Analysis of differences in the rumen microbiome and metabolic function in prepartum dairy cows with different body condition scores.

BACKGROUND: The rumen is a crucial digestive organ for dairy cows. The rumen microbiota assists in the digestion of plant feed through microbe-mediated fermentation, during which the plant feed is transformed into nutrients for the cow's use. Variations in the composition and function of the rumen microbiome affect the energy utilization efficiency of dairy cows, which is one of the reasons for the varying body condition scores (BCSs). This study focused on prepartum Holstein dairy cows to analyze differences in rumen microbiota and metabolites among cows with different BCSs. Twelve prepartum dairy cows were divided into two groups, low BCS (LBCS, BCS = 2.75, n = 6) and high BCS (HBCS, BCS = 3.5, n = 6), to explore differences in microbial composition and metabolites. RESULTS: In the HBCS group, the genera within the phylum Firmicutes exhibited stronger correlations and greater abundances. Phyla such as Firmicutes, Patescibacteria, Acidobacteriota, Euryarchaeota, and Desulfobacterota, in addition to most of their constituent microbial groups, were significantly more abundant in the HBCS group than in the LBCS group. At the genus level, the abundances of Anaerovibrio, Veillonellaceae_UCG_001, Ruminococcus_gauvreauii_group, Blautia, Eubacterium, Prevotellaceae_YAB2003_group, Schwartzia, and Halomonas significantly increased in the HBCS group. The citrate cycle, involved in carbohydrate metabolism, exhibited a significant enrichment trend, with a notable increase in the abundance of its key substrate, citrate, in the HBCS group. This increase was significantly positively correlated with the differential bacterial genera. CONCLUSION: In this study, prepartum dairy cows with higher BCS exhibited greater abundance of Firmicutes. This study provides theoretical support for microbiological research on dairy cows with different BCSs and suggests that regulating the rumen microbiome could help maintain prepartum dairy cows within an optimal BCS range.

Altered rumen microbiome and correlations of the metabolome in heat-stressed dairy cows at different growth stages.

IMPORTANCE: Heat stress is one of the main causes of economic losses in the dairy industry worldwide; however, the mechanisms associated with the metabolic and microbial changes in heat stress remain unclear. Here, we characterized both the changes in metabolites, rumen microbial communities, and their functional potential indices derived from rumen fluid and serum samples from cows at different growth stages and under different climates. This study highlights that the rumen microbe may be involved in the regulation of lipid metabolism by modulating the fatty acyl metabolites. Under heat stress, the changes in the metabolic status of growing heifers, heifers, and lactating cows were closely related to arachidonic acid metabolism, fatty acid biosynthesis, and energy metabolism. Moreover, this study provides new markers for further research to understand the effects of heat stress on the physiological metabolism of Holstein cows and the time-dependent changes associated with growth stages.

Effects of heat stress on 16S rDNA, metagenome and metabolome in Holstein cows at different growth stages.

Heat stress is an important issue in dairy cattle feeding management affecting summer health and economic efficiency. This experiment combined 16S rDNA sequencing(3,864,982 tags, 30 sequencing data), metagenomic sequencing(1,269,441,128 reads, 18 sequencing data), metabolomics analysis(72 sequencing data) and blood index analysis. Ten cows in each animal type (growing heifers, heifers, and lactating cows) were selected for sample collection in April and August. Here, we characterized both the changes in metabolites, rumen microbial communities and their functional potential and the effects of heat stress on serum biochemical, immune, oxidative stress, and hormonal indices derived from rumen fluid and serum samples from cows during different growth stages and in different climates. The generated data expand the resources for the rumen microbiome related to heat stress and age and provide useful datasets for research on developing therapeutic strategies to achieve high summer milk production in cows. These datasets will help researchers study the effects of heat stress on the physiological metabolism of Holstein cows and the time-dependent changes associated with growth stages.

Effect of Saccharomyces cerevisiae culture mitigates heat stress-related dame in dairy cows by multi-omics.

The effect of heat stress on ruminants is an important issue. In recent years, the growth of the Chinese dairy industry has rapidly increased, generating RMB 468,738 million revenue in 2021. A decreased milk yield is the most recognized impact of heat stress on dairy cows and results in significant economic loss to dairy producers. Heat stress also lowers immunity and antioxidant capacity and changes the bacterial composition and metabolites of the rumen. The purpose of this study was to investigate the effect of addition Saccharomyces cerevisiae culture on heat-stressed cows. The impact of S. cerevisiae culture on microbiota composition, functional profiles, and metabolomics was assessed in heat-stressed cows. A total of 45 Holstein cows in mid-lactation were selected and randomly divided into three groups (15 cows per group). Groups D-C, D-A, and D-B were fed with the basal diet, the basal diet + first S. cerevisiae culture 100 g/day, and the basal diet + second S. cerevisiae culture 30 g/day, respectively. The trial lasted 60 days. There was an increased abundance of the Phylum Firmicutes in the rumen of heat-stressed dairy cows fed with S. cerevisiae, of which four genera had significantly higher abundance, Ruminococcus_gauvreauii_group, Butyrivibrio_2, Moryella, and Ruminiclostridium_6. At the functional level, ten pathways differed significantly between the three groups (P < 0.05), with an increase in fatty acid biosynthesis, fatty acid metabolism, PPAR signaling pathway, ferroptosis, and biotin metabolism in the treatment groups. More differential metabolites were found in the D-C and D-A groups than in the D-C and D-B groups. These results indicate that S. cerevisiae cultures can influence the health status of heat-stressed cows by modulating rumen microbial composition, function, and metabolites, thereby improving rumen cellulolytic capacity. This study can provide or offer suggestions or recommendations for the development and utilization of feed additives.

Effects of Saccharomyces Cerevisiae Cultures on Performance and Immune Performance of Dairy Cows During Heat Stress.

The dairy farming industry is facing massive economic losses as heat stress continues to rise. The purpose of this study was to see how feeding Saccharomyces cerevisiae culture (SC) influences productive performance, lactation performance, serum biochemical indexes, hormonal level, antioxidant capacity, and immune function in mid-lactating cows during heat stress. Forty-five healthy mid-lactation dairy cows with comparable milk yield, lactation days, and parity were randomly divided into 3 groups (15 cows in each group). The control group (CON) was fed the basal diet, while the treatment groups were fed the basal diet + first Saccharomyces cerevisiae culture 100 g/d (SC-1) and the basal diet + second Saccharomyces cerevisiae culture 30 g/d (SC-2), respectively. The SC-1 and SC-2 groups with SC added in the treatment groups reduced rectal temperature and respiratory rate in heat-stressed cows (P < 0.05). The milk yield of SC-1 and SC-2 treatment groups was significantly higher than that of CON (P < 0.05). Except for somatic cell count, which was significantly lower in SC-1 and SC-2 than in CON (P < 0.05), there were no significant differences in the milk components. The addition of SC: (i) increased serum urea levels (P < 0.05), but there was no significant difference in glucose, total cholesterol, alanine transaminase, aspartate aminotransferase, total protein, albumin and alkaline phosphatase levels (P > 0.05); (ii) increased serum levels of immunoglobulin-A, immunoglobulin-G, immunoglobulin M, interleukin-4, interleukin-10 and heat shock protein-70 (P < 0.05), while decreasing serum levels of interleukin-1ß, interleukin-6, interleukin-2, interferon-γ and tumor necrosis factor-α (P < 0.05); (iii) increased total antioxidant capacity, glutathione peroxidase and superoxide dismutase in serum (P < 0.05), while decreasing malondialdehyde; (iv) increased serum levels of glucocorticoids, insulin, cortisol and prolactin (P < 0.05), while decreasing the serum levels of triiodothyronine and thyroxine (P < 0.05). In conclusion, under the current experimental conditions, the addition of SC can reduce rectal temperature and respiratory rate in heat-stressed mid-lactation cows, reduce the number of somatic cells in milk and improve the mid-lactation cow performance. In addition, SC addition to the diet can raise serum urea levels, regulate serum hormone levels, boost antioxidant capacity in mid-lactation cows, and boost overall immunity.

miR-181c protects CsA-induced renal damage and fibrosis through inhibiting EMT.

Cyclosporine A (CsA), a widely used immunosuppressive drug in organ transplantation and autoimmune disorders, frequently induces renal damage and fibrosis. Recent evidence has implicated epithelial-mesenchymal transition (EMT) in CsA-induced nephrotoxicity. Microarray analysis disclosed miR-181c as the microRNA most dramatically repressed by CsA. Downregulation of miR-181c expression at the transcriptional level by CsA is dependent on the transcription factor Nrf2. miR-181c mimics or inhibitors attenuate or aggravate CsA-induced EMT gene changes, respectively. Importantly, in Nrf2-/- mice, CsA-induced renal damage, fibrosis, and EMT gene changes are restored by miR-181c mimics. Mechanistically, we identified Notch2 as a potential target of miR-181c. Collectively, our data support the notion that miR-181c may serve as an important factor for protecting renal tissues from CsA-induced nephrotoxicity.

The differentiation of human multipotent adult progenitor cells into hepatocyte-like cells induced by coculture with human hepatocyte line L02.

PURPOSE: The aim of this study was to establish an in vitro method to purify human multipotent adult progenitor cells (hMAPCs) and assess their possible differentiation into hepatocytes by coculture with human hepatocyte line L02. METHODS: hMAPCs were isolated by magnetic activated cell sorting (MACS) depletion selection using CD45 and GlyA microbeads. After indirect or direct coculture of hMAPCs and human hepatocyte line L02, the expression of albumin (ALB), alpha-fetoprotein (AFP), cytokeratin (CK) 18, and CK19 by hMAPCs was detected by immunocytochemistry. RESULTS: With the MACS method, (5-10) × 10(4)/mL hMAPCs could be separated from 1 × 10(6)/mL bone marrow mononuclear cells. The purity of CD45-/GlyA- cells separated from bone marrow adherent cells was more than 98%, as determined by flow cytometry. In the coculture without cell-to-cell contact, hMAPCs expressed high AFP on day 1, and then tapered daily to low expression on day 7; ALB expression reached its peak on day 5, and remained high on day 7; CK18 was initially expressed on day 5 and was higher on day 7; CK19 was negative in all assays. In the coculture with cell-to-cell contact, ALB and CK18 were expressed by most cells while AFP appeared in only a few on day 5. CONCLUSION: hMAPCs were induced to differentiate into mature hepatocyte-like cells by coculture with a hepatocyte cell line, either with or without cell-to-cell contact, but the former seemed more effective.

The effects of ABCG2 on the viability, proliferation and paracrine actions of kidney side population cells under oxygen-glucose deprivation.

Bcrp1/ABCG2 is exclusively expressed in side population (SP) cells, however, it has not been fully elucidated whether it has an impact on the viability, proliferation and paracrine actions in kidney SP cells under oxygen-glucose deprivation (OGD) followed by reoxygenation. In this study, we found that 2-h OGD did not injure SP cells (sub-lethal OGD) but induced SP cells proliferation 48 and 72 h after reoxygenation; whereas 4-h OGD markedly injured the cells (lethal OGD) and led to apoptosis 24-72 h after reoxygenation. Fumitremorgin C, an inhibitor of ABCG2, attenuated both the proliferation and viability of SP cells. Sub-lethal and lethal OGD induced the increase in the secretion of vascular endothelial growth factor, insulin-like growth factor 1, hepatocyte growth factor, and stromal cell-derived factor-1α in kidney SP cells, which was inhibited by Fumitremorgin C. Collectively, these findings provide evidence for a crucial role for the ABCG2 expression in the viability, proliferation and paracrine actions of kidney SP cells after OGD.

Notch-RBP-J signaling is required by bone marrow stromal cells for the treatment of acute graft versus host disease.

Recent evidence has shown that bone marrow stromal cells (BMSCs) may exhibit immuno-suppression activities through soluble mediators and direct cell-cell contact, but how these processes are modulated has been poorly understood. In this study, we show that the Notch signaling pathway participates in the modulation of BMSCs to elicit their immuno-suppressive roles. In a murine lethal acute graft versus host disease (aGvHD) model, BMSCs deficient for RBP-J, the critical transcription factor mediating signaling from all four mammalian Notch receptors, failed to delay the development of the disease. RBP-J deficient BMSCs were not able to inhibit the proliferation and activation of allogenic T-cells. Moreover, RBP-J deficient BMSCs could not down-regulate the expression of MHC II and co-stimulation molecules CD80 and CD86 on dendritic cells (DCs). The antigen presentation capacity of DCs co-cultured with RBP-J deficient BMSCs was not impaired in contrast to wild type BMSCs. Furthermore, we showed that the productions of IL-6 and PGE2, two critical molecules mediating the immuno-suppressive activities of BMSCs, were reduced significantly in RBP-J deficient BMSCs. Both of the two molecules were importantly involved in the regulation of BMSCs by Notch signaling. In conclusion, our data suggests that the immuno-suppressive effects of BMSCs in aGvHD are dependent on Notch-RBP-J signaling, which regulates the productions of IL-6 and PGE2.

Protective effect of a polysaccharide from stem of Codonopsis pilosula against renal ischemia/reperfusion injury in rats.

In this study, we purified a homogeneous polysaccharide (S-CPPA1) with a molecular weight (Mw) of 133.2 kDa from the stem of Codonopsis pilosula for the first time. Gas chromatography (GC) analysis identified that S-CPPA1 contained glucose, galactose, and arabinose with a molar ratio of 10.5:3.4:1.7, along with a trace of mannose. Methylation analysis suggested S-CPPA1 was a branched polysaccharide, with five glucosidic linkage forms, namely (1→4)-linked Glcp (residue A), (1→6)-linked Galp (residue B), (1→2,6)-linked Glcp (residue C), (1→5)-linked Araf (residue D), and non-reducing terminal (1→)-linked Glcp (residue E). The protective effect of S-CPPA1 on kidney ischemia/reperfusion (I/R) injury was also evaluated. Blood urea nitrogen (BUN), creatinine and TNF-α levels, as well as lactate dehydrogenase (LDH) and alanine transaminase (AST) activities were elevated in the I/R group as compared to the sham group. On the other hand, S-CPPA1 treatment reversed all these biochemical indices, as well as histopathological alterations, which were induced by I/R. The findings imply that S-CPPA1 plays a causal role in the protection against I/R-induced renal injury and its renoprotective effect is probably mediated by inhibiting the proinflammatory cytokine TNF-α release.

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression.

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate- early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between- group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.

The PcG protein HPC2 inhibits RBP-J-mediated transcription by interacting with LIM protein KyoT2.

The DNA-binding protein recombination signal-binding protein-Jk (RBP-J) plays a key role in transcriptional regulation by targeting the intracellular domain of Notch (NIC) and the Epstein-Barr virus nuclear antigen 2 (EBNA2) to specific promoters. In the absence of the Notch signaling, RBP-J acts as a transcriptional suppressor through recruiting co-suppressors such as histone deacetylase (HDAC). KyoT2 is a LIM domain protein that suppresses the RBP-J-mediated transcriptional activation. In the current study, we show that the polycomb group (PcG) protein HPC2, which functions as a transcriptional suppressor, is a candidate of KyoT2-binding proteins. To confirm the physical and functional interaction between KyoT2 and HPC2, we carried out yeast two-hybrid, GST-pull down, co-immunoprecipitation, as well as mammalian two-hybrid assays. Our results showed HPC2 and KyoT2 interacted both in vitro and in vivo, probably through the C-terminal fragment of HPC2 and LIM domains of KyoT2. In addition, we also found that overexpression of HPC2, not only inhibited transactivation of a RBP-J-dependent promoter by NIC, but also transactivation by RBP-J-VP16, a constitutively active form of RBP-J. Taken together, our results suggested that KyoT2 might inhibit the RBP-J-mediated transactivation through NIC by recruiting co-suppressors such as HPC2.

Overexpression of mouse GlcNAc-1-phosphotransferase-gamma subunit in cells induced an I-cell-like phenotype of mucolipidosis.

In a screen of signal peptide-containing proteins from a mouse hypothetical protein library, we identified the mouse UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase-gamma chain (GlcNAc-1-phosphotransferase-gamma) (GenBank accession no. , HYP36 in this study). The mouse GlcNAc-1-phosphotransferase-gamma was localized in the Golgi complex in cells and was expressed ubiquitously in mouse tissues, as shown by fluorescence microscope analysis and a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay, respectively. Domain analysis showed that the mouse GlcNAc-1-phosphotransferase-gamma had a conserved mannose-6-phosphate (M-6-P)-binding domain. Interestingly, we found that overexpression of the mouse GlcNAc-1-phosphotransferase-gamma in fibroblast cell line NIH-3T3 induced accumulation of macromolecules, formation of large cytoplasmic vacuoles and decrease of lysosomal enzymes in cells. This phenotype was reminiscent of inclusion cells (I-cells) that were reported in mucolipidosis diseases caused by abnormal sorting of lysosomal proteins. Transient ectopic expression of GlcNAc-1-phosphotransferase-gamma in endoplasmic reticulum (ER) induced lowered lysosomal enzyme activity in cells. These results suggested on one hand that GlcNAc-1-phosphotransferase-gamma is an essential subunit of the GlcNAc-1-phosphotransferase, and on the other hand, the molecule might not only recognize the substrates of GlcNAc-1-phosphotransferase, but also the lysosomal proteins with M-6-P residuals.

HBV DNA vaccine with adjuvant cytokines induced specific immune responses against HBV infection.

AIM: To seek for an effective method to improve the immune responses induced by DNA vaccine expressing HBV surface antigen (pCR3.1-S) in Balb/c mice (H-2(d)). METHODS: The pCR3.1-S plasmid and the eukaryotic expression vectors expressing murine IL-2 (pDOR-IL-2) or IL-12 (pWRG3169) were injected into mice subcutaneously. The immune responses to pCR3.1-S and the adjuvant effect of the cytokines plasmid were studied. Meanwhile the effect of pCR3.1-S on anti-translated subcutaneous tumor of P815 mastocytoma cells stably expressing HBsAg (P815-HBV-S) was also studied. Anti-HBs in serum was detected by enzyme-linked immunoadsorbent assay (ELISA) and HBsAg specific cytotoxic T lymphocytes (CTLs) activity was measured by (51)Cr release assay. After three weeks of DNA immunization, the cells of P815-HBV-S were inoculated into mice subcutaneously and the tumor growth was measured every five days. The survival rate and living periods of mice were also calculated. RESULTS: After 8 wk DNA immunization, the A 450 nm values of sera in mice immunized with pCR3.1, pCR3.1-S and pCR3.1-S codeliveried with IL-2 or IL-12 plasmids were 0.03+/-0.01, 1.24+/-0.10, 1.98+/-0.17 and 1.67+/-0.12 respectively. Data in mice codeliveried pCR3.1-S with IL-2 or IL-12 plasmids were significantly higher than that of mice injected pCR3.1 or pCR3.1-S only. The HBsAg specific CTL activities in mice coinjected with pCR3.1-S and IL-2 or IL-12 eukaryotic expression vectors were (61.9+/-7.1) % and (73.3+/-8.8) %, which were significantly higher than that of mice injected with pCR3.1 (10.1+/-2.1) % or pCR3.1-S (50.5 +/-6.4) %. The HBsAg specific CTL activities in mice injected with pCR3.1, pCR3.1-S, pCR3.1-S combined with IL-2 or IL-12 eukaryotic expression vectors decreased significantly to (3.2+/-0.8) %, (10.6+/-1.4) %, (13.6+/-1.3) % and (16.9+/-2.3) % respectively after the spleen cells were treated by anti-CD8(+) monoclonal antibody, but presented no significant change to anti-CD4(+) monoclonal antibody or unrelated to monoclonal antibody. The HBV-S DNA vaccine (pCR3.1-S) could evidently inhibit the tumor growth, prolong the survival period of mice and improve the survival rate of mice and these effects could be improved by IL-12 gene codeliveried. CONCLUSION: HBV DNA vaccine has a strong antigenicity in humoral and cellular immunities, which can be promoted by plasmid expressing IL-2 or IL-12. CD8+ cells executed the CTL activities. DNA vaccine may be useful for both prophylaxis and treatment of HBV infection.