Genetic Structure of Duplicated Sequences Revealed by Genotyping Single Sperm
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Minjie Luo
PROVIDER: E-GEOD-11957 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Background. The bacterial foodborne pathogen Campylobacter jejuni is a common cause of acute gastroenteritis and is also associated with the postinfectious neuropathies, Guillain-Barré and Miller Fisher syndromes. This study described the use of multilocus sequence typing and DNA microarrays to examine the genetic content of a collection of South African C. jejuni strains, recovered from patients with enteritis, Guillain-Barré or Miller Fisher syndromes. Methodology/Principal Findings. The comparative genomic analysis by using multilocus sequence typing and DNA microarrays demonstrated that the South African strains with Penner heat-stable (HS) serotype HS:41 were clearly distinct from the other South African strains. Further analysis of the DNA microarray data demonstrated that the serotype HS:41 strains from South African GBS and enteritis patients are highly similar in gene content. Interestingly, the South African HS:41 strains were distinct in gene content when compared to serotype HS:41 strains from other geographical locations due to the presence of genomic islands, referred to as Campylobacter jejuni integrated elements. Only the genomic integrated element CJIE1, a Campylobacter Mu-like prophage, was present in the South African HS:41 strains whereas absent in the closely-related HS:41 strains from Mexico. A more distantly-related HS:41 strain from Canada possessed both genomic integrated elements CJIE1 and CJIE2. Conclusion/Significance. These findings demonstrated that these C. jejuni integrated elements may contribute to the differentiation of closely-related C. jejuni strains. In addition, the presence of bacteriophage-related genes in CJIE1 may probably contribute to increasing the genomic diversity of these C. jejuni strains. This comparative genomic analysis of the foodborne pathogen C. jejuni provides fundamental information that potentially could lead to improved methods for analyzing the epidemiology of disease outbreaks and their sources. The genomic diversity of 33 South African, 2 Mexican and 1 Canadian Campylobacter jejuni strains from humans were examined by microarray-based comparative genomic indexing (CGI) analysis. The CGI analysis allowed the assessment of CDS content for each C. jejuni strain relative to the C. jejuni DNA microarray, which comprises ORFs from strains NCTC 11168, RM1221. ORFs were spotted in duplicate. Genomic DNA from strains NCTC 11168/RM1221 were used as a reference DNA and competitively hybridized with genomic DNA from each of the other C. jejuni strains. Two or three replicates for each strain were performed. Data normalization was performed as in Parker et al. J Clin Microbiol 2006, 44(11):4125-4135.
2010-05-27 | E-GEOD-9862 | biostudies-arrayexpress
Project description:MicroRNAs (miRs) contribute to both neuronal and immune cell fate, but their involvement in inter-tissue communication remained unexplored. The brain, via vagal secretion of acetylcholine (ACh), suppresses peripheral inflammation by intercepting cytokine production; therefore, we predicted that microRNAs targeting acetylcholinesterase (AChE) can attenuate inflammation. Here, we report that inflammatory stimuli induce leukocyte over-expression of the AChE-targeting miR-132. Injected LNA-modified anti-miR-132 oligonucleotide depleted miR-132 levels while elevating AChE in mouse circulation and tissues. In transfected cells, a mutated 3âUTR miR-132 binding site increased AChE mRNA expression, whereas cells infected with a lentivirus expressing pre-miR-132 showed suppressed AChE. Transgenic mice over-expressing 3'UTR-null AChE showed excessive inflammatory mediators and impaired cholinergic anti-inflammatory regulation, in spite of substantial miR-132 up-regulation in brain and bone marrow. Our findings identify the AChE mRNA-targeting miR-132 as a functional regulator of the brain-to-body resolution of inflammation, opening new avenues for study and therapeutic manipulations of the neuro-immune dialogue. Pathogen-negative leukopacks (Buffy coat) obtained from the Blood Bank at the Hadassah Medical Center, Ein Kerem were transferred into 50 ml conical tubes (~10 ml/tube) and diluted 1:2 with Dulbecco's phosphate buffered saline (PBS, Biological Industries, Beit Haemek, Israel). The diluted blood was gently layered on top of 20 ml of lymphocyte separation medium (LSM)/Ficoll (MP Biomedicals, Solon, OH) without mixing the layers. Following centrifugation (500g, 30 min, 20°C, without brake), three layers were obtained (plasma & platelets, lymphocytes, erythrocytes & granulocytes). The upper phase was drawn with a Pasteur pipette until reaching 2 mm above the interphase cloud, and then 5 to 10 ml interphase lymphocytes were drawn gently with a 1ml pipette, washed once with 45 ml PBS, and centrifuged (400g , 10 min, room temp.). Pellets from all tubes were resuspended in a total of 40 ml PBS. An aliquot of cells was counted in a hemacytometer using 0.5% Trypan Blue Solution (Biological Industries) to determine cell number and viability. Cells were then centrifuged (200g, 5 min, room temp) and resuspended at a concentration of ~ 5 X 106 cells/ml in RPMI-1640 (Sigma, St. Louis, MO) supplemented with 2.5% heat-inactivated (56°C, 30 min) human serum (Sigma). 10 ml of cell suspension were plated in 75 ml flasks and incubated (2 hrs, 37°C, 5% CO¬2). Non-adherent cells were then removed and the adherent monocytes washed 3 times with warm PBS. Cells were incubated for 2hr in 10 ml per dish of RPMI-1640 supplemented with LPS-free 10% foetal bovine serum (FBS), 2 mM L-Glutamine and 0.5% penicillin/streptomycin (GIBCO, Carlsbad, CA) and washed 3 times with warm PBS and 10 ml of complete RPMI supplemented with 10 U/ml granulocyte-monocyte colony stimulating factor (GM-CSF, Sigma, G5035) added every 2-3 days. By ~day 7 monocytes were differentiated to macrophages. Escherichia coli LPS (Sigma) was added to the growth medium to a final concentration of 1μg/ml, which was found optimal to induce innate immune reaction. For comparison, 1 μM of the TLR9 ligand CpG 2006 (type B; Microsynth GmbH) were added for 24 hr. Cells were washed 2-3 times with cold PBS, then incubated in 5 ml of cold filtered PBS + EDTA 2 mM (pH = 7.2) for 10-15 min on ice. Flasks were then tapped firmly to loosen cell attachment, and cells were gently scraped and collected by centrifugation (4°C, 800 rpm, 10 min). Short RNAs isolated from LPS-treated, CpG-treated and control cells were labeled with CyDyes 3 and 5 (each with a dye-swap), and hybridized with slides with spotted miRvana probe set (Ambion) as described for each sample.
2010-05-19 | E-GEOD-19094 | biostudies-arrayexpress
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2010-06-09 | E-GEOD-2419 | biostudies-arrayexpress
Project description:RNA isolated from the cultures treated with 0 and 50 micromolar AFB1 at 15, 30, 60, 90, 120 min was used for microarray experiments. For each array hybridization experiment, RNAs from the treated sample and its corresponding time-matched control were co-hybridized to arrays and respectively quantified in different channels. A dye swap strategy was used to eliminate the dye bias. Keywords: time-course
2010-06-09 | E-GEOD-2420 | biostudies-arrayexpress
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2010-06-06 | E-GEOD-2112 | biostudies-arrayexpress
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2012-12-31 | E-GEOD-35137 | biostudies-arrayexpress
Project description:Three different experimental approaches were evaluated for discrimination of genomic variance in and between duplicated sequences using 48 markers in duplicon regions and 17 SNPs in unique sequences previously characterized in another study. We found only the method high-throughput single sperm typing could conclusively resolve the alleles of all markers. Resulting data from single sperm analysis were also used to examine the genetic structure of duplicon markers in the human population. Single sperm typing can be a rapid, efficient and accurate method for initial screening and assessment of genetic variation and for detailed genetic analysis of duplicon markers. Keywords: Genotyping
2008-07-25 | GSE11957 | GEO
Project description:Trichophyton rubrum, an anthropophilic and cosmopolitan fungus, is the most common agent of superficial mycoses, causing rarely deep dermatophytosis in immunocompromised hosts. In this study, an infection condition of T. rubrum was modeled by adding human skin sections into a limited medium containing glucose to monitor T. rubrum gene expression patterns using cDNA microarrays on a global level. We found that exposure to human skin resulted in up-regulation of the expression levels of T. rubrum genes related to many cellular and biological processes, including transcription and translation, metabolism and secondary transport, stress response, and signaling pathways. These results provide a reference set of T. rubrum genes whose expression patterns change upon infection and reveal previously unknown genes that probably corresponding to proteins that should be considered as virulence factor candidates and potential new drug targets for T. rubrum infections.
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Project description:Tropospheric ozone causes severe oxidative stress in plants. To investigate the transcriptional responsiveness of adult trees to ozone, fully-expanded sun and shade leaves of mature beech trees were harvested at four time points over the entire vegetation period in 2005 and 2006. Microarray analyses were conducted on leaves from trees grown in the field under ambient and twice-ambient ozone concentrations at Kranzberger Forst (Bavarian). Beech trees changed their transcript levels in response to ozone. In the years 2005 and 2006 different transcription patterns were observed; this may have been a result of different weather conditions and ozone uptake. Furthermore, we obtained differences in mRNA expression patterns between shade and sun leaves. In the ozone-treated sun leaves of 2005, slightly up- and down-regulated transcript levels were detected, particularly in the spring and autumn, whereas shade leaves clearly exhibited reduced mRNA-levels, particularly at the end of the vegetation period. In 2006, this pattern could not be confirmed, and in the autumn, four other transcripts were slightly up-regulated in ozone-treated shade leaves. In addition, two additional transcripts were found to be influenced in sun leaves in the spring/summer. While we detected changes in the levels of only a few transcripts, the observed effects were not identical in both years. In conclusion, elevated ozone exhibited very small influence on the transcription levels of genes of mature beech trees. The study was carried out at the Kranzberger Forst research site (near Freising, Germany: 48°25’08’’N, 11°39’41’””E, 485m (Pretzsch et al., 1998) in a mixed 60-year old stand (closed canopy) with about 30m high European beech (Fagus sylvatica) and Norway spruce (Picea abies) trees. Free-air ozone fumigation started in May 2000 at double the ambient ozone concentrations with a cut-off at 150 nl l-1 (Werner and Fabian, 2002), thereby avoiding acute damage to the leaves. The ozone concentrations were measured at four heights within the fumigated space and were additionally monitored with 200 passive samplers (Werner and Fabian, 2002). In 2005 the AOT40 value under twice ambient ozone was 64.3 μmol mol-1 h and in 2006 69.0 μmol mol-1 h. Detailed ozone concentration data over the growing seasons have been reported elsewhere (Gielen et al., 2007; Kitao et al., 2009). Sun and shade leaves from 60-year-old European beech trees were harvested in a total of 8 sampling campaigns in 2005 and 2006. Using scaffolding, five leaves of sun crown (height of about 25 m) and five shade (height of about 19 m) leaves were taken from each of five control and ozone-treated trees. The sampling was carried out in May, June, August, and September of 2005 and in June, August, September and October of 2006. To avoid diurnal effects, the samples were always taken around 11 a.m. For each tree, the four leaves (sun or shade) were combined, frozen in liquid nitrogen and stored at -80 °C until RNA isolation. For one time point we had five microarrays and five dye-swaps for each of sun and shade leaves. The probes of the trees under ambient ozone were labelled with Cy3 and probes of the trees under 2x ambient were labelled with Cy5. For every pair of trees a dye control were carried out, where the control trees were labelled with Cy5 and the ozone-treated one with Cy3. For statistical analysis a coefficient of variation about all microarrays of one time point was calculated using Acuity 4.0 microarray informatics software [Axon Instruments]. We used only those spots that had a coefficient of variation < 50 and were present on at least half of identical slides. The ozone-changed transcript level of genes were expressed in the median values as log2 ratios.
2011-10-20 | E-GEOD-17413 | biostudies-arrayexpress
Project description:Tropospheric ozone causes severe oxidative stress in plants. To investigate the transcriptional responsiveness of adult trees to ozone, fully-expanded sun and shade leaves of mature beech trees were harvested at four time points over the entire vegetation period in 2005 and 2006. Microarray analyses were conducted on leaves from trees grown in the field under ambient and twice-ambient ozone concentrations at Kranzberger Forst (Bavarian). Beech trees changed their transcript levels in response to ozone. In the years 2005 and 2006 different transcription patterns were observed; this may have been a result of different weather conditions and ozone uptake. Furthermore, we obtained differences in mRNA expression patterns between shade and sun leaves. In the ozone-treated sun leaves of 2005, slightly up- and down-regulated transcript levels were detected, particularly in the spring and autumn, whereas shade leaves clearly exhibited reduced mRNA-levels, particularly at the end of the vegetation period. In 2006, this pattern could not be confirmed, and in the autumn, four other transcripts were slightly up-regulated in ozone-treated shade leaves. In addition, two additional transcripts were found to be influenced in sun leaves in the spring/summer. While we detected changes in the levels of only a few transcripts, the observed effects were not identical in both years. In conclusion, elevated ozone exhibited very small influence on the transcription levels of genes of mature beech trees. The study was carried out at the Kranzberger Forst research site (near Freising, Germany: 48°25’08’’N, 11°39’41’””E, 485m (Pretzsch et al., 1998) in a mixed 60-year old stand (closed canopy) with about 30m high European beech (Fagus sylvatica) and Norway spruce (Picea abies) trees. Free-air ozone fumigation started in May 2000 at double the ambient ozone concentrations with a cut-off at 150 nl l-1 (Werner and Fabian, 2002), thereby avoiding acute damage to the leaves. The ozone concentrations were measured at four heights within the fumigated space and were additionally monitored with 200 passive samplers (Werner and Fabian, 2002). In 2005 the AOT40 value under twice ambient ozone was 64.3 μmol mol-1 h and in 2006 69.0 μmol mol-1 h. Detailed ozone concentration data over the growing seasons have been reported elsewhere (Gielen et al., 2007; Kitao et al., 2009). Sun and shade leaves from 60-year-old European beech trees were harvested in a total of 8 sampling campaigns in 2005 and 2006. Using scaffolding, five leaves of sun crown (height of about 25 m) and five shade (height of about 19 m) leaves were taken from each of five control and ozone-treated trees. The sampling was carried out in May, June, August, and September of 2005 and in June, August, September and October of 2006. To avoid diurnal effects, the samples were always taken around 11 a.m. For each tree, the four leaves (sun or shade) were combined, frozen in liquid nitrogen and stored at -80 °C until RNA isolation. For one time point we had five microarrays and five dye-swaps for each of sun and shade leaves. The probes of the trees under ambient ozone were labelled with Cy3 and probes of the trees under 2x ambient were labelled with Cy5. For every pair of trees a dye control were carried out, where the control trees were labelled with Cy5 and the ozone-treated one with Cy3. For statistical analysis a coefficient of variation about all microarrays of one time point was calculated using Acuity 4.0 microarray informatics software [Axon Instruments]. We used only those spots that had a coefficient of variation < 50 and were present on at least half of identical slides. The ozone-changed transcript level of genes were expressed in the median values as log2 ratios.
2011-10-20 | E-GEOD-17411 | biostudies-arrayexpress