Low-dose metformin targets the lysosomal AMPK pathway through PEN2.

Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.

Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome.

BACKGROUND: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. RESULTS: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. CONCLUSIONS: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

A comprehensive analysis of the WRKY family in soybean and functional analysis of GmWRKY164-GmGSL7c in resistance to soybean mosaic virus.

BACKGROUND: Soybean mosaic disease caused by soybean mosaic virus (SMV) is one of the most devastating and widespread diseases in soybean producing areas worldwide. The WRKY transcription factors (TFs) are widely involved in plant development and stress responses. However, the roles of the GmWRKY TFs in resistance to SMV are largely unclear. RESULTS: Here, 185 GmWRKYs were characterized in soybean (Glycine max), among which 60 GmWRKY genes were differentially expressed during SMV infection according to the transcriptome data. The transcriptome data and RT-qPCR results showed that the expression of GmWRKY164 decreased after imidazole treatment and had higher expression levels in the incompatible combination between soybean cultivar variety Jidou 7 and SMV strain N3. Remarkably, the silencing of GmWRKY164 reduced callose deposition and enhanced virus spread during SMV infection. In addition, the transcript levels of the GmGSL7c were dramatically lower upon the silencing of GmWRKY164. Furthermore, EMSA and ChIP-qPCR revealed that GmWRKY164 can directly bind to the promoter of GmGSL7c, which contains the W-box element. CONCLUSION: Our findings suggest that GmWRKY164 plays a positive role in resistance to SMV infection by regulating the expression of GmGSL7c, resulting in the deposition of callose and the inhibition of viral movement, which provides guidance for future studies in understanding virus-resistance mechanisms in soybean.

4-octyl itaconate ameliorates ventilator-induced lung injury.

Ventilator-induced lung injury (VILI) disturbs the disordered immune system and causes persistent inflammatory damage. 4-octyl itaconate (OI) is a synthetic cell-permeable itaconate derivative with antioxidant and anti-inflammatory effects. In this study, we assessed whether OI protects against VILI. OI was intraperitoneally injected for three days before mechanical ventilation (MV; 20 ml/kg at 70 breaths/min) for 2 h. Mouse lung vascular endothelial cells (MLVECs) were pretreated with OI (62.5, 125, and 250 µM) prior to cyclic stretch for 4 h. We found that OI attenuated VILI and inflammatory response. OI also increased superoxide dismutase, nuclear factor E2-related factor 2, and heme oxygenase-1 levels, and decreased reactive oxygen species and malondialdehyde levels. Furthermore, OI inhibited the expression of NLR family pyrin domain-containing 3 (NLRP3), caspase-1 p20, apoptosis-associated speck-like protein containing a CARD, and N-terminal fragment of gasdermin D. Therefore, OI attenuates VILI, potentially by suppressing oxidative stress and NLRP3 activation.

Cold tolerance SNPs and candidate gene mining in the soybean germination stage based on genome-wide association analysis.

KEY MESSAGE: Three QTLs associated with low-temperature tolerance were identified by genome-wide association analysis, and 15 candidate genes were identified by haplotype analysis and gene expression analyses. Low temperature is a critical factor affecting the geographical distribution, growth, development, and yield of soybeans, with cold stress during seed germination leading to substantial productivity loss. In this study, an association panel comprising 260 soybean accessions was evaluated for four germination traits and four cold tolerance index traits, revealing extensive variation in cold tolerance. Genome-wide association study (GWAS) identified 10 quantitative trait nucleotides (QTNs) associated with cold tolerance, utilizing 30,799 single nucleotide polymorphisms (SNPs) and four GWAS models. Linkage disequilibrium (LD) analysis positioned these QTNs within three cold-tolerance quantitative trait loci (QTL) and, with QTL19-1, was positioned by three multi-locus models, underscoring its importance as a key QTL. Integrative haplotype analysis, supplemented by transcriptome analysis, uncovered 15 candidate genes. The haplotypes within the genes Glyma.18G044200, Glyma.18G044300, Glyma.18G044900, Glyma.18G045100, Glyma.19G222500, and Glyma.19G222600 exhibited significant phenotypic variations, with differential expression in materials with varying cold tolerance. The QTNs and candidate genes identified in this study offer substantial potential for marker-assisted selection and gene editing in breeding cold-tolerant soybeans, providing valuable insights into the genetic mechanisms underlying cold tolerance during soybean germination.

Genome dosage alteration caused by chromosome pyramiding and shuffling effects on karyotypic heterogeneity, reproductive diversity, and phenotypic variation in Zea-Tripsacum allopolyploids.

KEY MESSAGE: We developed an array of Zea-Tripsacum tri-hybrid allopolyploids with multiple ploidies. We unveiled that changes in genome dosage due to the chromosomes pyramiding and shuffling of three species effects karyotypic heterogeneity, reproductive diversity, and phenotypic variation in Zea-Tripsacum allopolyploids. Polyploidy, or whole genome duplication, has played a major role in evolution and speciation. The genomic consequences of polyploidy have been extensively studied in many plants; however, the extent of chromosomal variation, genome dosage, phenotypic diversity, and heterosis in allopolyploids derived from multiple species remains largely unknown. To address this question, we synthesized an allohexaploid involving Zea mays, Tripsacum dactyloides, and Z. perennis by chromosomal pyramiding. Subsequently, an allooctoploid and an allopentaploid were obtained by hybridization of the allohexaploid with Z. perennis. Moreover, we constructed three populations with different ploidy by chromosomal shuffling (allopentaploid × Z. perennis, allohexaploid × Z. perennis, and allooctoploid × Z. perennis). We have observed 3 types of sexual reproductive modes and 2 types of asexual reproduction modes in the tri-species hybrids, including 2n gamete fusion (2n + n), haploid gamete fusion (n + n), polyspermy fertilization (n + n + n) or 2n gamete fusion (n + 2n), haploid gametophyte apomixis, and asexual reproduction. The tri-hybrids library presents extremely rich karyotype heterogeneity. Chromosomal compensation appears to exist between maize and Z. perennis. A rise in the ploidy of the trihybrids was linked to a higher frequency of chromosomal translocation. Variation in the degree of phenotypic diversity observed in different segregating populations suggested that genome dosage effects phenotypic manifestation. These findings not only broaden our understanding of the mechanisms of polyploid formation and reproductive diversity but also provide a novel insight into genome pyramiding and shuffling driven genome dosage effects and phenotypic diversity.

A preliminary prediction model of pediatric Mycoplasma pneumoniae pneumonia based on routine blood parameters by using machine learning method.

BACKGROUND: The prevalence and severity of pediatric Mycoplasma pneumoniae pneumonia (MPP) poses a significant threat to the health and lives of children. In this study, we aim to systematically evaluate the value of routine blood parameters in predicting MPP and develop a robust and generalizable ensemble artificial intelligence (AI) model to assist in identifying patients with MPP. METHODS: We collected 27 features, including routine blood parameters and hs-CRP levels, from patients admitted to The Affiliated Dazu's Hospital of Chongqing Medical University with or without MPP between January, 2023 and January, 2024. A classification model was built using seven machine learning (ML) algorithms to develop an integrated prediction tool for diagnosing MPP. It was evaluated on both an internal validation set (982 individuals) and an external validation set (195 individuals). The primary outcome measured the accuracy of the model in predicting MPP. RESULTS: The GBDT is state-of-the-art based on 27 features. Following inter-laboratory cohort testing, the GBDT demonstrated an AUC, accuracy, specificity, sensitivity, PPV, NPV, and F1-score of 0.980 (0.938-0.995), 0.928 (0.796-0.970), 0.929 (0.717-1.000), 0.926 (0.889-0.956), 0.922 (0.727-1.000), 0.937 (0.884-0.963), and 0.923 (0.800-0.966) in stratified 10-fold cross-validation. A GBDT-based AI Lab was developed to facilitate the healthcare providers in remote and impoverished areas. CONCLUSIONS: The GBDT-based AI Lab tool, with high sensitivity and specificity, could help discriminate between pediatric MPP infection and non-MPP infection based on routine blood parameters. Moreover, a user-friendly webpage tool for AI Lab could facilitate healthcare providers in remote and impoverished areas where advanced technologies are not accessible.

Dissecting the role of soybean rhizosphere-enriched bacterial taxa in modulating nitrogen-cycling functions.

Crop roots selectively recruit certain microbial taxa that are essential for supporting their growth. Within the recruited microbes, some taxa are consistently enriched in the rhizosphere across various locations and crop genotypes, while others are unique to specific planting sites or genotypes. Whether these differentially enriched taxa are different in community composition and how they interact with nutrient cycling need further investigation. Here, we sampled bulk soil and the rhizosphere soil of five soybean varieties grown in Shijiazhuang and Xuzhou, categorized the rhizosphere-enriched microbes into shared, site-specific, and variety-specific taxa, and analyzed their correlation with the diazotrophic communities and microbial genes involved in nitrogen (N) cycling. The shared taxa were dominated by Actinobacteria and Thaumarchaeota, the site-specific taxa were dominated by Actinobacteria in Shijiazhuang and by Nitrospirae in Xuzhou, while the variety-specific taxa were more evenly distributed in several phyla and contained many rare operational taxonomic units (OTUs). The rhizosphere-enriched taxa correlated with most diazotroph orders negatively but with eight orders including Rhizobiales positively. Each group within the shared, site-specific, and variety-specific taxa negatively correlated with bacterial amoA and narG in Shijiazhuang and positively correlated with archaeal amoA in Xuzhou. These results revealed that the shared, site-specific, and variety-specific taxa are distinct in community compositions but similar in associations with rhizosphere N-cycling functions. They exhibited potential in regulating the soybean roots' selection for high-efficiency diazotrophs and the ammonia-oxidizing and denitrification processes. This study provides new insights into soybean rhizosphere-enriched microbes and their association with N cycling. KEY POINTS: • Soybean rhizosphere affected diazotroph community and enriched nifH, amoA, and nosZ. • Shared and site- and variety-specific taxa were dominated by different phyla. • Rhizosphere-enriched taxa were similarly associated with N-cycle functions.

Long-term carbaryl exposure leads to behavioral abnormalities and reproductive toxicity in male marine medaka through apoptosis-mediated HPA and HPG axes dysregulation.

Carbaryl is a widely used carbamate pesticide that has been detected in the marine environment, but its effects on marine fish are still unknown. This study was aimed to investigate the effects of long-term exposure of carbaryl on male marine medaka. For this purpose, we set up five exposure concentration groups of 0, 0.1, 1, 10, and 100 µg/L for 180 days. On the one hand, we observed increased aggression and decreased ability to avoid predators in males after exposure, which was affected by the levels of HPA-axis hormones, especially decreased cortisol level. On the other hand, after exposure, HPG axis hormone levels and gene transcription levels were disturbed. Males exhibited a decreased gonadosomatic index and a notable reduction in mature sperm proportion and the F1 generation displayed a significant increase in malformation rate. Additionally, the number of apoptotic cells and the transcription level of apoptosis-related genes in the brains of male marine medaka substantially increased after exposure. Apoptosis of brain cells may be responsible for the disturbance of HPA and HPG axes, consequently leading to behavioral and reproductive abnormalities. These findings provide novel insights into evaluating the toxic effects of carbaryl on male marine medaka and emphasizing the criticality of exploring the potential environmental risks posed by carbaryl in the marine environment, thus providing toxicity value basis for further strengthening marine environmental monitoring and the protection of biological resources.

High-Efficiency Photo-Fenton-like Catalyst of FeOOH/g-C3N4 for the Degradation of PNP: Characterization, Catalytic Performance and Mechanism Exploration.

The composite photocatalyst FeOOH/g-C3N4 was prepared through thermal polycondensation and co-precipitation methods, followed by XRD, SEM and UV-vis characterization. The stability of FeOOH/g-C3N4 was explored by the recycling test. The active species in the reaction system were investigated by the capture experiment. The results indicated that the optimal preparation condition for g-C3N4 involved calcination at 600 °C for 4 h. XRD analysis revealed that g-C3N4 exhibits a high-purity phase, and Fe in FeOOH/g-C3N4 exists in a highly dispersed amorphous state. SEM analysis showed that FeOOH/g-C3N4 has a rough surface with an irregular layered structure. Element composition analysis confirmed that the content of elements in the prepared catalyst is consistent with the theoretical calculation. FeOOH/g-C3N4 possesses the largest specific surface area of 143.2 m2/g and a suitable pore distribution. UV-vis DRS analysis showed that the absorption intensity of FeOOH/g-C3N4 is stronger than that of g-C3N4. When the catalyst dosage was 1.0 g/L, the H2O2 dosage was 4 mmol/L, the PNP initial concentration was 10 mg/L and the initial pH value was 5, the PNP removal could reach 92% in 120 min. Even after 5 cycles, the efficiency of PNP removal by FeOOH/g-C3N4 remains nearly 80%. The capture experiment indicated that both •OH and •O2- play roles in the photocatalytic degradation of PNP, with •OH being more significant. These findings affirm that FeOOH has been successfully incorporated into g-C3N4, resulting in a conspicuous catalytic effect on the degradation of PNP in the visible light-assisted Fenton-like reaction.

A Novel Quercetin Encapsulated Glucose Modified Liposome and Its Brain-Target Antioxidative Neuroprotection Effects.

Neurodegenerative diseases (NDDs) are mainly induced by oxidative stress which produces excessive reactive oxygen species (ROS). Quercetin (QU) is a potent antioxidant with some effects on NDDs. This study prepared and characterized a novel glucose-modified QU liposome (QU-Glu-Lip), aiming not only to overcome QU's poor water solubility and bioavailability but also to deliver more QU to brain tissue to enhance its neuroprotective effect. QU-Glu-Lip possessed encapsulation efficiency (EE) of 89.9%, homogenous particle sizes (116-124 nm), small PDI value (<0.3), zeta value -1.363 ± 0.437 mV, proper pH and salt stability, and proper cytotoxicity. The glucose-modified liposome penetrated the blood-brain barrier (BBB) mediated via the glucose transporter 1 (GLUT1) and was taken by neuronal cells more efficiently than liposome without glucose, according to bEnd.3 and PC12 cell tests. QU-Glu-Lip attenuated H2O2-induced oxidative damage to PC12 with higher cell viability (88.42%) and lower intracellular ROS compared to that of QU. QU-Glu-Lip had higher brain target ability and delivered more QU to neuronal cells, effectively exerting the antioxidative neuroprotection effect. There is potential for the QU-Glu-Lip application for more effective treatment of NDDs.

Longitudinal Relationships Between School Climate, Academic Achievement, and Gaming Disorder Symptoms Among Chinese Adolescents.

Despite growing concerns regarding the development of gaming disorder symptoms among adolescents, the longitudinal relationship between school factors and gaming disorder symptoms remains far from being fully understood. This two-year longitudinal study examined the relationship between school climate perceptions, academic achievement, and gaming disorder symptoms among three distinct demographic cohorts: preadolescents (n = 1513; 46.9% girls, Mage = 10.64 years, SD = 0.56), early adolescents (n = 1771; 48.3% girls, Mage = 13.54 years, SD = 0.70), and late adolescents (n = 2385; 50.1% girls, Mage = 16.41 years, SD = 0.59). A four-wave study was conducted (six months apart) using random intercept cross-lagged panel models (RI-CLPMs) to separate the within-person (state level) from the between-person (trait level) effects. The results obtained from the RI-CLPMs indicated that fluctuations in school climate perceptions negatively predicted subsequent changes in gaming disorder symptoms among preadolescents at the within-person level, but not among early and late adolescents. Fluctuations relating to gaming disorder symptoms also negatively predicted subsequent changes regarding academic achievement in late adolescents, but not in preadolescents and early adolescents. The effect of school-related factors on gaming disorder symptoms varies across different developmental stages. While preadolescents may represent a particularly susceptible subgroup for gaming disorder in terms of being predicted by their school environment, late adolescents appear to be more vulnerable to predictors of gaming disorder symptoms. The current study also discusses the implications of school-wide programs aimed at improving school climate and preventing the development of gaming disorder symptoms during key developmental periods.

Long-term exposure to aryl hydrocarbon receptor agonist neburon induces reproductive toxicity in male zebrafish (Danio rerio).

Neburon is a phenylurea herbicide that is widely used worldwide, but its toxicity is poorly studied. In our previous study, we found that neburon has strong aryl hydrocarbon receptor (AhR) agonist activity, but whether it causes reproductive toxicity is not clear. In the present study, zebrafish were conducted as a model organism to evaluate whether environmental concentrations of neburon (0.1, 1 and 10 µg/L) induce reproductive disorder in males. After exposure to neburon for 150 days from embryo to adult, that the average spawning egg number in high concentration group was 106.40, which was significantly lower than 193.00 in control group. This result was mainly due to the abnormal male reproductive behavior caused by abnormal transcription of genes associated with reproductive behavior in the brain, such as secretogranin-2a. The proportions of spermatozoa in the medium and high concentration groups were 82.40% and 83.84%, respectively, which were significantly lower than 89.45% in control group. This result was mainly caused by hormonal disturbances and an increased proportion of apoptotic cells. The hormonal disruption was due to the significant changes in the transcription levels of key genes in the hypothalamus-pituitary-gonadal axis following neburon treatment. Neburon treatment also significantly activated the AhR signaling pathway, causing oxidative stress damage and eventually leading to a significant increase in apoptosis in the exposed group. Together, these data filled the currently more vacant profile of neburon toxicity and might provide information to assess the ecotoxicity of neburon on male reproduction at environmentally relevant concentrations.

OsRbohI Regulates Rice Growth and Development via Jasmonic Acid Signalling.

Reactive oxygen species (ROS) are highly reactive molecules, generated by nicotinamide adenine dinucleotide phosphate oxidases encoded by respiratory burst oxidase homologs. The functions of the OsRbohs gene family in rice are diverse and poorly understood. OsRbohI was recently identified as a newly evolved gene in the rice OsRbohs gene family. However, the function of OsRbohI in regulating rice growth is not yet reported. In this study, our results indicate that knockout (KO) OsRbohI mutants showed significantly shorter shoot and primary roots, along with lower ROS content than the control lines, whereas the overexpression (OE) lines displayed contrasting results. Further experiments showed that the abnormal length of the shoot and root is mainly caused by altered cell size. These results indicate that OsRbohI regulates rice shoot and root growth through the ROS signal. More importantly, RNA-seq analysis and jasmonic acid (JA) treatment demonstrated that OsRbohI regulates rice growth via the JA synthesis and signaling pathways. Compared with the control, the results showed that the KO mutants were more sensitive to JA, whereas the OE lines were less sensitive to JA. Collectively, our results reveal a novel pathway in which OsRbohI regulates rice growth and development by affecting their ROS homeostasis through JA synthesis and signaling pathway.

Comprehensive review and evaluation of computational methods for identifying FLT3-internal tandem duplication in acute myeloid leukaemia.

Internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3-ITD) constitutes an independent indicator of poor prognosis in acute myeloid leukaemia (AML). AML with FLT3-ITD usually presents with poor treatment outcomes, high recurrence rate and short overall survival. Currently, polymerase chain reaction and capillary electrophoresis are widely adopted for the clinical detection of FLT3-ITD, whereas the length and mutation frequency of ITD are evaluated using fragment analysis. With the development of sequencing technology and the high incidence of FLT3-ITD mutations, a multitude of bioinformatics tools and pipelines have been developed to detect FLT3-ITD using next-generation sequencing data. However, systematic comparison and evaluation of the methods or software have not been performed. In this study, we provided a comprehensive review of the principles, functionality and limitations of the existing methods for detecting FLT3-ITD. We further compared the qualitative and quantitative detection capabilities of six representative tools using simulated and biological data. Our results will provide practical guidance for researchers and clinicians to select the appropriate FLT3-ITD detection tools and highlight the direction of future developments in this field. Availability: A Docker image with several programs pre-installed is available at https://github.com/niu-lab/docker-flt3-itd to facilitate the application of FLT3-ITD detection tools.

Development of SNP marker panels for genotyping by target sequencing (GBTS) and its application in soybean.

A high-throughput genotyping platform with customized flexibility, high genotyping accuracy, and low cost is critical for marker-assisted selection and genetic mapping in soybean. Three assay panels were selected from the SoySNP50K, 40K, 20K, and 10K arrays, containing 41,541, 20,748, and 9670 SNP markers, respectively, for genotyping by target sequencing (GBTS). Fifteen representative accessions were used to assess the accuracy and consistency of the SNP alleles identified by the SNP panels and sequencing platform. The SNP alleles were 99.87% identical between technical replicates and 98.86% identical between the 40K SNP GBTS panel and 10× resequencing analysis. The GBTS method was also accurate in the sense that the genotypic dataset of the 15 representative accessions correctly revealed the pedigree of the accessions, and the biparental progeny datasets correctly constructed the linkage maps of the SNPs. The 10K panel was also used to genotype two parent-derived populations and analyze QTLs controlling 100-seed weight, resulting in the identification of the stable associated genetic locus Locus_OSW_06 on chromosome 06. The markers flanking the QTL explained 7.05% and 9.83% of the phenotypic variation, respectively. Compared with GBS and DNA chips, the 40K, 20K, and 10K panels reduced costs by 5.07% and 58.28%, 21.44% and 65.48%, and 35.74% and 71.76%, respectively. Low-cost genotyping panels could facilitate soybean germplasm assessment, genetic linkage map construction, QTL identification, and genomic selection. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01372-6.

A unified DNA- and RNA-based NGS strategy for the analysis of multiple types of variants at the dual nucleic acid level in solid tumors.

BACKGROUND: Targeted next-generation sequencing (NGS) is a powerful and suitable approach to comprehensively identify multiple types of variants in tumors. RNA-based NGS is increasingly playing an important role in precision oncology. Both parallel and sequential DNA- and RNA-based approaches are expensive, burdensome, and have long turnaround times, which can be impractical in clinical practice. A streamlined, unified DNA- and RNA-based NGS approach is urgently needed in clinical practice. METHODS: A DNA/RNA co-hybrid capture sequencing (DRCC-Seq) approach was designed to capture pre-capture DNA and RNA libraries in a single tube and convert them into one NGS library. The performance of the DRCC-Seq approach was evaluated by a panel of reference standards and clinical samples. RESULTS: The average depth, DNA data ratio, capture ratio, and target coverage 250 (×) of the DNA panel data had a negative correlation with an increase in the proportion of RNA probes. The SNVs, indels, fusions, and MSI status were not affected by the proportion of RNA probes, but the copy numbers of the target genes were higher than expected in the standard materials, and many unexpected gene amplifications were found using D:R (1:2) and D:R (1:4) probe panels. The optimal ratio of DNA and RNA probes in the combined probe panel was 1:1 using the DRCC-Seq approach. The DRCC-Seq approach was feasible and reliable for detecting multiple types of variants in reference standards and real-world clinical samples. CONCLUSIONS: The DRCC-Seq approach is more cost-effective, with a shorter turnaround time and lower labor requirements than either parallel or sequential targeted DNA NGS and RNA NGS. It is feasible to identify multiple genetic variations at the DNA and RNA levels simultaneously in clinical practice.

A novel RRGW derived peptide is a promising inhibitor of BoNT/A.

Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent biotoxins ever known. Its entry into neurons could block vesicle exocytosis to abolish the release of neurotransmitters from nerve terminals, thus leading to muscle paralysis. Although there are so many peptides, antibodies and chemical compounds claimed to have anti-toxin activity, no drug is available in the clinical application except equine antitoxin serum. In the present work, a short peptide inhibitor RRGW of BoNT/A was firstly identified by computer-aided ligand-receptor binding simulation, then an RRGW derived peptide was rational designed based on the fragment of SNAP-25 (141-206 aa). Proteolytic assay showed that the anti-toxin activity of the RRGW derived peptide was much higher than that of RRGW. Digit abduction score assay demonstrated that the derived peptide delayed BoNT/A-induced muscle paralysis at a lower concentration by 20-fold than RRGW. The results supported that RRGW derived peptide can be a potential BoNT/A inhibitor candidate for further treating botulism.

A situational analysis of human resource and non-communicable diseases management for community health workers in Chengdu, China: a cross-sectional study.

BACKGROUND: Non-communicable diseases (NCDs) pose a major challenge to health economic cost and residents' health status. Community health workers (CHWs) are the gatekeeper of primary health care. OBJECTIVE: This study aimed to conduct a situational analysis of current human resource and requirements of NCDs-related training among CHWs in Chengdu with regard to address to understand the suggestions for improvement of challenges and barriers. METHODS: A descriptive online cross-sectional survey was conducted among CHWs (doctors and nurses) from 23 districts and counties in Chengdu. Sociodemographic and NCDs-related variables were collected. Univariate analysis and multiple response analysis were used to describe the characteristics of these variables. RESULTS: 711 doctors and 637 nurses completely responded. There were significant differences among gender, age, educational levels, professional title, working year, type of institution, urban circle and registration in general practice between doctors and nurses (P < 0.001). 60.6% of doctors were female, compared to 98.0% for nurses. 58.2% of doctors held a bachelor's degree compared with 45.4% of nurses, while 48.3% of nurses held a junior college degree compared with 25.7% of doctors. Higher levels of professional title and registration in general practice were found in doctors compared with nurses. The proportions of NCDs' category, NCDs-related roles and tasks, NCDs-related training contents and forms that CHWs have attend and hoped to gain more were significantly different between doctors and nurses (P < 0.001). In general, the proportions in nurses were much lower than those of doctors (P < 0.05). The top five diseases managed by CHWs were hypertension, diabetes, cerebrovascular disease, chronic respiratory diseases and mental diseases. The five most reported roles performed among doctors included the distribution of health education (91.4%), following up (85.9%), establishing archives (71.3%), medicine adjustment (64.7%) and treatment implementation (52.0%). The top three diseases managed by nurses were same with doctors. The top four and five tasks were contact with patients or health services (39.6%) and referral (16.6%) in nurses. Most CHWs had received primary and common diseases-related trainings, but they had few opportunities to study in a tertiary hospital (40.4% in doctors and 20.9% in nurses, respectively), attend domestic academic conferences (26.9% in doctors vs. 9.7% in nurses), and take part in training courses (44.9% in nurses). CHWs hoped that the above-discussed training contents and forms could be provided more in the future. Besides basic skills related trainings, some specific skills related trainings should be strengthened. CONCLUSION: The qualifications in doctors were much better than those of nurses. The roles performed by CHWs in NCDs management are varied form common and frequent disease management to subsequent follow up and supervision. CHWs hope to receive more desired and oriented trainings. There is a need for building capacity of CHWs, optimizing and defining CHWs' role, facilitating postgraduate medical education support and strengthening multidisciplinary collaboration would be effective in NCDs management.

An R2R3 MYB transcription factor confers brown planthopper resistance by regulating the phenylalanine ammonia-lyase pathway in rice.

Brown planthopper (BPH) is one of the most destructive insects affecting rice (Oryza sativa L.) production. Phenylalanine ammonia-lyase (PAL) is a key enzyme involved in plant defense against pathogens, but the role of PAL in insect resistance is still poorly understood. Here we show that expression of the majority of PALs in rice is significantly induced by BPH feeding. Knockdown of OsPALs significantly reduces BPH resistance, whereas overexpression of OsPAL8 in a susceptible rice cultivar significantly enhances its BPH resistance. We found that OsPALs mediate resistance to BPH by regulating the biosynthesis and accumulation of salicylic acid and lignin. Furthermore, we show that expression of OsPAL6 and OsPAL8 in response to BPH attack is directly up-regulated by OsMYB30, an R2R3 MYB transcription factor. Taken together, our results demonstrate that the phenylpropanoid pathway plays an important role in BPH resistance response, and provide valuable targets for genetic improvement of BPH resistance in rice.