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This study examined the mechanisms by which Kaposi's Sarcoma associated Herpesvirus (KSHV) reactivates from latency in oral epithelial cells. The researchers exposed latently infected BCBL-1 cells to treatments that induce reactivation, including TPA, sodium butyrate, and Porphomonas gingivalis spent media. They found that TPA and P. gingivalis induced distinct gene expression profiles, suggesting P. gingivalis induces reactivation through a novel mechanism different from TPA. This supports the hypothesis that butyrate released by P. gingivalis can induce viral reactivation from latency in vivo in the oral cavity.
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This study analyzed genome and methylome variations in the bacterial pathogen Helicobacter pylori during experimental infection in humans. Researchers used single-molecule real-time sequencing to analyze H. pylori isolates obtained from human volunteers involved in a vaccine trial. The isolates came from individuals infected with a strain known as BCM-300, which has virulence factors associated with disease. The results showed changes in key virulence genes in the bacteria, suggesting an ability to adapt during infection, even in vaccinated individuals. However, the small number of volunteers limited conclusions about differences between vaccinated and unvaccinated groups.
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This document discusses Klebsiella pneumoniae, a pathogenic bacteria that can produce carbapenemase enzymes which make it resistant to beta-lactam antibiotics like carbapenems. It is found in human intestines as normal flora. The objective is to characterize carbapenemase-producing K. pneumoniae isolates and identify risk factors for acquisition. Methods described are PCR, MLST, and PFGE to analyze isolates genetically. Results show the distribution of isolates by genotype and identify specific sequence types associated with international spread. The discussion analyzes previous studies on risk factors like prior antibiotic use and links between isolates from the same hospital ward. Conclusions note that fecal transmission is a major risk factor and K. pneumoniae especially impacts patients
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In the first design of PCR primer pools, V-regions 2, 3, 4, 6-7, 8 and 9
were amplified as individual ~200 bp fragments in one of two multiplex
PCR reactions. The primer pools covered more than 80% of
eubacterial sequences in the GreenGenes database with perfectly
matched primer pairs for at least one V-region. In the second design
the amplicons are longer, 300-400 bp products
Our data analysis module classified individual reads by mapping them
to the reference libraries. With the fragment sizes ranging between
200-300 bp, we achieved genus and, in many cases species level
taxonomic resolution, depending on which V-region was evaluated.
In an initial evaluation we tested the complete solution (PCR
chemistry, workflow and software) on two mock community DNA
samples from BEI resources: HM-276D - even community, with equal
number of rDNA copies for each of 20 bacteria and HM-783D–
staggered community, with variable number of rDNA copies. Several
V-regions were amplified by our primer sets for every organism in the
mock community. The number of classified reads in each V-region for
every bacteria depended on both primer complementarity and ease of
sequencing of the particular PCR fragment. Our approach of
interrogating multiple V-regions and sequencing both amplicon strands
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biases.
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Increased sequencing depth (316v2 and 318v2) increased sensitivity
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human DNA and were able to identify and determine characteristic Vsignatures
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to increase the confidence in the organism presence and ID, but may
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simultaneously surveys multiple V-regions in the 16S rRNA gene.
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were amplified as individual ~200 bp fragments in one of two multiplex
PCR reactions. The primer pools covered more than 80% of
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matched primer pairs for at least one V-region. In the second design
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Our data analysis module classified individual reads by mapping them
to the reference libraries. With the fragment sizes ranging between
200-300 bp, we achieved genus and, in many cases species level
taxonomic resolution, depending on which V-region was evaluated.
In an initial evaluation we tested the complete solution (PCR
chemistry, workflow and software) on two mock community DNA
samples from BEI resources: HM-276D - even community, with equal
number of rDNA copies for each of 20 bacteria and HM-783D–
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mock community. The number of classified reads in each V-region for
every bacteria depended on both primer complementarity and ease of
sequencing of the particular PCR fragment. Our approach of
interrogating multiple V-regions and sequencing both amplicon strands
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in the staggered mock community with 10^4-10^6 rDNA copies/PCR).
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Karyotype variability in plant pathogenic fungi palm7016
This document summarizes a seminar presentation on karyotype variability in plant pathogenic fungi. It discusses types of karyotype variability including chromosome number polymorphisms and chromosome length polymorphisms. Mechanisms that can lead to karyotype variability are described, such as meiotic recombination, DNA repair pathways, parasexual recombination, transposon-associated rearrangements, and lateral DNA transfer. Case studies on fungi like Mycosphaerella graminicola and Alternaria alternata are provided that demonstrate karyotype changes through these mechanisms. The importance of karyotype variability in the evolution and emergence of new virulence strains in fungi is also noted.
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Design and Evaluation of a 16S-based Integrated Solution to Study Bacterial D...
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Karyotype variability in plant pathogenic fungi palm7016
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Pak Us Science And Technology Grant Project E Dited
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This study found that lysates from Gardnerella vaginalis, a bacteria commonly associated with bacterial vaginosis, significantly stimulated HIV expression in monocytoid cells and certain T cell lines. G. vaginalis lysates activated HIV long terminal repeat transcription and increased NF-kB binding activity in HIV-infected cells, indicating an effect on HIV transcription. The activation of HIV production by G. vaginalis suggests that G. vaginalis infection in the genital tract may increase the risk of HIV transmission by enhancing HIV expression levels in the genital tract. This could help explain the link between bacterial vaginosis and increased sexual transmission of HIV.New insight in gastric cancer
Calprotectin (CP) is a metal-chelating protein produced by neutrophils during inflammation. CP inhibits the growth of Helicobacter pylori, a major risk factor for gastric cancer, by sequestering the metals manganese and zinc. Studies show that CP reduces the activity of H. pylori's cag Type IV Secretion System (T4SS) in a zinc-dependent manner by inhibiting T4SS pilus biogenesis. This decreases H. pylori's ability to translocate the oncoprotein CagA into gastric cells and reduce inflammation. While CP limits H. pylori growth and virulence, it also allows the bacteria to persist by modulating the inflammatory response.
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In vitro transcription and transfection of HCV genomic replicon
Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 μg transcript using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR. Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
In vitro transcription and transfection of HCV genomic replicon
ABSTRACT:
Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 μg transcript using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR. Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
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Pak Us Science And Technology Grant Project E Dited
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In vitro transcription and transfection of HCV genomic replicon
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In vitro transcription and transfection of HCV genomic replicon
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Research - UNC
- 1. 3857 Determinants of KSHV Reactivation using a Viral pathogenesis based Microarray L.M. DOMINICK, V.G. LENTCHITSKY, and J. WEBSTER-CYRIAQUE, University of North Carolina, Chapel Hill, USAOur laboratory has established that both latent and lytic forms of Kaposi's Sarcoma associated Herpesvirus (KSHV) are present in oral epithelial cells. Objectives: The objective of this study was to develop methods to distinguish the mechanism by which KSHV reactivates from latency in the oral cavity in vivo. It is hypothesized that butyrate released by periodontal pathogens such as Porphomonas gingivalis induce viral reactivation from latency. Analyses of differential gene expression should then determine whether patterns of gene expression induced by P.gingivalis is similar to that of protein kinase C driven reactivation observed with the inducing agent 12-O- tetradecanoylphorbol-13-acetate (TPA), or histone deacetylase inhibition similar to sodium butyrate (n-bu). Methods: Latently KSHV infected BCBL-1 cells were exposed to 25ng/ml TPA, 3mM n-bu, P. gingivalis spent media or were left untreated. RNA was extracted from cells after 48 hours of treatment as well as from the uninfected lymphoid BJAB cell line. cDNA was synthesized, labeled with CY dyes and hybridized to a novel microarray containing 20,000 human genes and temporally regulated viral genes from distinct stages of the infectious cycle (immediate early, early, late, and latent) of each known human herpesvirus. Induction of viral replication was confirmed by detection of lytic gene products on the array, reverse transcriptase PCR, and by immunofluorescence detection of lytic antigen expression in the cultured cells. Results: We determined that the expression profile of cellular genes involved in signal transduction, cell cycle, immune regulation and cell adhesion during viral replication induced by TPA was distinct from that induced by the addition of P. gingivalis. Conclusion: These results support P. gingivalis as an inducer of gamma herpes viral replication, and suggest this novel mechanism of viral induction is central to viral reactivation in the oral cavity in vivo. Studies supported by R03 DE14444-01S. latres_dominick@dentistry.unc.edu Seq #388 - Microbial Pathogenesis and Inhibition 10:15 AM-11:30 AM, Saturday, 13 March 2004 Hawaii Convention Center Exhibit Hall 1-2 Back to the Oral Medicine & Pathology Program Back to the IADR/AADR/CADR 82nd General Session (March 10-13, 2004) https://iadr.confex.com/iadr/2004Hawaii/techprogram/abstract_47204.htm