A new variant of the colistin resistance gene MCR-1 with co-resistance to ß-lactam antibiotics reveals a potential novel antimicrobial peptide.

The emerging and global spread of a novel plasmid-mediated colistin resistance gene, mcr-1, threatens human health. Expression of the MCR-1 protein affects bacterial fitness and this cost correlates with lipid A perturbation. However, the exact molecular mechanism remains unclear. Here, we identified the MCR-1 M6 variant carrying two-point mutations that conferred co-resistance to ß-lactam antibiotics. Compared to wild-type (WT) MCR-1, this variant caused severe disturbance in lipid A, resulting in up-regulation of L, D-transpeptidases (LDTs) pathway, which explains co-resistance to ß-lactams. Moreover, we show that a lipid A loading pocket is localized at the linker domain of MCR-1 where these 2 mutations are located. This pocket governs colistin resistance and bacterial membrane permeability, and the mutated pocket in M6 enhances the binding affinity towards lipid A. Based on this new information, we also designed synthetic peptides derived from M6 that exhibit broad-spectrum antimicrobial activity, exposing a potential vulnerability that could be exploited for future antimicrobial drug design.

Prediction of Antibiotic Resistance Evolution by Growth Measurement of All Proximal Mutants of Beta-Lactamase.

The antibiotic resistance crisis continues to threaten human health. Better predictions of the evolution of antibiotic resistance genes could contribute to the design of more sustainable treatment strategies. However, comprehensive prediction of antibiotic resistance gene evolution via laboratory approaches remains challenging. By combining site-specific integration and high-throughput sequencing, we quantified relative growth under the respective selection of cefotaxime or ceftazidime selection in ∼23,000 Escherichia coli MG1655 strains that each carried a unique, single-copy variant of the extended-spectrum ß-lactamase gene blaCTX-M-14 at the chromosomal att HK022 site. Significant synergistic pleiotropy was observed within four subgenic regions, suggesting key regions for the evolution of resistance to both antibiotics. Moreover, we propose PEARP and PEARR, two deep-learning models with strong clinical correlations, for the prospective and retrospective prediction of blaCTX-M-14 evolution, respectively. Single to quintuple mutations of blaCTX-M-14 predicted to confer resistance by PEARP were significantly enriched among the clinical isolates harboring blaCTX-M-14 variants, and the PEARR scores matched the minimal inhibitory concentrations obtained for the 31 intermediates in all hypothetical trajectories. Altogether, we conclude that the measurement of local fitness landscape enables prediction of the evolutionary trajectories of antibiotic resistance genes, which could be useful for a broad range of clinical applications, from resistance prediction to designing novel treatment strategies.

Improving the High-Temperature Energy Storage Performance of Epoxy Films: Moderately Reducing Unsaturation for Extremely High Efficiency.

The rapid development of renewable energy systems, electric vehicles, and pulsed equipment requires energy storage media to have a high energy storage density and efficiency in a wide temperature range. The state-of-the-art biaxially oriented polypropylene (BOPP) film is insufficient to meet the growing demand for energy storage devices due to its low energy storage density and working temperature, which make it a research hotspot for developing dielectric energy storage materials. In this manuscript, based on the epoxy materials that have been shown as a potential energy storage medium, we aim to reduce the influence of the benzene ring delocalization structure on the energy storage losses and enhance the efficiency by gradually replacing them with cyclohexane structures to adjust the segment unsaturation of epoxy materials. The results show that by partially reducing the unsaturation of the curing agent, the epoxy material achieves an excellent high-temperature energy storage density of 2.21 J/cm3 at 150 °C and 300 MV/m while maintaining an extremely high energy storage efficiency of 99.2%. Leakage current density and high-voltage dielectric spectroscopy tests confirm that a moderate reduction of the segment unsaturation of epoxy materials can greatly inhibit polarization loss at high temperatures, which may explain their high energy storage efficiency.

Influences of water level fluctuation on water exchange and nutrient distribution in a bay: Evidence from the Xiangxi Bay, Three Gorges Reservoir.

Following the Three Gorges Reservoir (TGR) impoundment, many tributaries were turned into bays; hydrodynamic conditions of TGR profoundly changed the residence time, temperature, and nutrient distributions of bays, and nutrient enrichment occurred in these bays. However, little research has been done on the effects of water level qqfluctuations (WLFs) of TGR on the bay. In this study, Xiangxi Bay (XXB), one of the tributaries of TGR, was selected as the delegate to construct and calibrate a two-dimensional hydrodynamic-temperature-tracer-water quality model based on the CE-QUAL-W2. The results were the following: 1) In spring, as total nitrogen (TN) in the TGR tended to be higher than that in the XXB, the downward WLF increased water exchange, TGR-XXB nutrient flux and TN in the epilimnion of the XXB, and decreased the water exchange and TN in the hypolimnion of the XXB. The upward WLF did the opposite. The situation would be reversed in autumn. 2) Under a larger magnitude or a shorter period of WLF, its corresponding effects on the water exchange and TN increased. 2) Both the downward and upward modes of WLF helped to decrease the thermal stratification of XXB. 4) The upward/downward WLF could be used to decrease the epilimnetic TN of XXB in spring/autumn, and was suggested to reduce the local algal bloom. The WLFs by the TGR regulation could profoundly change the water exchange and nutrient distribution in the bay, which helped to control nutrient concentrations and prevent algal blooms.

Bacillus licheniformis prevents and reduces anxiety-like and depression-like behaviours.

As common mental disorders, depression and anxiety impact people all around the world. Recent studies have found that the gut microbiome plays an important role in mental health. It is becoming possible to treat mental disorders by regulating the composition of the gut microbiota. Bacillus licheniformis is a probiotic used to treat gut diseases through balancing the gut microbiome during lasting years. Considering the role of gut microbiota in the gut-brain axis, this study used chronic unpredictable mild stress (CUMS) model rats to explore whether Bacillus licheniformis can prevent and treat depression and anxiety. We found that B. licheniformis reduced the depressive-like and anxiety-like behaviours of the rats during the CUMS process. Meanwhile, B. licheniformis changed the gut microbiota composition; increased the short chain fatty acids (SCFAs) in the colon, decreased kynurenine, norepinephrine, and glutamate levels; and increased the tryptophan, dopamine, epinephrine, and γ-aminobutyric acid (GABA) in the brain. After correlation analysis, we found Parabacteroides, Anaerostipes, Ruminococcus-2, and Blautia showed significant correlation with neurotransmitters and SCFAs, indicating the gut microbiome plays an important role in B. licheniformis reducing depressive-like behaviours. Therefore, this study suggested B. licheniformis may prevent depressive-like and anxiety-like behaviours while regulating the gut microbiota composition and increasing the SCFA levels in the colon to alter the levels of the neurotransmitters in the brain. KEY POINTS: • B. licheniformis reduced depressive-like and anxiety-like behaviours induced by the chronic unpredictable mild stress. • GABA levels in the brain are assonated with B. licheniformis regulating depressive-like and anxiety-like behaviours. • Gut microbiota composition alteration followed by metabolic changes may play a role in the GABA levels increase.

The Effect of Various Training Variables on Developing Muscle Strength in Velocity-based Training: A Systematic Review and Meta-analysis.

Velocity-based training is an advanced auto-regulation method that uses objective indices to dynamically regulate training loads. However, it is unclear currently how to maximize muscle strength with appropriate velocity-based training settings. To fill this gap, we conducted a series of dose-response and subgroup meta-analyses to check the effects of training variables/parameters, such as intensity, velocity loss, set, inter-set rest intervals, frequency, period, and program, on muscle strength in velocity-based training. A systematic literature search was performed to identify studies via PubMed, Web of Science, Embase, EBSCO, and Cochrane. One repetition maximum was selected as the outcome to indicate muscle strength. Eventually, twenty-seven studies with 693 trained individuals were included in the analysis. We found that the velocity loss of 15 to 30%, the intensity of 70 to 80%1RM, the set of 3 to 5 per session, the inter-set rest interval of 2 to 4 min, and the period of 7 to 12 weeks could be appropriate settings for developing muscle strength. Three periodical programming models in velocity-based training, including linear programming, undulating programming, and constant programming, were effective for developing muscle strength. Besides, changing periodical programming models around every 9 weeks may help to avoid a training plateau in strength adaption.

Mss51 deletion increases endurance and ameliorates histopathology in the mdx mouse model of Duchenne muscular dystrophy.

Mitochondrial derangement is an important contributor to the pathophysiology of muscular dystrophies and may be among the earliest cellular deficits. We have previously shown that disruption of Mss51, a mammalian skeletal muscle protein that localizes to the mitochondria, results in enhanced muscle oxygen consumption rate, increased endurance capacity, and improved limb muscle strength in mice with wildtype background. Here, we investigate whether Mss51 deletion in the mdx murine model of Duchenne muscular dystrophy (mdx-Mss51 KO) counteracts the muscle pathology and mitochondrial irregularities observed in mdx mice. We found that mdx-Mss51 KO mice had increased myofiber oxygen consumption rates and an amelioration of muscle histopathology compared to mdx counterparts. This corresponded with greater treadmill endurance and less percent fatigue in muscle physiology, but no improvement in forelimb grip strength or limb muscle force production. These findings suggest that although Mss51 deletion ameliorates the skeletal muscle mitochondrial respiration defects in mdx and improves fatigue resistance in vivo, the lack of improvement in force production suggests that this target alone may be insufficient for a therapeutic effect.

Pcsk9 is associated with severity of coronary artery lesions in male patients with premature myocardial infarction.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (Pcsk9) correlated with incidence and prognosis of coronary heart disease. However, it is unclear whether Pcsk9 contributed to coronary artery lesion severity in patients with premature myocardial infarction (PMI). The present study investigated associations between Pcsk9 and coronary artery lesion severity in PMI patients who underwent coronary angiography (CAG). METHODS: This prospective cohort study included young men (age ≤ 45 years, n = 332) with acute MI who underwent CAG between January 2017 and July 2019. Serum Pcsk9 levels and clinical characteristics were evaluated. SYNTAX scores (SYNergy between percutaneous coronary intervention with [paclitaxel-eluting] TAXUS stent and cardiac surgery) were calculated to quantify coronary artery lesions. RESULTS: Serum Pcsk9 levels were positively associated with SYNTAX scores (r = 0.173, P < 0.05). The diagnostic cutoff value of PSCK9 level was 122.9 ng/mL, yielding an area under the curve (AUC) of 0.63, sensitivity 81%, and specificity 40%. Serum Pcsk9, LDL-C, Apob, NT-proBnp, CK level, and diabetes history were independent predictors of high SYNTAX scores (P < 0.05). After stratifying by serum LDL-C level (cutoff = 2.6 mmol/L), medium-high Pcsk9 levels had increased risk of high SYNTAX scores in patients with high LDL-C (P < 0.05), and higher serum Pcsk9 levels had increased risk of major adverse cardiac events (MACE) after adjusting for confounding factors (P < 0.05). CONCLUSION: Serum Pcsk9 levels correlates with severity of coronary artery lesion in PMI patients and may serve as a biomarker for severity of coronary artery stenosis in this patient population, which may contribute to risk stratification.

Integrated analysis of RNA-seq and microRNA-seq depicts miRNA-mRNA networks involved in stripe patterns of Botia superciliaris skin.

Botia superciliaris, an endemic cobitid fish in China, is widely accepted by Chinese consumers because its edibility. Recently, the black and yellow stripes of B. superciliaris skin have made this species increasingly popular as a novel ornamental fish. However, the genetic basis of the stripe patterns in B. superciliaris skin has not been extensively studied. In this study, Illumina sequencing was employed to identify the mRNAs and miRNAs involved in stripe pattern formation in B. superciliaris skin. A total of 147.25 and 155.15 million (M) high-quality transcriptome reads were generated from three black and yellow skin libraries respectively, which resulted in 159,327 unigenes that were used as reference sequences. A total of 3169 genes exhibited significantly differential expression patterns (fold-change ≥ 2 or ≤ 0.5 and q ≤ 0.05), including 1891 upregulated genes (59.67%) and 1278 downregulated genes (40.33%) in black vs yellow skin. These genes were enriched in 50 GO terms and 10 KEGG pathways (q ≤ 0.05), including melanogenesis, with 21 upregulated genes and 5 downregulated genes in black vs yellow skin. Based on the zebrafish genome, miRNA-seq identified a total of 355 miRNAs, which included 38 novel miRNAs. Furthermore, 87 differentially expressed miRNAs including 50 upregulated and 37 downregulated miRNAs were identified in different color skin (fold-change ≥ 2 or ≤ 0.5 and q ≤ 0.05). Then, target prediction revealed a variety of putative target genes; differentially expressed mRNAs and miRNAs patterns of high-throughput sequencing were validated in 5 mRNAs and miR-217-5p by qRT-PCR. In vivo tests and dual-luciferase reporter assay revealed that overexpression of miR-217-5p can inhibit pheomelanin formation by targeting Zgc. In this study, a comparative analysis was conducted to profile the transcriptome of black and yellow skin for B. superciliaris, and we detected key genes and important miRNAs involved in the B. superciliaris skin pigmentation process. These results will enhance understanding of molecular mechanisms underlying skin pigmentation and facilitate molecular-assisted selection of highly valued skin colors.

Transmission of mcr-1-Producing Multidrug-resistant Enterobacteriaceae in Public Transportation in Guangzhou, China.

Objectives: mcr-1-mediated colistin resistance in bacteria is concerning, as colistin is used in treating multidrug-resistant bacterial infections. And mcr-1-producing bacteria have been identified in multiple sources. Up to 248 million people use public transportation daily in China, however; public transportation hasn't been studied as a potential source of community-based transmission of mcr-1. Herein we investigated mcr-1-producing isolates from public transportation and explored the genomic characteristics of them. Methods: Surface samples were collected from public transportation in Guangzhou, China, from October 2016 to April 2017. Polymerase chain reaction was performed to detect mcr-1 gene, plasmid replicon type and phylogenetic group. Minimum inhibitory concentrations (MICs) were determined by microdilution method. S1-nuclease digestion and pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting were performed with mcr-1-harboring plasmids. Whole-genome sequencing was performed with mcr-1-producing isolates. Results: Of the 737 samples with bacterial growth, 26 isolates were positive for mcr-1, including 23 Escherichia coli and 3 Klebsiella pneumoniae isolates. The E. coli isolates belonged to phylogroups A and B1. Most mcr-1-producing isolates were resistant to ampicillin (25), cefotaxime (21), fosfomycin (16), and gentamicin (15). S1-PFGE, Southern blotting and replicon typing showed that mcr-1 was mainly located on ~33.3 kb to ~220 kb IncX4, IncI2 and IncHI2 plasmids in E. coli, while located on ~33.3 kb untyped plasmid in K. pneumoniae. Several sequence types (ST), including ST2253, ST101, ST10 complex and ST37, were revealed. Between 53 and 66 (mean = 61.8) resistance genes were identified among mcr-1-producing isolates. Conclusions: Public transportation may serve as a source of mcr-1-producing bacteria.

Exploration of exosomal microRNA expression profiles in pigeon 'Milk' during the lactation period.

BACKGROUND: Pigeon crop has the unique ability to produce a nutrient rich substance termed pigeon 'milk' (PM), which has functional resemblance with the mammalian milk. Previous researches have demonstrated that a large number of exosomes and exosomal miRNAs exist in mammalian milk, and many of them are associated with immunity, growth and development. However, to date, little is known about the exosomes and exosomal miRNAs in PM. RESULTS: In this study, we isolated the exosomes from PM and used small RNA sequencing to investigate the distribution and expression profiles of exosomal miRNAs. A total of 301 mature miRNAs including 248 conserved and 53 novel miRNAs were identified in five lactation stages i.e. 1d, 5d, 10d, 15d, and 20d. From these, four top 10 conserved miRNAs (cli-miR-21-5p, cli-miR-148a-3p, cli-miR-10a-5p and cli-miR-26a-5p) were co-expressed in all five stages. We speculate that these miRNAs may have important role in the biosynthesis and metabolism of PM. Moreover, similar to the mammalian milk, a significant proportion of immune and growth-related miRNAs were also present and enriched in PM exosomes. Furthermore, we also identified 41 orthologous miRNAs group (giving rise to 81 mature miRNA) commonly shared with PM, human, bovine and porcine breast milk. Additionally, functional enrichment analysis revealed the role of exosomal miRNAs in organ development and in growth-related pathways including the MAPK, Wnt and insulin pathways. CONCLUSIONS: To sum-up, this comprehensive analysis will contribute to a better understanding of the underlying functions and regulatory mechanisms of PM in squabs.

Hyaluronan negatively regulates vascular calcification involving BMP2 signaling.

Vascular calcification is a highly regulated biological process similar to bone formation involving osteogenic differentiation of vascular smooth muscle cells (VSMCs). Hyaluronan (HA), a major structural component of the extracellular matrix in cartilage, has been shown to inhibit osteoblast differentiation. However, whether HA affects osteogenic differentiation and calcification of VSMCs remains unclear. In the present study, we used in vitro and ex vivo models of vascular calcification to investigate the role of HA in vascular calcification. Both high and low molecular weight HA treatment significantly reduced calcification of rat VSMCs in a dose-dependent manner, as detected by alizarin red staining and calcium content assay. Ex vivo study further confirmed the inhibitory effect of HA on vascular calcification. Similarly, HA treatment decreased ALP activity and expression of bone-related molecules including Runx2, BMP2 and Msx2. By contrast, inhibition of HA synthesis by 4-methylumbelliferone (4MU) promoted calcification of rat VSMCs. In addition, adenovirus-mediated overexpression of HA synthase 2 (HAS2), a major HA synthase in VSMCs, also inhibited calcification of VSMCs, whereas CRISPR/Cas9-mediated HAS2 knockout promoted calcification of rat A10 cells. Furthermore, we found that BMP2 signaling was inhibited in VSMCs after HA treatment. Recombinant BMP2 enhanced high calcium and phosphate-induced VSMC calcification, which can be blocked by HA treatment. Taken together, these findings suggest that HA inhibits vascular calcification involving BMP2 signaling.

Guanidinoacetic Acid Regulates Myogenic Differentiation and Muscle Growth Through miR-133a-3p and miR-1a-3p Co-mediated Akt/mTOR/S6K Signaling Pathway.

Guanidinoacetic acid (GAA), an amino acid derivative that is endogenous to animal tissues including muscle and nerve, has been reported to enhance muscular performance. MicroRNA (miRNA) is a post-transcriptional regulator that plays a key role in nutrient-mediated myogenesis. However, the effects of GAA on myogenic differentiation and skeletal muscle growth, and the potential regulatory mechanisms of miRNA in these processes have not been elucidated. In this study, we investigated the effects of GAA on proliferation, differentiation, and growth in C2C12 cells and mice. The results showed that GAA markedly inhibited the proliferation of myoblasts, along with the down-regulation of cyclin D1 (CCND1) and cyclin dependent kinase 4 (CDK4) mRNA expression, and the upregulation of cyclin dependent kinase inhibitor 1A (P21) mRNA expression. We also demonstrated that GAA treatment stimulated myogenic differentiation 1 (MyoD) and myogenin (MyoG) mRNA expression, resulting in an increase in the myotube fusion rate. Meanwhile, GAA supplementation promoted myotube growth through increase in total myosin heavy chain (MyHC) protein level, myotubes thickness and gastrocnemius muscle cross-sectional area. Furthermore, small RNA sequencing revealed that a total of eight miRNAs, including miR-133a-3p and miR-1a-3p cluster, showed differential expression after GAA supplementation. To further study the function of miR-133a-3p and miR-1a-3p in GAA-induced skeletal muscle growth, we transfected miR-133a-3p and miR-1a-3p mimics into myotube, which also induced muscle growth. Through bioinformatics and a dual-luciferase reporter system, the target genes of miR-133a-3p and miR-1a-3p were determined. These two miRNAs were shown to modulate the Akt/mTOR/S6K signaling pathway by restraining target gene expression. Taken together, these findings suggest that GAA supplementation can promote myoblast differentiation and skeletal muscle growth through miR-133a-3p- and miR-1a-3p-induced activation of the AKT/mTOR/S6K signaling pathway.

Testosterone-Dependent miR-26a-5p and let-7g-5p Act as Signaling Mediators to Regulate Sperm Apoptosis via Targeting PTEN and PMAIP1.

Recent evidence suggests that testosterone deficiency can dramatically decrease the quality of sperm. MicroRNAs (miRNAs) are conserved mediators of post-transcriptional gene regulation in eukaryotes. However, the systemic regulation and function of miRNAs in sperm quality decline induced by testosterone deficiency has not been investigated. Here, we found that the sperm apoptosis was significantly enhanced and the sperm motility was dramatically decreased in hemicastrated pigs. We then used small RNA sequencing to detect miRNA profiles of sperm from pigs with prepubertal hemicastration (HC) and compared them with control libraries. We identified 16 differentially expressed (DE) miRNAs between the sperm of prepubertal HC and control (CT) pigs. Functional enrichment analysis indicated that the target genes of these DE miRNAs were mainly enriched in apoptosis-related pathways including the p53, mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin (mTOR) pathways. Furthermore, gain- and loss-of-function analyses demonstrated potential anti-apoptotic effects of the DE miRNAs miR-26a-5p and let-7g-5p on sperm cells. The luciferase reporter assay confirmed that PTEN and PMAIP1 are targets of miR-26a-5p and let-7g-5p, respectively. Spearman’s correlation analysis revealed significantly positive correlations between the sperm and its corresponding seminal plasma exosomes regarding the miRNA expression levels. In conclusion, testosterone deficiency-induced changes in the miRNA components of seminal plasma exosomes secreted by the genital tract may partially elucidate sperm miRNAome alterations, which are further responsible for the decline of sperm motility.

The Impact of Different Intensities of Physical Activity on Serum Urate and Gout: A Mendelian Randomization Study.

Physical activity is a potential protective factor against gout, but the role of exercise intensity in this context remains unclear. To overcome the limitations of observational studies in causal inference, this study employed a two-sample Mendelian randomization approach to explore the impact of different genetically proxied/predicted intensities of physical activity on serum urate concentration and the incidence of gout. Our data related to physical activity, serum urate, and gout were obtained from the UK Biobank, the Global Urate Genetics Consortium (GUGC), and the FinnGen dataset, respectively. Walking was included as representative of typical low-intensity physical activity in the analysis, and the other two types were moderate and vigorous physical activities. The estimation methods we used included the inverse-variance-weighted (IVW) method, MR-Egger regression, weighted-median method, simple-mode method, and weighted-mode method. Sensitivity analyses involved Rucker's framework, Cochran's Q test, funnel plots, MR-PRESSO outlier correction, and leave-one-out analysis. We found suggestive evidence from the inverse-variance-weighted method that moderate physical activity was a potential factor in reducing the incidence of gout (OR = 0.628, p = 0.034), and this association became more substantial in our subsequent sensitivity analysis (OR = 0.555, p = 0.006). However, we observed no distinctive effects of physical activity on serum urate concentration. In conclusion, our study supports some findings from observational studies and emphasizes the preventive role of moderate physical activity against gout. Given the limitations of the existing datasets, we call for future reexamination and expansion of our findings using new GWAS data.

Land degradation neutrality assessment and factors influencing it in China's arid and semiarid regions.

The ecosystems in China's arid and semiarid regions are notably fragile and experiencing dramatic land degradation. At the 12th Conference of the Parties (COP12) to the United Nations Convention to Combat Desertification (UNCCD) in October 2015, a definition for land degradation neutrality (LDN) was proposed and subsequently integrated into the Sustainable Development Goals (SDGs). Research on LDN has developed in terms of conceptual framework constructions, quantitative assessments, and empirical studies. However, LDN and its drivers must be clarified in China's arid and semiarid regions since some representative processes have yet to be fully considered in the assessment. Here, we develop an LDN indicator system specialised for the area, assess their LDN status, and determine the impacts of human activities and climate change on LDN. Our research aims to refine the LDN indicator system tailored for China's arid and semiarid regions by incorporating the trends of wind and water erosion. We also identify the influence of human activity and climate change on LDN, which provides insightful strategies for ecological restoration and sustainable development in drylands with climate-sensitive ecosystems. The results show that: (1) In 2020, more than half of areas of China's arid and semiarid regions achieved LDN, with more pronounced success in the southeastern areas compared to the central regions. (2) For LDN drivers, elevation shows negligible influence on LDN, whereas increased temperature promotes LDN achievement. Conversely, factors like vapour pressure deficit and v-direction wind speed hinder it. In conclusion, China's arid and semiarid regions achieved LDN, and the dominant factor that substantially influences LDN varies across geographical zones, with higher wind speeds and elevated GDP levels generally obstructing LDN in most areas.

A latent class analysis of depressive symptoms among rural Chinese adolescents and their association with psychological resilience.

Numerous studies have demonstrated the heterogeneity of depressive symptoms, but few studies have focused on the heterogeneity of depressive symptoms among rural Chinese adolescents. In November to December 2022, multistage sampling was employed to administer questionnaires to 1,816 rural adolescents aged 11-19 years from six schools in Henan Province, China. Depressive symptoms were measured using the Chinese version of the Children's Depression Inventory Scale. Latent class analysis (LCA) was utilized to identify subgroups of depressive symptoms. The investigation of subgroup characteristics and associated factors was conducted through χ2 tests, ANOVA, and multinomial logistic regression analyses. The findings revealed a 24.24 % detection rate of depressive symptoms among Chinese rural adolescents. LCA analysis of responses to the 27 items in the Depressive Symptoms Scale led to the classification of depressive symptoms into four subgroups based on severity: "no depressive symptoms group" (22.5 %), a "low depressive symptoms group" (35.7 %), a "transition group" (31.6 %), and a "high depressive symptoms group" (10.2 %). Gender, grade level, academic performance, academic stress, family environment, and level of psychological resilience are associated factors for subgroups of depressive symptoms among rural adolescents. There should be increased training of rural educators to enable early recognition of depressive characteristics and risk factors, facilitating targeted prevention and intervention strategies.

Assessment of the reversibility of resistance in the absence of antibiotics and its relationship with the resistance gene's fitness cost: a genetic study with mcr-1.

BACKGROUND: The intensive use of antibiotics has resulted in strong natural selection for the evolution of antimicrobial resistance (AMR), but whether, and under what circumstances, the removal of antibiotics would result in a rapid reduction in AMR has been insufficiently explored. We aimed to test the hypothesis that in the simple, yet common, case of AMR conferred by a single gene, removing antibiotics would quickly reduce the prevalence of resistance if the AMR gene imposes a high fitness cost and costless resistance is extremely rare among its proximal mutants. METHODS: In this genetic study, to test our hypothesis, we used the mcr-1 gene in Escherichia coli, which confers resistance to the last-resort antibiotic colistin, as a model. A high-throughput reverse genetics approach was used to evaluate mcr-1 variants for their fitness cost and resistance levels relative to a non-functional construct, by measuring relative growth rates in colistin-free media and at 2 µg/mL and 4 µg/mL colistin. We identified costless resistant mcr-1 mutants, and examined their properties within the context of the sequential organisation of mcr-1's functional domains as well as the evolutionary accessibility of these mutations. Finally, a simple population genetic model incorporating the measured fitness cost was constructed and tested against previously published real-world data of mcr-1 prevalence in colonised inpatients in China since the 2017 colistin ban in fodder additives. FINDINGS: We estimated the relative growth rates of 14 742 mcr-1 E coli variants (including the wild type), 3449 of which were single-nucleotide mutants. E coli showed 73·8% less growth per 24 h when carrying wild-type mcr-1 compared with the non-functional construct. 6252 (42·4%) of 14 741 mcr-1 mutants showed colistin resistance accompanied by significant fitness costs, when grown under 4 µg/mL colistin selection. 43 (0·3%) mcr-1 mutants exhibited costless resistance, most of which contained multiple mutations. Among the 3449 single mutants of mcr-1, 3433 (99·5%) had a fitness cost when grown in colistin-free media, with a mean relative growth of 0·305 (SD 0·193) compared with the non-functional variant. 3059 (88·7%) and 1833 (53·1%) of 3449 single mutants outgrew the non-functional mcr-1 in the presence of 2 µg/mL and 4 µg/mL colistin, respectively. Single mutations that gave rise to costless mutants were rare in all three domains of mcr-1 (transmembrane domain, flexible linker, and catalytic domain), but the linker domain was enriched with cost-reducing and resistance-enhancing mutations and depleted with cost-increasing mutations. The population genetics model based on the experimental data accurately predicts the rapid decline in mcr-1 prevalence in real-world data. INTERPRETATION: Many identified costless resistant variants that consist of multiple mutations are unlikely to evolve easily in nature. These findings for colistin and mcr-1 might be applicable to other cases in which AMR entails a substantial fitness cost that cannot be mitigated in proximal mutants. FUNDING: National Natural Science Foundation of China, and National Key Research and Development Program of China.

High-throughput mutagenesis and screening approach for the identification of drug-resistant mutations in the rifampicin resistance-determining region of mycobacteria.

Rifampicin is the most powerful first-line antibiotic for tuberculosis, which is caused by Mycobacterium tuberculosis. Although accumulating evidence from sequencing data of clinical M. tuberculosis isolates suggested that mutations in the rifampicin-resistance-determining region (RRDR) are strongly associated with rifampicin resistance, the comprehensive characterisation of RRDR polymorphisms that confer this resistance remains challenging. By incorporating I-SceI sites for I-SceI-based integrant removal and utilizing an L5 swap strategy, we efficiently replaced the integrated plasmid with alternative alleles, making mass allelic exchange feasible in mycobacteria. Using this method to establish a fitness-related gain-of function screen, we generated a mutant library that included all single-amino-acid mutations in the RRDR, and identified the important positions corresponding to some well-known rifampicin-resistance mutations (Q513, D516, S522, H525, R529, S531). We also detected a novel two-point mutation located in the RRDR confers a fitness advantage to M. smegmatis in the presence or absence of rifampicin. Our method provides a comprehensive insight into the growth phenotypes of RRDR mutants and should facilitate the development of anti-tuberculosis drugs.