[Huangdi Anxiao Capsules-containing serum protects cell model from cognitive dysfunction in diabetes via inhibiting NLRP3-mediated pyroptosis].

This study aims to investigate the effects and the molecular mechanism of Huangdi Anxiao Capsules(HDAX)-containing serum in protecting the rat adrenal pheochromocytoma(PC12) cells from diabetes-associated cognitive dysfunction induced by high glucose and whether the mechanism is related to the regulation of NOD-like receptor thermal protein domain associated protein 3(NLRP3)-mediated pyroptosis. The PC12 cell model of diabetes-associated cognitive dysfunction induced by high glucose was established and mcc950 was used to inhibit NLRP3. PC12 cells were randomized into control, model, HDAX-containing serum, mcc950, and HDAX-containing serum+mcc950 groups. Methyl thiazolyl tetrazolium(MTT) assay was employed to determine the viability, and Hoechst 33258/PI staining to detect pyroptosis of PC12 cells. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1 beta(IL-1ß) and IL-18. Western blot was employed to determine the protein levels of postsynaptic density protein 95(PSD-95), NLRP3, apoptosis-associated speck-like protein containing a CARD(ASC), gasdermin D(GSDMD), GSDMD-N, and cleaved cysteinyl aspartate specific proteinase-1(caspase-1), and RT-PCR to determine the mRNA levels of NLRP3, ASC, GSDMD, and caspase-1. The immunofluorescence assay was adopted to measure the levels and distribution of NLRP3 and GSDMD-N in PC12 cells. Compared with the control group, the model group showed decreased cell proliferation, increased PI positive rate, down-regulated protein level of PSD-95, up-regulated protein levels of NLRP3, ASC, GSDMD-N, GSDMD, and cleaved caspase-1, up-regulated mRNA levels of NLRP3, ASC, GSDMD, and caspase-1, and elevated levels of IL-1ß and IL-18. Compared with the model group, HDAX-containing serum, mcc950, and the combination of them improved cell survival rate and morphology, decreased the PI positive rate, down-regulated the protein levels of NLRP3, ASC, GSDMD-N, GSDMD, and cleaved caspase-1 and the mRNA levels of NLRP3, ASC, GSDMD, and caspase-1, and promoted the secretion of IL-1ß and IL-18. The findings demonstrated that HDAX-containing serum can inhibit the pyroptosis-mediated by NLRP3 and protect PC12 cells from the cognitive dysfunction induced by high glucose.

Traditional Banxia Xiexin decoction inhibits invasion, metastasis, and epithelial mesenchymal transition in gastric cancer by reducing lncRNA TUC338 expression.

Background: Banxia Xiexin decoction (BXD) is a classic traditional Chinese medicine (TCM) formula clinically used to treat chronic gastritis, gastric ulcers, gastric cancer, and many other gastrointestinal diseases. Long noncoding RNAs (lncRNAs) have been shown to play an important role in maintaining the malignant phenotype of tumors. However, no relevant studies have shown whether Banxia Xiexin decoction regulates and controls lncRNA TUC338, and the effect of TUC338 on the regulation of gastric cancer invasion and metastasis remains unclear. Purpose: To investigate the ability of the traditional Chinese medicine (TCM) Banxia Xiexin decoction (BXD) to inhibit the migration and invasion of human gastric cancer AGS cells by regulating the long noncoding RNA (lncRNA) TUC338. Methods: UHPLC‒MS/MS was used to analyze the chemical components of BXD. MTT was performed to determine the effects of BXD on the proliferation of AGS cells. qRT‒PCR was used to determine the expression of lncRNA TUC338 in gastric cancer tissues, paracarcinoma tissues, AGS human gastric cancer cells and GES-1 normal gastric mucosa cells and to evaluate the effects of BXD on the expression of lncRNA TUC338 in AGS cells. Lentiviral transfection was used to establish human gastric cancer AGS cells with knocked down lncRNA TUC338 expression. The effects of lncRNA TUC338 knockdown on the migration and invasion of AGS cells were observed by a scratch assay and Transwell migration assay, respectively. Western blotting was performed to analyze the effects of lncRNA TUC338 knockdown on epithelial-to-mesenchymal transition (EMT) in AGS cells. We performed quality control on three batches of BXD. We used UHPLC‒MS/MS to control the quality of three random batches of BXD used throughout the study. Results: Ninety-five chemical components were identified from the water extract of BXD, some of which have anticancer effects. The expression of TUC.338 in gastric cancer tissues was higher than that in para-carcinoma tissues. BXD inhibited the invasion and migration of gastric cancer cells by inhibiting the expression of lncRNA TUC338, which reduced EMT. After knockdown of lncRNA TUC338, the migration and invasion of AGS cells were reduced; the expression of the EMT-related protein E-cadherin was increased, and the expression of N-cadherin and vimentin was reduced. Conclusions: The present results suggest that BXD has potential as an effective treatment for gastric cancer through the inhibition of lncRNA TUC338 expression.

Transcriptomics analysis unveils key potential genes associated with brain development and feeding behavior in the hypothalamus of L-citrulline-fed broiler chickens.

High ambient temperature is a major environmental stressor affecting poultry production, especially in the tropical and subtropical regions of the world. Nutritional interventions have been adopted to combat thermal stress in poultry, including the use of amino acids. L-citrulline is a nonessential amino acid that is involved in nitric oxide generation and thermoregulation, however, the molecular mechanisms behind L-citrulline's regulation of body temperature are still unascertained. This study investigated the global gene expression in the hypothalamus of chickens fed either basal diet or L-citrulline-supplemented diets under different housing temperatures. Ross 308 broilers were fed with basal diet (CON) or 1% L-citrulline diet (LCT) from day-old, and later subjected to 2 environmental temperatures in a 2 by 2 factorial arrangement as follows; basal diet-fed chickens housed at 24°C (CON-TN); L-citrulline diet-fed chickens housed at 24°C (LCT-TN); basal diet-fed chickens housed at 35°C (CON-HS), and L-citrulline diet-fed chickens housed at 35°C (LCT-HS) from 22 to 42 d of age. At 42-days old, hypothalamic tissues were collected for mRNA analyses and RNA sequencing. A total of 1,019 million raw reads were generated and about 82.59 to 82.96% were uniquely mapped to genes. The gene ontology (GO) term between the CON-TN and LCT-TN groups revealed significant enrichments of pathways such as central nervous system development, and Wnt signaling pathway. On the other hand, GO terms between the CON-HS and LCT-HS groups revealed enrichments in the regulation of corticosteroid release, regulation of feeding behavior, and regulation of inflammatory response. Several potential candidate genes were identified to be responsible for central nervous system development (EMX2, WFIKKN2, SLC6A4 Wnt10a, and PHOX2B), and regulation of feed intake (NPY, AgRP, GAL, POMC, and NMU) in chickens. Therefore, this study unveils that L-citrulline can influence transcripts associated with brain development, feeding behavior, energy metabolism, and thermoregulation in chickens raised under different ambient temperatures.

Effect of exogenous bile salts supplementation on the performance and hepatic lipid metabolism of aged laying hens.

Bile acids (BA), a series of hydroxylated steroids secreted by the liver, are involved in the digestion and absorption of dietary fats. In the present study, the effect of exogenous BAs on the performance and liver lipid metabolism of laying hens was investigated. Three hundred and sixty 50-wk-old Hy-line Brown hens were randomly allocated into three groups and subjected to one of the following treatments: fed with the basal diet (control, Con), the basal diet supplemented with 0.1 g/kg (0.1 g/kg BAs), or 0.2 g/kg (0.2 g/kg BAs) porcine BAs. Laying performance, egg quality, and blood parameters were measured during the 8-wk experimental period. The expression of genes related to hepatic lipid metabolism was determined at the end of experiment. The results showed that BAs treatments had no influence (P > 0.05) on laying rate, egg weight, and feed efficiency. BAs treatment, however, significantly decreased mortality of hens (P = 0.006). BAs treatment showed a transient negative influence on eggshell quality at week 4 but not at week 8. The yolk color on week 8 was increased by BAs treatments (P < 0.0001) compared to control. The duodenum index showed a tendency to be increased (P = 0.053) and jejunum index were increased (P = 0.007) by BAs treatment. Compared to control, BAs treatments decreased lipid droplet content (P < 0.0001) and TG content (P = 0.002) of liver. Fatty acid synthase activity was also decreased as an effect of BAs dietary supplementation. Compared to the control group, 0.1 g/kg BAs treatment increased (P < 0.05) the mRNA expression of genes Farnesoid X receptor (FXR) (P = 0.042), cytochrome P450 family 7 subfamily A member 1 (CYP7A1) (P = 0.002), and cytochrome P450 family 8 subfamily B member 1 (CYP8B1) (P = 0.017), fatty acid synthase (FAS) (P = 0.020), acetyl-CoA carboxylase (ACC) (P = 0.032), sterol regulatory element binding protein-1c (SREBP-1c) (P = 0.037), proliferator-activated receptor gamma (PPARγ) (P = 0.002), apolipoprotein B (APO-B) (P = 0.020), and very low density lipoprotein receptor (VLDLR) (P = 0.024). In conclusion, the addition of exogenous BAs reduces lipid accumulation in liver. BA supplementation reduces the mortality of hens and improves egg yolk color, with no unfavorable effect on laying performance. The result suggests that suppressed FAS activity is involved in the reduced hepatic lipid accumulation by BAs treatment.

Fatty liver hemorrhagic syndrome is one of the most common diseases in laying hens and is a metabolic disease characterized by disorders of lipid metabolism in the liver, manifested by fatty liver degeneration and varying degrees of hemorrhage, which often occurs in caged hens in good condition and with high egg production rates. Bile acids (BA), a group of hydroxylated steroids synthesized from cholesterol in the liver, play an important role in lipid metabolism. This study aimed to examine the effects of dietary addition of different levels of BAs on the production performance and liver fat metabolism of 50-wk-old Hy-line Brown hens. The result indicates that the addition of exogenous BAs reduces lipid accumulation in liver. BAs supplementation reduces the mortality of hens and improves egg yolk color, with no other unfavorable side effects on laying performance. The results of the present study suggest that suppressed fatty acid synthase activity is involved in the reduced hepatic lipid accumulation as an effect of BAs dietary supplementation.

Efficacy and safety of ceftazidime-avibactam versus polymyxins in the treatment of carbapenem-resistant Enterobacteriaceae infection: a systematic review and meta-analysis.

OBJECTIVES: Carbapenem-resistant Enterobacteriaceae is increasingly recognised as a significant public health concern. Ceftazidime-avibactam (CAZ-AVI) and polymyxins are considered as the last therapeutic options worldwide. This is the first meta-analysis of recently published data to compare the clinical efficacy and safety of CAZ-AVI with polymyxins in the treatment of carbapenem-resistant Enterobacteriaceae infections. DESIGN: Systematic review and meta-analysis. DATA SOURCES: PubMed, Embase and the Cochrane Library were systematically searched, for publications in any language, from database inception to February 2023. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: Studies comparing the clinical efficacy and safety of CAZ-AVI with polymyxins were included. Mortality, clinical success, microbiological eradication and nephrotoxicity were assessed as the main outcomes. DATA EXTRACTION AND SYNTHESIS: Literature screening, data extraction and the quality evaluation of studies were conducted by two researchers independently, with disagreements resolved by another researcher. The Newcastle-Ottawa Scale was used to assess the bias risk for the included studies. Review Manager V.5.3 was employed for the meta-analysis. RESULTS: The meta-analysis included seven retrospective and four prospective cohort studies with 1111 patients enrolled. The CAZ-AVI groups demonstrated a lower 30-day mortality (risk ratio (RR)=0.48, 95% CI of 0.37 to 0.63, I2=10%, p<0.0001) in nine studies with 766 patients; higher clinical success (RR=1.71, 95% CI 1.33 to 2.20, I2=35%, p<0.0001) in four studies with 463 patients; and lower nephrotoxicity in seven studies with 696 patients (RR=0.42, 95% CI 0.23 to 0.77, I2=35%, p<0.05). However, no significant difference in microbiological eradication rates was observed in 249 patients from two studies (RR=1.16, 95% CI 0.97 to 1.39, I2=0, p>0.05). CONCLUSION: Available evidence suggested that CAZ-AVI treatment held a dominant position with respect to efficacy and safety compared with polymyxins in carbapenem-resistant Enterobacteriaceae infections. However, the analysis included only observational studies, and high-quality, large-scale, multicentre, double-blind randomised controlled trials are needed to confirm the advantage of CAZ-AVI.

Efficacy of Repeated Low-Level Red-Light Therapy for Slowing the Progression of Childhood Myopia: A Systematic Review and Meta-analysis.

PURPOSE: To evaluate the long-term efficacy and safety of repeated low-intensity red light (RLRL) treatment for childhood myopia. DESIGN: Systematic review and meta-analysis METHODS: We searched PubMed, Web of Science, CNKI, and Wanfang from inception to February 8, 2023. We used the RoB 2.0 and ROBINS-I tools to assess the risk of bias and then used a random-effect model to calculate the weighted mean difference (WMD) and 95% CIs. The primary outcomes were WMD in spherical equivalent refractive error (SER), WMD in axial length (AL), and WMD in subfoveal choroid thickness (SFChT). Subgroup analyses were performed to investigate the sources of heterogeneity based on variation in follow-up and study design. The Egger and Begg tests were used to assess publication bias. Sensitivity analysis was used to verify the stability. RESULTS: This analysis included 13 studies (8 randomized controlled trials, 3 non-randomized controlled trials, and 2 cohort studies) involving 1857 children and adolescents. Eight studies met the meta-analysis criteria, and the WMD for myopia progression between RLRL and the control group was 0.68 diopters (D) per 6 months (95% CI = 0.38 to 0.97 D; I2 = 97.7%; P < .001) for SER change; -0.35 mm per 6 months (95% CI = -0.51 to -0.19 mm; I2 = 98.0%; P < .001) for AL elongation; and 36.04 µm per 6 months (95% CI = 19.61 to 52.48 µm; I2 = 89.6%; P < .001) for SFChT change. CONCLUSIONS: Our meta-analysis shows that RLRL therapy may be effective for delaying the progression of myopia. The evidence is low certainty, and larger and better randomized clinical trials with 2-year follow-ups are needed to improve the existing state of knowledge to inform medical guidelines more comprehensively.

Effects of arginine replacement with L-citrulline on the arginine/nitric oxide metabolism in chickens: An animal model without urea cycle.

BACKGROUND: This study examined the efficacy of L-citrulline supplementation on the arginine/nitric oxide metabolism, and intestinal functions of broilers during arginine deficiency. A total of 288 day-old Arbor Acre broilers were randomly assigned to either an arginine deficient basal diet (NC diet), NC diet + 0.50% L-arginine (PC diet), or NC diet + 0.50% L-citrulline (NCL diet). Production performance was recorded, and at 21 days old, chickens were euthanized for tissue collection. RESULTS: The dietary treatments did not affect the growth performance of broilers (P > 0.05), although NC diet increased the plasma alanine aminotransferase, urate, and several amino acids, except arginine (P < 0.05). In contrast, NCL diet elevated the arginine and ornithine concentration higher than NC diet, and it increased the plasma citrulline greater than the PC diet (P < 0.05). The nitric oxide concentration in the kidney and liver tissues, along with the plasma and liver eNOS activities were promoted by NCL diet higher than PC diet (P < 0.05). In the liver, the activities of arginase 1, ASS, and ASL, as well as, the gene expression of iNOS and OTC were induced by PC diet greater than NC diet (P < 0.05). In the kidney, the arginase 1, ASS and ASL enzymes were also increased by PC diet significantly higher than the NC and NCL diets. Comparatively, the kidney had higher abundance of nNOS, ASS, ARG2, and OTC genes than the liver tissue (P < 0.05). In addition, NCL diet upregulated (P < 0.05) the mRNA expression of intestinal nutrient transporters (EAAT3 and PEPT1), tight junction proteins (Claudin 1 and Occludin), and intestinal mucosal defense (MUC2 and pIgR). The intestinal morphology revealed that both PC and NCL diets improved (P < 0.05) the ileal VH/CD ratio and the jejunal VH and VH/CD ratio compared to the NC fed broilers. CONCLUSION: This study revealed that NCL diet supported arginine metabolism, nitric oxide synthesis, and promoted the intestinal function of broilers. Thus, L-citrulline may serve as a partial arginine replacement in broiler's diet without detrimental impacts on the performance, arginine metabolism and gut health of chickens.

Effect of substitution of taurine for methionine and additional taurine supplementation on the performance and antioxidative capacity of laying hens.

Taurine (TAU), a sulfur-containing amino acid that synthesized from methionine and cystine, plays vital roles in maintenance of redox balance. The effect of substitution of TAU for methionine was evaluated in vivo and in vitro. The effects of replacing methionine with TAU and additional TAU supplementation on the performance and antioxidant capacity of laying hens were evaluated. The in vitro cultured chicken primary hepatocytes and intestinal epithelial cells were further employed. Two hubdred eighty-eight 40-wk-old Isa brown laying hens were divided into 4 groups and subjected one to the following treatments: fed with basal diet with 0.17% crystallized DL-Met (CON), the control diet and replace 25% (21% total Met, 21TAU) or 50% (42% total Met, 42TAU) of crystallized DL-Met with taurine, the control diet supplemented with 0.1% taurine (0.1% TAU). The laying rate, feed intake, egg weight, and feed efficiency were not influenced (P > 0.05) by TAU replacement or additional TAU supplementation. In the liver, 0.1% TAU decreased SOD but increased GSH-Px activity (P < 0.01). In duodenum, 42TAU decreased SOD activity (P < 0.05) while 0.1% TAU decreased GSH level and SOD activity (P < 0.05). In the hepatocytes, TAU treatment decreased (P < 0.05) the MDA and GSH contents, whereas increased SOD and GSH-Px activities (P < 0.05). Meanwhile, TAU treatment decreased (P < 0.05) the protein expression of Nrf2 while increase Keap1 expression. The mRNA expression of Nrf2, SOD1, SOD2, CAT, and GCLC were increased (P < 0.05) and GSR were decreased (P < 0.05) by 0.1% TAU. In the intestinal epithelial cells, TAU treatment decreased (P < 0.05) SOD activity, increased (P < 0.05) CAT activity, and decreased (P < 0.05) the mRNA and protein expression of Nrf2. In summary, partial substitution methionine for taurine (21-42%) has no influence on egg performance of hens. Taurine enhances the antioxidative capacity in hepatocyte but not in the enterocytes and if taurine could offer an improved effect on antioxidant capacity needs to be verified under oxidative stress-challenged conditions.

Dynamic change of oligopeptides and free amino acids composition in five types of tea with different fermentation degree processed from the same batch of fresh tea (Camelilia Sinensis. L.) leaves.

Our previous studies have confirmed oligopeptides could be meaningful to tea taste and biofunction. As the total content of oligopeptide among different tea types were varied. It is a natural speculation that certain tea processing step could be vital for oligopeptides enrichment. In current study, five types of traditional Chinese tea were produced from the same batch of fresh leaves. Step processing samples were acquired through which to profile the oligopeptides and free amio acids composition change during tea processed. As a result, firstly, withering was the vital step for oligopeptide enrichment, followed by fermentation, yellowing and drying. Secondly, as the enrichment of oligopeptides was often accompanied by the increase of protein amino acids, suggesting certain proteins degradation in fresh leaves could be the main source of oligopeptides. Thirdly, a total of 166 abundant oligopeptides were screened out, through which 14 high degradation protein were also located by protomic approaching.

Low-intensity red-light therapy in slowing myopic progression and the rebound effect after its cessation in Chinese children: a randomized controlled trial.

PURPOSE: To investigate the effect of low-intensity red-light (LRL) therapy on myopic control and the response after its cessation. METHODS: A prospective clinical trial. One hundred two children aged 6 to 13 with myopia were included in the LRL group (n = 51) and the single-focus spectacles (SFS) group (n = 51). In LRL group, subjects wore SFS and received LRL therapy provided by a laser device that emitted red-light of 635 nm and power of 0.35 ± 0.02 mW. One year after the control trial, LRL therapy was stopped for 3 months. The outcomes mainly included axial length (AL), spherical equivalent refraction (SER), subfoveal choroidal thickness (SFCT), and accommodative function. RESULTS: After 12 months of therapy, 46 children in the LRL group and 40 children in the SFS group completed the trial. AL elongation and myopic progression were 0.01 mm (95%CI: - 0.05 to 0.07 mm) and 0.05 D (95%CI: - 0 .08 to 0.19 D) in the LRL group, which were less than 0.39 mm (95%CI: 0.33 to 0.45 mm) and - 0.64 D (95%CI: - 0.78 to - 0.51 D) in the SFS group (p < 0.05). The change of SFCT in the LRL group was greater than that in the SFS group (p < 0.05). Accommodative response and positive relative accommodation in the LRL group were more negative than those in the SFS group (p < 0.05). Forty-two subjects completed the observation of LRL cessation, AL and SER increased by 0.16 mm (95%CI: 0.11 to 0.22 mm) and - 0.20 D (95%CI: - 0.26 to - 0.14 D) during the cessation (p < 0.05), and SFCT returned to baseline (p > 0.05). CONCLUSIONS: LRL is an effective measure for preventing and controlling myopia, and it may also have the ability to improve the accommodative function. There may be a slight myopic rebound after its cessation. The effect of long-term LRL therapy needs to be further explored. TRIAL REGISTRATION: Chinese Clinical Trial Registry: Chinese Clinical Trails registry: ChiCTR2100045250. Registered 9 April 2021; retrospectively registered. http://www.chictr.org.cn/showproj.aspx?proj=124250.

L-Arginine/nitric oxide regulates skeletal muscle development via muscle fibre-specific nitric oxide/mTOR pathway in chickens.

L-Arginine (L-Arg), the precursor of nitric oxide (NO), plays an important role in muscle function. Fast-twitch glycolytic fibres are more susceptible to age-related atrophy than slow-twitch oxidative fibres. The effect of L-Arg/NO on protein metabolism of fast- and slow-twitch muscle fibres was evaluated in chickens. In Exp. 1, 48 chicks at 1 day old were divided into 4 groups of 12 birds and subjected to 4 treatments: basal diet without supplementation or supplemented with 1% L-Arg, and water supplemented with or without L-nitro-arginine methyl ester (L-NAME, 18.5 mM). In Exp. 2, 48 chicks were divided into 4 groups of 12 birds fed with the basal diet and subjected to the following treatments: tap water (control), tap water supplemented with L-NAME (18.5 mM), or molsidomine (MS, 0.1 mM), or 18.5 mM L-NAME + 0.1 mM MS (NAMS). The regulatory effect of L-Arg/NO was further investigated in vitro with myoblasts obtained from chicken embryo pectoralis major (PM) and biceps femoris (BF). In vivo, dietary L-Arg supplementation increased breast (+14.94%, P < 0.05) and thigh muscle mass (+23.40%, P < 0.05); whereas, MS treatment had no detectable influence. However, L-NAME treatment blocked the beneficial influence of L-Arg on muscle development. L-Arg decreased (P < 0.05) protein synthesis rate, phosphorylated mTOR and ribosomal protein S6 kinase beta-1 (p70S6K) levels in breast muscle, which was recovered by L-NAME treatment. In vitro, L-Arg or sodium nitroprusside (SNP) reduced protein synthesis rate, suppressed phosphorylated mTOR/p70S6K and decreased atrogin-1 and muscle RING finger 1 (MuRF1) in myoblasts from PM muscle (P < 0.05). L-NAME abolished the inhibitory effect of L-Arg on protein synthesis and the mTOR/p70S6K pathway. However, myoblasts from BF muscle showed the weak influence. Moreover, blocking the mTOR/p70S6K pathway with rapamycin suppressed protein synthesis of the 2 types of myoblasts; whereas, the protein expression of atrogin-1 and MuRF1 levels were restricted only in myoblasts from PM muscle. In conclusion, L-Arg/NO/mTOR/p70S6K pathway enhances protein accumulation and muscle development in fast-twitch glycolytic muscle in chickens. L-Arg/NO regulates protein turnover in a muscle fibre specific way, which highlights the potential clinical application in fast-twitch glycolytic muscle fibres.

Effects and microbial mechanisms of phosphogypsum and medical stone on organic matter degradation and methane emissions during swine manure composting.

The degradation of organic matter (OM) and CH4 emissions during composting greatly influence the composting efficiency and greenhouse effect. This study evaluated the effects of adding phosphogypsum (PPG) and medical stone (MS) on OM breakdown, CH4 emissions, and their underlying mechanisms. MS accelerated the breakdown of OM in the early composting stage, whereas PPG increased it in the cooling and maturation periods. At the ending of composting, humification was also significantly promoted by PPG and MS (P < 0.05). Moreover, MS and PPG reduced CH4 emissions by 27.64% and 23.12%, respectively, and significantly inhibited the activities of methanogens in terms of their abundance (mcrA) and composition (dominant genera such as Methanobrevibacter, Methanocorpusculum, and Methanothermus) (P < 0.05). Interestingly, MS enhanced the activity of enzymes and bacterial metabolism related to OM degradation in the early composting stage, whereas PPG promoted them during the cooling and maturity stages. MS and PPG inhibited the activities of enzymes related to CH4 release during the cooling and maturity stages. Therefore, PPG and MS may have influenced OM degradation and CH4 releases during composting via changes in bacterial metabolism and enzyme activity levels. PPG and MS could have altered the activities of methanogens to influence the transformation of carbon and CH4 emissions according to network analysis and partial least-squares path modeling analysis. These findings provide insights at the molecular level into the effects of adding PPG and MS on OM degradation and CH4 emissions during composting, thereby facilitating the application of PPG and MS in composting systems.

Self-assembled DNA origami-based duplexed aptasensors combined with centrifugal filters for efficient and rechargeable ATP detection.

DNA origami technology has great potential for biosensor applications. Here, we described the construction of a self-assembled DNA origami biosensor for the precise localization of fluorescent aptamers. Due to the molecular weight difference between DNA origami and aptamer, centrifugal filters were used to quantitatively detect adenosine triphosphate (ATP). The ATP-specific aptamer labeled with fluorescence reporter 6-carboxyfluorescein FAM (FAM-aptamer) was selected as the recognition element and signal probe. ATP duplexed aptamers bound to triangular DNA origami by base-complementary pairing, resulting in high fluorescence signals on the origami arrays. The competitive binding of ATP toward the FAM-aptamer triggered the release of FAM-aptamer-ATP complexes from the surface of the origami array, resulting in weakened fluorescence signals. For ATP quantification, 100 kD centrifugal filters were employed, followed by measurement of the fluorescence signal trapped on the origami arrays of the filter device. The successful synthesis of origami-aptamer arrays was characterized by atomic force microscopy, laser confocal microscopy, and electrophoresis. Fluorescence measurements exhibited an excellent linear relationship with logarithms of ATP concentrations within 0.1-100 ng mL-1, with a detection limit of 0.29 ng mL-1. By replacing aptamers and complementary strands, we demonstrated the potential of this method for 17ß-estradiol detection. Considering that the detection mechanism is based on the hybridization and displacement of DNA strands, the detection system had the potential for recharging. Our study provides new insights into applying DNA origami technology in small molecule detection.

Estrogen valerate pretreatment with the antagonist protocol does not increase oocyte retrieval in patients with low ovarian response: a randomized controlled trial.

STUDY QUESTION: Does luteal phase estrogen valerate pretreatment improve oocyte yield and clinical outcomes in patients with low ovarian response during ovarian stimulation with the antagonist protocol? SUMMARY ANSWER: Pretreatment with oral estrogen valerate from Day 7 after ovulation to Day 2 of the next menstrual cycle did not increase oocyte yield in patients with a low ovarian response compared to no pretreatment. WHAT IS KNOWN ALREADY: Previous studies showed that patients with a normal ovarian response can obtain better clinical outcomes after pretreatment with estrogen in the antagonist protocol. For patients with advanced age and low ovarian response, it remains unclear if estrogen valerate pretreatment with the antagonist protocol yields more oocytes and improves pregnancy outcomes. STUDY DESIGN, SIZE, DURATION: This non-blinded randomized controlled trial (RCT) was conducted between November 2017 and March 2021. Participants were 552 women with low response who requested IVF treatment. The primary endpoint was comparison of the total number of retrieved oocytes between the two groups. The secondary endpoints were the total number of retrieved metaphase II (MII) oocytes, duration and total dosage of recombinant FSH (rFSH), good-quality embryo rate and clinical pregnancy rate. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted at a reproductive center. The RCT enrolled 552 infertile women with a low ovarian response (according to the Bologna criteria) who were undergoing IVF. In the study group, on Day 7 after ovulation patients were administered oral estrogen valerate (2 mg twice a day) until Day 2 of their next menstruation. Ovary stimulation was performed using rFSH, and a GnRH antagonist (0.25 mg/day) was started when a dominant follicle had a mean diameter ≥13 mm. MAIN RESULTS AND THE ROLE OF CHANCE: No significant difference was observed in the number (mean [SD]) of oocytes retrieved from the estrogen valerate pretreatment and control group (3.2 [2.8] versus 3.4 [2.6], respectively). The treatment difference was -0.18 (95% CI -0.67, 0.32, P = 0.49). No significant differences were observed in the number of MII oocytes (2.9 [2.5] versus 3.1 [2.4], mean difference -0.23, 95% CI (-0.69, 0.23), P = 0.16) and good-quality embryos (1.0 [1.3] versus 1.20 [1.6], mean difference -0.23, 95% CI (-0.50, 0.04), P = 0.19) between the two groups. The duration of rFSH treatment was significantly longer in the estrogen valerate pretreatment group than in the control group (10.3 [2.2] versus 8.6 [2.1] days, mean difference 1.7, 95% CI (1.3, 2.2), P = 0.00), and the total rFSH dosage was significantly higher in the estrogen valerate pretreatment group than in the control group (3081 [680] versus 2548 [649] IU, mean difference 553.7, 95% CI (405.8, 661.6), P = 0.00). The clinical pregnancy rate in the pretreatment group (19.3% [23/119]) was not significantly different from that in the control group (28.7% [43/150]). The mean difference was -0.09, 95% CI (-0.20, 0.01), P = 0.08. LIMITATIONS, REASONS FOR CAUTION: The major limitation was the high dropout rate of patients. Some patients did not return to the hospital for treatment because of predicted low success rates and for economic reasons. In addition, it is possible that the fixed dose of 300 IU rFSH was not sufficient to see differences in oocyte yield between the groups. WIDER IMPLICATIONS OF THE FINDINGS: Estrogen valerate pretreatment with an antagonist protocol did not increase oocyte yield in patients with low ovarian response. Similar to the number of retrieved oocytes, there was no significant difference in clinical pregnancy rate between estrogen pretreatment group and control group. More research is needed on whether patients with low ovarian response need pretreatment and which pretreatment is more appropriate. STUDY FUNDING/COMPETING INTEREST(S): This study was supported in part by a research grant from the Investigator-Initiated Studies Program of MSD (China) Holding Co., Ltd. and Organon (Shanghai) Pharmaceutical Technology Co., Ltd. (Grant number: IIS 56284). The authors declare that they have no competing interests regarding authorship or publication of this study. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT03300518. TRIAL REGISTRATION DATE: 28 September 2017. DATE OF FIRST PATIENT'S ENROLMENT: 15 November 2017.

Effects of dietary L-citrulline supplementation on nitric oxide synthesis, immune responses and mitochondrial energetics of broilers during heat stress.

L-Citrulline is a non-protein amino acid that promotes arginine recycling and muscle protein synthesis. This study investigated whether L-citrulline can exert these functions in heat-stressed chickens. Arbor acre broilers were fed either basal diets (Control) or basal diets supplemented with 1% L-citrulline (L-Cit). At 28 d old, broilers were subjected to two environmental temperatures, 35 °C for 8 h/d (HS) or 24 °C for 24 h/d (TNZ) for 2 weeks. The experiment was designed as a 2 by 2 factorial arrangement. Results showed that HS increased the core body temperature (CBT) and rectal temperature of broilers compared to the TNZ condition. The CBT and mean CBT decreased by ∼0.5oC in TNZ + L-Cit broilers compared to the TNZ + Control group (P < 0.05). L-Cit supplementation at TNZ significantly (P < 0.05) lowered the plasma malondialdehyde content but this was increased during HS. Exposure to HS significantly (P < 0.05) elevated plasma malondialdehyde compared to TNZ condition. Plasma immunoglobulin A, G, and M were increased (P < 0.05) by TNZ + L-Cit compared to the TNZ + Control group. Plasma nitric oxide and endothelial nitric oxide synthase (NOS) activity was increased during HS, whereas the total and inducible NOS enzymes were decreased. In addition, L-Cit supplementation increased both the inducible and endothelial NOS isoforms (P < 0.05). The mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-α, avian uncoupling protein, cytochrome C oxidase subunit 3, and ATP synthase F1 subunit beta (ATP5ß) in the breast muscle were downregulated (P < 0.05) during HS. However, L-Cit supplementation upregulated the mitochondrial transcription factor A and during HS, L-Cit increased ATP5ß expression similar to TNZ housed broilers. Therefore, this study demonstrates that dietary L-Cit can lower the body temperature, decrease lipid peroxidation and promote the immune status of broilers under thermoneutrality. Also, L-Cit would act to promote muscle ATP generation during heat stress in broilers.

Research Note: Effects of dietary L-arginine on the production performance and gene expression of reproductive hormones in laying hens fed low crude protein diets.

The present study was designed to investigate whether L-arginine (Arg) supplementation would influence the production performance and reproductive traits of laying hens fed low crude protein (LCP) diets. Hy-Line brown laying hens were randomly assigned to dietary treatments of control, LCP, and LCP supplemented with 0.05, 0.10, 0.15, or 0.20% Arg for 7 wk. The results showed no significant variations on the production performance, and relative organ indexes of hens. However, significant transcriptional and structural changes were evident along the HPG axis of hens. Arg supplementation linearly upregulated (P < 0.05) the gonadotropin-releasing hormone 1 (GnRH1), and gonadotropin inhibitory hormone in the hypothalamus. The pituitary growth hormone, GnRH receptor, and follicle-stimulating hormone (FSHß) were also increased (P < 0.05). In the ovary, GnRH1, and estrogen receptor ß were linearly increased by Arg, and the ovarian morphology revealed that LCP induced structural alterations which were minimally recovered by Arg supplementation. Serum insulin-like growth factor 1 (IGF-1) and nitric oxide (NO) were increased (P < 0.05) at higher levels of Arg supplementation. Therefore, supplementing high Arg (0.20%) to LCP hens influenced the ovarian morphology and modulated the gene expression of reproductive hormones in the hypothalamic-pituitary-gonadal axis of laying hens via actions that may be related to NO and IGF-1 activity.

Functional roles of taurine, L-theanine, L-citrulline, and betaine during heat stress in poultry.

Heat stress (HS) is an important environmental stress factor affecting poultry production on a global scale. With the rise in ambient temperature and increasing effects of global warming, it becomes pertinent to understand the effects of HS on poultry production and the strategies that can be adopted to mitigate its detrimental impacts on the performance, health, welfare, immunity, and survival of birds. Amino acids (AAs) have been increasingly adopted as nutritional modifiers in animals to ameliorate the adverse effects of HS. They are essential for protein synthesis, growth, maintenance, reproduction, immunity, stress response, and whole-body homeostasis. However, HS tends to adversely affect the availability, transport, absorption, and utilization of these AAs. Studies have investigated the provision of these AAs to poultry during HS conditions, and variable findings have been reported. Taurine, L-theanine, and L-citrulline are non-essential amino acids that are increasingly gaining attention as nutritional supplements in HS animals. Similarly, betaine is an amino acid derivative that possesses favorable biological properties which contributes to its role as a functional additive during HS. Of particular note, taurine is negligible in plants, while betaine, L-theanine, and L-citrulline can be found in selected plants. These nutrients are barely found in feed ingredients, but their supply has been shown to elicit important physiological roles including anti-stress effects, anti-oxidative, anti-inflammatory, gut promoting, and immunomodulatory functions. The present review provides information on the use of these nutritionally and physiologically beneficial nutrients as functional additives to poultry diets during HS conditions. Presently, although several studies have reported on the positive effects of these additives in human and murine studies, however, there is limited information regarding their utilization during heat stress in poultry nutrition. Therefore, this review aims to expound on the functional properties of these nutrients, their potentials for HS alleviation, and to stimulate further researches on their biological roles in poultry nutrition.

Stimulation of both inspiratory and expiratory muscles versus diaphragm-only paradigm for rehabilitation in severe chronic obstructive pulmonary disease patients: a randomized controlled pilot study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD), a progressive lung disease, might improve with neuromuscular electrical stimulation. No trials on inspiratory plus expiratory neuromuscular electrical stimulation have been conducted yet. AIM: The aim of this study was to evaluate the safety and effectiveness of inspiratory plus expiratory neuromuscular electrical stimulation in subjects with severe COPD. DESIGN: This was a multicenter, prospective, randomized controlled trial. SETTING: The subjects were outpatients enrolled from Beijing Chao-Yang Hospital affiliated with Capital Medical University, Tianjin Chest Hospital, and the First Hospital of Hebei Medical University. POPULATION: Subjects had stable COPD with severe respiratory impairment. METHODS: Using a computer statistical software, 120 stable subjects were randomly allocated (1:1) to receive inspiratory plus expiratory neuromuscular electrical stimulation (study group) and diaphragm pacing (control group). Demographic and clinical data were collected before, and after 2, and 4 weeks of the trial. The intention-to-treat analysis was conducted. The primary outcome was to analyze the changes in functional exercise capacity, estimated as six-minute walk distance (6MWD), following electrical stimulation for 4 weeks. The secondary outcomes were changes in modified Medical Research Council score, forced expiratory volume in 1 second (FEV1), FEV1% predicted, and FEV1 ratio forced vital capacity (FEV1/FVC) following electrical stimulation for 4 weeks. RESULTS: The change in 6MWD was greater in the study group (65.53±39.45 m) than in the control group (26.66±32.65 m). The mean between-group difference at the fourth week was 29.07 m (95% confidence interval, 16.098-42.035; P<0.001). There were no significant between-group differences in the secondary outcomes after 4 weeks of electrical stimulation. For GOLD-4 COPD subjects, FEV1 and FEV1/FVC improved in the study group (P<0.05). No electrical stimulation-related serious adverse events were observed in either group. CONCLUSIONS: 6MWD were increased significantly, without adverse events, after four weeks of treatment of inspiratory plus expiratory neuromuscular electrical stimulation in stable patients with severe COPD, suggesting that this protocol benefits COPD rehabilitation. CLINICAL REHABILITATION IMPACT: The results of this study suggest that the simultaneous use of inspiratory plus expiratory neuromuscular electrical stimulation as an adjunct therapy may improve the functional exercise capacity of severe stable COPD subjects.

Dietary Supplementation of Calcium Propionate and Calcium Butyrate Improves Eggshell Quality of Laying Hens in the Late Phase of Production.

The aim of this study was to evaluate the effects of dietary supplementation of calcium propionate and calcium butyrate on the laying performance, eggshell quality, and expression of genes related to calcium and phosphorus metabolism in the tibia. One hundred and twenty 70-week-old Isa Brown hens were randomly assigned to three treatments, and each treatment had four replicates of 10 birds fed a basal diet (control) or a basal diet supplemented with 0.5% calcium propionate (CP) or 0.5% calcium butyrate (CB) for 8 weeks. The CB and CP treatments had no significant effect (P>0.05) on the laying rate, egg production, egg weight, and feed efficiency. The eggshell percentage was increased from week 2 (P<0.05) and eggshell thickness was elevated at week 8 (P<0.01) by both CP and CB treatments. Compared to the control treatment, the CB treatment increased serum calcium and phosphorus levels at week 4 (P<0.05), whereas the CP and CB treatments decreased serum phosphorus at weeks 6 and 8, respectively (P<0.05). Dietary supplementation had no effect on the bone index and bending strength of the tibia (P>0.05). The calcium and phosphorus content of the tibia was decreased by the CB treatment (P<0.05). In the spleen, NF-κB and IL-6 transcript levels were not influenced (P>0.05) but TNF-α transcript levels were decreased by the CP treatment (P<0.05). In the tibia, the expression levels of NF-κB, TNF-α, and IL-17 were not affected by the CP or CB treatment (P>0.05). The CP and CB treatments had no significant effect on the transcript levels of RANKL, OPG, RNUX2, OPN, α-Clotho, and VDR (P>0.05). In contrast, PHEX transcript levels were increased by the CP treatment (P<0.05). The expression levels of osteocalcin (P=0.094) and FGF23 (P=0.087) tended to decrease under the CB treatment. In conclusion, dietary supplementation of 0.5% calcium butyrate or 0.5% calcium propionate improved the eggshell quality of aged laying hens, possibly as a result of decreased deposition or enhanced mobilization of bone calcium and phosphorus.

The temporal gene expression profiles of calcium and phosphorus transporters in Hy-Line Brown layers.

Calcium and phosphorus homeostasis is crucial for the performance and bone health of laying hens. The calcium and phosphorus transporters play an important role in calcium and phosphorus absorption, reabsorption, and excretion. In the present study, Hy-Line Brown layers were sampled at brooding period (1, 4, 6 wk), growing and developing period (12, 18 wk) and laying period (20, 28, 80 wk) respectively, and the calcium transporters CaBP-D28k and PMCA1b and phosphorus transporters NPt2a and NPt2b were respectively measured in duodenum, jejunum, ileum and kidney. The result showed that serum calcium increased (P < 0.0001) and phosphorus level fluctuated (P = 0.0019), while alkaline phosphatase activity decreased with age (P < 0.0001). The mRNA and protein expressions of CaBP-D28k in small intestine elevated after maturity (P ≤ 0.0001). In contrast, the PMCA1b mRNA showed a trend to increase with age in jejunum (P = 0.0059) and ileum (P = 0.0825) whereas there was a decrease for PMCA1b protein in 12-18 wk (P ≤ 0.0009). The peak of NPt2b mRNA were observed at 28 wk in duodenum (P = 0.0001) and jejunum (P = 0.0622) and 1 wk in ileum (P < 0.0001). The NPt2b protein expression reached the top point at 4 or 6 wk and 20 wk and decreased to the lowest point around 12 wk (P ≤ 0.0850). In kidney, CaBP-D28k mRNA was not influenced by age (P = 0.4999), while PMCA1b highly expressed in 6-12 wk (P = 0.0003). The protein expressions of CaBP-D28k (P = 0.0148) and PMCA1b (P = 0.0003) decreased with age and lowly expressed in 12-18 wk and increased thereafter. In contrast, NPt2a expression increased steadily with age and decreased at 80 wk (P < 0.0001). In conclusion, the expressions of intestinal calcium and phosphorus transporters were changed by age, intestinal CaBP-D28k and renal NPt2a showed a dramatic increase after maturity, which coincide with the increased calcium and phosphorus requirement for egg production.