Guanxiang Liang

Potential Regulatory Role of MicroRNAs in the Development of Bovine Gastrointestinal Tract during Early Life

This study aimed to investigate the potential regulatory role of miRNAs in the development of gastrointestinal tract (GIT) during the early life of dairy calves. Rumen and small intestinal (mid-jejunum and ileum) tissue samples were collected from newborn (30 min after birth; n = 3), 7-day-old (n = 6), 21-day-old (n = 6), and 42-day-old (n = 6) dairy calves. The miRNA profiling was performed using Illumina RNA-sequencing and the temporal and regional differentially expressed miRNAs were further validated using qRT-PCR. Analysis of 16S rRNA gene copy numbers was used to quantify total bacteria, Bifidobacterium and Lactobacillus species. The expression of miR-143 was abundant in all three gut regions, at all time points and it targets genes involved primarily in the proliferation of connective tissue cells and muscle cells, suggesting a role in regulating rapid tissue development during the early life of calves. The expression of miR-146, miR-191, miR-33, miR-7, miR-99/100, miR-486, miR-145, miR-196 and miR-211 displayed significant temporal differences (FDR <0.05), while miR-192/215, miR-194, miR-196, miR-205 and miR-31 revealed significant regional differences (FDR <0.05). The expression levels of miR-15/16, miR-29 and miR-196 were positively correlated with the copy numbers of 16S rRNA gene of Bifidobacterium or Lactobacillus species or both (P<0.05). Functional analysis using Ingenuity Pathway Analysis identified the above mentioned differentially expressed miRNAs as potential regulators of gut tissue cell proliferation and differentiation. The bacterial density-associated miRNAs were identified as modulators of the development of lymphoid tissues (miR-196), maturation of dendritic cells (miR-29) and development of immune cells (miR-15/16). The present study revealed temporal and regional changes in miRNA expression and a correlation between miRNA expression and microbial population in the GIT during the early life, which provides further evidence for another mechanism by which host-microbial interactions play a role in regulating gut development.

Heat-treated colostrum feeding promotes beneficial bacteria colonization in the small intestine of neonatal calves

The present study investigated the effect of heat-treated colostrum feeding on the bacterial colonization in calf small intestine of neonatal calves within the first 12h of life. Newborn Holstein bull calves (n=32) were assigned to 3 treatment groups and fed with either fresh colostrum (FC, n=12) or heat-treated (60°C, 60 min) colostrum (HC, n=12) soon after birth, whereas the control (NC, n=8) group did not receive colostrum or water. Small intestinal tissues and contents were collected from proximal jejunum, distal jejunum, and ileum at 6 and 12h after birth, following euthanasia. Quantitative real time-PCR was used to explore the colonization of total bacteria, Lactobacillus, Bifidobacterium, and Escherichia coli. The feeding of colostrum soon after birth increased the colonization of total bacteria in calf gut within the first 12h compared with NC. In contrast, the prevalence of Lactobacillus was lower in HC and FC compared to NC. Remarkable changes in the prevalence of small intestinal tissue-attached Bifidobacterium were observed with the feeding of HC, but not that in small intestinal contents. The prevalence of Bifidobacterium was 3.2 and 5.2 fold higher in HC than FC and NC, respectively, at 6h. Although the feeding of FC did not enhance the prevalence of tissue-attached Bifidobacterium at 6h compared with NC, it displayed a gradual increase over the time that was higher than NC, but similar to that of HC at 12h. Moreover, the colonization of E. coli was drastically reduced in HC calves compared with FC and NC. Thus, the present study suggests that the feeding of HC enhances the colonization of Bifidobacterium but lessens E. coli in the calf small intestine immediately postpartum compared with that of FC and NC. The increased colonization of beneficial bacteria along with the decreased colonization of potential pathogens in calf gut may also diminish the neonatal calf diarrhea when calves are fed heat-treated colostrum soon after birth.

Transcriptome profiling of the rumen epithelium of beef cattle differing in residual feed intake

BackgroundFeed efficient cattle consume less feed and produce less environmental waste than inefficient cattle. Many factors are known to contribute to differences in feed efficiency, however the underlying molecular mechanisms are largely unknown. Our study aimed to understand how host gene expression in the rumen epithelium contributes to differences in residual feed intake (RFI), a measure of feed efficiency, using a transcriptome profiling based approach.ResultsThe rumen epithelial transcriptome from highly efficient (low (L-) RFI, n = 9) and inefficient (high (H-) RFI, n = 9) Hereford x Angus steers was obtained using RNA-sequencing. There were 122 genes differentially expressed between the rumen epithelial tissues of L- and H- RFI steers (p < 0.05) with 85 up-regulated and 37 down-regulated in L-RFI steers. Functional analysis of up-regulated genes revealed their involvement in acetylation, remodeling of adherens junctions, cytoskeletal dynamics, cell migration, and cell turnover. Additionally, a weighted gene co-expression network analysis (WGCNA) identified a significant gene module containing 764 genes that was negatively correlated with RFI (r = −0.5, p = 0.03). Functional analysis revealed significant enrichment of genes involved in modulation of intercellular adhesion through adherens junctions, protein and cell turnover, and cytoskeletal organization that suggest possible increased tissue morphogenesis in the L-RFI steers. Additionally, the L-RFI epithelium had increased expression of genes involved with the mitochondrion, acetylation, and energy generating pathways such as glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation. Further qPCR analysis of steers with different RFI (L-RFI, n = 35; M-RFI, n = 34; H-RFI, n = 35) revealed that the relative mitochondrial genome copy number per cell of the epithelium was positively correlated with RFI (r = 0.21, p = 0.03).ConclusionsOur results suggest that the rumen epithelium of L-RFI (efficient) steers may have increased tissue morphogenesis that possibly increases paracellular permeability for the absorption of nutrients and increased energy production to support the energetic demands of increased tissue morphogenesis compared to those of H-RFI (inefficient) animals. Greater expression of mitochondrial genes and lower relative mitochondrial genome copy numbers suggest a greater rate of transcription in the rumen epithelial mitochondria of L-RFI steers. Understanding how host gene expression profiles are associated with RFI could potentially lead to identification of mechanisms behind this trait, which are vital to develop strategies for the improvement of cattle feed efficiency.

Egg ovotransferrin-derived ACE inhibitory peptide IRW increases ACE2 but decreases proinflammatory genes expression in mesenteric artery of spontaneously hypertensive rats

Scope Egg ovotransferrin‐derived angiotensin converting enzyme (ACE) inhibitory peptide IRW was previously shown to reduce blood pressure in spontaneously hypertensive rats through reduced vascular inflammation and increased nitric oxide‐mediated vasorelaxation. The main objective of the present study was to investigate the molecular mechanism of this peptide through transcriptome analysis by RNAseq technique. Methods and results Total RNA was extracted from kidney and mesenteric arteries; the RNAseq libraries (from untreated and IRW‐treated groups) were constructed and subjected to sequence using HiSeq 2000 system (Illumina) system. A total of 12 764 and 13 352 genes were detected in kidney and mesenteric arteries, respectively. The differentially expressed (DE) genes between untreated and IRW‐treated groups were identified and the functional analysis through ingenuity pathway analysis revealed a greater role of DE genes identified from mesenteric arteries than that of kidney in modulating various cardiovascular functions. Subsequent qPCR analysis further confirmed that IRW significantly increased the expression of ACE‐2, ABCB‐1, IRF‐8, and CDH‐1 while significantly decreased the expression ICAM‐1 and VCAM‐1 in mesenteric arteries. Conclusion Our research showed for the first time that ACE inhibitory peptide IRW could contribute to its antihypertensive activity through increased ACE2 and decreased proinflammatory genes expression.

Roles of small RNAs in the effects of nutrition on apoptosis and spermatogenesis in the adult testis

We tested whether reductions in spermatozoal quality induced by under-nutrition are associated with increased germ cell apoptosis and disrupted spermatogenesis, and whether these effects are mediated by small RNAs. Groups of 8 male sheep were fed for a 10% increase or 10% decrease in body mass over 65 days. Underfeeding increased the number of apoptotic germ cells (P < 0.05) and increased the expression of apoptosis-related genes (P < 0.05) in testicular tissue. We identified 44 miRNAs and 35 putative piRNAs that were differentially expressed in well-fed and underfed males (FDR < 0.05). Some were related to reproductive system development, apoptosis (miRNAs), and sperm production and quality (piRNAs). Novel-miR-144 (miR-98), was found to target three apoptotic genes (TP53, CASP3, FASL). The proportion of miRNAs as a total of small RNAs was greater in well-fed males than in underfed males (P < 0.05) and was correlated (r = 0.8, P < 0.05) with the proportion of piRNAs in well-fed and underfed males. In conclusion, the reductions in spermatozoal quality induced by under-nutrition are caused, at least partly, by disruptions to Sertoli cell function and increased germ cell apoptosis, mediated by changes in the expression of miRNAs and piRNAs.

Genome-wide whole blood microRNAome and transcriptome analyses reveal miRNA-mRNA regulated host response to foodborne pathogen Salmonella infection in swine.

To understand the role of miRNAs in regulating genes involved in host response to bacterial infection and shedding of foodborne pathogens, a systematic profiling of miRNAs and mRNAs from the whole blood of pigs upon Salmonella challenge was performed. A total of 62 miRNAs were differentially expressed post infection (false discovery rate <0.1). An integrative analysis of both the differentially expressed miRNAs and mRNAs using sequence-based miRNA target prediction and negative correlation of miRNA-mRNA profiles helped identify miRNA-mRNA networks that may potentially regulate host response to Salmonella infection. From these networks, miR-214 and miR-331-3p were identified as new candidates potentially associated with Salmonella infection. An miRNA seed sequence analysis suggested that these miRNAs regulate several critical immune-related genes including SLC11A1, PIGE-108A11.3 and VAV2. We showed that challenged pigs had reduced miR-214 expression and increased miR-331-3p expression in the whole blood. Furthermore, the expression of the proposed targets of miR-214 (SLC11A1 and PIGE-108A11.3) increased while that of the proposed target of miR-331-3p (VAV2) decreased following challenge (expression changes confirmed by in vitro assays). Based on these observations, we propose potential roles for miR-214 and miR-331-3p in regulation of immune responses to Salmonella infection.

Model systems to analyze the role of miRNAs and commensal microflora in bovine mucosal immune system development

Information is rapidly accumulating regarding the role of miRNAs as key regulators of immune system development and function. It is also increasingly evident that miRNAs play an important role in host-pathogen interactions through regulation of both innate and acquired immune responses. Little is known, however, about the specific role of miRNAs in regulating normal development of the mucosal immune system, especially during the neonatal period. Furthermore, there is limited knowledge regarding the possible role the commensal microbiome may play in regulating mucosal miRNAs expression, although evidence is emerging that a variety of enteric pathogens influence miRNA expression. The current review focuses on recent information that miRNAs play an important role in regulating early development of the bovine mucosal immune system. A possible role for the commensal microbiome in regulating mucosal development by altering miRNA expression is also discussed. Finally, we explore the potential advantages of using the newborn calf as a model to determine how interactions between developmental programming, maternal factors in colostrum, and colonization of the gastrointestinal tract by commensal bacteria may alter mucosal miRNA expression and immune development. Identifying the key factors that regulate mucosal miRNA expression is critical for understanding how the balance between protective immunity and inflammation is maintained to ensure optimal gastrointestinal tract function and health of the whole organism.

Comparative Transcriptomic Analysis of Rectal Tissue from Beef Steers Revealed Reduced Host Immunity in Escherichia coli O157:H7 Super-Shedders.

Super-shedder cattle are a major disseminator of E. coli O157:H7 into the environment, and the terminal rectum has been proposed as the primary E. coli O157:H7 colonization site. This study aimed to identify host factors that are associated with the super-shedding process by comparing transcriptomic profiles in rectal tissue collected from 5 super-shedder cattle and 4 non-shedder cattle using RNA-Seq. In total, 17,859 ± 354 genes and 399 ± 16 miRNAs were detected, and 11,773 genes were expressed in all animals. Fifty-eight differentially expressed (DE) genes (false discovery rate < 0.05) including 11 up-regulated and 47 down-regulated (log 2 (fold change) ranged from -5.5 to 4.2), and 2 up-regulated DE miRNAs (log 2 (fold change) = 2.1 and 2.5, respectively) were identified in super-shedders compared to non-shedders. Functional analysis of DE genes revealed that 31 down-regulated genes were potentially associated with reduced innate and adaptive immune functions in super-shedders, including 13 lymphocytes membrane receptors, 3 transcription factors and 5 cytokines, suggesting the decreased key host immune functions in the rectal tissue of super-shedders, including decreased quantity and migration of immune cells such as lymphocytes, neutrophils and dendritic cells. The up-regulation of bta-miR-29d-3p and the down regulation of its predicted target gene, regulator of G-protein signaling 13, suggested a potential regulatory role of this miRNA in decreased migration of lymphocytes in super-shedders. Based on these findings, the rectal tissue of super-shedders may inherently exhibit less effective innate and adaptive immune protection. Further study is required to confirm if such effect on host immunity is due to the nature of the host itself or due to actions mediated by E. coli O157:H7.

Altered microRNA expression and pre-mRNA splicing events reveal new mechanisms associated with early stage Mycobacterium avium subspecies paratuberculosis infection.

The molecular regulatory mechanisms of host responses to Mycobacterium avium subsp. paratuberculosis (MAP) infection during the early subclinical stage are still not clear. In this study, surgically isolated ileal segments in newborn calves (n = 5) were used to establish in vivo MAP infection adjacent to an uninfected control intestinal compartment. RNA-Seq was used to profile the whole transcriptome (mRNAs) and the microRNAome (miRNAs) of ileal tissues collected at one-month post-infection. The most related function of the differentially expressed mRNAs between infected and uninfected tissues was “proliferation of endothelial cells”, indicating that MAP infection may lead to the over-proliferation of endothelial cells. In addition, 46.2% of detected mRNAs displayed alternative splicing events. The pre-mRNA of two genes related to macrophage maturation (monocyte to macrophage differentiation-associated) and lysosome function (adenosine deaminase) showed differential alternative splicing events, suggesting that specific changes in the pre-mRNA splicing sites may be a mechanism by which MAP escapes host immune responses. Moreover, 9 miRNAs were differentially expressed after MAP infection. The integrated analysis of microRNAome and transcriptome revealed that these miRNAs might regulate host responses to MAP infection, such as “proliferation of endothelial cells” (bta-miR-196 b), “bacteria recognition” (bta-miR-146 b), and “regulation of the inflammatory response” (bta-miR-146 b).

Nutrition affects Sertoli cell function but not Sertoli cell numbers in sexually mature male sheep

We tested whether the reversible effects of nutrition on spermatogenesis in sexually mature sheep were mediated by Sertoli cells. Rams were fed with diets designed to achieve a 10% increase (High), no change (Maintenance) or a 10% decrease (Low) in body mass after 65 days. At the end of treatment, testes were lighter in the Low than the High group (PP<0.05) in the expression of seven Sertoli cell-specific genes. Under-nutrition appeared to reverse cellular differentiation leading to disruption of tight-junction morphology. In conclusion, in sexually mature sheep, reversible reductions in testis mass and spermatogenesis caused by under-nutrition were associated with impairment of basic aspects of Sertoli cell function but not with changes in the number of Sertoli cells.