, were fully inactivated by treatment (95°C for 45 seconds). DNA was isolated from bacterial suspensions of Gram-negative bacteria, including E. albertii, E. coli, Shigella dysenteriae, and S. flexneri, following inactivation via heat stabilization treatment and without treatment. DNA isolated following heat stabilization treatment was fully compatible with all downstream molecular applications tested, including next-generation sequencing, pulsed-field gel electrophoresis, multiplex polymerase chain reaction (PCR), and real-time PCR. Conclusions and Discussion: Heat stabilization treatment of Gram-negative and non-endospore-forming Gram-positive pathogens completely inactivates high-density bacterial suspensions. This treatment is compatible with downstream DNA molecular assays, including next-generation sequencing, pulsed-field gel electrophoresis, and PCR. Inactivation by heat stabilization is a rapid process that may increase safety by decreasing risks for laboratory-associated infections and risks associated with transportation of infectious materials.
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Habu, Tsuyoshi, Fumio Kishida, Miki Morikita, Akira Kitajima, Toshiaki Yamada, and Ryutaro Tao. "A Simple and Rapid Procedure for the Detection of Self-Compatible Individuals in Japanese Apricot (Prunus mume Sieb. et Zucc.) Using the Loop-Mediated Isothermal Amplification (LAMP) Method." HortScience 41, no. 5 (August 2006): 1156–58. http://dx.doi.org/10.21273/hortsci.41.5.1156.
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Japanese apricot (Prunus mume Sieb. et Zucc.) exhibits S-RNase-based gametophytic self-incompatibility as do other Prunus species. Both self-incompatible and self-compatible Japanese apricot cultivars are grown commercially in Japan. These self-compatible cultivars are shown to have a common S-haplotype called Sf that contains Sf-RNase and SFBf (S-haplotype-specific F-box protein). This study describes a simple and rapid detection of SFBf, in Japanese apricot, based on loop-mediated isothermal amplification (LAMP) method. A set of 4 primers, F3, B3, FIP, and BIP primer, were designed from the exon and the putative inserted sequence of SFBf. Optimal reaction time at 63 C was determined to be 90 minutes. It appeared that the LAMP method combined with the ultrasimple DNA extraction efficiently detected SFBf. The advantage of the marker-assisted selection of self-compatibility based on the LAMP method was discussed.
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Delaney, M. A., K. A. Terio, K. M. Colegrove, M. B. Briggs, and M. J. Kinsel. "Occlusive Fungal Tracheitis in 4 Captive Bottlenose Dolphins (Tursiops truncatus)." Veterinary Pathology 50, no. 1 (May 9, 2012): 172–76. http://dx.doi.org/10.1177/0300985812446153.
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Respiratory disease is common in dolphins, primarily affecting pulmonary parenchyma and sparing large airways. Over a 10-year period, 4 captive adult bottlenose dolphins succumbed to chronic, progressive respiratory disease with atypical recurrent upper respiratory signs. All dolphins had severe, segmental to circumferential fibrosing tracheitis that decreased luminal diameter. Histologically, tracheal cartilage, submucosa, and mucosa were distorted and replaced by extensive fibrosis and pyogranulomatous inflammation centered on fungal hyphae. In 3 of 4 cases, hyphae were morphologically compatible with Aspergillus spp and confirmed by culture in 2 cases. Amplification of fungal DNA from tracheal tissue was successful in one case, and sequences had approximately 98% homology to Aspergillus fumigatus. The remaining case had fungi compatible with zygomycetes; however, culture and polymerase chain reaction were unsuccessful. Lesions were evaluated immunohistochemically using antibodies specific to Aspergillus spp. Aspergillus-like hyphae labeled positively, while presumed zygomycetes did not. These cases represent a novel manifestation of respiratory mycoses in bottlenose dolphins.
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Oliveira, Luiz Carlos Marques de, Priscilla Dias Silva Abrahão, and Sergio Borges de Amorim. "Detection of hepatitis B virus DNA in sera from 18 alcoholic carriers of "anti-HBc alone" and response to a single dose of hepatitis B vaccine." Arquivos de Gastroenterologia 45, no. 3 (September 2008): 252–54. http://dx.doi.org/10.1590/s0004-28032008000300017.
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To evaluate the possibility of occult hepatitis B virus (HBV) infection in alcoholics carriers of "anti-HBc alone", and to verify the behavior of this serological pattern after a single dose of hepatitis B vaccine, 18 alcoholics who had this serological profile were evaluated by the polymerase chain reaction method, and 17 of them were vaccined. All were negative for HBV DNA. Nine (52.9%) of those vaccined had anamnestic response, mainly those with positive anti-HBe (8/10; 80%). "Anti-HBc alone" was compatible with low levels of anti-HBs in half of the patients, and probably with false positive results for anti-HBc in the others.
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Li, Qingbo, and Edward S. Yeung. "Simple Two-Color Base-Calling Schemes for DNA Sequencing Based on Standard Four-Label Sanger Chemistry." Applied Spectroscopy 49, no. 10 (October 1995): 1528–33. http://dx.doi.org/10.1366/0003702953965470.
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Two base-calling schemes for DNA sequencing are evaluated. Both are based on data collected from two broad-band emission channels derived from either one- or two-excitation channels. Standard four-dye Sanger reaction products are used in conjunction with capillary electrophoretic separation in a polymer matrix. Data acquisition is compatible with high light-throughput imaging and minimal data storage. In one scheme, commercial chromatographic software provides peak recognition and peak heights. The peak-height ratios from the two channels provide base-calling accuracies of 99.3% and 97.1% through 330 bp and 350 bp, respectively. In another scheme, ratiograms are derived from the two channels. The resulting step-like functions permit the calling of bases even when successive peaks are not resolved. The base-calling accuracy is 99% through 340 bp. Because of simplicity in implementation, either scheme should be readily applicable to high-speed, high-throughput DNA sequencing in capillary arrays.
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Andrade, Rosilene Viana de, Cesare Massone, Meline Nogueira Barbosa de Lucena, Anette Chusciak Talhari, Sinésio Talhari, Jorge Augusto de Oliveira Guerra, and Luiz Carlos de Lima Ferreira. "The use of polymerase chain reaction to confirm diagnosis in skin biopsies consistent with american tegumentary leishmaniasis at histopathology: a study of 90 cases." Anais Brasileiros de Dermatologia 86, no. 5 (October 2011): 892–96. http://dx.doi.org/10.1590/s0365-05962011000500005.
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BACKGROUND: Cutaneous leishmaniasis is a chronic, infectious disease caused by protozoa of the genus leishmania. The incidence of this disease is high in Brazil, with 19,746 new cases having been detected in 2008. The presence of amastigotes in the cytoplasm of histiocytes constitutes diagnosis of the disease; however, their presence is rarely found in late lesions, making histological diagnosis difficult. Polymerase chain reaction has been shown to represent a highly sensitive and specific technique for the diagnosis of cutaneous leishmaniasis. OBJECTIVES: To use polymerase chain reaction to evaluate paraffin-embedded skin biopsies with histopathological features consistent with cutaneous leishmaniasis. MATERIAL AND METHODS: Polymerase chain reaction amplification of a 120-base-pair fragment of Leishmania kinetoplast DNA (kDNA) minicircles was performed on 90 skin biopsies. The male/female ratio was 75/15. Mean age was 32.36 years, with a median of 31 years, range 4-72 years. Samples were histologically compatible with cutaneous leishmaniasis but a definitive diagnosis could not be made since amastigotes were not found. All cases were histologically classified according to the patterns described by de Magalhães. RESULTS: According to the de Magalhães classification, the most common histological pattern was type IV (exudative granulomatous reaction), which was found in 65.6% of cases (56/90), followed by type I (exudative cellular reaction) in 21.1% of cases (19/90) and type III (exudative and necrotic granulomatous reaction) in 12.2% of cases (11/90). Leishmania DNA was found in 96.7% of the biopsies (87/90). CONCLUSION: Polymerase chain reaction performed by amplifying kDNA is able to confirm a diagnosis of cutaneous leishmaniasis with a high degree of sensitivity in cases in which histopathology is consistent with a diagnosis of cutaneous leishmaniasis but not definitive.
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Dreaden, Tyler J., John M. Davis, Carrie L. Harmon, Randy C. Ploetz, Aaron J. Palmateer, Pamela S. Soltis, and Jason A. Smith. "Development of Multilocus PCR Assays for Raffaelea lauricola, Causal Agent of Laurel Wilt Disease." Plant Disease 98, no. 3 (March 2014): 379–83. http://dx.doi.org/10.1094/pdis-07-13-0772-re.
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Laurel wilt, caused by the fungus Raffaelea lauricola, is an exotic disease that affects members of the Lauraceae plant family in the southeastern United States. The disease is spreading rapidly in native forests and is now found in commercial avocado groves in south Florida, where an accurate diagnostic method would improve disease management. A polymerase chain reaction (PCR) method based on amplifying the ribosomal small-subunit DNA, with a detection limit of 0.0001 ng, was found to be suitable for some quantitative PCR applications; however, it was not taxon specific. Genomic sequencing of R. lauricola was used to identify and develop primers to amplify two taxon-specific simple-sequence repeat (SSR) loci, which did not amplify from related taxa or host DNA. The new SSR loci PCR assay has a detection limit of 0.1 ng of R. lauricola DNA, is compatible with traditional and real-time PCR, was tested in four labs to confirm consistency, and reduces diagnostic time from 1 week to 1 day. Our work illustrates pitfalls to designing taxon-specific assays for new pathogens and that undescribed fungi can limit specificity.
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Behrmann, Ole, Matthias Hügle, Franz Eckardt, Iris Bachmann, Cecilia Heller, Marina Schramm, Carrie Turner, Frank Hufert, and Gregory Dame. "3D Printed Monolithic Microreactors for Real-Time Detection of Klebsiella pneumoniae and the Resistance Gene blaNDM-1 by Recombinase Polymerase Amplification." Micromachines 11, no. 6 (June 17, 2020): 595. http://dx.doi.org/10.3390/mi11060595.
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We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-β-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.
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GUL, Sheraz, Richard BROWN, Earl MAY, Marie MAZZULLA, Martin G. SMYTH, Colin BERRY, Andrew MORBY, and David J. POWELL. "Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay." Biochemical Journal 383, no. 3 (October 26, 2004): 551–59. http://dx.doi.org/10.1042/bj20040054.
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DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the Km values for NAD+ (2.75±0.1 μM) and the acridinium-ester-labelled DNA substrate (2.5±0.2 nM). A study of the pH-dependencies of kcat, Km and kcat/Km has revealed values of kinetically influential ionizations within the enzyme–substrate complexes (kcat) and free enzyme (kcat/Km). In each case, the curves were shown to be composed of one kinetically influential ionization, for kcat, pKa=6.6±0.1 and kcat/Km, pKa=7.1±0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30±0.86 μM for doxorubicin and 1.40±0.07 μM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 μl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.
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Angeline, T., H. R. Krithiga, W. Isabel, A. J. Asirvatham, and A. Poornima. "Endothelial Nitric Oxide Synthase Gene Polymorphism (G894T) and Diabetes Mellitus (Type II) among South Indians." Oxidative Medicine and Cellular Longevity 2011 (2011): 1–4. http://dx.doi.org/10.1155/2011/462607.
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The objective of the study is to find out whether the endothelial nitric oxide synthase (eNOS) G894T single-nucleotide polymorphism is associated with type 2 diabetes mellitus in South Indian (Tamil) population. A total number of 260 subjects comprising 100 type 2 diabetic mellitus patients and 160 healthy individuals with no documented history of diabetes were included for the study. DNA was isolated, and eNOS G894T genotyping was performed using the polymerase chain reaction followed by restriction enzyme analysis usingBan II. The genotype distribution in patients and controls were compatible with the Hardy-Weinberg expectations (P>0.05). Odds ratio indicates that the occurrence of mutant genotype (GT/TT) was 7.2 times (95% CI = 4.09–12.71) more frequent in the cases than in controls. Thus, the present study demonstrates that there is an association of endothelial nitric oxide synthase gene (G894T) polymorphism with diabetes mellitus among South Indians.
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Fenton, James A. L., Jan-Willem Vaandrager, Wilhelmina M. Aarts, Richard J. Bende, Karel Heering, Martin van Dijk, Gareth Morgan, Carel J. M. van Noesel, Ed Schuuring, and Philip M. Kluin. "Follicular lymphoma with a novel t(14;18) breakpoint involving the immunoglobulin heavy chain switch mu region indicates an origin from germinal center B cells." Blood 99, no. 2 (January 15, 2002): 716–18. http://dx.doi.org/10.1182/blood.v99.2.716.
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Abstract With the use of DNA-fiber fluorescent in situ hybridization, a BCL2 protein positive follicular lymphoma with a novel BCL2 breakpoint involving the immunoglobulin heavy chain (IGH) switch mu (Sμ) region instead of the JH orDH gene segments was identified. Sequence analysis showed that the genomic breakpoint is localized between the Sμ region of the IGH complex and the first intron of BCL2. Reverse-transcriptase polymerase chain reaction showed expression of a unique hybrid IGH-BCL2 transcript involving the transcription initiation site Iμ. Sequence analysis of the VH region of the functional nontranslocatedIGH allele showed multiple shared somatic mutations but also a high intraclonal variation (53 differences in 15 clones), compatible with the lymphoma cells staying in or re-entering the germinal center. This is the first example of a t(14;18) translocation that results from an illegitimate IGH class-switch recombination during the germinal center B-cell stage.
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Abdelbaky, Ahmed S., Ivan A. Prokhorov, Igor P. Smirnov, Kristina M. Koroleva, Vitaliy I. Shvets, and Yulia G. Kirillova. "Synthesis of α-(R)-/γ-(S)-Dimethyl Substituted Peptide Nucleic Acid Submonomer Using Mitsunobu Reaction." Letters in Organic Chemistry 16, no. 5 (April 1, 2019): 437–46. http://dx.doi.org/10.2174/1570178616666190118155031.
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One of the major challenges facing modern biochemical and biomedical technologies are finding molecular tools for diagnosis and detection of genetic diseases. In this connection, several classes of oligonucleotides have been developed that can recognize and bind to DNA and RNA with high affinity and sequence selectivity and withstand enzymatic degradation by proteases and nucleases; however, few can traverse the cell membrane on their own. One such promising class of nucleic acid mimics developed in the last two decades which showed good results in vitro, are the peptide nucleic acids (PNAs). New chiral α- and γ-peptide Nucleic Acid (PNA) submonomer with methyl substituents in pseudopeptide backbone were synthesized via Mitsunobu reaction. The α-(R)-/γ-(S)-configuration of the chiral centres will ensure the preorganization of the PNA oligomer into a right-handed helix. The results obtained showed that Boc/Fmoc-submonomer compatible with Boc-protocol PNAs solid-phase synthesis on an MBHA resin. We synthesized simple and efficient α-R-, γ-S-disubstituted PNA submonomer based on L-Ala and D-Ala with the construction of the intermediate pseudopeptide moiety by Mitsunobu reaction for subsequent use in the Boc-Protocol of solid phase PNA synthesis.
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Davitkov, Darko, Milos Vucicevic, Jevrosima Stevanovic, Vanja Krstic, Snezana Tomanovic, Uros Glavinic, and Zoran Stanimirovic. "Clinical babesiosis and molecular identification of Babesia canis and Babesia gibsoni infections in dogs from Serbia." Acta Veterinaria Hungarica 63, no. 2 (June 2015): 199–208. http://dx.doi.org/10.1556/avet.2015.017.
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Canine babesiosis is a frequent and clinically significant tick-borne disease. Sixty symptomatic dogs with clinical findings compatible with babesiosis were included in this study conducted in Serbia. After clinical examination, blood samples were taken for microscopic examination, complete blood count (CBC), Canine SNAP 4Dx Test, DNA analyses and sequencing. The main clinical signs included apathy, anorexia, fever, brown/red discoloration of urine, pale mucous membranes, icterus, splenomegaly, and vomiting. The main clinicopathological findings in Babesia infections were a slight to severe thrombocytopenia and a mild to very severe normocytic normochromic anaemia. Microscopic evaluation revealed 58 positive samples with the presence of large and small intraerythrocytic piroplasms in 57 and 1 sample(s), respectively. No co-infections were found using SNAP test. Two Babesia species, B. canis (58/60) and B. gibsoni (2/60), were differentiated by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Species identification was further confirmed by sequencing PCR products of B. gibsoni samples and six randomly selected B. canis samples. All dogs were treated with imidocarb dipropionate (6.6 mg/kg of body weight), given intramuscularly twice at an interval of 14 days. This report presents the first molecular evidence of the occurrence of B. gibsoni and B. canis, confirmed by DNA sequencing, in sick dogs from Serbia.
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Lipej, Z., J. Segalés, I. Toplak, B. Šoštarić, Besi Roić, M. Lojkić, P. Hostnik, et al. "Postweaning multisystemic wasting syndrome (pmws) in pigs in Croatia: Detection and characterisation of porcine circovirus type 2 (pcv2)." Acta Veterinaria Hungarica 53, no. 3 (August 1, 2005): 385–96. http://dx.doi.org/10.1556/avet.53.2005.3.11.
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The objective of this study was to characterise porcine circovirus type 2 (PCV2) from pigs with naturally occurring postweaning multisystemic wasting syndrome (PMWS) in Croatia, and to determine the epizootiological, clinical and pathomorphological features of the disease. During a systematic health monitoring programme conducted in the period from January 2002 to June 2003, PMWS was suspected on eight different pig-producing farms in Croatia. The diagnosis of PMWS met all three key criteria: the presence of compatible clinical signs, the presence of the characteristic microscopic lymphoid lesions, and the detection of PCV2 within the lesions by polymerase chain reaction (PCR) and by in situ hybridisation (ISH). Moreover, PCV2 DNA from swine tissues was extracted and sequenced. The phylogenetic analysis of 4 Croatian PCV2 strains showed close relationship to PCV2 strains isolated in Slovenia, France, the Netherlands, the United Kingdom, China and Hungary. PCV2 was also demonstrated by electron microscopy in the lymph node of an affected animal. This is the first demonstration of PMWS in Croatia based on all scientifically accepted diagnostic criteria.
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Schlegel, PG, R. Aharoni, DE Smilek, LP Fernandez, HO McDevitt, N. Tran, M. Vaysburd, and NJ Chao. "Prevention of graft-versus-host disease by peptides binding to class II major histocompatibility complex molecules." Blood 84, no. 8 (October 15, 1994): 2802–10. http://dx.doi.org/10.1182/blood.v84.8.2802.2802.
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Abstract Graft-versus-host disease across minor histocompatibility barriers was induced in two different models by transplanting allogeneic bone marrow and spleen cells into irradiated H-2-compatible recipient mice. In this report, we show that administration of peptides with high binding affinity for the respective class II major histocompatibility complex molecules after transplantation is capable of preventing the development of graft-versus-host disease in two different murine models. The peptides used were myelin basic protein residues 1 through 11 with alanine at position 4 (Ac 1–11[4A]) for I-Au (A alpha uA beta u), and the antigenic core sequence 323 through 339 of ovalbumin with lysine and methionine extension (KM core) for I-As (A alpha sA beta s). In both systems, the mechanism of prevention was found to be major histocompatibility complex-associated, because nonbinding control peptides did not have any effect. Engraftment of allogeneic bone marrow cells was shown by polymerase chain reaction analysis of DNA polymorphisms in a microsatellite region within the murine interleukin- 5 gene.
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Schlegel, PG, R. Aharoni, DE Smilek, LP Fernandez, HO McDevitt, N. Tran, M. Vaysburd, and NJ Chao. "Prevention of graft-versus-host disease by peptides binding to class II major histocompatibility complex molecules." Blood 84, no. 8 (October 15, 1994): 2802–10. http://dx.doi.org/10.1182/blood.v84.8.2802.bloodjournal8482802.
Full textAbstract:
Graft-versus-host disease across minor histocompatibility barriers was induced in two different models by transplanting allogeneic bone marrow and spleen cells into irradiated H-2-compatible recipient mice. In this report, we show that administration of peptides with high binding affinity for the respective class II major histocompatibility complex molecules after transplantation is capable of preventing the development of graft-versus-host disease in two different murine models. The peptides used were myelin basic protein residues 1 through 11 with alanine at position 4 (Ac 1–11[4A]) for I-Au (A alpha uA beta u), and the antigenic core sequence 323 through 339 of ovalbumin with lysine and methionine extension (KM core) for I-As (A alpha sA beta s). In both systems, the mechanism of prevention was found to be major histocompatibility complex-associated, because nonbinding control peptides did not have any effect. Engraftment of allogeneic bone marrow cells was shown by polymerase chain reaction analysis of DNA polymorphisms in a microsatellite region within the murine interleukin- 5 gene.