Phosphoribosylpyrophosphate synthetase as a metabolic valve advances Methylobacterium/Methylorubrum phyllosphere colonization and plant growth.

The proficiency of phyllosphere microbiomes in efficiently utilizing plant-provided nutrients is pivotal for their successful colonization of plants. The methylotrophic capabilities of Methylobacterium/Methylorubrum play a crucial role in this process. However, the precise mechanisms facilitating efficient colonization remain elusive. In the present study, we investigate the significance of methanol assimilation in shaping the success of mutualistic relationships between methylotrophs and plants. A set of strains originating from Methylorubrum extorquens AM1 are subjected to evolutionary pressures to thrive under low methanol conditions. A mutation in the phosphoribosylpyrophosphate synthetase gene is identified, which converts it into a metabolic valve. This valve redirects limited C1-carbon resources towards the synthesis of biomass by up-regulating a non-essential phosphoketolase pathway. These newly acquired bacterial traits demonstrate superior colonization capabilities, even at low abundance, leading to increased growth of inoculated plants. This function is prevalent in Methylobacterium/Methylorubrum strains. In summary, our findings offer insights that could guide the selection of Methylobacterium/Methylorubrum strains for advantageous agricultural applications.

Endogenous Hormone Levels and Transcriptomic Analysis Reveal the Mechanisms of Bulbil Initiation in Pinellia ternata.

Pinellia ternata is a medicinal plant that has important pharmacological value, and the bulbils serve as the primary reproductive organ; however, the mechanisms underlying bulbil initiation remain unclear. Here, we characterized bulbil development via histological, transcriptomic, and targeted metabolomic analyses to unearth the intricate relationship between hormones, genes, and bulbil development. The results show that the bulbils initiate growth from the leaf axillary meristem (AM). In this stage, jasmonic acid (JA), abscisic acid (ABA), isopentenyl adenosine (IPA), and salicylic acid (SA) were highly enriched, while indole-3-acetic acid (IAA), zeatin, methyl jasmonate (MeJA), and 5-dexoxystrigol (5-DS) were notably decreased. Through OPLS-DA analysis, SA has emerged as the most crucial factor in initiating and positively regulating bulbil formation. Furthermore, a strong association between IPA and SA was observed during bulbil initiation. The transcriptional changes in IPT (Isopentenyltransferase), CRE1 (Cytokinin Response 1), A-ARR (Type-A Arabidopsis Response Regulator), B-ARR (Type-B Arabidopsis Response Regulator), AUX1 (Auxin Resistant 1), ARF (Auxin Response Factor), AUX/IAA (Auxin/Indole-3-acetic acid), GH3 (Gretchen Hagen 3), SAUR (Small Auxin Up RNA), GA2ox (Gibberellin 2-oxidase), GA20ox (Gibberellin 20-oxidase), AOS (Allene oxide synthase), AOC (Allene oxide cyclase), OPR (Oxophytodienoate Reductase), JMT (JA carboxy l Methyltransferase), COI1 (Coronatine Insensitive 1), JAZ (Jasmonate ZIM-domain), MYC2 (Myelocytomatosis 2), D27 (DWARF27), SMAX (Suppressor of MAX2), PAL (Phenylalanine Ammonia-Lyase), ICS (Isochorismate Synthase), NPR1 (Non-expressor of Pathogenesis-related Genes1), TGA (TGACG Sequence-specific Binding), PR-1 (Pathogenesis-related), MCSU (Molybdenium Cofactor Sulfurase), PP2C (Protein Phosphatase 2C), and SnRK (Sucrose Non-fermenting-related Protein Kinase 2) were highly correlated with hormone concentrations, indicating that bulbil initiation is coordinately controlled by multiple phytohormones. Notably, eight TFs (transcription factors) that regulate AM initiation have been identified as pivotal regulators of bulbil formation. Among these, WUS (WUSCHEL), CLV (CLAVATA), ATH1 (Arabidopsis Thaliana Homeobox Gene 1), and RAX (Regulator of Axillary meristems) have been observed to exhibit elevated expression levels. Conversely, LEAFY demonstrated contrasting expression patterns. The intricate expression profiles of these TFs are closely associated with the upregulated expression of KNOX(KNOTTED-like homeobox), suggesting a intricate regulatory network underlying the complex process of bulbil initiation. This study offers a profound understanding of the bulbil initiation process and could potentially aid in refining molecular breeding techniques specific to P. ternata.

Appropriate Stubble Height Can Effectively Improve the Rice Quality of Ratoon Rice.

Ratoon rice, the cultivation of a second crop from the stubble after the main harvest, is recognized as an eco-friendly and resource-saving method for rice production. Here, a field experiment was carried out in the Yangtze River region to investigate the impact of varying stubble heights on the grain quality of ratoon rice, as well as to compare the grain quality between the main and ratoon season. This study, which focused on 12 commonly cultivated rice varieties, conducted a comprehensive analysis assessing milling characteristics, appearance, and cooking quality. The results show that ratoon rice crops exhibited a higher milled rice rate and head rice rate compared to the main rice crops. Conversely, chalky rice percentage, chalkiness degree, and amylose content were lower in ratoon rice crops. Principal component analysis grouped eight relevant quality indicators of rice quality which were concentrated into three categories, with amylose content identified as the key indicator of rice quality for distinguishing between different stubble heights. Random forest results reveal a robust and significant correlation between appearance quality index and amylose content. Subordinate function analysis indicated that a stubble height of 30 cm resulted in optimal rice quality, with Lingliangyou 211 exhibiting the highest quality and Xiangzao Xian 32 the lowest. Overall, our study suggests that ratoon rice crops generally outperform main rice crops in terms of quality, with the optimal measurement at a stubble height of 30 cm. This study holds substantial importance for selecting appropriate stubble heights for ratoon rice crops and enhancing overall rice quality.

Preoperative serum cortisone levels are associated with cognition in preschool-aged children with tetralogy of Fallot after corrective surgery: new evidence from human populations and mice.

BACKGROUND: Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Children with TOF would be confronted with neurological impairment across their lifetime. Our study aimed to identify the risk factors for cerebral morphology changes and cognition in postoperative preschool-aged children with TOF. METHODS: We used mass spectrometry (MS) technology to assess the levels of serum metabolites, Wechsler preschool and primary scale of intelligence-Fourth edition (WPPSI-IV) index scores to evaluate neurodevelopmental levels and multimodal magnetic resonance imaging (MRI) to detect cortical morphological changes. RESULTS: Multiple linear regression showed that preoperative levels of serum cortisone were positively correlated with the gyrification index of the left inferior parietal gyrus in children with TOF and negatively related to their lower visual spaces index and nonverbal index. Meanwhile, preoperative SpO2 was negatively correlated with levels of serum cortisone after adjusting for all covariates. Furthermore, after intervening levels of cortisone in chronic hypoxic model mice, total brain volumes were reduced at both postnatal (P) 11.5 and P30 days. CONCLUSIONS: Our results suggest that preoperative serum cortisone levels could be used as a biomarker of neurodevelopmental impairment in children with TOF. Our study findings emphasized that preoperative levels of cortisone could influence cerebral development and cognition abilities in children with TOF.

Transcriptome and metabolome analysis reveals the effect of flavonoids on flower color variation in Dendrobium nobile Lindl.

Introduction: Dendrobium nobile L. is a rare orchid plant with high medicinal and ornamentalvalue, and extremely few genetic species resources are remaining in nature. In the normal purple flower population, a type of population material with a white flower variation phenotype has been discovered, and through pigment component determination, flavonoids were preliminarily found to be the main reason for the variation. Methods: This study mainly explored the different genes and metabolites at different flowering stages and analysed the flower color variation mechanism through transcriptome- and flavonoid-targeted metabolomics. The experimental materials consisted of two different flower color phenotypes, purple flower (PF) and white flower (WF), observed during three different periods. Results and discussion: The results identified 1382, 2421 and 989 differentially expressed genes (DEGs) in the white flower variety compared with the purple flower variety at S1 (bud stage), S2 (chromogenic stage) and S3 (flowering stage), respectively. Among these, 27 genes enriched in the ko00941, ko00942, ko00943 and ko00944 pathways were screened as potential functional genes affecting flavonoid synthesis and flower color. Further analysis revealed that 15 genes are potential functional genes that lead to flavonoid changes and flower color variations. The metabolomics results at S3 found 129 differentially accumulated metabolites (DAMs), which included 8 anthocyanin metabolites, all of which (with the exception of delphinidin-3-o-(2'''-o-malonyl) sophoroside-5-o-glucoside) were found at lower amounts in the WF variety compared with the PF variety, indicating that a decrease in the anthocyanin content was the main reason for the inability to form purple flowers. Therefore, the changes in 19 flavone and 62 flavonol metabolites were considered the main reasons for the formation of white flowers. In this study, valuable materials responsible for flower color variation in D. nobile were identified and further analyzed the main pathways and potential genes affecting changes in flavonoids and the flower color. This study provides a material basis and theoretical support for the hybridization and molecular-assisted breeding of D. nobile.

Characterization of C30 carotenoid and identification of its biosynthetic gene cluster in Methylobacterium extorquens AM1.

Methylobacterium species, the representative bacteria distributed in phyllosphere region of plants, often synthesize carotenoids to resist harmful UV radiations. Methylobacterium extorquens is known to produce a carotenoid pigment and recent research revealed that this carotenoid has a C30 backbone. However, its exact structure remains unknown. In the present study, the carotenoid produced by M. extorquens AM1 was isolated and its structure was determined as 4-[2-O-11Z-octadecenoyl-ß-glucopyranosyl]-4,4'-diapolycopenedioc acid (1), a glycosylated C30 carotenoid. Furthermore, the genes related to the C30 carotenoid synthesis were investigated. Squalene, the precursor of the C30 carotenoid, is synthesized by the co-occurrence of META1p1815, META1p1816 and META1p1817. Further overexpression of the genes related to squalene synthesis improved the titer of carotenoid 1. By using gene deletion and gene complementation experiments, the glycosyltransferase META1p3663 and acyltransferase META1p3664 were firstly confirmed to catalyze the tailoring steps from 4,4'-diapolycopene-4,4'-dioic acid to carotenoid 1. In conclusion, the structure and biosynthetic genes of carotenoid 1 produced by M. extorquens AM1 were firstly characterized in this work, which shed lights on engineering M. extorquens AM1 for producing carotenoid 1 in high yield.

GPX4 deficiency-dependent phospholipid peroxidation drives motor deficits of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by oxidative stress that triggers motor neurons loss in the brain and spinal cord. However, the mechanisms underlying the exact role of oxidative stress in ALS-associated neural degeneration are not definitively established. Oxidative stress-generated phospholipid peroxides are known to have extensive physiological and pathological consequences to tissues. Here, we discovered that the deficiency of glutathione peroxidase 4 (GPX4), an essential antioxidant peroxidase, led to the accumulation of phospholipid peroxides and resulted in a loss of motor neurons in spinal cords of ALS mice. Mutant human SOD1G93A transgenic mice were intrathecally injected with neuron-targeted adeno-associated virus (AAV) expressing GPX4 (GPX4-AAV) or phospholipid peroxidation inhibitor, ferrostatin-1. The results showed that impaired motor performance and neural loss induced by SOD1G93A toxicity in the lumbar spine were substantially alleviated by ferrostatin-1 treatment and AAV-mediated GPX4 delivery. In addition, the denervation of neuron-muscle junction and spinal atrophy in ALS mice were rescued by neural GPX4 overexpression, suggesting that GPX4 is essential for the motor neural maintenance and function. In comparison, conditional knockdown of Gpx4 in the spinal cords of Gpx4fl/fl mice triggered an obvious increase of phospholipid peroxides and the occurrence of ALS-like motor phenotype. Altogether, our findings underscore the importance of GPX4 in maintaining phospholipid redox homeostasis in the spinal cord and presents GPX4 as an attractive therapeutic target for ALS treatment.

Perioperative extracorporeal membrane oxygenation in pediatric congenital heart disease: Chinese expert consensus.

BACKGROUND: Congenital heart disease (CHD) is one of the main supportive diseases of extracorporeal membrane oxygenation in children. The management of extracorporeal membrane oxygenation (ECMO) for pediatric CHD faces more severe challenges due to the complex anatomical structure of the heart, special pathophysiology, perioperative complications and various concomitant malformations. The survival rate of ECMO for CHD was significantly lower than other classifications of diseases according to the Extracorporeal Life Support Organization database. This expert consensus aims to improve the survival rate and reduce the morbidity of this patient population by standardizing the clinical strategy. METHODS: The editing group of this consensus gathered 11 well-known experts in pediatric cardiac surgery and ECMO field in China to develop clinical recommendations formulated on the basis of existing evidences and expert opinions. RESULTS: The primary concern of ECMO management in the perioperative period of CHD are patient selection, cannulation strategy, pump flow/ventilator parameters/vasoactive drug dosage setting, anticoagulation management, residual lesion screening, fluid and wound management and weaning or transition strategy. Prevention and treatment of complications of bleeding, thromboembolism and brain injury are emphatically discussed here. Special conditions of ECMO management related to the cardiovascular anatomy, haemodynamics and the surgical procedures of common complex CHD should be considered. CONCLUSIONS: The consensus could provide a reference for patient selection, management and risk identification of perioperative ECMO in children with CHD. Video abstract (MP4 104726 kb).

Exogenous Betaine Enhances the Protrusion Vigor of Rice Seeds under Heat Stress by Regulating Plant Hormone Signal Transduction and Its Interaction Network.

Rice is an important food crop. Rice seedlings are mainly composed of root, coleoptile, mesocotyl and euphylla. The elongation of coleoptile and mesocotyl promotes the emergence of rice seedlings. Therefore, analyzing the mechanism of coleoptile and mesocotyl elongation is important for the cultivation of rice varieties. Due to global warming, heat stress is threatening rice yields. Betaine plays an important role in plant resistance to heat stress; however, we lack research on its regulation mechanism of rice seed germination under heat stress. Therefore, we explored the effects of soaking seeds with betaine at different concentrations on rice seed germination under heat stress. According to the results, soaking seeds with 10 mM of betaine could effectively improve the seeds' germination potential and rate under heat stress to promote the germination of rice seeds. To clarify the mitigation mechanism of betaine in heat stress, we measured the antioxidant enzyme activity, malondialdehyde content, soluble protein content and endogenous hormone content of seed protrusion under heat stress. We constructed the cDNA library for transcriptome sequencing. According to the results, 10 mM of betaine improved the activities of the superoxide dismutase, peroxidase and catalase of seed protrusion under heat stress to reduce the malondialdehyde content and increase the soluble protein content to alleviate the effect of heat stress on rice seed germination. The detection of the endogenous hormone content showed that soaking seeds with 10 mM of betaine increased the content of gibberellin and decreased the contents of auxin and abscisic acid of seed protrusion under heat stress. According to the transcriptome analysis, betaine can induce the expressions of key genes in the biosynthesis and metabolism of auxin, abscisic acid and gibberellins in the seed coleoptile and mesocotyl elongation stage, regulate the signal transduction of three hormones and promote the germination of rice seeds under heat stress. This study revealed, for the first time, the physiological and molecular regulation mechanism of betaine promotion of seed germination under heat stress.

Does Institutional Social Insurance Cause the Abandonment of Cultivated Land? Evidence from Rural China.

At present, the world's countryside needs to be revitalized urgently, and cultivated land is the critical factor in promoting the countryside's revitalization. The reduction of uncultivated land contributes to the efficient use of rural land resources, contributing to global rural revitalization. This study uses data from 3938 Chinese peasant households conducted in 2014 and the OLS method to investigate the quantitative impact of institutional social insurance on cultivated land abandonment. The empirical results point to the following conclusions: (1) Institutional social insurance will weaken the social insurance function of land, which will lead to the generation of peasant households' cultivated land abandonment. More specifically, for every 1% increase in the level of institutional social insurance of peasant households, the area of cultivated land abandoned increased by 0.002 mu; (2) The influence of institutional social insurance on peasant households' cultivated land abandonment is heterogeneous, that is, endowment insurance can weaken the social insurance function of land more than medical insurance, and households with a higher proportion of pensioners are more likely to abandon cultivated land. This study's conclusions may help understand the relationship between institutional and non-institutional social insurance and provide a reference for the effective use of cultivated land resources in the global rural revitalization.

Serum vitamin E concentration is negatively associated with body mass index change in girls not boys during adolescence.

BACKGROUND: Vitamin E is the most abundant lipid-soluble antioxidants present in plasma; however, the relationship between serum vitamin E and change in body mass index (BMI)-for-age Z scores in adolescents has not been well described. METHODS: This study is a cross-sectional study. Data were analyzed from 4014 adolescents who participated in the National Health and Nutrition Examination Survey. The nutritional status was calculated by BMI Z scores and was classified into normal weight, overweight, and obese. Multivariable-adjusted logistic regression was used to examine the association between serum vitamin E levels with overweight/obesity. Besides, the interaction effects between potential confounders and vitamin E on obesity were further evaluated. RESULTS: After adjusting potential confounders, serum vitamin E levels were negatively associated with overweight/obesity in girls but not in boys. Per standard deviation increment in vitamin E concentrations was associated with a 92% decreased risk of obesity in females. Besides, lower quartiles of serum vitamin E were associated with a higher risk of overweight/obesity in girls. Moreover, the inverse association between serum vitamin E levels and obesity was also found in most subgroups through subgroup analysis. CONCLUSIONS: Our study supports the negative association between serum vitamin E levels and overweight/obesity in adolescents. A higher serum vitamin E level may be associated with a reduced probability of obesity in girls, but not in boys.

Metabolomic analysis improves bioconversion of methanol to isobutanol in Methylorubrum extorquens AM1.

BACKGROUND: Methylorubrum extorquens AM1 can be engineered to convert methanol to value-added chemicals. Most of these chemicals derive from acetyl-CoA involved in the serine cycle. However, recent studies on methylotrophic metabolism have suggested that C3 pyruvate is a good potential precursor for broadening the types of synthesized products. METHODS AND RESULTS: In the present study, we found that isobutanol was a model chemical that could be generated from pyruvate through a 2-keto acid pathway. Initially, the engineered M. extorquens AM1 could only produce a trace amount of isobutanol at 0.62 mgL-1 after introducing the heterologous 2-ketoisovalerate decarboxylase and alcohol dehydrogenase. Furthermore, the metabolomic analysis revealed that insufficient carbon fluxes through 2-ketoisovalerate and pyruvate were the key limitation steps for efficient biosynthesis of isobutanol. Based on this analysis, the titer of isobutanol was improved by over 20-fold after overexpressing alsS gene encoding acetolactate synthase and deleting ldhA gene for lactate dehydrogenase. Moreover, substituting the cell chassis with the isobutanol-tolerant strain isolated from adaptive evolution of M. extorquens AM1 further increased the production of isobutanol by 1.7-fold, resulting in the final titer of 19 mgL-1 in flask cultivation. CONCLUSION: Our current findings provided promising insights into engineering methylotrophic cell factories capable of converting methanol to isobutanol or value-added chemicals using pyruvate as the precursor.

Rewiring the native methanol assimilation metabolism by incorporating the heterologous ribulose monophosphate cycle into Methylorubrum extorquens.

Methanol is assimilated through the serine cycle to generate acetyl-CoA without carbon loss. However, a highly active serine cycle requires high consumption of reducing equivalents and ATP, thereby leading to the impaired efficiency of methanol conversion to reduced chemicals. In the present study, a genome-scale flux balance analysis (FBA) predicted that the introduction of the heterologous ribulose monophosphate (RuMP) cycle, a more energy-efficient pathway for methanol assimilation, could theoretically increase growth rate by 31.3% for the model alphaproteobacterial methylotroph Methylorubrum extorquens AM1. Based on this analysis, we constructed a novel synergistic assimilation pathway in vivo by incorporating the RuMP cycle into M. extroquens metabolism with the intrinsic serine cycle. We demonstrated that the operation of the synergistic pathway could increase cell growth rate by 16.5% and methanol consumption rate by 13.1%. This strategy rewired the central methylotrophic metabolism through adjusting core gene transcription, leading to a pool size increase of C2 to C5 central intermediates by 1.2- to 3.6-fold and an NADPH cofactor improvement by 1.3-fold. The titer of 3-hydroxypropionic acid (3-HP), a model product in the newly engineered chassis of M. extorquens AM1, was increased to 91.2 mg/L in shake-flask culture, representing a 3.1-fold increase compared with the control strain with only the serine cycle. The final titer of 3-HP was significantly improved to 0.857 g/L in the fed-batch bioreactor, which was more competitive compared with the other 3-HP producers using methane and CO2 as C1 sources. Collectively, our current study demonstrated that engineering the synergistic methanol assimilation pathway was a promising strategy to increase the carbon assimilation and the yields of reduced chemicals in diverse host strains for C1 microbial cell factories.

Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis.

The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of Streptococcus pyogenes-derived deactivated cas9 (dcas9). When the dcas9 and sgRNA were respectively controlled by medium PR/tetO and strong PmxaF-g promoters, dynamic repression efficacy of cell growth through disturbing a central metabolism gene glyA was achieved from 41.9 to 96.6% dependent on the sgRNA targeting sites. Furthermore, the optimized CRISPRi system was shown to effectively decrease the abundance of exogenous fluorescent protein gene mCherry over 50% and to reduce the expression of phytoene desaturase gene crtI by 97.7%. We then used CRISPRi technology combined with 26 sgRNAs pool to rapidly discover a new phytoene desaturase gene META1_3670 from 2470 recombinant mutants. The gene function was further verified through gene deletion and complementation as well as phylogenetic tree analysis. In addition, we applied CRISPRi to repress the transcriptional level of squalene-hopene cyclase gene shc involved in hopanoid biosynthesis by 64.9%, which resulted in enhancing 1.9-fold higher of carotenoid production without defective cell growth. Thus, the CRISPRi system developed here provides a useful tool in mining functional gene of M. extorquens as well as in biotechnology for producing high-valued chemicals from methanol. KEY POINTS: Developing an efficient CRISPRi to knockdown gene expression in C1-utilizing bacteria CRISPRi combined with sgRNAs pool to rapidly discover a new phytoene desaturase gene Improvement of carotenoid production by repressing a competitive pathway.

Bioconversion of Methanol into Value-added Chemicals in Native and Synthetic Methylotrophs.

Methanol, commercially generated from methane, is a renewable chemical feedstock that is highly soluble, relatively inexpensive, and easy to handle. The concept of native methylotrophic bacteria serving as whole cell catalysts for production of chemicals and materials using methanol as a feedstock is highly attractive. In recent years, the available omics data for methylotrophic bacteria, especially for Methylobacterium extorquens, the most well-characterized model methylotroph, have provided a solid platform for rational engineering of methylotrophic bacteria for industrial production. In addition, there is a strong interest in converting the more traditional heterotrophic production platforms toward the use of single carbon substrates, including methanol, through metabolic engineering. In this chapter, we review the recent progress toward achieving the desired growth and production yields from methanol, by genetically engineered native methylotrophic strains and by the engineered synthetic methylotrophs.

Unusual and Highly Bioactive Sesterterpenes Synthesized by Pleurotus ostreatus during Coculture with Trametes robiniophila Murr.

Candida albicans and Cryptococcus neoformans, human-pathogenic fungi found worldwide, are receiving increasing attention due to high morbidity and mortality in immunocompromised patients. In the present work, 110 fungus pairs were constructed by coculturing 16 wood-decaying basidiomycetes, among which coculture of Trametes robiniophila Murr and Pleurotus ostreatus was found to strongly inhibit pathogenic fungi through bioactivity-guided assays. A combination of metabolomics and molecular network analysis revealed that 44 features were either newly synthesized or produced at high levels in this coculture system and that 6 of the features that belonged to a family of novel and unusual linear sesterterpenes contributed to high activity with MICs of 1 to 32 µg/ml against pathogenic fungi. Furthermore, dynamic 13C-labeling analysis revealed an association between induced features and the corresponding fungi. Unusual sesterterpenes were 13C labeled only in P. ostreatus in a time course after stimulation by the coculture, suggesting that these sesterterpenes were synthesized by P. ostreatus instead of T. robiniophila Murr. Sesterterpene compounds 1 to 3 were renamed postrediene A to C. Real-time reverse transcription-quantitative PCR (RT-qPCR) analysis revealed that transcriptional levels of three genes encoding terpene synthase, farnesyl-diphosphate farnesyltransferase, and oxidase were found to be 8.2-fold, 88.7-fold, and 21.6-fold higher, respectively, in the coculture than in the monoculture, indicating that biosynthetic gene cluster 10 was most likely responsible for the synthesis of these sesterterpenes. A putative biosynthetic pathway of postrediene A to postrediene C was then proposed based on structures of sesterterpenes and molecular network analysis.IMPORTANCE A number of gene clusters involved in biosynthesis of secondary metabolites are presumably silent or expressed at low levels under conditions of standard laboratory cultivation, resulting in a large gap between the pool of discovered metabolites and genome capability. This work mimicked naturally occurring competition by construction of an artificial coculture of basidiomycete fungi for the identification of secondary metabolites with novel scaffolds and excellent bioactivity. Unusual linear sesterterpenes of postrediene A to C synthesized by P. ostreatus not only were promising lead drugs against human-pathogenic fungi but also highlighted a distinct pathway for sesterterpene biosynthesis in basidiomycetes. The current work provides an important basis for uncovering novel gene functions involved in sesterterpene synthesis and for gaining insights into the mechanism of silent gene activation in fungal defense.

GroEL, a novel vaccine candidate of piscine Streptococcus agalactiae identified by immunoproteome.

Streptococcus agalactiae is the major etiological agent of streptococcosis, which is responsible for huge economic losses in fishery, particularly in tilapia (Oreochromis niloticus) aquaculture. A research priority to control streptococcosis is to develop vaccines, so we sought to figure out the immunogenic proteins of S. agalactiae and screen the vaccine candidates for streptococcosis in the present study. Immunoproteomics, a technique involving two-dimensional gel electrophoresis (2-DE) followed by immunoblotting and mass spectrometry (MS), was employed to investigate the immunogenic proteins of S. agalactiae THN0901. Whole-cell soluble proteins were separated using 2-DE, and the immunogenic proteins were detected by western blotting using rabbit anti-S. agalactiae sera. A total of 17 immunoreactive spots on the soluble protein profile, corresponding to 15 different proteins, were identified by MALDI-TOF/TOF MS. Among the immunogenic proteins, GroEL attracted our attention as it was demonstrated to be immunogenic and protective against other streptococci. Nevertheless, to date, there have been no published reports on the immunogenicity and protective efficacy of GroEL against piscine S. agalactiae. Therefore, recombinant GroEL (rGroEL) was expressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. Immunization of tilapia with rGroEL resulted in an increase in antibody titers and conferred protection against S. agalactiae, with the relative percentage survival of 68.61 ±â€¯7.39%. The immunoproteome in the present study narrows the scope of vaccine candidates, and the evaluation of GroEL immunogenicity and protective efficacy shows that GroEL forms an ideal candidate molecule in subunit vaccine against S. agalactiae.

Small-molecule-based generation of functional cardiomyocytes from human umbilical cord-derived induced pluripotent stem cells.

The purpose of this study was to investigate the cardiac-differentiation potential of induced pluripotent stem cells (iPSCs) generated from human umbilical cord-derived mesenchymal cells. Spontaneous beating colonies were observed at day 7 after the sequential addition of CHIR99021 and IWP-4. The combined use of CHIR99021 and IWP-4 downregulated the expression of pluripotency markers while upregulating cardiac transcription factors and cardiomyocyte-specific markers. The derived cardiomyocytes demonstrated typical sarcomeric structures and action-potential features; most importantly, the derived cells exhibited responsiveness to ß-adrenergic and muscarinic stimulations. The analyses of molecular, structural, and functional properties revealed that the derived cardiomyocytes were similar to cardiomyocytes derived from BJ foreskin fibroblast cells. In summary, our results demonstrate that functional cardiomyocytes can be generated from human umbilical cord-derived cells. The methodology described here has potential as a means for the production of functional cardiomyocytes from discarded human umbilical cord tissue.

Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor.

BACKGROUND: Butadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resources. Although the idea of biological synthesis of butadiene from sugars has been discussed in the literature, success for that goal has so far not been reported. As a model system for methanol assimilation, Methylobacterium extorquens AM1 can produce several unique metabolic intermediates for the production of value-added chemicals, including crotonyl-CoA as a potential precursor for butadiene synthesis. RESULTS: In this work, we focused on constructing a metabolic pathway to convert crotonyl-CoA into crotyl diphosphate, a direct precursor of butadiene. The engineered pathway consists of three identified enzymes, a hydroxyethylthiazole kinase (THK) from Escherichia coli, an isopentenyl phosphate kinase (IPK) from Methanothermobacter thermautotrophicus and an aldehyde/alcohol dehydrogenase (ADHE2) from Clostridium acetobutylicum. The Km and kcat of THK, IPK and ADHE2 were determined as 8.35 mM and 1.24 s-1, 1.28 mM and 153.14 s-1, and 2.34 mM and 1.15 s-1 towards crotonol, crotyl monophosphate and crotonyl-CoA, respectively. Then, the activity of one of rate-limiting enzymes, THK, was optimized by random mutagenesis coupled with a developed high-throughput screening colorimetric assay. The resulting variant (THKM82V) isolated from over 3000 colonies showed 8.6-fold higher activity than wild-type, which helped increase the titer of crotyl diphosphate to 0.76 mM, corresponding to a 7.6% conversion from crotonol in the one-pot in vitro reaction. Overexpression of native ADHE2, IPK with THKM82V under a strong promoter mxaF in M. extorquens AM1 did not produce crotyl diphosphate from crotonyl-CoA, but the engineered strain did generate 0.60 µg/mL of intracellular crotyl diphosphate from exogenously supplied crotonol at mid-exponential phase. CONCLUSIONS: These results represent the first step in producing a butadiene precursor in recombinant M. extorquens AM1. It not only demonstrates the feasibility of converting crotonol to key intermediates for butadiene biosynthesis, it also suggests future directions for improving catalytic efficiency of aldehyde/alcohol dehydrogenase to produce butadiene precursor from methanol.

miR­760 mediates hypoxia-induced proliferation and apoptosis of human pulmonary artery smooth muscle cells via targeting TLR4.

MicroRNAs (miRNAs) have a key role in the pathogenesis of pulmonary arterial hypertension (PAH), a disease characterized by enhanced proliferation and reduced apoptosis of pulmonary artery smooth muscle cells. In the present study, miR­760 was demonstrated to be downregulated in PAH lung tissues compared with normal lung tissues, an effect that may be associated with the development of PAH. Hypoxia is an important stimulus for human pulmonary artery smooth muscle cell (hPASMC) proliferation and the occurrence of PAH. Therefore, the effect of miR­760 in hypoxia­treated and normal hPASMCs was investigated. Expression of exogenous miR­760 decreased cell proliferation in hypoxia­induced hPASMCs, and promoted cell apoptosis with an increase in the BCL2 associated X/BCL2 ratio and the expression levels of Caspase­3 and Caspase­9. In addition, overexpression of miR­760 suppressed the migration of hPASMCs under hypoxic conditions. Furthermore, miR­760 was demonstrated to mediate its anti­proliferation effect via the regulation of toll­like receptor 4 (TLR4), a direct target of miR­760. The results revealed that knockdown of TLR4 restrained the hypoxia­induced hPASMC proliferation and induced cell apoptosis. The present study uncovered a novel regulatory pathway involving miR­760 and suggested that miR­760 may be explored as a potential therapy for PAH in the future.