Immunoprotective efficacy of 3 Klebsiella pneumoniae type I fimbriae proteins in a murine model.

Klebsiella pneumoniae is a primary cause of clinical mastitis in dairy cows, with prevention being crucial, as treatments often fail due to antimicrobial resistance. Recent studies identified type I fimbrial antigens of K. pneumoniae as promising vaccine candidates, but there are limited research data. In this study, 3 fimbriae genes (fimA, fimC and fimG) were cloned and recombinantly expressed in Escherichia coli and their protective efficacy against K. pneumoniae evaluated in a mouse model. All 3 recombinant fimbriae proteins elicited strong humoral immune responses in mice, significantly increasing IgG, IgG1 and IgG2a. Notably, using a model of mice challenged with an intraperitoneal injection of bacteria, FimG significantly reduced bacterial loads in the spleen and lung, whereas FimA and FimC had limited protection for these organs. Either active or passive immunization with FimG produced substantial protective effects in mice challenged with K. pneumoniae LD100; in contrast, the mortality rate in the FimA-immunized group was similar to that of the control group, whereas FimC had weak protection. We concluded that the FimG recombinant protein vaccine had a favorable protective effect, with potential for immunization against K. pneumoniae mastitis.

Epigallocatechin-3-gallate protects bovine ruminal epithelial cells against lipopolysaccharide-induced inflammatory damage by activating autophagy.

BACKGROUND: Subacute ruminal acidosis (SARA) causes an increase in endotoxin, which can induce immune and inflammatory responses in the ruminal epithelium of dairy cows. In non-ruminants, epigallocatechin-3-gallate (EGCG), a major bioactive ingredient of green tea, is well-known to alleviate inflammation. Whether EGCG confers protection against SARA-induced inflammation and the underlying mechanisms are unknown. RESULTS: In vivo, eight ruminally cannulated Holstein cows in mid-lactation were randomly assigned to either a low-concentrate (40%) diet (CON) or a high-concentrate (60%) diet (HC) for 3 weeks to induce SARA (n = 4). Cows with SARA had greater serum concentrations of tumor necrosis factor (TNF)-α and interleukin-6, and epithelium had histological signs of damage. In vitro, immortalized bovine ruminal epithelial cells (BREC) were treated with lipopolysaccharide (LPS) to imitate the inflammatory damage caused by SARA. Our data revealed that BREC treated with 10 µg/mL LPS for 6 h successfully induce a robust inflammatory response as indicated by increased phosphorylation of IκBα and nuclear factor kappa-B (NF-κB) p65. Pre-treatment of BREC with 50 µmol/L EGCG for 6 h before LPS challenge promoted the degradation of NLR family pyrin domain containing 3 (NLRP3) inflammasome through activation of autophagy, which further repressed activation of NF-κB pathway targeting Toll-like receptor 4 (TLR4). Analyses also revealed that the ECGG upregulated tight junction (TJ) protein expression upon incubation with LPS. CONCLUSIONS: Subacute ruminal acidosis causes ruminal epithelium injury and systemic inflammation in dairy cows. However, the anti-inflammatory effects of EGCG help preserve the integrity of the epithelial barrier through activating autophagy when BREC are exposed to LPS. Thus, EGCG could potentially serve as an effective therapeutic agent for SARA-associated inflammation.

Role of hypoxia-inducible-factor-1α (HIF-1α) in ferroptosis of adipose tissue during ketosis.

Postpartum cows experience lipolysis in adipose tissue due to negative energy balance (NEB), and accumulation of free fatty acids (FFA) leads to metabolic stress in adipose tissue. Ferroptosis is a type of cell death triggered by excessive buildup of iron-dependent lipid peroxides, which is involved in the occurrence and development of various metabolic diseases in nonruminants. However, it is still unclear whether ferroptosis occurs in the adipose tissue of ketotic cows and the regulatory mechanisms behind ferroptosis. Despite multiple studies demonstrating the significant involvement of hypoxia-inducible-factor-1α (HIF-1α) in regulating cellular dysfunction, its specific function in adipose tissue of ketotic dairy cows remains uncertain, particularly its regulation of oxidative stress and ferroptosis. The study aimed to explore the impact of HIF-1α on oxidative stress and ferroptosis in bovine subcutaneous adipose tissue and isolated adipocytes. The adipose tissue of clinical ketosis cows (n = 15) with a serum BHB concentration of 3.13 mM (interquartile range = 0.14) and healthy cows (n = 15) with a serum BHB concentration of and 0.58 mM (interquartile range = 0.13) was collected. The results showed that the concentrations of lipid peroxidation malondialdehyde (MDA), reactive oxygen species (ROS), Fe2+ and total iron were increased in adipose tissue of cows with ketosis, while the contents of glutathione (GSH) were reduced. Furthermore, the protein levels of HIF-1α, heme oxygenase 1 (HMOX1), catalase (CAT), superoxide dismutase 1 (SOD1), acyl-CoA synthetase 4 (ACSL4), and nuclear factor erythroid-derived 2-like 2 (NFE2L2) exhibited higher abundance in adipose tissue obtained from cows with ketosis, whereas the protein abundance of solute carrier family 7 member 11 (SLC7A11), glutamate cysteine ligase catalytic subunit (GCLC), kelch-like ECH-associated protein 1 (KEAP1), glutamate cysteine ligase regulatory subunit (GCLM) and glutathione peroxidase 4 (GPX4) were lower. To simulate the ferroptosis state of adipose tissue in ketotic cows, primary bovine adipocytes were isolated from the adipose tissue of healthy cows and cultured with erastin to construct ferroptosis model. Adipocytes were cultured with either an adenovirus overexpressing HIF-1α or small interfering RNA targeting HIF-for 48 h, followed by exposure to erastin (1 µM) for 24 h. Treatment with erastin led to higher protein abundance of CAT, SOD1, NFE2L2 and HMOX1, while it inhibited the protein expression levels of GCLC, SLC7A11, GCLM, GPX4 and KEAP1. Furthermore, erastin treatment elevated the levels of ROS, MDA, Fe2+, total iron and reduced the content of GSH. The overexpression of HIF-1α reversed the erastin-induced decreases in the protein abundance of GPX4 and SLC7A11, as well as the levels of MDA, ROS, Fe2+ and total iron, while significantly increasing protein abundance and content of CAT, SOD1, NFE2L2, HMOX1, GCLC, GCLM, GPX4, SLC7A11 and GSH. Conversely, the silencing of HIF-1α further exacerbated the erastin-induced levels of MDA, ROS, Fe2+ and total iron, while inhibiting the upregulation of SOD1, CAT, NFE2L2 and HMOX1. Collectively, these findings suggest that activation of HIF-1α may function as an adaptive mechanism to mitigate ferroptosis and alleviate oxidative stress in adipose tissue.

Diacylglycerol O-acyltransferase (DGAT) isoforms play a role in peridroplet mitochondrial fatty acid metabolism in bovine liver.

Hepatocellular lipid accumulation characterizes fatty liver in dairy cows. Lipid droplets (LD), specialized organelles that store lipids and maintain cellular lipid homeostasis, are responsible for the ectopic storage of lipids associated with several metabolic disorders. In recent years, non-ruminant studies have reported that LD-mitochondria interactions play an important role in lipid metabolism. Due to the role of diacylglycerol acyltransferase isoforms (DGAT1 and DGAT2) in LD synthesis, we explored mechanisms of mitochondrial fatty acid transport in ketotic cows using liver biopsies and isolated primary hepatocytes. Compared with healthy cows, cows with fatty liver had massive accumulation of LD and high protein expression of the triglyceride (TAG) synthesis-related enzymes DGAT1 and DGAT2, LD synthesis-related proteins perilipin 2 (PLIN2) and perilipin 5 (PLIN5), and the mitochondrial fragmentation-related proteins dynamin-related protein 1 (DRP1) and fission 1 (FIS1). In contrast, factors associated with fatty acid oxidation, mitochondrial fusion and mitochondrial electron transport chain complex were lower compared with those in the healthy cows. In addition, transmission electron microscopy revealed significant contacts between LD-mitochondria in liver tissue from cows with fatty liver. Compared with isolated cytoplasmic mitochondria, expression of carnitine palmitoyl transferase 1A (CPT1A) and DRP1 was lower, but mitofusin 2 (MFN2) and mitochondrial electron transport chain complex was greater in isolated peridroplet mitochondria from hepatic tissue of cows with fatty liver. In vitro data indicated that exogenous free fatty acids (FFA) induced hepatocyte LD synthesis and mitochondrial dynamics consistent with in vivo results. Furthermore, DGAT2 inhibitor treatment attenuated the FFA-induced upregulation of PLIN2 and PLIN5 and rescued the impairment of mitochondrial dynamics. Inhibition of DGAT2 also restored mitochondrial membrane potential and reduced hepatocyte reactive oxygen species production. The present in vivo and in vitro results indicated there are functional differences among different types of mitochondria in the liver tissue of dairy cows with ketosis. Activity of DGAT2 may play a key role in maintaining liver mitochondrial function and lipid homeostasis in dairy cows during the transition period.

Biological and genomic characterization of 4 novel bacteriophages isolated from sewage or the environment using non-aureus Staphylococci strains.

Non-aureus staphylococci (NAS) are an essential group of bacteria causing antimicrobial resistant intramammary infections in livestock, particularly dairy cows. Therefore, bacteriophages emerge as a potent bactericidal agent for NAS mastitis. This study aimed to obtain NAS-specific bacteriophages using bacterial strains isolated from cows with mastitis, subsequently evaluating their morphological, genomic, and lytic characteristics. Four distinct NAS bacteriophages were recovered from sewage or the environment of Chinese dairy farms; PT1-1, PT94, and PT1-9 were isolated using Staphylococcus chromogenes and PT1-4 using Staphylococcus gallinarum. Both PT1-1 (24/54, 44 %) and PT94 (28/54, 52 %) had broader lysis than PT1-4 (3/54, 6 %) and PT1-9 (10/54, 19 %), but PT1-4 and PT1-9 achieved cross-species lysis. All bacteriophages had a short latency period and good environmental tolerance, including surviving at pH=4-10 and at 30-60℃. Except for PT1-9, all bacteriophages had excellent bactericidal efficacy within 5 h of co-culture with host bacteria in vitro at various multiplicity of infection (MOIs). Based on whole genome sequencing, average nucleotide identity (ANI) analysis of PT1-1 and PT94 can be classified as the same species, consistent with whole-genome synteny analysis. Although motifs shared by the 4 bacteriophages differed little from those of other bacteriophages, a phylogenetic tree based on functional proteins indicated their novelty. Moreover, based on whole genome comparisons, we inferred that cross-species lysis of bacteriophage may be related to the presence of "phage tail fiber." In conclusion 4 novel NAS bacteriophages were isolated; they had good biological properties and unique genomes, with potential for NAS mastitis therapy.

ß-sitosterol alleviates high fatty acid-induced lipid accumulation in calf hepatocytes by regulating cholesterol metabolism.

A significant reduction in plasma concentration of cholesterol during early lactation is a common occurrence in high-yielding dairy cows. An insufficient synthesis of cholesterol in the liver has been linked to lipid accumulation caused by high concentrations of fatty acids during negative energy balance (NEB). As ruminant diets do not provide quantitative amounts of cholesterol for absorption, phytosterols such as ß-sitosterol may serve to mitigate the shortfall in cholesterol within the liver during NEB. To gain mechanistic insights, primary hepatocytes were isolated from healthy female 1-day old calves for in vitro studies with or without 1.2 mM fatty acids (FA) to induce metabolic stress. Furthermore, hepatocytes were treated with 50 µM ß-sitosterol with or without FA. Data were analyzed by one-way ANOVA with subsequent Bonferroni correction. Results revealed that calf hepatocytes treated with FA had greater content of non-esterified fatty acids (NEFA) and triacylglycerol (TAG), and greater mRNA and protein abundance of the lipid synthesis-related SREBF1 and FASN. In contrast, mRNA and protein of CPT1A (fatty acid oxidation) and the cholesterol metabolism-related targets SREBF2, HMGCR, ACAT2, APOA1, ABCA1 and ABCG5 was lower. Content of the antioxidant-related glutathione (GSH) and activities of superoxide dismutase (SOD) also was lower. Compared with FA challenge alone, 50 µM ß-sitosterol led to greater mRNA and protein abundance of SREBF2, HMGCR, ACAT2 and ABCG5, and greater content of GSH and activity of SOD. In contrast, compared with the FA group, the mRNA and protein abundance of SREBF1 and ACC1 and the content of TAG and NEFA in the ß-sitosterol + FA group were lower. Overall, ß-sitosterol can promote cholesterol metabolism and reduce oxidative stress while reducing lipid accumulation in hepatocytes challenged with high concentrations of fatty acids.

Fatty acids promote M1 polarization of monocyte-derived macrophages in healthy or ketotic dairy cows and a bovine macrophage cell line by impairing mTOR-mediated autophagy.

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In nonruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, our objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. We performed 4 experiments: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated for 24 h with 100 ng/mL LPS and 100 ng/mL IFN-γ or with 10 ng/mL IL4 and 10 ng/mL IL10; (2) Immortalized bovine macrophages were treated for 24 h with 0, 0.3, 0.6, or 1.2 mM FFA, LPS, and IFN-γ, or with IL4 and IL10; (3) Macrophages were pretreated with 2 µM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86+, but lower MFI of CD206+ and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and the MFI of CD86, whereas it downregulated the protein abundance of arginase 1 and the MFI of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α, IL1B, and IL6 mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate protein abundance and the number of autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows by impairing mTOR-mediated autophagy.

Sagittal balance parameters measurement on cervical spine MR images based on superpixel segmentation.

Introduction: Magnetic Resonance Imaging (MRI) is essential in diagnosing cervical spondylosis, providing detailed visualization of osseous and soft tissue structures in the cervical spine. However, manual measurements hinder the assessment of cervical spine sagittal balance, leading to time-consuming and error-prone processes. This study presents the Pyramid DBSCAN Simple Linear Iterative Cluster (PDB-SLIC), an automated segmentation algorithm for vertebral bodies in T2-weighted MR images, aiming to streamline sagittal balance assessment for spinal surgeons. Method: PDB-SLIC combines the SLIC superpixel segmentation algorithm with DBSCAN clustering and underwent rigorous testing using an extensive dataset of T2-weighted mid-sagittal MR images from 4,258 patients across ten hospitals in China. The efficacy of PDB-SLIC was compared against other algorithms and networks in terms of superpixel segmentation quality and vertebral body segmentation accuracy. Validation included a comparative analysis of manual and automated measurements of cervical sagittal parameters and scrutiny of PDB-SLIC's measurement stability across diverse hospital settings and MR scanning machines. Result: PDB-SLIC outperforms other algorithms in vertebral body segmentation quality, with high accuracy, recall, and Jaccard index. Minimal error deviation was observed compared to manual measurements, with correlation coefficients exceeding 95%. PDB-SLIC demonstrated commendable performance in processing cervical spine T2-weighted MR images from various hospital settings, MRI machines, and patient demographics. Discussion: The PDB-SLIC algorithm emerges as an accurate, objective, and efficient tool for evaluating cervical spine sagittal balance, providing valuable assistance to spinal surgeons in preoperative assessment, surgical strategy formulation, and prognostic inference. Additionally, it facilitates comprehensive measurement of sagittal balance parameters across diverse patient cohorts, contributing to the establishment of normative standards for cervical spine MR imaging.

Role of diacylglycerol O-acyltransferase 1 (DGAT1) in lipolysis and autophagy of adipose tissue from ketotic dairy cows.

High-yielding dairy cows in early lactation often encounter difficulties in meeting the energy requirements essential for maintaining milk production. This is primarily attributed to insufficient dry matter intake, which consequently leads to sustained lipolysis of adipose tissue. Fatty acids released by lipolysis can disrupt metabolic homeostasis. Autophagy, an adaptive response to intracellular environmental changes, is considered a crucial mechanism for regulating lipid metabolism and maintaining a proper cellular energy status. Despite its close relationship with aberrant lipid metabolism and cytolipotoxicity in animal models of metabolic disorders, the precise function of diacylglycerol o-acyltransferase 1 (DGAT1) in bovine adipose tissue during periods of negative energy balance is not fully understood, particularly regarding its involvement in lipolysis and autophagy. The objective of the present study was to assess the effect of DGAT1 on both lipolysis and autophagy in bovine adipose tissue and isolated adipocytes. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of BHB, which were 3.19 mM (interquartile range = 0.20) and 0.50 mM (interquartile range = 0.06), respectively. Protein abundance of DGAT1 and phosphorylation levels of unc-51-like kinase 1 (ULK1), were greater in adipose tissue from cows with ketosis, whereas phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were lower. Furthermore, when adipocytes isolated from the harvested adipose tissue of 15 healthy cows were transfected with DGAT1 overexpression adenovirus or DGAT1 small interfering RNA followed by exposure to epinephrine (EPI), it led to greater ratios and protein abundance of phosphorylated hormone-sensitive triglyceride lipase (LIPE) to total LIPE and adipose triglyceride lipase (ATGL), while inhibiting the protein phosphorylation levels of ULK1, PI3K, AKT, and mTOR. Overexpression of DGAT1 in EPI-treated adipocytes reduced lipolysis and autophagy, whereas silencing DGAT1 further exacerbated EPI-induced lipolysis and autophagy. Taken together, these findings indicate that upregulation of DGAT1 may function as an adaptive response to suppress adipocytes lipolysis, highlighting the significance of maintaining metabolic homeostasis in dairy cows during periods of negative energy balance.

Emerging role of extracellular vesicles in veterinary practice: novel opportunities and potential challenges.

Extracellular vesicles are nanoscale vesicles that transport signals between cells, mediating both physiological and pathological processes. EVs facilitate conserved intercellular communication. By transferring bioactive molecules between cells, EVs coordinate systemic responses, regulating homeostasis, immunity, and disease progression. Given their biological importance and involvement in pathogenesis, EVs show promise as biomarkers for veterinary diagnosis, and candidates for vaccine production, and treatment agents. Additionally, different treatment or engineering methods could be used to boost the capability of extracellular vesicles. Despite the emerging veterinary interest, EV research has been predominantly human-based. Critical knowledge gaps remain regarding isolation protocols, cargo loading mechanisms, in vivo biodistribution, and species-specific functions. Standardized methods for veterinary EV characterization and validation are lacking. Regulatory uncertainties impede veterinary clinical translation. Advances in fundamental EV biology and technology are needed to propel the veterinary field forward. This review introduces EVs from a veterinary perspective by introducing the latest studies, highlighting their potential while analyzing challenges to motivate expanded veterinary investigation and translation.

The Prediction of Clinical Mastitis in Dairy Cows Based on Milk Yield, Rumination Time, and Milk Electrical Conductivity Using Machine Learning Algorithms.

In commercial dairy farms, mastitis is associated with increased antimicrobial use and associated resistance, which may affect milk production. This study aimed to develop sensor-based prediction models for naturally occurring clinical bovine mastitis using nine machine learning algorithms with data from 447 mastitic and 2146 healthy cows obtained from five commercial farms in Northeast China. The variables were related to daily activity, rumination time, and daily milk yield of cows, as well as milk electrical conductivity. Both Z-standardized and non-standardized datasets pertaining to four specific stages of lactation were used to train and test prediction models. For all four subgroups, the Z-standardized dataset yielded better results than those of the non-standardized one, with the multilayer artificial neural net algorithm showing the best performance. Variables of importance had a similar rank in this algorithm, indicating the consistency of these variables as predictors for bovine mastitis in commercial farms with similar automatic systems. Moreover, the peak milk yield (PMY) of mastitic cows was significantly higher than that of healthy cows (p < 0.005), indicating that high-yielding cattle are more prone to mastitis. Our results show that machine learning algorithms are effective tools for predicting mastitis in dairy cows for immediate intervention and management in commercial farms.

Platelet-rich plasma-derived exosomes boost mesenchymal stem cells to promote peripheral nerve regeneration.

Peripheral nerve injury (PNI) remains a severe clinical problem with debilitating consequences. Mesenchymal stem cell (MSC)-based therapy is promising, but the problems of poor engraftment and insufficient neurotrophic effects need to be overcome. Herein, we isolated platelet-rich plasma-derived exosomes (PRP-Exos), which contain abundant bioactive molecules, and investigated their potential to increase the regenerative capacity of MSCs. We observed that PRP-Exos significantly increased MSC proliferation, viability, and mobility, decreased MSC apoptosis under stress, maintained MSC stemness, and attenuated MSC senescence. In vivo, PRP-Exo-treated MSCs (pExo-MSCs) exhibited an increased retention rate and heightened therapeutic efficacy, as indicated by increased axonal regeneration, remyelination, and recovery of neurological function in a PNI model. In vitro, pExo-MSCs coculture promoted Schwann cell proliferation and dorsal root ganglion axon growth. Moreover, the increased neurotrophic behaviour of pExo-MSCs was mediated by trophic factors, particularly glia-derived neurotrophic factor (GDNF), and PRP-Exos activated the PI3K/Akt signalling pathway in MSCs, leading to the observed phenotypes. These findings demonstrate that PRP-Exos may be novel agents for increasing the ability of MSCs to promote neural repair and regeneration in patients with PNI.

The Ameliorative Role of Lico A on Aflatoxin B1-Triggered Hepatotoxicity Partially by Activating Nrf2 Signal Pathway.

Aflatoxin B1 (AFB1) is one of the most harmful and toxic mycotoxins in foods and feeds, posing a serious health risk to both humans and animals, especially its hepatotoxicity. Nuclear factor-erythroid 2-related factor 2 (Nrf2), an important nuclear transcription factor, is generally recognized as a potential target for phytochemicals to ameliorate liver injury. The current study sought to elucidate the molecular processes by which licochalcone A (Lico A), a compound derived from Xinjiang licorice Glycyrrhiza inflate, protects against AFB1 toxicity. In vivo, male wild-type (WT) and Nrf2 knockout (Nrf2-/-) C57BL/6 mice were orally administered AFB1 at 1.5 mg/kg body weight (BW) with or without Lico A at 5 mg/kg. In vitro, AML12 cells were utilized to evaluate the protective effect and mechanism of Lico A against the AFB1-induced hepatotoxicity. Our findings demonstrated that AFB1 caused severe hepatotoxicity, while Lico A treatment successfully relieved the toxicity. Meanwhile, Lico A effectively improved liver injury, inflammatory mediators, oxidative insults, apoptosis, liver fibrosis, and pyroptosis, which contributed to the inhibition of toll receptor 4 (TLR4)-NF-κB/MAPK and NOD-like receptors protein 3 (NLRP3)/caspase-1/GSDMD signaling pathway activation. Furthermore, Lico A was able to enhance the Nrf2 antioxidant signaling pathway. Intriguingly, Lico A still had a protective effect on AFB1-caused liver injury in mice via the inhibition of inflammation and pyroptosis, while apoptosis and liver fibrosis were blocked in the absence of Nrf2. To sum up, the present study first elucidated that Lico A ameliorated AFB1-induced hepatotoxic effects and its main mechanism involved the inhibitory effects on oxidative stress, apoptosis, liver fibrosis, inflammation, and pyroptosis, which might be partially dependent on the regulation of Nrf2. The work may enrich the role and mechanism of Lico A's resistance to liver injury caused by various factors, and its application is promising.

Intervention of muscle-building and antifrailty exercise combined with Baduanjin for frailty of different functional levels: study protocol for a randomised controlled trial.

INTRODUCTION: Frailty has been currently considered as a multidimensional concept, including physical, cognitive and social frailty. Frailty has also been associated with a range of adverse events, which might increase the risks of disability, falls, fractures, delirium and death. Increasing evidence has shown that multicomponent exercise training can improve physical and cognitive function, delay or reverse frailty. However, there is still a lack of exercise intervention programmes for the frail older adults in China. This trial aims to investigate the effects of the muscle-building and antifrailty exercise combined with Baduanjin on the physical function of frail older adults, as well as the effectiveness and safety of the intervention. METHODS AND ANALYSIS: This study is a prospective randomised controlled trial. A total of 192 patients, aged 70 years or older, who are diagnosed as prefrailty or frailty based on the Fried criteria will be included. Prior written and informed consent will be obtained from every subject. These subjects will be randomly assigned to the exercise intervention group (n=96) and the control group (n=96). The exercise intervention group will undergo different exercise programmes for different levels of physical function. They will perform the muscle-building and antifrailty exercise three times per week for 30-60 min for 24 weeks. The control group will implement health education on frailty and maintain the old lifestyle without any intervention.The primary outcomes include the change in frailty and functional capacity, assessed according to the Fried Scale and the Short Physical Performance Battery. Secondary outcomes include the changes in body composition, Activities of daily living, Mini-Mental State Examination, The Geriatric Depression Scale-15 and the haematological indicators. ETHICS STATEMENT: The study has been approved by the Medical Ethics Committee of the PLA General Hospital (approval no.: S2022-600-02). TRIAL REGISTRATION NUMBER: ChiCTR2300070535.

Free Fatty Acids Induce Apoptosis of Mammary Epithelial Cells of Ketotic Dairy Cows via the Mito-ROS/NLRP3 Signaling Pathway.

Early lactation increases metabolic stress in ketotic dairy cows, leading to mitochondrial damage, apoptosis, and inflammatory response in mammary epithelial cells. The pyrin domain 3 (NLRP3) pathway involving the mitochondrial reactive oxygen species (Mito-ROS)-induced nucleotide-binding oligomerization domain-like receptor has been recognized as a key mechanism in this inflammatory response and cell apoptosis. This study aimed to elucidate the underlying regulatory mechanism of Mito-ROS-NLRP3 pathway-mediated mammary epithelial cell apoptosis in dairy cows with ketosis. Mitochondrial damage and cellular apoptotic program and NLRP3 inflammasome activation were observed in the mammary gland of ketotic cows. Similar damage was detected in MAC-T cells treated with exogenous fatty acids (FFAs). However, NLRP3 inhibitor MCC950 pretreatment or Mito-ROS scavenging by MitoTEMPO attenuated apoptosis in FFA-induced MAC-T cells by inhibiting the NLRP3 inflammasome pathway. These findings reveal that the Mito-ROS-NLRP3 pathway activation is a potent mechanism underlying mammary epithelial cell apoptosis in response to metabolic stress in ketotic dairy cows, which further contributes to reduced milk yield.

Prognostic impact of metastatic patterns and treatment modalities on overall survival in lung squamous cell carcinoma: A population-based study.

This study aimed to investigate the impact of distinct metastasis patterns on the overall survival (OS) of individuals diagnosed with organ metastatic lung squamous cell carcinoma (LUSC). OS was calculated using the Kaplan-Meier method, and univariate and multivariate Cox regression analyses were conducted to further assess prognostic factors. A total of 36,025 cases meeting the specified criteria were extracted from the Surveillance, Epidemiology, and End Results database. Among these patients, 30.60% (11,023/36,025) were initially diagnosed at stage IV, and 22.03% (7936/36,025) of these individuals exhibited metastasis in at least 1 organ, including the liver, bone, lung, and brain. Among the 4 types of single metastasis, patients with bone metastasis had the lowest mean OS, at 9.438 months (95% CI: 8.684-10.192). Furthermore, among patients with dual-organ metastases, those with both brain and liver metastases had the shortest mean OS, at 5.523 months (95% CI: 3.762-7.285). Multivariate Cox regression analysis revealed that metastatic site is an independent prognostic factor for OS in patients with single and dual-organ metastases. Chemotherapy was beneficial for patients with single and multiple-organ metastases; although surgery was advantageous for those with single and dual-organ metastases, it did not affect the long-term prognosis of patients with triple organ metastases. Radiotherapy only conferred benefits to patients with single-organ metastasis. LUSC patients exhibit a high incidence of metastasis at the time of initial diagnosis, with significant differences in long-term survival among patients with different patterns of metastasis. Among single-organ metastasis cases, lung metastasis is the most frequent and is associated with the longest mean OS. Regarding treatment options, patients with single-organ metastasis can benefit from chemotherapy, surgery, and radiotherapy, and those with metastasis in 2 organs can benefit from chemotherapy and surgery. Patients with metastasis in more than 2 organs, however, can only benefit from chemotherapy. Understanding the variations in metastasis patterns assists in guiding pretreatment assessments and in determining appropriate therapeutic interventions for LUSC.

Effects of Chromium Propionate and Calcium Propionate on Lactation Performance and Rumen Microbiota in Postpartum Heat-Stressed Holstein Dairy Cows.

Chromium propionate (Cr-Pro) and calcium propionate (Ca-Pro) are widely applied in dairy production, especially in the alleviation of heat stress (HS). HS can reduce the abundance of rumen microbiota and the lactation performance of dairy cows. The present work mainly focused on evaluating the effects of Cr-Pro and Ca-Pro on the performance, ruminal bacterial community, and stress of postpartum HS dairy cows as well as identifying the differences in their mechanisms. Fifteen multiparous postpartum Holstein cows with equivalent weights (694 ± 28 kg) and milk yields (41.2 ± 1.21 kg/day) were randomly divided into three groups: control (CON), Cr-Pro (CRPR), and Ca-Pro (CAPR). The control cows received the basal total mixed ration (TMR) diet, while the CRPR group received TMR with 3.13 g/day of Cr-Pro, and the CAPR group received TMR with 200 g/day of Ca-Pro. The rumen microbial 16S rRNA was sequenced using the Illumina NovaSeq platform along with the measurement of ruminal volatile fatty acids (VFAs) and milking performance. Cr-Pro and Ca-Pro improved lactation performance, increased the rumen VFA concentration, and altered the rumen microbiota of the HS dairy cows. Cr-Pro significantly improved the milk yield (p < 0.01). The richness and diversity of the microbial species significantly increased after feeding on Ca-Pro (p < 0.05). Gene function prediction revealed increased metabolic pathways and biological-synthesis-related function in the groups supplemented with Cr-Pro and Ca-Pro. Our results indicate that the application of Cr-Pro or Ca-Pro can provide relief for heat stress in dairy cows through different mechanisms, and a combination of both is recommended for optimal results in production.

Sodium Propionate Relieves LPS-Induced Inflammation by Suppressing the NF-ĸB and MAPK Signaling Pathways in Rumen Epithelial Cells of Holstein Cows.

Subacute ruminal acidosis (SARA) is a prevalent disease in intensive dairy farming, and the rumen environment of diseased cows acidifies, leading to the rupture of gram-negative bacteria to release lipopolysaccharide (LPS). LPS can cause rumentitis and other complications, such as liver abscess, mastitis and laminitis. Propionate, commonly used in the dairy industry as a feed additive, has anti-inflammatory effects, but its mechanism is unclear. This study aims to investigate whether sodium propionate (SP) reduces LPS-induced inflammation in rumen epithelial cells (RECs) and the underlying mechanism. RECs were stimulated with different time (0, 1, 3, 6, 9, 18 h) and different concentrations of LPS (0, 1, 5, 10 µg/mL) to establish an inflammation model. Then, RECs were treated with SP (15, 25, 35 mM) or 10 µM PDTC in advance and stimulated by LPS for the assessment. The results showed that LPS (6h and 10 µg/mL) could stimulate the phosphorylation of NF-κB p65, IκB, JNK, ERK and p38 MAPK through TLR4, and increase the release of TNF-α, IL-1ß and IL-6. SP (35 mM) can reduce the expression of cytokines by effectively inhibiting the NF-κB and MAPK inflammatory pathways. This study confirmed that SP inhibited LPS-induced inflammatory responses through NF-κB and MAPK in RECs, providing potential therapeutic targets and drugs for the prevention and treatment of SARA.

Molecular Characteristics of Bovine Viral Diarrhea Virus Strains Isolated from Persistently Infected Cattle.

In this study, we reported the isolation, identification, and molecular characteristics of nine BVDV strains that were isolated from the serum of persistently infected cattle. The new strains were designated as BVDV TJ2101, TJ2102, TJ2103, TJ2104, TJ2105, TJ2106, TJ2107, TJ2108 and TJ2109. The TJ2102 and TJ2104 strains were found to be cytopathic BVDV, and the other strains were non-cytopathic BVDV. An alignment and phylogenetic analysis showed that the new isolates share 92.2-96.3% homology with the CP7 strain and, thus, were classified as the BVDV-1b subgenotype. A recombination analysis of the genome sequences showed that the new strains could be recombined by the major parent BVDV-1a NADL strain and the minor parent BVDV-1m SD-15 strain. Some genome variations or unique amino acid mutations were found in 5'-UTR, E0 and E2 of these new isolates. In addition, a potential linear B cell epitopes prediction showed that the potential linear B cell epitope at positions 56-61 is highly variable in BVDV-1b. In conclusion, the present study has identified nine strains of BVDV from persistently infected cattle in China. Further studies on the virulence and pathogenesis of these new strains are recommended.

Alternatives to antibiotics for treatment of mastitis in dairy cows.

Mastitis is considered the costliest disease on dairy farms and also adversely affects animal welfare. As treatment (and to a lesser extent prevention) of mastitis rely heavily on antibiotics, there are increasing concerns in veterinary and human medicine regarding development of antimicrobial resistance. Furthermore, with genes conferring resistance being capable of transfer to heterologous strains, reducing resistance in strains of animal origin should have positive impacts on humans. This article briefly reviews potential roles of non-steroidal anti-inflammatory drugs (NSAIDs), herbal medicines, antimicrobial peptides (AMPs), bacteriophages and their lytic enzymes, vaccination and other emerging therapies for prevention and treatment of mastitis in dairy cows. Although many of these approaches currently lack proven therapeutic efficacy, at least some may gradually replace antibiotics, especially as drug-resistant bacteria are proliferating globally.