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Background: Little is known about the combined effect of bisphenol mixtures and metal mixtures on type 2 diabetes mellitus (T2DM) risk, and the mediating roles of metabolites. Methods: The study included 606 pairs of T2DM cases and controls matched by age and sex, and information of participants was collected through questionnaires and laboratory tests. Serum bisphenol and plasma metal concentrations were measured using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) and inductively coupled plasma-mass spectrometry (ICP-MS), respectively. Widely targeted metabolomics was employed to obtain the serum metabolomic profiles. Conditional logistic regression models were used to assess the single associations of bisphenols and metals with T2DM risk after multivariable adjustment. Additionally, the joint effects of bisphenol mixtures and metal mixtures were examined using quantile-based g-computation (QG-C) models. Furthermore, differential metabolites associated with T2DM were identified, and mediation analyses were performed to explore the role of metabolites in the associations of bisphenols and metals with T2DM risk. Results: The results showed bisphenol mixtures were associated with an increased T2DM risk, with bisphenol A (BPA) identified as the primary contributor. While the association between metal mixtures and T2DM remained inconclusive, cobalt (Co), iron (Fe), and zinc (Zn) showed the highest weight indices for T2DM risk. A total of 154 differential metabolites were screened between the T2DM cases and controls. Mediation analyses indicated that 9 metabolites mediated the association between BPA and T2DM, while L-valine mediated the association between Zn and T2DM risk. Conclusions: The study indicated that BPA, Co, Fe, and Zn were the primary contributors to increased T2DM risk, and metabolites played a mediating role in the associations of BPA and Zn with the risk of T2DM. Our findings contribute to a better understanding of the mechanisms underlying the associations of bisphenols and metals with T2DM.

Abstract Is childhood adversity associated with biological aging, and if so, does sex modify the association, and do lifestyle and mental health mediate the association? A lifespan analysis is conducted using data on 142 872 participants from the UK Biobank to address these questions. Childhood adversity is assessed through the online mental health questionnaire (2016), including physical neglect, physical abuse, emotional neglect, emotional abuse, sexual abuse, and a cumulative score. Biological aging is indicated by telomere length (TL) measured from leukocyte DNA using qPCR, and the shorter TL indicates accelerated biological aging; a lifestyle score is constructed using body mass index, physical activity, drinking, smoking, and diet; mental disorder is assessed using depression, anxiety, and insomnia at the baseline survey. The results reveal a sex‐specific association such that childhood adversity is associated with shorter TL in women after adjusting for covariates including polygenic risk score for TL, but not in men. Unhealthy lifestyle and mental disorder partially mediate the association in women. The proportions of indirect effects are largest for sexual and physical abuse. These findings highlight the importance of behavioral and psychological interventions in promoting healthy aging among women who experienced childhood adversity, particularly sexual and physical abuse.

The increasing concentration of Antimony (Sb) in ecological environments has raised serious concerns about its potential biotoxicological impact. This study investigated the toxicokinetics, Global DNA Methylation (GDM), biomarker expression, and Integrated Biological Response (IBR) of Sb at different concentrations in zebrafish. The toxic mechanism of Sb exposure was simulated using molecular dynamics (MD). The results showed that significant differences effect existed (BCFk: liver > ovary > gut > brain) and uptake saturation phenomenon of Sb among zebrafish tissues. Over a 54-day exposure period, the liver emerged as the main target site for Sb-induced GDM, and the restoration was slower than in other tissues during the 54-day recovery period. Moreover, the concentration of Sb had a significant impact on the normally expression of biomarkers, with GSTM1 inhibited and MTF2, MT1, TET3, and p53 showing varying degrees of activation at different Sb concentrations. This could be attributed to Sb3+ potentially occupying the active site or tightly binding to the deep cavity of these genes. The IBR and MD results highlighted DNMT1 as the most sensitive biomarker among those assessed. This heightened sensitivity can be attributed to the stable binding of Sb3+ to DNMT1, resulting in alterations in the conformation of DNMT1's catalytic domain and inhibition of its activity. Consequently, this disruption leads to damage to the integrity of GDM. The study suggests that DNA methylation could serve as a valuable biomarker for assessing the ecotoxicological impact of Sb exposure. It contributes to a better understanding of the toxicity mechanisms in aquatic environments caused potential pollutants.

Betula platyphylla, belonging to the cold-specialized lineage Betulaceae, exhibits a unique reproductive strategy where staminate catkins emerge in the first summer and undergo an overwintering process, culminating in flowering in the following year. However, the underlying regulatory mechanism remains unclear. In this study, we investigated the male germline development of B. platyphylla in four distinct stages: microsporocytes in Oct. (S1), uninuclear microspores from Dec. (S2) to Mar. of the following year (S3), and bicellular microspores in Apr. (S4). We performed RNA sequencing on mature pollen and the four stages of staminate catkins. Using weighted gene co-expression network analysis (WGCNA), we identified five highly correlated gene modules with distinct expression profiles. These modules exhibited strong correlations with sugar metabolism, cell cycle, flowering, and cell wall dynamics, highlighting their dynamic roles during male germline developmental stages. During the overwintering process, we observed that the expression of transcription factors such as BpDUO1 and BpAMS at the appropriate developmental stages, suggests their significant roles in male germline development. The expression patterns of BpFLC and BpFT suggest their potential involvement in temperature perception during male reproductive development. These findings offer valuable insights into the reproductive success of plants adapting to cold environments.

Hydrogen fuel cell tractors are emerging as a new power source for tractors. Currently, there is no mature energy management control method available. Existing methods mostly rely on engineers’ experience to determine the output power of the fuel cell and the power battery, resulting in relatively low energy utilization efficiency of the energy system. To address the aforementioned problems, a power optimization method for the energy system of hydrogen fuel cell wheel-driven electric tractor was proposed. A dynamic model of tractor ploughing conditions was established based on the system dynamics theory. From this, based on the equivalent hydrogen consumption theory, the charging and discharging of the power battery were equivalent to the fuel consumption of the hydrogen fuel cell, forming an equivalent hydrogen consumption model for the tractor. Using the state of charge (SOC) of the power battery as a constraint, and with the minimum equivalent hydrogen consumption as the objective function, an instantaneously optimized power allocation method based on load demand in the energy system is proposed by using a traversal algorithm. The optimization method was simulated and tested based on the MATLAB simulation platform, and the results showed under ploughing conditions, compared with the rule-based control strategy, the proposed energy system power optimization method optimized the power output of hydrogen fuel cells and power batteries, allowing the energy system to work in a high-efficiency range, reducing the equivalent hydrogen consumption of the tractor by 7.79%, and solving the energy system power distribution problem.

In response to the issues of hybrid tractors’ energy management strategies, such as reliance on experience, difficulty in achieving optimal control, and incomplete analysis of typical operating conditions of tractors, an energy management strategy based on dynamic programming is proposed in combination with various typical operating conditions of tractors. This is aimed at providing a reference for the modeling and energy management strategies of series hybrid tractors. Taking the series hybrid tractor as the research object, the tractor dynamics models under three typical working conditions of plowing, rotary tillage, and transportation were established. With the minimum total fuel consumption of the tractor as the optimization target, the engine power as the control variable, and the state of charge of the power battery as the state variable, an energy management strategy based on a dynamic programming algorithm was established and simulation experiments were conducted. The simulation results show that, compared with the power-following energy management strategy, the energy management strategy based on the dynamic programming algorithm can reasonably control the operating state of the engine. Under the three typical working conditions of plowing, rotary tillage, and transportation, the battery SOC consumption increased by approximately 8.37%, 7.24%, and 0.77%, respectively, while the total fuel consumption decreased by approximately 25.28%, 21.54%, and 13.24%, respectively.

Abstract The olivocerebellar projection is organized into an intricate topographical connection from the inferior olive (IO) subdivisions to the longitudinally-striped compartments of cerebellar Purkinje Cells (PCs), to play an essential role in cerebellar coordination and learning. However, the central mechanisms for forming topography need to be clarified. IO neurons and PCs are generated during overlapping periods of a few days in embryonic development. Therefore, we examined whether their neurogenic timing is specifically involved in the olivocerebellar topographic projection relationship. First, we mapped neurogenic timing in the entire IO by using the neurogenic-tagging system of neurog2-CreER (G2A) mice and specific labeling of IO neurons with FoxP2. IO subdivisions were classified into three groups depending on their neurogenic timing range. Then, we examined the relationships in the neurogenic-timing gradient between IO neurons and PCs by labeling topographic olivocerebellar projection patterns and PC neurogenic timing. Early, intermediate, and late groups of IO subdivisions projected to late, intermediate, and early groups of the cortical compartments, respectively, except for a few particular areas. The results indicated that the olivocerebellar topographic relationship is essentially arranged according to the reverse neurogenic-timing gradients of the origin and target.

BackgroundPerfluoroalkyl substances (PFASs) are classified as persistent organic pollutants and have been widely detected in human. Studies investigating the associations between PFASs exposure and estimated glomerular filtration rate (eGFR) yielded inconsistent results, and little is known about the effects of PFASs on eGFR in population without kidney disease. ObjectiveTo explore the associations of exposure to PFASs with eGFR and renal dysfunction in population without kidney disease. MethodsA total of 609 participants with an eGFR > 60 mL·min−1·1.73 m−2 and without renal impairment matched for sex and age (1∶1) were recruited from endocrinology department and medical examination center of two hospitals in Tianjin, China, from April 2021 to March 2022. Each subject was interviewed using a structured questionnaire to collect information about sex, age, height, weight, disease history, smoking, alcohol intake, etc. Clinical parameters were obtained from medical record, such as fasting blood glucose (FBG), creatinine (Cre), total cholesterol (TC), and triglyceride (TG). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by professionals using standard methods. The serum concentrations of PFASs were determined by liquid chromatography/mass spectrometry. Multivariable linear and logistic regression models were performed to evaluate the associations of PFASs exposure with eGFR and renal dysfunction, respectively. Subgroup analyses stratified by age and sex were also performed to assess the modified effects of covariates on the associations of PFASs exposure with eGFR. ResultsThere were 283 males, accounting for 46.5% of the total population. The mean age of the participants was (56.86±12.47) years, and the average body mass index (BMI) was (25.59±3.84) kg·m−2. Perfluoro-n-octanoic acid (PFOA), perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonic acid (PFOS), perfluoro-n-nonanoic acid (PFNA), perfluoro-n-decanoic acid (PFDA), perfluoro-n-undecanoic acid (PFUnDA), sodium 1H, 1H, 2H, 2H-perfluoro-1-octanesulfonate (6:2 FTS), and perfluoropentane sulfonic acid (PFPeS) were positive in more than 75% of serum samples, and the corresponding median concentrations were 9.50, 1.67, 17.22, 1.86, 1.41, 0.78, 0.42, and 0.43 μg·L−1, respectively. After full adjustments of sex, age, BMI, hypertension, diabetes, TC, TG, smoking, and drinking, the linear regression models showed that log2-transformed PFHxS concentration was negatively associated with eGFR (b=−1.160, 95%CI: −2.280, −0.410). Compared with the lowest exposure tertile, the estimated change of eGFR in the highest tertile for PFHxS was significantly decreased (b=−2.471, 95%CI: −4.574, −0.368). Furthermore, compared with males, the negative association of PFHxS with eGFR was strengthened among females (female: b=−1.281, 95%CI: −2.388, −0.174; male: b=−0.781, 95%CI: −2.823, 1.261, Pinteraction=0.043). ConclusionA significant negative association between serum PFHxS and eGFR is observed in the sampled population without kidney disease, and females are more susceptible to PFASs exposure than the males.

Abstract Background Comorbidities among cancer survivors remain a serious healthcare burden and require appropriate management. Using two widely used frailty indicators, this study aimed to evaluate whether frailty was associated with the incidence risk of cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM) among long-term cancer survivors. Methods We included 13,388 long-term cancer survivors (diagnosed with cancer over 5 years before enrolment) free of CVD and 6101 long-term cancer survivors free of T2DM, at the time of recruitment (aged 40–69 years), from the UK Biobank. Frailty was assessed by the frailty phenotype (FP_Frailty, range: 0–5) and the frailty index (FI_Frailty, range: 0–1) at baseline. The incident CVD and T2DM were ascertained through linked hospital data and primary care data, respectively. The associations were examined using Cox proportional hazards regression models. Results Compared with non-frail participants, those with pre-frailty (FP_Frailty [met 1–2 of the components]: hazard ratio [HR]=1.18, 95% confidence interval [CI]: 1.05, 1.32; FI_Frailty [0.10< FI ≤0.21]: HR=1.51, 95% CI: 1.32, 1.74) and frailty (FP_Frailty [met ≥3 of the components]: HR=2.12, 95% CI: 1.73, 2.60; FI_Frailty [FI >0.21]: HR=2.19, 95% CI: 1.85, 2.59) had a significantly higher risk of CVD in the multivariable-adjusted model. A similar association of FI_Frailty with the risk of incident T2DM was observed. We failed to find such an association for FP_Frailty. Notably, the very early stage of frailty (1 for FP_Frailty and 0.1-0.2 for FI_Frailty) was also positively associated with the risk of CVD and T2DM (FI_Frailty only). A series of sensitivity analyses confirmed the robustness of the findings. Conclusions Frailty, even in the very early stage, was positively associated with the incidence risk of CVD and T2DM among long-term cancer survivors, although discrepancies existed between frailty indicators. While the validation of these findings is required, they suggest that routine monitoring, prevention, and interventive programs of frailty among cancer survivors may help to prevent late comorbidities and, eventually, improve their quality of life. Especially, interventions are recommended to target those at an early stage of frailty when healthcare resources are limited.

When shifting gears around a tractor’s power shift transmission, it is necessary to coordinate the control of multiple clutches and formulate a reasonable clutch engagement law to ensure the reliability and power of the power system. This paper explores the impact of different clutch engagement patterns on shifting characteristics in a power-shift tractor with multiple clutches. Shifting performance is comprehensively evaluated using indicators such as shifting time, impact degree, slip energies, and transmission output torque. The aim is to enhance the quality of power shifting, operational efficiency, and the service life of the transmission system. This paper takes the YTO TX4A transmission system as the research object, analyzes its working principle, and establishes a mathematical model of a power shift transmission system under different clutch engagement rules. This model of power shift transmission is established based on Matlab2021/Simulink and the AMESim2021 software platform. A simulation analysis is carried out for four clutches working simultaneously in a tractor’s advance gear II and lift gear III. The simulation results indicate that switching rule C is superior to switching rule A and switching rule B. Compared with the engagement scheme in which the torque exchange overlap time for both sets of clutches is 0.3 s, the scheme with a torque exchange overlap time of 0.15 s indicated a shifting time increase of 0.2 s, a slip energy increase of 4%, and a minimum output torque of the transmission increase of 2%. In the scheme with a torque exchange overlap time of 0 s, the shifting time increased by 0.15 s, the slip energy decreased by 13.5%, and the minimum output torque of the transmission decreased by 17%. Through the study of shifting performance under three different clutch engagement patterns, it is concluded that, during the shift from forward gear II to gear III in the YTO TX4A transmission system, appropriately reducing the torque exchange overlap time for both sets of clutches and avoiding simultaneous torque exchange can reduce the complexity of clutch control. This leads to smoother, more powerful, and more comfortable power shifting, effectively prolonging the service life of the transmission system.

Objectives: Adults without diabetes are not completely healthy; they are probably heterogeneous with several potential health problems. The management of hemoglobin A1c (HbA1c) is crucial among patients with diabetes; but whether similar management strategy is needed for adults without diabetes is unclear. Thus, this study aimed to investigate the associations of visit-to-visit HbA1c variability with incident dementia and hippocampal volume among middle-aged and older adults without diabetes, providing potential insights into this question. Methods: We conducted a prospective analysis for incident dementia in 10,792 participants (mean age 58.9 years, 47.8 % men) from the UK Biobank. A subgroup of 3793 participants (mean age 57.8 years, 48.6 % men) was included in the analysis for hippocampal volume. We defined HbA1c variability as the difference in HbA1c divided by the mean HbA1c over the 2 sequential visits ([latter − former]/mean). Dementia was identified using hospital inpatient records with ICD-9 codes. T1-structural brain magnetic resonance imaging was conducted to derive hippocampal volume (normalized for head size). The nonlinear and linear associations were examined using restricted cubic spline (RCS) models, Cox regression models, and multiple linear regression models. Results: During a mean follow-up (since the second round) of 8.4 years, 90 (0.8 %) participants developed dementia. The RCS models suggested no significant nonlinear associations of HbA1c variability with incident dementia and hippocampal volume, respectively (All P > 0.05). Above an optimal cutoff of HbA1c variability at 0.08, high HbA1c variability (increment in HbA1c) was associated with an increased risk of dementia (Hazard Ratio, 1.88; 95 % Confidence Interval, 1.13 to 3.14, P = 0.015), and lower hippocampal volume (coefficient, −96.84 mm3, P = 0.037), respectively, in models with adjustment of covariates including age, sex, etc. Similar results were found for a different cut-off of 0. A series of sensitivity analyses verified the robustness of the findings. Conclusions: Among middle-aged and older adults without diabetes, increasing visit-to-visit HbA1c variability was associated with an increased dementia risk and lower hippocampal volume. The findings highlight the importance of monitoring and controlling HbA1c fluctuation in apparently healthy adults without diabetes.

The mechanisms underlying sex differences in response to stress are unclear. Here, the authors show that meningeal lymphatics dysfunction modulates the sex difference in the stress susceptibility to depression- and anxiety-like behaviours in mice.

Objectives: To evaluate a duplex droplet digital polymerase chain reaction (ddPCR) assay targeting Salmonella fimY and Shigella ipaH genes. Methods: The linear range, precision, analytical sensitivity, and analytical specificity of the ddPCR assay were analyzed. The ddPCR assay was compared with quantitative real-time PCR (qPCR) using 362 stool samples from 187 children with mild diarrhea and 175 children without diarrhea. Results: The duplex ddPCR assay showed good linearity in the range of 5.3 × 100 to 1.24 × 105 copies/reaction for Salmonella and 1.9 × 100 to 1.84 × 105 copies/reaction for Shigella. When analyzed with spiked stool samples, the limit of detection and limit of quantification were 550 and 5500 colony-forming units per mL of stool sample for Shigella, respectively, whereas both were 1.0 × 104 colony-forming units per mL of stool sample for Salmonella. Among 362 stool samples, more samples were detected as positive by ddPCR than by qPCR. Salmonella load was significantly higher in diarrheal samples than in non-diarrheal samples. Determined by receiver-operating characteristic analysis, the optimal cut-off value was 1.25 × 104 copies/mL of stool sample to distinguish between symptomatic and asymptomatic Salmonella infections. Conclusion: Salmonella and Shigella prevalence may have been underestimated in the past.

BackgroundCompared with adults with normal glucose metabolism, those with prediabetes tend to be frail. However, it remains poorly understood whether frailty could identify adults who are most at risk of adverse outcomes related to prediabetes. ObjectiveWe aimed to systematically evaluate the associations between frailty, a simple health indicator, and risks of multiple adverse outcomes including incident type 2 diabetes mellitus (T2DM), diabetes-related microvascular disease, cardiovascular disease (CVD), chronic kidney disease (CKD), eye disease, dementia, depression, and all-cause mortality in late life among middle-aged adults with prediabetes. MethodsWe evaluated 38,950 adults aged 40 years to 64 years with prediabetes using the baseline survey from the UK Biobank. Frailty was assessed using the frailty phenotype (FP; range 0-5), and participants were grouped into nonfrail (FP=0), prefrail (1≤FP≤2), and frail (FP≥3). Multiple adverse outcomes (ie, T2DM, diabetes-related microvascular disease, CVD, CKD, eye disease, dementia, depression, and all-cause mortality) were ascertained during a median follow-up of 12 years. Cox proportional hazards regression models were used to estimate the associations. Several sensitivity analyses were performed to test the robustness of the results. ResultsAt baseline, 49.1% (19,122/38,950) and 5.9% (2289/38,950) of adults with prediabetes were identified as prefrail and frail, respectively. Both prefrailty and frailty were associated with higher risks of multiple adverse outcomes in adults with prediabetes (P for trend

The massive axonal projection from the cerebrum to the cerebellum through the pontine nuclei supports the cerebrocerebellar coordination of motor and nonmotor functions. However, the cerebrum and cerebellum have distinct patterns of functional localization in their cortices. We addressed this issue by bidirectional neuronal tracing from 22 various locations of the pontine nuclei in the mouse in a comprehensive manner. Cluster analyses of the distribution patterns of labeled cortical pyramidal cells and cerebellar mossy fiber terminals classified all cases into six groups located in six different subareas of the pontine nuclei. The lateral (insular), mediorostral (cingulate and prefrontal), and caudal (visual and auditory) cortical areas of the cerebrum projected to the medial, rostral, and lateral subareas of the pontine nuclei, respectively. These pontine subareas then projected mainly to the crus I, central vermis, and paraflocculus divergently. The central (motor and somatosensory) cortical areas projected to the centrorostral, centrocaudal and caudal subareas of the pontine nuclei, which then projected mainly to the rostral and caudal lobules with a somatotopic arrangement. The results indicate a new pontine nuclei-centric view of the corticopontocerebellar projection: the generally parallel corticopontine projection to pontine nuclei subareas is relayed to the highly divergent pontocerebellar projection terminating in overlapping specific lobules of the cerebellum. Consequently, the mode of the pontine nuclei relay underlies the cerebellar functional organization.

Abstract Background Aging metrics incorporating cognitive and physical function are not fully understood, hampering their utility in research and clinical practice. This study aimed to determine the proportions of vulnerable persons identified by three existing aging metrics that incorporate cognitive and physical function and the associations of the three metrics with mortality. Methods We considered three existing aging metrics including the combined presence of cognitive impairment and physical frailty (CI-PF), the frailty index (FI), and the motoric cognitive risk syndrome (MCR). We operationalized them using data from the China Health and Retirement Longitudinal Study (CHARLS) and the US National Health and Nutrition Examination Survey (NHANES). Logistic regression models or Cox proportional hazards regression models, and receiver operating characteristic curves were used to examine the associations of the three metrics with mortality. Results In CHARLS, the proportions of vulnerable persons identified by CI-PF, FI, and MCR were 2.2, 16.6, and 19.6%, respectively. Each metric predicted mortality after adjustment for age and sex, with some variations in the strength of the associations (CI-PF, odds ratio (OR) (95% confidence interval (CI)) 2.87 (1.74–4.74); FI, OR (95% CI) 1.94 (1.50–2.50); MCR, OR (95% CI) 1.27 (1.00–1.62)). CI-PF and FI had additional predictive utility beyond age and sex, as demonstrated by integrated discrimination improvement and continuous net reclassification improvement (all P

BackgroundEarly identification and intervention of diabetic peripheral neuropathy is beneficial to improve clinical outcome.ObjectiveTo establish a risk prediction model for diabetic peripheral neuropathy (DPN) in patients with type 2 diabetes mellitus (T2DM).MethodsThe derivation cohort was from a meta-analysis. Risk factors and the corresponding risk ratio (RR) were extracted. Only risk factors with statistical significance were included in the model and were scored by their weightings. An external cohort were used to validate this model. The outcome was the occurrence of DPN.ResultsA total of 95,604 patients with T2DM from 18 cohorts were included. Age, smoking, body mass index, duration of diabetes, hemoglobin A1c, low HDL-c, high triglyceride, hypertension, diabetic retinopathy, diabetic kidney disease, and cardiovascular disease were enrolled in the final model. The highest score was 52.0. The median follow-up of validation cohort was 4.29 years. The optimal cut-off point was 17.0, with a sensitivity of 0.846 and a specificity of 0.668, respectively. According to the total scores, patients from the validation cohort were divided into low-, moderate-, high- and very high-risk groups. The risk of developing DPN was significantly increased in moderate- (RR 3.3, 95% CI 1.5–7.2, P = 0.020), high- (RR 15.5, 95% CI 7.6–31.6, P < 0.001), and very high-risk groups (RR 45.0, 95% CI 20.5–98.8, P < 0.001) compared with the low-risk group.ConclusionA risk prediction model for DPN including 11 common clinical indicators were established. It is a simple and reliable tool for early prevention and intervention of DPN in patients with T2DM.

Abstract DNA assembly is a vital process in biotechnology and synthetic biology research, during which DNA plasmids are designed and constructed using bioparts to engineer microorganisms for a wide range of applications. Here, we present an enzymatic homology-based DNA assembly method, SENAX (Stellar ExoNuclease Assembly miX), that can efficiently assemble multiple DNA fragments at ambient temperature from 30 to 37 °C and requires homology overlap as short as 12–18 base pairs. SENAX relies only on a 3′–5′ exonuclease, XthA (ExoIII), followed by Escherichia coli transformation, enabling easy scaling up and optimization. Importantly, SENAX can efficiently assemble short fragments down to 70 bp into a vector, overcoming a key shortcoming of existing commonly used homology-based technologies. To the best of our knowledge, this has not been reported elsewhere using homology-based methods. This advantage leads us to develop a framework to perform DNA assembly in a more modular manner using reusable promoter-RBS short fragments, simplifying the construction process and reducing the cost of DNA synthesis. This approach enables commonly used short bioparts (e.g., promoter, RBS, insulator, terminator) to be reused by the direct assembly of these parts into intermediate constructs. SENAX represents a novel accurate, highly efficient, and automation-friendly DNA assembly method.

Abstract Background Exposure to general anesthesia (GA) during the postnatal period is associated with neuroinflammation and long-term neurocognitive impairment in preclinical and clinical settings. Pyroptosis is a novel type of programmed cell death that, along with inflammation, has been found to play an important role in the mechanism of diverse neurological diseases. However, its roles in GA-induced neuroinflammation and neurocognitive impairment in the developing brain have not been investigated. Methods Rats at postnatal day 6 or primary hippocampal neurons at 9 days in vitro received 3% sevoflurane for 2 h daily for three consecutive days. A pharmacological inhibitor of nuclear factor (NF)-κB (BAY 11-7082) was administered to suppress NF-κB activation. Histological and biochemical analyses were performed to assess the pyroptosis as well as neuronal and synaptic damage both in vivo and in vitro. In addition, behavioral tests were performed to evaluate neurocognitive ability in rats. Results Repeated sevoflurane exposure activated NF-κB-mediated pyroptosis and neuroinflammation in the hippocampus in developing rats, damaged the neuronal morphology and synaptic integrity, and induced neurocognitive impairment in rats. BAY 11-7082 treatment suppressed the activation of pyroptosis, attenuated the neuronal and synaptic damage, and ameliorated the neurocognitive impairment induced by repeated sevoflurane administration to developing rats. Conclusions Repeated sevoflurane GA may induce neuroinflammation and neurocognitive impairment in developing rats via the activation of NF-κB-mediated pyroptosis. Our findings characterize a novel role of pyroptosis as a potential therapeutic target in neuroinflammation after repeated neonatal GA.

ObjectivePoor sleep quality is highly prevalent in patients with chronic diarrheal symptoms or inflammatory bowel disease (IBD). This study aimed to evaluate the associations of chronic diarrheal symptoms and IBD with sleep quality in the general US population.Methods14,696 adults (≥20 years) from the National Health and Nutrition Examination Survey (2005–2010) were included in the study. Chronic diarrheal symptoms and IBD were defined by self-reports. Sleep quality was assessed by sleep disorder, sleep trouble, and sleep duration. Multivariable logistic regression models were used to examine the associations.ResultsAfter adjustment of a series of covariates, we found that participants with chronic diarrheal symptoms or IBD had higher odds of sleep disorder [chronic diarrheal symptoms: odds ratio (OR) = 1.20, 95% confidence interval (CI) = 1.04–1.38; IBD: OR = 3.86, 95% CI = 1.92–7.77] and sleep trouble (chronic diarrheal symptoms: OR = 1.19, 95% CI = 1.09–1.30; IBD: OR = 2.32, 95% CI = 1.30–4.14), respectively. Sleep duration for participants with IBD was significantly shorter than that for those without IBD (β = −0.39, 95% CI = −0.78 to 0.01, P = 0.045). Subgroup analyses revealed that the associations of chronic diarrheal symptoms and IBD with sleep disorder and sleep trouble were more pronounced among women.ConclusionsIn this large sample of US adults, we found that chronic diarrheal symptoms and IBD were significantly associated with sleep quality, particularly in women. The findings highlight the importance of managing bowel health to promote high quality of sleep; and thus, improve quality of life in this subpopulation.

BackgroundThis study aimed to explore the relationship between iron markers and metabolic obesity phenotypes and the role of age.MethodsData were from the China Health and Nutrition Survey 2009. Metabolic obesity phenotypes included metabolically healthy with normal weight (MHNW), metabolically unhealthy with normal weight (MUNW), metabolically healthy with overweight/obesity (MHO), and metabolically unhealthy with overweight/obesity (MUO). Iron markers including ferritin, transferrin, and soluble transferrin receptor were calculated as Log and quartered. The linear regression and multinomial logistic regression were used to explore the association of iron markers with age and metabolic obesity phenotypes, respectively.ResultsFerritin was linearly related with age, with β (95% confidence interval, CI) of 0.029 (0.027 to 0.032) and -0.005 (-0.007 to -0.002) for women and men. Transferrin was negatively associated with age in both men and women (β < -0.011). Furthermore, compared with participants in the quartile 1 ferritin group, those in the quartile 4 had increased odds of MUNW, MHO, and MUO, with odds ratio and 95% confidence interval (OR, 95% CI) of 3.06 (2.20 to 4.25), 1.66 (1.35 to 2.05), and 5.27 (4.17 to 6.66). Transferrin showed similar relationships with MUNW, MUO, and MHO; whereas transferrin receptor showed no significance. We also found joint associations of ferritin and transferrin with MUNW, MUO, and MHO. The interactive effect of ferritin and transferrin on MUO was significant (P = 0.015).ConclusionIncreased ferritin and transferrin were associated with MUNW, MHO, and MUO. Age should be considered when investigating iron.

ABSTRACT When exposed to adverse conditions, many bacterial pathogens, including Vibrio cholerae, can adapt to the environment by entering the viable but nonculturable (VBNC) state. Cells in this state cannot grow on conventional media but still survive. The VBNC state is a significant threat to aquatic safety and public health due to the enhanced ability of the bacteria to remain in the environment and escape from monitoring. Detecting and quantifying VBNC cells and distinguishing them from nonviable cells is necessary for microbiological studies and pathogen monitoring. Cell staining and microscopy are used for the observation of VBNC cells, but it is difficult to quantify VBNC cells accurately. In this study, we developed droplet digital PCR (ddPCR) with a chromosomal single-copy gene as an internal reference combined with Propidium monoazide (PMA) treatment to enumerate VBNC cells of V. cholerae. In this method, bacterial cells, but not extracted chromosomal DNA, were used directly to form oil-enveloped droplets in the ddPCR procedure. One bacterial cell possesses one copy of the chromosome. Thus, enumeration of a single-copy gene on the chromosome can be used to count VBNC cells. ddPCR showed greater accuracy and sensitivity than qPCR. This study showed that the oil-enveloped bacterial method can reduce the number of steps needed and improve the accuracy of VBNC cells quantification and has the potential to be extended to quantify bacterial VBNC cells and assess pathogen survival in the environment. IMPORTANCE The viable but nonculturable (VBNC) state of bacteria represents an important life state for their survival in adverse environments. The VBNC cells of the pathogenic bacteria in the environment and food will be a potential threat to public health because these pathogens cannot be found by the detection of culture. We developed a sensitive molecular method to detect and enumerate the VBNC cells of V. cholerae, which can distinguish the VBNC and dead cells, and count the VBNC cells in the sample without the step of DNA extraction from cells. It can be used to improve the sensitivity of pathogen detection in the surveillance, risk assessment of environment and food contamination, and outbreak warning. The accurate identification and numeration of VBNC cells will also facilitate the microbiological and genetic studies on the development, adaptation, resuscitation, and elimination of the VBNC state.

Existing evidence has showed that exposure to polycyclic aromatic hydrocarbons (PAHs) increases the risk of many chronic diseases. Given the close connection between aging (a major risk factor) and chronic diseases, however, very few studies have evaluated the association between PAHs and aging. Furthermore, whether modifiable healthy lifestyle could attenuate the detrimental effect of PAHs on aging remains unknown. Therefore, we conducted this study, aiming to: (1) examine the associations of urinary monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs) and lifestyle with Phenotypic Age Acceleration (PhenoAge.Accel), a novel aging measure that captures morbidity and mortality risk; and (2) evaluate the potential interaction effects of OH-PAHs and lifestyle on PhenoAge.Accel. Cross-sectional data of 2,579 participants (aged 20–84 years, n = 1,292 females) from the National Health and Nutrition Examination Survey for years 2001–2010 were analyzed. A lifestyle index was constructed based on five components (drinking, smoking, body mass index, physical activity, and diet), ranging from 0 to 5. We calculated PhenoAge.Accel using algorithms developed previously. General linear regression models were used to examine the associations. We observed strong associations of OH-PAHs and lifestyle with PhenoAge.Accel. For instance, one unit increase in ∑NAP (sum of 1- and 2-hydroxynaphthalene) was associated with 0.37 year (95% confidence interval [CI]: 0.26, 0.48) increase in PhenoAge.Accel. We did not observe statistically significant interaction effects between OH-PAHs and lifestyle on PhenoAge.Accel. After stratified by sex, we observed strong associations as well as statistically significant interactions of OH-PAHs and lifestyle with PhenoAge.Accel among females. In conclusion, both OH-PAHs and lifestyle were independently associated with phenotypic aging and there were statistically significant interactions between OH-PAHs and lifestyle on phenotypic aging among females. The findings highlight the importance of adherence to a healthy lifestyle to attenuate the detrimental effects of exposures to PAHs on phenotypic aging among females.

Vulvovaginal candidiasis (VVC) is characterized by symptomatic inflammatory responses in the vagina caused by Candida albicans and non-albicans Candida (NAC) species. The epidermal growth factor receptor (EGFR) -mitogen-activated protein kinase (MAPK) signaling pathway has been linked to immune responses of oral mucosa after C. albicans exposure, but whether this pathway plays a similar response in vaginal epithelial cells is not known. Here, we observed that phosphorylation of EGFR and p38 was continuously activated in vaginal epithelial cells by C. albicans strain SC5314. This differs markedly from oral epithelial cells, which respond in a biphasic manner in order to properly discriminate the morphology of C. albicans. When compared with SC5314, a highly azole-resistant C. albicans isolate 1052 can induce a stronger phosphorylated signal of EGFR and p38, while clinically-isolated NAC strains including C. tropicalis, C. glabrata, C. parapsilosis and C. auris trigger higher levels of phosphorylated ERK1/2 and c-Fos than C. albicans. Inhibition of EGFR significantly reduces inflammatory response and epithelial damage induced by C. albicans both in vitro and in vivo, while inhibition of p38 leads to significant repair of epithelial damage triggered by both C. albicans and NAC species. These results confirm the importance of the EGFR-MAPK signaling in VVC pathogenesis and highlight the remarkable immunogenic differences between C. albicans and NAC species in host-microbe interactions.

BackgroundAging, as a multi-dimensional process, can be measured at different hierarchical levels including biological, phenotypic, and functional levels. The aims of this study were to: (1) compare the predictive utility of mortality by three aging measures at three hierarchical levels; (2) develop a composite aging measure that integrated aging measures at different hierarchical levels; and (3) evaluate the response of these aging measures to modifiable life style factors.MethodsData from National Health and Nutrition Examination Survey 1999–2002 were used. Three aging measures included telomere length (TL, biological level), Phenotypic Age (PA, phenotypic level), and frailty index (FI, functional level). Mortality information was collected until December 2015. Cox proportional hazards regression and multiple linear regression models were performed.ResultsA total of 3,249 participants (20–84 years) were included. Both accelerations (accounting for chronological age) of PA and FI were significantly associated with mortality, with HRs of 1.67 [95% confidence interval (CI) = 1.41–1.98] and 1.59 (95% CI = 1.35–1.87), respectively, while that of TL showed non-significant associations. We thus developed a new composite aging measure (named PC1) integrating the accelerations of PA and FI, and demonstrated its better predictive utility relative to each single aging measure. PC1, as well as the accelerations of PA and FI, were responsive to several life style factors including smoking status, body mass index, alcohol consumption, and leisure-time physical activity.ConclusionThis study demonstrates that both phenotypic (i.e., PA) and functional (i.e., FI) aging measures can capture mortality risk and respond to modifiable life style factors, despite their inherent differences. Furthermore, the PC1 that integrated phenotypic and functional aging measures outperforms in predicting mortality risk in comparison with each single aging measure, and strongly responds to modifiable life style factors. The findings suggest the complementary of aging measures at different hierarchical levels and highlight the potential of life style-targeted interventions as geroprotective programs.

Objectives: To compare the performance of two syndromic panels: Luminex xTAG Gastrointestinal Pathogen Panel (GPP) and FilmArray Gastrointestinal (GI) panel. Methods: A total of 243 diarrhea specimens were detected by two panels in parallel, and the inconsistent results were analyzed by real-time PCR or reverse transcription PCR (RT-PCR). The target concentration in specimens was examined by comparing the crossing point values of FilmArray, the median fluorescence intensity of xTAG and the cycle threshold values in any discrepancies. Results: For pathogens detected by both panels, the positive rates of FilmArray GI and xTAG GPP were 65.0% and 48.6%, respectively. The two panels showed high consistency (kappa ≥0.74) in detecting norovirus, rotavirus and Campylobacter, while there was low consistency (kappa ≤0.40) in detecting Cryptosporidium, Salmonella, Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic Escherichia coli (ETEC). Samples with low concentration targets were more often detected by FilmArray than with xTAG GPP. The xTAG GPP was more likely to be affected by amplification inhibitors. Several defects of xTAG GPP were found in detecting ETEC. Conclusions: FilmArray was more sensitive. For specimens with low target concentrations or containing ETEC heat stable enterotoxin, the false negatives of xTAG GPP need to be considered.

There is a growing demand for rapid, sensitive, field-deployable nucleic acid tests for cholera, which usually occurs in rural areas. In this study, we developed a Cas12a-assisted rapid isothermal detection (CARID) system for the detection of toxigenic V. cholerae serogroups O1 and O139 by combining recombinase-aided amplification and CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). The results can be determined by fluorescence signal and visualized by lateral flow dipstick. We identified 154 V. cholerae strains and 129 strains of other intestinal diarrheagenic bacteria with a 100% coincidence rate. The limit of detection of CARID was 20 copies/reaction of V. cholerae genomic DNA, which is comparable to that of polymerase chain reaction (PCR) and qPCR. Multiple-CARID was also established for efficiency and economic considerations with an acceptable decrease in sensitivity. Simulated sample tests showed that CARID is suitable for complex samples. In conclusion, CARID is a rapid, sensitive, economically efficient, and portable method for the detection of V. cholerae, which makes it suitable for field responses to cholera.

BackgroundExisting aging metrics incorporating cognitive and physical function are often not feasible for application in research and clinical practice. Therefore, this study aimed to develop and validate a new simple functional score based on self-reported cognitive and physical function in the older Chinese population.MethodsThe development sample included 3,929 older adults aged 60–95 years from the China Health and Retirement Longitudinal Study (CHARLS). The validation sample included 1,345 older adults aged 60–87 years from the Rugao Longitudinal Aging study (RLAS). Logistic regression models and receiver operating characteristic curves were used to examine the associations of the new functional score with all-cause mortality risk.ResultsSix items were selected to construct the new functional score in CHARLS. This functional score was associated with all-cause mortality risk, with an adjusted odds ratio of 1.10 (95% confidence interval = 1.07, 1.13). This functional score presented additional predictive utility beyond age and sex, as demonstrated by the significantly increased C-statistic, integrated discrimination improvement (IDI), and continuous net reclassification improvement (NRI) (all P < 0.001). Furthermore, this functional score was further validated in RLAS, such that adding the new functional score to a model of age and sex improved all-cause mortality risk discrimination (IDI = 0.036, P < 0.001; NRI = 0.485, P < 0.001). To facilitate the quick screening of the older population with deteriorations in cognitive and physical function, we introduced a publicly available online tool designed for this new functional score.ConclusionsA new functional score based on six self-reported items was developed and validated in the older Chinese population, and was demonstrated to be a simple and practical tool to assess functional deterioration, showing good feasibility, and performance.

ObjectiveTo examine the associations of serum albumin, a nutrition indicator, with disability in activities of daily living (ADL), mobility, and objective physical functioning among Chinese older adults.Materials and MethodsCross-sectional data of 2233 older adults (≥65 years) who participated in the 2011/2012 main survey of the Chinese Longitudinal Healthy Longevity Survey (CLHLS) and the 2012 biomarker sub-study was used. Serum albumin was measured by immunoturbidimetric assay. Physical functioning included subjectively (ADL and mobility) and objectively measured disability (standing up from a chair, picking up a book from the floor, and turning around 360°). Multivariable logistic regression models were performed.ResultsAfter adjusting for age and sex, compared with participants in the lowest quartile group of serum albumin, those in the highest quartile group had 45% lower odds of disability in ADL (odds ratio [OR]: 0.55; 95% confidence interval [CI]: 0.38, 0.80); 48% lower odds of disability in mobility (OR: 0.52; 95% CI: 0.38, 0.71); 46% lower odds of disability in standing up from a chair (OR: 0.54; 95% CI: 0.34, 0.85); and 37% lower odds of disability in picking up a book from the floor (OR: 0.63; 95% CI: 0.40, 0.97). We did not observe a statistically significant interaction effect between serum albumin and vitamin D on disability in physical functioning.ConclusionSerum albumin level was associated with physical functioning among Chinese older adults, regardless of vitamin D level. The findings indicate that appropriate management of poor nutritional status, in particular low serum albumin levels, may contribute to maintaining physical functioning in older adults.

Abstract Background With the inherent high density and durable preservation, DNA has been recently recognized as a distinguished medium to store enormous data over millennia. To overcome the limitations existing in a recently reported high-capacity DNA data storage while achieving a competitive information capacity, we are inspired to explore a new coding system that facilitates the practical implementation of DNA data storage with high capacity. Result In this work, we devised and implemented a DNA data storage scheme with variable-length oligonucleotides (oligos), where a hybrid DNA mapping scheme that converts digital data to DNA records is introduced. The encoded DNA oligos stores 1.98 bits per nucleotide (bits/nt) on average (approaching the upper bound of 2 bits/nt), while conforming to the biochemical constraints. Beyond that, an oligo-level repeat-accumulate coding scheme is employed for addressing data loss and corruption in the biochemical processes. With a wet-lab experiment, an error-free retrieval of 379.1 KB data with a minimum coverage of 10x is achieved, validating the error resilience of the proposed coding scheme. Along with that, the theoretical analysis shows that the proposed scheme exhibits a net information density (user bits per nucleotide) of 1.67 bits/nt while achieving 91% of the information capacity. Conclusion To advance towards practical implementations of DNA storage, we proposed and tested a DNA data storage system enabling high potential mapping (bits to nucleotide conversion) scheme and low redundancy but highly efficient error correction code design. The advancement reported would move us closer to achieving a practical high-capacity DNA data storage system.

Many bacterial species, including Vibrio cholerae (the pathogen that causes cholera), enter a physiologically viable but non-culturable (VBNC) state at low temperature or in conditions of low nutrition; this is a survival strategy to resist environmental stress. Identification, detection, and differentiation of VBNC cells and nonviable cells are essential for both microbiological study and disease surveillance/control. Enumeration of VBNC cells requires an accurate method. Traditional counting methods do not allow quantification of VBNC cells because they are not culturable. Morphology-based counting cannot distinguish between live and dead cells. A bacterial cell possesses one copy of the chromosome. Hence, counting single-copy genes on the chromosome is a suitable approach to count bacterial cells. In this study, we developed quantitative PCR-based methods, including real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR), to enumerate VBNC V. cholerae cells by counting the numbers of single-copy genes in samples during VBNC-state development. Propidium monoazide (PMA) treatment was incorporated to distinguish dead cells from viable cells. Both PCR methods could be used to quantify the number of DNA copies/mL and determine the proportion of dead cells (when PMA was used). The methods produced comparable counts using three single-copy genes (VC1376, thyA, and recA). However, ddPCR showed greater accuracy and sensitivity than qPCR. ddPCR also allows direct counting without the need to establish a standard curve. Our study develops a PMA-ddPCR method as a new tool to quantify VBNC cells of V. cholerae. The method can be extended to other bacterial species.

The multi-model ensemble (MME) forecast for meteorological elements has been proved many times to be more skillful than the single model. It improves the forecast quality by integrating multiple sets of numerical forecast results with different spatial-temporal characteristics. Currently, the main numerical forecast results present a grid structure formed by longitude and latitude lines in space and a special two-dimensional time structure in time, namely the initial time and the lead time, compared with the traditional one-dimensional time. These characteristics mean that many MME methods have limitations in further improving forecast quality. Focusing on this problem, we propose a deep MME forecast method that suits the special structure. At spatial level, our model uses window self-attention and shifted window attention to aggregate information. At temporal level, we propose a recurrent like neural network with rolling structure (Roll-RLNN) which is more suitable for two-dimensional time structure that widely exists in the institutions of numerical weather prediction (NWP) with running service. In this paper, we test the MME forecast for sea level pressure as the forecast characteristics of the essential meteorological element vary clearly across institutions, and the results show that our model structure is effective and can make significant forecast improvements.

Background Gliomas are the most common primary tumors of the central nervous system. The complexity and heterogeneity of the tumor makes it difficult to obtain good biomarkers for drug development. In this study, through The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA), we analyze the common diagnostic and prognostic moleculer markers in Caucasian and Asian populations, which can be used as drug targets in the future. Methods The RNA-seq data from Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) were analyzed to identify signatures. Based on the signatures, the prognosis index (PI) of every patient was constructed to predict the prognostic risk. Also, gene ontology (GO) functional enrichment analysis and KEGG analysis were conducted to investigate the biological functions of these mRNAs. Glioma patients’ data in the CGGA database were introduced to validate the effectiveness of the signatures among Chinese populations. Excluding the previously reported prognostic markers of gliomas from this study, the expression of HSPA5 and MTPN were examined by qRT-PCR and immunohistochemical assay. Results In total, 20 mRNAs were finally selected to build PI for patients from TCGA, including 16 high-risk genes and four low-risk genes. For Chinese patients, the log-rank test p values of PI were both less than 0.0001 in two independent datasets. And the AUCs were 0.831 and 0.907 for 3 years of two datasets, respectively. Moreover, among these 20 mRNAs, 10 and 15 mRNAs also had a significant predictive effect via univariate COX analysis in CGGA_693 and CGGA_325, respectively. qRT-PCR and Immunohistochemistry assay indicated that HSPA5 and MTPN over-expressed in Glioma samples compared to normal samples. Conclusion The 20-gene signature can forecast the risk of Glioma in TCGA effectively, moreover it can also predict the risks of Chinese patients through validation in the CGGA database. HSPA5 and MTPN are possible biomarkers of gliomas suitable for all populations to improve the prognosis of these patients.

Attachment and specific binding to the receptor on the host cell surface is the first step in the process of bacteriophage infection. The lytic phage VP2 is used in phage subtyping of the Vibrio cholerae biotype El Tor of the O1 serogroup; however, its infection mechanism is poorly understood. In this study, we aimed to identify its receptor on V. cholerae. The outer membrane protein EpsD in the type II secretory system (T2SS) was found to be related to VP2-specific adsorption to V. cholerae, and the T2SS inner membrane protein EpsM had a role in successful VP2 infection, although it was not related to adsorption of VP2. The tail fiber protein gp20 of VP2 directly interacts with EpsD. Therefore, we found that in V. cholerae, in addition to the roles of the T2SS as the transport apparatus of cholera toxin secretion and filamentous phage release, the T2SS is also used as the receptor for phage infection and probably as the channel for phage DNA injection. Our study expands the understanding of the roles of the T2SS in bacteria.

Abstract Background Of the hundreds of Vibrio cholerae serogroups, O1 and O139 are the main epidemic-causing ones. Although non-O1/non-O139 serogroups rarely cause epidemics, the possibility exists for strains within them to have pathogenic potential. Results We selected 25 representative strains within 16 V. cholerae serogroups and examined their genomic and functional characteristics. We tentatively constructed a gene pool containing 405 homologous gene clusters, which is well organized and functions in O-antigen polysaccharide (O-PS) synthesis. Our network analysis indicate that great diversity exists in O-PS among the serogroups, and several serogroup pairs share a high number of homologous genes (e.g., O115 and O37; O170 and O139; O12 and O39). The phylogenetic analysis results suggest that a close relationship exists between serogroups O170, O89 and O144, based on neighbor-joining (NJ) and gene trees, although serogroup O159 showed an inconsistent phylogenetic relationship between the NJ tree and the gene tree, indicating that it may have undergone extensive recombination and horizontal gene transfer. Different phylogenetic structures were observed between the core genes, pan genes, and O-PS genes. The virulence gene analysis indicated that the virulence genes from all the representative strains may have their sources from four particular bacteria (Pseudomonas aeruginosa, V. vulnificus, Haemophilus somnus and H. influenzae), which suggests that V. cholerae may have exchanged virulence genes with other bacterial genera or species in certain environments. The mobile genetic element analysis indicated that O159 carries nearly complete VSP-II and partial VPI-1 and VPI-2, O170 carries partial VPI-1 and VPI-2, and several non-O1/non-O139 strains contain full or partial VPI-1 and VPI-2. Several genes showing evidence of positive selection are involved in chemotaxis, Na + resistance, or cell wall synthesis, suggestive of environmental adaptation. Conclusions This study reports on the newly sequenced O159 and O170 genomes and their comparisons with other V. cholerae serogroups. The complicated O-PS network of constituent genes highlights the detailed recombination mechanisms that have acted on the serogroups’ genomes. The serogroups have different virulence-related gene profiles, and there is evidence of positive selection acting on other genes, possibly during adaptation to different environments and hosts.

Antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) have been detected in human-impacted habitats, especially in densely populated cities. The Qinghai–Tibet Plateau is located far from the heavily populated regions of China, and Tibetan residents have distinct dietary habits and gut microbes. Antibiotic-resistance monitoring in the Tibetan population is rare. Here, we collected stool samples from Tibetan outpatients with diarrhea. From 59 samples, 48 antibiotic-resistant Enterobacteriaceae isolates were obtained, including 19 extended-spectrum beta lactamase (ESBL)-producing isolates from 16 patients and 29 polymyxin-resistant isolates from 22 patients. Either ESBL or mcr genes were found in 17 Escherichia coli isolates, approximately 58.8% of which were multidrug-resistant, and ten incompatible plasmid types were found. The gene blaCTX-M was a common genotype in the ESBL-producing E. coli isolates. Four E. coli isolates contained mcr-1. The same mcr-1-carrying plasmid was found in distinct E. coli isolates obtained from the same sample, thus confirming horizontal transmission of mcr-1 between bacteria. Genomic clustering of E. coli isolates obtained from Lhasa, with strains from other regions providing evidence of clone spreading. Our results reveal a strong presence of ARB and ARGs in Tibetan outpatients with diarrhea, implying that ARB and ARGs should be monitored in the Tibetan population.

Abstract Background Chinese traditional herbal medicine Fuzhengkangai (FZKA) formulation combination with gefitinib can overcome drug resistance and improve the prognosis of lung adenocarcinoma patients. However, the pharmacological and molecular mechanisms underlying the active ingredients, potential targets, and overcome drug resistance of the drug are still unclear. Therefore, it is necessary to explore the molecular mechanism of FZKA. Methods A systems pharmacology and bioinformatics-based approach was employed to investigate the molecular pathogenesis of EGFR-TKI resistance with clinically effective herb formula. The differential gene expressions between EGFR-TKI sensitive and resistance cell lines were calculated and used to find overlap from targets as core targets. The prognosis of core targets was validated from the cancer genome atlas (TCGA) database by Cox regression. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment is applied to analysis core targets for revealing mechanism in biology. Results The results showed that 35 active compounds of FZKA can interact with eight core targets proteins (ADRB2, BCL2, CDKN1A, HTR2C, KCNMA1, PLA2G4A, PRKCA and LYZ). The risk score of them were associated with overall survival and relapse free time (HR = 6.604, 95% CI: 2.314–18.850; HR = 5.132, 95% CI: 1.531–17.220). The pathway enrichment suggested that they involved in EGFR-TKI resistance and non-small cell lung cancer pathways, which directly affect EGFR-TKI resistance. The molecular docking showed that licochalcone a and beta-sitosterol can closely bind two targets (BCL2 and PRKCA) that involved in EGFR-TKI resistance pathway. Conclusions This study provided a workflow for understanding mechanism of CHM for against drug resistance.

Vibrio cholerae and Vibrio parahaemolyticus are common diarrheal pathogens of great public health concern. A multiplex TaqMan-based real-time PCR assay was developed on the BD MAX platform; this assay can simultaneously detect and differentiate V. cholerae and V. parahaemolyticus directly from human fecal specimens. The assay includes two reactions. One reaction, BDM-VC, targets the gene ompW, the cholera toxin (CT) coding gene ctxA, the O1 serogroup specific gene rfbN, and the O139 serogroup specific gene wbfR of V. cholerae. The other, BDM-VP, targets the gene toxR and the toxin coding genes tdh and trh of V. parahaemolyticus. In addition, each reaction contains a sample process control. When evaluated with spiked stool samples, the detection limit of the BD MAX assay was 195–780 CFU/ml for V. cholerae and 46–184 CFU/ml for V. parahaemolyticus, and the amplification efficiency of all genes was between 95 and 115%. The assay showed 100% analytical specificity, using 63 isolates. The BD MAX assay was evaluated for its performance compared with conventional real-time PCR after automated DNA extraction steps, using 164 retrospective stool samples. The overall percent agreement between the BD MAX assay and real-time PCR was ≥ 98.8%; the positive percent agreement was 85.7% for ompW, 100% for toxR/tdh, and lower (66.7%) for trh because of a false negative. This is the first report to evaluate the usage of the BD MAX open system in detection and differentiation of V. cholerae and V. parahaemolyticus directly from human samples.

The cyclic adenosine 3′,5′-monophosphate (cAMP)/protein kinase A (PKA) pathway of Candida albicans responds to nutrient availability to coordinate a series of cellular processes for its replication and survival. The elevation of cAMP for PKA signaling must be both transitory and tightly regulated. Otherwise, any abnormal cAMP/PKA pathway would disrupt metabolic potential and ergosterol synthesis and promote a stress response. One possible mechanism for controlling cAMP levels is direct induction of the phosphodiesterase PDE2 gene by cAMP itself. Our earlier studies have shown that most single-gene-deletion mutants of the mitochondrial electron transport chain (ETC) complex I (CI) are hypersensitive to fluconazole. To understand the fluconazole hypersensitivity observed in these mutants, we focused upon the cAMP/PKA-mediated ergosterol synthesis in CI mutants. Two groups of the ETC mutants were used in this study. Group I includes CI mutants. Group II is composed of CIII and CIV mutants; group II mutants are known to have greater respiratory loss. All mutants are not identical in cAMP/PKA-mediated ergosterol response. We found that ergosterol levels are decreased by 47.3% in the ndh51Δ (CI core subunit mutant) and by 23.5% in goa1Δ (CI regulator mutant). Both mutants exhibited a greater reduction of cAMP and excessive trehalose production compared with other mutants. Despite the normal cAMP level, ergosterol content decreased by 33.0% in the CIII mutant qce1Δ as well, thereby displaying a cAMP/PKA-independent ergosterol response. While the two CI mutants have some unique cAMP/PKA-mediated ergosterol responses, we found that the degree of cAMP reduction correlates linearly with a decrease in total nicotinamide adenine dinucleotide (NAD) levels in all mutants, particularly in the seven CI mutants. A mechanism study demonstrates that overactive PDE2 and cPDE activity must be the cause of the suppressive cAMP-mediated ergosterol response in the ndh51Δ and goa1Δ. While the purpose of this study is to understand the impact of ETC proteins on pathogenesis-associated cellular events, our results reveal the importance of Ndh51p in the regulation of the cAMP/PKA pathway through Pde2p inhibition in normal physiological environments. As a direct link between Ndh51p and Pde2p remains elusive, we suggest that Ndh51p participates in NAD homeostasis that might regulate Pde2p activity for the optimal cAMP pathway state.

Attached Vibrio cholerae biofilms are essential for environmental persistence and infectivity. The vps loci (vpsU, vpsA-K, and vpsL-Q) are required for mature biofilm formation and are responsible for the synthesis of exopolysaccharide. Transcription of vps genes is activated by the signaling molecule bis-(3′–5′)-cyclic di-GMP (c-di-GMP), whose metabolism is controlled by the proteins containing the GGDEF and/or EAL domains. The ferric uptake regulator (Fur) plays key roles in the transcription of many genes involved in iron metabolism and non-iron functions. However, roles for Fur in Vibrio biofilm production have not been documented. In this study, phenotypic assays demonstrated that Fur, independent of iron, decreases in vivo c-di-GMP levels and inhibits in vitro biofilm formation by Vibrio cholerae. The Fur box-like sequences were detected within the promoter-proximal DNA regions of vpsU, vpsA-K, vieSAB, and cdgD, suggesting that transcription of these genes may be under the direct control of Fur. Indeed, the results of luminescence, quantitative PCR (qPCR), electrophoretic mobility shift assay (EMSA), and DNase I footprinting assays demonstrated Fur to bind to the promoter-proximal DNA regions of vpsU, vpsA-K, and cdgD to repress their transcription. In contrast, Fur activates the transcription of vieSAB in a direct manner. The cdgD and vieSAB encode proteins with GGDEF and EAL domains, respectively. Thus, data presented here highlight a new physiological role for Fur wherein it acts as a repressor of V. cholerae biofilm formation mediated by decreasing the production of exopolysaccharide and the intracellular levels of c-di-GMP.

It is known that complex growth environments often induce inhomogeneity in two-dimensional (2D) materials and significantly restrict their applications. In this paper, we proposed an efficient method to analyze the inhomogeneity of 2D materials by combination of Raman spectroscopy and unsupervised k-means clustering analysis. Taking advantage of k-means analysis, it can provide not only the characteristic Raman spectrum for each cluster but also the cluster spatial maps. It has been demonstrated that inhomogeneities and their spatial distributions are simultaneously revealed in all CVD-grown MoS2, WS2 and WSe2 samples. Uniform p-type doping and varied tensile strain were found in polycrystalline monolayer MoS2 from the grain boundary and edges to the grain center (single crystal). The bilayer MoS2 with AA and AB stacking are shown to have relatively uniform p-doping but a gradual increase of compressive strain from center to the periphery. Irregular distribution of 2LA(M)/E2g1 mode in WS2 and E2g1 mode in WSe2 is revealed due to defect and strain, respectively. All the inhomogeneity could be directly characterized in color-coded Raman imaging with correlated characteristic spectra. Moreover, the influence of strain and doping in the MoS2 can be well decoupled and be spatially verified by correlating with the clustered maps. Our k-means clustering Raman analysis can dramatically simplify the inhomogeneity analysis for large Raman data in 2D materials, paving the way towards direct evaluation for high quality 2D materials.

Cholera toxin (CT) and toxin coregulated pilus (TCP, TcpA is the major subunit) are two major virulence factors of Vibrio cholerae, both of which play critical roles in developing severe diarrhea in human. Expression of CT and TCP is under the tight control of the regulatory cascade known as the ToxR virulence regulon, which is composed of three regulators ToxR, TcpP, and ToxT. Besides, their expression is also regulated by the quorum sensing (QS) master regulator HapR and the regulatory protein Fur. Though transcription of tcpP, toxT, and/or tcpA are reported to be regulated by HapR and Fur, to date there are no studies to verify their direct regulations. In the present study, we showed that HapR directly repress the transcription of tcpP and tcpA by binding to their promoter regions, and possibly repress toxT transcription in an indirect manner. Fur directly activated the transcription of tcpP, toxT, and tcpA by binding to their promoters. Taking account of the sequential expression of hapR, fur, tcpP, toxT, and tcpA in the different growth phases of V. cholerae, we deduce that at the early mid-logarithmic growth phase, Fur binds to the promoters of tcpP, toxT, and tcpA to activate their transcription; while at the later mid-logarithmic growth phase, HapR can bind to the promoters of tcpP and tcpA to repress their transcription. Our study reveals the new recognition in the virulence regulatory pathways in V. cholerae and suggests the complicated and subtle regulation network with the growth density dependence.

Since the real estate market reform in 1998, China's house prices have been rising. High housing prices have exerted negative impacts on labour mobility, the investment environment and resident consumption, all of which may affect the overall resource allocation efficiency and the improvement of the global value chain position of China's manufacturing industry. However, there is little research on the mechanism of the impact of housing prices on the status of manufacturing global value chain. Based on the matching data of China and the OECD-TiVA from 2005 to 2016, the parallel multiple mediator model is adopted to empirically test the transmission mechanism of housing price fluctuation on the global value chain position of China's manufacturing industry in this study using human capital level, resident consumption level, resident consumption structure, and R&D investment level as the mediating variables. This article finds that although housing price fluctuations do not have a direct impact on the global value chain position of the manufacturing industry, human capital level and resident consumption structure do have positive and negative mediating effects, respectively. Finally, we put forward some suggestions to promote the global value chain position of China's manufacturing industry, which provides useful reference for policy makers.

Different analytical methods or models can often find completely different prognostic biomarkers for the same cancer. In the study of prognostic molecular biomarkers of ovarian cancer (OvCa), different studies have reported a variety of prognostic gene signatures. In the current study, based on geometric concepts, the linearity-clustering phase diagram with integrated P-value (LCP) method was used to comprehensively consider three indicators that are commonly employed to estimate the quality of a prognostic gene signature model. The three indicators, namely, concordance index, area under the curve, and level of the hazard ratio were determined via calculation of the prognostic index of various gene signatures from different datasets. As evaluation objects, we selected 13 gene signature models (Cox regression model) and 16 OvCa genomic datasets (including gene expression information and follow-up data) from published studies. The results of LCP showed that three models were universal and better than other models. In addition, combining the three models into one model showed the best performance in all datasets by LCP calculation. The combination gene signature model provides a more reliable model and could be validated in various datasets of OvCa. Thus, our method and findings can provide more accurate prognostic biomarkers and effective reference for the precise clinical treatment of OvCa.

Objectives: To conduct a one-year pathogen surveillance of acute diarrheal disease based on outpatient clinics in township hospitals in rural Hongta District of Yunnan Province, China. Methods: Fecal specimens of acute diarrhea cases and relevant epidemiological information were collected. Salmonella, Shigella, Vibrio, Aeromonas, Plesiomonas shigelloides and diarrheogenic Escherichia coli (DEC) were examined. Results: Among the 797 stool specimens sampled, 198 samples (24.8%) were positive in pathogen isolation, and 223 strains were isolated. The order of isolation rates from high to low were DEC, Aeromonas, P. shigelloides, Salmonella, Shigella and Vibrio. The overall positive rate in middle school students and preschool children was relatively high; while the overall positive rate of less than 1-year-old infants and above 55 years olds was relatively low. The isolates were analyzed by pulsed-field gel electrophoresis (PFGE). Some cases had the same or very close onset time, and the isolates had similar PFGE patterns, suggesting a possible outbreak once occurred but was not detected by the current infectious disease reporting system. Conclusions: Pathogen infection and transmission in rapidly urbanized rural areas is a serious issue. There is a great need for a more sensitive and accurate mode of monitoring, reporting and outbreak identification of diarrheal disease. Keywords: Diarrheal disease, Diarrheogenic pathogen, Molecular typing, Surveillance, Bacterial pathogen

Abstract As the first known Weyl semimetals, binary compounds including TaP, TaAs, NbAs, and NbP have received worldwide interest. This work explored the phase behaviours of NbAs and NbP under high pressure up to 200 GPa via first-principles calculations combined with intelligent particle swarm optimization. Upon compression, a new phase of NbAs with P-6m2 symmetry appeared above 23 GPa and remained stable until 38 GPa, whereupon a monoclinic structure with space group P2 1 /c became more energetically favourable. This lasted until 73 GPa, when a Pm-3m phase followed. Surprisingly, NbP underwent a single phase transition around 63.5 GPa to a new phase with Cmcm symmetry that was completely distinct from the structures shown by TaAs-class compounds such as NbAs and TaAs. All these newly pressure-stabilized structures were dynamically stable at both high and ambient pressure. Electronic band structure calculations revealed a transition from semimetal to metal under high pressure. This work is meaningful and fundamental for future studies and applications of TaAs-class Weyl semimetals under compression or extreme conditions.

Stone cells are a characteristic trait of pear fruit, but the contents and sizes of stone cells negatively correlate with fruit texture and flavor. Secondary cell wall thickening and lignification have been established as key steps of stone cell development. KNOTTED-LIKE HOMEOBOX (KNOX) proteins play important roles in plant cell growth and development, including cell wall formation and lignification. Although the characteristics and biological functions of KNOX proteins have been investigated in other plants, this gene family has not been functionally characterized in pear. Eighteen PbKNOX genes were identified in the present study, and all of the identified family members contained the KNOX I and/or KNOX II domains. Based on the phylogenetic tree and chromosomal localization, the 18 PbKNOX genes were divided into five subfamilies [SHOOT MERISTEMLESS (STM)-like, BREVIPEDICELLUS (BP)-like, KNOTTED ARABIDOPSIS THALIANA 2/6 (KNAT2/6)-like, KNAT7-like, and KNAT3-5-like] and were distributed among 10 chromosomes. In addition, we identified 9, 11, and 11 KNOX genes in the genomes of grape, mei, and strawberry, respectively, and the greatest number of collinear KNOX gene pairs formed between pears and peaches. Analyses of the spatiotemporal expression patterns showed that the tissue specificity of PbKNOX gene expression was not very significant and that the level of the PbKNOX1 transcript showed an opposite trend to the levels of stone cells and lignin accumulation. Furthermore, PbKNOX1 has high sequence identity and similarity with Arabidopsis BP. Compared with wild-type Arabidopsis, plants overexpressing PbKNOX1 not only showed an approximately 19% decrease in the secondary cell wall thickness of vessel cells but also exhibited an approximately 13% reduction in the lignin content of inflorescence stems. Moreover, the expression of several genes involved in lignin biosynthesis was downregulated in transgenic lines. Based on our results, PbKNOX1/BP participates in cell wall-thickening and lignin biosynthesis and represses the transcription of key structural genes involved in lignin synthesis, providing genetic evidence for the roles of KNOX in cell wall thickening and lignin biosynthesis in pear.

Pathogenic marine bacteria are found in environments and food sources with high salt concentrations, which the bacteria must effectively manage for their survival. Several mechanisms, such as the transport of ions and compatible solutes as well as changes in aerobic and anaerobic respiration, confer salt tolerance to bacteria. In this study, we found that the outer membrane protein OmpW was related to salt stress in Vibrio cholerae and that ompW gene transcription and expression were up-regulated in cultures containing high NaCl concentrations. Deletion of ompW resulted in reduced V. cholerae growth in hypersaline culture conditions. Supplements of the compatible solutes betaine, L-carnitine, or L-lysine enhanced the growth of V. cholerae in hypersaline media. Supplements of betaine or L-lysine had the same growth enhancement effect on the ompW-deletion mutant cultured in hypersaline media, whereas L-carnitine supplementation did not restore mutant growth. In addition, the uptake of L-carnitine was decreased in the ompW-deletion mutant. Our study showed that among the multiplex factors that enhance the hypersaline tolerance of V. cholerae, OmpW also plays a role by transporting L-carnitine.