VP3 protein of Senecavirus A promotes viral IRES-driven translation and attenuates innate immunity by specifically relocalizing hnRNPA2B1.

Viruses deploy sophisticated strategies to hijack the host's translation machinery to favor viral protein synthesis and counteract innate cellular defenses. However, little is known about the mechanisms by which Senecavirus A (SVA) controls the host's translation. Using a series of sophisticated molecular cell manipulation techniques, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as an essential host factor involved in translation control in SVA-infected cells. It was also determined that the SVA structural protein, VP3, binds to and relocalizes hnRNPA2B1, which interferes with the host's protein synthesis machinery to establish a cellular environment that facilitates viral propagation via a two-pronged strategy: first, hnRNPA2B1 serves as a potent internal ribosome entry site (IRES) trans-acting factor, which is selectively co-opted to promote viral IRES-driven translation by supporting the assembly of translation initiation complexes. Second, a strong repression of host cell translation occurs in the context of the VP3-hnRNPA2B1 interaction, resulting in attenuation of the interferons response. This is the first study to demonstrate the interaction between SVA VP3 and hnRNPA2B1, and to characterize their key roles in manipulating translation. This novel dual mechanism, which regulates selective mRNA translation and immune evasion of virus-infected cells, highlights the VP3-hnRNPA2B1 complex as a potential target for the development of modified antiviral or oncolytic reagents. IMPORTANCE: Viral reproduction is contingent on viral protein synthesis, which relies entirely on the host's translation machinery. As such, viruses often need to control the cellular translational apparatus to favor viral protein production and avoid host innate defenses. Senecavirus A (SVA) is an important virus, both as an emerging pathogen in the pork industry and as a potential oncolytic virus for neuroendocrine cancers. Here, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as a critical regulator of the translational landscape during SVA infection. This study supports a model whereby the VP3 protein of SVA efficiently subverts the host's protein synthesis machinery through its ability to bind to and relocalize hnRNPA2B1, not only selectively promoting viral internal ribosome entry site-driven translation but also resulting in global translation shutdown and immune evasion. Together, these data provide new insights into how the complex interactions between translation machinery, SVA, and innate immunity contribute to the pathogenicity of the SVA.

Body mass index, waist circumference, and mortality in subjects older than 80 years: a Mendelian randomization study.

BACKGROUND AND AIMS: Emerging evidence has raised an obesity paradox in observational studies of body mass index (BMI) and health among the oldest-old (aged ≥80 years), as an inverse relationship of BMI with mortality was reported. This study was to investigate the causal associations of BMI, waist circumference (WC), or both with mortality in the oldest-old people in China. METHODS: A total of 5306 community-based oldest-old (mean age 90.6 years) were enrolled in the Chinese Longitudinal Healthy Longevity Survey (CLHLS) between 1998 and 2018. Genetic risk scores were constructed from 58 single-nucleotide polymorphisms (SNPs) associated with BMI and 49 SNPs associated with WC to subsequently derive causal estimates for Mendelian randomization (MR) models. One-sample linear MR along with non-linear MR analyses were performed to explore the associations of genetically predicted BMI, WC, and their joint effect with all-cause mortality, cardiovascular disease (CVD) mortality, and non-CVD mortality. RESULTS: During 24 337 person-years of follow-up, 3766 deaths were documented. In observational analyses, higher BMI and WC were both associated with decreased mortality risk [hazard ratio (HR) 0.963, 95% confidence interval (CI) 0.955-0.971 for a 1-kg/m2 increment of BMI and HR 0.971 (95% CI 0.950-0.993) for each 5 cm increase of WC]. Linear MR models indicated that each 1 kg/m2 increase in genetically predicted BMI was monotonically associated with a 4.5% decrease in all-cause mortality risk [HR 0.955 (95% CI 0.928-0.983)]. Non-linear curves showed the lowest mortality risk at the BMI of around 28.0 kg/m2, suggesting that optimal BMI for the oldest-old may be around overweight or mild obesity. Positive monotonic causal associations were observed between WC and all-cause mortality [HR 1.108 (95% CI 1.036-1.185) per 5 cm increase], CVD mortality [HR 1.193 (95% CI 1.064-1.337)], and non-CVD mortality [HR 1.110 (95% CI 1.016-1.212)]. The joint effect analyses indicated that the lowest risk was observed among those with higher BMI and lower WC. CONCLUSIONS: Among the oldest-old, opposite causal associations of BMI and WC with mortality were observed, and a body figure with higher BMI and lower WC could substantially decrease the mortality risk. Guidelines for the weight management should be cautiously designed and implemented among the oldest-old people, considering distinct roles of BMI and WC.

An entropy-based approach for assessing the directional persistence of cell migration.

Cell migration, which is primarily characterized by directional persistence, is essential for the development of normal tissues and organs, as well as for numerous pathological processes. However, there is a lack of simple and efficient tools to analyze the systematic properties of persistence based on cellular trajectory data. Here, we present a novel approach, the entropy of angular distribution , which combines cellular turning dynamics and Shannon entropy to explore the statistical and time-varying properties of persistence that strongly correlate with cellular migration modes. Our results reveal the changes in the persistence of multiple cell lines that are tightly regulated by both intra- and extracellular cues, including Arpin protein, collagen gel/substrate, and physical constraints. Significantly, some previously unreported distinctive details of persistence have also been captured, helping to elucidate how directional persistence is distributed and evolves in different cell populations. The analysis suggests that the entropy of angular distribution-based approach provides a powerful metric for evaluating directional persistence and enables us to better understand the relationships between cellular behaviors and multiscale cues, which also provides some insights into the migration dynamics of cell populations, such as collective cell invasion.

Element Screening of High-Entropy Silicon Anodes for Superior Li-Storage Performance of Li-Ion Batteries.

The high-entropy silicon anodes are attractive for enhancing electronic and Li-ionic conductivity while mitigating volume effects for advanced Li-ion batteries (LIBs), but are plagued by the complicated elements screening process. Inspired by the resemblances in the structure between sphalerite and diamond, we have selected sphalerite-structured SiP with metallic conductivity as the parent phase for exploring the element screening of high-entropy silicon-based anodes. The inclusion of the Zn in the sphalerite structure is crucial for improving the structural stability and Li-storage capacity. Within the same group, Li-storage performance is significantly improved with increasing atomic number in the order of BZnSiP3 < AlZnSiP3 < GaZnSiP3 < InZnSiP3. Thus, InZnSiP3-based electrodes achieved a high capacity of 719 mA h g-1 even after 1,500 cycles at 2,000 mA g-1, and a high-rate capacity of 725 mA h g-1 at 10,000 mA g-1, owing to its superior lithium-ion affinity, faster electronic conduction and lithium-ion diffusion, higher Li-storage capacity and reversibility, and mechanical integrity than others. Additionally, the incorporation of elements with larger atomic sizes leads to greater lattice distortion and more defects, further facilitating mass and charge transport. Following these screening rules, high-entropy disordered-cation silicon-based compounds such as GaCuSnInZnSiP6, GaCu(or Sn)InZnSiP5, and CuSnInZnSiP5, as well as high-entropy compounds with mixed-cation and -anion compositions, such as InZnSiPSeTe and InZnSiP2Se(or Te), are synthesized, demonstrating improved Li-storage performance with metallic conductivity. The phase formation mechanism of these compounds is attributed to the negative formation energies arising from elevated entropy.

Multicolor Tuning of Perylene Diimides Dyes for Targeted Organelle Imaging In Vivo.

The adjustment of the emission wavelengths and cell permeability of the perylene diimides (PDI) for multicolor cell imaging is a great challenge. Herein, based on a bay-region substituent engineering strategy, multicolor perylene diimides (MCPDI) were rationally designed and synthesized by introducing azetidine substituents on the bay region of PDIs. With the fine-tuned electron-donating ability of the azetidine substituents, these MCPDI showed high brightness, orange, red, and near infrared (NIR) fluorescence along with Stokes shifts increasing from 35 to 110 nm. Interestingly, azetidine substituents distorted to the plane of the MCPDI dyes, and the twist angle of monosubstituted MCPDI was larger than that of disubstituted MCPDI, which might efficiently decrease their π-π stacking. Moreover, all of these MCPDI dyes were cell-permeable and selectively stained various organelles for multicolor imaging of multiple organelles in living cells. Two-color imaging of lipid droplets (LDs) and other organelles stained with MCPDI dyes was performed to reveal the interaction between the LDs and other organelles in living cells. Furthermore, a NIR-emitting MCPDI dye with a mitochondria-targeted characteristic was successfully applied for tumor-specific imaging. The facile synthesis, excellent stability, high brightness, tunable fluorescence emission, and Stokes shifts make these MCPDI promising fluorescent probes for biological applications.

Superior Strength, Toughness, and Damage-Tolerance Observed in Microlattices of Aperiodic Unit Cells.

Characterized by periodic cellular unit cells, microlattices offer exceptional potential as lightweight and robust materials. However, their inherent periodicity poses the risk of catastrophic global failure. To address this limitation, a novel approach, that is to introduce microlattices composed of aperiodic unit cells inspired by Einstein's tile, where the orientation of cells never repeats in the same orientation is proposed. Experiments and simulations are conducted to validate the concept by comparing compressive responses of the aperiodic microlattices with those of common periodic microlattices. Indeed, the microlattices exhibit stable and progressive compressive deformation, contrasting with catastrophic fracture of periodic structures. At the same relative density, the microlattices outperform the periodic ones, exhibiting fracture strain, energy absorption, crushing stress efficiency, and smoothness coefficients at least 830%, 300%, 130%, and 160% higher, respectively. These improvements can be attributed to aperiodicity, where diverse failure thresholds exist locally due to varying strut angles and contact modes during compression. This effectively prevents both global fracture and abrupt stress drops. Furthermore, the aperiodic microlattice exhibits good damage tolerance with excellent deformation recoverability, retaining 76% ultimate stress post-recovery at 30% compressive strain. Overall, a novel concept of adopting aperiodic cell arrangements to achieve damage-tolerant microlattice metamaterials is presented.

Operation-robust SiO2-based memristive application in air.

In recent years, neuromorphic computing is recognized as a promising path to further improve the efficiency of integrated computing system in the post-Moore era, relying on its high parallelism. As a key fundamental element in hardware-implementing neuromorphic system, the synaptic device has made substantial research progress. Among these, SiO2 trapping-based memristive devices generally have systematically integrated merits, such as ease of fabrication and high CMOS process compatibility, but electrochemical activity to oxygen makes them unreliable for operating in air. Here, by using ultrathin Si3N4 as a physical isolation layer, we have obtained a robust memristive device based on SiO2 trapping although operating in air. Further study of Si3N4 thickness dependence has demonstrated that 7 nm is suggested as the most favorable thickness for reliable and flexible programming, and that an inherent isolating mechanism is 'switching-on' for an electron but 'switching-off' for large-sized oxygen molecules. Based on a device with 7 nm Si3N4, we have mimicked various modes of synaptic plasticities. These results could thus not only increase the prospects of using SiO2 trapping in memristive applications but also provide an effective path to improve the robustness of these SiO2-based applications against ambient air.

Serum 25-Hydroxyvitamin D and Risk of Disability in Activities of Daily Living among the Oldest-Old: An Observational and Mendelian Randomization Study.

BACKGROUND: Vitamin D deficiency and disability are both prevalent among older adults. However, the association between them has rarely been investigated in the oldest-old subjects (aged ≥80 y), and the causality remains unclear. OBJECTIVE: This study aimed to elucidate the causal effect of vitamin D on the incident risk of disability in activities of daily living (ADL) among Chinese oldest-old based on the 2012-2018 Chinese Healthy Ageing and Biomarkers Cohort Study. METHODS: Serum 25-hydroxyvitamin D [25(OH)D] concentrations and ADL status at baseline and follow-up interviews were documented. Cox regression models were applied among 1427 oldest-old (mean age, 91.2 y) with normal baseline ADL status. One sample Mendelian randomization (MR) analyses were performed on a subset of 941 participants with qualified genetic data, using a 25(OH)D-associated genetic risk score as the genetic instrument. RESULTS: During a median follow-up of 3.4 y, 231 participants developed disability in ADL. Serum 25(OH)D concentration was inversely associated with the risk of disability in ADL [per 10 nmol/L increase hazard ratio (HR) 0.85; 95% CI: 0.75, 0.96]. Consistent results from MR analyses showed that a 10 nmol/L increment in genetically predicted 25(OH)D concentration corresponded to a 20% reduced risk of ADL disability (HR 0.80; 95% CI: 0.68, 0.94). Nonlinear MR demonstrated a monotonic declining curve, with the HRs exhibiting a more pronounced reduction among individuals with 25(OH)D concentrations below 50 nmol/L. Subgroup analyses showed that the associations were more distinct among females and those with poorer health conditions. CONCLUSIONS: Our study supports an inverse causal relationship between serum 25(OH)D concentration and the risk of disability in ADL among Chinese oldest-old. This protective effect was more distinct, especially for participants with vitamin D deficiency. Appropriate measures for improving vitamin D might help reduce the incidence of physical disability in this specific age group.

Flexible Full-Inorganic Ultrathin Films with Stable Circularly Polarized Luminescence Covering the Visible to Near-Infrared Region.

Circularly polarized luminescence (CPL) materials hold significant value in various fields, including information storage, secure communication, three-dimensional displays, biological detection, and optoelectronic devices. Using the Langmuir-Schaeffer (LS) assembly technique, we successfully construct a series of large-area flexible optical ultrathin films. Impressively, the inorganic assembled ultrathin films exhibit excellent CPL optical activity covering the visible to near-infrared (NIR) region, with the luminescence asymmetry factor glum ranging from 0.59 to 0.72. Moreover, such ultrathin films also display outstanding mechanical flexibility, the optical activity of which even after 240 bending cycles shows almost no difference compared to the unbent samples. Owing to the ultra-broadband optical activity and ultra-stable optical activity of such full-inorganic assembled materials on flexible substrates, coupled with their excellent processability and outstanding mechanical flexibility, we anticipate they will find use in many fields such as communication technology and flexible optoelectronics.

Design and synthesis of luotonin A-derived topoisomerase targeting scaffold with potent antitumor effect and low genotoxicity.

Conventional topoisomerase (Topo) inhibitors typically usually exert their cytotoxicity by damaging the DNAs, which exhibit high toxicity and tend to result in secondary carcinogenesis risk. Molecules that have potent topoisomerase inhibitory activity but involve less DNA damage provide more desirable scaffolds for developing novel chemotherapeutic agents. In this work, we broke the rigid pentacyclic system of luotonin A and synthesized thirty-three compounds as potential Topo inhibitors based on the devised molecular motif. Further investigation disclose that two compounds with the highest antiproliferation activity against cancer cells, 5aA and 5dD, had a distinct Topo I inhibitory mechanism different from those of the classic Topo I inhibitors CPT or luteolin, and were able to obviate the obvious cellular DNA damage typically associated with clinically available Topo inhibitors. The animal model experiments demonstrated that even in mice treated with a high dosage of 50 mg/kg 5aA, there were no obvious signs of toxicity or loss of body weight. The tumor growth inhibition (TGI) rate was 54.3 % when 20 mg/kg 5aA was given to the T24 xenograft mouse model, and 5aA targeted the cancer tissue precisely without causing damage to the liver and other major organs.

Establishment of tumor marker reference intervals for different age and gender groups in the healthy population of South China.

To establish age- and sex-specific reference intervals (RIs) for serum tumor markers (AFP, CEA, CA125, CA199, CA153, HE4, CA724, CYFRA21-1, PSA, and NSE) among a cohort of healthy individuals in South China, a retrospective analysis was conducted on 51,353 samples collected from 2015 to 2020, during health assessments at Guangdong Provincial People's Hospital. The influence of age and gender on serum tumor markers was investigated. New RIs were determined using non-parametric rank-based methods per CLSI EP28-A3C guidelines. Significant differences were detected across age groups for AFP, CEA, CA125, CA199, HE4, CYFRA21-1, PSA, and NSE (p < 0.05). The upper reference limits (URLs) for CA153 and HE4 are significantly lower compared to our current laboratory standards. The URL for CA125 exceeds these limits in individuals under 50 but decreases in those aged 50 and above. For CA199, CEA, and PSA, the URLs are below current standards in individuals younger than 60 but exceed them in those aged 60 and older. Noteworthy elevations were observed in CA724, CYFRA21-1, and NSE levels. Our study establishes age- and sex-specific RIs for ten serum tumor markers among healthy individuals from South China, providing a fundamental resource for the prevention, early detection, and management of tumor-related disorders.

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.

Expression of hepatic genes involved in bile acid metabolism in dairy cows with fatty liver.

Bile acids are cholesterol-derived molecules that are primarily produced in the liver. In nonruminants with fatty liver, overproduction of bile acids is associated with liver injury. During the transition period, fatty liver is a metabolic disorder that can affect up to 50% of high-producing dairy cows. The purpose of this study was to provide a comprehensive evaluation of hepatic bile acid metabolism in dairy cows with fatty liver by assessing the expression changes of genes involved in bile acid synthesis, export, and uptake. The serum activities of aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase and the concentration of total bile acids were all greater, whereas the serum concentration of total cholesterol was lower in cows with fatty liver than in healthy cows. The content of total bile acids was higher, but total cholesterol was slightly lower in liver tissues from fatty liver cows than from healthy cows. The hepatic mRNA abundance of cholesterol 7a-hydroxylase (CYP7A1); hydroxy-delta-5-steroid dehydrogenase, 3 ß- and steroid delta-isomerase 7 (HSD3B7); and sterol 12α-hydroxylase (CYP8B1), enzymes involved in the classic pathway of bile acid synthesis, was higher in fatty liver cows than in healthy cows. Compared with healthy cows, the hepatic mRNA abundance of alternative bile acid synthesis pathway-related genes sterol 27-hydroxylase (CYP27A1) and oxysterol 7α-hydroxylase (CYP7B1) did not differ in cows with fatty liver. The protein and mRNA abundances of bile acid transporter bile salt efflux pump (BSEP) were lower in the liver of dairy cow with fatty liver. Compared with healthy cows, the hepatic mRNA abundance of bile acid transporters solute carrier family 51 subunit α (SLC51A) and ATP binding cassette subfamily C member 1 (ABCC1) and 3 (ABCC3) was greater in cows with fatty liver, whereas the solute carrier family 51 subunit ß (SLC51B) did not differ. The expression of genes involved in bile acid uptake, including solute carrier family 10 member 1 (NTCP), solute carrier organic anion transporter family member 1A2 (SLCO1A2) and 2B1 (SLCO2B1) was upregulated in dairy cows with fatty liver. Furthermore, the hepatic protein and mRNA abundance of bile acid metabolism regulators farnesoid X receptor (FXR) and small heterodimer partner (SHP) were lower in cows with fatty liver than in healthy cows. Overall, these data suggest that inhibition of the FXR signaling pathway may lead to increased bile acid synthesis and uptake and decreased secretion of bile acids from hepatocytes to the bile, which elevates hepatic bile acid content in dairy cows with fatty liver. Because the hepatotoxicity of bile acids has been demonstrated on nonruminant hepatocytes, it is likely that liver injury is induced by increased hepatic bile acid content in dairy cows with fatty liver.

Evaluation of hepatic AMPK, mTORC1, and autophagy-lysosomal pathway in cows with mild or moderate fatty liver.

The aim of the present study was to investigate the activity of AMPK and mTORC1 as well as TFEB transcriptional activity and autophagy-lysosomal function in the liver of dairy cows with mild fatty liver (FL) and cows with moderate FL. Liver and blood samples were collected from healthy dairy cows (n = 10; hepatic triglyceride content <1% wet weight) and cows with mild FL (n = 10; 1% ≤ hepatic triglyceride content < 5% wet weight) or moderate FL (n = 10; 5% ≤ hepatic triglyceride content < 10% wet weight) that had a similar number of lactations (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9). Blood parameters were determined using a Hitachi 3130 autoanalyzer with commercially available kits. Protein and mRNA abundances were determined using western blotting and quantitative real-time PCR, respectively. Activities of calcineurin and ß-N-acetylglucosaminidase were measured with commercial assay kits. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Blood concentrations of glucose were lower in moderate FL cows (3.03 ± 0.21 mM) than in healthy (3.71 ± 0.14 mM) and mild FL cows (3.76 ± 0.14 mM). Blood concentrations of ß-hydroxybutyrate (BHB, 1.37 ± 0.15 mM in mild FL, 1.88 ± 0.17 mM in moderate FL) and free fatty acids (FFA, 0.69 ± 0.05 mM in mild FL, 0.96 ± 0.09 mM in moderate FL) were greater in FL cows than in healthy cows (BHB, 0.76 ± 0.12 mM; FFA, 0.42 ± 0.04 mM). Compared with healthy cows, phosphorylation of AMPK was greater and phosphorylation of its downstream target acetyl-CoA carboxylase 1 was lower in cows with mild and moderate FL. Phosphorylation of mTOR was lower in cows with mild FL compared with healthy cows. In cows with moderate FL, phosphorylation of mTOR and its downstream effectors was greater than in healthy cows and cows with mild FL. The mRNA abundance of TFEB was downregulated in cows with moderate FL compared with healthy cows and mild FL cows. In mild FL cows, the mRNA and protein abundances of TFEB were greater than in healthy cows. Compared with healthy cows, the mRNA abundances of autophagy markers sequestosome-1 and microtubule-associated protein 1 light chain 3-II, and the protein and mRNA abundances of lysosome-associated membrane protein 1 and cathepsin D were increased in mild FL cows but decreased in moderate FL cows. Compared with healthy cows, the mRNA abundance of mucolipin 1 and activities of ß-N-acetylglucosaminidase and calcineurin were higher in cows with mild FL but lower in cows with moderate FL. These data demonstrate that hepatic AMPK signaling pathway, TFEB transcriptional activity, and autophagy-lysosomal function are increased in dairy cows with mild FL; the hepatic mTORC1 signaling pathway is inhibited in mild FL cows but activated in moderate FL cows; and activities of AMPK and TFEB as well as autophagy-lysosomal function are impaired in moderate FL cows.

Nuciferine protects bovine hepatocytes against free fatty acid-induced oxidative damage by activating the transcription factor EB/peroxisome proliferator-activated receptor γ coactivator 1 alpha pathway.

Excessive free fatty acid (FFA) oxidation and related metabolism are the major cause of oxidative stress and liver injury in dairy cows during the early postpartum period. In nonruminants, activation of transcription factor EB (TFEB) can improve cell damage and reduce the overproduction of mitochondrial reactive oxygen species. As a downstream target of TFEB, peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α, gene name PPARGC1A) is a critical regulator of oxidative metabolism. Nuciferine (Nuc), a major bioactive compound isolated from the lotus leaf, has been reported to possess hepatoprotective activity. Therefore, the objective of this study was to investigate whether Nuc could protect bovine hepatocytes from FFA-induced lipotoxicity and the underlying mechanisms. A mixture of FFA was diluted in RPMI-1640 basic medium containing 2% low fatty acid bovine serum albumin to treat hepatocytes. Bovine hepatocytes were isolated from newborn calves and treated with various concentrations of FFA mixture (0, 0.3, 0.6, or 1.2 mM) or Nuc (0, 25, 50, or 100 µM), as well as co-treated with 1.2 mM FFA and different concentrations of Nuc. For the experiments of gene silencing, bovine hepatocytes were transfected with small interfering RNA targeted against TFEB or PPARGC1A for 36 h followed by treatment with 1.2 mM FFA for 12 h in presence or absence of 100 µΜ Nuc. The results revealed that FFA treatment decreased protein abundance of nuclear TFEB, cytosolic TFEB, total (t)-TFEB, lysosome-associated membrane protein 1 (LAMP1) and PGC-1α and mRNA abundance of LAMP1, but increased phosphorylated (p)-TFEB. In addition, FFA treatment increased the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) and decreased the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) in bovine hepatocytes. Moreover, FFA administration enhanced the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactose dehydrogenase (LDH) in the medium of FFA-treated hepatocytes, but reduced the content of urea. In FFA-treated bovine hepatocytes, Nuc administration increased TFEB nuclear localization and the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, decreased the contents of MDA and H2O2 and the protein abundance of p-TFEB, and enhanced the activities of CAT and GSH-Px in a dose-dependent manner. Consistently, Nuc administration reduced the activities of ALT, AST, and LDH and increased the content of urea in the medium of FFA-treated hepatocytes. Importantly, knockdown of TFEB reduced the protein abundance of p-TFEB, t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, and impeded the beneficial effects of Nuc on FFA-induced oxidative damage in bovine hepatocytes. In addition, PPARGC1A silencing did not alter Nuc-induced nuclear translocation of TFEB, increase of the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, or decrease of the protein abundance of p-TFEB, whereas it partially reduced the beneficial effects of Nuc on FFA-caused oxidative injury. Taken together, Nuc exerts protective effects against FFA-induced oxidative damage in bovine hepatocytes through activation of the TFEB/PGC-1α signaling pathway.

Inositol-requiring enzyme 1α and c-Jun N-terminal kinase axis activation contributes to intracellular lipid accumulation in calf hepatocytes.

During the perinatal period, dairy cows undergo negative energy balance, resulting in elevated circulating levels of nonesterified fatty acids (NEFA). Although increased blood NEFA concentrations are a physiological adaptation of early lactation, excessive NEFA in dairy cows is a major cause of fatty liver. Aberrant lipid metabolism leads to hepatic lipid accumulation and subsequently the development of fatty liver. Both inositol-requiring enzyme 1α (IRE1α) and c-Jun N-terminal kinase (JNK) have been validated for their association with hepatic lipid accumulation, including their regulatory functions in calf hepatocyte insulin resistance, oxidative stress, and apoptosis. Meanwhile, both IRE1α and JNK are involved in lipid metabolism in nonruminants. Therefore, the aim of this study was to investigate how IRE1α and JNK regulate lipid metabolism in bovine hepatocytes. An experiment was conducted on randomly selected 10 healthy cows (hepatic triglyceride [TG] content <1%) and 10 cows with fatty liver (hepatic TG content >5%). Liver tissue and blood samples were collected from experimental cows. Serum concentrations of NEFA and ß-hydroxybutyrate (BHB) were greater, whereas serum concentrations of glucose and milk production were lower in cows with fatty liver. The western blot results revealed that dairy cows with fatty liver had higher phosphorylation levels of JNK, c-Jun, and IRE1α in the liver tissue. Three in vitro experiments were conducted using primary calf hepatocytes isolated from 5 healthy calves (body weight: 30-40 kg; 1 d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h, which showed that the phosphorylated levels of JNK, c-Jun, and IRE1α increased in both linear and quadratic effects. In the second experiment, hepatocytes were treated with high concentrations of NEFA (1.2 mM) for 12 h with or without SP600125, a canonical inhibitor of JNK. Western blot results showed that SP600125 treatment could decrease the expression of lipogenesis-associated proteins (PPARγ and SREBP-1c) and increase the expression of fatty acid oxidation (FAO)-associated proteins (CPT1A and PPARα) in NEFA-treated hepatocytes. The perturbed expression of lipogenesis-associated genes (FASN, ACACA, and CD36) and FAO-associated gene ACOX1 were also recovered by JNK inhibition, indicating that JNK reduced excessive NEFA-induced lipogenesis and FAO dysregulation in calf hepatocytes. Third, short hairpin RNA targeting IRE1α (sh-IRE1α) was transfected into calf hepatocytes to silence IRE1α, and KIRA6 was used to inhibit the kinase activity of IRE1α. The blockage of IRE1α could at least partially suppressed NEFA-induced JNK activation. Moreover, the blockage of IRE1α downregulated the expression of lipogenesis genes and upregulated the expression of FAO genes in NEFA-treated hepatocytes. In conclusion, these findings indicate that targeting the IRE1α-JNK axis can reduce NEFA-induced lipid accumulation in bovine hepatocytes by modulating lipogenesis and FAO. This may offer a prospective therapeutic target for fatty liver in dairy cows.

Targeting PHB2-mediated mitophagy alleviates nonesterified fatty acid-induced mitochondrial dysfunction in bovine mammary epithelial cells.

Mitochondrial dysfunction has been reported to occur in the mammary gland of dairy cows suffering from ketosis. Prohibitin 2 (PHB2) plays a crucial role in regulating mitophagy, which clears impaired mitochondria to maintain normal mitochondrial function. Therefore, the current study aimed to investigate how PHB2 mediates mitophagy, thereby influencing mitochondrial function in the immortalized bovine mammary epithelial cell line (MAC-T cells). First, mammary gland tissue and blood samples were collected from healthy cows (n = 15, BHB <0.6 mM) and cows with clinical ketosis (n = 15, BHB >3.0 mM). Compared with healthy cows, cows with clinical ketosis exhibited lower DMI, milk production, milk protein, milk lactose, and serum glucose. In contrast, milk fat, serum nonesterified fatty acids (NEFA) and BHB were greater in cows with clinical ketosis. The protein abundance of PHB2, peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α), mitofusin 2 (MFN2) in whole cell lysates (WCL), as well as PHB2, sequestosome-1 (SQSTM1, also called p62), microtubule-associated protein 1 light chain 3-II (MAP1LC3-II, also called LC3-II), and ubiquitinated proteins in mitochondrial fraction were significantly lower in cows with clinical ketosis. The ATP content of mammary gland tissue in cows with clinical ketosis was lower than that of healthy cows. Second, MAC-T were cultured and treated with NEFA (0, 0.3, 0.6, 1.2 mM). The MAC-T treated with 1.2 mM NEFA displayed decreased protein abundance of PHB2, PGC-1α, and MFN2 in WCL, as well as protein abundance of PHB2, p62, LC3-II, and ubiquitinated proteins in mitochondrial fraction. The content of ATP and JC-1 aggregates in 1.2 mM NEFA group were lower than in the 0 mM NEFA group. Additionally, 1.2 mM NEFA disrupted the fusion between mitochondria and lysosomes. The MAC-T were then pretreated with 100 nM rapamycin, followed by treatment with or without NEFA. Rapamycin alleviated impaired mitophagy and mitochondria dysfunction induced by 1.2 mM NEFA. Third, MAC-T were transfected with small interfering RNA to silence PHB2 or a plasmid for overexpression of PHB2, followed by treatment with or without NEFA. The silencing of PHB2 aggravated 1.2 mM NEFA-induced impaired mitophagy and mitochondrial dysfunction, whereas the overexpression of PHB2 alleviated these effects. Overall, this study provides evidence that PHB2, in regulation of mitophagy, is a mechanism for bovine mammary epithelial cells to counteract NEFA-induced mitochondrial dysfunction.

Thioredoxin-2 suppresses hydrogen peroxide-activated nuclear factor kappa B signaling via alleviating oxidative stress in bovine adipocytes.

During the periparturient period, both oxidative stress, and inflammation of adipose tissue are considered high risk factors for metabolic disorder of dairy cows. Oxidative stress can activate transcription factor nuclear factor kappa B (NF-κB), which lead to the upregulation of genes involved in inflammatory pathways. Thioredoxin-2 (TXN2) is a mitochondrial protein that regulates cellular redox by suppressing mitochondrial reactive oxygen species (ROS) generation in nonruminant, whereas the function of TXN2 in bovine adipocytes was unclear. Thus, the objective of this study was to evaluate how or by which mechanisms TXN2 regulates oxidative stress and NF-κB signaling pathway in bovine adipocytes. Bovine pre-adipocytes isolated from 5 healthy Holstein cows were differentiated and used for (1) treatment with different concentrations of hydrogen peroxide (H2O2; 0, 25, 50, 100, 200, or 400 µM) for 2 h; (2) transfection with or without TXN2 small interfering RNA (si-TXN2) for 48 h and then treated with or without 200 µM H2O2 for 2 h; (3) transfection with scrambled negative control siRNA (si-control) or si-TXN2 for 48 h, and then treatment with or without 10 mM N-acetylcysteine (NAC) for 2 h; (4) transfection with or without TXN2-overexpressing plasmid for 48 h and then treatment with or without 200 µM H2O2 for 2 h. High concentrations of H2O2 (200 and 400 µM) decreased protein and mRNA abundance of TXN2, reduced total antioxidant capacity (T-AOC) and ATP content in adipocytes. Moreover, 200 and 400 µM H2O2 reduced protein abundance of inhibitor of kappa B α (IκBα), increased phosphorylation of NF-κB and upregulated mRNA abundance of tumor necrosis factor-α (TNFA) and interleukin-1B (IL-1B), suggesting that H2O2-induced oxidative stress and activated NF-κB signaling pathway. Silencing of TXN2 increased intracellular ROS content, phosphorylation of NF-κB and mRNA abundance of TNFA and IL-1B, decreased ATP content and protein abundance of IκBα in bovine adipocytes. Knockdown of TXN2 aggravated H2O2-induced oxidative stress and inflammation. In addition, treatment with antioxidant NAC ameliorated oxidative stress and inhibited NF-κB signaling pathway in adipocytes transfected with si-TXN2. In bovine adipocytes treated with H2O2, overexpression of TXN2 reduced the content of ROS and elevated the content of ATP and T-AOC. Overexpression of TXN2 alleviated H2O2-induced inflammatory response in adipocytes, as demonstrated by decreased expression of phosphorylated NF-κB, TNFA, IL-1B, as well as increased expression of IκBα. Furthermore, the protein and mRNA abundance of TXN2 was lower in adipose tissue of dairy cows with clinical ketosis. Overall, our studies contribute to the understanding of the role of TXN2 in adipocyte oxidative stress and inflammatory response.

Caveolin 1 ameliorates nonesterified fatty acid-induced oxidative stress via the autophagy regulator Beclin 1 in bovine mammary gland epithelial cells.

High blood concentrations of nonesterified fatty acids (NEFA) during ketosis enhance uptake by the mammary gland and impair autophagy while causing oxidative stress. Caveolin 1 (CAV1) is closely related to autophagy and plays a role in regulating oxidative stress. The aim of this study was to explore the potential role of CAV1 on oxidative stress and autophagy during a high NEFA challenge in the immortalized bovine mammary epithelial cell line MAC-T. Mammary gland tissue biopsies and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) Holstein cows at 3 to 10 (average = 6) days in milk. Compared with healthy cows, ketotic cows had lower dry matter intake (DMI), daily milk yield, serum glucose and greater serum NEFA and BHBA, accompanied by greater milk fat and lower milk protein. Malondialdehyde (MDA) was greater but activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH) were lower in cows with clinical ketosis. A lower protein abundance of CAV1, Beclin 1, autophagy relative gene 5 (ATG5), and microtubule-associated protein 1 light chain 3 (LC3) as well as greater protein abundance of sequestosome-1 (SQSTM1, also called p62) were detected in mammary tissue of cows with clinical ketosis. In vitro, the MAC-T cells were treated with 0, 0.6 and 1.2 mM NEFA for 12 h or treated with 1.2 mM NEFA for various time points (0, 0.5, 1, 2, 4, 8, 12 and 24 h). Compared with 0 mM NEFA, protein abundance of CAV1, Beclin 1, ATG5 and LC3 was greater in the MAC-T challenged with 0.6 mM NEFA, but lower in the 1.2 mM NEFA group. Protein abundance of p62 was lower with 0.6 mM NEFA, but higher with 1.2 mM NEFA. In response to increasing doses of NEFA, mRNA abundance of CAV1, total antioxidant capacity (T-AOC) and SOD activity decreased while the level of reactive oxygen species (ROS) and content of MDA increased. The protein abundance of CAV1, Beclin 1, ATG5 and LC3 peaked at 0.5 h and 1 h, resulting in both linear and quadratic effects. The protein abundance of p62 decreased, reaching a nadir at 4 h in both a linear and quadratic manner. The silencing of CAV1 in MAC-T cells aggravated the 1.2 mM NEFA-induced decrease in Beclin 1 expression, impaired autophagy, and increase in oxidative stress, whereas the overexpression of CAV1 alleviated these effects. Pretreatment of MAC-T cells with Beclin 1 siRNA (si-Beclin 1) and overexpressing CAV1 followed by challenged with 1.2 mM NEFA reversed the CAV1 induced autophagy, thereby enhancing oxidative stress. Overall, these data suggest that CAV1 protects bovine mammary epithelial cells from NEFA-induced oxidative stress through enhancing the expression of Beclin 1 and activating autophagy.

Causal association of gastroesophageal reflux disease with chronic sinusitis and chronic disease of the tonsils and adenoids.

BACKGROUND: Exploring bidirectional causal associations between gastroesophageal reflux disease (GERD) and chronic disease of the tonsils and adenoids and chronic sinusitis, respectively. METHODS: We first conducted a TSMR (two-sample mendelian randomization) study using the results of the inverse variance weighting method as the primary basis and bidirectional MR to rule out reverse causation. Subsequently, MVMR (multivariate MR) analysis was performed to identify phenotypes associated with SNPs and to explore the independent effect of GERD on two outcomes. Finally, we calculated MR-Egger intercepts to assess horizontal polytropy and Cochran's Q statistic to assess heterogeneity and ensure the robustness of the study. RESULTS: For each standard deviation increase in genetically predicted GERD rate, there was an increased risk of chronic disease of the tonsils and adenoids (OR 1.162, 95% CI 1.036-1.304, P: 1.06E-02) and of developing chronic sinusitis (OR 1.365, 95% CI 1.185-1.572, P: 1.52E-05), and there was no reverse causality. Causality for TSMR was obtained on the basis of IVW (inverse variance weighting) and appeared to be reliable in almost all sensitivity analyses, whereas body mass index may be a potential mediator of causality between GERD and chronic sinusitis. CONCLUSION: There is a causal association between GERD and chronic disease of the tonsils and adenoids and chronic sinusitis, respectively, and the occurrence of GERD increases the risk of developing chronic disease of the tonsils and adenoids and chronic sinusitis.