Programming and inheritance of parental DNA methylomes in mammals.

The reprogramming of parental methylomes is essential for embryonic development. In mammals, paternal 5-methylcytosines (5mCs) have been proposed to be actively converted to oxidized bases. These paternal oxidized bases and maternal 5mCs are believed to be passively diluted by cell divisions. By generating single-base resolution, allele-specific DNA methylomes from mouse gametes, early embryos, and primordial germ cell (PGC), as well as single-base-resolution maps of oxidized cytosine bases for early embryos, we report the existence of 5hmC and 5fC in both maternal and paternal genomes and find that 5mC or its oxidized derivatives, at the majority of demethylated CpGs, are converted to unmodified cytosines independent of passive dilution from gametes to four-cell embryos. Therefore, we conclude that paternal methylome and at least a significant proportion of maternal methylome go through active demethylation during embryonic development. Additionally, all the known imprinting control regions (ICRs) were classified into germ-line or somatic ICRs.

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.

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.

Tumor necrosis factor-α reduces adiponectin production by decreasing transcriptional activity of peroxisome proliferator-activated receptor-γ in calf adipocytes.

Adiponectin (encoded by ADIPOQ) is an adipokine that orchestrates energy homeostasis by modulating glucose and fatty acid metabolism in peripheral tissues. During the periparturient period, dairy cows often develop adipose tissue inflammation and decreased plasma adiponectin levels. Proinflammatory cytokine tumor necrosis factor-α (TNF-α) plays a pivotal role in regulating the endocrine functions of adipocytes, but whether it affects adiponectin production in calf adipocytes remains obscure. Thus, the present study aimed to determine whether TNF-α could affect adiponectin production in calf adipocytes and to identify the underlying mechanism. Adipocytes isolated from Holstein calves were differentiated and used for (1) BODIPY493/503 staining; (2) treatment with 0.1 ng/mL TNF-α for different times (0, 8, 16, 24, or 48 h); (3) transfection with peroxisome proliferator-activated receptor-γ (PPARG) small interfering RNA for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h; and (4) overexpression of PPARG for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h. After differentiation, obvious lipid droplets and secretion of adiponectin were observed in adipocytes. Treatment with TNF-α did not alter mRNA abundance of ADIPOQ but reduced the total and high molecular weight (HMW) adiponectin content in the supernatant of adipocytes. Quantification of mRNA abundance of endoplasmic reticulum (ER)/Golgi resident chaperones involved in adiponectin assembly revealed that ER protein 44 (ERP44), ER oxidoreductase 1α (ERO1A), and disulfide bond-forming oxidoreductase A-like protein (GSTK1) were downregulated in TNF-α-treated adipocytes, while 78-kDa glucose-regulated protein and Golgi-localizing γ-adaptin ear homology domain ARF binding protein-1 were unaltered. Moreover, TNF-α diminished nuclear translocation of PPARγ and downregulated mRNA abundance of PPARG and its downstream target gene fatty acid synthase, suggesting that TNF-α suppressed the transcriptional activity of PPARγ. In the absence of TNF-α, overexpression of PPARG enhanced the total and HMW adiponectin content in supernatant and upregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. However, knockdown of PPARG reduced the total and HMW adiponectin content in supernatant and downregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. In the presence of TNF-α, overexpression of PPARG decreased, while knockdown of PPARG further exacerbated TNF-α-induced reductions in total and HMW adiponectin secretion and gene expression of ERP44, ERO1A, and GSTK1. Overall, TNF-α reduces adiponectin assembly in the calf adipocyte, which may be partly mediated by attenuation of PPARγ transcriptional activity. Thus, locally elevated levels of TNF-α in adipose tissue may be one reason for the decrease in circulating adiponectin in periparturient dairy cows.

Tumor necrosis factor-α promotes lipolysis and reduces insulin sensitivity by activating nuclear factor kappa B and c-Jun N-terminal kinase in primary bovine adipocytes.

Sustained lipolysis and insulin resistance increase the risk of metabolic dysfunction in dairy cows during the transition period. Proinflammatory cytokines are key regulators of adipose tissue metabolism in nonruminants, but biological functions of these molecules in ruminants are not well known. Thus, the objective of this study was to investigate whether tumor necrosis factor-α (TNF-α) could affect insulin sensitivity and lipolysis in bovine adipocytes as well as the underlying mechanisms. Bovine adipocytes (obtained from the omental and mesenteric adipose depots) isolated from 5 Holstein female calves (1 d old) with similar body weight (median: 36.9 kg, range: 35.5-41.2 kg) were differentiated and used for (1) treatment with different concentrations of TNF-α (0, 0.1, 1, or 10 ng/mL) for 12 h; (2) pretreatment with 10 µM lipolytic agonist isoproterenol (ISO) for 3 h, followed by treatment with or without 10 ng/mL TNF-α for 12 h; and (3) pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (20 µM for 2 h) and nuclear factor kappa B (NF-κB) inhibitor BAY 11-7082 (10 µM for 1 h) followed by treatment with or without 10 ng/mL TNF-α for 12 h. The TNF-α increased glycerol content in supernatant, decreased triglyceride content and insulin-stimulated phosphorylation of protein kinase B suggesting activation of lipolysis and impairment of insulin sensitivity. The TNF-α reduced cell viability, upregulated mRNA abundance of Caspase 3 (CASP3), an apoptosis marker, and increased activity of Caspase 3. In addition, increased phosphorylation of NF-κB and JNK, upregulation of mRNA abundance of interleukin-6 (IL-6), TNFA, and suppressor of cytokine signaling 3 (SOCS3) suggested that TNF-α activated NF-κB and JNK signaling pathways. Furthermore, ISO plus TNF-α-activated NF-κB and JNK signaling pathway to a greater extent than TNF-α alone. Combining TNF-α and ISO aggravated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. In the absence of TNF-α, inhibition of NF-κB and JNK did not alter glycerol content in supernatant, triglyceride content or insulin-stimulated phosphorylation of protein kinase B. In the presence of TNF-α, inhibition of NF-κB and JNK alleviated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. Overall, TNF-α impairs insulin sensitivity and induces lipolysis and apoptosis in bovine adipocytes, which may be partly mediated by activation of NF-κB and JNK. Thus, the data suggested that NF-κB and JNK are potential therapeutic targets for alleviating lipolysis dysregulation and insulin resistance in adipocytes.

Disruption of endoplasmic reticulum homeostasis exacerbates liver injury in clinically ketotic cows.

Disruption of endoplasmic reticulum (ER) homeostasis, a condition termed "ER stress," contributes to the development of liver injury in nonruminants. Because liver injury is a prominent pathological feature associated with overproduction of ketone bodies in dairy cows with ketosis, understanding the ER stress state and its functional consequences on liver injury is of particular interest. Here, 30 multiparous cows (within 3 wk postpartum) classified based on blood ß-hydroxybutyrate (BHB) as healthy (n = 15, BHB <0.6 mM) or clinically ketotic (n = 15, BHB >3.0 mM) were used. Compared with healthy cows, ketotic cows had greater levels of serum fatty acids and activities of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyl transferase, and glutamate dehydrogenase but lower serum glucose. Furthermore, dairy cows with ketosis had greater protein abundance of ER stress markers in liver tissue, including protein kinase RNA-like ER kinase (PERK), inositol-requiring protein-1α (IRE1α), and cleaved activating transcription factor-6 (ATF6). Cows with ketosis also had higher mRNA levels of hepatic 78-kDa glucose-regulated protein (GRP78) and spliced X-box binding protein 1 (sXBP1). These data confirmed an enhanced ER stress state in clinically ketotic cows. To explore whether enhanced hepatic ER stress was induced by elevated ketone bodies and the possible contribution of ER stress to liver injury, in vitro experiments were then performed using isolated primary calf hepatocytes treated with incremental concentrations of BHB (0, 0.6, 1.2, 3.0, and 4.8 mM) for 12 h with or without overexpression of GRP78 (the master regulator of unfolded protein response). Phosphorylation levels of PERK and IRE1α proteins, level of cleaved ATF6 protein, and mRNA abundance of GRP78 and sXBP1 in hepatocytes increased after treatment with high (3.0 and 4.8 mM) BHB, indicating a mechanistic link between excessive BHB and enhanced hepatic ER stress. Furthermore, treatment with 3.0 and 4.8 mM BHB markedly elevated activities of aspartate aminotransferase and alanine aminotransferase in cell supernatant, indicating exacerbated hepatocyte damage after ER stress was enhanced. Overexpression of GRP78 attenuated both BHB-induced ER stress and the ensuing cellular damage, suggesting that hepatocyte damage caused by excessive BHB can be mediated via enhanced ER stress. Overall, the present study revealed that ER stress may exacerbate liver injury development in clinically ketotic cows, underscoring the biological relevance of this pathway in the context of liver injury.

Reshaping the substrate binding region of (R)-selective ω-transaminase for asymmetric synthesis of (R)-3-amino-1-butanol.

(R)-Selective ω-transaminase (ω-TA) is a key enzyme for the asymmetric reductive amination of carbonyl compounds to produce chiral amines which are essential parts of many therapeutic compounds. However, its practical industrial applications are hindered by the low catalytic efficiency and poor thermostability of naturally occurring enzymes. In this work, we report the molecular modification of (R)-selective ω-TA from Aspergillus terreus (AtTA) to allow asymmetric reductive amination of 4-hydroxy-2-butanone, producing (R)-3-amino-1-butanol. Based on substrate docking analysis, 4 residues in the substrate tunnel and binding pocket of AtTA were selected as mutation hotspots. The screening procedure was facilitated by the construction of a "small-intelligent" library and the use of thin-layer chromatography for preliminary screening. The resulting mutant AtTA-M5 exhibited a 9.6-fold higher kcat/Km value and 9.4 °C higher [Formula: see text] than that of wild-type AtTA. Furthermore, the conversion of 20 and 50 g L-1 4-hydroxy-2-butanone by AtTA-M5 reached 90.8% and 79.1%, suggesting significant potential for production of (R)-3-amino-1-butanol. Under the same conditions, wild-type AtTA achieved less than 5% conversion. Moreover, the key mutation (S215P in AtTA) was validated in 7 other (R)-selective ω-TAs, indicating its general applicability in improving the catalytic efficiency of homologous (R)-selective ω-TAs.

An extracellular aminopeptidase encoded by the ywaD gene plays an important role in supplying nitrogen nutrition for the growth of Bacillus subtilis 168.

To investigate the physiological role of an extracellular aminopeptidase (BSAP168) encoded by the ywaD gene in Bacillus subtilis 168, we constructed the ywaD-deletion mutant (BS-AP-K). Compared with that of the wild-type strain, the maximum growth rate of BS-AP-K was reduced by 28% when grown in soybean protein medium at 37 °C, but not in Luria-Bertani medium. The impaired growth rate was more marked at higher temperature and could be compensated by supplementation of amino acid to the culture media. Further studies showed that in regards to the amino acid compositions and peptide distribution in the culture supernatants, there was an obvious difference between the culture supernatants of wild-type and BS-AP-K strains. In addition, another mutant strain (BS-AP-R) was constructed by replacing ywaD with ywaD-ΔPA to evaluate the effect of a protease-associated domain in BSAP168 on growth. All these findings indicated that BSAP168 played an important role in supplying the amino acids required for growth.

An androgen receptor-microrna-29a regulatory circuitry in mouse epididymis.

MicroRNAs are involved in a number of cellular processes; thus, their deregulation is usually apt to the occurrence of diverse diseases. Previous studies indicate that abnormally up-regulated miR-29a is associated with several diseases, such as human acute myeloid leukemia and diabetes; therefore, the proper level of miR-29a is critical for homeostasis. Herein, we observed that miR-29a was repressed by androgen/androgen receptor signaling in mouse epididymis by targeting a conserved androgen response element located 8 kb upstream of miR-29b1a loci. It is well known that multiple regulatory programs often form a complicated network. Here, we found that miR-29a reversibly suppressed androgen receptor and its target genes by targeting IGF1 and p53 pathways. miR-29b1a-overexpressing transgenic mice displayed epididymis hypoplasia partially similar to the phenotype of those mice with an impaired androgen-androgen receptor signal system. Taken together, the results demonstrated that there is a regulatory circuitry between the androgen signaling pathway and miR-29a in mouse epididymis that may be vital for epididymal development and functions.

Pluronic-Based Nanoparticles for Delivery of Doxorubicin to the Tumor Microenvironment by Binding to Macrophages.

The active targeting drug delivery system based on special types of endogenous cells such as macrophages has emerged as a promising strategy for tumor therapy, owing to its tumor homing property and biocompatibility. In this work, the active tumor-targeting drug delivery system carrying doxorubicin-loaded nanoparticles (DOX@MPF127-MCP-1, DMPM) on macrophage (RAW264.7) surfaces via the mediation of interaction with the CCR2/MCP-1 axis was exploited. Initially, the amphiphilic block copolymer Pluronic F127 (PF127) was carboxylated to MPF127 at the hydroxyl terminus. Subsequently, MPF127 was modified with MCP-1 peptide to prepare MPF127-MCP-1 (MPM). The DOX was wrapped in MPM to form DMPM nanomicelles (approximately 100 nm) during the self-assembly process of MPM. The DMPM spontaneously bound to macrophages (RAW264.7), which resulted in the construction of an actively targeting delivery system (macrophage-DMPM, MA-DMPM) in vitro and in vivo. The DOX in MA-DMPM was released in the acidic tumor microenvironment (TME) in a pH-responsive manner to increase DOX accumulation and enhance the tumor treatment effect. The ratio of MA-DMPM homing reached 220% in vitro compared with the control group, indicating that the MA-DMPM was excellently capable of tumor-targeting delivery. In in vivo experiments, nonsmall cell lung cancer cell (NCI-H1299) tumor models were established. The results of the fluorescence imaging system (IVIS) showed that MA-DMPM demonstrated tremendous tumor-targeting ability in vivo. The antitumor effects of MA-DMPM in vivo indicated that the proportion of tumor cell apoptosis in the DMPM-treated group was 63.33%. The findings of the tumor-bearing mouse experiment proved that MA-DMPM significantly suppressed tumor cell growth, which confirmed its immense potential and promising applications in tumor therapy.

Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis.

The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg(-1) and the k cat/K m was 1.9 × 10(4) mM(-1) s(-1), exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.

Over-expression, secretion, biochemical characterisation, and structure analysis of Bacillus subtilis aminopeptidase.

BACKGROUND: Aminopeptidases have great application in the food industry. Current research on the expression of aminopeptidases mainly focuses on the Escherichia coli expression system. However, the application of recombinant E. coli in the food industry is restricted due to its pathogenicity and low secretory efficiency, which should be concerned in the industrial production of aminopeptidases. RESULTS: The gene of aminopeptidase from Bacillus subtilis Zj016 (BSAP) was identified. Over-expression and secretion of BSAP were achieved in a B. subtilis expression system with the signal peptide of itself. The yield researched 52 ± 1.9 U mL(-1) , which was 18 times that of the wild-type microbe. The purified enzyme was stable at pH 7.5-9.0 and below 60°C, and was inhibited by several metal ions except appropriate Co(2+) . BSAP was most active toward p-nitroaniline derivatives of Leu, Arg and Lys. Homology modelling and structure analysis showed that there was a flexible protease-associated domain in the predicted structure of BSAP. CONCLUSIONS: The study presented a simple procedure for over-expression and purification of BSAP. The substrate specificity and structure information were indicated based on the characterisation and homology modelling. This will be useful for further research of aminopeptidases not only from an academic standpoint but also from an applied point of view.

Advanced heart failure patients and family caregivers health and function: randomised controlled pilot trial of online dignity therapy.

OBJECTIVES: This research investigated the effectiveness of the caregiver-mediated online dignity therapy in enhancing dyadic health and family function. METHODS: Heart failure (HF) family dyads were recruited between May and December 2021 from a university-affiliated hospital in China. The dyads (N=70) were randomly allocated to the intervention group and the control group. We assessed patients' outcomes (hope, well-being, Family APGAR Index and quality of life (QoL)) and their family caregivers' outcomes (anxiety, depression and Family APGAR Index) at baseline (T0), 1 week (T1), 4 weeks (T2) and 8 weeks (T3) after discharge. RESULTS: For patients, the difference over time was significant in QoL (p<0.001). The interaction effects were significant for hope (p<0.001), well-being (p<0.001), Family APGAR Index (p<0.001) and QoL (p=0.007). For family caregivers, a significant difference in depression (p=0.001) was found within groups. Meanwhile, the interaction effects were significant on anxiety (p=0.002) and depression (p=0.016). CONCLUSIONS: Caregiver-mediated online dignity therapy among patients with advanced HF had potential to enhance patient outcomes (level of hope, well-being, family function and QoL) and alleviate caregiver outcomes (anxiety, depression) at 4-week and 8-week follow-up. Thus, we provided scientific evidence for palliative care for advanced HF. TRIAL REGISTRATION NUMBER: ChiCTR2100053758.

Engineering Corynebacterium glutamicum for de novo production of 2-phenylethanol from lignocellulosic biomass hydrolysate.

BACKGROUND: 2-Phenylethanol is a specific aromatic alcohol with a rose-like smell, which has been widely used in the cosmetic and food industries. At present, 2-phenylethanol is mainly produced by chemical synthesis. The preference of consumers for "natural" products and the demand for environmental-friendly processes have promoted biotechnological processes for 2-phenylethanol production. Yet, high 2-phenylethanol cytotoxicity remains an issue during the bioproduction process. RESULTS: Corynebacterium glutamicum with inherent tolerance to aromatic compounds was modified for the production of 2-phenylethanol from glucose and xylose. The sensitivity of C. glutamicum to 2-phenylethanol toxicity revealed that this host was more tolerant than Escherichia coli. Introduction of a heterologous Ehrlich pathway into the evolved phenylalanine-producing C. glutamicum CALE1 achieved 2-phenylethanol production, while combined expression of the aro10. Encoding 2-ketoisovalerate decarboxylase originating from Saccharomyces cerevisiae and the yahK encoding alcohol dehydrogenase originating from E. coli was shown to be the most efficient. Furthermore, overexpression of key genes (aroGfbr, pheAfbr, aroA, ppsA and tkt) involved in the phenylpyruvate pathway increased 2-phenylethanol titer to 3.23 g/L with a yield of 0.05 g/g glucose. After introducing a xylose assimilation pathway from Xanthomonas campestris and a xylose transporter from E. coli, 3.55 g/L 2-phenylethanol was produced by the engineered strain CGPE15 with a yield of 0.06 g/g xylose, which was 10% higher than that with glucose. This engineered strain CGPE15 also accumulated 3.28 g/L 2-phenylethanol from stalk hydrolysate. CONCLUSIONS: In this study, we established and validated an efficient C. glutamicum strain for the de novo production of 2-phenylethanol from corn stalk hydrolysate. This work supplied a promising route for commodity 2-phenylethanol bioproduction from nonfood lignocellulosic feedstock.

Direct Oral Anticoagulants vs. Vitamin K Antagonists in Atrial Fibrillation Patients at Risk of Falling: A Meta-Analysis.

Background: Direct oral anticoagulants (DOACs) and warfarin are usually used for people with atrial fibrillation (AF). However, for the AF patients at risk of falling, the effectiveness and safety outcomes of DOACs compared with warfarin remain unclear. Therefore, we performed a meta-analysis regarding the effectiveness and safety of DOACs vs. warfarin in AF patients at risk of falling. Methods: A search of the PubMed and Embase databases until November 2021 was performed. We included studies if they satisfied the following criteria: (1) study type: randomized clinical trials or observational cohort studies. (2) Comparisons: effectiveness and/or safety of DOACs (dabigatran, rivaroxaban, apixaban, or edoxaban) compared with warfarin. (3) Study data: the sample size, the number of events in the VKAs or DOACs groups, adjusted risk ratios (RRs), and 95% confidence intervals (CIs). (4) Study outcomes: stroke or systemic embolism (SSE), ischemic stroke, myocardial infarction (MI), all-cause death, and cardiovascular death; major bleeding, major or clinically relevant non-major (CRNM) bleeding, intracranial bleeding, gastrointestinal bleeding, and any bleeding. (5) Study population: patients at risk of falling. According to the Morse Fall Scale, the risk of falling relates to the history of falling, secondary diagnosis, ambulatory aids, intravenous therapy, type of gait, and mental status. In this meta-analysis, if the patient's MFS score is ≥25 points, he will be thought of as having the risk of falling. The adjusted risk ratios (RRs) and 95% confidence intervals (CIs) were pooled by a random-effects model with an inverse variance method. Results: Three cohort studies were included in our study. For the effectiveness outcomes, the use of DOACs was only associated with a significantly reduced risk of hemorrhagic stroke (RR = 0.28, 95%CI:0.10-0.75) compared with warfarin, but there were no significant differences in stroke or systemic embolism (SSE) (RR = 0.87, 95%CI:0.70-1.08), cardiovascular death (RR = 0.97, 95%CI:0.73-1.29) and all-cause death (RR = 0.90, 95%CI:0.72-1.11). For the safety outcomes, the use of DOACs was significantly associated with reduced risks of major or clinically relevant non-major bleeding (RR = 0.77, 95%CI:0.61-0.98) and intracranial bleeding (RR = 0.26, 95%CI:0.11-0.66) but not major bleeding (RR = 0.78, 95%CI:0.58-1.06). Conclusions: Compared with warfarin, the use of DOACs in AF patients at risk of falling is significantly associated with reduced risks of hemorrhagic stroke, major or clinically relevant non-major bleeding, and intracranial bleeding.

Activation of Transcription Factor EB Is Associated With Adipose Tissue Lipolysis in Dairy Cows With Subclinical Ketosis.

Excessive lipid mobilization for adipose tissue caused by severe negative energy balance is the pathological basis for subclinical ketosis (SCK) in dairy cows. In non-ruminants, transcription factor EB (TFEB) was reported to play a role in the regulation of lipid catabolism, but its role in the control of lipolysis in the bovine is unknown. The present study aimed to determine whether the enhanced TFEB transcriptional activity contributes to lipolysis of adipose tissue in SCK cows, and to explore the possibility of establishing a therapeutic strategy by using TFEB as a target to control lipolysis. Thirty cows with similar lactation number (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9) were selected into a healthy control (n = 15) and SCK (n = 15) group, and used for subcutaneous adipose tissue biopsies and blood sampling. Adipocytes from healthy Holstein calves were used as a model for in vitro studies involving treatment with 10 µM isoproterenol (ISO) for 0, 1, 2 and 3 h, 250 nM of the TFEB activator Torin1 for 3 h, or used for transfection with TFEB small interfering RNA for 48 h followed by treatment with 10 µM ISO for 3 h. Compared with healthy cows, adipose tissue in SCK cows showed increased lipolysis accompanied by enhanced TFEB transcriptional activity. In vitro, ISO and Torin1 treatment increased lipolysis and enhanced TFEB transcriptional activity in calf adipocytes. However, knockdown of TFEB attenuated ISO-induced lipolysis in adipocytes. Overall, these findings indicated that enhanced transcriptional activity of TFEB may contribute to lipolysis of adipose tissue in dairy cows with SCK. The regulation of TFEB activity may be an effective therapeutic strategy for controlling overt lipolysis in ketotic cows.

Diagnostic Accuracy of the HAS-BLED Bleeding Score in VKA- or DOAC-Treated Patients With Atrial Fibrillation: A Systematic Review and Meta-Analysis.

Background: Several bleeding risk assessment models have been developed in atrial fibrillation (AF) patients with oral anticoagulants, but the most appropriate tool for predicting bleeding remains uncertain. Therefore, we aimed to assess the diagnostic accuracy of the Hypertension, Abnormal liver/renal function, Stroke, Bleeding history or predisposition, Labile international normalized ratio, Elderly, Drugs/alcohol concomitantly (HAS-BLED) score compared with other risk scores in anticoagulated patients with AF. Methods: We comprehensively searched the PubMed and Embase databases until July 2021 to identify relevant pieces of literature. The predictive abilities of risk scores were fully assessed by the C-statistic, net reclassification improvement (NRI) and integrated discrimination improvement (IDI) values, calibration data, and decision curve analyses. Results: A total of 39 studies met the inclusion criteria. The C-statistic of the HAS-BLED score for predicting major bleeding was 0.63 (0.61-0.65) in anticoagulated patients regardless of vitamin k antagonists [0.63 (0.61-0.65)] and direct oral anticoagulants [0.63 (0.59-0.67)]. The HAS-BLED had the similar C-statistic to the Hepatic or renal disease, Ethanol abuse, Malignancy, Older, Reduced platelet count or function, Re-bleeding risk, Hypertension (uncontrolled), Anemia, Genetic factors, Excessive fall risk, Stroke (HEMORR2HAGES), the Anticoagulation and Risk Factors in Atrial Fibrillation (ATRIA), the Outcomes Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT), the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF), or the Age, Biomarkers, Clinical History (ABC) scores, but significantly higher C-statistic than the Congestive heart failure, Hypertension, Age ≥75 years, Diabetes mellitus, Stroke/transient ischemic attack history (CHADS2) or the Congestive heart failure/left ventricular ejection fraction ≤ 40%, Hypertension, Age ≥75 years, Diabetes mellitus, Stroke/transient ischemic attack/thromboembolism history, Vascular disease, Age 65-74 years, Sex (female) (CHA2DS2-VASc) scores. NRI and IDI values suggested that the HAS-BLED score performed better than the CHADS2 or the CHA2DS2-VASc scores and had similar or superior predictive ability compared with the HEMORR2HAGES, the ATRIA, the ORBIT, or the GARFIELD-AF scores. Calibration and decision curve analyses of the HAS-BLED score compared with other scores required further assessment due to the limited evidence. Conclusion: The HAS-BLED score has moderate predictive abilities for bleeding risks in patients with AF regardless of type of oral anticoagulants. Current evidence support that the HAS-BLED score is at least non-inferior to the HEMORR2HAGES, the ATRIA, the ORBIT, the GARFIELD-AF, the CHADS2, the CHA2DS2-VASc, or the ABC scores.

GnRH Antagonist Protocol With Cessation of Cetrorelix on Trigger Day Improves Embryological Outcomes for Patients With Sufficient Ovarian Reserve.

Objective: To evaluate the efficiency and validity of cessation of cetrorelix on trigger day during gonadotropin releasing hormone antagonist (GnRH-ant)-controlled ovarian stimulation of in vitro fertilization (IVF) cycles. Methods: In this retrospective study, a total of 1271 patients undergoing initial IVF cycles following the GnRH-ant protocol were enrolled; 832 patients received cetrorelix on trigger day (Group A) and 439 patients ceased cetrorelix on trigger day (Group B). We compared demographic characteristics, embryological and clinical outcomes between the two groups. A Poisson regression model was used to identify factors that significantly affected embryological outcomes. Patients were further divided into subgroups according to anti-Mullerian hormone (AMH) and age, to assess associations between ceasing cetrorelix on trigger day and embryological outcomes. Results: There was a significant improvement on embryological outcomes in patients who ceased cetrorelix on trigger day, and there were no significant differences in clinical outcomes or preovulation rates between the two groups. Furthermore, for patients with 1.1 ≤ AMH ≤ 4.7 ng/ml, all embryological outcomes were significantly higher in Group B compared with Group A. For patients with AMH > 4.7 ng/ml, the number of oocytes retrieved, fertilization rate (2PN) of IVF cycles and proportion of day 3 good quality embryos were all significantly higher in Group B. For patients with age < 35 years, all the embryological outcomes, besides the number of available embryos, were significantly higher in Group B than in Group A. There were no differences in embryological outcomes between the two groups when patients were stratified based on age > 35 years or AMH < 1.1 ng/ml. Conclusion: GnRH-ant protocol with cessation of cetrorelix on trigger day improved embryological outcomes for young patients or patients with sufficient ovarian reserve, and was effective at preventing preovulation.

Nuciferine improves high-fat diet-induced obesity via reducing intestinal permeability by increasing autophagy and remodeling the gut microbiota.

Nuciferine (NF) has received extensive attention due to its medicinal value in the treatment of metabolic diseases, such as obesity; however, to date, the effects of NF on obesity-related intestinal permeability, autophagy and the gut microbiota have not been investigated. Herein, C57BL/6J mice were fed either a chow or a high-fat diet (HFD) with or without NF for 8 weeks. The results showed that NF supplement reduced weight gain, fat accumulation and intestinal permeability in the HFD mice accompanied by improved autophagy. Subsequently, an in vitro experiment was performed using Caco-2 and HT-29 cells, which showed that NF supplement not only promoted the formation of autophagosomes and autophagolysosomes, but also alleviated LPS-increased intestinal permeability. Importantly, NF supplement protected from LPS-induced paracellular permeability impairment after the administration of autophagy-related gene (Atg) 5 small-interfering RNA (siRNA). These results demonstrate that NF exerts beneficial effects on the intestinal permeability by improving autophagy. Furthermore, we also found that NF supplement lowered the abundance of Butyricimonas and increased the abundance of Akkermansia, an anti-obesity bacterium. Thus, overall, we demonstrated that NF supplement confers reduced intestinal permeability by improving autophagy and alters the composition of the gut microbiota in HFD-fed mice, thereby producing an anti-obesity effect.

Recent progress in Keap1-Nrf2 protein-protein interaction inhibitors.

Therapeutic targeting the protein-protein interaction (PPI) of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its main regulator, Kelch-like ECH-Associating protein 1 (Keap1) has been emerged as a feasible way to combat oxidative stress related diseases, due to the key role of Nrf2 in oxidative stress regulation. In recent years, many efforts have been made to develop potent Keap1-Nrf2 inhibitors with new chemical structures. Various molecules with diverse chemical structures have been reported and some compounds exhibit high potency. This review summarizes peptide and small molecule Keap1-Nrf2 inhibitors reported recently. We also highlight the pharmacological effects and discuss the possible therapeutic application of Keap1-Nrf2 inhibitors.