Dietary carbohydrate sources differently prime the microbial ecosystem but not the epithelial gene expression profile along the complete gut of young calves.

BACKGROUND: Recent data indicated similar growth performance of young calves fed solely high-quality hay instead of a starter diet based on starchy ingredients. Yet, providing exclusively such distinct carbohydrate sources during early life might specifically prime the microbiota and gene expression along the gut of young calves, which remains to be explored. We investigated the effects of starter diets differing in carbohydrate composition, that is medium- or high-quality hay and without or with 70% concentrate supplementation (on fresh matter basis), across the gastrointestinal tract (GIT) of weaned Holstein calves (100 ± 4 days of age) using 16 S rRNA gene sequencing and analyses of short-chain fatty acids and host epithelial gene expressions. RESULTS: The concentrate supplementation drastically decreased microbial diversity throughout the gut, which was also true to a much lesser extent for high-quality hay when compared to medium-quality hay in the foregut. Similarly, the factor concentrate strongly shaped the diet-associated common core microbiota, which was substantially more uniform along the gut with concentrate supplementation. The fermentation profile shifted towards less acetate but more propionate with concentrate supplementation in almost all gut sections, corresponding with higher abundances of starch-utilizing bacteria, while major fibrolytic clusters declined. Noteworthy, the n-butyrate proportion decreased in the rumen and increased in the colon with concentrate, showing an opposite, gut site-dependent effect. Both dietary factors modestly influenced the host epithelial gene expression. CONCLUSIONS: Concentrate supplementation clearly primed the microbial ecosystem on a starch-targeted fermentation with characteristic genera occupying this niche along the entire GIT of calves, whereas the microbial differentiation due to hay quality was less distinct. Overall, changes in the microbial ecosystem were only marginally reflected in the targeted transcriptional profile of the host epithelium.

MET receptor serves as a promising target in melanoma brain metastases.

The development of brain metastases hallmarks disease progression in 20-40% of melanoma patients and is a serious obstacle to therapy. Understanding the processes involved in the development and maintenance of melanoma brain metastases (MBM) is critical for the discovery of novel therapeutic strategies. Here, we generated transcriptome and methylome profiles of MBM showing high or low abundance of infiltrated Iba1high tumor-associated microglia and macrophages (TAMs). Our survey identified potential prognostic markers of favorable disease course and response to immune checkpoint inhibitor (ICi) therapy, among them APBB1IP and the interferon-responsive gene ITGB7. In MBM with high ITGB7/APBB1IP levels, the accumulation of TAMs correlated significantly with the immune score. Signature-based deconvolution of MBM via single sample GSEA revealed enrichment of interferon-response and immune signatures and revealed inflammation, stress and MET receptor signaling. MET receptor phosphorylation/activation maybe elicited by inflammatory processes in brain metastatic melanoma cells via stroma cell-released HGF. We found phospho-METY1234/1235 in a subset of MBM and observed a marked response of brain metastasis-derived cell lines (BMCs) that lacked druggable BRAF mutations or developed resistance to BRAF inhibitors (BRAFi) in vivo to MET inhibitors PHA-665752 and ARQ197 (tivantinib). In summary, the activation of MET receptor in brain colonizing melanoma cells by stromal cell-released HGF may promote tumor self-maintenance and expansion and might counteract ICi therapy. Therefore, therapeutic targeting of MET possibly serves as a promising strategy to control intracranial progressive disease and improve patient survival.

Integrated microbiota-host-metabolome approaches reveal adaptive ruminal changes to prolonged high-grain feeding and phytogenic supplementation in cattle.

Diets rich in readily fermentable carbohydrates primarily impact microbial composition and activity, but can also impair the ruminal epithelium barrier function. By combining microbiota, metabolome, and gene expression analysis, we evaluated the impact of feeding a 65% concentrate diet for 4 weeks, with or without a phytogenic feed additive (PFA), on the rumen ecosystem of cattle. The breaking point for rumen health seemed to be the second week of high grain (HG) diet, with a dysbiosis characterized by reduced alpha diversity. While we did not find changes in histological evaluations, genes related with epithelial proliferation (IGF-1, IGF-1R, EGFR, and TBP) and ZO-1 were affected by the HG feeding. Integrative analyses allowed us to define the main drivers of difference for the rumen ecosystem in response to a HG diet, identified as ZO-1, MyD88, and genus Prevotella 1. PFA supplementation reduced the concentration of potentially harmful compounds in the rumen (e.g. dopamine and 5-aminovaleric acid) and increased the tolerance of the epithelium toward the microbiota by altering the expression of TLR-2, IL-6, and IL-10. The particle-associated rumen liquid microbiota showed a quicker adaptation potential to prolonged HG feeding compared to the other microenvironments investigated, especially by the end of the experiment.

Probiotic Bacillus Strains Enhance T Cell Responses in Chicken.

Banning antibiotic growth promotors and other antimicrobials in poultry production due to the increasing antimicrobial resistance leads to increased feeding of potential alternatives such as probiotics. However, the modes of action of those feed additives are not entirely understood. They could act even with a direct effect on the immune system. A previously established animal-related in vitro system using primary cultured peripheral blood mononuclear cells (PBMCs) was applied to investigate the effects of immune-modulating feed additives. Here, the immunomodulation of different preparations of two probiotic Bacillus strains, B. subtilis DSM 32315 (BS), and B. amyloliquefaciens CECT 5940 (BA) was evaluated. The count of T-helper cells and activated T-helper cells increased after treatment in a ratio of 1:3 (PBMCs: Bacillus) with vital BS (CD4+: p < 0.05; CD4+CD25+: p < 0.01). Furthermore, vital BS enhanced the proliferation and activation of cytotoxic T cells (CD8+: p < 0.05; CD8+CD25+: p < 0.05). Cell-free probiotic culture supernatants of BS increased the count of activated T-helper cells (CD4+CD25+: p < 0.1). UV-inactivated BS increased the proportion of cytotoxic T cells significantly (CD8+: p < 0.01). Our results point towards a possible involvement of secreted factors of BS in T-helper cell activation and proliferation, whereas it stimulates cytotoxic T cells presumably through surface contact. We could not observe any effect on B cells after treatment with different preparations of BS. After treatment with vital BA in a ratio of 1:3 (PBMCs:Bacillus), the count of T-helper cells and activated T-helper cells increased (CD4+: p < 0.01; CD4+CD25+: p < 0.05). Cell-free probiotic culture supernatants of BA as well as UV-inactivated BA had no effect on T cell proliferation and activation. Furthermore, we found no effect of BA preparations on B cells. Overall, we demonstrate that the two different Bacillus strains enhanced T cell activation and proliferation, which points towards an immune-modulating effect of both strains on chicken immune cells in vitro. Therefore, we suggest that administering these probiotics can improve the cellular adaptive immune defense in chickens, thereby enabling the prevention and reduction of antimicrobials in chicken farming.

MicroRNAs in Ruminants and Their Potential Role in Nutrition and Physiology.

The knowledge of how diet choices, dietary supplements, and feed intake influence molecular mechanisms in ruminant nutrition and physiology to maintain ruminant health, is essential to attain. In the present review, we focus on the role of microRNAs in ruminant health and disease; additionally, we discuss the potential of circulating microRNAs as biomarkers of disease in ruminants and the state of technology for their detection, also considering the major difficulties in the transition of biomarker development from bench to clinical practice. MicroRNAs are an inexhaustible class of endogenous non-protein coding small RNAs of 18 to 25 nucleotides that target either the 3' untranslated (UTR) or coding region of genes, ensuring a tight post-transcriptionally controlled regulation of gene expression. The development of new "omics" technologies facilitated a fresh perspective on the nutrition-to-gene relationship, incorporating more extensive data from molecular genetics, animal nutrition, and veterinary sciences. MicroRNAs might serve as important regulators of metabolic processes and may present the inter-phase between nutrition and gene regulation, controlled by the diet. The development of biomarkers holds the potential to revolutionize veterinary practice through faster disease detection, more accurate ruminant health monitoring, enhanced welfare, and increased productivity. Finally, we summarize the latest findings on how microRNAs function as biomarkers, how technological paradigms are reshaping this field of research, and how platforms are being used to identify novel biomarkers. Numerous studies have demonstrated a connection between circulating microRNAs and ruminant diseases such as mastitis, tuberculosis, foot-and-mouth disease, fasciolosis, and metabolic disorders. Therefore, the identification and analysis of a small number of microRNAs can provide crucial information about the stage of a disease, etiology, and prognosis.

Diet and phytogenic supplementation substantially modulate the salivary proteome in dairy cows.

Phytogenic compounds may influence salivation or salivary properties. However, their effects on the bovine salivary proteome have not been evaluated. We investigated changes in the bovine salivary proteome due to transition from forage to high-concentrate diet, with and without supplementation with a phytogenic feed additive. Eight non-lactating cows were fed forage, then transitioned to a 65% concentrate diet (DM basis) over a week. Cows were control (n = 4, CON) or supplemented with a phytogenic feed additive (n = 4, PHY). Proteomic analysis was conducted using liquid chromatography coupled with mass spectrometry. We identified 1233 proteins; 878 were bovine proteins, 189 corresponded to bacteria, and 166 were plant proteins. Between forage and high-concentrate, 139 proteins were differentially abundant (P < 0.05), with 48 proteins having a log2FC difference > |2|. The salivary proteome reflected shifts in processes involving nutrient utilization, body tissue accretion, and immune response. Between PHY and CON, 195 proteins were differently abundant (P < 0.05), with 37 having a log2FC difference > |2|; 86 proteins were increased by PHY, including proteins involved in smell recognition. Many differentially abundant proteins correlated (r > |0.70|) with salivary bicarbonate, total mucins or pH. Results provide novel insights into the bovine salivary proteome using a non-invasive approach, and the association of specific proteins with major salivary properties influencing rumen homeostasis. SIGNIFICANCE: Phytogenic compounds may stimulate salivation due to their olfactory properties, but their effects on the salivary proteome have not been investigated. We investigated the effect of high-concentrate diets and supplementation with a phytogenic additive on the salivary proteome of cows. We show that analysis of cows' saliva can be a non-invasive approach to detect effects occurring not only in the gut, but also systemically including indications for gut health and immune response. Thus, results provide unique insights into the bovine salivary proteome, and will have a crucial contribution to further understand animal response in terms of nutrient utilization and immune activity due to the change from forage to a high-energy diet. Additionally, our findings reveal changes due to supplementation with a phytogenic feed additive with regard to health and olfactory stimulation. Furthermore, findings suggest an association between salivary proteins and other components like bicarbonate content.

Decoding molecular programs in melanoma brain metastases.

Melanoma brain metastases (MBM) variably respond to therapeutic interventions; thus determining patient's prognosis. However, the mechanisms that govern therapy response are poorly understood. Here, we use a multi-OMICS approach and targeted sequencing (TargetSeq) to unravel the programs that potentially control the development of progressive intracranial disease. Molecularly, the expression of E-cadherin (Ecad) or NGFR, the BRAF mutation state and level of immune cell infiltration subdivides tumors into proliferative/pigmented and invasive/stem-like/therapy-resistant irrespective of the intracranial location. The analysis of MAPK inhibitor-naive and refractory MBM reveals switching from Ecad-associated into NGFR-associated programs during progression. NGFR-associated programs control cell migration and proliferation via downstream transcription factors such as SOX4. Moreover, global methylome profiling uncovers 46 differentially methylated regions that discriminate BRAFmut and wildtype MBM. In summary, we propose that the expression of Ecad and NGFR sub- classifies MBM and suggest that the Ecad-to-NGFR phenotype switch is a rate-limiting process which potentially indicates drug-response and intracranial progression states in melanoma patients.

Characterization of presence and activity of microRNAs in the rumen of cattle hints at possible host-microbiota cross-talk mechanism.

MicroRNAs (miRNAs), as important post-transcriptional regulators, are ubiquitous in various tissues. The aim of this exploratory study was to determine the presence of miRNAs in rumen fluid, and to investigate the possibility of miRNA-mediated cross-talk within the ruminal ecosystem. Rumen fluid samples from four cannulated Holstein cows were collected during two feeding regimes (forage and high-grain diet) and DNA and RNA were extracted for amplicon and small RNA sequencing. Epithelial biopsies were simultaneously collected to investigate the co-expression of miRNAs in papillae and rumen fluid. We identified 377 miRNAs in rumen fluid and 638 in rumen papillae, of which 373 were shared. Analysis of microbiota revealed 20 genera to be differentially abundant between the two feeding regimes, whereas no difference in miRNAs expression was detected. Correlations with at least one genus were found for 170 miRNAs, of which, 39 were highly significant (r > |0.7| and P < 0.01). Both hierarchical clustering of the correlation matrix and WGCNA analysis identified two main miRNA groups. Putative target and functional prediction analysis for the two groups revealed shared pathways with the predicted metabolic activities of the microbiota. Hence, our study supports the hypothesis of a cross-talk within the rumen at least partly mediated by miRNAs.

Bovine rumen epithelial miRNA-mRNA dynamics reveals post-transcriptional regulation of gene expression upon transition to high-grain feeding and phytogenic supplementation.

The rumen epithelium has a pivotal role in nutrient uptake and host health. This study aimed to explore the role of microRNAs (miRNAs) in the epithelial transcriptome during diet transition from forage to high-grain feeding and the modulation through supplementation with a phytogenic feed additive. Rumen biopsies were collected from 9 ruminally-cannulated non-lactating Holstein cows fed a baseline forage diet (FD) and then transitioned to high-grain feeding (HG; 65% concentrate on a dry matter basis). Cows were randomly allocated into a control group (CON, n = 5) and a group supplemented with a phytogenic feed additive (PHY, n = 4). MiRNA and mRNA sequencing was performed in parallel and transcripts were analyzed for differential expression, pathway enrichment analysis, and miRNA-mRNA interaction networks. We identified 527 miRNAs shared by all samples of the rumen epithelium, from which, bta-miR-21-5p, bta-miR-143 and bta-miR-24-3p were the most expressed. Six miRNAs were differentially expressed between CON and PHY and 8 miRNAs between FD and HG feeding, which were mainly associated with fat metabolism. Transcriptome analysis identified 9481 differentially expressed genes (DEGs) between FD and HG, whereas PHY supplementation resulted in 5 DEGs. DEGs were mainly involved in epithelium development and morphogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with tricarboxylic acid and short chain fatty acid (SCFA) metabolism were enriched in DEGs between diets. MiRNA target prediction and anti-correlation analysis was used to construct networks and identify DEGs targeted by DE miRNAs responsive to diet or PHY. This study allowed the identification of potential miRNA regulation mechanisms of gene expression during transition from FD to HG feeding and phytogenic supplementation, evidencing a direct role of miRNAs in host responses to nutrition.

Chicken Immune Cell Assay to Model Adaptive Immune Responses In Vitro.

Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, gained through controlled but animal-related in vitro systems using primary cultured peripheral blood mononuclear cells (PBMCs) will allow the development of targeted nutrition strategies. Moreover, it could contribute to the prevention of infectious diseases and the usage of antimicrobials, and further promote the health of the animals. However, to our knowledge, a protocol for the isolation of PBMCs with reduced thrombocyte count from chicken blood and subsequent cell culture over several days to assess the effects of immunomodulating compounds is not available. Therefore, we established an optimized protocol for blood sampling and immune cell isolation, culture, and phenotyping for chicken PBMCs. For blood sampling commercial Na-citrate tubes revealed the highest count of vital cells compared to commercial Li-heparin (p < 0.01) and K3EDTA (p < 0.05) tubes. Using combined dextran and ficoll density gradient separation, the thrombocyte count was significantly reduced (p < 0.01) compared to slow-speed centrifugation with subsequent ficoll. For cell culture, the supplementation of RPMI-1640 medium with 10% chicken serum resulted in the lowest relative cell count of thrombocytes compared to fetal calf serum (FCS) (p < 0.05). To validate the ability of the cell culture system to respond to stimuli, concanavalin A (conA) was used as a positive control. The optimized protocol allows the isolation and cultivation of vital PBMCs with reduced thrombocyte count from chicken blood for subsequent investigation of the modes of action of immunomodulating compounds.

High dosage of zinc modulates T-cells in a time-dependent manner within porcine gut-associated lymphatic tissue.

Zn serves as a powerful feed additive to reduce post-weaning diarrhoea in pigs. However, the mechanisms responsible for Zn-associated effects on the adaptive immune responses following feeding of a very high dosage of Zn remain elusive. In this study, we examined the T-cell response in gut-associated lymphatic tissues of seventy-two weaned piglets. Piglets received diets with 57 mg Zn/kg (low Zn concentration, LZn), 164 mg Zn/kg (medium Zn concentration, MZn) or 2425 mg Zn/kg (high Zn concentration, HZn) mg Zn/kg feed for 1, 2 or 4 weeks. We observed that feeding the HZn diet for 1 week increased the level of activated T-helper cells (CD4+ and CD8α dim) compared with feeding MZn and LZn (P<0·05). In addition, we observed higher transcript amounts of interferon γ and T-box 21 (TBET) in the HZn group compared with the MZn and LZn groups (P<0·05). A gene set enrichment analysis revealed an over-representation of genes associated with 'cytokine signalling in immune system'. Remarkably, feeding of a very high Zn dosage led to a switch in the immune response after 2 weeks. We detected higher relative cell counts of CD4+CD25high regulatory T-helper cells (P<0·05) and a higher expression of forkhead box P3 (FOXP3) transcripts (P<0·05). After 4 weeks of feeding a high-dosage Zn diet, the relative CD4+ T-cell count (P<0·05) and the relative CD8ß + T-cell count (P<0·1) were reduced compared with the MZn group. We hypothesise that after 1 week the cellular T-helper 1 response is switched on and after 2 weeks it is switched off, leading to decreased numbers of T-cells.

Feeding a high dosage of zinc oxide affects suppressor of cytokine gene expression in Salmonella Typhimurium infected piglets.

Suppressor of cytokine signaling (SOCS) proteins play an important role in the regulation of the immune response by inhibiting cytokines. Here we investigated the effects of zinc oxide fed at three different dosages (LZN=57ppm, MZN=167ppm, HZN=2425ppm) to weaned piglets that were or were not orally infected with Salmonella enterica serovar Typhimurium DT 104. We detected higher expression of SOCS3 six days after weaning for all analyzed piglets, regardless of the infection or the zinc feeding, suggesting a stress induced immune response. Whereas, SOCS1 showed only higher transcript amounts in S. Typhimurium infected piglets, especially the LZN group. This might indicate an infection regulating effect of zinc oxide in the infection model. After 42days of infection, the expression of SOCS2, SOCS4, and SOCS7 was increased only in animals fed the highest concentrations of zinc oxide, while non-infected piglets at the age of 56days showed no regulation for these genes. The up-regulation of SOCS genes in the mesenteric lymph nodes of piglets fed a diet with a very high concentration of zinc over 6 weeks suggests that such treatments may impair the immune response.

Feeding of Enterococcus faecium NCIMB 10415 Leads to Intestinal miRNA-423-5p-Induced Regulation of Immune-Relevant Genes.

Probiotics are widely used in human and animal health, but little is known about the mode of action of probiotics. One possible mechanism at the molecular level could be an influence on microRNAs (miRNAs) and the related immune-relevant target genes. Here, we analyzed differential expression of miRNA and potential target genes of ileal and jejunal lymphatic tissues from Enterococcus faeciumNCIMB 10415-fed piglets versus untreated controls by using next-generation sequencing. We identified miR-423-5p as being greatly affected by the treatment group (2.32-fold;P= 0.014). Validation by reverse transcription-quantitative PCR (RT-qPCR) confirmed a significant upregulation of miR-423-5p (2.11-fold;P= 0.03) and, additionally, downregulation of the important immune-relevant immunoglobulin lambda light C region (IGLC) (0.61-fold;P= 0.03) and immunoglobulin kappa constant (IGKC) (0.69-fold;P= 0.04) target genes. Expression analysis of miR-423-5p and IGLC at different age points shows a clear anti correlated relationship. Luciferase reporter assays with a HeLa cell line verified IGLC as a target of miR-423-5p. The results provided evidence for an effect of feeding of E. faeciumon the expression of miR-423-5p and on the regulation of the IGLC gene through miR-423-5p. This might be a possible mode of action of E. faeciumon immune cell regulation in the small intestine.

The Zinc Concentration in the Diet and the Length of the Feeding Period Affect the Methylation Status of the ZIP4 Zinc Transporter Gene in Piglets.

High doses of zinc oxide are commonly used in weaned pig diets to improve performance and health. Recent reports show that this may also lead to an imbalanced zinc homeostasis in the animal. For a better understanding of the regulatory mechanisms of different zinc intakes, we performed a feeding experiment to assess potential epigenetic regulation of the ZIP4 gene expression via DNA methylation in the small intestine of piglets. Fifty-four piglets were fed diets with 57 (LZn), 164 (NZn) or 2,425 (HZn) mg Zn/kg feed for one or four weeks. The ZIP4 expression data provided significant evidence for counter-regulation of zinc absorption with higher dietary zinc concentrations. The CpG +735 in the second exon had a 56% higher methylation in the HZn group compared to the others after one week of feeding (8.0·10-4 < p < 0.035); the methylation of this CpG was strongly negatively associated with the expression of the long ZIP4 transcripts (p < 0.007). In the LZn and NZn diets, the expression of the long ZIP4 transcripts were lower after four vs. one week of feeding (2.9·10-4 < p < 0.017). The strongest switch leading to high DNA methylation in nearly all analysed regions was dependent on feeding duration or age in all diet groups (3.7·10-10 < p < 0.099). The data suggest that DNA methylation serves as a fine-tuning mechanism of ZIP4 gene regulation to maintain zinc homeostasis. Methylation of the ZIP4 gene may play a minor role in the response to very high dietary zinc concentration, but may affect binding of alternate zinc-responsive transcription factors.

Probiotic Treatment Decreases the Number of CD14-Expressing Cells in Porcine Milk Which Correlates with Several Intestinal Immune Parameters in the Piglets.

Modulating the mucosal immune system of neonates by probiotic treatment of their mothers is a promising approach which can only be investigated through the use of animal models. Here, we used sows and their piglets to investigate the impact of a bacterial treatment on the sow's milk and on the neonate piglet intestinal immune system. In previous experiments, feed supplementation of sows with the probiotic Enterococcus faecium NCIMB 10415 during pregnancy and lactation had been shown to affect intestinal microbiota and cytokine expression of the offspring during the suckling and weaning periods. We therefore investigated the composition of the milk from treated sows in comparison to samples from a control group. In treated sows, the amount of lactose increased, and the somatic cell numbers were reduced. In all milk samples, the percentage of cells expressing membranous CD14 (mCD14) was greater than the fractions of immune cells, indicating expression of mCD14 on mammary epithelial cells. However, in the milk of E. faecium-treated sows, mCD14(+) cells were reduced. Furthermore, the number of CD14(+) milk cells was positively correlated with the percentages of B cells and activated T cells in the ileal MLN of the piglets. This study provides evidence for the expression of mCD14 by the porcine mammary epithelium, and suggests an immunological effect of mCD14(+) milk cells on the piglets' intestinal immune system. Our study further suggests that mCD14(+) mammary epithelial cell populations can be modulated by probiotic feed supplementation of the sow.