Functional annotation of novel lineage-specific genes using co-expression and promoter analysis

<p>Abstract</p> <p>Background</p> <p>The diversity of placental architectures within and among mammalian orders is believed to be the result of adaptive evolution. Although, the genetic basis for these differences is unknown, some may arise from rapidly diverging and lineage-specific genes. Previously, we identified 91 novel lineage-specific transcripts (LSTs) from a cow term-placenta cDNA library, which are excellent candidates for adaptive placental functions acquired by the ruminant lineage. The aim of the present study was to infer functions of previously uncharacterized lineage-specific genes (LSGs) using co-expression, promoter, pathway and network analysis.</p> <p>Results</p> <p>Clusters of co-expressed genes preferentially expressed in liver, placenta and thymus were found using 49 previously uncharacterized LSTs as seeds. Over-represented composite transcription factor binding sites (TFBS) in promoters of clustered LSGs and known genes were then identified computationally. Functions were inferred for nine previously uncharacterized LSGs using co-expression analysis and pathway analysis tools. Our results predict that these LSGs may function in cell signaling, glycerophospholipid/fatty acid metabolism, protein trafficking, regulatory processes in the nucleus, and processes that initiate parturition and immune system development.</p> <p>Conclusions</p> <p>The placenta is a rich source of lineage-specific genes that function in the adaptive evolution of placental architecture and functions. We have shown that co-expression, promoter, and gene network analyses are useful methods to infer functions of LSGs with heretofore unknown functions. Our results indicate that many LSGs are involved in cellular recognition and developmental processes. Furthermore, they provide guidance for experimental approaches to validate the functions of LSGs and to study their evolution.</p

Old and New Stories: Revelations from Functional Analysis of the Bovine Mammary Transcriptome during the Lactation Cycle

The cow mammary transcriptome was explored at −30, −15, 1, 15, 30, 60, 120, 240, and 300 d relative to parturition. A total of 6,382 differentially expressed genes (DEG) at a false discovery rate ≤0.001 were found throughout lactation. The greatest number of DEG (>3,500 DEG) was observed at 60 and 120 d vs. −30 d with the largest change between consecutive time points observed at −15 vs. 1 d and 120 vs. 240 d. Functional analysis of microarray data was performed using the Dynamic Impact Approach (DIA). The DIA analysis of KEGG pathways uncovered as the most impacted and induced ‘Galactose metabolism’, ‘Glycosylphosphatidylinositol (GPI)-anchor biosynthesis’, and ‘PPAR signaling’; whereas, ‘Antigen processing and presentation’ was among the most inhibited. The integrated interpretation of the results suggested an overall increase in metabolism during lactation, particularly synthesis of carbohydrates and lipid. A marked degree of utilization of amino acids as energy source, an increase of protein export, and a decrease of the protein synthesis machinery as well cell cycle also were suggested by the DIA analysis. The DIA analysis of Gene Ontology and other databases uncovered an induction of Golgi apparatus and angiogenesis, and the inhibition of both immune cell activity/migration and chromosome modifications during lactation. All of the highly-impacted and activated functions during lactation were evidently activated at the onset of lactation and inhibited when milk production declined. The overall analysis indicated that the bovine mammary gland relies heavily on a coordinated transcriptional regulation to begin and end lactation. The functional analysis using DIA underscored the importance of genes associated with lactose synthesis, lipid metabolism, protein synthesis, Golgi, transport, cell cycle/death, epigenetic regulation, angiogenesis, and immune function during lactation

Gene network and pathway analysis of bovine mammary tissue challenged with Streptococcus uberis reveals induction of cell proliferation and inhibition of PPARγ signaling as potential mechanism for the negative relationships between immune response and lipid metabolism

<p>Abstract</p> <p>Background</p> <p>Information generated via microarrays might uncover interactions between the mammary gland and <it>Streptococcus uberis </it>(<b><it>S. uberis</it></b>) that could help identify control measures for the prevention and spread of <it>S. uberis </it>mastitis, as well as improve overall animal health and welfare, and decrease economic losses to dairy farmers. The main objective of this study was to determine the most affected gene networks and pathways in mammary tissue in response to an intramammary infection (<b>IMI</b>) with <it>S. uberis </it>and relate these with other physiological measurements associated with immune and/or metabolic responses to mastitis challenge with <it>S. uberis </it>O140J.</p> <p>Results</p> <p><it>Streptococcus uberis </it>IMI resulted in 2,102 (1,939 annotated) differentially expressed genes (<b>DEG</b>). Within this set of DEG, we uncovered 20 significantly enriched canonical pathways (with 20 to 61 genes each), the majority of which were signaling pathways. Among the most inhibited were <it>LXR/RXR Signaling </it>and <it>PPARα/RXRα Signaling</it>. Pathways activated by IMI were <it>IL-10 Signaling </it>and <it>IL-6 Signaling </it>which likely reflected counter mechanisms of mammary tissue to respond to infection. Of the 2,102 DEG, 1,082 were up-regulated during IMI and were primarily involved with the immune response, e.g., <it>IL6</it>, <it>TNF</it>, <it>IL8, IL10, SELL, LYZ</it>, and <it>SAA3</it>. Genes down-regulated (1,020) included those associated with milk fat synthesis, e.g., <it>LPIN1, LPL, CD36</it>, and <it>BTN1A1</it>. Network analysis of DEG indicated that <it>TNF </it>had positive relationships with genes involved with immune system function (e.g., <it>CD14, IL8, IL1B</it>, and <it>TLR2</it>) and negative relationships with genes involved with lipid metabolism (e.g., <it>GPAM</it>, <it>SCD</it>, <it>FABP4</it>, <it>CD36</it>, and <it>LPL</it>) and antioxidant activity (<it>SOD1</it>).</p> <p>Conclusion</p> <p>Results provided novel information into the early signaling and metabolic pathways in mammary tissue that are associated with the innate immune response to <it>S. uberis </it>infection. Our study indicated that IMI challenge with <it>S. uberis </it>(strain O140J) elicited a strong transcriptomic response, leading to potent activation of pro-inflammatory pathways that were associated with a marked inhibition of lipid synthesis, stress-activated kinase signaling cascades, and PPAR signaling (most likely PPARγ). This latter effect may provide a mechanistic explanation for the inverse relationship between immune response and milk fat synthesis.</p

Functional and gene network analyses of transcriptional signatures characterizing pre-weaned bovine mammary parenchyma or fat pad uncovered novel inter-tissue signaling networks during development

<p>Abstract</p> <p>Background</p> <p>The neonatal bovine mammary fat pad (<b>MFP</b>) surrounding the mammary parenchyma (<b>PAR</b>) is thought to exert proliferative effects on the PAR through secretion of local modulators of growth induced by systemic hormones. We used bioinformatics to characterize transcriptomics differences between PAR and MFP from ~65 d old Holstein heifers. Data were mined to uncover potential crosstalk through the analyses of signaling molecules preferentially expressed in one tissue relative to the other.</p> <p>Results</p> <p>Over 9,000 differentially expressed genes (<b>DEG</b>; False discovery rate ≤ 0.05) were found of which 1,478 had a ≥1.5-fold difference between PAR and MFP. Within the DEG highly-expressed in PAR vs. MFP (n = 736) we noted significant enrichment of functions related to cell cycle, structural organization, signaling, and DNA/RNA metabolism. Only actin cytoskeletal signaling was significant among canonical pathways. DEG more highly-expressed in MFP vs. PAR (n = 742) belong to lipid metabolism, signaling, cell movement, and immune-related functions. Canonical pathways associated with metabolism and signaling, particularly immune- and metabolism-related were significantly-enriched. Network analysis uncovered a central role of <it>MYC</it>, <it>TP53</it>, and <it>CTNNB1 </it>in controlling expression of DEG highly-expressed in PAR vs. MFP. Similar analysis suggested a central role for <it>PPARG</it>, <it>KLF2</it>, <it>EGR2</it>, and <it>EPAS1 </it>in regulating expression of more highly-expressed DEG in MFP vs. PAR. Gene network analyses revealed putative inter-tissue crosstalk between cytokines and growth factors preferentially expressed in one tissue (e.g., <it>ANGPTL1</it>, <it>SPP1</it>, <it>IL1B </it>in PAR vs. MFP; <it>ADIPOQ</it>, <it>IL13</it>, <it>FGF2</it>, <it>LEP </it>in MFP vs. PAR) with DEG preferentially expressed in the other tissue, particularly transcription factors or pathways (e.g., <it>MYC</it>, <it>TP53</it>, and actin cytoskeletal signaling in PAR vs. MFP; <it>PPARG </it>and LXR/RXR Signaling in MFP vs. PAR).</p> <p>Conclusions</p> <p>Functional analyses underscored a reciprocal influence in determining the biological features of MFP and PAR during neonatal development. This was exemplified by the potential effect that the signaling molecules (cytokines, growth factors) released preferentially (i.e., more highly-expressed) by PAR or MFP could have on molecular functions or signaling pathways enriched in the MFP or PAR. These bidirectional interactions might be required to coordinate mammary tissue development under normal circumstances or in response to nutrition.</p

Systems Biology Analysis of Brucella Infected Peyers Patch Reveals Rapid Invasion with Modest Transient Perturbations of the Host Transcriptome

Brucella melitensis causes the most severe and acute symptoms of all Brucella species in human beings and infects hosts primarily through the oral route. The epithelium covering domed villi of jejunal-ileal Peyer’s patches is an important site of entry for several pathogens, including Brucella. Here, we use the calf ligated ileal loop model to study temporal in vivo Brucella-infected host molecular and morphological responses. Our results document Brucella bacteremia occurring within 30 min after intraluminal inoculation of the ileum without histopathologic traces of lesions. Based on a system biology Dynamic Bayesian Network modeling approach (DBN) of microarray data, a very early transient perturbation of the host enteric transcriptome was associated with the initial host response to Brucella contact that is rapidly averted allowing invasion and dissemination. A detailed analysis revealed active expression of Syndecan 2, Integrin alpha L and Integrin beta 2 genes, which may favor initial Brucella adhesion. Also, two intestinal barrier-related pathways (Tight Junction and Trefoil Factors Initiated Mucosal Healing) were significantly repressed in the early stage of infection, suggesting subversion of mucosal epithelial barrier function to facilitate Brucella transepithelial migration. Simultaneously, the strong activation of the innate immune response pathways would suggest that the host mounts an appropriate protective immune response; however, the expression of the two key genes that encode innate immunity anti-Brucella cytokines such as TNF-a and IL12p40 were not significantly changed throughout the study. Furthermore, the defective expression of Toll-Like Receptor Signaling pathways may partially explain the lack of proinflammatory cytokine production and consequently the absence of morphologically detectable inflammation at the site of infection. Cumulatively, our results indicate that the in vivo pathogenesis of the early infectious process of Brucella is primarily accomplished by compromising the mucosal immune barrier and subverting critical immune response mechanisms.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

Systems Biology Analysis of Gene Expression during In Vivo Mycobacterium avium paratuberculosis Enteric Colonization Reveals Role for Immune Tolerance

Survival and persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in the intestinal mucosa is associated with host immune tolerance. However, the initial events during MAP interaction with its host that lead to pathogen survival, granulomatous inflammation, and clinical disease progression are poorly defined. We hypothesize that immune tolerance is initiated upon initial contact of MAP with the intestinal Peyer's patch. To test our hypothesis, ligated ileal loops in neonatal calves were infected with MAP. Intestinal tissue RNAs were collected (0.5, 1, 2, 4, 8 and 12 hrs post-infection), processed, and hybridized to bovine gene expression microarrays. By comparing the gene transcription responses of calves infected with the MAP, informative complex patterns of expression were clearly visible. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis, and genes were grouped into the specific pathways and gene ontology categories to create a holistic model. This model revealed three different phases of responses: i) early (30 min and 1 hr post-infection), ii) intermediate (2, 4 and 8 hrs post-infection), and iii) late (12 hrs post-infection). We describe here the data that include expression profiles for perturbed pathways, as well as, mechanistic genes (genes predicted to have regulatory influence) that are associated with immune tolerance. In the Early Phase of MAP infection, multiple pathways were initiated in response to MAP invasion via receptor mediated endocytosis and changes in intestinal permeability. During the Intermediate Phase, perturbed pathways involved the inflammatory responses, cytokine-cytokine receptor interaction, and cell-cell signaling. During the Late Phase of infection, gene responses associated with immune tolerance were initiated at the level of T-cell signaling. Our study provides evidence that MAP infection resulted in differentially regulated genes, perturbed pathways and specifically modified mechanistic genes contributing to the colonization of Peyer's patch

昭和20年代, 教室の日常と研究余話(第922回千葉医学会例会・第13回千葉精神科集談会)

<p>Unique MAA Arcs and Mechanistic Genes in Tight Junction Pathway.</p

Role of SPI-1 Secreted Effectors in Acute Bovine Response to Salmonella enterica Serovar Typhimurium: A Systems Biology Analysis Approach

Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis with diarrhea and polymorphonuclear cell (PMN) influx into the intestinal mucosa in humans and calves. The Salmonella Type III Secretion System (T3SS) encoded at Pathogenicity Island I translocates Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into epithelial cells and is required for induction of diarrhea. These effector proteins act together to induce intestinal fluid secretion and transcription of C-X-C chemokines, recruiting PMNs to the infection site. While individual molecular interactions of the effectors with cultured host cells have been characterized, their combined role in intestinal fluid secretion and inflammation is less understood. We hypothesized that comparison of the bovine intestinal mucosal response to wild type Salmonella and a SipA, SopABDE2 effector mutant relative to uninfected bovine ileum would reveal heretofore unidentified diarrhea-associated host cellular pathways. To determine the coordinated effects of these virulence factors, a bovine ligated ileal loop model was used to measure responses to wild type S. Typhimurium (WT) and a ΔsipA, sopABDE2 mutant (MUT) across 12 hours of infection using a bovine microarray. Data were analyzed using standard microarray analysis and a dynamic Bayesian network modeling approach (DBN). Both analytical methods confirmed increased expression of immune response genes to Salmonella infection and novel gene expression. Gene expression changes mapped to 219 molecular interaction pathways and 1620 gene ontology groups. Bayesian network modeling identified effects of infection on several interrelated signaling pathways including MAPK, Phosphatidylinositol, mTOR, Calcium, Toll-like Receptor, CCR3, Wnt, TGF-β, and Regulation of Actin Cytoskeleton and Apoptosis that were used to model of host-pathogen interactions. Comparison of WT and MUT demonstrated significantly different patterns of host response at early time points of infection (15 minutes, 30 minutes and one hour) within phosphatidylinositol, CCR3, Wnt, and TGF-β signaling pathways and the regulation of actin cytoskeleton pathway

Pharmacogenetic allele nomenclature: International workgroup recommendations for test result reporting

This manuscript provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward

Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular “uncoupling”. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way