Discovery of 4-amino-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one derivatives as potential receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitors.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important regulatory factor in the necroptosis signaling pathway, and is considered an attractive therapeutic target for treating multiple inflammatory diseases. Herein, we describe the design, synthesis, and structure-activity relationships of 4-amino-1,6-dihydro-7H-pyrrolo [2,3-d]pyridazin-7-one derivatives as RIPK1 inhibitors. Among them, 13c showed favorable RIPK1 kinase inhibition activity with an IC50 value of 59.8 nM, and high RIPK1 binding affinity compared with other regulatory kinases of necroptosis (RIPK1 Kd = 3.5 nM, RIPK3 Kd = 1700 nM, and MLKL Kd > 30,000 nM). 13c efficiently blocked TNFα-induced necroptosis in both human and murine cells (EC50 = 1.06-4.58 nM), and inhibited TSZ-induced phosphorylation of the RIPK1/RIPK3/MLKL pathway. In liver microsomal assay studies, the clearance rate and half-life of 13c were 18.40 mL/min/g and 75.33 min, respectively. 13c displayed acceptable pharmacokinetic characteristics, with oral bioavailability of 59.55%. In TNFα-induced systemic inflammatory response syndrome, pretreatment with 13c could effectively protect mice from loss of body temperature and death. Overall, these compounds are promising candidates for future optimization studies.

[Effects and mechanisms of total flavones of Abelmoschus manihot in inhibiting podocyte necroptosis and renal fibrosis in diabetic kidney disease].

Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD). Therein, necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF). To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases, on podocyte necroptosis and RF in DKD, and to further reveal its scientific connotation with multi-pathway and multi-target, the authors randomly divided all rats into four groups: a namely normal group, a model group, a TFA group and a rapamycin(RAP) group. After the modified DKD rat models were successfully established, four group rats were given double-distilled water, TFA suspension and RAP suspension, respectively by gavage every day. At the end of the 4th week of drug treatment, all rats were sacrificed, and the samples of their urine, blood and kidneys were collected. And then, the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed, detected and analyzed, respectively. The results indicated that, general condition, body weight(BW), serum creatinine(Scr), urinary albumin(UAlb), and kidney hypertrophy index(KHI) in these modified DKD model rats were both improved by TFA and RAP. Indicators of RF, including glomerular histomorphological characteristics, fibronectin(FN) and collagen type Ⅰ(collagen Ⅰ) staining extent in glomeruli, as well as the protein expression levels of FN, collagen Ⅰ, transforming growth factor-ß1(TGF-ß1) and Smad2/3 in the kidneys were improved respectively by TFA and RAP. Podocyte damage, including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP. In addition, tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys, including the morphological characteristics of podocyte necroptosis, the extent and levels of the protein expression of TNF-α and phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL) was improved respectively by TFA and RAP. Among them, RAP had the better effect on p-MLKL. More importantly, the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys, including the expression levels of its key signaling molecules, such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1), p-RIPK3, p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8) was improved respectively by TFA and RAP. Among them, the effect of TFA on p-RIPK1 was superior. On the whole, in this study, the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. The authors' findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.

The Existing Drug Nifuroxazide as an Antischistosomal Agent: In Vitro, In Vivo, and In Silico Studies of Macromolecular Targets.

Schistosomiasis is a parasitic disease that afflicts approximately 250 million people worldwide. There is an urgent demand for new antiparasitic agents because praziquantel, the only drug available for the treatment of schistosomiasis, is not universally effective and may derail current progress toward the WHO goal of eliminating this disease as a public health problem by 2030. Nifuroxazide (NFZ), an oral nitrofuran antibiotic, has recently been explored to be repurposed for parasitic diseases. Here, in vitro, in vivo, and in silico studies were conducted to evaluate the activity of NFZ on Schistosoma mansoni. The in vitro study showed significant antiparasitic activity, with 50% effective concentration (EC50) and 90% effective concentration (EC90) values of 8.2 to 10.8 and 13.7 to 19.3 µM, respectively. NFZ also affected worm pairing and egg production and induced severe damage to the tegument of schistosomes. In vivo, a single oral dose of NFZ (400 mg/kg of body weight) to mice harboring either prepatent or patent S. mansoni infection significantly reduced the total worm burden (~40%). In patent infection, NFZ achieved a high reduction in the number of eggs (~80%), but the drug caused a low reduction in the egg burden of animals with prepatent infection. Finally, results from in silico target fishing methods predicted that serine/threonine kinases could be one of the potential targets for NFZ in S. mansoni. Overall, the present study revealed that NFZ possesses antischistosomal properties, mainly in terms of egg burden reduction in animals with patent S. mansoni infection. IMPORTANCE The increasing recognition of the burden imposed by helminthiasis, associated with the limited therapeutic arsenal, has led to initiatives and strategies to research and develop new drugs for the treatment of schistosomiasis. One of these strategies is drug repurposing, which considers low-risk compounds with potentially reduced costs and shorter time for development. In this study, nifuroxazide (NFZ) was evaluated for its anti-Schistosoma mansoni potential through in vitro, in vivo, and in silico studies. In vitro, NFZ affected worm pairing and egg production and induced severe damage to the tegument of schistosomes. In vivo, a single oral dose of NFZ (400 mg/kg) to mice harboring either prepatent or patent S. mansoni infection significantly reduced the total worm burden and egg production. In silico investigations have identified serine/threonine kinases as a molecular target for NFZ. Collectively, these results implied that NFZ might be a potential therapeutic candidate for the treatment of schistosomiasis.

Increased dietary Trp, Thr, and Met supplementation improves growth performance and protein deposition of salmonella-challenged growing pigs under poor housing conditions.

Highly intensified rearing conditions and precarious sanitary management predispose pigs to immune system activation, altered amino acid (AA) metabolism, and decreased growth performance. Thus, the main objective of this study was to evaluate the effects of increased dietary tryptophan (Trp), threonine (Thr), and methionine + cysteine (Met + Cys) supplementation on performance, body composition, metabolism, and immune responses of group-housed growing pigs under challenging sanitary conditions. A hundred and twenty pigs (25.4 ± 3.7 kg) were randomly assigned to a 2 × 2 factorial arrangement, consisting of two sanitary conditions (SC, good [GOOD] or salmonella-challenge and poor housing condition [Salmonella Typhimurium (ST) + POOR]) and two diets, control (CN) or supplemented with AA (Trp, Thr, and Met + Cys:Lys ratios 20% higher than those of the CN diet [AA>+]). Pigs were followed during the growing phase (25-50 kg) and the trial lasted 28 d. The ST + POOR SC pigs were challenged with Salmonella Typhimurium and raised in a poor housing condition. The ST + POOR SC increased rectal temperature, fecal score, serum haptoglobin, and urea concentration (P < 0.05) and decreased serum albumin concentration (P < 0.05) compared with GOOD SC. Body weight, average daily feed intake, average daily gain (ADG), feed efficiency (G:F), and protein deposition (PD) were greater in GOOD SC than in ST + POOR SC (P < 0.01). However, pigs housed in ST + POOR SC fed with AA+ diet had lower body temperature (P < 0.05), increased ADG (P < 0.05) and nitrogen efficiency (P < 0.05), and a tendency for improved PD and G:F (P < 0.10) compared with CN diet fed pigs. Regardless of the SC, pigs fed AA+ diet had lower serum albumin (P < 0.05) and tended to decrease serum urea levels (P < 0.10) compared with CN diet. The results of this study suggest that the ratio of Trp, Thr, and Met + Cys to Lys for pigs are modified by sanitary conditions. Furthermore, supplementation of diets with a blend of Trp, Thr, and Met + Cys improves performance, especially under salmonella-challenge and poor housing conditions. Dietary tryptophan, threonine, and methionine supplementation can modulate immune status and influence resilience to sanitary challenges.

Immune system activation alters pigs' physiology and metabolism, increasing maintenance requirements and reducing voluntary feed intake and weight gain. Dietary functional amino acid supplementation (tryptophan, threonine, and methionine) is a strategy to support the immune system activation for immune components production, maintenance of the gut barrier integrity, and reduction of the oxidative status. Additionally, amino acid supplementation may mitigate growth performance losses. In this context, this study was conducted to investigate the effect of diets with or without tryptophan, threonine, and methionine supplementation on the performance and immune system activation of growing pigs under a sanitary challenge. The amino acid supplementation mitigated the immune system activation of challenged growing pigs and improved growth performance when compared to pigs fed diets with no supplementation. The functional amino acid supplementation may be an efficient nutritional strategy to optimize health and growth performance of immune-challenged pigs.

Serine Threonine-Protein Kinase-Derived IW13 Improves Lipid Metabolism via C/EBP-α/SREBP1/FAS Signaling Pathways in HFD-Induced Zebrafish In Vivo Larval Model.

Obesity is linked to the development of major metabolic disorders such as type 2 diabetes, cardiovascular disease, and cancer. Recent research has focused on the molecular link between obesity and oxidative stress. Obesity impairs antioxidant function, resulting in dramatically increased reactive oxygen levels and apoptosis. In this study, we investigated the effect of IW13 peptide on inhibiting lipid accumulation and regulating the antioxidant mechanism to normalize the lipid metabolism in HFD induced zebrafish larvae. Our results showed that co-treatment with IW13 peptide showed a protective effect in HFD zebra fish larvae by increasing the survival and heart rate. However, IW13 peptide co-treatment reduced triglycerides and cholesterol levels while also restoring the SOD and CAT antioxidant enzymes. In addition, IW13 co-treatment inhibited the formation of lipid peroxidation and superoxide anion by regulating the glutathione level. Also, the results showed that IW13 specifically downregulated the expression of the lipogenic-specific genes (C/EBP-α, SREBP1, and FAS). The findings exhibited that the IW13 peptide with effective antioxidant and anti-obesity activity could act as a futuristic drug to treat obesity and oxidative stress-related diseases.

Nutritional geometry framework of sucrose taste in Drosophila.

Dietary protein (P) and carbohydrate (C) have a major impact on the sweet taste sensation. However, it remains unclear whether the balance of P and C influences the sweet taste sensitivity. Here, we use the nutritional geometry framework (NGF) to address the interaction of protein and carbohydrates on sweet taste using Drosophila as a model. Our results reveal that high-protein, low-carbohydrate (HPLC) diets sensitize to sweet taste and low-protein, high-carbohydrate (LPHC) diets desensitize sweet taste in both male and female flies. We further investigate the underlying mechanisms of the effects of two diets on sweet taste using RNA sequencing. When compared to the LPHC diet, the mRNA expression of genes involved in the metabolism of glycine, serine, and threonine is significantly upregulated in the HPLC diet group, suggesting these amino acids may mediate sweet taste perception. We further find that sweet sensitization occurs in flies fed with the LPHC diet supplemented with serine and threonine. Our study demonstrates that sucrose taste sensitivity is affected by the balance of dietary protein and carbohydrates possibly through changes in serine and threonine.

Akt substrate of 160 kDa is essential for the calorie restriction-induced increase in insulin-stimulated glucose uptake by skeletal muscle of female rats.

We evaluated effects of calorie restriction (CR; consuming 65% of ad libitum (AL) intake) for 8 weeks on female wildtype (WT) and Akt substrate of 160 kDa knockout (AS160-KO) rats. Insulin-stimulated glucose uptake (ISGU) was determined in isolated epitrochlearis muscles incubated with 0, 50, 100, or 500 µU/mL insulin. Phosphorylation of key insulin signaling proteins that control ISGU (Akt and AS160) was assessed by immunoblotting (Akt phosphorylation on Threonine-308, pAktThr308 and Serine-473, pAktSer473; AS160 phosphorylation on Serine-588, pAS160Ser588, and Threonine-642, pAS160Thr642). Abundance of proteins that regulate ISGU (GLUT4 glucose transporter protein and hexokinase II) was also determined by immunoblotting. The major results were as follows: (i) WT-CR versus WT-AL rats had greater ISGU with 100 and 500 µU/mL insulin; (ii) CR versus WT-AL rats had greater GLUT4 protein abundance; (iii) WT-CR versus WT-AL rats had greater pAktThr308 with 500 µU/mL insulin; (iv) WT-CR versus WT-AL rats did not differ for pAktSer473, pAS160Ser588, or pAS160Thr642 at any insulin concentration; (v) AS160-KO versus WT rats with each diet had lower ISGU at each insulin concentration, but not lower pAkt on either phosphosite; (vi) AS160-KO versus WT rats had lower muscle GLUT4 abundance regardless of diet; and (vii) AS160-KO-CR versus AS160-KO-AL rats did not differ for ISGU, GLUT4 abundance, pAkt on either phosphosite, or pAS160 on either phosphosite. These novel results demonstrated that AS160 expression, but not greater pAS160 on key phosphosites, was essential for the CR-induced increases in muscle ISGU and GLUT4 abundance of female rats.

Impacts of a bacterial algicide on metabolic pathways in Chlorella vulgaris.

Chlorella is a dominant species during harmful algal blooms (HABs) worldwide, which bring about great environmental problems and are also a serious threat to drinking water safety. Application of bacterial algicides is a promising way to control HABs. However, the identified bacterial algicides against Chlorella and the understanding of their effects on algal metabolism are very limited. Here, we isolated a novel bacterium Microbacterium paraoxydans strain M1 that has significant algicidal activities against Chlorella vulgaris (algicidal rate 64.38 %, at 120 h). Atrazine-desethyl (AD) was then identified from strain M1 as an effective bacterial algicide, with inhibition or algae-lysing concentration values (EC50) of 1.64 µg/mL and 1.38 µg/mL, at 72 h and 120 h, respectively. LAD (2 µg/mL AD) or HAD (20 µg/mL AD) causes morphology alteration and ultrastructure damage, chlorophyll a reduction, gene expression regulation (for example, psbA, 0.05 fold at 24 h, 2.97 fold at 72 h, and 0.23 fold of the control in HAD), oxidative stress, lipid oxidation (MDA, 2.09 and 3.08 fold of the control in LAD and HAD, respectively, at 120 h) and DNA damage (average percentage of tail DNA 6.23 % at 120 h in HAD, slight damage: 5∼20 %) in the algal cells. The impacts of AD on algal metabolites and metabolic pathways, as well as the algal response to the adverse effects were investigated. The results revealed that amino acids, amines, glycosides and urea decreased significantly compared to the control after 24 h exposure to AD (p < 0.05). The main up-regulated metabolic pathways implied metabonomic resistance and defense against osmotic pressure, oxidative stress, photosynthesis inhibition or partial cellular structure damage, such as phenylalanine metabolism, arginine biosynthesis. The down-regulated glycine, serine and threonine metabolism is a major lead in the algicidal mechanism according to the value of pathway impact. The down-regulated glycine, and serine are responsible for the downregulation of glyoxylate and dicarboxylate metabolism, aminoacyl-tRNA biosynthesis, glutathione metabolism, and sulfur metabolism, which strengthen the algae-lysing effect. It is the first time to highlight the pivotal role of glycine, serine and threonine metabolism in algicidal activities, which provided a new perspective for understanding the mechanism of bacterial algicides exerting on algal cells at the metabolic level.

Inhibition of 26S proteasome enhances AKAP3-mediated cAMP-PKA signaling during boar sperm capacitation.

This study investigates the effects of the ubiquitin-proteasome pathway (UPP) on porcine sperm capacitation and its interactions with the cAMP-PKA pathway. The semen of adult Landrace boars was divided into four groups: non-capacitated, capacitated, 10 µM/mL MG132, and 10 µM/mL DMSO groups. We characterized the parameters related to sperm dynamics using a computer-assisted sperm analysis system. The level of sperm protein tyrosine phosphorylation was detected using Western blotting, and the change of zinc ion signal was detected via flow cytometry. The relationship between A-kinase-anchor protein 3 (AKAP3), ubiquitin (Ub), and protein kinase A (PKA) was assessed by co-precipitation assays; to evaluate the interactions between the UPP and cAMP-PKA pathway, threonine, serine, and tyrosine phosphorylation were detected using Western blotting to evaluate the interaction between the UPP and cAMP-PKA pathway; Hoechst staining was used to detect the sperm-egg binding state and evaluate the effects of UPP inhibition. During capacitation, the levels of protein tyrosine, serine, and threonine phosphorylation and ubiquitination of porcine sperm increased, and sperm-egg binding was inhibited (P < 0.05). AKAP3 was degraded by UPP, and after inhibiting the 26 S proteasome, ubiquitinated AKAP3 accumulated in large quantities. Our findings indicate that, after the 26 S proteasome was inhibited, PKA was uncoupled from AKAP3 and degraded by UPP; the level of tyrosine phosphorylation induced by PKA-AKAP3 was reduced, the level of serine threonine phosphorylation increased, and the ubiquitination pathway interacted with the phosphorylation pathway and was involved in sperm capacitation.

[Effect of heat-reinforcing needling on expression of serum inflammatory factors and autophagy of knee joint synovial tissue in rheumatoid arthritis rabbits with cold syndrome].

OBJECTIVE: To observe the effect of heat-reinforcing needling on the expression of serum inflammatory factors and autophagy of knee synovial tissue in rheumatoid arthritis (RA) rabbits with cold syndrome, so as to explore its mechanism of anti-inflammatory in the treatment of RA. METHODS: Fifty rabbits were randomly divided into normal, model, heat-reinforcing needling, inhibitor and agonist groups (n=10 rabbits in each group). The model of RA with cold syndrome was established by Freund's adjuvant and ovalbumin mixed solution injection combined with freezing and wind-cold dampness method. Heat-reinforcing needling was applied at "Zusanli" (ST36) for 30 min, once a day for 14 days. Rabbits of the inhibitor and agonist groups were given intraperitoneally injected with autophagy inhibitor 3-methyladenine (3-MA) or autophagy agonist rapamycin, once every 2 days for 7 days. The knee circumference and skin temperature of the rabbits in each group were measured. Color doppler ultrasonography was applied to examine the synovial membrane, joint effusion and blood flow signals in the knee joints of the rabbits in each group. Serum tumor necrosis factor (TNF) -α, interleukin (IL)-1ß, IL-6 and C-creactive protein (CRP) were detected by ELISA. Transmission electron microscopy was applied to observe the ultrastructure and autophagosomes of synovial cells. The protein expressions of autophagy-related protein Atg5, serine/threonine protein kinase-dysregulated 51-like kinase 1 (ULK1), microtubule-associated protein light chain 3B (LC3B), and Beclin-1 were detected by Western blot. Fluorescence quantitative PCR was used to detect the mRNA expressions of NOD-like receptor 3 (NLRP3) and nuclear factor-κB (NF-κB). RESULTS: Compared with the normal group, the circumference of the knee joint was increased (P<0.01), the skin temperature was decreased (P<0.01), the knee joint synovium was thickened and the blood flow signal was abundant, the contents of serum TNF-α, IL-1ß, IL-6, and CRP were increased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3BⅡ/LC3BⅠof synovial tissue were significantly decreased (P<0.01), the mRNA expressions of NLRP3 and NF-κB were increased (P<0.01) in the model group. In comparison with the model and inhibitor groups, the circumference of the knee joint was decreased (P<0.01), whlie the skin temperature was increased (P<0.01), the synovial membrane became thinner and the blood flow signal was wea-kened, the contents of TNF-α, IL-1ß, IL-6 and CRP were decreased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3B Ⅱ/LC3B Ⅰ were increased (P<0.01), and the mRNA expressions of NLRP3 and NF-κB were decreased (P<0.01) in the heat-reinforcing needling and agonist groups. CONCLUSION: Heat-reinforcing needling can alleviate the inflammatory response of the knee joint synovium in RA rabbits with cold syndrome, which may be related to its function in enhancing the autophagy activity of synovial cells and inhibiting the synthesis and release of inflammatory factors TNF-α, IL-1ß, IL-6 and CRP.

GpsB Promotes PASTA Kinase Signaling and Cephalosporin Resistance in Enterococcus faecalis.

Enterococci are opportunistic pathogens that can cause severe bacterial infections. Treatment of these infections is challenging because enterococci possess intrinsic and acquired mechanisms of resistance to commonly used antibiotics, including cephalosporins. The transmembrane serine/threonine PASTA kinase, IreK, is an important determinant of enterococcal cephalosporin resistance. Upon exposure to cephalosporins, IreK becomes autophosphorylated, which stimulates its kinase activity to phosphorylate downstream substrates and drive cephalosporin resistance. However, the molecular mechanisms that modulate IreK autophosphorylation in response to cell wall stress, such as that induced by cephalosporins, remain unknown. A cytoplasmic protein, GpsB, promotes signaling by PASTA kinase homologs in other bacterial species, but the function of enterococcal GpsB has not been previously investigated. We used in vitro and in vivo approaches to test the hypothesis that enterococcal GpsB promotes IreK signaling in response to cephalosporins to drive cephalosporin resistance. We found that GpsB promotes IreK activity both in vivo and in vitro. This effect is required for cephalosporins to trigger IreK autophosphorylation and activation of an IreK-dependent signaling pathway, and thereby is also required for enterococcal intrinsic cephalosporin resistance. Moreover, analyses of GpsB mutants and a ΔireK gpsB double mutant suggest that GpsB has an additional function, beyond regulation of IreK activity, which is required for optimal growth and full cephalosporin resistance. Collectively, our data provide new insights into the mechanism of signal transduction by the PASTA kinase IreK and the mechanism of enterococcal intrinsic cephalosporin resistance. IMPORTANCE Enterococci are opportunistic pathogens that can cause severe bacterial infections. Treatment of these infections is challenging because enterococci possess intrinsic and acquired resistance to commonly used antibiotics. In particular, enterococci are intrinsically resistant to cephalosporin antibiotics, a trait that requires the activity of a transmembrane serine/threonine kinase, IreK, which belongs to the bacterial PASTA kinase family. The mechanisms by which PASTA kinases are regulated in cells are poorly understood. Here, we report that the cytoplasmic protein GpsB directly promotes IreK signaling in enterococci to drive cephalosporin resistance. Thus, we provide new insights into PASTA kinase regulation and control of enterococcal cephalosporin resistance, and suggest that GpsB could be a promising target for new therapeutics to disable cephalosporin resistance.

Characterization of a new Bacillus velezensis as a powerful biocontrol agent against tomato gray mold.

Biocontrol microbes are environment-friendly and safe for humans and animals. To seek biocontrol microbes effective in suppressing tomato gray mold is important for tomato production. Therefore, serial experiments were conducted to characterize the antagonism of Bacillus velezensis HY19, a novel self-isolated biocontrol bacterium, against Botrytis cinerea in vitro and the control on tomato gray mold in greenhouse. This bacterium produced extracellular phosphatase, protease, cellulose and siderophores, and considerably inhibited the growth of B. cinerea. A liquid chromatography-mass spectrometry (LC-MS) detected salicylic acid and numerous antifungal substances present in B. velezensis HY19 fermentation liquid (BVFL). When B. cinerea was grown on potato glucose agar, BVFL crude extract remarkably suppressed the fungal growth and reduced protein content and the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD). Transcriptome studies showed that BVFL crude extract significantly induced different expression of numerous genes in B. cinerea, most of which were down-regulated. Theses differently expressed genes were involved in the biological process, cell compartment, molecular functions, and metabolisms of glycine, serine, threonine, and sulfur in pathogen hyphae. Thus, this biocontrol bacterium antagonized B. cinerea in multiple ways due to the production of numerous antifungal substances that acted on multiple targets in the cells. BVFL significantly increased antioxidant enzyme activities in tomato leaves and decreased the incidence of tomato gray mold, with the control efficacies of 73.12-76.51%. Taken together, B. velezensis HY19 showed a promising use potential as a powerful bioagent against tomato gray mold.

[Effect and mechanism of Dahuang Zhechong Pills against testicular aging in rats by inhibiting necroptosis signaling pathway].

To explore the effect and mechanism of Dahuang Zhechong Pills(DHZCP), a classical prescription, in improving testicular aging(TA) in vivo, the authors randomly divided 24 male rats into four groups: the normal, model, DHZCP and vitamin E(VE) groups. The TA rat model was established by continuous gavage of D-galactose(D-gal). During the experiment, the rats in the DHZCP and VE groups were given DHZCP suspension and VE suspension, respectively by gavage, while those in the normal and model groups were gavaged saline separately every day. After the co-administration of D-gal and various drugs for 60 days, all rats were sacrificed, and their blood and testis were collected. Further, various indexes related to TA and necroptosis of testicular cells in the model rats were examined and investigated, which included the aging phenotype, total testicular weight, testicular index, histopathological features of testis, number of spermatogenic cells, sex hormone level, expression characteristics of reactive oxygen species(ROS) in testis, expression levels and characteristics of cyclins in testis, and protein expression levels of the key molecules in receptor-interacting serine/threonine-protein kinase 1(RIPK1)/receptor-interacting serine/threonine-protein kinase 3(RIPK3)/mixed lineage kinase domain like pseudokinase(MLKL) signaling pathway in each group. The results showed that, for the TA model rats, both DHZCP and VE improved their aging phenotype, total testicular weight, testicular index, pathological features of testis, number of spermatogenic cells, serum testosterone and follicle stimulating hormone levels, expression characteristics of ROS and protein expression levels and characteristics of P21 and P53 in testis. In addition, DHZCP and VE improved the protein expression levels of the key molecules in RIPK1/RIPK3/MLKL signaling pathway in testis of the model rats. Specifically, DHZCP was better than VE in the improvement of RIPK3. In conclusion, in this study, the authors found that DHZCP, similar to VE, ameliorated D-gal-induced TA in model rats in vivo, and its mechanism was related to reducing necroptosis of testicular cells by inhibiting the activation of RIPK1/RIPK3/MLKL signaling pathway. This study provided preliminary pharmacological evidence for the development and application of classical prescriptions in the field of men's health.

LncRNA-MEG3 attenuates hyperglycemia-induced damage by enhancing mitochondrial translocation of HSP90A in the primary hippocampal neurons.

The diabetic cognitive impairments are associated with high-glucose (HG)-induced mitochondrial dysfunctions in the brain. Our previous studies demonstrated that long non-coding RNA (lncRNA)-MEG3 alleviates diabetic cognitive impairments. However, the underlying mechanism has still remained elusive. Therefore, this study was designed to investigate whether the mitochondrial translocation of HSP90A and its phosphorylation are involved in lncRNA-MEG3-mediated neuroprotective effects of mitochondrial functions in HG-treated primary hippocampal neurons and diabetic rats. The primary hippocampal neurons were exposed to 75 mM glucose for 72 h to establish a HG model in vitro. Firstly, the RNA pull-down and RNA immunoprecipitation (RIP) assays clearly indicated that lncRNA-MEG3-associated mitochondrial proteins were Annexin A2, HSP90A, and Plectin. Although HG promoted the mitochondrial translocation of HSP90A and Annexin A2, lncRNA-MEG3 over-expression only enhanced the mitochondrial translocation of HSP90A, rather than Annexin A2, in the primary hippocampal neurons treated with or without HG. Meanwhile, Plectin mediated the mitochondrial localization of lncRNA-MEG3 and HSP90A. Furthermore, HSP90A threonine phosphorylation participated in regulating mitochondrial translocation of HSP90A, and lncRNA-MEG3 also enhanced mitochondrial translocation of HSP90A through suppressing HSP90A threonine phosphorylation. Finally, the anti-apoptotic role of mitochondrial translocation of HSP90A was found to be associated with inhibiting death receptor 5 (DR5) in HG-treated primary hippocampal neurons and diabetic rats. Taken together, lncRNA-MEG3 could improve mitochondrial functions in HG-exposed primary hippocampal neurons, and the underlying mechanisms were involved in enhanced mitochondrial translocation of HSP90A via suppressing HSP90A threonine phosphorylation, which may reveal a potential therapeutic target for diabetic cognitive impairments.

Dibutyl phthalate-induced oxidative stress and apoptosis in swine testis cells and therapy of naringenin via PTEN/PI3K/AKT signaling pathway.

Dibutyl phthalate (DBP) is a phthalic acid ester (PAE) that has posed a health hazard to the organisms. Naringenin (NRG) is a flavanone compound that has shown protection against several environmental chemicals through suppression of oxidative stress and activation of phosphatidylinositol 3-kinase/threonine kinase (PI3K/AKT) signaling pathway. Herein, swine testis (ST) cells were treated with 1.8 µM of DBP or/and 25.39 nM of NRG for 24 h, we described the discovery path of NRG inhibition on apoptosis in DBP-exposed ST cells through targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN). We first found that the anti-apoptosis effect of NRG is dependent on mitochondrial pathway through flow cytometry and related gene/protein expression, and then we detected PI3K/AKT pathway-related gene/protein expression, and established a computational docking assay between NRG and PTEN. We found that NRG specifically binds to three basic residues (His93, Lys125, Lys128) of P loop in PTEN, as well as phosphatase domains (Asp92, His93, Cys124, Lys125, Ala126, Lys128, and Arg130) in active dephosphorylation pockets, thereby reducing PTEN level and activating PI3K/AKT signaling pathway, and further inhibiting oxidative stress and mitochondrial pathway apoptosis. Taken together, our results push forward that NRG deserves further attention as a potential antagonistic therapy against DBP through targeting PTEN to inhibit oxidative stress and activate PI3K/AKT signaling pathway.

Nutritional interventions to support broiler chickens during Eimeria infection.

Different combinations of gut health-promoting dietary interventions were tested to support broilers during different stages of Eimeria infection. One-day-old male Ross 308 broilers (n = 720) were randomly assigned to one of 6 dietary treatments, with 6 pens per treatment and 20 birds per pen, for 35 d. At 7 d of age (d7), all birds were inoculated with 1000, 100, and 500 sporulated oocysts of E. acervulina, E. maxima, and E. tenella, respectively. A 4-phase feeding schedule was provided. The dietary treatments (TRT) 1 to 4 included the basal diet supplemented with multispecies probiotics from d0 to 9 and coated butyrate and threonine from d28 to 35 but received four different combinations of prebiotics and phytochemicals from d9 to 18 and d18 to 28. The basal diet for the positive control (PC, TRT5) included diclazuril as a anticoccidial. The negative control (NC, TRT6) contained no anticoccidial. Performance was assessed for each feeding phase, and oocyst output, Eimeria lesion scores, cecal weight, litter quality, and footpad lesions were assessed at d14, d22, d28, and d35. Body weight gain (BWG) and feed intake (FI) were not affected by dietary treatment. PC broilers had the best feed conversion ratio (FCR) of all treatments from d0 to 35 (P < 0.001). None of the dietary treatments resulted in better litter quality or reduced footpad lesions compared to the PC. Moreover, the PC was most effective in reducing oocyst output and lesion scores compared to all other treatments. However, broilers that received the multispecies probiotics (d0 to 9), saponins (d9 to 18), saponins, artemisin, and curcumin (d18 to 28), and coated butyrate and threonine (d28 to 35) had the best FCR (P < 0.001) and lowest oocyst output and lesion scores compared to other dietary treatments. This study suggests that although the tested compounds did not perform as well as the anticoccidial, when applied in the proper feeding period, they may support bird resilience during coccidiosis infection.

In ovo threonine supplementation affects ileal gene expression of nutrient transporters in broilers inoculated post-hatch with Salmonella Enteritidis.

The effect of in ovo threonine (Thr) supplementation on the ileal expression of glucose, peptide and amino acid transporters was assessed in Salmonella Enteritidis-challenged broiler chicks. At 17.5 days of incubation, fertile eggs were supplemented in the amniotic fluid with sterile saline or 3.5% threonine. Hatchlings were individually weighed, and Salmonella Enteritidis negative status was confirmed. At 2 days of age, half of the birds of each group were inoculated with sterile nutrient broth or Salmonella Enteritidis inoculum. Relative expression of sodium-dependent glucose transporter 1 (SGLT1), glucose transporter 2 (GLUT2), di- and tri-peptide transporter 1 (PepT1) and alanine, serine, cysteine, threonine transporter (ASCT1) was assessed at hatch, 2 and 9 days of age, i.e., before inoculation and 7 days post-inoculation (dpi). At 9 days of age (7dpi), threonine increased SGLT1 and GLUT2 expression, whereas GLUT2 expression decreased in Salmonella-challenged birds. There was a significant interaction between threonine and Salmonella for PepT1 and ASCT1. Threonine increased PepT1 expression only in non-challenged birds. In addition, in ovo supplementation increased expression of ASCT1 regardless of post-hatch inoculation; Salmonella inoculation resulted in decreased expression of ASCT1 only in supplemented birds. The results suggest that while intra-amniotic threonine administration in broiler embryos increases the expression of genes related to the absorption of monosaccharides and amino acids, Salmonella challenge may negatively affect the expression of protein related transporters in the ileum of broilers.

L-Threonine upregulates the expression of ß-defensins by activating the NF-κB signaling pathway and suppressing SIRT1 expression in porcine intestinal epithelial cells.

The use of antimicrobial peptide (AMP), found in all forms of life and playing a pivotal role in the innate immune system, has been developed as a new strategy for maintaining intestinal health and reducing antibiotic usage due to its ability to resist pathogens and commensal microbes. The current study investigated the effects of l-threonine on ß-defensin expression, the intestinal mucosal barrier and inflammatory cytokine expression in porcine intestinal epithelial cell lines (IPEC-J2). The results revealed that in IPEC-J2 cells, l-threonine significantly increased the expression of ß-defensin (including pBD-1, pBD-2, and pBD-3) in a dose- and time-dependent manner (P < 0.05). By using different concentrations and treatment times of l-threonine, the results showed that the expression of ß-defensin was upregulated to the greatest extent in IPEC-J2 cells cultured with 1 mM l-threonine for 24 h. Although the mRNA expression levels of ß-defensins were markedly increased (P < 0.05), there was relatively little inducible pBD-1, pBD-2 and pBD-3 mRNA expression at the sub-confluent and confluent densities in comparison with post-confluent densities. Furthermore, we found that l-threonine enhanced the ß-defensin expression by suppressing the expression of SIRT1, which increased acetylated p65 expression, and activating the NF-κB signaling pathway, which induced the translocation of p65 from the cytoplasm to the nucleus. In addition, l-threonine significantly prevented LPS-induced intestinal mucosal barrier damage by attenuating the decreasing tendency of the mRNA expression of Mucin1 and Mucin2 (P < 0.05). Simultaneously, l-threonine enhanced the expression of ß-defensins upon LPS challenge in IPEC-J2 cells (P < 0.05). l-Threonine obviously decreased the mRNA expression of inflammatory cytokines compared to that in untreated cells (P < 0.05). In conclusion, l-threonine can upregulate ß-defensin expression and reduce inflammatory cytokine expression in IPEC-J2 cells; meanwhile, l-threonine alleviates LPS-induced intestinal mucosal barrier damage in porcine intestinal epithelial cells. The l-threonine-mediated modulation of endogenous defensin expression may be a promising approach to reduce antibiotic use, enhance disease resistance and intestinal health in animals.

Protective role of l-threonine against cadmium toxicity in Saccharomyces cerevisiae.

Environment and food contamination with cadmium (Cd) can cause serious toxicity, posing a severe threat to agricultural production and human health. However, how amino acids contribute to defenses against oxidative stress caused by Cd in cells is not fully understood. As a model eukaryote with a relatively clear genetic background, Saccharomyces cerevisiae has been commonly used in Cd toxicity research. To gain insight into Cd toxicity and cell defenses against it, 20 amino acids were screened for protective roles against Cd stress in S. cerevisiae. The results showed that threonine (Thr, T) had the strongest protective effect against Cd-induced mortality and membrane damage in the cells. Compared to the antioxidant vitamin C (VC), Thr exhibited a higher efficacy in restoring the superoxide dismutase (SOD) activity that was inhibited by Cd but not by H2 O2 in vivo. Thr exhibited evident DPPH (2,2-diphenyl-1-picrylhydrazyl) activity but weak ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-9 sulfonic acid)) scavenging activity, giving it a weaker effect against Cd-induced lipid peroxidation and superoxide radical O2- , compared to VC. More importantly, compared to the chelating agent EDTA, Thr showed stronger chelation of Cd, giving it a stronger protective effect on SOD against Cd than VC in vitro. The results of the in vivo and in vitro experiments revealed that the role Thr plays in cell defenses against Cd may be attributed to its protection of the SOD enzyme, predominantly through the preferential chelation of Cd. Our results provide insights into the protective mechanisms of amino acid Thr that ameliorate Cd toxicity and suggest that a supplement of Thr might help to reduce Cd-induced oxidative damage.

Regulation of the First Committed Step in Lipopolysaccharide Biosynthesis Catalyzed by LpxC Requires the Essential Protein LapC (YejM) and HslVU Protease.

We previously showed that lipopolysaccharide (LPS) assembly requires the essential LapB protein to regulate FtsH-mediated proteolysis of LpxC protein that catalyzes the first committed step in the LPS synthesis. To further understand the essential function of LapB and its role in LpxC turnover, multicopy suppressors of ΔlapB revealed that overproduction of HslV protease subunit prevents its lethality by proteolytic degradation of LpxC, providing the first alternative pathway of LpxC degradation. Isolation and characterization of an extragenic suppressor mutation that prevents lethality of ΔlapB by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated lapC, suggesting LapB and LapC act antagonistically. The same lapC gene was identified during selection for mutations that induce transcription from LPS defects-responsive rpoEP3 promoter, confer sensitivity to LpxC inhibitor CHIR090 and a temperature-sensitive phenotype. Suppressors of lapC mutants that restored growth at elevated temperatures mapped to lapA/lapB, lpxC and ftsH genes. Such suppressor mutations restored normal levels of LPS and prevented proteolysis of LpxC in lapC mutants. Interestingly, a lapC deletion could be constructed in strains either overproducing LpxC or in the absence of LapB, revealing that FtsH, LapB and LapC together regulate LPS synthesis by controlling LpxC amounts.