This study investigated the role of the DNA damage checkpoint kinase Chk1 in sister chromatid cohesion (SCC) and genome stability in Saccharomyces cerevisiae. The results showed that unlike SCC mutants, loss of CHK1 did not increase spontaneous or damage-induced allelic recombination or aneuploidy. Exposure of G2-arrested cells to ionizing radiation also did not increase allelic recombination or reduce survival in chk1 mutant cells as seen in SCC mutants. This suggests that Chk1 has a redundant role in controlling damage-induced SCC or that damage-induced SCC is redundant for maintaining genome stability in yeast.
Institute of Learning in Retirement - Miami University (Ohio)Andor Kiss
CRISPR/Cas9 is a new genetic engineering technique that uses a bacterial immune system to edit DNA. It involves using an RNA guide sequence and Cas9 protein to cut DNA at a targeted location. This allows genes to be knocked out or altered. CRISPR has many advantages over older techniques and has greatly improved efficiency of genetic engineering. However, it also raises ethical concerns about its applications, including the first reported use in human embryos.
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
This document summarizes the discovery of duplicated VegfA and KDR receptor genes in zebrafish that mediate vascular development. Specifically:
- The researchers identified a duplicated zebrafish VegfA gene (VegfAb) that encodes 171- and 210-amino acid isoforms not found in the single VegfA gene.
- They also found a duplicated KDR receptor gene (Kdrb) that encodes a receptor similar to mammalian KDR.
- Knockdown experiments in zebrafish showed that both VegfAb and the duplicated KDR receptor genes play important roles in vascular development.
- Further experiments demonstrated that the VegfAb isoforms are poorly secreted compared to VegfA isoforms
Refining Gene Ontology to Include Terms and Relationships Relevant to exRNA C...exrna
The document discusses efforts to standardize terminology related to extracellular vesicles (EVs) and exRNA-containing particles through developing new terms and relationships within the Gene Ontology (GO). It describes work with domain experts to propose additions to GO's Cellular Component branch to accurately describe and classify EVs and exRNA communications. The goal is to strike a balance between overgeneralizing and being too specific when defining terms in this emerging research area.
1) The study analyzed the effects of Csk knockouts on development of the initial segment of the mouse epididymis. Csk knockout was expected to promote cell proliferation and differentiation through increased ERK pathway activity due to lack of inhibition of SRC kinases.
2) A tissue-specific Csk conditional knockout mouse model was generated using Cre/lox recombination. Genotyping identified one mouse with the desired genotype.
3) Preliminary results found increased vasculogenesis in the initial segment of Csk knockout mice, suggesting effects on differentiation through the ERK pathway. Immunofluorescence found decreased activity of phospho-SRC in knockouts while other markers were similar to controls.
This document summarizes Mohd Kyum's PhD research on using CRISPR/Cas9 genome editing to induce haploidy in rice. The objectives were to generate knockout mutants of the OsMATL gene, which is responsible for haploid induction in maize, using CRISPR/Cas9 RNP complexes delivered via particle bombardment. Embryos of the rice variety Purple Line were bombarded with three different sgRNAs targeting OsMATL. Regenerated T0 plants were analyzed for mutations in the target gene using techniques like T7E1 assay, PCR-RFLP, and sequencing. Putative mutant plants showed a transformation efficiency of around 12.5% and are being further analyzed to characterize mutations induced
The document summarizes a study that used live cell imaging to visualize hepatitis C virus (HCV) entry into human liver cells. Key findings include:
1) HCV was labeled with fluorescent membrane dyes and found to move along actin stress fibers in an actin-dependent manner after internalization, suggesting clathrin-mediated endocytosis.
2) Inhibition of clathrin machinery or related host factors blocked HCV entry. Immunofluorescence showed HCV co-localizing with clathrin and early endosomes.
3) While tight junction proteins were thought to mediate HCV entry, live imaging found internalization occurring outside of tight junctions, indicating their role may be overstated.
4
This study investigated the role of the DNA damage checkpoint kinase Chk1 in sister chromatid cohesion (SCC) and genome stability in Saccharomyces cerevisiae. The results showed that unlike SCC mutants, loss of CHK1 did not increase spontaneous or damage-induced allelic recombination or aneuploidy. Exposure of G2-arrested cells to ionizing radiation also did not increase allelic recombination or reduce survival in chk1 mutant cells as seen in SCC mutants. This suggests that Chk1 has a redundant role in controlling damage-induced SCC or that damage-induced SCC is redundant for maintaining genome stability in yeast.
Institute of Learning in Retirement - Miami University (Ohio)Andor Kiss
CRISPR/Cas9 is a new genetic engineering technique that uses a bacterial immune system to edit DNA. It involves using an RNA guide sequence and Cas9 protein to cut DNA at a targeted location. This allows genes to be knocked out or altered. CRISPR has many advantages over older techniques and has greatly improved efficiency of genetic engineering. However, it also raises ethical concerns about its applications, including the first reported use in human embryos.
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
This document summarizes the discovery of duplicated VegfA and KDR receptor genes in zebrafish that mediate vascular development. Specifically:
- The researchers identified a duplicated zebrafish VegfA gene (VegfAb) that encodes 171- and 210-amino acid isoforms not found in the single VegfA gene.
- They also found a duplicated KDR receptor gene (Kdrb) that encodes a receptor similar to mammalian KDR.
- Knockdown experiments in zebrafish showed that both VegfAb and the duplicated KDR receptor genes play important roles in vascular development.
- Further experiments demonstrated that the VegfAb isoforms are poorly secreted compared to VegfA isoforms
Refining Gene Ontology to Include Terms and Relationships Relevant to exRNA C...exrna
The document discusses efforts to standardize terminology related to extracellular vesicles (EVs) and exRNA-containing particles through developing new terms and relationships within the Gene Ontology (GO). It describes work with domain experts to propose additions to GO's Cellular Component branch to accurately describe and classify EVs and exRNA communications. The goal is to strike a balance between overgeneralizing and being too specific when defining terms in this emerging research area.
1) The study analyzed the effects of Csk knockouts on development of the initial segment of the mouse epididymis. Csk knockout was expected to promote cell proliferation and differentiation through increased ERK pathway activity due to lack of inhibition of SRC kinases.
2) A tissue-specific Csk conditional knockout mouse model was generated using Cre/lox recombination. Genotyping identified one mouse with the desired genotype.
3) Preliminary results found increased vasculogenesis in the initial segment of Csk knockout mice, suggesting effects on differentiation through the ERK pathway. Immunofluorescence found decreased activity of phospho-SRC in knockouts while other markers were similar to controls.
This document summarizes Mohd Kyum's PhD research on using CRISPR/Cas9 genome editing to induce haploidy in rice. The objectives were to generate knockout mutants of the OsMATL gene, which is responsible for haploid induction in maize, using CRISPR/Cas9 RNP complexes delivered via particle bombardment. Embryos of the rice variety Purple Line were bombarded with three different sgRNAs targeting OsMATL. Regenerated T0 plants were analyzed for mutations in the target gene using techniques like T7E1 assay, PCR-RFLP, and sequencing. Putative mutant plants showed a transformation efficiency of around 12.5% and are being further analyzed to characterize mutations induced
The document summarizes a study that used live cell imaging to visualize hepatitis C virus (HCV) entry into human liver cells. Key findings include:
1) HCV was labeled with fluorescent membrane dyes and found to move along actin stress fibers in an actin-dependent manner after internalization, suggesting clathrin-mediated endocytosis.
2) Inhibition of clathrin machinery or related host factors blocked HCV entry. Immunofluorescence showed HCV co-localizing with clathrin and early endosomes.
3) While tight junction proteins were thought to mediate HCV entry, live imaging found internalization occurring outside of tight junctions, indicating their role may be overstated.
4
CRISPR is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments from viruses that have previously infected the prokaryote and are used to detect and destroy DNA from similar viruses during subsequent infections.
C. trachoamtis detection and genotypings assayquint
This document describes the development of assays for amplifying, detecting, and genotyping Chlamydia trachomatis (Ct). It includes a Ct amplification assay using multiplex PCR, a Ct detection assay using a DNA enzyme immunoassay to detect conserved probes, and a Ct genotyping assay using reverse hybridization to identify Ct serovars. Clinical testing showed the assays had sensitivity comparable to other tests and allowed identification of multiple Ct infections and determination of serovar distribution patterns.
This document provides an overview of CRISPR/Cas9 genome editing. It discusses how CRISPR/Cas9 enables precise modification of DNA sequences, outlines the timeline of key discoveries in CRISPR research, and describes the molecular mechanism and potential applications of this technology, including in microbial research, crop improvement, and human gene therapy. It also notes some limitations of the CRISPR/Cas9 system and concludes by emphasizing the opportunities it provides to advance research and address challenges like food security.
"Estimation of Divergence Times in Asparagales in the Presence of Hybridization," presented in symposium "Insights and Benefits from Monocot Palaeobiology: Fossils, DNA, and Phylogenies" at Monocots V (5th International Conference on Comparative Biology of Monocotyledons, The New York Botanical Garden, July 2013).
As the COVID-19 pandemic continues, people are becoming infected at an alarming rate, individuals are unknowingly spreading disease, and more lives are lost every day. There is
an immediate need for a simple, rapid, early, and sensitive point-of-care testing for COVID-
19 disease. Recently,
clustered regularly interspaced short palindromic repeats (CRISPR)-based detection methods have received substantial attention for nucleic acid-based molecular testing due to their simplicity, high sensitivity and specificity. This review explores the various CRISPR-based COVID-19 detection methods and related diagnostic devices. As with any emerging technology, CRISPR/Cas-based nucleic acid testing methods have several
challenges that must be overcome for practical applications in clinics and hospitals. More importantly, these detection methods are not limited to COVID-19 but can be applied to detect any type of pathogen, virus, and fungi that may threaten humans, agriculture, and food industries in resource-limited settings. CRISPR/Cas-based detection methods have the potential to become simpler, more reliable, more affordable, and faster in the near future, which is highly important for achieving point-of-care diagnostics.
This document describes a method for performing RNA sequencing on single nuclei. Key points:
1) The authors demonstrate that double-stranded cDNA can be synthesized from single mouse neural progenitor cell nuclei and hippocampal tissue nuclei, allowing for whole transcriptome sequencing.
2) On average, sequencing of single nuclei detected over 16,000 of the approximately 24,000 mouse protein-coding genes.
3) Analysis of single nuclei avoids issues with dissociating intact cells from complex tissues, is applicable across eukaryotic species, and provides insight into nuclear gene regulation.
4) RNA sequencing of single nuclei is a powerful new method for investigating gene expression at the single cell level without disrupting cells.
The ASR Zebrafish facility provides several services including fish housing, embryo production, gene manipulation, and phenotypic analysis. Gene expression can be manipulated through RNA, DNA, and morpholino microinjections. Phenotypic analysis includes visualization of morphology, histology, fluorescence, and apoptosis. The facility also supports toxicity and drug screening in zebrafish using 96 or 384 well formats with wildtype or transgenic lines. Shared resources are partially supported by Georgetown and Howard University.
1. The document discusses quantitative modeling of genetic circuits that integrate both transcriptional and sRNA-mediated gene regulation.
2. It provides examples of genetic circuits involving sRNA regulation in bacteria, including the RyhB circuit for iron homeostasis in E. coli and quorum sensing circuits in Vibrio species.
3. The document introduces mathematical models for transcription factor-mediated gene regulation and sRNA-target mRNA interactions, describing the basic equations used to model such genetic circuits.
This study reports on the development of 14 polymorphic microsatellite loci for the red sea urchin (Strongylocentrotus franciscanus). The loci were highly polymorphic, with the number of alleles ranging from 7 to 62. However, 12 of the 14 loci showed significant heterozygote deficits, which is likely due to the presence of null alleles. While these microsatellites can be useful markers, caution should be used and some loci may need redesign of primers or sequencing of alleles to address issues like null alleles and size homoplasy.
1) The document discusses the CRISPR-Cas9 system of genome editing and its applications.
2) CRISPR-Cas9 allows for accurate and multiplex gene modification guided by RNA and is an advanced technique compared to earlier tools like ZFNs and TALENs.
3) The document covers the mechanism of CRISPR-Cas9 immunity in bacteria, the general protocol for genome editing using CRISPR-Cas9, and new developments like modified Cas9 enzymes and the Cpf1 protein.
This study analyzed faecal specimens from 2,495 diarrhoea cases in Kolkata, India between 2007-2009 to determine the seasonal distribution and characteristics of norovirus (NoV) infections. NoV was detected in 78 cases, mostly children under 2 years old, sometimes as the sole pathogen but often along with other enteric pathogens. Sequencing of the NVGII strains showed clustering with GII.4, GII.13 and GII.6 NoV types. NoV infections occurred year-round and were associated with mild dehydration in children and adults in Kolkata.
Gene editing uses tools like CRISPR to precisely alter DNA sequences. CRISPR uses guide RNA to target specific DNA sites for editing. Early gene editing tools included ZFNs and TALENs but CRISPR is easier to implement. While CRISPR shows promise for curing diseases, it also raises ethical concerns about altering human heredity that require open discussion.
CRISPR: Opportunities and Challenges WebinarPreScouter
CRISPR is a genome-editing technique that provides a simple yet versatile method for making targeted changes to the genome of living cells. Researchers have already applied CRISPR to reverse disease-causing mutations and to engineer pest-resistant crops.
In this CRISPR webinar, PreScouter will be joined by experts and researchers to review CRISPR's opportunities and challenges. We will touch upon key challenges, such as reducing off-target effects, as well as new advances set to overcome some of the current limitations, such as novel CRISPR systems. Additionally, we'll highlight potential first applications of the technology within medicine and agriculture.
This is a great opportunity to not only learn from experts about how this disruptive technology is changing gene editing but also ask your personal questions about CRISPR.
Moderator:
This CRISPR discussion will be moderated by Charles Wright, the Medical Project Architect at PreScouter.
Panelists:
John G. Doench, Ph.D., is Associate Director of the Genetic Perturbation Platform at the Broad Institute.
C. B. Gurumurthy (Guru), BVSC, MVSC, Ph.D. Exec. MBA, is an Assistant Professor at the Department of Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, and he serves as the Director of UNMC Mouse Genome Engineering Core Facility at the University of Nebraska Medical Center.
Shuibing Chen is an Assistant Professor in the Department of Surgery and Biochemistry at Weill Cornell Medical College, New York.
The document describes an experiment using CRISPR/Cas9 to introduce mutations into the PNPLA3 gene in HepG2 cells. Sequencing of genomic DNA and mRNA from the cells showed the presence of SNP1, which changes an amino acid, but not SNP2. The researcher will use this sequence as a template to generate four combinations of the SNPs in order to study their effects on fat accumulation when transfected into HepG2 cells. This could provide insight into how genetic variants influence susceptibility to NAFLD in different ethnic groups.
This document summarizes a student research project that analyzed the frequency of the CCR5 Delta 32 allele in a population in Northeastern Ohio. The students mapped the CCR5 gene, developed a DNA collection and analysis protocol, and tested 50 samples. Their results found that 5 out of the 50 samples were heterozygous for the Delta 32 allele, indicating a gene frequency of 5% in the population. The students propose continuing their research and exploring using the Delta 32 mutation for potential gene therapy applications.
This document summarizes a student research project that analyzed the frequency of the CCR5 Δ32 allele in a population in Northeastern Ohio. The students mapped primers for the CCR5 gene, developed a DNA collection and analysis protocol, and tested 50 samples from their population. Their results found that 45 samples were homozygous wild-type, 5 were heterozygous for the Δ32 mutation, and none were homozygous. They conclude that the Δ32 allele frequency in this population is 5%. Future plans include further testing at their college and using the Δ32 mutation for potential gene therapy.
Metasystem to Study Emergence of Infectious DiseasesSuresh Gopalan
Part of my work that uses model organisms to study emergence of infectious diseases. (* previous presentation got deleted by accident). This has relevance to nosocomial infections, immune-compromised state, necrotizing fasciitis and systems approach to study such emergence of new infectious disease and manipulate host responses for many immune-modulated diseases.
The document describes a lab experiment that tests how the addition of a pGLO plasmid affects the growth and characteristics of E. coli bacteria. The experiment involves transforming E. coli bacteria with the pGLO plasmid by adding it to a solution containing the bacteria. One solution receives the pGLO plasmid (+pGLO) while the other does not (-pGLO). The bacteria are then observed under UV light and incubated under various conditions to analyze effects on growth and gene expression.
This document discusses the analysis of microbial communities through sequencing of the 16S rRNA gene. It presents WATERS, a workflow system that automates and bundles various software tools for analyzing 16S rRNA sequence data. The goals of WATERS are to simplify the analysis process for users without specialized bioinformatics expertise and to facilitate reproducibility through tracking of data provenance. WATERS guides users through the typical sequence analysis steps of alignment, chimera filtering, OTU clustering, taxonomy assignment, phylogeny tree building, and ecological analyses and visualization. By integrating existing tools into a single automated workflow, WATERS aims to reduce the effort required for 16S rRNA data analysis and allow researchers to focus on biological interpretation of results.
CRISPR is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments from viruses that have previously infected the prokaryote and are used to detect and destroy DNA from similar viruses during subsequent infections.
C. trachoamtis detection and genotypings assayquint
This document describes the development of assays for amplifying, detecting, and genotyping Chlamydia trachomatis (Ct). It includes a Ct amplification assay using multiplex PCR, a Ct detection assay using a DNA enzyme immunoassay to detect conserved probes, and a Ct genotyping assay using reverse hybridization to identify Ct serovars. Clinical testing showed the assays had sensitivity comparable to other tests and allowed identification of multiple Ct infections and determination of serovar distribution patterns.
This document provides an overview of CRISPR/Cas9 genome editing. It discusses how CRISPR/Cas9 enables precise modification of DNA sequences, outlines the timeline of key discoveries in CRISPR research, and describes the molecular mechanism and potential applications of this technology, including in microbial research, crop improvement, and human gene therapy. It also notes some limitations of the CRISPR/Cas9 system and concludes by emphasizing the opportunities it provides to advance research and address challenges like food security.
"Estimation of Divergence Times in Asparagales in the Presence of Hybridization," presented in symposium "Insights and Benefits from Monocot Palaeobiology: Fossils, DNA, and Phylogenies" at Monocots V (5th International Conference on Comparative Biology of Monocotyledons, The New York Botanical Garden, July 2013).
As the COVID-19 pandemic continues, people are becoming infected at an alarming rate, individuals are unknowingly spreading disease, and more lives are lost every day. There is
an immediate need for a simple, rapid, early, and sensitive point-of-care testing for COVID-
19 disease. Recently,
clustered regularly interspaced short palindromic repeats (CRISPR)-based detection methods have received substantial attention for nucleic acid-based molecular testing due to their simplicity, high sensitivity and specificity. This review explores the various CRISPR-based COVID-19 detection methods and related diagnostic devices. As with any emerging technology, CRISPR/Cas-based nucleic acid testing methods have several
challenges that must be overcome for practical applications in clinics and hospitals. More importantly, these detection methods are not limited to COVID-19 but can be applied to detect any type of pathogen, virus, and fungi that may threaten humans, agriculture, and food industries in resource-limited settings. CRISPR/Cas-based detection methods have the potential to become simpler, more reliable, more affordable, and faster in the near future, which is highly important for achieving point-of-care diagnostics.
This document describes a method for performing RNA sequencing on single nuclei. Key points:
1) The authors demonstrate that double-stranded cDNA can be synthesized from single mouse neural progenitor cell nuclei and hippocampal tissue nuclei, allowing for whole transcriptome sequencing.
2) On average, sequencing of single nuclei detected over 16,000 of the approximately 24,000 mouse protein-coding genes.
3) Analysis of single nuclei avoids issues with dissociating intact cells from complex tissues, is applicable across eukaryotic species, and provides insight into nuclear gene regulation.
4) RNA sequencing of single nuclei is a powerful new method for investigating gene expression at the single cell level without disrupting cells.
The ASR Zebrafish facility provides several services including fish housing, embryo production, gene manipulation, and phenotypic analysis. Gene expression can be manipulated through RNA, DNA, and morpholino microinjections. Phenotypic analysis includes visualization of morphology, histology, fluorescence, and apoptosis. The facility also supports toxicity and drug screening in zebrafish using 96 or 384 well formats with wildtype or transgenic lines. Shared resources are partially supported by Georgetown and Howard University.
1. The document discusses quantitative modeling of genetic circuits that integrate both transcriptional and sRNA-mediated gene regulation.
2. It provides examples of genetic circuits involving sRNA regulation in bacteria, including the RyhB circuit for iron homeostasis in E. coli and quorum sensing circuits in Vibrio species.
3. The document introduces mathematical models for transcription factor-mediated gene regulation and sRNA-target mRNA interactions, describing the basic equations used to model such genetic circuits.
This study reports on the development of 14 polymorphic microsatellite loci for the red sea urchin (Strongylocentrotus franciscanus). The loci were highly polymorphic, with the number of alleles ranging from 7 to 62. However, 12 of the 14 loci showed significant heterozygote deficits, which is likely due to the presence of null alleles. While these microsatellites can be useful markers, caution should be used and some loci may need redesign of primers or sequencing of alleles to address issues like null alleles and size homoplasy.
1) The document discusses the CRISPR-Cas9 system of genome editing and its applications.
2) CRISPR-Cas9 allows for accurate and multiplex gene modification guided by RNA and is an advanced technique compared to earlier tools like ZFNs and TALENs.
3) The document covers the mechanism of CRISPR-Cas9 immunity in bacteria, the general protocol for genome editing using CRISPR-Cas9, and new developments like modified Cas9 enzymes and the Cpf1 protein.
This study analyzed faecal specimens from 2,495 diarrhoea cases in Kolkata, India between 2007-2009 to determine the seasonal distribution and characteristics of norovirus (NoV) infections. NoV was detected in 78 cases, mostly children under 2 years old, sometimes as the sole pathogen but often along with other enteric pathogens. Sequencing of the NVGII strains showed clustering with GII.4, GII.13 and GII.6 NoV types. NoV infections occurred year-round and were associated with mild dehydration in children and adults in Kolkata.
Gene editing uses tools like CRISPR to precisely alter DNA sequences. CRISPR uses guide RNA to target specific DNA sites for editing. Early gene editing tools included ZFNs and TALENs but CRISPR is easier to implement. While CRISPR shows promise for curing diseases, it also raises ethical concerns about altering human heredity that require open discussion.
CRISPR: Opportunities and Challenges WebinarPreScouter
CRISPR is a genome-editing technique that provides a simple yet versatile method for making targeted changes to the genome of living cells. Researchers have already applied CRISPR to reverse disease-causing mutations and to engineer pest-resistant crops.
In this CRISPR webinar, PreScouter will be joined by experts and researchers to review CRISPR's opportunities and challenges. We will touch upon key challenges, such as reducing off-target effects, as well as new advances set to overcome some of the current limitations, such as novel CRISPR systems. Additionally, we'll highlight potential first applications of the technology within medicine and agriculture.
This is a great opportunity to not only learn from experts about how this disruptive technology is changing gene editing but also ask your personal questions about CRISPR.
Moderator:
This CRISPR discussion will be moderated by Charles Wright, the Medical Project Architect at PreScouter.
Panelists:
John G. Doench, Ph.D., is Associate Director of the Genetic Perturbation Platform at the Broad Institute.
C. B. Gurumurthy (Guru), BVSC, MVSC, Ph.D. Exec. MBA, is an Assistant Professor at the Department of Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, and he serves as the Director of UNMC Mouse Genome Engineering Core Facility at the University of Nebraska Medical Center.
Shuibing Chen is an Assistant Professor in the Department of Surgery and Biochemistry at Weill Cornell Medical College, New York.
The document describes an experiment using CRISPR/Cas9 to introduce mutations into the PNPLA3 gene in HepG2 cells. Sequencing of genomic DNA and mRNA from the cells showed the presence of SNP1, which changes an amino acid, but not SNP2. The researcher will use this sequence as a template to generate four combinations of the SNPs in order to study their effects on fat accumulation when transfected into HepG2 cells. This could provide insight into how genetic variants influence susceptibility to NAFLD in different ethnic groups.
This document summarizes a student research project that analyzed the frequency of the CCR5 Delta 32 allele in a population in Northeastern Ohio. The students mapped the CCR5 gene, developed a DNA collection and analysis protocol, and tested 50 samples. Their results found that 5 out of the 50 samples were heterozygous for the Delta 32 allele, indicating a gene frequency of 5% in the population. The students propose continuing their research and exploring using the Delta 32 mutation for potential gene therapy applications.
This document summarizes a student research project that analyzed the frequency of the CCR5 Δ32 allele in a population in Northeastern Ohio. The students mapped primers for the CCR5 gene, developed a DNA collection and analysis protocol, and tested 50 samples from their population. Their results found that 45 samples were homozygous wild-type, 5 were heterozygous for the Δ32 mutation, and none were homozygous. They conclude that the Δ32 allele frequency in this population is 5%. Future plans include further testing at their college and using the Δ32 mutation for potential gene therapy.
Metasystem to Study Emergence of Infectious DiseasesSuresh Gopalan
Part of my work that uses model organisms to study emergence of infectious diseases. (* previous presentation got deleted by accident). This has relevance to nosocomial infections, immune-compromised state, necrotizing fasciitis and systems approach to study such emergence of new infectious disease and manipulate host responses for many immune-modulated diseases.
The document describes a lab experiment that tests how the addition of a pGLO plasmid affects the growth and characteristics of E. coli bacteria. The experiment involves transforming E. coli bacteria with the pGLO plasmid by adding it to a solution containing the bacteria. One solution receives the pGLO plasmid (+pGLO) while the other does not (-pGLO). The bacteria are then observed under UV light and incubated under various conditions to analyze effects on growth and gene expression.
This document discusses the analysis of microbial communities through sequencing of the 16S rRNA gene. It presents WATERS, a workflow system that automates and bundles various software tools for analyzing 16S rRNA sequence data. The goals of WATERS are to simplify the analysis process for users without specialized bioinformatics expertise and to facilitate reproducibility through tracking of data provenance. WATERS guides users through the typical sequence analysis steps of alignment, chimera filtering, OTU clustering, taxonomy assignment, phylogeny tree building, and ecological analyses and visualization. By integrating existing tools into a single automated workflow, WATERS aims to reduce the effort required for 16S rRNA data analysis and allow researchers to focus on biological interpretation of results.
Investigation of phylogenic relationships of shrew populations using genetic...Juan Barrera
This study investigated the phylogenetic relationships of shrew populations using genetic markers. DNA was extracted from tissue samples of shrews from different geographic regions. Two mitochondrial genes, cytochrome c oxidase subunit 1 (COI) and cytochrome b (cyt-b), were amplified via PCR and sequenced. Genetic markers were used to identify species and construct a phylogenetic tree. The cyt-b gene was found to be more accurate for species identification and phylogenetic analysis of shrews. Some inconsistencies between the COI and cyt-b trees and BLAST results suggest that cyt-b data is more abundant in genetic databases for shrew species comparison.
Investigation of phylogenic relationships of shrew populations using genetic...Juan Barrera
This study investigated the phylogenetic relationships of shrew populations using genetic markers. DNA was extracted from tissue samples of shrews from different geographic regions. Two mitochondrial genes, COI and cyt-b, were amplified via PCR and sequenced. The genetic markers were used to identify species and construct a phylogenetic tree. Cyt-b was found to be more accurate for species identification and phylogenetic analysis of shrews. Some inconsistencies between the COI and cyt-b trees and BLAST results suggest that the genetic databases are more complete for cyt-b in shrews. The study provides insights into shrew diversity and evolution at the DNA level.
This study investigated the phylogenetic relationships of shrew populations using genetic markers. DNA was extracted from tissue samples of shrews from different geographic regions. Two mitochondrial genes, COI and cyt-b, were amplified and sequenced. The genetic markers were used to identify species and construct a phylogenetic tree. The study found that cyt-b more accurately identified species and analyzed phylogeny in shrews compared to COI. Some inconsistencies between the COI and cyt-b BLAST results and phylogenetic trees were observed, possibly due to limited shrew sequence data available in databases. The study provided insights into shrew diversity and evolutionary relationships at the DNA level.
It has long been assumed that the individual cisternal stacks that comprise the plant Golgi apparatus multiply
by some kind of fission process. However, more recently, it has been demonstrated that the Golgi apparatus
can be experimentally disassembled and the reformation process from the ER (endoplasmic reticulum)
monitored sequentially using confocal fluorescence and electron microscopy. Some other evidence suggests
that Golgi stacks may arise de novo in cells. In the present paper, we review some of the more recent
findings on plant Golgi stack biogenesis and propose a new model for their growth de novo from ER exit
sites.
Presentation summarising the 2013 ICSB conference in Copenhagen, a requirement of James Hutton Institute Visits Abroad funding. Presented at the Cellular and Molecular Sciences seminar series.
Pablo Gracia is interested in molecular biophysics and how biomolecular structures influence biological systems. He enjoys collaborative academic research and using new ideas to solve longstanding biological mysteries. Gracia has experience in single-molecule fluorescence spectroscopy and applying it to study membrane protein folding mechanisms. He is currently working on membrane protein folding and the unfolded state of membrane proteins using this technique.
This study implemented two Design-Build-Test-Learn cycles to optimize production of the biofuel precursor 1-dodecanol in E. coli. The approach resulted in a 21% increase in dodecanol titer between cycles, up to 0.83 g/L. Beyond lessons for dodecanol production, the study highlighted the importance of sequencing checks on plasmids and the need for more accurate protein expression prediction tools in synthetic biology.
Improving Animal Modeling with 24/7 Home Cage Monitoring in Bioexclusion & Bi...InsideScientific
https://insidescientific.com/webinar/improving-animal-modeling-24/7-home-cage-monitoring-bioexclusion-biocontainment-mouse-housing-system-tecniplast
Recently, a surging response to the COVID-19 pandemic has led to an exponential increase in study support for biocontainment and bioexclusion research. Mouse models are being rapidly developed in both areas, and biosafe housing of these animal models is critical. Additionally, non-invasive home cage monitoring can improve the translational value of these research models.
Locomotor activity patterns can be monitored 24/7 as a diagnostic tool for biosecurity studies. Researchers, staff and animals alike will also benefit from a decreased need for animal handling, caging manipulations and animal monitoring.
This webinar will be most valuable for institutions where biocontainment and bioexclusion work is being considered or conducted, and for researchers who wish to better understand what can be achieved through continuous measurement of animal welfare based of use of non-invasive activity monitoring.
IN-SILICO CHARACTERISATION OF PROTEIN CODED BY CYT-B GENE OF Radopholus simil...Amit Yadav
Of the more than 30 species in the genus Radopholus, the burrowing nematode, Radopholus similis, is the only pathogen of widespread economic importance (Duncan and Moens, 2006). Radopholus similis is a migratory endoparasitic nematode that is known to be a destructive pest of citrus crops, pepper and, most importantly, banana, on which it causes toppling disease. The nematode causes economic problems throughout the world, most notably in warmer regions, including South America, the Caribbean, Africa, Asia and the Pacific.
DNA Barcoding and its application in species identificationsupriya k
1) The document introduces a seminar on DNA barcoding and its role in discriminating plant species, given by Kaldate Supriya.
2) DNA barcoding is a technique that uses short, standardized gene sequences from organisms as a genetic barcode for species identification and discrimination.
3) The most common plant barcoding markers are chloroplast genes matK and rbcL, which provide sufficient variability to discriminate most plant species.
Potency of Neural Stem Cells within the Retina of Japanese Rice FishCorbett Hall
The document summarizes research on the potency of neural stem cells within the retina of the Japanese rice fish. It finds that retinal stem cells are multipotent and can develop into all three layers of the neural retina. Additionally, it determines that the neural retina has a single clonal origin, with each arched continuous stripe originating from one retinal stem cell. The developmental progression in the fish does not affect the multipotency of retinal stem cells. The Japanese rice fish provides an ideal model for studying vertebrate retina development.
As the bioreporters we would like to use to detect environmental arsenic is a genetically modified bacteria, we proposed a mode of using this bacteria in the field and to discuss the safety issues with the Swiss governmental agency responsible for the environment.
Advances in Molecular Cytogenetics: Potential for Crop Improvement.pptxKanshouwaModunshim
Title: Exploring Advances in Cytogenetics and Molecular Cytogenetics
Description:
Delve into the intricate world of cytogenetics and its cutting-edge counterpart, molecular cytogenetics, through this insightful presentation. Understand the profound relationship between chromosome structure, behavior, and gene function, with a particular focus on their relevance to crop improvement programs.
Key Points:
Introduction to Cytogenetics: Explore the fundamental principles of cytogenetics, its historical significance, and the recent influence of molecular tools, leading to the emergence of molecular cytogenetics.
Importance in Crop Improvement: Uncover the pivotal role of molecular cytogenetics in crop improvement programs, offering insights into the structural and functional organization of genomes within chromosomes.
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Exploring the role of Campylobacter. coli GT-42 enzymes on LOS biosynthesis
1. Eläinlääketieteellinen tiedekunta
Associate Professor Mirko Rossi
CHRO 2017, Nantes
”Pathogenicity and Virulence factors”
Exploring the role of C. coli GT-42
enzymes on LOS biosynthesis
12/09/2017CHRO2017/ Mirko Rossi 1
2. Eläinlääketieteellinen tiedekunta
• Are sialyltransferases involved in the
biosythesis of several LOS structures in
C. jejuni related to ganglioside mimicry
• Associated with the development of GBS
• In C. jejuni: Cst-I, Cst-II and Cst-III have
been characterized as mono/bi alfa-
2,3/alfa-2,8 sialyltransferases
• GT-42 genes were detected also in C. coli
(Skap-De Haan et al., 2014) but the
function is still unknown
12/09/2017CHRO2017/ Mirko Rossi 2
Glycosyltransferases family 42 in Campylobacter
LOS locus class A of C. jejuni
Cst-II
Gangliosides
3. Eläinlääketieteellinen tiedekunta
• BLAST C. jejuni sialyltransferases vs
NCBI nr
• 7 phylogenetically distinct
glycosyltransferases GT-42 in C. coli
• Clustered into two monophyletic
clusters:
• C. jejuni and C. coli GT-42
• C. coli specific GT-42
12/09/2017CHRO2017/ Mirko Rossi 3
Campylobacter coli GT-42
NJ phylogeny of nt sequences of GT-42
homologues found in C. coli
Skap-De Haan et al., 2014
Culebro et al., 2016
Richard et al., 2013
4. Eläinlääketieteellinen tiedekunta
• 2574 genomes from ENA mapped against
the 7 C. coli GT-42 groups and neuB using
ReMatCh
• 818 genomes possess one or multiple GT-
42 genes
• C. coli-specific GT-42 are dominant;
present in Clade 1a and Clade 3
• neuB is frequently not detected in GT-42
positive strains
12/09/2017CHRO2017/ Mirko Rossi 3
Frequency of GT-42 in C. coli
Phylogeny based on atpA genes
hierBAPS clustering (inner ring)
Clade 3 Clade 2 Clade1bc Clade1a
5. Eläinlääketieteellinen tiedekunta
LOS locus classes in GT-42 positive C. coli
• 818 GT-42 + C. coli genomes mapped vs
genes of C. coli LOS locus classes I to XII
using ReMatCh (~100 genes)
• LOS locus class I, II, III and IX; in addition
to unknown classes
• Strains having C. coli-specific GT-42 II,
III and IX
12/09/2017CHRO2017/ Mirko Rossi 5
LOS locus class IX
LOS locus class III
LOS locus class II
Cst-V
Cst-IV
Cst-VI
Culebro et al., 2016
6. Eläinlääketieteellinen tiedekunta
Role of GT-42 in C. coli LOS biosynthesis
12/09/2017CHRO2017/ Mirko Rossi 6
cst-V neuA
LOS locus class IX
LOS locus class III
LOS locus class II cst-IV
cst-VI
neuCneuB
C. coli 76339 (Clade 3)
• LOS locus class IX: Cst-V
• Contains also Cst-I on CPS
• NANA detected by Dionex
C. coli 65 (Clade 1a)
• LOS locus class II: Cst-IV
• Does not contain neuB
• NANA not detected on LOS by ms/ms
Skap-De Haan et al., 2014 Culebro et al., 2016
pseudogene
7. Eläinlääketieteellinen tiedekunta
Cst-V is involved in LOS biosynthesis but not Cst-I
12/09/2017CHRO2017/ Mirko Rossi 7
WT ΔcstV→ ΔcstV←
GGT16SrRNA
cst-V CAT
GGT
WT ΔcstVΔggt::cstV
Deletion of cstV induce a change in the
electrophoresis motility of LOS in SDSPAGE
Phenotype is partially restored after insertion
of cstV within ggt locus
cst-I
… CPS locuscst-V neuALOS locus class IX neuCneuB
C. coli 76339
Deletion of cstI does not produce any changes
in the LOS
8. Eläinlääketieteellinen tiedekunta
NeuB is involved in LOS biosynthesis
12/09/2017CHRO2017/ Mirko Rossi 8
cst-I
… CPS locus
Deletion of neuB produce same change in
the electrophoresis motility of LOS in SDS-
PAGE as observed in ΔcstV
cst-V neuALOS locus class IX neuCneuB
C. coli 76339
C. coli 76339 C. jejuni 81-176
LOS of C. coli 76339 isn’t sensitive to the
action of C. perfringens neuroaminidase,
indicating that NANA (=Sialic Acid), if
present, is not terminal
9. Eläinlääketieteellinen tiedekunta
Cst-IV is involved in LOS biosynthesis
12/09/2017CHRO2017/ Mirko Rossi 9
LOS locus class II cst-IV
C. coli 65
Deletion of cstIV induce a change in the
electrophoresis motility of LOS in SDS-
PAGE
neuB2
… Flagella locus
WT WTΔcstIV→
… neuB3
Missing of NANA biosynthesis genes possible role of Leg and Pse
Deletion of neuB2 (Leg) does not change
LOS phenotype.
Deletion of neuB3 (Pse) was unsuccessful
(lethal?)
10. Eläinlääketieteellinen tiedekunta
Testing in vitro activity of C. coli GT-42
• Enzymatic activity using:
• DONOR CMP-NANA
• ACCEPTORS BODIPY or FCHASE-labeled Lac, LacNAc, Sialyl-Lactose, alpha/beta-GalNAc, GM3,
alpha-Gal, beta-GlcNAc, LewisX
• Mobility on TLC
• Enzyme:
• Native using the cell extracts of WTs and mutants
• Overexpression using pCW and pCWmalET
12/09/2017CHRO2017/ Mirko Rossi 10
11. Eläinlääketieteellinen tiedekunta
Testing in vitro activity of C. coli GT-42
12/09/2017CHRO2017/ Mirko Rossi 11
C. coli 76339 WT
C. coli 76339 ΔcstV
C. coli 76339 ΔcstI
C. coli 65
Cst-I is very active on Lac, LacNAc, Sialyl-Lactose and alpha/beta-GalNAc
Cst-V and Cst-IV are not active unknown donors and acceptors
12. Eläinlääketieteellinen tiedekunta
Structural prediction of Cst-V and Cst-IV
• Three- dimensional structures of Cst-V
and Cst-IV were built with Phyre2 server
• Both Cst-IV and Cst-V protein models
were built with 100% confidence
• Several substitutions at amino acids
important for the interaction with CMP-
NANA in Cst-II were identified
12/09/2017CHRO2017/ Mirko Rossi 12
Change of Cst-II
activity
Chiu et al., 2004
13. Eläinlääketieteellinen tiedekunta
CONCLUSION
• C. coli possess at least 7 types of glycosyltransferases belonging to CAZy family 42 probably
involved both in LOS and CPS biosynthesis, clustering into two clusters:
• C. jejuni and C. coli GT-42 (rarely found in the population)
• C. coli specific GT-42 (most frequent)
• C. coli specific GT-42 Cst-V and Cst-IV are involved in LOS biosynthesis of C. coli, but are unable to
transfer NANA
• C. coli specific GT-42 have acquired a different unknown function compared to others GT-
42 enzymes, diverging from C. jejuni sialyltransferase
12/09/2017CHRO2017/ Mirko Rossi 13
14. Eläinlääketieteellinen tiedekunta
ACKNOWLEDGMENTS
12/09/2017CHRO2017/ Mirko Rossi 14
PhD student Alejandra Culebro
Prof. Warren Wakarchuk, Ryerson University, Canada
Prof. Emerita Marja-Liisa Hänninen (University of Helsinki, Finland) for providing the strains
Dr. Michel Gilbert (NRC, Canada) for helping in the in vitro activity on FCHASE acceptors
Dr. João Carriço and Miguel Machado (University of Lisbon, Portugal) for giving us the
opportunity to use ReMatCh
Funding:
University of Helsinki Research grant
MBDP doctoral program
Walter Ehrströmin säätiö
15. Eläinlääketieteellinen tiedekunta 12/09/2017CHRO2017/ Mirko Rossi 15
THANKS FOR THE ATTENTION
Contact me on twitter
@happygipsy
Slides are available on Slideshare
If you are interested in ReMatCh:
https://github.com/B-UMMI/ReMatCh
16. Eläinlääketieteellinen tiedekunta
• Chiu et al. 2004. Structural analysis of the sialyltransferase CstII from
Campylobacter jejuni in complex with a substrate analog. Nat Struct Mol Biol.
11(2):163-70
• Richards et al. 2013. Comparative characterization of the virulence gene clusters
(lipooligosaccharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter
coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species. Infect
Genet Evol 14:200–213
• Skarp-de Haan, et al. 2014. Comparative genomics of unintrogressed
Campylobacter coli clades 2 and 3. BMC Genomics 15:129
• Culebro et al. 2016. Large sequence diversity within biosynthesis locus and common
biochemical features of Campylobacter coli lipooligosaccharides. J Bacteriol
198(20):2829-40. doi: 10.1128/JB.00347-16 (preprint
http://dx.doi.org/10.1101/050328)
• .
12/09/2017CHRO2017/ Mirko Rossi 16
REFERENCES
Editor's Notes
Glycosyltransferases belonging to the family 42 as classified in the Cazy database, are sialyltransferases involved in the synthesis of LOS structure of C. jejuni related to ganglioside mimicry and associated with the development of the Guillain-Barré syndrome. Up to date three different GT-42 enzymes named Cst-I, Cst-II and Cst-III have been characterized in C. jejuni as monofunctional or bifunctional alpha-2,3 or 2,8 sialyltransferases. On the right a graphical representation of the biosynthesis of ganglioside-like structure on the LOS of C. jejuni expressing LOS locus class A which contains Cst-II.
Our group and other have already detected orthologues of GT-42 enzymes in C. coli, but their role in the LOS biosynthesis in this species is still obscure. My presentation will focus on GT-42 enzyme in C. coli. I will present an overview of the types of GT-42 found in this species, their frequency in the population and their possible role in the LOS biosynthesis.
Blasting the C. jejuni Csts against NCBI nr database, we retrieved 45 C. coli sequences forming 7 phylogenetically distinct GT-42 groups. They group into two separate monophyletic clusters: cluster A including C. jejuni orthologues Cst-I, Cst-II and Cst-III sialyltransferases, and cluster B which contains proteins exclusively found in C. coli including group 5, 6 and 7. Group 5 includes sequences similar to Cst-V while group 7 consists of sequences similar to Cst-IV, both GT-42 previously described in C. coli by our group.
To investigate the frequency of the presence of GT-42 in C. coli population, we mapped all C. coli genomes currently available in ENA against the 45 sequences corresponding to the 7 GT-42 groups. We used ReMatCh for mining ENA and performing the mapping.
The tree on the right represents the phylogeny of the strains based on atpA gene which clearly showed the division of the C. coli population in its main three monophyletic clades: Clades 1abc (red and violet), 2 (yellow) and 3 (green). As results, 818 over 2574 genomes possess one or multiple GT-42 genes. C. coli specific GT-42 genes of cluster B are more frequently detected than genes of GT-42 cluster A. In addition, GT-42 are practically restricted to Clade 1a and Clade3 C. coli strains. It is also interesting to notice that although NANA synthesis gene neuB is frequently found in C. coli, it is often absent in those strains possessing GT-42 enzyme of cluster B.
We further analyzed the GT-42 positive genomes for predicting which type of LOS locus classes were linked to the detected GT-42 genes. Using ReMatCh we mapped the reads of the 818 genomes against all the genes of the known 11 C. coli LOS locus classes. We then summarized the results by showing the percentage of positive map of genes for each class in the two heatmaps; here showed on the right. The upper heatmap displays the results for all the 818 genomes, showing that the majority of the strains have predicted LOS classes I, II, III and IX and few have possibly novel classes (click).
Limiting the analysis to those genomes possessing the C. coli specific GT-42 of the cluster B (lower heatmap), strains are positive for LOS locus class II, III and IX. Specifically Cst-V is restricted to class IX, Cst-VI to class III and Cst-IV to class II.
A schematic representation of the three LOS locus classes containing C. coli specific GT-42 are represented here. LOS class IX is the only one including also the three genes involved in the biosynthesis of Sialic Acid; in yellow. (click) Since the Cst-VI of LOS class III is likely a pseudogene (at least in the genomes we did manually check) we focused on the other two classes (click) selecting two strains. Cst-V positive strain C. coli 76339 belonging to Clade 3 which possesses also a GT-42 of cluster A in the capsule locus and detectable NANA on phenol extracted LOS using Dionex. We also selected the Cst-IV positive C. coli 65 strain which is characterized by the absence of NANA biosynthesis locus and for which NANA was not detected using ms/ms. We then performed mutational studies to investigate the possible role of Cst-V and Cst-IV in the biosynthesis of LOS. We also performed in vitro activity test to investigate the functionalities of these enzymes on several acceptors.
Deleting cstV (click) by inserting the cassette either in the direction of the gene or in opposite direction, we observed a clear effect on the electrophoresis motility of LOS in SDSPAGE. (click) On the contrary, the deletion of Cst-I does not produce any detectable changes in the LOS. (click) The phenotype was partially restored after inserting the cstV within ggt gene under the its promoter, confirming that cstV is involved in decorating the LOS of C. coli 76339.
(click) Similar changes in the electrophoresis motility of LOS of C. coli 76339 in SDS-PAGE have been showed after deleting neuB (the NANA synthase), indicating that most likely the LOS have been decorating with NANA. (click) Since the LOS of C. coli 76339 isn’t sensitive to the action of Clostridium perfringens neuraminidase, sialic acid is probably not a terminal sugar residue on the oligosaccharide chain, as also shown for C. jejuni 11168, or alternatively CstV transfers a sialidase resistant unidentified nonulosonate
As observed for LOS class IX, deleting cstIV (click) by inserting the cassette in the same direction of the gene, we observed a clear effect on the electrophoresis motility in SDS-PAGE of the LOS of the strain C. coli 65. However, (click) differently from C. coli 76339, C. coli 65 does not possess NANA biosynthesis genes. We therefore investigated the possible role of other synthases locate in the Flagella region involved in the production of other nonulosonic acids such as Legionaminic and Pseudaminic acid. (click) Deletion of neuB2 does not produce any changes in the LOS, while it was not possible to obtain neuB3 mutants. Remain therefore unknown the possible substrate of cstIV and the type of decoration of the LOS class II.
To compare with well characterized GT-42 enzymes, we performed enzymatic activity of C. coli GT-42 Cst-I, Cst-V and Cst-IV using CMP-NANA as donor and a long list of BODIPY or FCHASE labeled acceptors found to be substrates for GT-42 enzymes. We tested using both native proteins obtained from cell extract of C. coli strains and after overexpressing the proteins using pCW and pCWmalET as vectors.
Here is shown the TLC mobility assay on the three major acceptors for C. jejuni Csts (Lac, LacNac and 3’Sialyllactose) tested using native proteins from cell extracts. C. coli 76339 showed activity on these acceptors due to action of Cst-I which resulted also very active on alpha and beta-GalNAc. On the contrary, C. coli specific GT-42 Cst-V and Cst-IV does not show any activity in any acceptors using CMP-NANA as donor. This results clearly showed that C. coli GT-42 enzymes of cluster B have functionally diverged from other characterized GT-42 by being specific for unknown acceptors (Cst-V) or both unknown donor and acceptors (Cst-IV).
Comparing the predicted structure of Cst-IV and Cst-V with C. jejuni Cst-II we identified several amino acid substitutions that have demonstrated to be important in the interaction between Cst-II and CMP-NANA, supporting the theory of different donors (especially for Cst-IV).
In conclusion. At least 7 types of glycosyltransferases belonging to CAZy family 42 have been found in C. coli. These enzymes are probably involved both in LOS and CPS biosynthesis. They form two major clusters. Cluster A including both C. jejuni and C. coli sialyltransferases which are relative infrequent in the C. coli population; and Cluster B composed by glycosyltranferases involved exclusively in the LOS biosynthesis of C. coli which are quite common among the available strains. Our study showed that C. coli specific GT-42 Cst-V and Cst-IV have acquired different unknown function (in term of donor and/or acceptor specificity) compared to others GT-42 enzymes, diverging from C. jejuni sialyltransferases. The possible implications for the ecology of this species and virulence are objectives for ongoing and future studies.