Delineating Recombination Frequency Between Methicillin Resistant and Susceptible Homologous Strains and the Relationship with Levels of Antibiotic Resistance Genes in Staphylococcus aureus
Resistance to antibiotics can occur either by mutation or by acquisition of resistance conferring genes via horizontal gene transfer (HGT), of which the latter is considered to be the most important factor in the current pandemic of antimicrobial resistance genes1. Pathogenic Staphylococcus aureus is an adept bacteria that becomes more dangerous with a strain’s procurement of the SCCmec complex. This provides multiple antibiotic resistance features rendering methicillin and most other beta-lactams useless in the fight against this pathogen. Utilizing computational methods, this study investigates methicillin resistant and methicillin susceptible bacteremia to elucidate the relationship between frequencies of recombination events and horizontally acquired antibiotic resistant genes. We hypothesized that methicillin resistant (R) strains will experience homologous recombination more frequent than methicillin susceptible (S) strains and therefore have a positive correlation with the number of antibiotic resistant genes present in the genome. Using a collection of patient blood samples, diagnosed with Staphylococcus aureus bacteremia, and computational biology to infer parameters of recombination. We examined the genomes for antibiotic resistance genes known to be gained through recombination. In clusters that were analyzed, R strains showed that sample diversity of genomes are greater than S strains and that a greater percentage of the genome is from recombination, respectively. Phylogenetic sequence cluster’s (SC) R genomes had more median recombination divergence than the SC S genomes, (SC5R = 0.15 nt, SC5S = 0.025 nt) and (SC8R = 0.048 nt SC8S = 0.044 nt) per locus. These same methicillin resistant SC genomes contained two and a half times and two times as many antibiotic resistant genes than its methicillin susceptible SC genomes, on average, (SC5R = 12, SC5S =5) and (SC8R =10, SC8S = 6).
Stem Cells in A New Era of Cell based Therapies - Creative BiolabsCreative-Biolabs
This document discusses stem cells and their applications in cell-based therapies. It defines stem cells as having endless self-renewal and the ability to differentiate into any cell type. Stem cells are classified by potency and origin, and can be embryonic, adult, or induced. The document outlines how different stem cell types like mesenchymal stem cells can treat diseases by regenerating tissues. Current stem cell therapy research aims to treat cancer, autoimmune diseases, and genetic disorders by using stem cells as drug delivery vehicles or for immunotherapy.
Widespread human T cell receptor beta variable gene polymorphism: implication...Thermo Fisher Scientific
Polymorphism within the TCRB variable gene (TRBV) has been linked to chronic autoimmune diseases such as Type 1 Diabetes, Rheumatoid Arthritis, Psoriatic Arthritis, Multiple Sclerosis and Asthma (1-8), and may also be mechanistically linked to immune mediated adverse events (IMAEs) during immunotherapy (9-11). Here we use the Ion-AmpliSeq™ Immune Repertoire Plus TCRB assay to evaluate TRBV gene polymorphism in a group of 85 Caucasians with melanoma. The assay provides coverage of all three CDR domains to enable detection of TRBV polymorphism. We find evidence of extensive genetic diversity within the TRBV gene, including 15 nonsynonymous variants that are absent from the IMGT database (12). TRBV gene allele typing may provide rich biomarker information for the prediction of IMAEs and chronic autoimmune disease.
Insights into the tumor microenvironment and therapeutic T cell manufacture r...Thermo Fisher Scientific
TCRβ immune repertoire analysis by next-generation sequencing is emerging as a valuable tool for research studies of the tumor microenvironment and potential immune responses to cancer immunotherapy1-4. Here we describe a multiplex PCR-based TCRβ sequencing assay (Ion AmpliSeqTM Immune Repertoire Assay Plus – TCRβ) that leverages Ion AmpliSeq library construction chemistry and the long read capability of the Ion S5 530TM chip to provide coverage of all three CDR domains of the human TCRβ chain. We demonstrate use of the assay to evaluate tumor-infiltrating T cell repertoire features and monitor manufacture of therapeutic T cells.
This document summarizes a research project investigating essential genes and β-lactam resistance mechanisms in Klebsiella pneumoniae. The student conducted transposon mutagenesis and next-generation sequencing to identify 374 essential genes under laboratory conditions, including 50 hypothetical genes with no homologs in other bacteria. Certain transposon insertions were associated with resistance to clinically relevant β-lactams like cefotaxime and meropenem. Specifically, insertions in ramR, bamB and hupA conferred resistance, with hupA not previously known to be involved in β-lactam resistance. The results provide new antibiotic targets and resistance genes warranting further study to address the growing problem of multidrug
The document discusses a clinical trial comparing the monoclonal antibodies obinutuzumab and rituximab for treating diffuse large B-cell lymphoma. Obinutuzumab is a glycoengineered, humanized antibody that clusters the CD20 antigen more effectively than rituximab. In a phase III trial, obinutuzumab plus chemotherapy did not significantly improve progression-free survival over rituximab plus chemotherapy. However, obinutuzumab's glycoengineering may enhance its antibody-dependent cellular cytotoxicity and direct cell death mechanisms relative to rituximab.
NIH3T3 cells are mouse embryonic fibroblast cells isolated in 1962 and used widely in research. They were obtained from desegregated NIH Swiss mouse embryo fibroblasts and form an immortal cell line after 20-30 generations of growth in culture. NIH3T3 cells are adherent, contact inhibited fibroblasts with a murine hypertriploid karyotype that are receptive to viral transformation and commonly used to study calcium signaling, viral titer, and effects on adipocyte differentiation. They are cultured in DMEM with calf serum and split 1:10 every 4-6 days using trypsin.
Investigation of genetic modification in maize and soymilkFrank Soto
This document summarizes a student's experiment to identify genetically modified organisms (GMOs) using DNA extraction and polymerase chain reaction (PCR). The student aimed to identify three genes (35S promoter, NOS terminator, and PSII) in samples of corn, soy milk, papaya, and corn chips. DNA was extracted from the samples and PCR was performed to amplify the target genes. Electrophoresis revealed bands at 203bp and 225bp in the corn sample, indicating it contains the 35S and NOS genes and is genetically modified. The results for soy milk, papaya, and corn chips were inconclusive. The experimenter concluded the maize was genetically modified but the methods need improvement through repetition.
Chimeric Antigen Receptors (paper with corresponding power point)Kevin B Hugins
Gene therapy was first conceptualized to alter debilitating fates of genetic diseases. Gene therapy technology can help introduce new functional DNA to replace mutated genes. The idea first arose in 1972 when Friedmann and Roblin authored a paper, “Gene therapy for human genetic disease?”, demonstrating that exogenous DNA can be taken up by mammalian cells (1). They proposed that the same procedure could be done on humans to correct genetic defects by introducing therapeutic DNA. Currently, genetic modification of T lymphocytes has been the major area of research for treating malignant tumors. This technique seeks to create chimeric antigen receptor (CAR) in T cells by genetically modifying them in vitro and reintroduce them back into blood circulation. The T cells are unique to every patient and the chimeric antigen receptors are unique to the tumor that it is targeting.
Stem Cells in A New Era of Cell based Therapies - Creative BiolabsCreative-Biolabs
This document discusses stem cells and their applications in cell-based therapies. It defines stem cells as having endless self-renewal and the ability to differentiate into any cell type. Stem cells are classified by potency and origin, and can be embryonic, adult, or induced. The document outlines how different stem cell types like mesenchymal stem cells can treat diseases by regenerating tissues. Current stem cell therapy research aims to treat cancer, autoimmune diseases, and genetic disorders by using stem cells as drug delivery vehicles or for immunotherapy.
Widespread human T cell receptor beta variable gene polymorphism: implication...Thermo Fisher Scientific
Polymorphism within the TCRB variable gene (TRBV) has been linked to chronic autoimmune diseases such as Type 1 Diabetes, Rheumatoid Arthritis, Psoriatic Arthritis, Multiple Sclerosis and Asthma (1-8), and may also be mechanistically linked to immune mediated adverse events (IMAEs) during immunotherapy (9-11). Here we use the Ion-AmpliSeq™ Immune Repertoire Plus TCRB assay to evaluate TRBV gene polymorphism in a group of 85 Caucasians with melanoma. The assay provides coverage of all three CDR domains to enable detection of TRBV polymorphism. We find evidence of extensive genetic diversity within the TRBV gene, including 15 nonsynonymous variants that are absent from the IMGT database (12). TRBV gene allele typing may provide rich biomarker information for the prediction of IMAEs and chronic autoimmune disease.
Insights into the tumor microenvironment and therapeutic T cell manufacture r...Thermo Fisher Scientific
TCRβ immune repertoire analysis by next-generation sequencing is emerging as a valuable tool for research studies of the tumor microenvironment and potential immune responses to cancer immunotherapy1-4. Here we describe a multiplex PCR-based TCRβ sequencing assay (Ion AmpliSeqTM Immune Repertoire Assay Plus – TCRβ) that leverages Ion AmpliSeq library construction chemistry and the long read capability of the Ion S5 530TM chip to provide coverage of all three CDR domains of the human TCRβ chain. We demonstrate use of the assay to evaluate tumor-infiltrating T cell repertoire features and monitor manufacture of therapeutic T cells.
This document summarizes a research project investigating essential genes and β-lactam resistance mechanisms in Klebsiella pneumoniae. The student conducted transposon mutagenesis and next-generation sequencing to identify 374 essential genes under laboratory conditions, including 50 hypothetical genes with no homologs in other bacteria. Certain transposon insertions were associated with resistance to clinically relevant β-lactams like cefotaxime and meropenem. Specifically, insertions in ramR, bamB and hupA conferred resistance, with hupA not previously known to be involved in β-lactam resistance. The results provide new antibiotic targets and resistance genes warranting further study to address the growing problem of multidrug
The document discusses a clinical trial comparing the monoclonal antibodies obinutuzumab and rituximab for treating diffuse large B-cell lymphoma. Obinutuzumab is a glycoengineered, humanized antibody that clusters the CD20 antigen more effectively than rituximab. In a phase III trial, obinutuzumab plus chemotherapy did not significantly improve progression-free survival over rituximab plus chemotherapy. However, obinutuzumab's glycoengineering may enhance its antibody-dependent cellular cytotoxicity and direct cell death mechanisms relative to rituximab.
NIH3T3 cells are mouse embryonic fibroblast cells isolated in 1962 and used widely in research. They were obtained from desegregated NIH Swiss mouse embryo fibroblasts and form an immortal cell line after 20-30 generations of growth in culture. NIH3T3 cells are adherent, contact inhibited fibroblasts with a murine hypertriploid karyotype that are receptive to viral transformation and commonly used to study calcium signaling, viral titer, and effects on adipocyte differentiation. They are cultured in DMEM with calf serum and split 1:10 every 4-6 days using trypsin.
Investigation of genetic modification in maize and soymilkFrank Soto
This document summarizes a student's experiment to identify genetically modified organisms (GMOs) using DNA extraction and polymerase chain reaction (PCR). The student aimed to identify three genes (35S promoter, NOS terminator, and PSII) in samples of corn, soy milk, papaya, and corn chips. DNA was extracted from the samples and PCR was performed to amplify the target genes. Electrophoresis revealed bands at 203bp and 225bp in the corn sample, indicating it contains the 35S and NOS genes and is genetically modified. The results for soy milk, papaya, and corn chips were inconclusive. The experimenter concluded the maize was genetically modified but the methods need improvement through repetition.
Chimeric Antigen Receptors (paper with corresponding power point)Kevin B Hugins
Gene therapy was first conceptualized to alter debilitating fates of genetic diseases. Gene therapy technology can help introduce new functional DNA to replace mutated genes. The idea first arose in 1972 when Friedmann and Roblin authored a paper, “Gene therapy for human genetic disease?”, demonstrating that exogenous DNA can be taken up by mammalian cells (1). They proposed that the same procedure could be done on humans to correct genetic defects by introducing therapeutic DNA. Currently, genetic modification of T lymphocytes has been the major area of research for treating malignant tumors. This technique seeks to create chimeric antigen receptor (CAR) in T cells by genetically modifying them in vitro and reintroduce them back into blood circulation. The T cells are unique to every patient and the chimeric antigen receptors are unique to the tumor that it is targeting.
Rituximab added to CHOP chemotherapy for elderly patients with diffuse large B-cell lymphoma significantly improves outcomes including complete response rates, decreases treatment failure and relapse rates, and improves event-free and overall survival compared to CHOP alone, without significantly increasing toxicity. These benefits are sustained over long-term follow up of 10 years. The addition of rituximab improves progression-free and disease-free survival in patients who initially had a complete remission.
Evaluating Ozoralizumab (ATN-203) as a Novel Biotherapeutic Agent for the Tre...jake9606
Poster Presentation which focuses on the background, production and direction of novel Nanobody Technology as a potential biotherapeutic. Technology has been developed by Ablynx (Belgium) and has many benefits particularly in production compared to Antibodies.
Assessment of immunomolecular_expression_and_prognostic_role_of_tlr7_among_pa...dr.Ihsan alsaimary
This document summarizes a research study that assessed the immunomolecular expression and prognostic role of Toll-like receptor 7 (TLR7) in patients with prostatitis. The study included 135 confirmed prostatitis patients and 50 control patients. DNA was extracted from blood samples and amplified using PCR to assess TLR7 expression. Results showed the PCR product for TLR7 was 149bp, indicating a high percentage of TLR7 presence. The study suggests TLR alleles may be associated with risk of prostatitis.
1) The C-terminal carboxylate group of integrin β1 is necessary for its association with the kindlin-2 adapter protein. Affinity measurements indicate this interaction is coordinated by a putative carboxylate-binding motif in the FERM subdomain F3 of kindlin-2.
2) Injection of mRNA encoding mutant integrin β1 cytoplasmic tails that lack the C-terminal carboxylate group perturbed laterality organ development in zebrafish, indicating the carboxylate group is required for physiological functions.
3) The unusual interaction between integrin β1 and kindlin-2 identified here represents a novel protein-protein interaction mode governed mainly by the integrin
This document discusses allele mining, which aims to identify allelic variations in gene banks that could have important traits for crops. It summarizes that identifying these variations could help in tracing the evolution of alleles, developing markers for selection, and providing access to alleles that confer stress resistance, nutrient use efficiency, yield, and quality. The document also mentions that the TILLING method is used for allele mining, which treats seeds with mutagens, analyzes pooled DNA samples, and identifies variations using Cel I enzyme cleavage and gel electrophoresis.
The document discusses a new technology for analyzing the three-dimensional conformational structure of monoclonal antibodies (mAbs). It describes how the technology was developed using an antibody array to measure epitope exposure on mAbs. Case studies show the technology can detect minor conformational differences between biosimilar and reference mAbs, and distinguish between mAbs that were clinical successes versus failures. The technology provides a unique conformational signature for each mAb.
Nuhu et al_Poster NAPA2016 correction and observationNuhu Tanko
The study determined the prevalence and genetic profiles of ESBL-producing uropathogens among members of the Enterobacteriaceae family at Specialist Hospital Sokoto, Nigeria. A total of 64 Gram-negative uropathogens were isolated from 365 urine samples, with E. coli and Salmonella arizonae being most prevalent. The isolates showed high resistance to cotrimoxazole, nalidixic acid, ciprofloxacin and norfloxacin. 64.1% of isolates were multidrug resistant. ESBL production was detected in 23.4% of isolates. PCR analysis showed 73.3% of ESBL producers contained the blaCTX-M gene and 26.7
October 8, 2015
From the event "Synthetic Biology: Science, Policy, and Ethics."
Sponsored by the Petrie-Flom Center for Health Law Policy, Biotechnology, and Bioethics at Harvard Law School.
For more information, visit our website at http://petrieflom.law.harvard.edu/events/details/synthetic-biology.
The document describes experiments conducted to induce apoptosis in non-invasive retinoblastoma cell lines using tissue-specific promoter guided suicide gene transfection. Key objectives included propagating the suicide gene plasmid, transfecting it into a non-invasive retinoblastoma cell line, validating transfection efficiency through various assays, and studying the apoptotic effects induced. Transfection was confirmed at the mRNA and protein level. Assays showed over 60% of transfected cells undergoing early apoptosis and 35% undergoing late apoptosis, demonstrating the suicide gene successfully induced apoptosis in the targeted retinoblastoma cells.
STR DNA profiling is now a powerful, inexpensive tool that can generate unique DNA signatures that can be used to authenticate cell lines and detect contamination of more than one cell type. This presentation will talk about why scientists need cell authentication, what is STR profile and STR profile workflow from Creative Bioarray.
Candidemia in HIV-positive patients in Dschang District Hospital (West Region...Claude Nangwat
Candidemia has been identified as a public health problem in HIV-infected patients. The evaluation of CD4 count, transaminases and blood glucose, are being used as a means to monitor the health of HIV-infected patients, without excluding the diagnosis of candidemia and other opportunistic infections. In order to contribute in improving the care of HIV-infected patients attending Dschang District Hospital and later on, in other hospitals in Cameroon, we conducted from June to September 2014 a cross-sectional study, with general objective; to determine the association between candidemia and selected biochemical and haematological parameter changes in HIV-infected patients, as a possible indicator in monitoring HIV disease progression.
To do this, blood samples were collected from HIV-infected patients assigned to the UPEC of Dschang District Hospital for follow up, and haemogram report, CD4 counts, ALAT level, ASAT level, and glucose level in blood were evaluated by cytometric and spectrophotometric assays. Candida species were isolated from some blood samples, and then identified using CHROMagar Candida culture medium. The broth microdilution method was afterwards used to test the susceptibility of the fungal isolates vis-a-vis three conventional antifungal agents.
Mycological analysis of blood samples showed that eight (08) patients had candidemia, a prevalence of 6.11%. Eight (08) isolates were obtained from these eight (08) candidemic HIV-infected patients; this consisted of 4(50%) Candida albicans, 3(37.5%) Candida parapsilosis and 1(12.5%) Candida glabrata. All these isolates were resistant (MICs ranged from 2 to >256 µg/mL) to the antifungals used, that is, ketoconazole, amphotericin B and nystatin.
A significant correlation was found between candidemia and white blood cell count, with a correlation coefficient of r = 0.240 (p < 0.05). Based on the results obtained, the systematic diagnosis of candidemia should be performed in patients infected with HIV in Cameroon in order to improve on their care.
Key words: Candidemia, HIV, biochemical parameters, hematological parameters, Antifungals activities.
The document discusses hypoxia-inducible factor (HIF) activation by hypoxia. It describes how hypoxia leads to the activation of HIF, which upregulates genes like VEGF and EPO. It discusses the role of the von Hippel-Lindau tumor suppressor protein and prolyl hydroxylases in the HIF pathway. The document also summarizes VEGF structure and signaling, how it promotes angiogenesis, and the role of differential splicing in producing pro-angiogenic and anti-angiogenic isoforms. Finally, it discusses how anti-angiogenic therapies target tumor vasculature and their limitations.
The researchers developed a novel non-viral single doxycycline inducible system that can efficiently regulate gene expression in stem cells both in vitro and in vivo. They generated stable clonal cell lines from mouse neuroblastoma cells (N2a) and human embryonic stem cells (hNSC-ESC) that responded to doxycycline by eliminating GFP expression within 3-8 days. GFP expression was restored 9-10 days after doxycycline removal. When transplanted hNSC-ESCs labeled with quantum dots were subjected to doxycycline in mice, GFP expression was reduced by 61-56% compared to controls. This suggests the system can control gene expression in stem cells without using viral vectors
Paternal radiation exposure led to increased expression of miR-29a and miR-29b in the male germline. This upregulation of miR-29 caused decreased expression of the de novo methyltransferase DNMT3a and profound hypomethylation of LINE1 and SINE B2 transposable elements in the germline. Hypomethylation of these transposable elements was also found in the thymus tissue of offspring conceived from exposed fathers and was associated with decreased expression of the LSH chromatin remodeling protein. Furthermore, miR-468 expression was significantly upregulated in the offspring thymus and directly targeted LSH, contributing to its decreased expression and hypomethylation of transposable
Effect of stemregenin1 and sb431542 small molecules on ex vivo expansion of u...Liberty University (LU)
This study investigated the ex vivo expansion of umbilical cord blood hematopoietic stem cells (HSCs) using three-dimensional (3D) polyethersulfone (PES) nanofiber scaffolds and two small molecules, Stemregenin1 (SR1) and SB431542. Isolated HSCs were cultured in 3D PES scaffolds or 2D conditions with SR1 and/or SB431542 and growth factors. On days 5 and 10, the cells were analyzed for marker expression, colony formation ability, and gene expression of stemness genes. SR1 increased HSC expansion while SB431542 induced more lineage-committed cells. SR1 upregulated stemness genes and
Humanisation of antibodies & immunotherapeutics in clinical practice Aaqib Naseer
This document discusses humanization of monoclonal antibodies and immunotherapeutics used in clinical practice. It describes the techniques used to humanize antibodies, including CDR grafting, phage display, and transgenic animals. It then discusses various immunotherapeutics used clinically such as monoclonal antibodies, cytokines, cancer vaccines, and cell-based therapies. Monoclonal antibodies target specific antigens on cancer cells and can be naked or conjugated. Checkpoint inhibitors like anti-CTLA-4 and anti-PD-1 antibodies work by releasing brakes on the immune system. Cytokines such as interferons and interleukins modulate immune responses. Cancer vaccines aim to stimulate immunity against tumor antigens.
Functional profile of the pre- to post-mortem transition in bloodJoaquin Dopazo
This document summarizes research analyzing gene expression data from pre-mortem and post-mortem blood samples to characterize the functional changes that occur after death. Five main functional responses were identified, including immune response deactivation, cell division arrest, and metabolism deactivation. Specific signaling pathways involved in processes like response to hypoxia, hemostasis, and apoptosis were found to change activity levels. Computational models were used to quantify changes in signaling activity over cell functions, providing a more informative analysis than examining single genes. This approach could help understand post-mortem tissue preservation for transplantation.
This document discusses allele mining as a technique for improving crops. It defines allele mining as identifying allelic variation within genetic resources collections to find superior alleles. There are two main approaches - TILLING based allele mining which uses mutagenized populations and enzymatic cleavage to find mutations, and sequencing-based allele mining which uses PCR and sequencing to identify natural variation. Both have benefits and limitations. Applications of allele mining include finding alleles for resistance, abiotic stress tolerance, and improved yield and quality. Overall, allele mining is a promising approach for utilizing genetic resources to discover variants that can aid crop breeding.
This document summarizes a study that used multilocus sequence typing (MLST) to analyze the evolutionary history and origins of major methicillin-resistant Staphylococcus aureus (MRSA) clones. The researchers analyzed 912 MRSA and methicillin-susceptible S. aureus isolates from 20 countries. They identified 11 major MRSA clones within 5 groups of related genotypes. Analysis of the methicillin resistance genes and the most parsimonious patterns of descent identified the likely ancestral genotype and MSSA progenitor of each major MRSA clone. Major MRSA clones have repeatedly emerged from successful epidemic MSSA strains through acquisition of the methicillin resistance gene. Isolates with decreased susceptibility to vancomycin,
The Molecular Diagnostics Laboratory processes around 20,000 specimens annually for various testing categories including infectious diseases, hematological malignancies, solid tumor malignancies, and inherited disorders. Next generation sequencing is proposed as a solution to provide more comprehensive genetic testing by simultaneously evaluating variations in multiple genes from a single sample. While next generation sequencing offers advantages over traditional testing methods, there are also technical and clinical challenges to address in implementation, including optimizing detection of structural variations and interpretation of variants with unclear clinical significance.
Rituximab added to CHOP chemotherapy for elderly patients with diffuse large B-cell lymphoma significantly improves outcomes including complete response rates, decreases treatment failure and relapse rates, and improves event-free and overall survival compared to CHOP alone, without significantly increasing toxicity. These benefits are sustained over long-term follow up of 10 years. The addition of rituximab improves progression-free and disease-free survival in patients who initially had a complete remission.
Evaluating Ozoralizumab (ATN-203) as a Novel Biotherapeutic Agent for the Tre...jake9606
Poster Presentation which focuses on the background, production and direction of novel Nanobody Technology as a potential biotherapeutic. Technology has been developed by Ablynx (Belgium) and has many benefits particularly in production compared to Antibodies.
Assessment of immunomolecular_expression_and_prognostic_role_of_tlr7_among_pa...dr.Ihsan alsaimary
This document summarizes a research study that assessed the immunomolecular expression and prognostic role of Toll-like receptor 7 (TLR7) in patients with prostatitis. The study included 135 confirmed prostatitis patients and 50 control patients. DNA was extracted from blood samples and amplified using PCR to assess TLR7 expression. Results showed the PCR product for TLR7 was 149bp, indicating a high percentage of TLR7 presence. The study suggests TLR alleles may be associated with risk of prostatitis.
1) The C-terminal carboxylate group of integrin β1 is necessary for its association with the kindlin-2 adapter protein. Affinity measurements indicate this interaction is coordinated by a putative carboxylate-binding motif in the FERM subdomain F3 of kindlin-2.
2) Injection of mRNA encoding mutant integrin β1 cytoplasmic tails that lack the C-terminal carboxylate group perturbed laterality organ development in zebrafish, indicating the carboxylate group is required for physiological functions.
3) The unusual interaction between integrin β1 and kindlin-2 identified here represents a novel protein-protein interaction mode governed mainly by the integrin
This document discusses allele mining, which aims to identify allelic variations in gene banks that could have important traits for crops. It summarizes that identifying these variations could help in tracing the evolution of alleles, developing markers for selection, and providing access to alleles that confer stress resistance, nutrient use efficiency, yield, and quality. The document also mentions that the TILLING method is used for allele mining, which treats seeds with mutagens, analyzes pooled DNA samples, and identifies variations using Cel I enzyme cleavage and gel electrophoresis.
The document discusses a new technology for analyzing the three-dimensional conformational structure of monoclonal antibodies (mAbs). It describes how the technology was developed using an antibody array to measure epitope exposure on mAbs. Case studies show the technology can detect minor conformational differences between biosimilar and reference mAbs, and distinguish between mAbs that were clinical successes versus failures. The technology provides a unique conformational signature for each mAb.
Nuhu et al_Poster NAPA2016 correction and observationNuhu Tanko
The study determined the prevalence and genetic profiles of ESBL-producing uropathogens among members of the Enterobacteriaceae family at Specialist Hospital Sokoto, Nigeria. A total of 64 Gram-negative uropathogens were isolated from 365 urine samples, with E. coli and Salmonella arizonae being most prevalent. The isolates showed high resistance to cotrimoxazole, nalidixic acid, ciprofloxacin and norfloxacin. 64.1% of isolates were multidrug resistant. ESBL production was detected in 23.4% of isolates. PCR analysis showed 73.3% of ESBL producers contained the blaCTX-M gene and 26.7
October 8, 2015
From the event "Synthetic Biology: Science, Policy, and Ethics."
Sponsored by the Petrie-Flom Center for Health Law Policy, Biotechnology, and Bioethics at Harvard Law School.
For more information, visit our website at http://petrieflom.law.harvard.edu/events/details/synthetic-biology.
The document describes experiments conducted to induce apoptosis in non-invasive retinoblastoma cell lines using tissue-specific promoter guided suicide gene transfection. Key objectives included propagating the suicide gene plasmid, transfecting it into a non-invasive retinoblastoma cell line, validating transfection efficiency through various assays, and studying the apoptotic effects induced. Transfection was confirmed at the mRNA and protein level. Assays showed over 60% of transfected cells undergoing early apoptosis and 35% undergoing late apoptosis, demonstrating the suicide gene successfully induced apoptosis in the targeted retinoblastoma cells.
STR DNA profiling is now a powerful, inexpensive tool that can generate unique DNA signatures that can be used to authenticate cell lines and detect contamination of more than one cell type. This presentation will talk about why scientists need cell authentication, what is STR profile and STR profile workflow from Creative Bioarray.
Candidemia in HIV-positive patients in Dschang District Hospital (West Region...Claude Nangwat
Candidemia has been identified as a public health problem in HIV-infected patients. The evaluation of CD4 count, transaminases and blood glucose, are being used as a means to monitor the health of HIV-infected patients, without excluding the diagnosis of candidemia and other opportunistic infections. In order to contribute in improving the care of HIV-infected patients attending Dschang District Hospital and later on, in other hospitals in Cameroon, we conducted from June to September 2014 a cross-sectional study, with general objective; to determine the association between candidemia and selected biochemical and haematological parameter changes in HIV-infected patients, as a possible indicator in monitoring HIV disease progression.
To do this, blood samples were collected from HIV-infected patients assigned to the UPEC of Dschang District Hospital for follow up, and haemogram report, CD4 counts, ALAT level, ASAT level, and glucose level in blood were evaluated by cytometric and spectrophotometric assays. Candida species were isolated from some blood samples, and then identified using CHROMagar Candida culture medium. The broth microdilution method was afterwards used to test the susceptibility of the fungal isolates vis-a-vis three conventional antifungal agents.
Mycological analysis of blood samples showed that eight (08) patients had candidemia, a prevalence of 6.11%. Eight (08) isolates were obtained from these eight (08) candidemic HIV-infected patients; this consisted of 4(50%) Candida albicans, 3(37.5%) Candida parapsilosis and 1(12.5%) Candida glabrata. All these isolates were resistant (MICs ranged from 2 to >256 µg/mL) to the antifungals used, that is, ketoconazole, amphotericin B and nystatin.
A significant correlation was found between candidemia and white blood cell count, with a correlation coefficient of r = 0.240 (p < 0.05). Based on the results obtained, the systematic diagnosis of candidemia should be performed in patients infected with HIV in Cameroon in order to improve on their care.
Key words: Candidemia, HIV, biochemical parameters, hematological parameters, Antifungals activities.
The document discusses hypoxia-inducible factor (HIF) activation by hypoxia. It describes how hypoxia leads to the activation of HIF, which upregulates genes like VEGF and EPO. It discusses the role of the von Hippel-Lindau tumor suppressor protein and prolyl hydroxylases in the HIF pathway. The document also summarizes VEGF structure and signaling, how it promotes angiogenesis, and the role of differential splicing in producing pro-angiogenic and anti-angiogenic isoforms. Finally, it discusses how anti-angiogenic therapies target tumor vasculature and their limitations.
The researchers developed a novel non-viral single doxycycline inducible system that can efficiently regulate gene expression in stem cells both in vitro and in vivo. They generated stable clonal cell lines from mouse neuroblastoma cells (N2a) and human embryonic stem cells (hNSC-ESC) that responded to doxycycline by eliminating GFP expression within 3-8 days. GFP expression was restored 9-10 days after doxycycline removal. When transplanted hNSC-ESCs labeled with quantum dots were subjected to doxycycline in mice, GFP expression was reduced by 61-56% compared to controls. This suggests the system can control gene expression in stem cells without using viral vectors
Paternal radiation exposure led to increased expression of miR-29a and miR-29b in the male germline. This upregulation of miR-29 caused decreased expression of the de novo methyltransferase DNMT3a and profound hypomethylation of LINE1 and SINE B2 transposable elements in the germline. Hypomethylation of these transposable elements was also found in the thymus tissue of offspring conceived from exposed fathers and was associated with decreased expression of the LSH chromatin remodeling protein. Furthermore, miR-468 expression was significantly upregulated in the offspring thymus and directly targeted LSH, contributing to its decreased expression and hypomethylation of transposable
Effect of stemregenin1 and sb431542 small molecules on ex vivo expansion of u...Liberty University (LU)
This study investigated the ex vivo expansion of umbilical cord blood hematopoietic stem cells (HSCs) using three-dimensional (3D) polyethersulfone (PES) nanofiber scaffolds and two small molecules, Stemregenin1 (SR1) and SB431542. Isolated HSCs were cultured in 3D PES scaffolds or 2D conditions with SR1 and/or SB431542 and growth factors. On days 5 and 10, the cells were analyzed for marker expression, colony formation ability, and gene expression of stemness genes. SR1 increased HSC expansion while SB431542 induced more lineage-committed cells. SR1 upregulated stemness genes and
Humanisation of antibodies & immunotherapeutics in clinical practice Aaqib Naseer
This document discusses humanization of monoclonal antibodies and immunotherapeutics used in clinical practice. It describes the techniques used to humanize antibodies, including CDR grafting, phage display, and transgenic animals. It then discusses various immunotherapeutics used clinically such as monoclonal antibodies, cytokines, cancer vaccines, and cell-based therapies. Monoclonal antibodies target specific antigens on cancer cells and can be naked or conjugated. Checkpoint inhibitors like anti-CTLA-4 and anti-PD-1 antibodies work by releasing brakes on the immune system. Cytokines such as interferons and interleukins modulate immune responses. Cancer vaccines aim to stimulate immunity against tumor antigens.
Functional profile of the pre- to post-mortem transition in bloodJoaquin Dopazo
This document summarizes research analyzing gene expression data from pre-mortem and post-mortem blood samples to characterize the functional changes that occur after death. Five main functional responses were identified, including immune response deactivation, cell division arrest, and metabolism deactivation. Specific signaling pathways involved in processes like response to hypoxia, hemostasis, and apoptosis were found to change activity levels. Computational models were used to quantify changes in signaling activity over cell functions, providing a more informative analysis than examining single genes. This approach could help understand post-mortem tissue preservation for transplantation.
This document discusses allele mining as a technique for improving crops. It defines allele mining as identifying allelic variation within genetic resources collections to find superior alleles. There are two main approaches - TILLING based allele mining which uses mutagenized populations and enzymatic cleavage to find mutations, and sequencing-based allele mining which uses PCR and sequencing to identify natural variation. Both have benefits and limitations. Applications of allele mining include finding alleles for resistance, abiotic stress tolerance, and improved yield and quality. Overall, allele mining is a promising approach for utilizing genetic resources to discover variants that can aid crop breeding.
Similar to Delineating Recombination Frequency Between Methicillin Resistant and Susceptible Homologous Strains and the Relationship with Levels of Antibiotic Resistance Genes in Staphylococcus aureus
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Delineating Recombination Frequency Between Methicillin Resistant and Susceptible Homologous Strains and the Relationship with Levels of Antibiotic Resistance Genes in Staphylococcus aureus
1. Delineating Recombination Frequency Between Methicillin
Resistant and Susceptible Homologous Strains and the
Relationship with Levels of Antibiotic Resistance Genes in
Staphylococcus aureus
By
J. R. Matthews
July 27, 2021
A Thesis
Submitted to the University at Albany, State University of New York
In Partial Fulfillment of
the Requirements for the Degree of
Master of Science
3. INTRODUCTION
• Transfer of DNA
• Three ways to obtain foreign genes.
• Homologous Recombination
• Relationship Among Recombination
& Antibiotic Resistance
• Two concepts
• SCCmec Complex
• Rationale
• Hypothesis
• Goal of this Study
• Two Questions to be Answered
4. • Many bacterial species
have the unique ability to
acquire DNA from their
neighbors, regardless of
whether they are related
or not.
• HGT (Horizontal Gene
Transfer)
• The processes that
enable bacteria to
directly obtain foreign
DNA are:
Soucy et al. 2015
Conjugation:
transfer of DNA
through direct cell-
to-cell contact
Transduction:
phages carry DNA
from cell to cell
Transformation:
uptake of naked
DNA
5. Homologous recombination: Genetic material that is exchanged
between two strands of DNA containing stretches of similar
base sequences.
Incoming DNA
Host genome
Recombinant host
genome
Gene A
Gene A
Gene A
Gene C
Gene C
Gene C
Gene B’
Gene B
Gene B’
Incoming DNA
Recombinant
host genome
Host genome
6. Homologous recombination
• Results in a mosaic chromosome consisting of DNA segments from
different sources
• Recombination does not occur similarly among strains of the same
species.
• Some strains are hyper-recombinants they are able to acquire foreign DNA
more often than others
• This process has the potential to provide different allelic variants
which may introduce new phenotypes, such as virulence or antibiotic
resistance, more rapidly than by mutation alone.
7. 2 scenarios to explain the association between
antibiotic resistance and recombination:
1. Different strains have different resistance levels because they are
able to acquire resistance genes at different recombination rates
(Hanage et al. 2009)
2. The distribution of resistance genes among strains is the result of
these genes conferring different fitness benefits on different strains
and not because of recombination (Lehtinen et al. 2020)
• The two scenarios have been investigated in the bacterial pathogen
Streptococcus pneumoniae.
8. Staphylococcal Chromosomal Cassette (SCCmec)
• Mobile genetic element (MGE) that carries the mecA gene
• confers resistance to most beta-lactams, including methicillin
Lakhundi and Zhang. 2018 Clin Microbiol Rev
mec complex ccr complex
9. Rationale
• Methicillin resistance is acquired through a mobile genetic element
(SCCmec) that can move from cell to cell.
• Major event in the evolution of S. aureus.
• Independent acquisition by several multi-drug resistant strains in 1960’s
(penicillin, streptomycin, tetracycline, and erythromycin) (Crisostomo, M. I. et al., 2001)
• When SCCmec is acquired, other DNA segments may also be
integrated into the recipient chromosome at the same time.
• (Hypothesis) It is likely then, that methicillin resistant strains will have
more recombined DNA.
• Therefore, they will have greater number of antibiotic resistant genes.
10. The aim of my study
• Analyze the frequency of recombination and mutation in MRSA and
MSSA isolates.
• Does MRSA genomes experience recombination more frequent than MSSA
genomes.
• Analyze the distribution of antibiotic resistance (ABR) genes within
MRSA and MSSA isolates.
• Does MRSA genomes posses a greater amount of ABR genes than MSSA
genomes.
12. • There is 323 Staphylococcus aureus genomes obtained from bacteremia
infected patients at Dartmouth-Hitchcock Med Center.
• Isolates were collected from 2010-2018.
• They consists of a mix of methicillin resistant (MRSA) and methicillin
susceptible (MSSA) strains.
• Selected the largest phylogenetic clusters, two consisting of a mix of
MRSA and MSSA strains, and one solely made up of MSSA strains.
Data Set to be Used
13. • Phylogenetic tree showing the
phenotypic results of MRSA
and MSSA (outer ring).
• Multi-locus sequence typing
(MLST) shows that sequence
types (ST) (inner ring) were
mixed with each cluster having
at least two dominant STs.
• Eight clades considered closely
related genomes were found
(indicated by the colors on the
tree branches).
14. • Divided the dataset into
sequence clusters (SC1-8),
which consists of closely
related genomes.
• MRSA strains are mostly
found in SC5 and SC8.
• The analysis is predominantly
on these two dominant SCs.
• SC3 has only MSSA strains
and used for additional
comparative analysis for ABR
gene levels.
15. Pan Genome
ML phylogenetic tree used only those genes that are common to all
strains (called core genes).
Shared Gene Percentage
Total pan
genome #
SC3 pan
genome #
SC5 pan
genome #
SC8 pan
genome #
(99% <= strains <= 100%) Core genes 1692Core genes 1971Core genes 1909Core genes 2012
(95% <= strains < 99%) Soft core genes 151Soft core genes 121
Soft core
genes 297Soft core genes 236
(15% <= strains < 95%) Shell genes 1381Shell genes 791Shell genes 594Shell genes 525
(0% <= strains < 15%) Cloud genes 5180Cloud genes 1367Cloud genes 2279Cloud genes 1710
(0% <= strains <= 100%) Total genes 8404Total genes 4250Total genes 5079Total genes 4483
16. Breakdown of Isolates
• Measure & compare different recombination parameters between:
• MRSA (n = 50 genomes) and MSSA (n = 39 genomes) in SC5
• MRSA (n = 52 genomes) and MSSA (n = 24 genomes) in SC8
• Determine & analyze the level of antimicrobial resistance between:
• MRSA (n = 50 genomes) and MSSA (n = 39 genomes) in SC5
• MRSA (n = 52 genomes) and MSSA (n = 24 genomes) in SC8
• MRSA (n = 0 genomes) and MSSA (n = 41 genomes) in SC3
18. MRSA strains have significantly higher sequence diversity
than MSSA in both clusters. (p < 0.001, SC5 & SC8)
MRSA in SC5 MSSA in SC5 MRSA in SC8 MSSA in SC8
*** ***
0.004
0.0025
0.0025 0.0011
19. Mean number of mutations per locus differed significantly
between MRSA and MSSA in both clusters. (p < 0.001, SC5 & SC8)
MRSA in SC5 MSSA in SC5 MRSA in SC8 MSSA in SC8
*** ***
0.074
0.062
0.057
0.042
20. *** ***
Significantly greater mean number of recombination events
per locus occurs in MRSA than in MSSA. (p < 0.001, SC5 & SC8)
MRSA in SC5 MSSA in SC5 MRSA in SC8 MSSA in SC8
0.043
0.049
0.136
0.031
21. *** ***
Contrasting results in the relative rate of recombination (ϕ/θ) to
mutation between SC5 & SC8. (p < 0.001, SC5 & SC8)
MRSA in SC5 MSSA in SC5 MRSA in SC8 MSSA in SC8
1.84
0.492
0.042
0.043
22. *** ***
The fraction of the genome acquired through recombination was
significantly larger in MRSA than in MSSA. (p < 0.001, SC5 & SC8)
MRSA in SC5 MSSA in SC5 MRSA in SC8 MSSA in SC8
4.5% of sites in any one
genome were consequent
of recombination
2.6% of sites in any one
genome were derived
from recombination
4.3% of sites in any one
genome were derived
from recombination
5.8% of sites in any one
genome were consequent
of recombination
23. Summary Results of Recombination
• Recombination provides a wider range of genomic sample diversity in MRSA than in
MSSA in both SC5 and SC8.
• Mutational divergence is very pronounced in methicillin resistant genomes compared
to methicillin susceptible genomes.
• More mutations take place per locus in MRSA genomes than that of MSSA genomes
within its associated SC.
• Greater occurrences of recombinational divergence in MRSA genomes than its MSSA
counterpart.
• Recombination events occur significantly more within MRSA genomes than MSSA
genomes within both SC5 and SC8.
• Relative rate of recombination to mutation (ratio) displays opposing results.
• SC5 demonstrated that MRSA genomes experience greater recombination events
than mutation events significantly more then MSSA genomes.
• SC8 shows contrasting results that the MRSA genomes have experienced less
recombination to mutation events since divergence of LCA than MSSA genomes.
24. Antibiotic Resistance
• Comparison of MRSA vs.
MSSA isolates within SCs
• MRSA vs. MRSA isolates
between SCs
• MSSA vs. MSSA isolates
between SCs
• SCs average & maximum
antibiotic resistance genes
• Each resistant & susceptible
group
• Genes Present
• Similar & different ABR genes
• Summary
26. SC3 (MSSA) has a mean of 5.85 ABR genes per genome and 1 isolate has 8 ABR genes, the
most of any within SC3.
SC5 (MRSA) has a mean of more than 10.88 ABR genes per genome and 1 isolate has a total
of 18 associated ABR genes in SC5 resistant strains.
SC5 (MSSA) possess a mean of 4.95 ABR genes per genome and 2 isolates possess 12 ABR
genes, the maximum within SC5 susceptible strains.
SC8 (MRSA) has a mean of 9.62 ABR genes per genome and a total of 5 isolates have a
maximum of 12 ABR genes in SC8 MRSA.
SC8 (MSSA) has a mean of 6.29 ABR genes per genome, with only one isolate having a
maximum of 11 ABR genes in SC8 MSSA.
Amount of ABR Genes
27. Genes Present
fosB – protein coding gene which makes
a fosfomycin-inactivating enzyme.
• Catalyzes l-cys or BSH (bacillithiol) to the
antibiotic by nucleophilic addition.
• All isolates
tet(31) – codes for an energy-
dependent efflux protein.
• A membrane assoc. protein which
exports tetracycline out of the bacterial
cell, thus protecting the ribosomes
• All isolates
bleO – codes for a high affinity
bleomycin binding protein.
• Potentially conferred in niche
environments.
• (56% SC5 MRSA & 5% MSSA) ( 0% all
SC8 isolates).
tet(K) – another gene which codes an
efflux protein.
• SC5 MSSA 3%, MRSA 0%
• SC8 MSSA 4%, MRSA 0%
28. Summary of Antibiotic Resistance results
SC5 & SC8 showed that MRSA genomes have, on average, a significantly greater
number of ABR genes compared to MSSA genomes.
• There was more than a 2-fold difference in the number of ABR genes present in MRSA
isolates than MSSA isolates in SC5.
• More than a 1.5-fold difference exists between the number of ABR genes in resistant
strains than in susceptible strains in SC8.
There is a slight difference among MSSA genomes between SC3 & SC5 (p = 0.046),
reject null hypothesis if p-value < 0.05.
• MSSA SC5 v. SC8 no significant difference, null accepted.
• MSSA SC3 v. SC8 no significant difference, null accepted.
SC5 MRSA and SC8 MRSA have no difference of significance when comparing the
amount of ABR genes.
Though some ABR genes in MSSA strains only, majority of genes present in only
MRSA strains or in both MRSA & MSSA, w/more MRSA strains possessing the genes.
29. Conclusions
• The characteristics of homologous recombination has been defined for two
major lineages of S. aureus.
• Genomes that were resistant to methicillin had more point mutations when
compared to the susceptible genomes within its cluster.
• Both SC5 and SC8 had more instances of recombination in methicillin resistant
isolates than the susceptible counterparts within the clusters.
• MRSA strains have a larger percentage of its genome that consists of foreign
DNA than that of MSSA strains from recombination.
• Results demonstrate that MRSA strains possessed greater numbers of HGT
antibiotic resistance genes, which may in part be due to those strains
experiencing more recombination events than the MSSA strains.
30. • The mecA gene being acquired by recombination is not the determining factor
for having more ABR genes.
• But it has been shown in other papers to be a co-contributor for Staphylococcus
aureus obtaining a particular antibiotic resistant designation (beta-lactum tolerant).
• Incorporation of the mecA gene typically is by acquisition of the SCCmec
complex, which potentially brings numerous resistance determinants in a single
instance.
• This could be ABR genes, virulence factors, associated transcription proteins, and
mechanisms pertaining to the proper function of the related determinants.
• The presence of mecA seems to positively correlate with greater recombination
frequencies
• Recombinational divergence is continuously greater within strains that are
methicillin resistant.
Factoring in the SCCmec
31. Outstanding Questions
• What factors can explain the contrasting results in the relative rates of recombination
to mutation:
• Environment which SC8 MRSA or MSSA infection was acquired?
• Length of time either strain type infection was present?
• Are the genes that frequently recombine similar between SC5 and SC8?
• What percentage of core genes overlap? Accessory genes?
• Are the genes that frequently recombine similar between MSSA and MRSA?
• How many genes are shared in the pan-genome of resistant & susceptible strains?
• Does the amount of virulence factors present in isolates follow the same trend as
antibiotic gene enumeration?
• With over 64 known virulent factors in S. aureus, does MRSA posses more than
MSSA?