Project description:Chikungunya virus (CHIKV) infection is characterized by alterations in gene expression profile on host cells that consequently lead to an immune response. Here, we used RNA sequencing to analyze the mRNA expression profile in human monocyte-derived macrophages (MDMs) infected with a Colombian clinical isolate of CHIKV at 6 and 24 hpi. analyze the mRNA expression profile in the human monocyte-derived macrophages infected at 6 and 24 hrs with a Colombian clinical isolate of Chikungunya virus.
Project description:A knockout cell library in Huh7.5.1 cells was generated by introducing a genome-scale CRISPR library (GeCKOv2, Addgene #1000000049) and subjected to hepatitis A virus infection (HM175/18f) to isolate virus-resistant mutant cells. Genomic DNA was isolated from the original and virus-selected mutant cell populations and abundance of guideRNA encoding sequences were measured by sequencing on an Illumina NextSeq (High Output).
Project description:The pathogen Clostridioides difficile causes toxin-mediated diarrhea and is the leading cause of hospital-acquired infection in the United States. Due to growing antibiotic resistance and recurrent infection, targeting C. difficile metabolism presents a new approach to combat this infection. Genome-scale metabolic network reconstructions (GENREs) have been used to identify therapeutic targets and uncover properties that determine cellular behaviors. Thus, we constructed C. difficile GENREs for a hypervirulent isolate (strain [str.] R20291) and a historic strain (str. 630), validating both with in vitro and in vivo data sets. Growth simulations revealed significant correlations with measured carbon source usage (positive predictive value [PPV] ≥ 92.7%), and single-gene deletion analysis showed >89.0% accuracy. Next, we utilized each GENRE to identify metabolic drivers of both sporulation and biofilm formation. Through contextualization of each model using transcriptomes generated from in vitro and infection conditions, we discovered reliance on the pentose phosphate pathway as well as increased usage of cytidine and N-acetylneuraminate when virulence expression is reduced, which was subsequently supported experimentally. Our results highlight the ability of GENREs to identify novel metabolite signals in higher-order phenotypes like bacterial pathogenesis.
Project description:A few transformed cell lines and two historic strains have been extensively used to study respiratory syncytial virus (RSV). We report a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with four strains of RSV representing both major subgroups as well as historic and more contemporary genotypes -- [RSV/A/Tracy (GA1), RSV/A/Ontario (ON), RSV/B/18537 (GB1), RSV/B/Buenos Aires (BA)] -- via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response including transcriptional changes and levels of secreted cytokines and growth factors. Our findings strongly suggest 1) the existence of a conserved difference in gene expression between RSV subgroups A and B; 2) the A549 cell line is a more stringent and natural host of replicating RSV than the HEp-2 cell line; and 3) consistent with previous studies, determining the full effects of viral genetic variation in RSV pathogenesis requires model systems as tractable as transformed cell lines but better representative of the human host.
Project description:The enamel proteome includes a range of proteins which are well-preserved in archaeological settings but have so far received less study than the sex-specific sequencing of enamel. We look beyond sex-specific sequencing of amelogenin to investigate the potential of several serum proteins, including immunoglobulin gamma (IgG), the major immunoglobulin found in blood serum, and C-reactive protein (CRP) which is associated with inflammatory response, to provide insight into the health and stresses experienced by individuals in the past. We apply this approach to enamel samples from Mission-Period ancestral Ohlone interred at Asistencia San Pedro y San Pablo (CA-SMA-71/H; n=11). For comparison, we also examine enamel from historic-period European-Americans interred in the City Cemetery in San Francisco, and extracted third molars from present-day military cadets. Results indicate that IgG is elevated among individuals at the asistencia relative to samples from present-day military cadets (n=8), or historic City Cemetery individuals (ANOVA with post-hoc Tukey Kramer tests, p < 0.02). Further, the inflammatory protein CRP, normally expressed at much lower levels than IgG, was present in 55% (6 of 11) of the asistencia samples, and in 17% (2 of 12) of the historic City Cemetery samples, but was not detected in enamel samples from contemporary military cadets. While more studies are needed, we argue that the difference in IgG could reflect higher levels of chronic diseases such as tuberculosis among Ohlone living within the Mission system, while the presence of measurable amounts of CRP could relate to high degrees of physical, social, and emotional stresses. To our knowledge, this is the first paleoproteomic study of immune proteins in tooth enamel. The ability to track immune responses during tooth formation could provide valuable and high-resolution information on ancient health and disease at the level of the individual over archaeological time-scales.
Project description:Purpose: The goals of this study are to monitor the evolution pattern of Shaan virus in depending host cells by viral transcriptome sequencing analysis of MARC145, A549, HEK293, and HRT18 cells infected with Shaan virus. Methods: The original isolate of Shaan virus (B16-40, Genbank accession no. MG230624.1) was passaged in MARC-145 cells. Low-passaged virus was obtained from the virus 4 times serially passaged in MARC-145 cells. The shaan virus which was 44 times serially passaged in MARC-145 cells was used for high-passaged virus. The prepared low- (p4) and high passaged shaan virus (p44) in MARC-145 cells were inoculated in triplicate to different host cells, HEK-293, A549, and HRT18 cell lines at M.O.I of 0.5 for 2 hours. The infected cells were incubated with the maintenance medium for each cell line for 24hrs. The infected cell pellets were resuspended to 250µl with fresh medium, to extract RNA for the high-throughput sequencing. The RNA samples were sequenced with illumine TruSeq Strand Total RNA LT kit and illumine NovaSeq6000 plaform form Macrogen, Inc (Seoul, Korea) for high throughput sequencing. The raw reads were trimmed with BBDuk and mapped the isolate Bat-ParaV/B16-40 (Genebank accession number. MG230624.1) with Bowtie 2 using Geneious program 2021.2.2. Transcript expression level in cells infected with shaan virus were calculated based on annotation on a reference shaan virus. Result: The total reads counts were between 50,285,454 and 76,298,278. The reference mapping of the trimmed reads with a reference genome (Genbank accession no. MG230624.1) showed different coverage values. The mapped reads were normalized and expressed as Transcript per Million (TPM) value. There were no noticeable differences of TPM values of each gene between the cells infected with low- and high-passaged shaan virus. However, the nucleocapsid (N) and matrix (M) gene-associated transcripts were shown to be differently measured among the host cells. Conclusion: In this regard, we tried to investigate certain selected mutation patterns by host switching using shaan virus isolate in MARC-145 cells. Therefore, this study provided potential evidence for host-specific selective mutation patterns by cell types as well as host of cells for shaan virus evolution.
Project description:The transcriptional response of the Mexican Lime to two different isolates of the Citrus Tristeza Virus was evaluated. Virus Isolates were T305, which provokes severe symptoms in Lime plants, and isolate T385 which does not result in any visible symptoms. Five Lime plants were inoculated with each virus isolate, and four healthy plants were used to create a pooled control. Citrus custom arrays were hybridized with an infected sample and the pooled control, using a dyeswap design. In total, the experiment comprised hybridizations with twenty microarrays, ten for each virus isolate, including five biological replicates, each technically duplicated by dye-swapping.
Project description:Affymetrix single nucleotide polymorphism (SNP) array data were collected to study genome-wide patterns of genomic variation across a broad geographical range of Island Southeast Asian populations. This region has experienced an extremely complex admixture history. Initially settled ~50,000 years ago, Island Southeast Asia has since been the recipient of multiple waves of population movements, most recently by Austronesian-speaking groups ultimately from Neolithic mainland Asia and later arrivals during the historic era from India and the Middle East. We have genotyped SNPs in ~500 individuals from 30 populations spanning this entire geographical region, from communities close to mainland Asia through to New Guinea. Particular attention has been paid to genomic data that are informative for population history, including the role of recent arrivals during the historic era and admixture with archaic hominins.