Ting Yang

In plants, K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) is the largest potassium (K) transporter family; however, few of the members have had their physiological functions characterized in planta. Here, we studied OsHAK5 of the KT/HAK/KUP family in rice (Oryza sativa). We determined its cellular and tissue localization and analyzed its functions in rice using both OsHAK5 knockout mutants and overexpression lines in three genetic backgrounds. A β-glucuronidase reporter driven by the OsHAK5 native promoter indicated OsHAK5 expression in various tissue organs from root to seed, abundantly in root epidermis and stele, the vascular tissues, and mesophyll cells. Net K influx rate in roots and K transport from roots to aerial parts were severely impaired by OsHAK5 knockout but increased by OsHAK5 overexpression in 0.1 and 0.3 mm K external solution. The contribution of OsHAK5 to K mobilization within the rice plant was confirmed further by the change of K concentration in the xylem sap and K distribution in the transgenic lines when K was removed completely from the external solution. Overexpression of OsHAK5 increased the K-sodium concentration ratio in the shoots and salt stress tolerance (shoot growth), while knockout of OsHAK5 decreased the K-sodium concentration ratio in the shoots, resulting in sensitivity to salt stress. Taken together, these results demonstrate that OsHAK5 plays a major role in K acquisition by roots faced with low external K and in K upward transport from roots to shoots in K-deficient rice plants.

Potassium (K) absorption and translocation in plants rely upon multiple K transporters for adapting varied K supply and saline conditions. Here, we report the expression patterns and physiological roles of OsHAK1, a member belonging to the KT/KUP/HAK gene family in rice (Oryza sativa L.). The expression of OsHAK1 is up-regulated by K deficiency or salt stress in various tissues, particularly in the root and shoot apical meristem, the epidermises and steles of root, and vascular bundles of shoot. Both oshak1 knockout mutants in comparison to their respective Dongjin or Manan wild types showed a dramatic reduction in K concentration and stunted root and shoot growth. Knockout of OsHAK1 reduced the K absorption rate of unit root surface area by ∼50-55 and ∼30%, and total K uptake by ∼80 and ∼65% at 0.05-0.1 and 1 mm K supply level, respectively. The root net high-affinity K uptake of oshak1 mutants was sensitive to salt stress but not to ammonium supply. Overexpression of OsHAK1 in rice increased K uptake and K/Na ratio. The positive relationship between K concentration and shoot biomass in the mutants suggests that OsHAK1 plays an essential role in K-mediated rice growth and salt tolerance over low and high K concentration ranges.

Some previous studies have identified bacteria in semen as being a potential factor in male infertility. However, only few types of bacteria were taken into consideration while using PCR-based or culturing methods. Here we present an analysis approach using next-generation sequencing technology and bioinformatics analysis to investigate the associations between bacterial communities and semen quality. Ninety-six semen samples collected were examined for bacterial communities, measuring seven clinical criteria for semen quality (semen volume, sperm concentration, motility, Kruger's strict morphology, antisperm antibody (IgA), Atypical, and leukocytes). Computer-assisted semen analysis (CASA) was also performed. Results showed that the most abundant genera among all samples were Lactobacillus (19.9%), Pseudomonas (9.85%), Prevotella (8.51%) and Gardnerella (4.21%). The proportion of Lactobacillus and Gardnerella was significantly higher in the normal samples, while that of Prevotella was significantly higher in the low quality samples. Unsupervised clustering analysis demonstrated that the seminal bacterial communities were clustered into three main groups: Lactobacillus, Pseudomonas, and Prevotella predominant group. Remarkably, most normal samples (80.6%) were clustered in Lactobacillus predominant group. The analysis results showed seminal bacteria community types were highly associated with semen health. Lactobacillus might not only be a potential probiotic for semen quality maintenance, but also might be helpful in countering the negative influence of Prevotella and Pseudomonas. In this study, we investigated whole seminal bacterial communities and provided the most comprehensive analysis of the association between bacterial community and semen quality. The study significantly contributes to the current understanding of the etiology of male fertility.

Chronic stress is a key risk factor for depression, and microglia have been implicated in the pathogenesis of the disease. Recent studies show that the Nod-like receptor protein 3 (NLRP3) inflammasome is expressed in microglia and may play a crucial role in depression. However, the mechanism of NLRP3 inflammasome activation in hippocampal microglia and its role in depressive-like behaviors remain poorly understood. In this study, rats were subjected to 6 h of restraint stress per day for 21 days to produce a model of stress-induced depression. Behavioral tests and serum corticosterone were used to assess the success of the model. Furthermore, HAPI cells were pretreated with dexamethasone (5 × 10-7 M) to assess stress-induced changes in microglial cells in culture. The microglial marker Iba-1, reactive oxygen species (ROS), nuclear factor kappa B (NF-κB) and key components of the NLRP3 inflammasome and its downstream inflammatory effectors (IL-1β and IL-18) were measured. Chronic stress induced depressive-like behavior, increased serum corticosterone levels and produced hippocampal structural changes. Chronic stress and dexamethasone both increased Iba-1 expression and ROS formation and also elevated levels of NF-κB, NLRP3, cleaved caspase-1, IL-1β and IL-18. After use of the NF-κB inhibitor BAY 117082 and knocked out NLRP3 in vitro decreased ROS formation and the expression of Iba-1, NF-κB and NLRP3 as well as levels of cleaved caspase-1, IL-1β and IL-18. These findings suggest that activation of the glucocorticoid receptor-NF-κB-NLRP3 pathway in hippocampal microglia mediates chronic stress-induced hippocampal neuroinflammation and depression-like behavior.

Relying on exclusionary confounder terms to define non-alcoholic fatty liver disease (NAFLD) is a limitation of the term. Recent consensus has advocated for diagnosis to be based on affirmative criteria rather than exclusionary standards, emphasizing the strong epidemiological and pathogenic link between NAFLD, metabolic dysfunction and insulin resistance.1 However, in predicting insulin resistance, high-density lipoprotein cholesterol (HDL-C) and diastolic blood pressure are less effective compared to elevated blood glucose, hypertriglyceridemia, and overweight or obesity.

Vitamin A (VA) plays an essential role in modulating both the gut microbiota and gut barrier function. Short-chain fatty acids (SCFAs), as metabolites of the gut microbiota, protect the physiological intestinal barrier; however, they are compromised when VA is deficient. Thus, there is an urgent need to understand how and which SCFAs modulate colonic epithelial barrier integrity in VA deficiency (VAD). Herein, compared with normal VA rats (VAN), at the beginning of pregnancy, we confirmed that the colonic desmosome junction was impaired in the VAD group, and the amounts of acetate, propionate, and butyrate declined because of the decreased abundance of SCFA-producing bacteria (Romboutsia, Collinsella, and Allobaculum). The differentially expressed genes correlated with the gut barrier and the histone deacetylase complex between the VAD and VAN groups were enriched by RNA sequencing. In the VAD group, the expression levels of colonic CEA cell adhesion molecule 1 (CEACAM1) were down-regulated, and the levels of histone deacetylase 1 (HDAC1) and HDAC3 were up-regulated. Intriguingly, the above changes in the VAD groups were rescued by VA supplementation in the early postnatal period. Further study indicated that in Caco-2 cells, butyrate treatment significantly repressed the enrichment of HDAC3 on the promoter of the CEACAM1 gene to induce its expression. Our findings support that butyrate intervention can alleviate the impairment of colonic barrier function caused by VAD, and timely postnatal VA intervention may reverse the damage caused by VAD on gut barrier integrity during pregnancy.

Abstract Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity and the fourth leading malignancy and cause of cancer-related death in the male population of Taiwan. Most cases are detected at advanced stages, resulting in poor prognosis. Therefore, improved detection of early oral health disorders is indispensable. The involvement of oral bacteria in inflammation and their association with OSCC progression provide a feasible target for diagnosis. Due to the nature of oral neoplasms, the diagnosis of epithelial precursor lesions is relatively easy compared with that of other types of cancer. However, the transition from an epithelial precursor lesion to cancer is slow and requires further and continuous follow-up. In this study, we investigated microbiota differences between normal individuals, epithelial precursor lesion patients, and cancer patients with different lifestyle habits, such as betel chewing and smoking, using next-generation sequencing. Overall, the oral microbiome compositions of five genera, Bacillus , Enterococcus , Parvimonas , Peptostreptococcus , and Slackia , revealed significant differences between epithelial precursor lesion and cancer patients and correlated with their classification into two clusters. These composition changes might have the potential to constitute a biomarker to help in monitoring the oral carcinogenesis transition from epithelial precursor lesion to cancer.

Epigallocatechin gallate (EGCG) is the most abundant and effective green tea catechin and has been reported to attenuate diabetic nephropathy (DN). However, the mechanism by which EGCG ameliorates DN, till now, has remained unclear. EGCG is known as a potent activator of nuclear factor erythroid 2-related factor 2 (NRF2), which plays a key role in cellular defense against diabetes-induced oxidative stress and in the prevention of DN. In the present study, we tested whether NRF2 is required for EGCG protection against DN. Therefore, C57BL/6 wild type (WT) and Nrf2 knockout mice were induced to diabetes by streptozotocin, in the presence or absence of a 24-week treatment with EGCG. In the WT mice, EGCG activated Nrf2 expression and function without altering the expression of Kelch-like ECH-associated protein 1 (Keap1). Diabetes-induced renal oxidative damage, inflammation, fibrosis and albuminuria were significantly prevented by EGCG. Notably, deletion of the Nrf2 gene completely abrogated these actions of EGCG. To further determine the effect of EGCG on KEAP1/NRF2 signaling, mouse mesangial cells were treated with high glucose, in the presence of both Keap1 siRNA and EGCG. Interestingly, EGCG failed to enhance NRF2 signaling and alleviate oxidative, inflammatory and fibrotic indicators, in the presence of Keap1 siRNA. The present study demonstrated, for the first time, that NRF2 plays a critical role in EGCG protection against DN. Other findings indicated that inactivation of KEAP1 protein by EGCG may mediate EGCG function in activating NRF2.

Summary WRKY comprises a large family of transcription factors in plants, but most WRKY members are still poorly understood. In this study, we report the identification and functional characterization of PbrWRKY53 isolated from Pyrus betulaefolia . PbrWRKY53 was greatly up‐regulated by drought and abscisic acid, but slightly induced by salt and cold. Subcellar localization analyses showed that PbrWRKY53 was located in the nucleus. Ectopic expression of PbrWRKY53 in tobacco and Pyrus ussuriensis conferred enhanced tolerance to drought stress. The transgenic plants exhibited better water status, less reactive oxygen species generation and higher levels of antioxidant enzyme activities and metabolites than the wild type. In addition, overexpression of PbrWRKY53 in transgenic tobacco resulted in enhanced expression level of PbrNCED1 , and led to the increase in larger amount of vitamin C accumulation in comparison to WT. Knock‐down of PbrWRKY53 in P. ussuriensis down‐regulated PbrNCED1 abundance, accompanied by compromised drought tolerance. Yeast one‐hybrid assay, EMSA and transient expression analysis demonstrated that PbrWRKY53 could bind to the W‐box element in the promoter region of PbrNCED1 . Taken together, these results demonstrated that PbrWRKY53 plays a positive role in drought tolerance, which might be, at least in part, promoting production of vitamin C via regulating PbrNCED1 expression.

Restricted diets and inadequate nutrient intake of children with autism spectrum disorder (ASD) have been reported. This study examined the nutritional statuses of children with ASD and the relationships between their behaviors and nutritional intake. A total of 154 children with ASD (age = 5.21 ± 1.83 years) and 73 typically-developing (TD) children (age = 4.83 ± 0.84 years) from Chongqing, China, were enrolled. The severity of ASD was evaluated using the Childhood Autism Rating Scale (CARS). The serum ferritin, folate, vitamin B12, 25(OH) vitamin D, and vitamin A concentrations in the children with ASD were determined. All participants underwent anthropometric examinations, dietary assessments, and questionnaire assessments about their feeding behaviors, and gastrointestinal symptoms. The ZHA, ZWA, and ZBMIA were found to be significantly lower in the children with ASD compared with those without ASD. In addition, the percentages of children exhibiting severe picky eating and severe resistance to new foods, as well as those with a reported general impression of severe eating problems and constipation, were higher among the children with ASD. These children consumed significantly fewer macronutrients compared with the children without ASD. In addition, the children with ASD had the highest rate of vitamin A deficiency, followed by iron deficiency. After adjusting for sex, the vitamin A concentration was found to be negatively correlated with the CARS score (rs = −0.222, p = 0.021). No correlation between the ferritin, folate, vitamin D, or vitamin B12 concentration and the CARS score was found. These results suggest that reduced macronutrient intakes, severe feeding behavior issues, constipation, and vitamin A deficiency are quite common among children with ASD. Further, a low serum vitamin A level may be a risk factor for symptoms of ASD. However, the underlying mechanism should be further studied.

Eighty-one stool samples from Taiwanese were collected for analysis of the association between the gut flora and obesity. The supervised analysis showed that the most, abundant genera of bacteria in normal samples (from people with a body mass index (BMI) ≤ 24) were Bacteroides (27.7%), Prevotella (19.4%), Escherichia (12%), Phascolarctobacterium (3.9%), and Eubacterium (3.5%). The most abundant genera of bacteria in case samples (with a BMI ≥ 27) were Bacteroides (29%), Prevotella (21%), Escherichia (7.4%), Megamonas (5.1%), and Phascolarctobacterium (3.8%). A principal coordinate analysis (PCoA) demonstrated that normal samples were clustered more compactly than case samples. An unsupervised analysis demonstrated that bacterial communities in the gut were clustered into two main groups: N-like and OB-like groups. Remarkably, most normal samples (78%) were clustered in the N-like group, and most case samples (81%) were clustered in the OB-like group (Fisher's P value = 1.61E - 07). The results showed that bacterial communities in the gut were highly associated with obesity. This is the first study in Taiwan to investigate the association between human gut flora and obesity, and the results provide new insights into the correlation of bacteria with the rising trend in obesity.

Theanine, a unique non-proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H+ -dependent. The tissue-specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root-to-bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root-to-shoot delivery in the tea plant.

Acute kidney injury (AKI) is often secondary to sepsis. Previous studies suggest that damaged mitochondria and the inhibition of autophagy results in AKI during sepsis, but dexmedetomidine (DEX) alleviates lipopolysaccharide (LPS)-induced AKI. However, it is uncertain whether the renoprotection of DEX is related to autophagy or the clearance of damaged mitochondria in sepsis-induced AKI.In this study, AKI was induced in rats by injecting 10 mg/kg of LPS intraperitoneally (i.p.). The rats were also pretreated with DEX (30 μg/kg, i.p.) 30 min before the injection of LPS. The structure and function of kidneys harvested from the rats were evaluated, and the protein levels of autophagy-related proteins, oxidative stress levels, and apoptosis levels were measured. Further, atipamezole (Atip) and 3-Methyladenine (3-MA), which are inhibitors of DEX and autophagy, respectively, were administered before the injection of DEX to examine the protective mechanism of DEX.Pretreatment with DEX ameliorated kidney structure and function. DEX decreased the levels of blood urea nitrogen (BUN) and creatinine (Cre), urine kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), reactive oxygen species (ROS), and apoptosis proteins (such as cleaved caspase-9 and cleaved caspase-3). However, DEX upregulated the levels of autophagy and mitophagy proteins, such as Beclin-1, LC3 II and PINK1. These results suggest that DEX ameliorated LPS-induced AKI by reducing oxidative stress and apoptosis and enhancing autophagy. To promote autophagy, DEX inhibited the phosphorylation levels of PI3K, AKT, and mTOR. Furthermore, the administration of Atip and 3-MA inhibitors blocked the renoprotection effects of DEX.Here, we demonstrate a novel mechanism in which DEX protects against LPS-induced AKI. DEX enhances autophagy, which results in the removal of damaged mitochondria and reduces oxidative stress and apoptosis in LPS-induced AKI through the α2-AR and inhibition of the PI3K/AKT/mTOR pathway.

Dysbiosis of gut microbiota are commonly reported in autism spectrum disorder (ASD) and may contribute to behavioral impairment. Vitamin A (VA) plays a role in regulation of gut microbiota. This study was performed to investigate the role of VA in the changes of gut microbiota and changes of autism functions in children with ASD. Sixty four, aged 1 to 8 years old children with ASD completed a 6-month follow-up study with VA intervention. High-performance liquid chromatography was used to assess plasma retinol levels. The Autism Behaviour Checklist (ABC), Childhood Autism Rating Scale (CARS) and Social Responsiveness Scale (SRS) were used to assess autism symptoms. CD38 and acid-related orphan receptor alpha (RORA) mRNA levels were used to assess autism-related biochemical indicators’ changes. Evaluations of plasma retinol, ABC, CARS, SRS, CD38 and RORA mRNA levels were performed before and after 6 months of intervention in the 64 children. Illumina MiSeq for 16S rRNA genes was used to compare the differences in gut microbiota before and after 6 months of treatment in the subset 20 of the 64 children. After 6 months of intervention, plasma retinol, CD38 and RORA mRNA levels significantly increased (all P < 0.05); the scores of ABC, CARS and SRS scales showed no significant differences (all P > 0.05) in the 64 children. Meanwhile, the proportion of Bacteroidetes/Bacteroidales significantly increased and the proportion of Bifidobacterium significantly decreased in the subgroup of 20 (all false discovery rate (FDR) q < 0.05). Bacteroidetes/Bacteroidales were the key taxa related to VA. Moreover, VA played a role in the changes in autism biomarkers. It remains unclear whether the VA concentration is associated with autism symptoms. The study protocol was peer reviewed and approved by the institutional review board of Children’s Hospital, Chongqing Medical University in 2013 and retrospectively registered in Chinese Clinical Trial Registry (ChiCTR) on November 6, 2014 (TRN: ChiCTR-ROC-14005442 ).

Acute kidney injury (AKI) is a frequent and serious complication of sepsis; however, there are currently no effective therapies. Inflammation and oxidative stress are the major mechanisms implicated in lipopolysaccharide (LPS)-induced AKI. Dexmedetomidine (DEX) has been reported to have remarkable anti-inflammatory and antioxidant effects. Here, we examined the renoprotective effects of DEX and potential underlying mechanisms in rats with LPS-induced AKI. We analyzed renal function and structure; serum inflammatory cytokine; renal oxidant and antioxidant levels; and renal expression of glycogen synthase kinase-3β (GSK-3β)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway-related proteins in rats 4 hr after administration of LPS. Pretreatment with DEX improved renal function and significantly reduced the levels of inflammatory cytokines and oxidative stress markers. Treatment with DEX and the GSK-3β inhibitor SB216367 promoted phosphorylation of GSK-3β, induced Nrf2 nuclear translocation, and increased transcription of the Nrf2 target genes heme oxygenase-1 and NAD(P)H quinone oxidoreductase-1, primarily in renal tubules. Alpha-2-adrenergic receptor (α2-AR) antagonist atipamezole and imidazoline I 2 receptor (I 2 R) antagonist idazoxan reversed the effects of DEX. These results suggest that the renoprotective effects of DEX are mediated via α2-AR and I 2 R-dependent pathways that reduce inflammation and oxidative stress through GSK-3β/Nrf2 signaling.

Autism spectrum disorder (ASD) is a type of neurodevelopmental disease that is frequently accompanied by sleep disorder. Herein, we investigated changes in the gut microbiota and its metabolites correlated with core symptoms and sleep problems in children with ASD.One hundred and twenty children diagnosed with ASD based on Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria were enrolled in our study. The Autism Behavior Checklist (ABC), Social Responsiveness Scale (SRS), and Childhood Autism Rating Scale (CARS) were used to assess autism symptoms, and the Children Sleep Habits Questionnaire (CSHQ) was employed to evaluate sleep problems in children with ASD. The 120 children were divided into a sleep disorder group (n = 60) and a control group without sleep disorder (n = 60) according to the CSHQ answers. Illumina MiSeq analysis of 16S rRNA genes was used to compare differences in gut microbiota, and metabolomics analysis was employed to asses associated metabolites.SRS and CARS scores for the sleep disorder group were significantly higher than for the control group (p < 0.05). The abundances of butyrate-producing bacteria Faecalibacterium and Agathobacter were reduced significantly in the sleep disorder group (p < 0.05), and this was negatively correlated with CSHQ score (p = 0.007 and p = 0.014, respectively). The abundance of Agathobacter was also negatively associated with the ABC language score (p = 0.044). Furthermore, levels of 3-hydroxybutyric acid and melatonin were significantly lower (p < 0.05) while serotonin levels were higher (p < 0.05) in the sleep disorder group. The 3-hydroxybutyric acid level was positively associated with Faecalibacterium abundance (p = 0.000), and melatonin was positively associated with the abundance of Faecalibacterium (p = 0.036) and Agathobacter (p = 0.041). We also observed negative correlations between 3-hydroxybutyric acid and CSHQ (p = 0.000) and CARS (p = 0.009), between melatonin and CSHQ (p = 0.002) and ABC sensory score (p = 0.021), and a positive correlation between serotonin and CSHQ (p = 0.002) and ABC sensory score (p = 0.025).ASD children with sleep disorder exhibited declines in the abundance of Faecalibacterium and Agathobacter, decreased levels of 3-hydroxybutyric acid and melatonin, and an increase in serotonin. These changes may aggravate sleep problems and core symptoms in children with ASD.

Abstract Free amino acids, including theanine, glutamine and glutamate, contribute greatly to the pleasant taste and multiple health benefits of tea. Amino acids in tea plants are mainly synthesized in roots and transported to new shoots, which are significantly affected by nitrogen (N) level and forms. However, the regulatory amino acid metabolism genes have not been systemically identified in tea plants. Here, we investigated the dynamic changes of free amino acid contents in response to N deficiency and forms in tea plant roots, and systemically identified the genes associated amino acid contents in individual metabolism pathways. Our results showed that glutamate-derived amino acids are the most dynamic in response to various forms of N and N deficiency. We then performed transcriptomic analyses of roots treated with N deficiency and various forms of N, and differentially expressed amino acid metabolic genes in each pathway were identified. The analyses on expression patterns and transcriptional responses of metabolic genes to N treatments provided novel insights for the molecular basis of high accumulation of theanine in tea plant root. These analyses also identified potential regulatory genes in dynamic amino acid metabolism in tea plant root. Furthermore, our findings indicated that the dynamic expression levels of CsGDH, CsAlaDC, CsAspAT, CsSDH, CsPAL, CsSHMT were highly correlated with changes of amino acid contents in their corresponding pathways. Herein, this study provides comprehensive insights into transcriptional regulation of amino acid metabolism in response to nitrogen deficiency and nitrogen forms in tea plant root.

To determine the relationship of the gut microbiota and its metabolites with autism spectrum disorder (ASD)-like behaviors and preliminarily explore the potential molecular mechanisms, the fecal microbiota from donors with ASD and typically developing (TD) donors were transferred into germ-free (GF) mice to obtain ASD-FMT mice and TD-FMT mice, respectively. Behavioral tests were conducted on these mice after 3 weeks. 16S rRNA gene sequencing of the cecal contents and untargeted metabolomic analysis of the cecum, serum, and prefrontal cortex were performed. Untargeted metabolomics was also used to analyze fecal samples of TD and ASD children. Western blotting detected the protein expression levels of tryptophan hydroxylase 1 (TPH1), serotonin transporter (SERT), and serotonin 1A receptor (5-HT1AR) in the colon and TPH2, SERT, and 5-HT1AR in the prefrontal cortex of mice. ASD-FMT mice showed ASD-like behavior and a microbial community structure different from that of TD-FMT mice. Tryptophan and serotonin metabolisms were altered in both ASD and TD children and ASD-FMT and TD-FMT mice. Some microbiota may be related to tryptophan and serotonin metabolism. Compared with TD-FMT mice, ASD-FMT mice showed low SERT and 5-HT1AR and high TPH1 expression levels in the colon. In the prefrontal cortex, the expression levels of TPH2 and SERT were increased in the ASD-FMT group relative to the TD-FMT group. Therefore, the fecal microbiome of ASD children can lead to ASD-like behaviors, different microbial community structures, and altered tryptophan and serotonin metabolism in GF mice. These changes may be related to changes in some key proteins involved in the synthesis and transport of serotonin.IMPORTANCE The relationship between the gut microbiota and ASD is not yet fully understood. Numerous studies have focused on the differences in intestinal microbial and metabolism profiles between TD and ASD children. However, it is still not clear if these microbes and metabolites cause the development of ASD symptoms. Here, we collected fecal samples from TD and ASD children, transplanted them into GF mice, and found that the fecal microbiome of ASD children can lead to ASD-like behaviors, different microbial community structures, and altered tryptophan and serotonin metabolism in GF mice. We also demonstrated that tryptophan and serotonin metabolism was also altered in ASD and TD children. Together, these findings confirm that the microbiome from children with ASD may lead to ASD-like behavior of GF mice through metabolites, especially tryptophan and serotonin metabolism.

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of clenbuterol (CLB), salbutamol (SAL) and ractopamine (RAC) in milk by ultra high performance liquid chromatography–positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) with isotope dilution. Samples were directly purified through HLB cartridge. Then the eluate was dried under nitrogen and residues were redissolved in mobile phase. Samples were analyzed by LC–MS/MS on an Acquity UPLC® BEH C18 column with gradient elution. The samples were quantified using clenbuterol-D9, salbutamol-D3 and ractopamine-D6 as internal standards. The proposed method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity, robustness and stability. CCα values were 0.054, 0.006 and 0.008 μg/kg for CLB, SAL and RAC, respectively. CCβ values were 0.058, 0.007 and 0.009 μg/kg for CLB, SAL and RAC, respectively. The mean recoveries, repeatability (expressed as coefficient of variation, CVr), and reproducibility (CVR) varied from 95.8 to 106.2%, from 3.60 to 6.44% (CVr), and from 4.77 to 7.53% (CVR), respectively. The method is demonstrated to be suitable for the determination of clenbuterol, salbutamol and ractopamine in milk. The total time required for the analysis of one sample, including sample preparation, was about 45 min.

To cite this article: Kim YH, Yang TY, Park C‐S, Ahn S‐H, Son BK, Kim JH, Lim DH, Jang TY. Anti‐IL‐33 antibody has a therapeutic effect in a murine model of allergic rhinitis. Allergy 2012; 67 : 183–190. Abstract Background: Interleukin (IL)‐33 is involved in the Th2 immune response and could play an essential role in nasal allergy. Therefore, we aimed to investigate the therapeutic potential of anti‐IL‐33 for allergic rhinitis (AR). Methods: Twenty‐four BALB/c mice were used. In group A (control group, n = 6), mice were sensitized and challenged with saline. Group B [ovalbumin (OVA) group, n = 6] mice received intraperitoneal and intranasal OVA challenge. In group C (control IgG group, n = 6), mice were injected intraperitoneally with rabbit control IgG before OVA challenge. In group D (anti‐IL‐33 group, n = 6), anti‐IL‐33 was injected before challenge. We evaluated the number of nose‐scratching events and external morphology; serum total and OVA‐specific IgE; number of eosinophils, neutrophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid; histopathologic examination of nasal cavity; and IL‐4, IL‐5, and IL‐13 in BAL fluid. Results: Anti‐IL‐33 treatment significantly reduced the nose‐scratching events and ameliorated skin denudation. Serum total and OVA‐specific IgE was significantly decreased in group D. The number of eosinophils in BAL fluid was also significantly decreased. Eosinophilic infiltration in the nasal cavity was significantly decreased in group D. IL‐4, IL‐5, and IL‐13 in BAL fluid were also significantly decreased after treatment. Conclusions: Anti‐IL‐33 antibody has a therapeutic potential for experimental AR.

A simple and inexpensive pretreatment procedure was developed for 10 β2-agonists (clenbuterol, ractopamine, salbutamol, bambuterol, penbuterol, tulobuterol, clorprenaline, mabuterol, cimaterol and terbutaline) in swine urine using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). The sample was analysed after purification by ultra high performance liquid chromatography–positive electrospray ionisation tandem mass spectrometry (UHPLC-ESI–MS/MS). The pH value of the swine urine, extraction time, type and amount of MWCNTs and type of eluent were optimised to increase the sample throughput and sensitivity. The samples were quantified using clenbuterol-D9, ractopamine-D6 and salbutamol-D3 as internal standards. The recoveries of the target compounds from swine urine samples at pH 10.0 were most efficient when using 20 mg of MWCNTs with a 30–50 nm outer diameter and a length of 10–30 μm, while a mixture of water/methanol (90:10, v/v) with 0.5% formic acid was shown to be the most suitable solvent for desorbing the compounds from the MWCNTs. The proposed method was validated according to the European Commission Decision 2002/657/EC, which determines linearity, specificity, decision limit (CCα), detection capability (CCβ), recovery, precision and stability.

Many terpenoids are known to have antifungal properties and overexpression of these compounds in crops is a potential tool in disease control. In this study, 15 different mono- and sesquiterpenoids were tested in vitro against two major pathogenic fungi of maize (Zea mays), Colletotrichum graminicola and Fusarium graminearum. Among all tested terpenoids, geranic acid showed very strong inhibitory activity against both fungi (MIC<46 μM). To evaluate the possibility of enhancing fungal resistance in maize by overexpressing geranic acid, we generated transgenic plants with the geraniol synthase gene cloned from Lippia dulcis under the control of a ubiquitin promoter. The volatile and non-volatile metabolite profiles of leaves from transgenic and control lines were compared. The headspaces collected from intact seedlings of transgenic and control plants were not significantly different, although detached leaves of transgenic plants emitted 5-fold more geranyl acetate compared to control plants. Non-targeted LC–MS profiling and LC–MS–MS identification of extracts from maize leaves revealed that the major significantly different non-volatile compounds were 2 geranic acid derivatives, a geraniol dihexose and 4 different types of hydroxyl-geranic acid-hexoses. A geranic acid glycoside was the most abundant, and identified by NMR as geranoyl-6-O-malonyl-β-d-glucopyranoside with an average concentration of 45 μM. Fungal bioassays with C. graminicola and F. graminearum did not reveal an effect of these changes in secondary metabolite composition on plant resistance to either fungus. The results demonstrate that metabolic engineering of geraniol into geranic acid can rely on the existing default pathway, but branching glycosylation pathways must be controlled to achieve accumulation of the aglycones.

Feed efficiency appears to vary even within closely related pigs, and may be partly affected by the diversity in the composition and function of gut microbes. To investigate the components and functional differences of gut microbiota of low and high feed efficiency (FE) pigs, high throughput sequencing and de novo metagenomics were performed on pig cecal contents. Pigs were selected in pairs with low and high feed conversion ratio. The microorganisms of individuals with different feed efficiency were clustered according to diversity. The genus Prevotella was the most enriched in both groups, and the abundance of species Prevotella sp. CAG:604 was significantly increased in low efficiency individuals compared to that in animals showing high efficiency. In contrast, other differential species, including lactic acid bacteria, were all enriched in the group with good feeding characteristics. Functional analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases demonstrated that differential genes for the metabolism of carbohydrates were most abundant in both groups, but pathways of pyruvate-related metabolism were more intense in pigs with higher feed efficiency. All these data indicated that the microbial environment was closely related to the growth traits of pigs, and regulating microbial composition could aid developing strategies to improve feed efficiency for pigs.

A LC-MS/MS method for determination of eight pesticides (triadimefon, sulfoxaflor, flusilazole, tebuconazole, difenoconazole, amitraz, azoxystrobin, and thiophanate-methyl) in Lycium barbarum was established. The samples were extracted with acetonitrile, and then cleaned up by primary secondary amine. The extracts were diluted with 0.1% formic acid in water. The results showed that at the fortified levels of 0.01-10mg/kg, the average recoveries of these pesticides ranged from 82.1% to 96.2% with the relative standard deviations lower than 7%. The half-lives of eight pesticides were 1.3-5.0days in Lycium barbarum fruits. The pre-harvest interval of all pesticides mentioned above were investigated. Tebuconazole (14days), sulfoxaflor (14days) and flusilazole (28days) have longer pre-harvest interval than the others which have 7days. The dietary risks, assessed as hazard quotients, were far below 100%. The results showed that the eight pesticides applied to Lycium barbarum were comparably safe for the consumer.

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 38 veterinary drugs (18 sulfonamides, 11 quinolones and 9 benzimidazoles) and 8 metabolites of benzimidazoles in bovine milk by ultra high performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS). Samples were extracted with acidified acetonitrile, cleaned up with Oasis® MCX cartridges, and analyzed by LC–MS/MS on an Acquity UPLC® BEH C18 column with gradient elution. The method allows such multi-analyte measurements within a 13 min runtime while the specificity is ensured through the MRM acquisition mode. The method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity and stability. For compounds which have MRLs in bovine milk, the CCα values fall into a range from 11 to 115 μg/kg, and the CCβ values fall within a range of 12–125 μg/kg. For compounds which have not MRLs in bovine milk, the CCα values fall into a range from 0.01 to 0.08 μg/kg, and the CCβ values fall within a range of 0.02–0.11 μg/kg. The mean recoveries of the 46 analytes were between 87 and 119%. The calculated RSD values of repeatability and within-laboratory reproducibility experiments were below 11% and 15% for the 46 compounds, respectively. The method was demonstrated to be suitable for the simultaneous determination of sulfonamides, quinolones and benzimidazoles in bovine milk.

Autism spectrum disorders (ASD) are complicated neurodevelopmental disorders. Many studies have demonstrated that children with autism have multiple nutritional deficiencies and increased serum 5-hydroxytryptamine (5-HT) levels. In our previous study, 77.9% of autistic children were found to have vitamin A deficiency, and the concentration of vitamin A was negatively associated with the CARS score. In the present study, we sought to test whether vitamin A supplementation could improve autistic symptoms and decrease serum 5-HT levels. The DSM-V criteria and CARS score were used for symptom description and symptom assessment of the patients, respectively, before and after vitamin A supplementation (VAS). Serum retinol and 5-HT levels, mRNA levels of RAR α, β, and γ and TpH 1 expression were detected in autistic children before and after VAS and in normal children. Serum retinol levels in children with ASD were significantly lower than in control children. Serum 5-HT levels in children with ASD were higher than in control children, which were correlated with symptom severity of children with autism. After VA supplementation, the children with ASD exhibited significant improvement in autism symptoms. Serum retinol concentrations of children with ASD were significantly increased, and serum 5-HT levels were decreased. Moreover, statistically significant changes were observed in mRNA expression levels of RAR α, RAR γ and TpH 1 after VAS compared to baseline. This study suggested that VA supplementation may improve symptoms and reduce 5-HT levels in children with ASD, indicating that VA supplementation is a reasonable therapy at least for a subset of children with autism.

The aim of this study is to investigate the specific effects of vitamin A (VA) on diarrhea in rats and its potential targets to protect the intestinal mucosa.Specific pathogen-free Sprague Dawley rats were fed a VA deficient (VAD) or VA normal (VAN) diet for 4 wk. Then, half of the VAN rats were treated with a VAN diet and the other half with a lactose VAN diet. VAD rats were randomly assigned to one of four groups and fed a VAD diet, lactose VAD diet, VAN diet with VA supplementation (VAS) via daily intragastric administration, or a lactose VAN diet with daily VAS. Rat weight and degree of diarrhea were evaluated daily. After 15 d, the serum retinol level was measured by high-performance liquid chromatography, and the serum diamine oxidase (DAO) and zonulin concentrations were analyzed by enzyme-linked immunosorbent assays. The small intestine mucosal pathology was observed by hematoxylin and eosin staining. Western blotting was performed to detect the protein expression levels of occludin and claudin-1 in the intestinal mucosa, and the zonula-occludens 1 expression was assessed using immunohistochemistry.VAD limited weight gain in rats and increased the degree of diarrhea. The serum retinol levels and the level of tight junction (TJ) proteins claudin-1 and occludin and grip strength were affected by the interaction between lactose-induced diarrhea and the VA diet. Diarrhea, independent of VAD, significantly decreased rat weight, increased serum DAO levels, damaged small intestine villi, and impaired zonula-occludens 1 protein expression. VAD significantly increased the concentration of zonulin independently of diarrhea, but VAS increased the serum retinol level, reduced the severity of diarrhea, increased the expression levels of the TJ proteins, facilitated the restoration of the small intestine villi that were damaged by the diarrhea, and decreased the concentrations of serum DAO and zonulin.VAD may aggravate the degree of diarrhea and intestinal mucosal damage during the duration of diarrhea, and VAS helps relieve diarrhea and improves intestinal damage likely by regulating the expression of TJ proteins. Therefore, VA plays a pivotal role in the protection of the intestinal mucosa during instances of diarrhea.

β-Amylase (BAM) is involved in sugar metabolism, but the role of BAM genes in cold tolerance remains poorly understood. Here, we report the identification and functional characterization of the chloroplast-localized BAM-encoding gene PbrBAM3 isolated from Pyrus betulaefolia. The transcript levels of PbrBAM3 were up-regulated under cold, dehydration and ABA, but repressed by maltose. Overexpression of PbrBAM3 in tobacco (Nicotiana tabacum) and pear (P. ussuriensis) conferred increased BAM activity, promoted starch degradation after chilling treatments and enhanced tolerance to cold. Under the chilling stress, the transgenic tobacco and P. ussuriensis exhibited lessened reactive oxygen species (ROS) generation, higher levels of antioxidant enzymes activity, and greater accumulation of soluble sugars (specially maltose) than the corresponding wild type plants. Taken together, these results demonstrate that PbrBAM3 plays an important role in cold tolerance, at least in part, by raising the levels of soluble sugars capable of acting as osmolytes or antioxidants.

To investigate whether gut microbiota is associated with vitamin A nutritional levels in children with persistent diarrhea, a total of 59 pediatric patients with persistent diarrhea aged 1-12 months were selected from the Department of Gastroenterology at the Children's Hospital of Chongqing Medical University, China. Subjects were hospitalized and divided into VA-deficient (n = 30) and VA-normal (n = 29) groups according to their venous serum retinol levels. Fecal samples from all 59 subjects were collected immediately after admission and analyzed by Illumina MiSeq for 16S rRNA genes to characterize the overall microbiota of the samples. The gut microbiota of the VA-deficient and VA-normal groups were compared using a bioinformatic statistical approach. The Shannon index (p = 0.02), Simpson index (p = 0.01) and component diagram data indicated significantly lower diversity in the VA-deficient than the VA-normal group. A metagenome analysis (LEfSe) and a differentially abundant features approach using Metastats revealed that Escherichia coli and Clostridium butyricum were the key phylotypes of the VA-normal group, while Enterococcus predominated the VA-deficient group. In conclusion, the diversity of gut microbiota and the key phylotypes are significantly different in children with persistent diarrhea at different VA nutritional levels.

The present study sought to identify a panel of DNA markers for noninvasive diagnosis using cell-free DNA (cfDNA) from urine supernatant or cellular DNA from urine sediments of hematuria patients. A panel of 48 bladder cancer-specific genes was selected. A next-generation sequencing-based assay with a cfDNA barcode-enabled single-molecule test was employed. Mutation profiles of blood, urine, and tumor sample from 16 bladder cancer patients were compared. Next, urinary cellular DNA and cfDNA were prospectively collected from 125 patients (92 bladder cancer cases and 33 controls) and analyzed using the 48-gene panel. The individual gene markers and combinations of markers were validated according to the pathology results. The mean areas under the receiver operating characteristic (ROC) curves (AUCs) obtained with the various modeling approaches were calculated and compared.This pilot study of 16 bladder cancer patients demonstrated that gene mutations in urine supernatant and sediments had better concordance with cancer tissue as compared with plasma. Logistic analyses suggested two powerful combinations of genes for genetic diagnostic modeling: five genes for urine supernatant (TERT, FGFR3, TP53, PIK3CA, and KRAS) and seven genes for urine sediments (TERT, FGFR3, TP53, HRAS, PIK3CA, KRAS, and ERBB2). The accuracy of the five-gene panel and the seven-gene panel in the validation cohort yielded AUCs of 0.94 [95% confidence interval (CI) 0.91-0.97] and 0.91 (95% CI 0.86-0.96), respectively. With the addition of age and gender, the diagnostic power of the urine supernatant five-gene model and the urine sediment seven-gene model improved as the revised AUCs were 0.9656 (95% CI 0.9368-0.9944) and 0.9587 (95% CI 0.9291-0.9883).cfDNA from urine bears great diagnostic potential. A five-gene panel for urine supernatant and a seven-gene panel for urine sediments are promising options for identifying bladder cancer in hematuria patients.

Abstract Bacterial degradation of organosulfonates plays an important role in sulfur recycling, and has been extensively studied. However, this process in anaerobic bacteria especially gut bacteria is little known despite of its potential significant impact on human health with the production of toxic H 2 S. Here, we describe the structural and biochemical characterization of an oxygen-sensitive enzyme that catalyzes the radical-mediated C-S bond cleavage of isethionate to form sulfite and acetaldehyde. We demonstrate its involvement in pathways that enables C2 sulfonates to be used as terminal electron acceptors for anaerobic respiration in sulfate- and sulfite-reducing bacteria. Furthermore, it plays a key role in converting bile salt-derived taurine into H 2 S in the disease-associated gut bacterium Bilophila wadsworthia . The enzymes and transporters in these anaerobic pathways expand our understanding of microbial sulfur metabolism, and help deciphering the complex web of microbial pathways involved in the transformation of sulfur compounds in the gut.

Acute kidney injury (AKI) is a severe complication of sepsis; however, no effective drugs have been found. Activation of the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome is a major pathogenic mechanism of AKI induced by lipopolysaccharide (LPS). Autophagy, a process of intracellular degradation related to renal homeostasis, effectively restricts inflammatory responses. Herein, we explored the potential protective mechanisms of dexmedetomidine (DEX), which has confirmed anti-inflammatory effects, on LPS-induced AKI.AKI was induced in rats by injecting 10 mg/kg of LPS intraperitoneally (i.p.). Wistar rats received intraperitoneal injections of DEX (30 µg/kg) 30 min before an intraperitoneal injection of LPS. Atipamezole (ATI) (250 µg/kg) and 3-methyladenine (3-MA) (15 mg/kg) were intraperitoneally injected 30 min before the DEX injection.DEX significantly attenuated renal injury. Furthermore, DEX decreased activation of the NLRP3 inflammasome and expression of interleukins 1β and 18. In addition, autophagy-related protein and gene analysis indicated that DEX could significantly enhance autophagy. Finally, we verified the pharmacological effects of DEX on the 5'-adenosine monophosphate-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) pathway. Atip and 3-MA significantly reversed the protective effects of DEX.Our results suggest that the protective effects of DEX were mediated by enhanced autophagy via the α2-adrenoreceptor/AMPK/mTOR pathway, which decreased activation of the NLRP3 inflammasome. Above all, we verified the renal protective effects of DEX and offer a new treatment strategy for AKI.

Objective: Detection of aberrant methylated genes in feces has been developed as an early screening method for colorectal cancer.The aim of this study was to probe the methylation status of SEPT9, BMP3, NDRG4, and SDC2 in stool and study whether methylation of these genes is associated with colorectal cancer.Materials and Methods: DNAs were isolated and purified from cancerous and non-cancerous stool samples and colorectal cancer tissue.Gene methylation levels were quantified by methylation-specific PCR on SEPT9, BMP3, NDRG4, and SDC2 and analyzed by a diagnostic model.Results: DNA methylation of SEPT9, NDRG4 and SDC2, but not BMP3, had diagnostic potential for detecting colorectal cancer.Moreover, integration of SEPT9, NDRG4, and SDC2 methylation demonstrated high feasibility for detecting colorectal cancer and adenoma, with better performance on colorectal cancer than adenoma. Conclusion:The methylation of SEPT9, NDRG4, and SDC2 in stool may be a potential biomarker for early screening of colorectal cancer.

Plant HAK/KUP/KT family members function as plasma membrane (PM) H+/K+ symporters and may modulate chemiosmotically-driven polar auxin transport (PAT). Here, we show that inactivation of OsHAK5, a rice K+ transporter gene, decreased rootward and shootward PAT, tiller number, and the length of both lateral roots and root hairs, while OsHAK5 overexpression increased PAT, tiller number, and root hair length, irrespective of the K+ supply. Inhibitors of ATP-binding-cassette type-B transporters, NPA and BUM, abolished the OsHAK5-overexpression effect on PAT. The mechanistic basis of these changes included the OsHAK5-mediated decrease of transmembrane potential (depolarization), increase of extracellular pH, and increase of PM-ATPase activity. These findings highlight the dual roles of OsHAK5 in altering cellular chemiosmotic gradients (generated continuously by PM H+-ATPase) and regulating ATP-dependent auxin transport. Both functions may underlie the prominent effect of OsHAK5 on rice architecture, which may be exploited in the future to increase crop yield via genetic manipulations.

To explore possible associations between maternal use of micronutrient supplements and the subsequent vitamin levels and symptoms in offspring with autism spectrum disorder (ASD), a total of 416 children with ASD and 201 typically developing (TD) children were enrolled. The children born to mothers without folic acid (FA) and micronutrient supplementation during pregnancy had more severe social cognition impairments, social communication impairments, autism behaviour mannerisms, developmental delays in adaptive and gross motor behaviour and gastrointestinal problems than children born to mothers who used FA and micronutrient supplements (P＜0.05). Interestingly, there was an association between maternal micronutrient supplementation and vitamin A (VA), vitamin D (VD) and folate levels in the ASD children (P<0.05), and levels of these vitamins also were associated with symptoms of ASD. Maternal FA and/or micronutrient supplementation may potentially moderate the symptoms of ASD. Interrupting the chain of micronutrient deficiencies between pregnant mothers and children may be beneficial in improving symptoms of ASD.

Abstract Background High prevalence of sleep problems have been reported in children with Autism Spectrum Disorder (ASD). This study aims to investigate the sleep conditions of ASD children in China, and explore the relationship between the common sleep problems and core symptoms and developmental levels. Methods Using a cross-sectional design, we included 2 to 7-year-old children from 13 cities in China: 1310 with ASD and 1158 with typically-developing (TD) children. The neurodevelopmental level was evaluated with the revised Children Neuropsychological and Behavior Scale (CNBS-R2016). ASD were diagnosed with DSM-5 and Child Autism Rating Scale (CARS). the Social Responsiveness Scale (SRS), the Autism Behavior Checklist (ABC) and the communication warning behavior sub-scale in CNBS-R2016 valued autism behaviors. The children’ s sleep habits questionnaire (CSHQ) assessed sleep conditions. Results The prevalence of sleep disorders in ASD children was significantly higher than that in TD (67.4% vs. 51%, p &lt; 0.01), and among them the four dimensions with the highest prevalence of sleep problems were bedtime resistance (25.6%), sleep anxiety (22.7%), sleep onset delay (17.9%) and daytime sleepiness (14.7%). ASD children with sleep onset delay or sleep anxiety had higher ABC, SRS scores and higher scores on communication warning behavior with sleep anxiety, with daytime sleepiness had higher ABC, SRS and CARS scores, and with bedtime resistance had higher SRS total scores. Differences in the neurodevelopmental level were not significant. Conclusion Children with ASD have a higher prevalence of sleep problems. Bedtime resistance, anxiety, sleep onset delay and daytime sleepiness may be related to the core symptoms, but not be related to the developmental level in ASD children. In the clinic, sleep assessment should be included in the routine of ASD visits, and during the intervention, sleep hygiene education is as important as the treatment of biological factors. Trial registration The study was approved by the ethics committee of the Children’s Hospital of Chongqing Medical University, Approval Number: (2018) IRB (STUDY) NO. 121, and registered in the Chinese Clinical Trial Registry (Registration number: ChiCTR2000031194 ).

Shading was thought as an effective approach to increase theanine in harvested tea shoots. Previous studies offered conflicting findings, perhaps since the integration of theanine metabolism and transport in different tissues was not considered. Theanine is synthesized primarily in the roots and is then transported, via the vascular system, to new vegetative tissues. Here, we found that theanine increased in the stem, was reduced in the leaf, and remained stable in the roots, under shading conditions. Notably, in tea roots, shading significantly increased ethylamine and activated the theanine biosynthesis pathway and theanine transporter genes. Furthermore, shading significantly increased the expression of theanine transporter genes, CsAAP2/4/5/8, in the stem, while decreasing the expression of CsAAP1/2/4/5/6 in the leaf, in accordance with shading effects on theanine levels in these tissues. These findings reveal that shading of tea plants promotes theanine biosynthesis and allocation in different tissues, processes which appear to involve the theanine biosynthesis pathway enzymes and AAP family of theanine transporters.

Tea (Camellia sinensis (L.) O. Kuntze) is predominantly grown in acidic soils with severe limitations of P-nutrition. The present study evaluated the effects of an arbuscular mycorrhizal (AM) fungus, Claroideoglomus etunicatum, on root morphology, leaf and root P concentration, phosphatase activity in soils and roots, and the relative expression of root P transporter genes (PT1 and PT4) in Camellia sinensis ‘Fuding Dabaicha’ seedlings in sands under two levels of P viz., 0.5 μM (P0.5) and 50 μM (P50) through Hoagland solutions (pH 4.2) for 24 weeks. Under both P0.5 and P50 conditions, AM seedlings displayed a higher growth performance (shoot biomass, root biomass, and plant height), root morphology (total length, taproot length, 2nd- and 3rd-lateral root number, and volume), and root-hair length, but lower root-hair density than non-AM seedlings. Mycorrhizal inoculation significantly increased root P concentrations in P0.5 and P50 and leaf P concentrations in P0.5 over non-mycorrhizal treatment. The AM seedlings recorded considerably higher soil acid, neutral, and total phosphatase activity under P0.5 treatment, and soil neutral and total phosphatase activity under P50 treatment, compared with the non-AM seedlings. Root acid phosphatase activity was reduced due to mycorrhization at both of P0.5 and P50. AM fungal inoculation up-regulated the relative expression of root CsPT1, while down-regulated the CsPT4 expression in P0.5 and P50 treatments. It was concluded that AM fungal inoculation significantly promoted P acquisition capacity of tea plants, especially in roots through improving root growth and enhancing soil acid phosphatase activity and root CsPT1 expression.

Plant Biotechnology JournalVolume 19, Issue 12 p. 2395-2397 Brief CommunicationOpen Access CsAlaDC and CsTSI work coordinately to determine theanine biosynthesis in tea plants (Camellia sinensis L.) and confer high levels of theanine accumulation in a non-tea plant Biying Zhu, Biying Zhu State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, China These authors contributed equally to this work.Search for more papers by this authorJiayi Guo, Jiayi Guo State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, China These authors contributed equally to this work.Search for more papers by this authorChunxia Dong, Chunxia Dong State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, China These authors contributed equally to this work.Search for more papers by this authorFang Li, Fang Li State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, China College of Horticulture, Nanjing Agricultural University, Nanjing, ChinaSearch for more papers by this authorSiming Qiao, Siming Qiao State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, ChinaSearch for more papers by this authorShijia Lin, Shijia Lin State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, ChinaSearch for more papers by this authorTianyuan Yang, Tianyuan Yang State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, ChinaSearch for more papers by this authorYingling Wu, Yingling Wu State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, ChinaSearch for more papers by this authorShilai Bao, Shilai Bao State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China School of Life Sciences, University of Chinese Academy of Sciences, Beijing, ChinaSearch for more papers by this authorWilliam J. Lucas, William J. Lucas Department of Plant Biology, College of Biological Sciences, University of California, Davis, CA, USASearch for more papers by this authorZhaoliang Zhang, Corresponding Author Zhaoliang Zhang zhlzhang@ahau.edu.cn orcid.org/0000-0001-6615-1598 State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, China *Correspondence (Tel: +86 551 657 86401; Fax: +86 551 657 86765; Email: zhlzhang@ahau.edu.cn)Search for more papers by this author Biying Zhu, Biying Zhu State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, China These authors contributed equally to this work.Search for more papers by this authorJiayi Guo, Jiayi Guo State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, China These authors contributed equally to this work.Search for more papers by this authorChunxia Dong, Chunxia Dong State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, China These authors contributed equally to this work.Search for more papers by this authorFang Li, Fang Li State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, China College of Horticulture, Nanjing Agricultural University, Nanjing, ChinaSearch for more papers by this authorSiming Qiao, Siming Qiao State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, ChinaSearch for more papers by this authorShijia Lin, Shijia Lin State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, ChinaSearch for more papers by this authorTianyuan Yang, Tianyuan Yang State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, ChinaSearch for more papers by this authorYingling Wu, Yingling Wu State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, ChinaSearch for more papers by this authorShilai Bao, Shilai Bao State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China School of Life Sciences, University of Chinese Academy of Sciences, Beijing, ChinaSearch for more papers by this authorWilliam J. Lucas, William J. Lucas Department of Plant Biology, College of Biological Sciences, University of California, Davis, CA, USASearch for more papers by this authorZhaoliang Zhang, Corresponding Author Zhaoliang Zhang zhlzhang@ahau.edu.cn orcid.org/0000-0001-6615-1598 State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui, China *Correspondence (Tel: +86 551 657 86401; Fax: +86 551 657 86765; Email: zhlzhang@ahau.edu.cn)Search for more papers by this author First published: 09 October 2021 https://doi.org/10.1111/pbi.13722Citations: 1 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Tea, the second most popular nonalcoholic beverage consumed worldwide, is favoured by billions of consumers due to its special flavour and numerous health benefits (Higdon and Frei, 2003). Theanine, a unique secondary metabolite in the tea plant (Camellia sinensis L.), confers the umami taste of the tea infusion. Theanine also has many physiological beneficial effects, including promoting relaxation, improving sleep quality and immunity and protecting the cardiovascular system (Kanarek et al., 2011). Theanine only accumulates at a high level in tea plants (Tadahiro and Shinsuke, 1984). This might be controlled by the specific presence of ethylamine in tea plants (Cheng et al., 2017), as theanine is primarily biosynthesized from ethylamine and glutamate by theanine synthetase in tea roots, with ethylamine being synthesized from alanine, by alanine decarboxylase (AlaDC) (Figure 1a,b), and CsAlaDC being specifically expressed in tea roots (Figure 1c). Although CsAlaDC exhibited AlaDC activity, in vitro (Bai et al., 2019), the in vivo role in tea plants has not been characterized. Figure 1Open in figure viewerPowerPoint CsAlaDC expression was highly correlated with ethylamine and theanine contents in different tea plant cultivars and conferred a high level of theanine accumulation when co-expressed with CsTSI in Nicotiana benthamiana. (a) Roots harvested for analysis. (b) Theanine biosynthesis pathway in the tea plant. CsAlaDC, alanine decarboxylase; CsTSI, theanine synthetase. (c) Tissue-specific expression pattern of CsAlaDC. MV, major vein; VB, vascular bundle, peeled from the stem. (d–f) Theanine (d), glutamate (e), and ethylamine (f) contents in the roots of 7 tea plant cultivars. ZC108, Zhongcha 108; HJYA, Huangjinya; LJ43, Longjing 43; JXY, Jingxiangyu; HJYE, Huangjinye; BC1, Baicha 1; JB, Jibai. (g) The correlation coefficient of glutamate and ethylamine contents with theanine contents. *P < 0.05, **P < 0.01. (h, i) The relative expression of CsAlaDC (H) and CsTSI (I). CsGAPDH was used as an internal control. (j, k) The correlation coefficient of CsAlaDC expression levels with ethylamine contents (j), and the correlation coefficient of CsAlaDC and CsTSI expression levels with theanine contents (k). **P < 0.01. (l–o) Ectopic expression of CsAlaDC in N. benthamiana leaves. WT, wild-type N. benthamiana without infiltration. EV, empty vector control; CsAlaDC, overexpression of CsAlaDC in N. benthamiana. NbGAPDH was used as an internal control. (p–t) Theanine contents in N. benthamiana leaves co-expressing CsAlaDC and CsTSI. CsAlaDC, pK7WGF2+pCAMBIA1305-CsAlaDC; CsTSI, pCAMBIA1305+pK7WGF2-CsTSI; CsAlaDC+CsTSI, pCAMBIA1305-CsAlaDC+pK7WGF2-CsTSI. Theanine in leaves of N. benthamiana was identified by mass chromatogram (UPLC-QQQ-MS). Characteristic theanine ions are located at m/z 158. Data represent means ± SD (n = 3), significant difference (P < 0.05) was labelled with different letters, according to Duncan's multiple range test. Theanine content varies greatly among the different cultivars, and theanine levels appear to be genetically regulated in tea leaves (Fang et al., 2021). To investigate the in vivo role of ethylamine in theanine accumulation, we quantified the key metabolites in the theanine metabolic pathway, in the roots of seven tea plant cultivars (Figure 1d–f). As shown in Figure 1d, theanine contents in the roots of these cultivars varied greatly. Importantly, the ethylamine contents exhibited a similar pattern as the theanine contents (Figure 1F). The correlation coefficient between the contents of ethylamine and theanine was 0.883 (P < 0.01) (Figure 1g). Taken together, these findings provided in vivo evidence for the pivotal role of ethylamine in determining theanine accumulation in tea plants. Next, we examined CsAlaDC expression in the roots of the seven cultivars (Figure 1h,i). Here, we found that its expression level was highly and positively correlated with the ethylamine contents, and the correlation coefficient reached 0.903 (P < 0.01) (Figure 1j). We also analysed the correlation between CsAlaDC expression and theanine content. Our findings indicated that CsAlaDC expression, in roots, was highly and positively correlated with the theanine contents, and the correlation coefficient was 0.881 (P < 0.01) (Figure 1k). Unexpectedly, the CsTSI expression level was not correlated with the theanine contents (Figure 1k). Taken together, these results suggested that CsAlaDC expression plays a critical role in determining theanine accumulation in roots of tea plants. We used the Nicotiana benthamiana transient expression system to further characterize the function of CsAlaDC, in planta. To this end, A. tumefaciens strain GV3101 (pSoup-p19), carrying pCAMBIA1305-CsAlaDC-GFP plasmid, was infiltrated into leaves of 5-week-old N. benthamiana plants (Figure 1l); the pCAMBIA1305 empty vector was used as the control (EV). We detected CsAlaDC expression at the mRNA level (Figure 1m) by qRT-PCR, and a high level of ethylamine (Figure 1n; >100 μg/g dry weight) was also identified in the CsAlaDC-expressing tobacco leaves. As anticipated, no ethylamine product was detected in WT tobacco leaves and those infiltrated with EV. Taken together, these findings offer support for the hypothesis that CsAlaDC has the capacity to synthesize ethylamine, in planta. Theanine is highly demanded, by the market, due to its health effects and medicinal value, as a food constituent, in cosmetics and in other fields (Cheng et al., 2017). The theanine obtained both by direct extraction and chemical synthesis is of very poor quality (Gu et al., 2004). In addition, plant cell culture is disadvantaged by high cost, poor genetic stability and the low content of metabolites, hampering its use for industrial theanine production. An alternative, and intriguing possibility, would be to synthesize theanine in other crops to improve their health-promoting effects of foods. Until now, however, the synthesis of theanine in non-tea plants through synthetic biology has not been achieved. Tobacco (N. benthamiana) is generally used as the model plant for synthetic biology research (Forestier et al., 2021; Li et al., 2019). Thus, we felt it would be important to synthesize theanine in tobacco, as a model for theanine biosynthesis in non-tea plants. Given the biosynthesis of ethylamine in tobacco, we speculated whether theanine could be produced in the tobacco leaves in the presence of ethylamine. Unexpectedly, theanine production was not detected (Figure 1o), indicating that the presence of ethylamine did not lead to theanine synthesis, at least not in detectable quantities. We then speculated that the combination of CsAlaDC and CsTSI, in tobacco leaves, could synthesize theanine. Based on this notion, we co-infiltrated A. tumefaciens strains carrying pCAMBIA1305-CsAlaDC and pK7WGF2-CsTSI plasmids (CsAlaDC+CsTSI) into tobacco leaves. A. tumefaciens strains containing pCAMBIA1305 and pK7WGF2-CsTSI (CsTSI) and pK7WGF2 and pCAMBIA1305-CsAlaDC (CsAlaDC) were infiltrated as the controls. Both CsAlaDC and CsTSI were being expressed in the infiltrated tobacco leaves (Figure 1p,q). Ethylamine was produced in CsAlaDC- and CsAlaDC+CsTSI-expressing leaves (Figure 1r). High levels of theanine were produced in leaves co-expressing CsAlaDC and CsTSI, and the theanine content reached to ˜4 mg/g (Figure 1s,t), which is comparable with the theanine contents in tea plant leaves. Thus, theanine biosynthesis requires both the presence of ethylamine and the co-action of CsAlaDC and CsTSI. To our knowledge, this is the first report of theanine biosynthesis, by synthetic biology, in a non-tea plant. In summary, we established that CsAlaDC works coordinately with CsTSI in determining the unique high theanine accumulation in the roots of tea plants. More importantly, we reported the first biosynthesis of theanine in a model plant, tobacco. Theanine production in this system does not require the provision of additional substrates, thereby greatly reducing costs and avoids causing environmental pollution. As theanine confers the umami taste, but also has various health effects, engineering its synthesis in non-tea plants, including crops, may well contribute to improve the health-promoting quality of the foods derived from such crops, as well as meeting the market demand for theanine. Acknowledgement This work was supported by grants from the National Natural Science Foundation of China (32072624, 31770731), the Base of Introducing Talents for Tea Plant Biology and Quality Chemistry (D20026) and Outstanding Youth Project of the Natural Science Foundation of Anhui Province (2008085J18). Conflict of interest The authors declare that they have no conflict of interest. Author contributions ZZ, TY, YW and SB conceived the study and designed the experiments; BZ, JG, CD, FL, SQ and SL carried out the experiments; BZ and ZZ wrote the manuscript, with editing by WJL; SB and WJL provided instructive comments. All authors reviewed and approved the final manuscript. 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(2021) Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation. Plant Biotechnol. J., 19(1), 98– 108. PubMedWeb of Science®Google Scholar Gu, H., Jiang, Y. and Wang, J. (2004) A practical synthesis of ethyl-L-glutamine (L-Theanine). Org. Prep. Proced. Int., 36(2), 182– 185. CrossrefCASWeb of Science®Google Scholar Higdon, J.V. and Frei, B. (2003) Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions. Crit. Rev. Food Sci. Nutr., 43(1), 89– 143. CrossrefCASPubMedWeb of Science®Google Scholar Kanarek, R., Mahoney, C., Brunye, T. and Giles, G. (2011) Theanine mitigates caffeine-accentuated stress response and reduces attentional processing biases following exposure to stress. Eur. J. Pharmacol., 668, e16. CrossrefGoogle Scholar Li, J., Mutanda, I., Wang, K., Yang, L., Wang, J. and Wang, Y. (2019) Chloroplastic metabolic engineering coupled with isoprenoid pool enhancement for committed taxanes biosynthesis in Nicotiana benthamiana. Nat. Commun., 10(1), 4850. CrossrefPubMedWeb of Science®Google Scholar Tadahiro, N. and Shinsuke, S. (1984) Differences in caffeine, flavanols and amino acids contents in leaves of cultivated species of Camellia. Japan J. Breed., 34(4), 459– 467. CrossrefGoogle Scholar Citing Literature Volume19, Issue12December 2021Pages 2395-2397 FiguresReferencesRelatedInformation

Chronic stress causes duodenal damage, in which iron death is likely to play an important role. Chlorogenic acid (CGA), one of the most widely consumed dietary polyphenols, has been shown to protect the intestine. However, it is unclear whether CGA exerts a duodenoprotective effect in chronic stress by inhibiting ferroptosis. In this work, rats were daily exposed to restraint stress for 6 h over 21 consecutive days, with/without CGA (100 mg/kg, gavage). CGA reduced blood hepcidin, iron, reactive oxygen species (ROS), and ferroportin 1 (FPN1) levels and upregulated the levels of ferroptosis-related biomarkers (GPX4, GSH, NADPH, etc.). These results confirmed that CGA inhibited ferroptosis in the duodenum. Furthermore, the use of S3I-201 (STAT3 inhibitor) helped to further clarify the mechanism of action of CGA. Overall, CGA could reduce hepcidin production by inhibiting the IL-6/JAK2/STAT3 pathway in the liver to increase the expression of FPN1 in the duodenum, which restored iron homeostasis and inhibited ferroptosis, alleviating chronic stress-induced duodenal injury.

This study aims to discover novel autoantibodies against tumor-associated antigens (TAAs) and establish diagnostic models for assisting in the diagnosis of lung cancer and discrimination of pulmonary nodules (PNs). Ten autoantibodies to TAAbs (TAAbs) were discovered by means of protein microarray and their serum level was also higher in 212 LC patients than that in 212 NC of validation cohort 1 (P < 0.05). The model 1 comprising 4 TAAbs and CEA reached an AUC of 0.813 (95%CI: 0.762–0.864) for diagnosing LC from normal individuals. Five TAAbs existed a significant difference between 105 malignant pulmonary nodules (MPNs) and 105 benign pulmonary nodules (BPNs) patients in validation cohort 2 (P < 0.05). Model 2 could distinguish MPNs from BPNs with an AUC of 0.845. High-throughput protein microarray is an efficient approach in discovering novel TAAbs which could be used as biomarkers in lung cancer diagnosis.

The culturable endophytic fungus Serendipita indica has many beneficial effects on plants, but whether and how it affects physiological activities and phosphorus (P) acquisition of tea seedlings at low P levels is unclear. The objective of this study was to analyze the effects of inoculation with S. indica on growth, gas exchange, chlorophyll fluorescence, auxins, cytokinins, P levels, and expressions of two phosphate transporter (PT) genes in leaves of tea (Camellia sinensis L. cv. Fudingdabaicha) seedlings grown at 0.5 μM (P0.5) and 50 μM (P50) P levels. Sixteen weeks after the inoculation, S. indica colonized roots of tea seedlings, with root fungal colonization rates reaching 62.18% and 81.34% at P0.5 and P50 levels, respectively. Although plant growth behavior, leaf gas exchange, chlorophyll values, nitrogen balance index, and chlorophyll fluorescence parameters of tea seedlings were suppressed at P0.5 versus P50 levels, inoculation of S. indica mitigated the negative effects to some extent, along with more prominent promotion at P0.5 levels. S. indica inoculation significantly increased leaf P and indoleacetic acid concentrations at P0.5 and P50 levels and leaf isopentenyladenine, dihydrozeatin, and transzeatin concentrations at P0.5 levels, coupled with the reduction of indolebutyric acid at P50 levels. Inoculation of S. indica up-regulated the relative expression of leaf CsPT1 at P0.5 and P50 levels and CsPT4 at P0.5 levels. It is concluded that S. indica promoted P acquisition and growth in tea seedlings under P deficit conditions by increasing cytokinins and indoleacetic acid and CsPT1 and CsPT4 expression.

A simple and sensitive analytical method was developed for the simultaneous determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk by isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Samples were directly purified through HLB cartridge. The organic phase was dried under nitrogen and residues were redissolved in mobile phase. Samples were analyzed by UPLC-MS/MS on an Acquity UPLC® BEH C(18) column with gradient elution. The samples were quantified using clenbuterol-D(9), chloramphenicol-D(5) and diethylstilbestrol-D(8) as internal standards. The proposed method was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), trueness, precision, linearity and stability. The method is demonstrated to be suitable for the determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk. The total time required for the analysis of one sample was about 50min.

A simple and cost-effective pre-treatment procedure was developed for 18 sulfonamides in pork using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). The sample was analysed after purification by ultra high-performance liquid chromatography-positive electrospray ionisation tandem mass spectrometry (UHPLC-ESI-MS/MS). After extraction with phosphate buffer (pH 6.0), a dSPE procedure was conducted with MWCNTs. The pH value of the extract, extraction time with MWCNTs, type and amount of MWCNTs and type of eluent were optimised to increase the sample throughput and sensitivity. The samples were quantified using sulfamethazine-(13)C6 as an internal standard. The recoveries of the target compounds from the pork samples were most efficient when 150mg of MWCNTs with an outer diameter of less than 8nm and a length of 0.5-2μm was used. A mixture of acetonitrile/50mM ammonium acetate (95:5, v/v) was shown to be the most suitable solvent for desorbing the compounds from the MWCNTs. The proposed method was validated according to the European Commission Decision 2002/657/EC, which determines linearity, specificity, decision limit (CCα), detection capability (CCβ), recovery, precision and stability.

The daisy-like flowers of pyrethrum (Tanacetum cinerariifolium) are used to extract pyrethrins, a botanical insecticide with a long history of safe and effective use. Pyrethrum flowers also contain other potential defense compounds, particularly sesquiterpene lactones (STLs), which represent problematic allergenic residues in the extracts that are removed by the pyrethrum industry. The STLs are stored in glandular trichomes present on the pyrethrum achenes, and have been shown to be active against herbivores, micro-organisms and in the below-ground competition with other plants. Despite these reported bioactivities and industrial significance, the biosynthetic origin of pyrethrum sesquiterpene lactones remains unknown. In the present study, we show that germacratrien-12-oic acid is most likely the central precursor for all sesquiterpene lactones present in pyrethrum. The formation of the lactone ring depends on the regio- (C6 or C8) and stereo-selective (α or β) hydroxylation of germacratrien-12-oic acid. Candidate genes implicated in three committed steps leading from farnesyl diphosphate to STL and other oxygenated derivatives of germacratrien-12-oic acid were retrieved from a pyrethrum trichome EST library, cloned, and characterized in yeast and in planta. The diversity and distribution of sesquiterpene lactones in different tissues and the correlation with the expression of these genes are shown and discussed.

Herbivore-induced plant volatiles are often involved in direct and indirect plant defence against herbivores. Linalool is a common floral scent and found to be released from leaves by many plants after herbivore attack. In this study, a linalool/nerolidol synthase, FaNES1, was overexpressed in the plastids of chrysanthemum plants (Chrysanthemum morifolium). The volatiles of FaNES1 chrysanthemum leaves were strongly dominated by linalool, but they also emitted small amount of the C11-homoterpene, (3E)-4,8-dimethyl-1,3,7-nonatriene, a derivative of nerolidol. Four nonvolatile linalool glycosides in methanolic extracts were found to be significantly increased in the leaves of FaNES1 plants compared to wild-type plants. They were putatively identified by LC-MS-MS as two linalool-malonyl-hexoses, a linalool-pentose-hexose and a glycoside of hydroxy-linalool. A leaf-disc dual-choice assay with western flower thrips (WFT, Frankliniella occidentalis) showed, initially during the first 15 min of WFT release, that FaNES1 plants were significantly preferred. This gradually reversed into significant preference for the control, however, at 20-28 h after WFT release. The initial preference was shown to be based on the linalool odour of FaNES1 plants by olfactory dual-choice assays using paper discs emitting pure linalool at similar rates as leaf discs. The reversal of preference into deterrence could be explained by the initial nonvolatile composition of the FaNES1 plants, as methanolic extracts were less preferred by WFT. Considering the common occurrence of linalool and its glycosides in plant tissues, it suggests that plants may balance attractive fragrance with 'poor taste' using the same precursor compound.

Vitamin A (VA) protects the intestinal epithelial barrier by improving cell migration and proliferation. Our previous studies demonstrated that VA deficiency (VAD) during pregnancy suppresses the systemic and mucosal immune responses in the intestines of offspring and that VA supplementation (VAS) during early life can increase immune cell counts. However, little is known about the mechanisms by which VA regulates intestinal epithelial barrier function.Caco-2 cells were treated with all-trans retinoic acid (ATRA) for 24 hours to determine the optimum ATRA concentration to which the cells in question respond. Caco-2 cells were infected with recombinant adenoviruses carrying retinoic acid receptor beta (Ad-RARβ) and small interfering RARβ(siRARβ) to assess the effects of RARβ signalling on the expression of specific proteins. A siTLR4 lentivirus was used to knockdown Toll-like receptor 4 (TLR4) in Caco-2 cells to determine its role in the protective effects of VA on the intestinal epithelial barrier, and experiments involving TLR4-knock-out mice were performed to verify the effect of TLR4. VA normal (VAN), VAD and VAS rat models were established to confirm that changes in RARβ, TLR4 and ZO-2 expression levels that occurred following decreases or increases in retinol concentrations in vivo, and the permeability of the Caco-2 cell monolayer, as well as that of the epithelial barrier of the rat intestine was detected by measuring transepithelial resistance (TER) or performing enzyme-linked immunosorbent assay (ELISA). Retinoic acid receptor (RAR), toll like receptor (TLR) and tight junction (TJ) mRNA and protein expression levels in Caco-2 cells and the colon monolayers in the rat and mouse models were measured by PCR and western blotting, respectively. Co-immunoprecipitation (co-IP) and immunofluorescence staining were performed to assess the interactions among RARβ, TLR4 and zonula occluden-2 (ZO-2) in Caco-2 cells, and chromatin immunoprecipitation (ChIP) assay was performed to assess the binding between RARβ and the TLR4 promoter sequence in Caco-2 cells.In the present study, ATRA treatment not only increased the TER of the Caco-2 monolayer but also up-regulated the expression levels of RARβ, TLR4 and ZO-2 in Caco-2 cells. The expression levels of these three proteins were significantly decreased in the colonic epithelial monolayers of VAD rats compared with those of VAN rats and were significantly increased following VAS in the corresponding group compared with the control group. Furthermore, the above changes in TLR4 and ZO-2 expression levels were augmented or attenuated by Ad-RARβ or siRARβ infection, respectively, in Caco-2 cells. Interestingly, siTLR4 down-regulated ZO-2 expression but did not affect RARβ expression in Caco-2 cells, and in VAD mice the lack of TLR4 did not affect ZO-2 expression. We noted direct interactions between RARβ and TLR4, TLR4 and ZO-2 in Caco-2 cells, and ChIP assay showed that RARβ could bind to the TLR4 promoter but not the ZO-2 promoter in Caco-2 cells.Taken together, our results indicate that RARβ enhanced ZO-2 expression by regulating TLR4 to improve intestinal epithelial barrier function in Caco-2 cells, as well as in rat and mouse models, but not in humans.

Methyl salicylate (MeSA) is one of the volatile organic compounds (VOCs) that releases floral scent and plays an important role in the sweet flowery aroma of tea. During the withering process for white tea producing, MeSA was generated by salicylic acid carboxyl methyltransferase (SAMT) with salicylic acid (SA), and the specific floral scent was formed. In this study, we first cloned a CsSAMT from tea leaves (GenBank accession no. MG459470) and used Escherichia coli and Saccharomyces cerevisiae to express the recombinant CsSAMT. The enzyme activity in prokaryotic and eukaryotic expression systems was identified, and the protein purification, substrate specificity, pH, and temperature optima were investigated. It was shown that CsSAMT located in the chloroplast, and the gene expression profiles were quite different in tea organs. The obtained results might give a new understanding for tea aroma formation, optimization, and regulation and have great significance for improving the specific quality of white tea.

Nanofibrous materials produced by electrospinning have attracted considerable attention from researchers in regenerative medicine. A combination of nanofibrous scaffold and chondrocytes is considered promising for repair of cartilage defect or damage. In the present study, we fabricated a poly(l-lactic-acid) (PLLA)/silk fibroin (SF) nanofibrous scaffold by electrospinning and evaluated its chondrogenic potential. The PLLA/SF nanofibers were characterized for diameter, surface wettability, swelling ratio, and tensile strength. Throughin vitroexperiments, PLLA/SF scaffold-chondrocyte interactions were investigated relative to the unmodified PLLA scaffold with regard to cellular adhesion, spreading, and proliferation by scanning electron microscopy and confocal laser scanning microscopy, and through analyses of DNA, sulfated glycosaminoglycan, and collagen. In addition, hematoxylin-eosin and Alcian blue-nuclear fast red staining were used to observe growth of chondrocytes, and secretion and distribution of cartilage-specific extracellular matrices in the scaffolds. Expressions of cartilage-related genes (collagen II, aggrecan, sox9, collagen I, and collagen X) were detected by real-time quantitative PCR. The PLLA/SF scaffold had better hydrophilicity, and could support chondrocytes adhesion and spreading more effectively than the unmodified PLLA scaffold. Chondrocytes secreted more cartilage-specific extracellular matrices and maintained their phenotype on the PLLA/SF scaffold. So it is concluded that the PLLA/SF scaffold is more conducive toin vitroformation of cartilage-like new tissues than the unmodified PLLA scaffold, and may be a promising material in cartilage tissue engineering.

Autophagy regulates cell survival (or cell death in several cases), whereas apoptosis regulates cell death. However, the relationship between autophagy and apoptosis and the regulative mechanism is unclear. We report that steroid hormone 20-hydroxyecdysone (20E) promotes switching from autophagy to apoptosis by increasing intracellular calcium levels in the midgut of the lepidopteran insect Helicoverpa armigera. Autophagy and apoptosis sequentially occurred during midgut programmed cell death under 20E regulation, in which lower concentrations of 20E induced microtubule-associated protein 1 light chain 3–phosphatidylethanolamine (LC3–II, also known as autophagy-related gene 8, ATG8) expression and autophagy. High concentrations of 20E induced cleavage of ATG5 to NtATG5 and pro-caspase-3 to active caspase-3, which led to a switch from autophagy to apoptosis. Blocking autophagy by knockdown of ATG5, ATG7, or ATG12, or with the autophagy inhibitor 3-methyladenine, inhibited 20E-induced autophagy and apoptosis. Blocking apoptosis by using the apoptosis inhibitor Ac-DEVD-CHO did not prevent 20E-induced autophagy, suggesting that apoptosis relies on autophagy. ATG5 knockdown resulted in abnormal pupation and delayed pupation time. High concentrations of 20E induced high levels of intracellular Ca2+, NtATG5, and active caspase-3, which mediated the switch from autophagy to apoptosis. Blocking 20E-mediated increase of cellular Ca2+ caused a decrease of NtATG5 and active caspase-3 and repressed the transformation from autophagy to apoptosis, thereby promoting cell survival. 20E induces an increase in the concentration of intracellular Ca2+, thereby switching autophagic cell survival to apoptotic cell death.

Objectives: This study was designed to investigate the vitamin A (VA) and vitamin D (VD) levels in children with autism spectrum disorders (ASD) and to determine whether co-deficiency of VA and VD exacerbates clinical symptoms in autistic children.Methods: The Autism Behavior Checklist, Childhood Autism Rating Scale (CARS), and Social Responsiveness Scale (SRS) were used to assess the symptoms of 332 children diagnosed as ASD. And the Gesell Developmental Scale (GDS) was used to evaluate neurodevelopment in children with ASD. Anthropometric measurement and questionnaire results were compared for all autistic children and 197 age- and gender-matched control children. Serum retinol levels were detected with high-performance liquid chromatography, and serum levels of 25-OH vitamin D were measured with an immunoassay method in the two groups.Results: The ZHA, ZWA, and ZBMIA of the children with ASD were significantly lower than those of the control children. Furthermore, higher proportions of children with picky eating, resistance to new foods, and eating problems were observed in the ASD group when compared with the control group. Serum retinol and 25-OH vitamin D levels in autistic children were significantly lower than those in the control children. Additionally, VA and VD co-deficiency impacts more on the symptoms and development in autistic children.Conclusions: We found that children with autism have more VA and VD deficiencies than control children, and VA and VD co-deficiency may exacerbate the symptoms of children with ASD.

The wheat aphids, Rhopalosiphum padi (Linnaeus) and Sitobion avenae (Fabricius), are key pests on wheat crops worldwide. Management practices rely primarily on insecticides. The pirimicarb (carbamate) is used extensively as an effective insecticide to control these two aphids. In addition to the mortality caused by pirimicarb, various sublethal effects may occur in aphids when exposed to low lethal or sublethal doses. Understanding the general effect of pirimicarb on aphids could help increasing rational use of this insecticide. Under laboratory conditions, we assessed the sublethal effects of a low lethal concentration of pirimicarb (LC25) on biological traits and acetylcholinesterase (AChE) activity of R. padi and S. avenae. Both direct and transgenerational effects, i.e. on parent and the F1 generations were assessed, respectively. We found that R. padi and S. avenae responded differentially to the LC25 of pirimicarb. The parent generation of R. padi showed a 39% decrease in fecundity and multiple transgenerational effects were observed in the F1 generation; overall juvenile development, reproductive period, adult longevity and lifespan were longer than those of the control group. By contrast, LC25 of pirimicarb showed almost no effects on S. avenae biological traits in both the parent and F1 generations; only the pre-reproductive duration was reduced in F1 generations. Demographic parameter estimates (e.g. rm) showed similar trend, i.e. significant negative effect on R. padi population growth and no effect on S. avenae. However, AChE activity decreased in both R. padi and S. avenae treated by the LC25 of pirimicarb. We demonstrated sublethal and transgenerational effects of pirimicarb in the two wheat aphid species; it hinted at the importance of considering sublethal effects (including hormesis) of pirimicarb for optimizing Integrated Pest Management (IPM) of wheat aphids.

Lupins (Lupinus spp.) are nitrogen-fixing legumes that accumulate toxic alkaloids in their protein-rich beans. These anti-nutritional compounds belong to the family of quinolizidine alkaloids (QAs), which are of interest to the pharmaceutical and chemical industries. To unleash the potential of lupins as protein crops and as sources of QAs, a thorough understanding of the QA pathway is needed. However, only the first enzyme in the pathway, lysine decarboxylase (LDC), is known. Here, we report the transcriptome of a high-QA variety of narrow-leafed lupin (L. angustifolius), obtained using eight different tissues and two different sequencing technologies. In addition, we present a list of 33 genes that are closely co-expressed with LDC and that represent strong candidates for involvement in lupin alkaloid biosynthesis. One of these genes encodes a copper amine oxidase able to convert the product of LDC, cadaverine, into 1-piperideine, as shown by heterologous expression and enzyme assays. Kinetic analysis revealed a low KM value for cadaverine, supporting a role as the second enzyme in the QA pathway. Our transcriptomic data set represents a crucial step towards the discovery of enzymes, transporters, and regulators involved in lupin alkaloid biosynthesis.

Scope Vitamin A (VA) is an essential nutrient for the development of the brain. We previously found that children with autism spectrum disorder (ASD) have a significant rate of VA deficiency (VAD). In the current study, we aim to determine whether VAD is a risk factor for the generation of autistic‐like behaviors via the transcription factor retinoic acid receptor beta (RARβ)‐regulated cluster of differentiation 38 (CD38)‐oxytocin (OXT) axis. Methods and results Gestational VAD or VA supplementation (VAS) rat models are established, and the autistic‐like behaviors in the offspring rats are investigated. The different expression levels of RARβ and CD38 in hypothalamic tissue and serum retinol and OXT concentration are tested. Primary cultured rat hypothalamic neurons are treated with all‐trans retinoic acid (atRA), and recombinant adenoviruses carrying the rat RARβ (AdRARβ) or RNA interference virus RARβ‐siRNA (siRARβ) are used to infect neurons to change RARβ signal. Western blotting, chromatin immunoprecipitation (ChIP), and intracellular Ca 2+ detections are used to investigate the primary regulatory mechanism of RARβ in the CD38‐OXT signaling pathway. We found that gestational VAD increases autistic‐like behaviors and decreases the expression levels of hypothalamic RARβ and CD38 and serum OXT levels in the offspring. VAS ameliorates these autistic‐like behaviors and increases the expression levels of RARβ, CD38, and OXT in the gestational VAD pups. In vitro, atRA increases the Ca 2+ excitability of neurons, which might further promote the release of OXT. Different CD38 levels are induced in the neurons by infection with different RARβ adenoviruses. Furthermore, atRA enhances the binding of RARβ to the proximal promoter of CD38, indicating a potential upregulation of CD38 transcriptional activity by RARβ. Conclusions Gestational VAD might be a risk factor for autistic‐like behaviors due to the RARβ signal suppression of CD38 expression in the hypothalamus of the offspring, which improves with VAS during the early‐life period. The nutritional status during pregnancy and the early‐life period is important in rats.

Mesenchymal stem cells (MSCs) treatment is an effective strategy for the functional repair of central nervous system (CNS) insults through the production of bioactive molecules. We have previously demonstrated that the interleukin-6 (IL-6) secreted by MSCs plays an anti-apoptotic role in injured astrocytes and partly promotes functional recovery in neonatal rats with hypoxic-ischemic brain damage (HIBD). However, the mechanisms of IL-6 underlying the proliferation of injured astrocytes have not been fully elucidated. In this study, we investigated the therapeutic effects of MSCs on astrocyte proliferation in neonatal rats subjected to HIBD. A HIBD model was established in Sprague Dawley (SD) rats, and MSCs were administered by intracerebroventricular injection 5 days after HIBD. Rat primary astrocytes were cultured, subjected to oxygen glucose deprivation (OGD) injury and then immediately co-cultured with MSCs in vitro. Immunofluorescence staining, Cell Counting Kit (CCK)-8, flow cytometry, Ca2+ imaging, enzyme-linked immunosorbent assay (ELISA), western blotting, and co-immunoprecipitation (Co-IP) were performed. We found that MSCs transplantation not only promoted the recovery of learning and memory function in HIBD rats but also significantly reduced the number of Ki67+/glial fibrillary acidic protein (GFAP)+ cells in the hippocampi 7–14 days after HIBD. In addition to increasing IL-6 expression in both the hippocampi of HIBD rats and astrocyte culture medium, MSCs treatment in vitro significantly increased the expression levels of glycoprotein (gp) 130 and phosphorylated AMP-activated protein kinase α (p-AMPKα) and decreased the expression levels of p-mammalian target of rapamycin (mTOR) and its downstream targets. Furthermore, MSCs treatment induced a protein-protein interaction between gp130 and p-AMPKα. Suppression of IL-6 expression in MSCs reversed the above regulatory functions of MSCs in hippocampal astrocytes. The utilization of rapamycin further confirmed that mTOR participated in the proliferation of reactive astrocytes. These findings suggest that endogenous IL-6 produced by MSCs in the HIBD microenvironment provides therapeutic advantages by activating AMPK/mTOR signaling, thus reducing the proliferation of reactive astrocytes.

Abstract Fatness traits are important in pigs because of their implications for fattening efficiency, meat quality, reproductive performance and immunity. Songliao black pigs and Landrace pigs show important differences in production and meat quality traits, including fatness and muscle growth. Therefore, we used a high-throughput massively parallel RNA-seq approach to identify genes differentially expressed in backfat tissue between these two breeds (six pigs in each). An average of 37.87 million reads were obtained from the 12 samples. After statistical analysis of gene expression data by edgeR, a total of 877 differentially expressed genes were detected between the two pig breeds, 205 with higher expression and 672 with lower expression in Songliao pigs. Candidate genes (LCN2, CES3, DGKB, OLR1, LEP, PGM1, PCK1, ACACB, FADS1, FADS2, MOGAT2, SREBF1, PPARGC1B) with known effects on fatness traits were included among the DEGs. A total of 1071 lncRNAs were identified, and 85 of these lncRNAs were differentially expressed, including 53 up-regulated and 32 down-regulated lncRNAs, respectively. The differentially expressed genes and lncRNAs involved in glucagon signaling pathway, glycolysis/gluconeogenesis, insulin signaling pathway, MAPK signaling pathway and so on. Integrated analysis potential trans-regulating or cis-regulating relation between DEGs and DE lncRNAs, suggested lncRNA MSTRG.2479.1 might regulate the expressed level of VLDLR affecting porcine fat metabolism. These results provide a number of candidate genes and lncRNAs potentially involved in porcine fat deposition and provide a basis for future research on the molecular mechanisms underlying in fat deposition.

Theanine, a unique and abundant non-proteinogenic amino acid in tea, confers to the tea infusion its umami taste and multiple health benefits. Its content in new tea shoots is dynamic in winter and spring. However, its seasonal accumulation pattern and the underlying regulation mechanism of tea plants remain largely unknown. In this study, we measured the theanine contents in the roots and leaf buds of 13 tea cultivars at four time points from winter to spring (Dec. 12, Mar. 1, Mar. 23, and Apr. 13). We found theanine accumulated significantly in the roots to as high as ∼6% dry weight. We found theanine content in the roots was constant or slightly decreased on Mar. 1 compared with Dec.12 but increased consistently on Mar. 23 and then decreased on Apr. 13 in all 13 cultivars. In the leaf buds, theanine content kept increasing from Mar. 1 to Mar. 23 and decreasing from Apr. 13 in most of the 13 cultivars, meaning it was probably both season- and developmental stage-dependent. The expression of theanine biosynthesis and amino acid transporter genes in the roots and buds at the four time points was then examined. The correlation analyses between the gene expression and theanine content suggested the expression of theanine-biosynthesis genes was generally and negatively correlated with theanine content; however, the expression of amino acid transporter genes including CsLHT was generally and positively correlated with theanine contents. Finally, we showed that CsLHT has theanine transport activity. Taken together, this study provided insight into the seasonal regulation of theanine biosynthesis and transport in tea plants during winter and spring.

The goal of this study was to evaluate the microbial communities in the gut and feces from female finishing Landrace pigs with high and low feed conversion ratio (FCR) by 16S rRNA gene amplicon sequencing. Many potential biomarkers can distinguish between high and low FCR groups in the duodenum, ileum, cecum, colon, and rectum, according to linear discriminant analysis effect sizes. The relative abundance of microbes were tested by Mann-Whitney test between the high and low FCR groups in different organs: Campylobacter, Prevotella and Sphaerochaeta were different in the duodenum (P < 0.05); Sanguibacter, Kingella and Anaeroplasma in jejunum; Anaeroplasma, Arthrobacter, Kingella, Megasphaera and SMB53 in the ileum; Butyricicoccus, Campylobacter, Mitsuokella, and Coprobacillus in the cecum; Lactococcus and Peptococcus in the colon; Staphylococcus in the rectum; and Rothia in feces. The prevalence of microbial genera in certain locations could potentially be used as biomarkers to distinguish between high and low FCR. Functional prediction clustering analysis suggested that bacteria in the hindgut mainly participated in carbohydrate metabolism and amino acid metabolism, and different in the relative abundance of metabolic pathways, as predicted from the microbial taxa present, were identified by comparing the high and low groups of each location. The results may provide insights for the alteration of the intestinal microbial communities to improve the growth rate of pigs.

Objectives: To investigate the specific role of TLR4 in the regulation of the intestinal mucosa-associated flora by vitamin A (VA). Methods: Both TLR4-/- (KO) and wild-type (WT) female mice were randomly fed a VA normal (VAN) or VA deficiency (VAD) diet for 4 weeks to establish the following four mouse model groups: TLR4-/- mice fed a VAN diet (KO VAN), TLR4-/- mice fed a VAD diet (KO VAD), WT mice fed a VAN diet (WT VAN), and WT mice fed a VAD diet (WT VAD) . Then, the female mice from each experimental group were mated with male mice with the same genetic background. The pups in the KO VAD and WT VAD groups were subsequently fed the VAD diet after weaning, while the pups in the KO VAN and WT VAN groups were fed the VAN diet continuously after weaning. The serum retinol levels of 7-week-old offspring were determined using high-performance liquid chromatography, and the colons from each group of mice were collected and analyzed by using an Illumina MiSeq platform for 16S rRNA gene sequencing to characterize the overall microbiota of the samples. Results: The abundance and evenness of the colon mucosa-associated microflora were unaffected by dietary VA and TLR4-/-. VAD decreased the abundances of Anaerotruncus (Firmicutes), Oscillibacter (Firmicutes), Lachnospiraceae _NK4A136 _group (Firmicutes) and Mucispirillum (Deferribacteres) and increased the abundance of Parasutterella (Proteobacteria). TLR4-/- decreased the abundances of Bacteroides (Bacteroidetes) and Alloprevotella (Bacteroidetes). However, the abundances of Allobaculum (Firmicutes), Ruminiclostridium_9 (Firmicutes), Alistipes (Bacteroidetes) and Rikenellaceae_RC9 (Bacteroidetes) impacted the interaction between VA and TLR4. Conclusions: TLR4 may play a pivotal role in the regulation of the intestinal mucosa-associated flora by VA to maintain the intestinal microecology.

Objective: The study aimed to compare the nutritional status and symptoms of preschool children with autism spectrum disorder (ASD) from two regions of China, and to analyze the association between nutritional status and symptoms of ASD. Methods: In this cross-sectional study, 738 ASD children and 302 typically developing children (TD) were recruited from Chongqing and Hainan of China. Symptoms of ASD children were evaluated with the Autism Behavior Checklist (ABC), Social Responsiveness Scale (SRS), and Childhood Autism Rating Scale (CARS). Neurodevelopment of ASD children was assessed with the Gesell Developmental Scale (GDS). Nutritional status was evaluated by anthropometric measures, biochemical detection of micronutrients, and providing questionnaire and food frequency questionnaire (FFQ) to caregivers. Results: Comparing ASD children with local TD children, ASD children consumed fewer whole grains, milk and dairy products, beans and soy products, vegetables, and fruits than local TD children in both regions. The serum concentrations of folate, vitamin B12 (VB12), and vitamin D (VD) were consistently lower in ASD children in both regions. Comparing the ASD children between the two regions, the ASD children in Chongqing had significantly higher mean scores of CARS, SRS, and ABC than those in Hainan. The ASD children in Chongqing consumed fewer whole grains, seafood, and fruits than those in Hainan. The serum concentrations of ferritin, vitamin A (VA), VB12, and VD were reduced in the ASD children of Chongqing than those in Hainan, and the ASD children in Chongqing had higher deficiency rates of zinc, ferritin, VA, and VD than those in Hainan. The serum levels of VA, VD, and folate showed a negative association with symptom scores of ASD children. VD and zinc levels had a positive association with the GDS scores of ASD children. Conclusions: ASD children exhibit a higher risk of nutrient deficiencies than neurotypical children, and there are regional differences in the nutritional status of ASD children. Micronutrients VA, VD, folate, and zinc levels were correlated with symptoms and development of ASD children. Therefore, it is essential to provide detailed nutrition evaluation and individualized nutrition interventions for ASD children from different backgrounds.

Plant symptoms are derived from specific interactions between virus and host components. However, little is known about viral or host factors that participate in the establishment of systemic necrosis. Here, we showed that helper component proteinase (HCPro), encoded by Chilli veinal mottle virus (ChiVMV), could directly interact with catalase 1 (CAT1) and catalase 3 (CAT3) in the cytoplasm of tobacco (Nicotiana tabacum) plants to facilitate viral infection. In vitro, the activities of CAT1 and CAT3 were inhibited by the interaction between HCPro and CATs. The C-terminus of HCPro was essential for their interaction and was also required for the decrease of enzyme activities. Interestingly, the mRNA and protein level of CATs were up-regulated in tobacco plants in response to ChiVMV infection. Nicotiana tabacum plants with HCPro overexpression or CAT1 knockout were more susceptible to ChiVMV infection, which was similar to the case of H2O2-pre-treated plants, and the overexpression of CAT1 inhibited ChiVMV accumulation. Also, neither CAT1 nor CAT3 could affect the RNA silencing suppression (RSS) activity of HCPro. Our results showed that the interaction between HCPro and CATs promoted the development of plant systemic necrosis, revealing a novel role for HCPro in virus infection and pathogenicity.

Abstract Background Tea plant is one of the most important non-alcoholic beverage crops worldwide. While potassium (K + ) is an essential macronutrient and greatly affects the growth and development of plants, the molecular mechanism underlying K + uptake and transport in tea plant root, especially under limited-K + conditions, is still poorly understood. In plants, HAK/KUP/KT family members play a crucial role in K + acquisition and translocation, growth and development, and response to stresses. Nevertheless, the biological functions of these genes in tea plant are still in mystery, especially their roles in K + uptake and stress responses. Results In this study, a total of 21 non-redundant HAK/KUP/KT genes (designated as CsHAKs ) were identified in tea plant. Phylogenetic and structural analysis classified the CsHAKs into four clusters (I, II, III, IV), containing 4, 8, 4 and 5 genes, respectively. Three major categories of cis -acting elements were found in the promoter regions of CsHAKs . Tissue-specific expression analysis indicated extremely low expression levels in various tissues of cluster I CsHAKs with the exception of a high root expression of CsHAK4 and CsHAK5 , a constitutive expression of clusters II and III CsHAKs , and a moderate cluster IV CsHAKs expression. Remarkably, the transcript levels of CsHAKs in roots were significantly induced or suppressed after exposure to K + deficiency, salt and drought stresses, and phytohormones treatments. Also notably, CsHAK7 was highly expressed in all tissues and was further induced under various stress conditions. Therefore, functional characterization of CsHAK7 was performed, and the results demostrated that CsHAK7 locates on plasma membrane and plays a key role in K + transport in yeast. Taken together, the results provide promising candidate CsHAKs for further functional studies and contribute to the molecular breeding for new tea plants varieties with highly efficient utilization of K + . Conclusion This study demonstrated the first genome-wide analysis of CsHAK family genes of tea plant and provides a foundation for understanding the classification and functions of the CsHAKs in tea plants.

Abstract Organic tea is more popular than conventional tea that originates from fertilized plants. Amino acids inorganic soils constitute a substantial pool nitrogen (N) available for plants. However, the amino-acid contents in soils of tea plantations and how tea plants take up these amino acids remain largely unknown. In this study, we show that the amino-acid content in the soil of an organic tea plantation is significantly higher than that of a conventional tea plantation. Glutamate, alanine, valine, and leucine were the most abundant amino acids in the soil of this tea plantation. When 15 N-glutamate was fed to tea plants, it was efficiently absorbed and significantly increased the contents of other amino acids in the roots. We cloned seven CsLHT genes encoding amino-acid transporters and found that the expression of CsLHT1 , CsLHT2, and CsLHT6 in the roots significantly increased upon glutamate feeding. Moreover, the expression of CsLHT1 or CsLHT6 in a yeast amino-acid uptake-defective mutant, 22∆10α, enabled growth on media with amino acids constituting the sole N source. Amino-acid uptake assays indicated that CsLHT1 and CsLHT6 are H + -dependent high- and low-affinity amino-acid transporters, respectively. We further demonstrated that CsLHT1 and CsLHT6 are highly expressed in the roots and are localized to the plasma membrane. Moreover, overexpression of CsLHT1 and CsLHT6 in Arabidopsis significantly improved the uptake of exogenously supplied 15 N-glutamate and 15 N-glutamine. Taken together, our findings are consistent with the involvement of CsLHT1 and CsLHT6 in amino-acid uptake from the soil, which is particularly important for tea plants grown inorganic tea plantations.

Objective: To investigate the role of TLR4 on the microglia activation in the pre-frontal cortex, which leads to autism-like behavior of the offspring induced by maternal lipopolysaccharide (LPS) exposure. Methods: Pregnant TLR4-/- (knockout, KO) and WT (wild type, WT) dams were intraperitoneally injected with LPS or PBS, respectively. The levels of TNFα, IL-1β, and IL-6 in the maternal serum and fetal brain were assessed with ELISA following LPS exposure. The gestation period, litter size and weight of the offspring were evaluated. Three-chamber sociability test, open field test and olfactory habituation/dishabituation test were used to assess the offspring's autism-like behavior at 7 weeks of age. Western blotting was performed to examine the levels of TLR4, Phospho-NFκB p65, IKKα, IBA-1, iNOS, Arg-1, C3, CR3A, NMDAR2A, and Syn-1 expression in the pre-frontal cortex. The morphological changes in the microglia, the distribution and expression of TLR4 were observed by immunofluorescence staining. Golgi-Cox staining was conducted to evaluate the dendritic length and spine density of the neurons in 2-week-old offspring. Results: Maternal LPS stimulation increased serum TNFα and IL-6, as well as fetal brain TNFα in the WT mice. The litter size and the weight of the WT offspring were significantly reduced following maternal LPS treatment. LPS-treated WT offspring had lower social and self-exploration behavior, and greater anxiety and repetitive behaviors. The protein expression levels of TLR4 signaling pathways, including TLR4, Phospho-NFκB p65, IKKα, and IBA-1, iNOS expression were increased in the LPS-treated WT offspring, whereas Arg-1 was decreased. Maternal LPS treatment resulted in the significant reduction in the levels of the synaptic pruning-related proteins, C3 and CR3A. Moreover, the neuronal dendritic length and spine density, as well as the expression levels of the synaptic plasticity-related proteins, NMDAR2A and Syn-1 were reduced in the WT offspring; however, gestational LPS exposure had no effect on the TLR4-/- offspring. Conclusion: Activation of TLR4 signaling pathway following maternal LPS exposure induced the abnormal activation of microglia, which in turn was involved in excessive synaptic pruning to decrease synaptic plasticity in the offspring. This may be one of the reasons for the autism-like behavior in the offspring mice.

Insoluble dietary fibers derived from <italic>Laminaria japonica</italic> attenuate high-fat diet-induced obesity associated with restoration of <italic>Akkermansia</italic> to the normal level.

Dysregulation of DNA methylation in B cells has been observed during their neoplastic transformation and therefore closely associated with various B-cell malignancies including multiple myeloma (MM), a malignancy of terminally differentiated plasma cells. Emerging evidence has unveiled pronounced alterations in DNA methylation in MM, including both global and gene-specific changes that can affect genome stability and gene transcription. Moreover, dysregulated expression of DNA methylation-modifying enzymes has been related with myelomagenesis, disease progression, and poor prognosis. However, the functional roles of the epigenetic abnormalities involving DNA methylation in MM remain elusive. In this article, we review current understanding of the alterations in DNA methylome and DNA methylation modifiers in MM, particularly focusing on DNA methyltransferases (DNMTs) and tet methylcytosine dioxygenases (TETs). We also discuss how these DNA methylation modifiers may be regulated and function in MM cells, therefore providing a rationale for developing novel epigenetic therapies targeting DNA methylation in MM.

Microglial dysfunction has been reported in the valproic acid (VPA)-induced autism spectrum disorder (ASD) rat models. However, how does prenatal VPA exposure affect microglia remains to be elucidated. The triggering receptor expressed on myeloid cells 2 (TREM2) is revealed to be implicated in a range of microglia functions. However, reports on the association between TREM2 and VPA-induced ASD rat models are scarce. Our results showed that prenatal VPA exposure induced autistic-like behaviors, downregulated the levels of TREM2, up-regulated microglial activation, dysregulated microglial polarization, and altered synapse in offspring. TREM2 overexpression partly ameliorated microglia dysfunction and autistic-like behaviors in prenatal VPA-exposed rats. Our findings demonstrated that prenatally VPA exposure is likely to cause autistic-like behavior in the rat offspring via TREM2 down-regulation to affect the microglial activation, microglial polarization and synaptic pruning of microglia for the first time.

ABSTRACT Short-chain fatty acids (SCFAs, especially butyric acid) have been demonstrated to play a promising role in the development of autism spectrum disorders (ASD). Recently, the hypothalamic–pituitary–adrenal (HPA) axis is also suggested to increase the risk of ASD. However, the mechanism underlying SCFAs and HPA axis in ASD development remains unknown. Here, we show that children with ASD exhibited lower SCFA concentrations and higher cortisol levels, which were recaptured in prenatal lipopolysaccharide (LPS)-exposed rat model of ASD. These offspring also showed decreased SCFA-producing bacteria and histone acetylation activity as well as impaired corticotropin-releasing hormone receptor 2 (CRHR2) expression. Sodium butyrate (NaB), which can act as histone deacetylases inhibitors, significantly increased histone acetylation at the CRHR2 promoter in vitro and normalized the corticosterone as well as CRHR2 expression level in vivo . Behavioral assays indicated ameliorative effects of NaB on anxiety and social deficit in LPS-exposed offspring. Our results imply that NaB treatment can improve ASD-like symptoms via epigenetic regulation of the HPA axis in offspring; thus, it may provide new insight into the SCFA treatment of neurodevelopmental disorders like ASD. IMPORTANCE Growing evidence suggests that microbiota can affect brain function and behavior through the “microbiome–gut–brain’’ axis, but its mechanism remains poorly understood. Here, we show that both children with autism and LPS-exposed rat model of autism exhibited lower SCFA concentrations and overactivation of HPA axis. SCFA-producing bacteria, Lactobacillus , might be the key differential microbiota between the control and LPS-exposed offspring. Interestingly, NaB treatment contributed to the regulation of HPA axis (such as corticosterone as well as CRHR2) and improvement of anxiety and social deficit behaviors in LPS-exposed offspring. The potential underlying mechanism of the ameliorative effect of NaB may be mediated via increasing histone acetylation to the CRHR2 promoter. These results enhance our understanding of the relationship between the SCFAs and the HPA axis in the development of ASD. And gut microbiota-derived SCFAs may serve as a potential therapeutic agent to neurodevelopmental disorders like ASD.

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report a new assembly of the N. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated three to four times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and uncovers the crucial role of BBLs in the stereoselectivity of nicotine biosynthesis.

In recent years, the incidence rate of diabetes mellitus (DM), including type 1 diabetes mellitus(T1DM), type 2 diabetes mellitus(T2DM), and gestational diabetes mellitus (GDM), has increased year by year and has become a major global health problem. DM can lead to serious complications of macrovascular and microvascular. Tryptophan (Trp) is an essential amino acid for the human body. Trp is metabolized in the body through the indole pathway, kynurenine (Kyn) pathway and serotonin (5-HT) pathway, and is regulated by intestinal microorganisms to varying degrees. These three metabolic pathways have extensive regulatory effects on the immune, endocrine, neural, and energy metabolism systems of the body, and are related to the physiological and pathological processes of various diseases. The key enzymes and metabolites in the Trp metabolic pathway are also deeply involved in the pathogenesis of DM, playing an important role in pancreatic function, insulin resistance (IR), intestinal barrier, and angiogenesis. In DM and its complications, there is a disruption of Trp metabolic balance. Several therapy approaches for DM and complications have been proven to modify tryptophan metabolism. The metabolism of Trp is becoming a new area of focus for DM prevention and care. This paper reviews the impact of the three metabolic pathways of Trp on the pathogenesis of DM and the alterations in Trp metabolism in these diseases, expecting to provide entry points for the treatment of DM and its complications.

Preeclampsia (PE) is a pregnancy-related vascular disorder which is the leading cause of maternal morbidity and mortality. We sought to identify novel serological protein markers to diagnose PE with a multi-'omics' based discovery approach.Seven previous placental expression studies were combined for a multiplex analysis, and in parallel, two-dimensional gel electrophoresis was performed to compare serum proteomes in PE and control subjects. The combined biomarker candidates were validated with available ELISA assays using gestational age-matched PE (n=32) and control (n=32) samples. With the validated biomarkers, a genetic algorithm was then used to construct and optimize biomarker panels in PE assessment.In addition to the previously identified biomarkers, the angiogenic and antiangiogenic factors (soluble fms-like tyrosine kinase (sFlt-1) and placental growth factor (PIGF)), we found 3 up-regulated and 6 down-regulated biomakers in PE sera. Two optimal biomarker panels were developed for early and late onset PE assessment, respectively.Both early and late onset PE diagnostic panels, constructed with our PE biomarkers, were superior over sFlt-1/PIGF ratio in PE discrimination. The functional significance of these PE biomarkers and their associated pathways were analyzed which may provide new insights into the pathogenesis of PE.

Litchi chinensis pericarp from litchi processing waste is an important plant source of A-type procyanidins, which were considered a natural dietary supplement because of their high biological activity in vivo. Litchi pericarp oligomeric procyanidins (LPOPCs) did not selectively modify the growth of Streptococcus thermophilus and Lactobacillus casei -01 at concentrations of 0.25 and 0.5 mg/mL, and it was demonstrated that the two strains could transform procyanidins during their log period of growth by two different pathways. S. thermophilus was able to metabolize procyanidin A2 to its isomer, and L. casei could decompose flavan-3-ols into 3,4-hydroxyphenylacetic acid, 4-hydroxyphenylpropionic acid, m-coumaric acid, and p-coumaric acid. The total antioxidant capability (T-AOC) of LPOPCs before and after microbial incubation was estimated, and the results suggested that probiotic bacteria bioconversion is a feasible and efficient method to convert litchi pericarp procyanidins to a more effective antioxidant agent.

Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12–0.16 μg h−1 g−1 fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.Chrysanthemyl diphosphate synthase (CDS) is known to catalyze the formation of the irregular terpenoid, chrysanthemyl diphosphate (CPP).ResultsCDS also catalyzes the next step from CPP to chrysanthemol.ConclusionCDS is actually a chrysanthemol synthase (CHS) with bifunctional enzyme activity.SignificanceIdentification of CHS increases our understanding in terpene biosynthesis and paves the way for engineering biosynthesis of the most widely used natural pesticide, pyrethrins. Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12–0.16 μg h−1 g−1 fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.Chrysanthemyl diphosphate synthase (CDS) is known to catalyze the formation of the irregular terpenoid, chrysanthemyl diphosphate (CPP). CDS also catalyzes the next step from CPP to chrysanthemol. CDS is actually a chrysanthemol synthase (CHS) with bifunctional enzyme activity.

A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method using multi-walled carbon nanotubes (MWCNTs) as a reversed-dispersive solid phase extraction (r-dSPE) material combined with ultra-high liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) was developed for the simultaneous determination of amantadine, rimantadine and memantine in chicken muscle. The satisfactory separation of isomers (rimantadine and memantine) was obtained on an Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm) after optimization of mobile phase composition, column temperature and flow rate. The method involved an acetonitrile-based sample preparation and a dSPE clean-up procedure with MWCNTs material. Variations in the type and amount of MWCNTs, the pH value of the extract, the extraction time for MWCNTs, and the type of eluent were used to determine the optimal parameters for increasing the sample throughput and the sensitivity. The samples were quantified using amantadine-D15, rimantadine-D4 and memantine-D6 as the internal standards. Under the optimized conditions, recoveries of 96.8–104.6% and the values of coefficient of variation (CV) of 3.8–6.4% were obtained for the three drugs in chicken muscle at three spiked levels (0.5, 1.0 and 1.5 μg/kg), and the decision limits (CCα) and detection capabilities (CCβ) were 0.15–0.20 μg/kg and 0.20–0.25 μg/kg, respectively. Positive results were obtained from local supermarket using this method, and the concentrations obtained from the newly developed method compared well to the previously reported method.

We sought to identify serological markers capable of diagnosing preeclampsia (PE). We performed serum peptide analysis (liquid chromatography mass spectrometry) of 62 unique samples from 31 PE patients and 31 healthy pregnant controls, with two-thirds used as a training set and the other third as a testing set. Differential serum peptide profiling identified 52 significant serum peptides, and a 19-peptide panel collectively discriminating PE in training sets (n = 21 PE, n = 21 control; specificity = 85.7% and sensitivity = 100%) and testing sets (n = 10 PE, n = 10 control; specificity = 80% and sensitivity = 100%). The panel peptides were derived from 6 different protein precursors: 13 from fibrinogen alpha (FGA), 1 from alpha-1-antitrypsin (A1AT), 1 from apolipoprotein L1 (APO-L1), 1 from inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), 2 from kininogen-1 (KNG1), and 1 from thymosin beta-4 (TMSB4). We concluded that serum peptides can accurately discriminate active PE. Measurement of a 19-peptide panel could be performed quickly and in a quantitative mass spectrometric platform available in clinical laboratories. This serum peptide panel quantification could provide clinical utility in predicting PE or differential diagnosis of PE from confounding chronic hypertension.

Identification of maternal environmental factors influencing preterm birth risks is important to understand the reasons for the increase in prematurity since 1990. Here, we utilized a health survey, the US National Health and Nutrition Examination Survey (NHANES) to search for personal environmental factors associated with preterm birth. 201 urine and blood markers of environmental factors, such as allergens, pollutants, and nutrients were assayed in mothers (range of N: 49-724) who answered questions about any children born preterm (delivery <37 weeks). We screened each of the 201 factors for association with any child born preterm adjusting by age, race/ethnicity, education, and household income. We attempted to verify the top finding, urinary bisphenol A, in an independent study of pregnant women attending Lucile Packard Children's Hospital. We conclude that the association between maternal urinary levels of bisphenol A and preterm birth should be evaluated in a larger epidemiological investigation.

Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a transmembrane zinc-endopeptidase that breaks down extracellular matrix components, including several collagens, during tissue development and physiological remodeling. MT1-MMP-deficient mice (MT1-MMP(-/-)) feature severe defects in connective tissues, such as impaired growth, osteopenia, fibrosis, and conspicuous loss of molar tooth eruption and root formation. In order to define the functions of MT1-MMP during root formation and tooth eruption, we analyzed the development of teeth and surrounding tissues in the absence of MT1-MMP. In situ hybridization showed that MT1-MMP was widely expressed in cells associated with teeth and surrounding connective tissues during development. Multiple defects in dentoalveolar tissues were associated with loss of MT1-MMP. Root formation was inhibited by defective structure and function of Hertwig's epithelial root sheath (HERS). However, no defect was found in creation of the eruption pathway, suggesting that tooth eruption was hampered by lack of alveolar bone modeling/remodeling coincident with reduced periodontal ligament (PDL) formation and integration with the alveolar bone. Additionally, we identified a significant defect in dentin formation and mineralization associated with the loss of MT1-MMP. To segregate these multiple defects and trace their cellular origin, conditional ablation of MT1-MMP was performed in epithelia and mesenchyme. Mice featuring selective loss of MT1-MMP activity in the epithelium were indistinguishable from wild type mice, and importantly, featured a normal HERS structure and molar eruption. In contrast, selective knock-out of MT1-MMP in Osterix-expressing mesenchymal cells, including osteoblasts and odontoblasts, recapitulated major defects from the global knock-out including altered HERS structure, short roots, defective dentin formation and mineralization, and reduced alveolar bone formation, although molars were able to erupt. These data indicate that MT1-MMP activity in the dental mesenchyme, and not in epithelial-derived HERS, is essential for proper tooth root formation and eruption. In summary, our studies point to an indispensable role for MT1-MMP-mediated matrix remodeling in tooth eruption through effects on bone formation, soft tissue remodeling and organization of the follicle/PDL region.

Piglet uniformity (PU) and farrowing interval (FI) are important reproductive traits related to production and economic profits in the pig industry. However, the genetic architecture of the longitudinal trends of reproductive traits still remains elusive. Herein, we performed a genome-wide association study (GWAS) to detect potential genetic variation and candidate genes underlying the phenotypic records at different parities for PU and FI in a population of 884 Large White pigs. In total, 12 significant SNPs were detected on SSC1, 3, 4, 9, and 14, which collectively explained 1~1.79% of the phenotypic variance for PU from parity 1 to parity 4, and 2.58~4.11% for FI at different stages. Of these, 7 SNPs were located within 16 QTL regions related to swine reproductive traits. One QTL region was associated with birth body weight (related to PU) and contained the peak SNP MARC0040730, and another was associated with plasma FSH concentration (related to FI) and contained the SNP MARC0031325. Finally, some positional candidate genes for PU and FI were identified because of their roles in prenatal skeletal muscle development, fetal energy substrate, pre-implantation and the expression of mammary gland epithelium. Identification of novel variants and candidate genes will greatly advance our understanding of the genetic mechanisms of PU and FI, and suggest a specific opportunity for improving marker assisted selection or genomic selection in pigs.

Pyrus ussuriensis is extremely cold hardy when fully acclimated, but knowledge relevant to the molecular mechanisms underlying this economically valuable trait is still limited so far. In this study, global transcriptome profiles of Pyrus ussuriensis under cold conditions (4 °C) over a time course were generated by high-throughput sequencing. In total, >57,121,199 high quality clean reads were obtained with approximately 11.0 M raw data for each library. Among them, the values of 66.84%-72.03% of clean reads in the digital transcript abundance measurement could be well mapped to the pear genome database, resulting in the identification of 8544 differentially expressed genes (DEGs) having 43 Gene Ontology (GO) terms and 17 clusters of orthologous groups (COG) involved in 385 Kyoto Encyclopedia of Genes and Genomes (KEGG) defined pathways. These comprised 3124 (1033 up-regulated, 2091 down-regulated), 1243 (729 up-regulated, 514 down-regulated), and 750 (458 up-regulated, 292 down-regulated) genes from the cold-treated samples at 5, 12 and 24 h, respectively. The accuracy of the RNA-Seq derived transcript expression data was validated by analyzing the expression patterns of 16 DGEs by quantitative real-time PCR. Plant-pathogen interaction, plant hormone signal transduction, Photosynthesis, signal transduction, innate immune response and response to biotic stimulus were the most significantly enriched GO categories among in the DEGs. A total of 335 transcription factors were shown to be cold responsive. In addition, a number of genes involved in the catabolism and signaling of hormones were significantly affected by the cold stress. The RNA-Seq and digital expression profiling provides valuable insights into the understanding the molecular events related to cold responses in Pyrus ussuriensis and dataset may help guide future identification and functional analysis of potential genes that are important for enhancing cold hardiness.

Light signaling and phytohormones play important roles in plant growth, development, and biotic and abiotic stress responses. However, the roles of phytochromes and cross-talk between these two signaling pathways in response to salt stress in tobacco plants remain underexplored. Here, we explored the defense response in phytochrome-defective mutants under salt stress. We monitored the physiological and molecular changes of these mutants under salt stress conditions. The results showed that phytochrome A (phyA), phytochrome B (phyB) and phyAphyB (phyAB) mutants exhibited improved salt stress tolerance compared with wild-type (WT) plants. The mutant plants had a lower electrolyte leakage (EL) and malondialdehyde (MDA) concentration than WT plants, and the effect was clearly synergistic in the phyAB double mutant plants. Furthermore, the data showed that the transcript levels of defense-associated genes and the activities of some antioxidant enzymes in the mutant plants were much higher than those in WT plants. Additionally, the results indicated that phytochrome signaling strongly modulates the expression of endogenous abscisic acid (ABA) and jasmonic acid (JA) of Nicotiana tobacum in response to salt stress. To illustrate further the relationship between phytochrome and phytohormone, we measured the expression of defense genes and phytochrome. The results displayed that salt stress and application of methyl jasmonate (MeJA) or ABA up-regulated the transcript levels of salt response-associated genes and inhibited the expression of NtphyA and NtphyB. Foliar application of inhibitors of ABA and JA further confirmed that JA co-operated with ABA in phytochrome-mediated salt stress tolerance.

Narrow-leafed lupin (NLL, Lupinus angustifolius) is a promising legume crop that produces seeds with very high protein content. However, NLL accumulates toxic quinolizidine alkaloids (QAs) in most of its tissues, including the seeds. To determine the level of in situ biosynthesis in the seeds, we compared the accumulation of QAs with the expression of the biosynthetic gene lysine decarboxylase (LDC) in developing seeds and pods of a bitter (high-QA) variety of NLL. While QAs accumulated steadily in seeds until the drying phase, LDC expression was comparatively very low throughout seed development. In contrast, both QA accumulation and LDC expression peaked early in pods and decreased subsequently, reaching background levels at the onset of drying. We complemented these studies with MS imaging, which revealed the distribution patterns of individual QAs in cross-sections of pods and seeds. Finally, we show that a paternal bitter genotype does not influence the QA levels of F1 seeds grown on a maternal, low-QA genotype. We conclude that the accumulation of QAs in seeds of bitter NLL is mostly, if not exclusively, transported from other tissues. These results open the possibility of using transport engineering to generate herbivore-resistant bitter NLL varieties that produce QA-free seeds.

The natural carotenoid lycopene (LYC) has strong antioxidant and neuroprotective capacities. This study investigated the effects and mechanisms of LYC on chronic stress-induced hippocampal lesions and learning and memory dysfunction. Rats were administered LYC and/or chronic restraint stress (CRS) for 21 days. Morris water maze results demonstrated that LYC prevented CRS-induced learning and memory dysfunction. Histopathological staining and transmission electron microscopy observation revealed that LYC ameliorated CRS-induced hippocampal microstructural and ultrastructural damage. Furthermore, LYC alleviated CRS-induced oxidative stress by reducing reactive oxygen species (ROS) production and enhancing antioxidant enzyme activities. LYC also improved CRS-induced hippocampal mitochondrial dysfunction by recovering mitochondrial membrane potential, and complex I (NADH dehydrogenase) and II (succinate dehydrogenase) activities. Moreover, LYC reduced CRS-induced apoptosis via the mitochondrial apoptotic pathway, and decreased the number of terminal deoxynucleotidyl transferase dUTP nick-end-labeled positive cells. Additionally, western blot analysis demonstrated that LYC inhibited CRS-induced activation of the c-Jun N-terminal kinase (JNK) signaling pathway. Correlation analysis indicated that ROS levels, JNK activation, and the mitochondrial apoptotic pathway were positively correlated. Further investigation of the underlying mechanisms revealed that the ROS scavenger N-acetyl-l-cysteine inhibited CRS-induced JNK activation. Furthermore, the JNK inhibitor SP600125 relieved CRS-induced hippocampal mitochondrial dysfunction, apoptosis via the mitochondrial apoptotic pathway, and learning and memory dysfunction. Together, these results suggest that LYC alleviates hippocampal oxidative stress, mitochondrial dysfunction, and apoptosis by inhibiting the ROS/JNK signaling pathway, thereby improving CRS-induced hippocampal injury and learning and memory dysfunction. This study provides a theoretical basis and new therapeutic strategies for the application of LYC to relieve chronic stress encephalopathy.

In this study, the effects of a polysaccharide derived from Laminaria japonica (LJP) on obesity were investigated in mice fed a high-fat diet (HFD). LJP significantly attenuated obesity-related features, lowering serum triglycerides, glucose, total cholesterol and low-density lipoprotein cholesterol levels. HFD-induced liver steatosis and hepatocellular ballooning were significantly attenuated by LJP. Additionally, LJP was found to significantly modulate hepatic gene expressions of AMPK and HMGCR, which are key regulators of lipid and cholesterol metabolism. We further found that LJP ameliorated HFD-induced gut microbiota (GM) dysbiosis by significantly reducing the obesity-related Firmicutes to Bacteroidetes ratio, meanwhile promoting the growth of Verrucomicrobia at the phylum level. At the genus level, propionate-producing bacteria Bacteroides and Akkermansia were elevated by LJP, which might explain the result that LJP elevated fecal propionate concentration. Taken together, these findings suggest that dietary intake of LJP modulates hepatic energy homeostasis to alleviate obesity-related nonalcoholic fatty liver disease associated with GM regulation.

Bisphenol A (BPA) is extensively used in plastic products and epoxy resins. The epigenetic response to the environmental chemical BPA was involved in multiple dysfunctional categories, such as cancer, the reproductive system, metabolism, pubertal development, peripheral arterial disease, infant and childhood growth, and neurodevelopment outcomes. In this mini-review, we described the recent progress of the epigenetic effects of the environmental chemical BPA, including DNA methylation, histone methylation, and toxic epigenomics. Notably, the histone modification changes under BPA exposure are summarized in this review. DNA methylation accompanied by transcriptional changes in key genes affected by BPA exposure is related to various processes, including neural development, cancer pathways, and generational transmission. In addition, BPA could also affect histone modifications in many species, such as humans, rats, and zebrafish. Finally, we reviewed recent studies of the toxico-epigenomics approach to reveal the epigenetic effect of BPA exposure genome-wide.

Methyl jasmonate (MeJA), a volatile organic compound, is a principal flowery aromatic compound in tea. During the processing of black tea, MeJA is produced by jasmonic acid carboxyl methyltransferase (JMT) of the jasmonic acid (JA) substrate, forming a specific floral fragrance. CsJMT was cloned from tea leaves; the three-dimensional structure of CsJMT was predicted. Enzyme activity was identified, and protein purification was investigated. Site-directed deletions revealed that N-10, S-22, and Q-25 residues in the beginning amino acids played a key functional role in enzyme activity. The expression patterns of CsJMT in tea organs differed; the highest expression of CsJMT was observed in the fermentation process of black tea. These results aid in further understanding the synthesis of MeJA during black tea processing, which is crucial for improving black tea quality using specific fragrances and could be applied to the aromatic compound regulation and tea breeding improvement in further studies.

Metabolic disturbance may be implicated in the pathogenesis of autism. This study aimed to investigate the gut metabolomic profiles of autistic children and to analyze potential interaction between gut metabolites with autistic symptoms and neurodevelopment levels. We involved 120 autistic and 60 neurotypical children. Autistic symptoms and neurodevelopment levels were assessed. Fecal samples were analyzed using untargeted liquid chromatography-tandem mass spectrometry methods. Our results showed the metabolic disturbances of autistic children involved in multiple vitamin and amino acid metabolism pathways, with the strongest enrichment identified for tryptophan metabolism, retinol metabolism, cysteine-methionine metabolism, and vitamin digestion and absorption. Differential gut metabolites were correlated to autistic symptoms and neurodevelopment levels. Our findings improved the understanding of the perturbations of metabolome networks in autism.

Theanine and flavonoids (especially proanthocyanidins) are the most important and abundant secondary metabolites synthesized in the roots of tea plants. Nitrogen promotes theanine and represses flavonoid biosynthesis in tea plant roots, but the underlying mechanism is still elusive. Here, we analyzed theanine and flavonoid metabolism in tea plant roots under nitrogen deficiency and explored the regulatory mechanism using proteome and ubiquitylome profiling together with transcriptome data. Differentially expressed proteins responsive to nitrogen deficiency were identified and found to be enriched in flavonoid, nitrogen, and amino acid metabolism pathways. The proteins responding to nitrogen deficiency at the transcriptional level, translational level, and both transcriptional and translational levels were classified. Nitrogen-deficiency-responsive and ubiquitinated proteins were further identified. Our results showed that most genes encoding enzymes in the theanine synthesis pathway, such as CsAlaDC, CsGDH, and CsGOGATs, were repressed by nitrogen deficiency at transcriptional and/or protein level(s). While a large number of enzymes in flavonoid metabolism were upregulated at the transcriptional and/or translational level(s). Importantly, the ubiquitylomic analysis identified important proteins, especially the hub enzymes in theanine and flavonoid biosynthesis, such as CsAlaDC, CsTSI, CsGS, CsPAL, and CsCHS, modified by ubiquitination. This study provided novel insights into the regulation of theanine and flavonoid biosynthesis and will contribute to future studies on the post-translational regulation of secondary metabolism in tea plants.

Abstract Background We aimed to compare differences in infant feeding patterns (breastfeeding and complementary food supplementation) between children with the autism spectrum disorder (ASD) and typically developing (TD) children through a multicentre study. The relationship between these patterns and later core symptoms and neurodevelopment in children with ASD was also investigated. Methods We analysed breastfeeding and complementary feeding patterns in 1389 children with ASD and 1190 TD children. The Children Neuropsychological and Behavior Scale-Revision 2016 (CNBS-R2016) was used to assess neurodevelopmental levels. The Autism Behavior Checklist (ABC), Social Responsiveness Scale (SRS), Childhood Autism Rating Scale (CARS), and ASD Warning Behavior Subscale of the CNBS-R2016 were used to assess ASD symptoms. Results Children with ASD had a shorter breastfeeding duration in infancy (8 (3–12) months vs. 10 (6–14) months, P &lt; 0.001), later introduction of complementary foods ( P &lt; 0.001), and poorer acceptance of complementary foods ( P &lt; 0.001) than TD children. Total ABC and CARS scores were lower in the group of children with ASD who had been breastfed for 12 months or more than in the group who had been breastfed for less than 6 months. Children with ASD who were given complementary food after 6 months had lower general quotient (GQ), adaptive ability, fine motor and language scores than those who were given complementary food within 4–6 months. Children with ASD with poor acceptance of complementary foods had higher ABC and SRS scores and lower gross motor scores than those who had good acceptance. Conclusions Children with ASD have a shorter duration of breastfeeding, a later introduction of complementary foods, and poorer acceptance of complementary foods than TD children. These feeding patterns may be related to the symptoms and growth of children with ASD. The research suggests that continued breastfeeding for longer than 12 months may be beneficial in reducing ASD symptoms and that infants who have difficulty introducing complementary foods should be followed up for neurodevelopment. Trial registration The ethics committee of the Children’s Hospital of Chongqing Medical University approved the study. Approval Number: (2018) IRB (STUDY) NO. 121, and registered in the Chinese Clinical Trial Registry (Registration number: ChiCTR2000031194, registered on 23/03/2020).

Chronic stress can cause intestinal barrier damage. MAPK and NF-κB are closely related to it. Chlorogenic acid (CGA), a dietary polyphenol, has been shown to have intestinal protective effects, but whether by regulating MAPK and NF-κB is not known. Therefore, in this experiment, 24 Wistar rats were randomly divided into 4 groups (C group, CS group, CS + SB203580, and CS + CGA group). Rats in the CS group were restrained stress for 6 h per day for 21 days. Rats in the CS + SB203580 group were given SB203582 (0.5 mg/kg, intraperitoneal injection) 1 h before restraint stress every other day. Rats in the CS + CGA group were given CGA (100 mg/kg, gavage) 1 h before restraint stress. In chronic stress, intestinal barrier damage was evident, while being restored after CGA treatment. After chronic stress, the levels of p-P38 were increased (P < 0.01), while the levels of p-JNK and p-ERK were not changed. The levels of p-p38 were elevated after CGA treatment (P < 0.01). These results suggested that p38MAPK played an important role in chronic stress-induced intestinal injury, and CGA could inhibit p38MAPK activity. Therefore, we chose SB203582 (P38MAPK inhibitor) to elucidate the role of p38. After chronic stress, intestinal tight junction key proteins Occludin, ZO-1, and Claudin3 protein and gene expression were reduced (P < 0.01), while being elevated after CGA or SB203582 intervention (P < 0.05). After CGA treatment, the levels of p-IκB, p-p65, p-p38, and TNF-α were reduced (P < 0.01). SB203582 intervention reduced p-p65 and TNF-α levels significantly (P < 0.01). These results suggested that CGA could inhibit the NF-κB pathway by suppressing p38MAPK, thereby alleviating chronic stress-induced intestinal damage.

A simple, sensitive and reliable analytical method for the rapid simultaneous determination of dexamethasone and betamethasone in milk by high performance liquid chromatography–negative electrospray ionization tandem mass spectrometry (HPLC–NESI-MS/MS) with isotope dilution was developed. Samples were directly purified through C18 cartridge. Then the eluate was dried under nitrogen and residues were dissolved in mobile phase. Samples were analyzed by HPLC–MS/MS on a Hypercarb graphite column with a mixture of acetonitrile–water–formic acid as mobile phase. The samples were quantified using dexamethasone-D4 as an internal standard. The procedure was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), trueness, precision, linearity and stability. The method is demonstrated to be suitable for the determination of dexamethasone and betamethasone in milk. The total time required for the analysis of one sample was about 35 min.

Pyrethrins are active ingredients extracted from pyrethrum flowers (Tanacetum cinerariifolium), and are the most widely used botanical insecticide. However, several thrips species are commonly found on pyrethrum flowers in the field, and are the dominant insects found inside the flowers. Up to 80% of western flower thrips (WFT, Frankliniella occidentalis) adults died within 3 days of initiating feeding on leaves of pyrethrum, leading us to evaluate the role of pyrethrins in the defense of pyrethrum leaves against WFT. The effects of pyrethrins on WFT survival, feeding behavior, and reproduction were measured both in vitro and in planta (infiltrated leaves). The lethal concentration value (LC50) for pyrethrins against WFT adults was 12.9 mg/ml, and pyrethrins at 0.1% (w/v) and 1% (w/v) had significantly negative effects on feeding, embryo development, and oviposition. About 20-70% of WFT were killed within 2 days when they were fed chrysanthemum leaves containing 0.01-1% pyrethrins. Chrysanthemum leaves containing 0.1% or 1% pyrethrins were significantly deterrent to WFT. In a no-choice assay, the reproduction of WFT was reduced significantly when the insects were fed leaves containing 0.1% pyrethrins, and no eggs were found in leaves containing 1% pyrethrins. Our results suggest that the natural concentrations of pyrethrins in the leaves may be responsible for the observed high mortality of WFT on pyrethrum.

The fate of pymetrozine was studied in rice field ecosystem, and a simple and reliable analytical method for determination of pymetrozine in soil, rice straw, paddy water and brown rice was developed. Pymetrozine residues were extracted from samples, cleaned up by solid phase extraction (SPE) and then determined by high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). The average recovery was 81.2-88.1% from soil, 83.4-88.6% from rice straw, 87.3-94.1% from paddy water and 82.9-85.3% from brown rice. The relative standard deviation (RSD) was less than 15%. The limits of detection (LODs) of pymetrozine calculated as a sample concentration were 0.0003 mg kg(-1) (mg L(-1)) for soil and paddy water, 0.001 mg kg(-1) for brown rice and rice straw. The results of kinetics study of pymetrozine residue showed that pymetrozine degradation in water, soil, and rice straw coincided with C=0.194e(-0.986t), C=0.044e(-0.099t), and C=0.988e(-0.780t), respectively; the half-lives were about 0.70 d, 7.0 d and 0.89 d, respectively. The degradation rate of pymetrozine in water was the fastest, followed by rice straw. The highest final pymetrozine residues in brown rice were 0.01 mg kg(-1), which was lower than the EU's upper limit of 0.02 mg kg(-1) in rice. Therefore, a dosage of 300-600 g a.i.hm(-2) was recommended, which could be considered as safe to human beings and animals.

Tea plant (Camellia sinensis (L.) O. kuntze) is known to be a fluoride (F) and aluminum (Al(3+)) hyper-accumulator. Previous study showed that pre-treatment of Al(3+) caused a significant increase of F accumulation in tea plants. However, less is known about the intricate network of Al(3+) promoted F accumulation in tea plants. In this study, the involvement of endogenous Ca(2+) and CaM in Al(3+) pretreatment-promoted F accumulation in tea plants was investigated. Our results showed that Al(3+) induced the inverse change of intracellular Ca(2+) fluorescence intensity and stimulated Ca(2+) trans-membrane transport in the mature zone of tea root. Also, a link between internal Ca(2+) and CaM was found in tea roots under the presence of Al(3+). In order to investigate whether Ca(2+) and CaM were related to F accumulation promoted by Al(3+) pretreatment, Ca(2+) chelator EGTA and CaM antagonists CPZ and TFP were used. EGTA, CPZ, and TFP pretreatment inhibited Al(3+)-induced increase of Ca(2+) fluorescence intensity and CaM content in tea roots, and also significantly reduced Al(3+)-promoted F accumulation in tea plants. Taken together, our results suggested that the endogenous Ca(2+) and CaM are involved in Al(3+) pretreatment-promoted F accumulation in tea roots.

For successful tissue engineering of articular cartilage, a scaffold with mechanical properties that match those of natural cartilage as closely as possible is needed. In the present study, we prepared a fibrous silk fibroin (SF)/poly(L-lactic acid) (PLLA) scaffold via electrospinning and investigated the morphological, mechanical, and degradation properties of the scaffolds fabricated using different electrospinning conditions, including collection distance, working voltage, and the SF:PLLA mass ratio. In addition, in vitro cell-scaffold interactions were evaluated in terms of chondrocyte adhesion to the scaffolds as well as the cytotoxicity and cytocompatibility of the scaffolds. The optimum electrospinning conditions for generating a fibrous SF/PLLA scaffold with the best surface morphology (ordered alignment and suitable diameter) and tensile strength (~1.5 MPa) were a collection distance of 20 cm, a working voltage of 15 kV, and a SF:PLLA mass ratio of S50P50. The degradation rate of the SF/PLLA scaffolds was found to be determined by the SF:PLLA mass ratio, and it could be increased by reducing the PLLA proportion. Furthermore, chondrocytes spread well on the fibrous SF/PLLA scaffolds and secreted extracellular matrix, indicating good adhesion to the scaffold. The cytotoxicity of SF/PLLA scaffold extract to chondrocytes over 24 and 48 h in culture was low, indicating that the SF/PLLA scaffolds are biocompatible. Chondrocytes grew well on the SF/PLLA scaffold after 1, 3, 5, and 7 days of direct contact, indicating the good cytocompatibility of the scaffold. These results demonstrate that the fibrous SF/PLLA scaffold represents a promising composite material for use in cartilage tissue engineering.

Alpha-momorcharin (α-MMC) is a type-I ribosome inactivating protein (RIP) with a molecular weight of 29 kDa found in plants. This protein has been shown to be effective against a broad range of human viruses and also has anti-tumor activities. However, the mechanism by which α-MMC induces plant defense responses and regulates the N gene to promote resistance to the Tobacco mosaic virus (TMV) is still not clear. By using pharmacological and infection experiments, we found that α-MMC enhances TMV resistance of tobacco plants containing the N gene (tobaccoNN). Our results showed that plants pretreated with 0.5 mg/ml α-MMC could relieve TMV-induced oxidative damage, had enhanced the expression of the N gene and increased biosynthesis of jasmonic acid (JA) and salicylic acid (SA). Moreover, transcription of JA and SA signaling pathway genes were increased, and their expression persisted for a longer period of time in plants pretreated with α-MMC compared with those pretreated with water. Importantly, exogenous application of 1-Aminobenzotriazole (ABT, SA inhibitor) and ibuprofen (JA inhibitor) reduced α-MMC induced plant resistance under viral infection. Thus, our results revealed that α-MMC enhances TMV resistance of tobaccoNN plants by manipulating JA-SA crosstalk.