Limosilactobacillus johnsoni and Limosilactobacillus mucosae and Their Extracellular Vesicles Alleviate Gut Inflammatory Injury by Mediating Macrophage Polarization in a Lipopolysaccharide-Challenged Piglet Model.

BACKGROUND: Limosilactobacillus johnsoni (L. j) and Limosilactobacillus mucosae (L. m) can alleviate the inflammatory response. OBJECTIVES: This study aimed to elucidate the underlying mechanisms by which L. j- and L. m-derived extracellular vesicles (EVs) mitigate lipopolysaccharide (LPS)-induced intestinal injury. METHODS: Piglets were assigned to 4 groups: oral phosphate-buffered saline inoculation for 2 wk prior to intraperitoneal injection of physiological saline or LPS, and oral L. j/L. m inoculation for 2 wk prior to intraperitoneal injection of LPS. The intestinal integrity, macrophage markers, cytokine levels, and microbiota were determined. The cytokine levels and macrophage phenotype were detected after L. j/L. m and their EVs were coincubated with macrophages. The levels of cytokines, tight junction proteins, and apoptosis were measured after intestinal epithelial cells were cocultured with macrophages. RESULTS: LPS challenge decreased jejunal villus length; expression levels of zonula occludens-1 (ZO-1), occludin, arginase-1 (Arg1), and interleukin (IL)-10; and number of CD163+ cells and increased the expression levels of inducible nitric oxide synthase (iNOS), IL-1ß, IL-6, and tumor necrosis factor (TNF)-α compared with that in the control. L. j and L. m pretreatment rescued the aforementioned indicators compared with LPS challenge. Pretreatment of L. j and L. m and their EVs reversed the levels of IL-1ß, IL-6, TNF-α, and IL-10 and the gene expression of iNOS and Arg1 in the LPS group in macrophages. Pretreatment with L. j and L. m-derived EVs increased ZO-1 and occludin mRNA expression and reduced IL-1ß, caspase-3, and bax gene expression in intestinal epithelial cells of the coculture system. Enzyme-treated EVs were less effective than native EVs. CONCLUSIONS: This study suggests that EVs secreted by L. j and L. m control inflammation by modulating macrophage polarization, thereby improving intestinal barrier function.

N-(3-oxododecanoyl)-l-homoserine lactone disrupts intestinal epithelial barrier through triggering apoptosis and collapsing extracellular matrix and tight junction.

Microbes employ autoinducers of quorum sensing (QS) for population communication. Although the autoinducer of Pseudomonas aeruginosa LasI-LasR system, N-(3-oxododecanoyl)- l-homoserine lactone (3OC12), has been reported with deleterious effects on host cells, its biological effects on integrity of the intestinal epithelium and epithelial barrier are still unclear and need further investigation. In the present study, flow cytometry, transcriptome analysis and western blot technology have been adopted to investigate the potential molecular mechanisms of 3OC12 and its structurally similar analogs damage to intestinal epithelial cells. Our results indicated that 3OC12 and 3OC14 trigger apoptosis rather than necrosis and ferroptosis in intestinal epithelial cells. RNA-sequencing combined with bioinformatics analysis showed that 3OC12 and 3OC14 reduced the expression of genes from extracellular matrix (ECM)-receptor interaction pathway. Consistently, protein expressions from ECM and tight junction-associated pathway were significantly reduced after 3OC12 and 3OC14 challenge. In addition, 3OC12 and 3OC14 led to blocked cell cycle, decreased mitochondrial membrane potential, increased reactive oxygen species level and elevated Ca2+ concentration. Reversely, the antioxidant NAC could effectively mitigate the reduced expression of ECM and tight junction proteins caused by 3OC12 and 3OC14 challenge. Collectively, this study demonstrated that QS autoinducer exposure to intestinal epithelial cells ablates the ECM and tight junctions by triggering oxidative stress and apoptosis, and finally disrupts the intestinal epithelial barrier. These findings provide a rationale for defensing QS-dependent bacterial infections and potential role of NAC for alleviating the syndrome.

N-Acyl-Homoserine Lactones May Affect the Gut Health of Low-Birth-Weight Piglets by Altering Intestinal Epithelial Cell Barrier Function and Amino Acid Metabolism.

BACKGROUND: In piglets, low birth weight (LBW) is associated with intestinal dysfunction, which affects their growth performance and causes economic losses. OBJECTIVES: This study was designed to test whether microbial quorum sensing (QS) affects LBW-induced intestinal developmental defects in piglets. METHODS: Seven normal-birth-weight (NBW; 1.36 ± 0.01 kg) and 7 LBW (0.89 ± 0.01 kg) piglets were selected. Feces were collected from piglets on 2, 21, and 50 days of age for detection of the QS signaling molecules, N-acyl-homoserine lactones (AHLs), and microbiota analysis. The associations between 2 long-chain AHLs [N-3-oxo-dodecanoyl-l-homoserine lactone (3OC12-HSL) and N-3-oxo-tetradecanoyl-l-homoserine lactone (3OC14-HSL)] and the microbes were tested using Spearman correlation coefficients. The effect of 3OC12-HSL and 3OC14-HSL on intestinal porcine epithelial cell-jejunum 2 (IPEC-J2) cell viability was investigated by cholecystokinin octapeptide assay. Transcriptomic analysis was performed by RNA sequencing on cells treated with 3OC12-HSL. RESULTS: The concentrations of 3OC12-HSL and 3OC14-HSL in the feces of LBW piglets were higher than those in NBW piglets at age 50 d by 2.5- and 2.24-fold, respectively (P < 0.05). The microbial α diversity (observed species, abundance-based coverage estimator, and Shannon index) of LBW piglets was 81-91% lower than that of NBW piglets (P < 0.05). The relative abundance of Ruminococcaceae UCG-002/UCG-013 was 43.0% and 30.0% lower, respectively, in feces from LBW compared with NBW piglets (P < 0.05). 3OC12-HSL and Ruminococcaceae UCG-002/UCG-005/UCG-010 abundance were negatively correlated (ρ  ≤  -0.58). Treatment with 400 µM 3OC12-HSL markedly reduced IPEC-J2 cell viability by 47.5%. Transcriptomic data showed that 3OC12-HSL mainly changed the "import across plasma membrane" and "arginine and proline metabolism" of IPEC-J2 cells. CONCLUSIONS: 3OC12-HSL is a QS signaling molecule with an ability to impair gut health of LBW piglets. This finding adds to our understanding of the mechanisms responsible for gut injury in LBW piglets.

Effects of dietary fibers with different physicochemical properties on fermentation kinetics and microbial composition by fecal inoculum from lactating sows in vitro.

BACKGROUND: Efficient utilization of dietary fibers (DFs) is important for optimizing feed resource utilization and animal health. The aim of the current study was to assess the effects of DFs with varying physicochemical properties (bulky, viscous, and fermentable) on fermentation kinetics and microbial composition during in vitro fermentation by fecal inoculum from lactating sow. According to the physicochemical properties, three different DFs, lignocellulose (LC), modified cassava starch (MCS) and konjac flour (KF) were selected as bulky fiber, fermentable fiber and viscous fiber respectively. Gas production, short-chain fatty acids (SCFAs) profiles and microbial composition were monitored during the fermentation. RESULTS: Results showed that the gas production in 72 h (GP72h ) ranked as: KF > MCS > LC (P < 0.05). The halftime of asymptotic gas production ranked as: KF < MCS = LC (P < 0.001). At 36 h of fermentation, MCS group showed higher concentrations of formic acid and lactate than LC and KF groups, whereas KF group showed higher concentrations of propionate and butyrate than LC and MCS groups (P < 0.05). At 72 h of fermentation, KF group showed higher concentrations of formic acid, lactate and propionate than LC and MCS groups, whereas MCS group showed higher concentrations of acetate and butyrate than LC and KF groups (P < 0.05). At 36 h of fermentation, Anaerovibrio and Erysipelatoclostridium abundances were higher in KF group, whereas Proteiniclasticum abundance was higher in MCS group. At 72 h of fermentation, the abundance of Fibrobacter in LC group was higher than that in MCS and KF groups. In addition, we also observed that the abundances of certain specific bacteria (Anaerovibrio and Erysipelatoclostridium) were closely related to the SCFAs production (propionate and butyrate) at different fermentation times. CONCLUSION: Collectively, the present study revealed that KF is a fast fermentation fiber which could produce propionate and butyrate rapidly, whereas LC is difficult to be fermented by bacteria. In addition, the fermentation of DFs with different physicochemical properties had divergent impacts on microbial composition and SCFA production. These findings deepen our understanding of the mechanisms of interaction between DFs and intestinal microbiota, and provide new ideas for the rational use of fiber resources in lactating sows. © 2020 Society of Chemical Industry.

Original low birth weight deteriorates the hindgut epithelial barrier function in pigs at the growing stage.

The deteriorative effect of low birth weight (LBW) on the mucosal permeability of the small intestine in piglets has been widely confirmed. However, whether the hindgut epithelial barrier function in LBW pigs is deteriorated during the growing stage is still unclear. Our study investigated differences in the hindgut epithelial barrier function between LBW and normal birth weight pigs during the growing stage (d 90). Our data demonstrated that the hindgut epithelium of LBW pigs has a high histopathological score, accompanied by decreased antioxidant capacity and increased apoptosis rate, as well as elevated expression and activity of caspase-3. In addition, the number of intestinal goblet cells and gene expression of mucin 2 were significantly down-regulated in LBW pigs. The expression of tight junction proteins (ZO-1 and occludin) was markedly inhibited by the LBW. The mRNA abundance of inflammatory cytokines such as TNF-α, IL-1ß, and IL-8 was significantly increased in the hindgut mucosa of LBW pigs. Furthermore, our data revealed that LBW altered the intestinal microbial community in the hindgut mucosa of pigs. Collectively, these finding add to our understanding of the mechanisms responsible for hindgut epithelial barrier dysfunction in LBW pigs during the growing stage and facilitate the development of nutritional intervention strategies.-Tao, S., Bai, Y., Li, T., Li, N., Wang, J. Original low birth weight deteriorates the hindgut epithelial barrier function in pigs at the growing stage.

Effect of dietary fiber fermentation on short-chain fatty acid production and microbial composition in vitro.

BACKGROUND: The efficient utilization of fiber-rich co-products is important for optimizing feed resource utilization and animal health. This study was conducted to evaluate the fermentation characteristics of fiber-rich co-products, which had equal quantities of total dietary fiber (TDF), at different time points using batch in vitro methods. It considered their gas production, short-chain fatty acid (SCFA) production, and microbial composition. RESULTS: The fermentation of wheat bran (WB) and oat bran (OB) showed higher and faster (P < 0.05) gas and SCFA production than corn bran (CB), sugar beet pulp (SBP), and soybean hulls (SH). The α-diversity was higher in the CB, SBP, and SH groups than in the WB and OB groups (P < 0.05). At the phylum level, OB and WB fermentation showed lower (P < 0.05) relative abundance of Actinobacteria than the CB, SBP, and SH groups. At the genus level, OB and WB fermentation increased the Enterococcus population in comparison with the CB, SBP, and SH groups, whereas CB and SBP fermentation improved the relative abundance of the Christensenellaceae R-7 group more than the WB, OB, and SH groups (P < 0.05). CONCLUSION: Overall, WB and OB were rapidly fermented by fecal microbiota, in contrast with SBP, SH, and CB. Fermentation of different fiber-rich co-products with an equal TDF content gives different responses in terms of microbial composition and SCFA production due to variations in their physicochemical properties and molecular structure. © 2020 Society of Chemical Industry.

Caspase-1-dependent mechanism mediating the harmful impacts of the quorum-sensing molecule N-(3-oxo-dodecanoyl)-l-homoserine lactone on the intestinal cells.

N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), a quorum-sensing (QS) molecule produced by Gram-negative bacteria in the gastrointestinal tract, adversly impacts host cells. Our previous study demonstrated that 3-oxo-C12-HSL induced a decrease in cell viability via cell apoptosis and eventually disrupted mucin synthesis from LS174T goblet cells. However, the molecular mechanism underlying cell apoptosis and whether pyroptosis was involved in this process are still unknown. In this study, we emphasized on the caspases signal pathway and sterile inflammation to reveal the harmful effects of 3-oxo-C12-HSL on LS174T goblet cells. Our data showed that 3-oxo-C12-HSL is a major inducer of oxidative stress indicated by a high level of intracellular reactive oxygen species (ROS). However, TQ416, an inhibitor of paraoxonase 2, can effectively block oxidative stress. A higher ROS level is the trigger for activating the caspase-1 and 3 cascade signal pathways. Blockade of ROS synthesis and caspase-1 and 3 cascades can obviously rescue the viability of LS174T cells after 3-oxo-C12-HSL treatment. We also found that paralleled with a higher level of ROS and caspases activation, an abnormal expression of proinflammatory cytokines was induced by 3-oxo-C12-HSL treatment; however, the blockage of TLRs-NF-κB pathway cannot restore cell viability and secretary function. These data collectively indicate that 3-oxo-C12-HSL exposure induces damages to cell viability and secretary function of LS174T goblet cells, which is mediated by oxidative stress, cell apoptosis, and sterile inflammation. Overall, the data in this study will provide a better understanding of the harmful impacts of some QS molecules on host cells and their underlying mechanism.

Chronic Dexamethasone exposure activates the TLR4-Mediated inflammation pathway and induces epithelial apoptosis in the goat colon.

Chronic stress has a profound effect on health in both animals and humans. Dexamethasone (Dex), a synthetic glucocorticoid, is used to induce chronic stress in many studies. The impact of chronic stress on epithelial cells of hindgut of ruminants is still unknown. In this study, we investigated the effect of chronic stress induced by long term injection of low dosage of Dex on the colonic epithelium of goats. The results showed that Dex exposure increased the number of TUNEL-positive cells, upregulated caspase-3 and caspase-8 enzyme activity, but decreased protein expression of cell proliferation markers proliferating cell nuclear antigen (PCNA) and Cyclin D2(CCND2). It also activated TLR-4 and NF-κB pathway and increased the transcription levels of vital inflammatory cytokines such as interleukin-10 (IL-10), interleukin-1ß (IL-1ß), and inducible nitric oxide synthase 2 (iNOS2). Chronic stress down-regulated the methylation level of total DNA, suggesting a mechanism for the transcriptional activation of genes, such as claudin-1, claudin-4, ZO-1, and cell cycle-related genes. Taken together, long-term injection of a low dosage of Dex caused damage to the colon epithelium accompanied with the inhibition of cell proliferation and the activation of cell apoptosis and inflammation. However, a general up-regulation of genes expression induced by Dex is due to a lower level of genomic DNA methylation.

Combined supplementation of Lactobacillus fermentum and Pediococcus acidilactici promoted growth performance, alleviated inflammation, and modulated intestinal microbiota in weaned pigs.

BACKGROUND: Probiotics are important for pigs to enhance health and intestinal development, which are potential alternative to antibiotics. Many studies have reported the functions of single bacterial strain as probiotic on the animals. In this study, we evaluated effects of combined probiotics on growth performance, inflammation and intestinal microbiota in weaned pigs. One hundred and eight pigs, weaned at 28 day old (7.12 ± 0.08 kg), were randomly divided into the 3 dietary treatments with 6 pens and 6 pigs per pen (half male and half female). The experimental period lasted for 28 days and treatments were as follows: i. CONTROL: basal diet; ii. Antibiotic: the basal diet plus 75 mg· kg- 1 chlortetracycline; and iii. Probiotics: basal diet plus 4% compound probiotics. RESULTS: Supplementation probiotics improved average daily gain over the entire 28 days (P < 0.01) and feed efficiency in the last 14 days (P < 0.05) compared with the other two groups. Both probiotics and antibiotic supplementation decreased concentrations of serum pro-inflammatory cytokines interleukin-6 (P < 0.05) and interferon-γ (P < 0.01). Probiotics group had greater abundance of Lactobacillus in the caecal digesta and Firmicutes in the colonic digesta, while both probiotics and antibiotic supplementation inhibited Treponema_2 and Anaerovibrio in the caecal digesta. Caecal acetic and propionic acid (P < 0.05) of probiotics group were higher than the other two groups, whereas concentrations of colonic lactic acid and propionic acid (P < 0.05) of antibiotic group were lower than control and probiotics groups. CONCLUSIONS: These findings suggest that combined supplementation of Lactobacillus fermentum and Pediococcus acidilactici regulate the gut health and improve the host ADG and F/G by decreasing serum pro-inflammatory factors (IL-6, IFN-γ), promoting beneficial bacteria (Lactobacillus in the caecal digesta and Firmicutes in the colonic digesta), enhancing production of short chain fatty acids, and inhibiting pathogens (Treponema_2, Anaerovibrio in the caecal digesta).

Adverse effects of bisphenol A on Sertoli cell blood-testis barrier in rare minnow Gobiocypris rarus.

Bisphenol A (BPA), an environmental contaminant, has been shown to disturb the dynamics of Sertoli cell blood-testis barrier (BTB) in mammal testis. However, the effects of BPA on Sertoli cell barrier (SC barrier) were little known in fish to date. To evaluate the potential mechanism of reproductive toxicity of BPA, we studied the damage of SC barrier using in vivo models. In this study, male adult rare minnow Gobiocypris rarus were exposed to 15 µg/L BPA for 7-35 days. Gonadal histology and the integrity of SC barrier were analyzed. Meanwhile, the expressions of SC barrier -associated proteins, tumor necrosis factor (TNFα) content, and the mRNA expressions of genes in the mitogen activated protein kinase (MAPK) pathway were detected. Histological analysis demonstrated 15 µg/L BPA promoted the infiltration of inflammatory cells in fish testes after 7-days exposure. The biotin tracer assay showed that 7-days BPA exposure increased permeability for spermatid cysts. In addition, the BPA treatment caused increased TNFα in testis, which was reportedly related to SC barrier impairment. The expressions of Occludin and ß-Catenin protein were significantly decreased in the testes after 7- and 21-days exposure. BPA also altered the mRNA expressions of occludin, ß-catenin, p38 MAPK and JNK. Therefore, the detrimental effects of BPA on reproduction of male fish may attribute to the disturbed expressions of SC junction proteins.

A Single-Amino-Acid Substitution at Position 225 in Hemagglutinin Alters the Transmissibility of Eurasian Avian-Like H1N1 Swine Influenza Virus in Guinea Pigs.

Efficient transmission from human to human is the prerequisite for an influenza virus to cause a pandemic; however, the molecular determinants of influenza virus transmission are still largely unknown. In this study, we explored the molecular basis for transmission of Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses by comparing two viruses that are genetically similar but differ in their transmissibility in guinea pigs: the A/swine/Guangxi/18/2011 virus (GX/18) is highly transmissible by respiratory droplet in guinea pigs, whereas the A/swine/Heilongjiang/27/2012 virus (HLJ/27) does not transmit in this animal model. We used reverse genetics to generate a series of reassortants and mutants in the GX/18 background and tested their transmissibility in guinea pigs. We found that a single-amino-acid substitution of glycine (G) for glutamic acid (E) at position 225 (E225G) in the HA1 protein completely abolished the respiratory droplet transmission of GX/18, whereas the substitution of E for G at the same position (G225E) in HA1 enabled HLJ/27 to transmit in guinea pigs. We investigated the underlying mechanism and found that viruses bearing 225E in HA1 replicated more rapidly than viruses bearing 225G due to differences in assembly and budding efficiencies. Our study indicates that the amino acid 225E in HA1 plays a key role in EAH1N1 swine influenza virus transmission and provides important information for evaluating the pandemic potential of field influenza virus strains.IMPORTANCE Efficient transmission among humans is a prerequisite for a novel influenza virus to cause a human pandemic. Transmissibility of influenza viruses is a polygenic trait, and understanding the genetic determinants for transmissibility will provide useful insights for evaluating the pandemic potential of influenza viruses in the field. Several amino acids in the hemagglutinin (HA) protein of influenza viruses have been shown to be important for transmissibility, usually by increasing virus affinity for human-type receptors. In this study, we explored the genetic basis of the transmissibility difference between two Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses in guinea pigs and found that the amino acid glutamic acid at position 225 in the HA1 protein plays a critical role in the transmission of EAH1N1 virus by increasing the efficiency of viral assembly and budding.

Chronic dexamethasone exposure retards growth without altering the digestive tract microbiota composition in goats.

BACKGROUND: Dexamethasone (Dex), an artificially synthetic cortisol substitute, is commonly used as an anti-inflammatory drug, and is also employed to mimic the stress state experimentally. It is well known that chronic stress disturbs the gut microbiota community and digestive functions. However, no relevant studies have been conducted in ruminants. RESULTS: In this study, a low dosage of Dex (0.2 mg/kg body weight, Dex group, n = 5) was consecutively injected intramuscularly for 21 days to simulate chronic stress in growing goats. Goats were injected with saline (0.2 mg/kg body weight) as the control group (Con, n = 5). Dex-treated goats showed a higher number of white blood cells and blood glucose levels (p < 0.01), but lower dry matter intake (DMI) and body weight (p < 0.01) than those of saline-injected goats. Plasma cortisol concentration decreased significantly in response to the Dex injection compared to the control (p < 0.05). The Dex treatment did not change most ruminal volatile fatty acid (VFAs) concentrations before the morning feeding after 1-21 days of treatment (p > 0.05); however, ruminal VFA concentrations decreased dramatically 2, 4, 6, and 8 h after the morning feeding on day 21 of the Dex injections. In this study, chronic Dex exposure did not alter the community structure of microbes or methanogenes in the rumen, caecum, or colonic digesta. Only Prevotella increased on days 7 and 14 of Dex treatment, but decreased on day 21, and Methanosphaera was the only genus of methanogene that decreased. CONCLUSIONS: Our results suggest that chronic Dex exposure retards growth by decreasing DMI, which may be mediated by higher levels of blood glucose and lower ruminal VFA production. Microbiota in the digestive tract was highly resistant to chronic Dex exposure.

Chronic dexamethasone exposure markedly decreased the hepatic triglyceride accumulation in growing goats.

Chronic stress seriously threatens welfare and health in animals and humans. Consecutive dexamethasone (Dex) injection was used to mimic chronic stress previously. In order to investigate the effect of chronic stress on hepatic lipids metabolism, in this study, 10 healthy male goats were randomly allocated into two groups, one received a consecutive injection of Dex via intramuscularly for 3 weeks (Dex group), the other received the same volume of saline as the control group (Con group). Hepatic health and triglyceride (TG) metabolism were analyzed and compared between two groups. The data showed that a significant decrease of TG in plasma and the liver was significantly decreased by Dex (P < .05), while the hepatic nonesterified fatty acid (NEFA) concentration was increased compared to the Con group (P < .05). Consistent with the decrease of TG level, the activity of hepatic lipoprotein lipase (LPL) and hepatic lipase (HL) enzymes activities were significantly enhanced by Dex. Real-time PCR results showed that the mRNA expression of sterol regulatory element binding transcription factor 1 (SREBP-1), acyl-CoA dehydrogenase long chain (ACADL) and acyl-CoA synthetase bubblegum family member 1 (ACSBG1) genes in liver was significantly up-regulated by chronic Dex injection (P < .05), whereas perilipin 2 (PLIN2) and adipose triglyceride lipase (ATGL) mRNA expression was significantly decreased by Dex (P < .05). In addition, no obvious damages were observed in the liver in both Con and Dex groups demonstrating by the sirius red staining, HE staining as well as several biochemical parameters related to the functional status of hepatocytes. Our data indicate that chronic Dex exposure decreases TG levels in the circulation and the liver through activating lipolysis and inhibiting lipogenesis without causing hepatic damages in the growing goats.

Effects of Oat Bran on Nutrient Digestibility, Intestinal Microbiota, and Inflammatory Responses in the Hindgut of Growing Pigs.

Oat bran has drawn great attention within human research for its potential role in improving gut health. However, research regarding the impact of oat bran on nutrient utilization and intestinal functions in pigs is limited. The purpose of this study was to investigate the effects of oat bran on nutrient digestibility, intestinal microbiota, and inflammatory responses in the hindgut of growing pigs. Twenty-six growing pigs were fed either a basal diet (CON) or a basal diet supplemented with 10% oat bran (OB) within a 28 day feeding trial. Results showed that digestibility of dietary gross energy, dry matter, organic matter, and crude protein were lower in the OB group compared to the CON group on day 14, but no differences were observed between the two groups on day 28. In the colon, the relative abundance of operational taxonomic units (OTUs) associated with Prevotella, Butyricicoccus, and Catenibacterium were higher, while those associated with Coprococcus and Desulfovibrio were lower in the OB group compared to the CON group. Oat bran decreased mRNA expression of caecal interleukin-8 (IL-8), as well as colonic IL-8, nuclear factor-κB (NF-κB), and tumor necrosis factor-α (TNF-α) of the pigs. In summary, oat bran treatment for 28 day did not affect dietary nutrient digestibility, but promoted the growth of cellulolytic bacteria and ameliorated inflammatory reactions in the hindgut of growing pigs.

Glycine at Position 622 in PB1 Contributes to the Virulence of H5N1 Avian Influenza Virus in Mice.

UNLABELLED: We isolated two H5N1 viruses, A/duck/Hunan/S4020/2008 (DK/08) and A/chicken/Guangxi/S2039/2009 (CK/09), from live-bird markets during routine surveillance and found that these two viruses are genetically similar but differ in their replication and virulence in mice. The CK/09 virus is lethal for mice with a 50% mouse lethal dose (MLD50) of 1.6 log10 50% egg infectious doses (EID50), whereas the DK/08 virus is nonpathogenic for mice with an MLD50 value of 6.2 log10 EID50. We explored the genetic basis of the virulence difference of these two viruses by generating a series of reassortant viruses and mutants in the lethal virus CK/09 background and evaluating their virulence in mice. We found that the PB1 gene of the DK/08 virus dramatically attenuated the virulence of the CK/09 virus and that the amino acid at position 622 in PB1 made an important contribution. We further demonstrated that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impaired the binding of PB1 to viral RNA, thereby dramatically decreasing the polymerase activity and attenuating H5N1 virus virulence in mice. Our results identify a novel virulence-related marker of H5N1 influenza viruses and provide a new target for live attenuated vaccine development. IMPORTANCE: H5N1 avian influenza viruses have caused the deaths of nearly 60% of the humans that they have infected since 1997 and clearly represent a threat to public health. A thorough understanding of the genetic basis of virulence determinants will provide important insights for antiviral drug and live attenuated vaccine development. Several virulence-related markers in the PB2, PA, M1, and NS1 proteins of H5N1 viruses have been identified. In this study, we isolated two H5N1 avian influenza viruses that are genetically similar but differ in their virulence in mice, and we identified a new virulence-related marker in the PB1 gene. We found that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impairs the binding of PB1 to viral RNA, thereby attenuating H5N1 virus virulence in mice. This newly identified virulence-related marker could be applied to the development of live attenuated vaccines against H5N1 influenza.

Downregulation of prostaglandin E2 is involved in hindgut mucosal damage in lactating goats fed a high-concentrate diet.

NEW FINDINGS: What is the central question of this study? A high-concentrate (HC) diet results in damage to the hindgut mucosa. The aim of the study was to investigate the status of epithelial proliferation in the hindgut mucosa of goats with subacute ruminal acidosis and, simultaneously, to evaluate prostaglandin E2 synthesis and the downstream signalling pathways. What is the main finding and its importance? The downregulation of local prostaglandin E2 synthesis and its downstream signalling pathway are involved in the process of inhibiting epithelial proliferation in the hindgut epithelium of HC-fed goats. Our results provide new insight into the relationship between abnormal fermentation in the hindgut and damage to the intestinal mucosal barrier. Our previous data demonstrated that feeding a high-concentrate (HC) diet to lactating goats for a long time causes severe damage to the hindgut mucosa and parallels the activation of cell apoptosis and local oxidative stress. In the present study, changes in production of prostaglandin E2 (PGE2 ) and its signalling pathway were evaluated in the process of epithelial barrier disruption in the hindgut. Twelve goats in mid-lactation were randomly assigned to either a HC diet group or a low-concentrate (LC) diet group for 10 weeks. Cell proliferation markers, cyclooxygenase-2 activity, PGE2 content and the relative signalling pathway, including CREB and AKT, were analysed by enzyme-linked immunosorbent assay and Western blot, respectively. The mRNA and protein expressions of MKI67 and CCND2 (two proliferation markers) were significantly decreased in the caecal mucosa of HC- compared with LC-fed goats (P < 0.05). The protein content of interleukin-10 and ß-defensin in the caecal mucosa was also downregulated in HC-fed goats (P < 0.05). The HC-fed goats showed a tendency to a decrease in cyclooxygenase-2 enzyme activity (P = 0.081) and a significant decrease of local PGE2 content and EP4 (PGE2 receptor) protein expression in caecal mucosa (P < 0.05). Moreover, the protein abundance of p-CREB (P = 0.069) and p-AKT (P < 0.05) and the mRNA expression of epidermal growth factor receptor (P < 0.05) were downregulated in caecal mucosa of HC- compared with LC-fed goats. These results indicate that a reduction in epithelial cell proliferation was partly responsible for the damage to the epithelial barrier, which might be associated with the downregulation of PGE2 synthesis and its downstream signalling pathway.

Global DNA methylation in gonads of adult zebrafish Danio rerio under bisphenol A exposure.

Altered DNA methylation is pervasively associated with changes in gene expression and signal transduction after exposure to a wide range of endocrine disrupting chemicals. As a weak estrogenic chemical, bisphenol A (BPA) has been extensively studied for reproductive toxicity. In order to explore the effects of BPA on epigenetic modification in gonads of zebrafish Danio rerio, we measured the global DNA methylation together with the gene expression of DNA methyltransferase (dnmts), glycine N-methyltransferase (gnmt), and ten-eleven translocation (tets) in gonads of D. rerio under BPA exposure by ELISA and quantitative real-time PCR method, respectively. The global level of DNA methylation was significantly decreased in ovaries after exposed to BPA for 7 days, and testes following 35-day exposure. Moreover, the global level of DNA methylation was also significantly reduced in testes after exposed to 15µg/L BPA for 7 days. Besides the alteration of the global level of DNA methylation, varying degrees of transcriptional changes of dnmts, gnmt and tets were detected in gonads of D. rerio under BPA exposure. The present study suggested that BPA might cause the global DNA demethylation in gonads of zebrafish by regulating the transcriptional changes of the DNA methylation/demethylation-associated genes (dnmts, gnmt, and tets).

Oxidative stress and immunotoxic effects of bisphenol A on the larvae of rare minnow Gobiocypris rarus.

Bisphenol A (BPA), a known endocrine disrupting chemical, is ubiquitous in the aquatic environment and can pose risk to the health of aquatic organisms. Studies on immunotoxicity of BPA in aquatic organisms are limited. In this study, rare minnow (Gobiocypris rarus) larvae were exposed to 1, 225 and 1000µg/L BPA for 7 days. Inflammatory effects of BPA exposure were assessed from the increased production of nitric oxide (NO) and reactive oxygen species (ROS), the change of iNOS mRNA and other TLRs-associated immune gene expression. Our findings provide evidences that different concentrations of BPA can induce a toxic response in fish to produce reactive free radicals which can affect the function of T lymphocytes and decrease the transcription levels of cytokine genes. The excess production of H2O2, induced oxidative stress and suppressed TLR4/NF-κB signaling, leading to immunosuppressive effects in fish larvae. The present results suggest that BPA has the potential to induce oxidative stress accompanied by immunosuppression in rare minnow larvae.

Activation of cellular apoptosis in the caecal epithelium is associated with increased oxidative reactions in lactating goats after feeding a high-concentrate diet.

NEW FINDINGS: What is the central question of this study? What are the ultrastructural changes of the caecal mucosa and the status of epithelial cellular apoptosis and oxidative reactions in lactating goats after prolonged feeding with a high-concentrate diet? What is the main finding and its importance? High-concentrate diet results in ultrastructural damage to the caprine caecal epithelium. Increased oxidative and decreased antioxidative reactions are involved in the process of activating epithelial apoptosis in the caecal epithelium of goats fed a high-concentrate diet. Our results provide new insight into the relationship between abnormal fermentation in the hindgut and damage to the intestinal mucosal barrier. The effect of feeding a high-concentrate diet (HC) to lactating ruminants on their hindgut epithelial structure remains unknown. In this study, 12 lactating goats were randomly assigned to either HC (65% of dry matter as concentrate; n = 6) or a low-concentrate diet (LC; 35% of dry matter as concentrate; n = 6). After 10 weeks, the epithelial ultrastructure and cell apoptotic status in the caecal mucosa were determined by transmission electron microscopy and TUNEL, respectively. The results showed that the level of free lipopolysaccharide (P < 0.05), total volatile fatty acid concentrations (P < 0.1) and starch content (P < 0.05) in the caecal digesta were significantly increased in HC- compared with LC-fed goats. The HC-fed goats exhibited obvious epithelial cellular damage, with widened tight junction spaces, nuclear breakdown and mitochondrial swelling. Compared with their LC-fed counterparts, HC-fed goats showed greater apoptosis in the caecal epithelium, as evidenced by more TUNEL-positive apoptotic cells. Western blot analysis showed that there was no significant difference in activated caspase-3, Bax protein expression in caecal epithelial mucosa between HC- and LC-fed goats (P > 0.05). However, the level of malondialdehyde content in the caecal epithelium from HC-fed goats was markedly higher than that in LC-fed goats (P < 0.05), whereas the level of glutathione peroxidase and the superoxide dismutase activity were significantly decreased. Gene expressions of cytokines, including interleukin-1ß, interleukin-6, interleukin-10, tumour necrosis factor-α and interferon-γ, as well as myeloperoxidase activity in the caecal mucosa did not show any significant difference between HC- and LC-fed goats. These results indicate that feeding a high-concentrate diet to lactating goats for a prolonged period results in abnormal fermentation and structural disruption in the hindgut, which is accompanied by greater cellular apoptosis and an enhanced oxidative stress response.

A high-concentrate diet induced colonic epithelial barrier disruption is associated with the activating of cell apoptosis in lactating goats.

BACKGROUND: In ruminants, lower ruminal pH causes massive disruption of ruminal epithelial structure during periods of feeding high-concentrate diets. However, the influence of excessive organic fatty acids in the lumen of hindgut on the epithelial structure is unclear. In this study, twelve mid-lactating goats were randomly assigned to either a HC diet group (65% concentrate of dry matter; n = 6) or a LC diet group (35% concentrate of dry matter; n = 6) for 10 weeks. The colonic epithelial structure was detected by HE staining and transmission electron microscopy (TEM), and the apoptotic status of epithelial cells was estimated by TUNEL method and caspase activities. RESULTS: HC goats showed higher level of free lipopolysaccharide (LPS) in rumen fluid (p < 0.01) but not in colonic digesta (p > 0.05), and higher total volatile fatty acid (VFA) concentrations in rumen fluid (p < 0.05) and in colonic digesta (p < 0.01), and higher content of starch in colonic digesta (p < 0.05) compared to LC goats. HC goats demonstrated profound alterations in the colonic epithelial structure and tight junctions (TJ), apparently due to damage of the epithelium with widened TJs space and nuclear breakdown and mitochondrial swelling. HC goats showed higher level of apoptosis in the colonic epithelium with higher proportion of TUNEL-positive apoptotic cells and increases of caspase-3 and -3/7 activities, as well as the lower ratio of bcl-2/bax mRNA expression in the colonic mucosa (p < 0.05). However, ß-defense mRNA was significantly down-regulated in the colonic mucosa of HC goats compared to LC (p < 0.05). HC goats showed higher level of TJ proteins including claudin-1 and claudin-4 in the colonic mucosa than LC (p < 0.05). Neither free LPS content in the colonic digesta nor NF-κ B protein expression in tissues showed significant difference between HC and LC goats (p > 0.05). CONCLUSIONS: Our results reveal that long-term feeding HC diet to lactating goats causes severe damages to the colonic mucosa barrier associated with activating cells apoptosis.