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Peripheral blood mononuclear cells (PBMCs) were obtained from a patient diagnosed with Familial Hemiplegic Migraine Type 3, who carried a heterozygous A > C mutation in the SCN1A gene and reprogrammed using CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit. The iPSC line maintained the mutation while expressing markers of pluripotency. Additionally, it exhibited a normal karyotype and demonstrated potential for in vitro differentiation into cells representing all three embryonic germ layers.

Abstract Background Village doctors are the main health service providers in China’s rural areas. Compared with other rural groups, they will have a sense of relative deprivation, which has an impact on their practice mentality and job stability. This study aims to analyze the changes and causes of relative deprivation among village doctors, so as to improve the stability of them. Methods The data were collected from two surveys conducted in Shandong Province in 2015 and 2021. In 2015, 322 village doctors were surveyed and 307 questionnaires were collected, with a recovery rate of 95.3%. In 2021, 394 village doctors were surveyed and 366 questionnaires were collected, with a recovery rate of 92.9%. Descriptive and univariate analysis were used to compare the changes before and after the survey. Results The scores of vertical deprivation of village doctors increased from 2.77 ± 0.81 in 2015 to 3.04 ± 0.83 in 2021, with a statistically significant difference (P

Abstract Background Migraine stands as a prevalent primary headache disorder, with prior research highlighting the significant involvement of oxidative stress and inflammatory pathways in its pathogenesis and chronicity. Existing evidence indicates the capacity of Dl-3-n-butylphthalide (NBP) to mitigate oxidative stress and inflammation, thereby conferring neuroprotective benefits in many central nervous system diseases. However, the specific therapeutic implications of NBP in the context of migraine remain to be elucidated. Methods We established a C57BL/6 mouse model of chronic migraine (CM) using recurrent intraperitoneal injections of nitroglycerin (NTG, 10 mg/kg), and prophylactic treatment was simulated by administering NBP (30 mg/kg, 60 mg/kg, 120 mg/kg) by gavage prior to each NTG injection. Mechanical threshold was assessed using von Frey fibers, and photophobia and anxious behaviours were assessed using a light/dark box and elevated plus maze. Expression of c-Fos, calcitonin gene-related peptide (CGRP), Nucleus factor erythroid 2-related factor 2 (Nrf2) and related pathway proteins in the spinal trigeminal nucleus caudalis (SP5C) were detected by Western blotting (WB) or immunofluorescence (IF). The expression of IL-1β, IL-6, TNF-α, Superoxide dismutase (SOD) and malondialdehyde (MDA) in SP5C and CGRP in plasma were detected by ELISA. A reactive oxygen species (ROS) probe was used to detect the expression of ROS in the SP5C. Results At the end of the modelling period, chronic migraine mice showed significantly reduced mechanical nociceptive thresholds, as well as photophobic and anxious behaviours. Pretreatment with NBP attenuated nociceptive sensitization, photophobia, and anxiety in the model mice, reduced expression levels of c-Fos and CGRP in the SP5C and activated Nrf2 and its downstream proteins HO-1 and NQO-1. By measuring the associated cytokines, we also found that NBP reduced levels of oxidative stress and inflammation. Most importantly, the therapeutic effect of NBP was significantly reduced after the administration of ML385 to inhibit Nrf2. Conclusions Our data suggest that NBP may alleviate migraine by activating the Nrf2 pathway to reduce oxidative stress and inflammation in migraine mouse models, confirming that it may be a potential drug for the treatment of migraine. Graphical Abstract

Objective To understand the impact of chronic disease comorbidity on healthcare institution-seeking behavior of elderly people in China, and to provide a theoretical basis for optimizing healthcare order and enhancing the management capacity of primary care for chronic diseases. MethodsThe data on 4 508 elderly people aged ≥ 60 years in 25 provincial-level administrative divisions across China were collected from the China Family Panel Studies (CFPS) conducted in 2020. The Stata 17.0 statistical software was used to analyze the impact of chronic disease comorbidity on healthcare facility-seeking behavior of elderly people in China using the probit regression model and the propensity score matching (PSM) method. ResultsOf the 4 508 elderly people surveyed, 748 (16.59%) reported having chronic disease comorbidity (suffering from two or more chronic diseases); 2 536 (56.26%) and 1 972 (43.74%) reported seeking medical treatment at primary and non-primary healthcare institutions during past six months. After adjusting for predisposing factors (gender, age, education level, marital status), enabling factors (household registration type, monthly retirement pension, satisfaction with medical treatment, participation in medical insurance, emotional relationship with children, financial support from children, caregiving provided by children), and need factors (self-rated health status, changes in health conditions, physical discomfort in the past two weeks, the ability to take care of oneself), the results of probit regression analysis showed that the elderly people with comorbid chronic diseases were more inclined to seek medical treatment at non-primary healthcare institutions (β = – 0.130, 95% confidence interval: – 0.237 – – 0.022). The estimated values of average treatment effect on the treated (ATT) of chronic disease comorbidity on the healthcare institution selection of the elderly people were – 0.052, – 0.048, and – 0.047, respectively, based on matched propensity scores of predisposing, enabling, and need factors with K-nearest neighbor, kernel, and radius matching (all P < 0.05), indicating that chronic disease comorbidity can reduce the probability of elderly people seeking medical treatment at primary healthcare institutions. The heterogeneity test results showed that the impact of chronic disease comorbidity on females (ATT = – 0.078 – – 0.063, all P < 0.05) and the elderly people with a junior high school education or below (ATT = – 0.058 – – 0.052, all P < 0.05) was more significant, but there was no statistically significant difference in the impact on males and elderly people with a high school education or above (all P > 0.05). ConclusionChronic disease comorbidity may affect the selection of medical institutions for elderly people in China to some extent, and its influence differs by gender and education level of the elderly.

Abstract Background Both epidemiological and clinical studies have indicated that headache and sleep disturbances share a complex relationship. Although headache and sleep share common neurophysiological and anatomical foundations, the mechanism underlying their interaction remains poorly understood. The structures of the diencephalon and brainstem, particularly the locus coeruleus (LC), are the primary sites where the sleep and headache pathways intersect. To better understand the intricate nature of the relationship between headache and sleep, our study focused on investigating the role and function of noradrenergic neurons in the LC during acute headache and acute sleep disturbance. Method To explore the relationship between acute headache and acute sleep disturbance, we primarily employed nitroglycerin (NTG)-induced migraine-like headache and acute sleep deprivation (ASD) models. Initially, we conducted experiments to confirm that ASD enhances headache and that acute headache can lead to acute sleep disturbance. Subsequently, we examined the separate roles of the LC in sleep and headache. We observed the effects of drug-induced activation and inhibition and chemogenetic manipulation of LC noradrenergic neurons on ASD-induced headache facilitation and acute headache-related sleep disturbance. This approach enabled us to demonstrate the bidirectional function of LC noradrenergic neurons. Results Our findings indicate that ASD facilitated the development of NTG-induced migraine-like headache, while acute headache affected sleep quality. Furthermore, activating the LC reduced the headache threshold and increased sleep latency, whereas inhibiting the LC had the opposite effect. Additional investigations demonstrated that activating LC noradrenergic neurons further intensified pain facilitation from ASD, while inhibiting these neurons reduced this pain facilitation. Moreover, activating LC noradrenergic neurons exacerbated the impact of acute headache on sleep quality, while inhibiting them alleviated this influence. Conclusion The LC serves as a significant anatomical and functional region in the interaction between acute sleep disturbance and acute headache. The involvement of LC noradrenergic neurons is pivotal in facilitating headache triggered by ASD and influencing the effects of headache on sleep quality.

IntroductionCoronavirus disease 2019 occurred unexpectedly in late December 2019, it was difficult to immediately develop an effective vaccine or propose targeted medical interventions in the early stages of the outbreak. At this point, non-pharmaceutical interventions (NPIs) are essential components of the public health response to COVID-19. How to combine different NPIs in the early stages of an outbreak to control the spread of epidemics and ensure that the policy combination does not incur high socio-economic costs became the focus of this study.MethodsWe mainly used the fuzzy set qualitative comparative analysis to assess the impact of different combinations of NPIs on the effectiveness of control in the COVID-19 pandemic early stage, using open datasets containing case numbers, country populations and policy responses.ResultsWe showed that the configuration of high morbidity results includes one, which is the combination of non-strict face covering, social isolation and travel restrictions. The configuration of non-high morbidity results includes three, one is strict mask wearing measures, which alone constitute sufficient conditions for interpreting the results; the second is strict testing and contact tracing, social isolation; the third is strict testing and contact tracing, travel restriction. The results of the robustness test showed that the number, components and consistency of the configurations have not changed after changing the minimum case frequency, which proved that the analysis results are reliable.ConclusionIn the early stages of the epidemic, the causes of high morbidity are not symmetrical with the causes of non-high morbidity. Strict face covering is the most basic measure required to prevent and control epidemics, and the combination of non-strict face covering and containment is the most important factor leading to poor prevention and control, and the combination of strict containment and proactive pursuit is the way to achieve superior prevention and control, timely and proactive containment strategies have better prevention and control, and should mobilize the public to cooperate.

The root systems of Brassica species are complex. Eight root system architecture (RSA) traits, including total root length, total root surface area, root average diameter, number of tips, total primary root length, total lateral root length, total tertiary root length, and basal link length, were phenotyped across 379 accessions representing six Brassica species (B. napus, B. juncea, B. carinata, B. oleracea, B. nigra, and B. rapa) using a semi-hydroponic system and image analysis software. The results suggest that, among the assessed species, B. napus and B. oleracea had the most intricate and largest root systems, while B. nigra exhibited the smallest roots. The two species B. juncea and B. carinata shared comparable root system complexity and had root systems with larger root diameters. In addition, 313 of the Brassica accessions were genotyped using a 19K Brassica single nucleotide polymorphism (SNP) array. After filtering by TASSEL 5.0, 6,213 SNP markers, comprising 5,103 markers on the A-genome (covering 302,504 kb) and 1,110 markers on the C-genome (covering 452,764 kb), were selected for genome-wide association studies (GWAS). Two general linear models were tested to identify the genomic regions and SNPs associated with the RSA traits. GWAS identified 79 significant SNP markers associated with the eight RSA traits investigated. These markers were distributed across the 18 chromosomes of B. napus, except for chromosome C06. Sixty-five markers were located on the A-genome, and 14 on the C-genome. Furthermore, the major marker-trait associations (MTAs)/quantitative trait loci (QTLs) associated with root traits were located on chromosomes A02, A03, and A06. Brassica accessions with distinct RSA traits were identified, which could hold functional, adaptive, evolutionary, environmental, pathological, and breeding significance.

Chromosome karyotyping is an important yet labor-intensive procedure for diagnosing genetic diseases. Automating such a procedure drastically reduces the manual work of cytologists and increases congenital disease diagnosing precision. Researchers have contributed to chromosome segmentation and classification for decades. However, very few studies integrate the two tasks as a unified, fully automatic procedure or achieved a promising performance. This paper addresses the gap by presenting: 1) A novel chromosome segmentation module named ChrRender, with the idea of rendering the chromosome instances by combining rich global features from the backbone and coarse mask prediction from Mask R-CNN; 2) A devised chromosome classification module named ChrNet4 that pays more attention to channel-wise dependencies from aggregated informative features and calibrating the channel interdependence; 3) An integrated Render-Attention-Architecture to accomplish fully automatic karyotyping with segmentation and classification modules; 4) A strategy for eliminating differences between training data and segmentation output data to be classified. These proposed methods are implemented in three ways on the public Q-band BioImLab dataset and a G-band private dataset. The results indicate promising performance: 1) on the joint karyotyping task, which predicts a karyotype image by first segmenting an original microscopical image, then classifying each segmentation output with a precision of 89.75% and 94.22% on the BioImLab and private dataset, respectively; 2) On the separate task with two datasets, ChrRender obtained AP50 of 96.652% and 96.809% for segmentation, ChrNet4 achieved 95.24% and 94.07% for classification, respectively. The COCO format annotation files of BioImLab used in this paper are available at https://github.com/Alex17swim/BioImLab The study introduces an integrated workflow to predict a karyotyping image from a Microscopical Chromosome Image. With state-of-the-art performance on a public dataset, the proposed Render-Attention-Architecture has accomplished fully automatic chromosome karyotyping.

In this study to accurately simulate the entire collapse process of steel-reinforced concrete (SRC) building structures subjected to seismic actions, a segment fiber model of SRC components (SRC-SFM) was constructed based on the segment fiber model of the discrete element method (DEM) with the introduction of steel fiber bundles. The internal steel and reinforcement fiber bundles were modeled using a uniaxial steel constitutive model, while the concrete fiber bundles were represented by a uniaxial concrete constitutive model that considered the confinement provided by the steel and stirrups. Subsequently, the hysteretic performance of the SRC components and frames was simulated, and the results indicate that the model can effectively capture their hysteretic behavior. A dynamic elastoplastic analysis was conducted on an SRC structure model, and the results obtained from the discrete element software DEM-COLLAPSE were compared with those generated using the finite element software ABAQUS6.96.9. The results indicate that when the acceleration amplitude is relatively low, the computational results are highly consistent with those of the ABAQUS6.9-SRC structural model. At higher acceleration amplitudes, while some numerical differences are observed, the overall trend of the curves re-mains consistent. This demonstrates that the results of dynamic elastic-plastic analysis obtained from DEM-COLLAPSE are of significant reference value, which lays a solid foundation for the application of DEM-COLLAPSE in subsequent collapse simulations of SRC building structures.

ObjectiveThe aim of this study is to understand the job burnout of village doctors during the COVID-19 epidemic and its influencing factors, and to provide a reference for effectively alleviating the job burnout of village doctors.MethodsA cross-sectional survey was conducted among village doctors in S province in December 2021. The survey included a general information questionnaire and the CMBI Burnout Scale. Epidata was used for dual input, and descriptive analysis, t-test, chi-square test, and binary Logistic regression for statistical analysis were used.ResultsA total of 993 village doctors participated in the survey. Most of them were male village doctors (62.84%), with an average age of 46.57 (SD = 7.50). Village doctors believed that the impact of the epidemic on work was serious, with a score of 3.87 ± 0.91. The economic support was small, with a score of 2.31 ± 0.99. The development space was low, with a score of 2.62 ± 0.98. The overall incidence of burnout was 53.47%. In the burnout group, 54.05% were mild, 33.14% were moderate, and 12.81% were severe. The high degree of difficulty in using WeChat (OR = 1.436, 95%CI: 1.229–1.679), high work pressure (OR = 1.857, 95%CI: 1.409–2.449), high risk of practice (OR = 1.138, 95%CI: 1.004–1.289), less economic support (OR = 0.825, 95%CI: 0.684–0.995), less technical support (OR = 0.696, 95%CI: 0.565–0.858), and poor emotional support (OR = 0.632, 95%CI: 0.513–0.780) were more likely to have job burnout.ConclusionBurnout is a common phenomenon among village doctors during the COVID-19 pandemic, which needs to be prevented and alleviated by various measures.

BackgroundHemorrhagic fever with renal syndrome (HFRS) is a zoonotic infectious disease commonly found in Asia and Europe, characterized by fever, hemorrhage, shock, and renal failure. China is the most severely affected region, necessitating an analysis of the temporal incidence patterns in the country.MethodsWe employed Autoregressive Integrated Moving Average (ARIMA), Long Short-Term Memory (LSTM), Convolutional Neural Network (CNN), Nonlinear AutoRegressive with eXogenous inputs (NARX), and a hybrid CNN-LSTM model to model and forecast time series data spanning from January 2009 to November 2023 in the mainland China. By comparing the simulated performance of these models on training and testing sets, we determined the most suitable model.ResultsOverall, the CNN-LSTM model demonstrated optimal fitting performance (with Root Mean Square Error (RMSE), Mean Absolute Percentage Error (MAPE), and Mean Absolute Error (MAE) of 93.77/270.66, 7.59%/38.96%, and 64.37/189.73 for the training and testing sets, respectively, lower than those of individual CNN or LSTM models).ConclusionThe hybrid CNN-LSTM model seamlessly integrates CNN’s data feature extraction and LSTM’s recurrent prediction capabilities, rendering it theoretically applicable for simulating diverse distributed time series data. We recommend that the CNN-LSTM model be considered as a valuable time series analysis tool for disease prediction by policy-makers.

Abstract Background Migraine, a complex brain disorder, is regarded as a possible clinical manifestation of brain energy dysfunction. The trigeminovascular system is considered the basis for the pathogenesis of migraine, hence we depicted the proteomics profiling of key regions in this system, then focusing on protein alterations related to mitochondrial function. The aim of this study is to illustrate the role of mitochondria in migraine. Methods A mouse model of chronic migraine (CM) was established by repeated nitroglycerin (NTG) stimulation and evaluated by von-Frey filaments, a hot plate and a light-dark box. Differentially expressed proteins (DEPs) in some subcortical brain regions of the trigeminovascular system were screened through liquid chromatography-tandem mass spectrometry (LC‒MS/MS) to analyse the specificity of key signaling pathways in different brain regions. And then mitochondrial function, structure and dynamics were determined by qPCR, ELISA, and transmission electron microscope (TEM). Finally, the effect of mitochondrial intervention-Urolithin A (UA) on CM was investigated. Results Repeated NTG injection triggered photophobia, periorbital and hind paw allodynia in mice. The proteomics profiling of CM model showed that 529, 109, 163, 152 and 419 DEPs were identified in the thalamus, hypothalamus, periaqueductal grey (PAG), trigeminal ganglion (TG) and trigeminocervical complex (TCC), respectively. The most significant changes in the brain region-specific pathways pointed to thalamic mitochondrial impairment. NTG induced mitochondrial structural disruption, dysfunction and homeostatic dysregulation, which could be partially attenuated by UA intervention. Conclusion Our findings highlight the involvement of mitochondrial damage in the thalamus in central sensitization of CM, which provides evidence of possible metabolic mechanisms in migraine pathophysiology.

Abstract Background Bladder cancer (BLCA) is a malignancy that frequently metastasizes and leads to poor patient prognosis. It is essential to understand the molecular mechanisms underlying the progression and metastasis of BLCA and identify potential biomarkers. Methods The expression of peptidase inhibitor 16 (PI16) was analysed using quantitative PCR, immunoblotting and immunohistochemistry assays. The functional roles of PI16 were evaluated using wound healing, transwell, and human umbilical vein endothelial cell tube formation assays, as well as in vivo tumour models. The effects of PI16 on nuclear factor κB (NF-κB) signalling activation were examined using luciferase reporter gene systems, immunoblotting and immunofluorescence assays. Co-immunoprecipitation was used to investigate the interaction of PI16 with annexin-A1 (ANXA1) and NEMO. Results PI16 expression was downregulated in bladder cancer tissues, and lower PI16 levels correlated with disease progression and poor survival in patients with BLCA. Overexpressing PI16 inhibited BLCA cell growth, motility, invasion and angiogenesis in vitro and in vivo, while silencing PI16 had the opposite effects. Mechanistically, PI16 inhibited the activation of the NF-κB pathway by interacting with ANXA1, which inhibited K63 and M1 ubiquitination of NEMO. Conclusions These results indicate that PI16 functions as a tumour suppressor in BLCA by inhibiting tumour growth and metastasis. Additionally, PI16 may serve as a potential biomarker for metastatic BLCA.

Abstract Background Migraine is a highly disabling health burden with multiple symptoms; however, it remains undertreated because of an inadequate understanding of its neural mechanisms. Neuropeptide Y (NPY) has been demonstrated to be involved in the modulation of pain and emotion, and may play a role in migraine pathophysiology. Changes in NPY levels have been found in patients with migraine, but whether and how these changes contribute to migraine is unknown. Therefore, the purpose of this study was to investigate the role of NPY in migraine-like phenotypes. Methods Here, we used intraperitoneal injection of glyceryl trinitrate (GTN, 10 mg/kg) as a migraine mouse model, which was verified by light-aversive test, von Frey test, and elevated plus maze test. We then performed whole-brain imaging with NPY-GFP mice to explore the critical regions where NPY was changed by GTN treatment. Next, we microinjected NPY into the medial habenula (MHb), and further infused Y1 or Y2 receptor agonists into the MHb, respectively, to detect the effects of NPY in GTN-induced migraine-like behaviors. Results GTN effectively triggered allodynia, photophobia, and anxiety-like behaviors in mice. After that, we found a decreased level of GFP+ cells in the MHb of GTN-treated mice. Microinjection of NPY attenuated GTN-induced allodynia and anxiety without affecting photophobia. Furthermore, we found that activation of Y1—but not Y2—receptors attenuated GTN-induced allodynia and anxiety. Conclusions Taken together, our data support that the NPY signaling in the MHb produces analgesic and anxiolytic effects through the Y1 receptor. These findings may provide new insights into novel therapeutic targets for the treatment of migraine. Graphical Abstract

ObjectivesThe proportion of middle-aged and older adult people exposed to the Internet continues to grow. Internet use may have an impact on the mental health of the older adult, especially loneliness. This study analyzed the relationship between Internet use and presence of loneliness.MethodsA total of 550 person aged 45 years and above were randomly selected from a province in eastern China at the end of 2022. The outcome variable was presence of loneliness, as measured by self-report. Descriptive analysis, chi-square test and binary logistic analysis were used to analyze the data.Results58.3% of respondents use the Internet. Internet use could reduce the possible of reported loneliness in middle-aged and older adult people (OR = 0.652, 95%CI: 0.465, 0.940), and residence played a moderating role in the relationship between them. Middle-aged and older adults who used the Internet for 1–3 h (OR = 0.464, 95%CI: 0.275, 0.784) and 3–5 h (OR = 0.484, 95%CI: 0.247, 0.946) were less likely to felt lonely than those who used the Internet for less than 1 h per day. In addition, middle-aged and older adult people using the Internet to contact relatives and friends (OR = 0.488, 95%CI:0.292, 0.818), read the news (OR = 0.485, 95%CI:0.277, 0.848), assets management (OR = 0.297, 95%CI:0.109, 0.818) were less likely to report loneliness, while those who made online payment (OR = 3.101, 95%CI:1.413, 6.807) were more likely to report loneliness.ConclusionThere is a significant negative correlation between Internet use and presence of loneliness, but different Internet duration and content have different effects on loneliness in middle-aged and older adult people. We should pay attention to the impact of Internet use on loneliness in middle-aged and older adult people.

[This corrects the article DOI: 10.1371/journal.pbio.3001134.].

Summary: Medication overuse headache (MOH) is a serious global condition. The interaction between headache attacks and medication overuse complicates the understanding of its pathophysiology. In this study, we developed a preclinical MOH model that incorporates these two key factors by overusing rizatriptan benzoate (RIZ, 4 mg/kg, i.g.) in a glyceryl trinitrate (GTN, 10 mg/kg, i.p.) induced chronic migraine mouse model. We observed that RIZ overuse aggravated GTN-induced cutaneous allodynia and caused a prolonged state of latent sensitization. We also detected a significant upregulation of Annexin-A1 (ANXA1), a protein mainly expressed in the microglia of the spinal trigeminal nucleus caudalis (SPVC), in GTN+RIZ mice. Intracerebroventricular injection of ANXA1-derived peptide Ac2-26 trifluoroacetic acid (TFA) (5 μg/mouse) inhibited bright light stress (BLS) induced acute allodynia via the formyl peptide receptor (FPR) in GTN+RIZ mice. These results suggest that ANXA1 may have an analgesic effect in triptan-associated MOH and could potentially serve as a therapeutic target.

Abstract Background Hair follicle stem cells (HFSCs) are considered as a promising cell type in the stem cell transplantation treatment of neurological diseases because of their rich sources, easy access, and the same ectoderm source as the nervous system. Hepatocyte growth factor (HGF) is a pleiotropic cytokine that shows neuroprotective function in ischemic stroke. Here we assessed the therapeutic effects of HFSCs on ischemic stroke injury and the synthetic effect of HGF along with HFSCs. Methods Rat HFSCs were intravenously transplanted into a middle cerebral artery ischemia/reperfusion (I/R) rat model. Neurological scoring and TTC staining were performed to assess the benefits of HFSC transplantation. Inflammatory cytokines, blood–brain barrier integrity and angiogenesis within penumbra were estimated by Western blot and immunohistochemistry. The differentiation of HFSCs was detected by immunofluorescence method 2 weeks after transplantation. Results HFSC transplantation could significantly inhibit the activation of microglia, improve the integrity of blood–brain barrier and reduce brain edema. Moreover, the number of surviving neurons and microvessels density in the penumbra were upregulated by HFSC transplantation, leading to better neurological score. The combination of HFSCs and HGF could significantly improve the therapeutic benefit. Conclusion Our results indicate for the first time that HGF modified HFSCs can reduce I/R injury and promote the neurological recovery by inhibiting inflammatory response, protecting blood–brain barrier and promoting angiogenesis.

This paper endeavors to identify the causal effects between the smart city transition and the provision of healthcare services while uncovering potential pathways of influence. This study first constructs a logical analytical framework and posits five hypotheses for examination. Subsequently, leveraging the quasi-natural experiment of the China Smart City Pilot Policy (CSCPP), empirical tests are conducted utilizing a Difference-in-Differences (DD) two-way fixed effects model. The findings suggest that the CSCPP has significantly enhanced the provision of healthcare services. Even after addressing the formidable challenges of endogeneity, sample self-selection, and spatial spillovers, the conclusion remains robust. Mechanism tests indicate that the CSCPP primarily operates through two avenues: augmenting human resources and institutional services. Heterogeneity tests reveal that the efficacy of CSCPP is heightened in cities boasting administrative approval service centers, experiencing diminished financial constraints, and exhibiting elevated healthcare provision levels and situated in the eastern region. The theoretical and empirical analysis of this paper demonstrates that smart city transitions can facilitate the enhancement of healthcare services. The potential contribution of this paper is to enrich the conceptualization of governance frameworks for smart city transition while providing empirical evidence from China.

Abstract Omeprazole and sodium bicarbonate dry suspension are effective treatments for acid-related disorders. This study compared the bioequivalence and safety of the two formulations of omeprazole and sodium bicarbonate powder and assessed how CYP2C19 gene polymorphisms affect pharmacokinetics (PK). A single-center, randomized, single-dose, 2-sequence and 2-period crossover method was performed in forty healthy Chinese subjects. Blood samples were collected after a single dose for PK (AUC0–∞, AUC0–t, and Cmax) analysis. The concentrations of Omeprazole in human plasma were determined by HPLC–MS/MS. Besides, the gene polymorphisms of CYP2C19 were assessed by Sanger sequencing. The geometric mean ratios (90% confidence interval) [GMR (95% CI)] of Test/Reference preparation for Cmax: 95.2% (88.48%, 102.43%), AUC0–t: 97.47% (94.4%, 101.02%), AUC0–∞: 97.68% (94.27%, 101.21%) were within the range of 80.00–125.00%. The non-parametric test showed no statistical difference in Tmax between the two groups (p > 0.05). All drugs were well tolerated, no severe adverse reactions occurred, and no significant differences in adverse events between the two drugs. For CYP2C19 gene polymorphisms, the results showed that of 40 subjects, 12 subjects were extensive metabolizers, 24 were intermediate metabolizers, and 4 were poor metabolizers, the frequency of metabolic genotypes were 30%, 60%, and 10%. And the allele distributions for CYP2C19 were *1, *2, and *3 at 60%, 38.75%, and 1.25%. Both the CYP2C19 alleles and metabolic genotypes were consistent with other studies in Chinese. The results of PK parameters showed that different genotypes of CYP2C19 lead to significant differences in t1/2, AUC0–t, AUC0–∞ and Cmax, but no significant differences in Tmax in each group. At the same time, we confirmed that the PK parameters of the test and reference had no differences between the males and females. This study has shown that the pharmacokinetic parameters of the two formulations are not significantly different, which showed bioequivalence and exemplary safety. CYP2C19 gene polymorphism significantly differed in the PK parameters of omeprazole sodium bicarbonate powder.

Background Chronic exposure to elevated levels of saturated fatty acids results in pancreatic β-cell senescence. However, targets and effective agents for preventing stearic acid-induced β-cell senescence are still lacking. Although melatonin administration can protect β-cells against lipotoxicity through anti-senescence processes, the precise underlying mechanisms still need to be explored. Therefore, we investigated the anti-senescence effect of melatonin on stearic acid-treated mouse β-cells and elucidated the possible role of microRNAs in this process. Methods β-Cell senescence was identified by measuring the expression of senescence-related genes and senescence-associated β-galactosidase staining. Gain- and loss-of-function approaches were used to investigate the involvement of microRNAs in stearic acid-evoked β-cell senescence and dysfunction. Bioinformatics analyses and luciferase reporter activity assays were applied to predict the direct targets of microRNAs. Results Long-term exposure to a high concentration of stearic acid-induced senescence and upregulated miR-146a-5p and miR-8114 expression in both mouse islets and β-TC6 cell lines. Melatonin effectively suppressed this process and reduced the levels of these two miRNAs. A remarkable reversibility of stearic acid-induced β-cell senescence and dysfunction was observed after silencing miR-146a-5p and miR-8114. Moreover, V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) was verified as a direct target of miR-146a-5p and miR-8114. Melatonin also significantly ameliorated senescence and dysfunction in miR-146a-5pand miR-8114-transfected β-cells. Conclusion These data demonstrate that melatonin protects against stearic acid-induced β-cell senescence by inhibiting miR-146a- 5p and miR-8114 and upregulating Mafa expression. This not only provides novel targets for preventing stearic acid-induced β-cell dysfunction, but also points to melatonin as a promising drug to combat type 2 diabetes progression.

Highlights 1. We established an optogenetic mouse CSD model without irritation dura, which could mimic the behaviors of headache and photophobia. 2. It was the first time to depict a broad, unbiased neuronal activity map throughout the mouse brain after CSD. 3. Results of whole brain image and immunofluorescence staining showed that optogenetic CSD could activate multiple sensory-related networks without activating thalamus or TNC to induce cortical pain.

Abstract Background Gut microbial dysbiosis and gut-brain axis dysfunction have been implicated in the pathophysiology of migraine. However, it is unclear whether migraine-related cephalic allodynia could induce the alteration of gut microbial composition. Methods A classic migraine rat model was established by repeated dural infusions of inflammatory soup (IS). Periorbital mechanical threshold and nociception-related behaviors were used to evaluate IS-induced cephalic allodynia and the preventive effect of topiramate. The alterations in gut microbial composition and potential metabolic pathways were investigated based on the results of 16 S rRNA gene sequencing. Microbiota-related short-chain fatty acids and tryptophan metabolites were detected and quantified by mass spectrometry analysis. Results Repeated dural IS infusions induced cephalic allodynia (decreased mechanical threshold), migraine-like behaviors (increased immobility time and reduced moving distance), and microbial composition alteration, which were ameliorated by the treatment of topiramate. Decreased Lactobacillus was the most prominent biomarker genus in the IS-induced alteration of microbial composition. Additionally, IS infusions also enhanced metabolic pathways of the gut microbiota in butanoate, propanoate, and tryptophan, while the increased tryptophan-related metabolites indole-3-acetamide and tryptophol in feces could be the indicators. Conclusions Inflammatory dural stimulation-induced cephalic allodynia causes the alterations of gut microbiota profile and microbial metabolic pathways.

The etiology of numerous metabolic disorders is characterized by hepatic insulin resistance (IR). Uncertainty surrounds miR-34a’s contribution to high-fat-induced hepatic IR and its probable mechanism. The role and mechanism of miR-34a and its target gene ENO3 in high-fat-induced hepatic IR were explored by overexpressing/suppressing miR-34a and ENO3 levels in in vivo and in vitro experiments. Moreover, as a human hepatic IR model, the miR-34a/ENO3 pathway was validated in patients with non-alcoholic fatty liver disease (NAFLD). The overexpression of hepatic miR-34a lowered insulin signaling and altered glucose metabolism in hepatocytes. In contrast, reducing miR-34a expression significantly reversed hepatic IR indices induced by palmitic acid (PA)/HFD. ENO3 was identified as a direct target gene of miR-34a. Overexpression of ENO3 effectively inhibited high-fat-induced hepatic IR-related indices both in vitro and in vivo. Moreover, the expression patterns of members of the miR-34a/ENO3 pathway in the liver tissues of NAFLD patients was in line with the findings of both cellular and animal studies. A high-fat-induced increase in hepatic miR-34a levels attenuates insulin signaling and impairs glucose metabolism by suppressing the expression of its target gene ENO3, ultimately leading to hepatic IR. The miR-34a/ENO3 pathway may be a potential therapeutic target for hepatic IR and related metabolic diseases.

Background: A long-term consumption of saturated fat significantly increases the concentration of saturated fatty acids in serum, which accelerates the appearance of senescence markers in β-cells and leads to their dysfunction. An understanding of the mechanisms underlying β-cell senescence induced by stearic acid and the exploration of effective agents preventing it remains largely unclear. Here, we aimed to investigate the protective effect of metformin against stearic acid-treated β-cell senescence and to assess the involvement of miR-297b-5p in this process. Methods: To identify senescence, we measured senescence-associated β-galactosidase activity and the expression of senescence-related genes. Gain and loss of function approaches were applied to explore the role of miR-297b-5p in stearic acid-induced β-cell senescence. Bioinformatics analysis and a luciferase activity assay were used to predict the downstream targets of miR-297b-5p. Results: Stearic acid markedly induced senescence and suppressed miR-297b-5p expression in mouse β-TC6 cells, which were significantly alleviated by metformin. After transfection of miR-297b-5p mimics, stearic acid-evoked β-cell senescence was remarkably prevented. Insulin-like growth factor-1 receptor was identified as a direct target of miR-297b-5p. Inhibition of the insulin-like growth factor-1 receptor prevented stearic acid-induced β-cell senescence and dysfunction. Moreover, metformin alleviates the impairment of the miR-297b-5p inhibitor in β-TC6 cells. Additionally, long-term consumption of a high-stearic-acid diet significantly increased senescence and reduced miR-297b-5p expression in mouse islets. Conclusions: These findings imply that metformin alleviates β-cell senescence by stearic acid through upregulating miR-297b-5p to suppress insulin-like growth factor-1 receptor expression, thereby providing a potential target to not only prevent high fat-diet-induced β-cell dysfunction but also for metformin therapy in type 2 diabetes.

BackgroundIntestinal microbiota has been confirmed to influencing the pharmacokinetic processes of a variety of oral drugs. However, the pharmacokinetic effects of the gut microbiota on cyclosporine A, a drug with a narrow therapeutic window, remain to be studied.MethodTwenty-one rats were randomly divided into three groups: (a) control group (CON), (b) antibiotic treatment group (ABT) and (c) fecal microbe transplantation group (FMT). The ABT group was administrated with water containing multiple antibiotics to deplete microorganisms. FMT was with the same treatment, followed by oral administration of conventional rat fecal microorganisms for normalization.ResultThe bioavailability of CSA increased by 155.6% after intestinal microbes were consumed by antibiotics. After intestinal microbiota reconstruction by fecal transplantation, the increased bioavailability was significantly reduced and basically returned to the control group level. Changes in gut microbiota alter the protein expression of CYP3A1, UGT1A1 and P-gp in liver. The expressions of these three proteins in ABT group were significantly lower than those in CON and FMT groups. The relative abundance of Alloprevolleta and Oscillospiraceae UCG 005 was negatively correlated with CSA bioavailability while the relative abundance of Parasutterella and Eubacterium xylanophilum group was negatively correlated with CSA bioavailability.ConclusionIntestinal microbiota affects the pharmacokinetics of CSA by regulating the expression of CYP3A1, UGT1A1 and P-GP.

Henosepilachna vigintioctopunctata is an emerging horticultural pest of Solanaceae plants in Asia. Here, we employed transcriptome sequencing of three female and three male H. vigintioctopunctata libraries to identify sex-related genes. We recorded 281 599 294 bp of clean reads, and de novo assembly generated 85 206 unigenes, with N50 of 1 311 bp and average length of 763 bp. We found 5 002 genes highly expressed in males and 2 179 genes highly expressed in females. Expression profiles of six unigenes specific to females, six unigenes specific to males, and ten unigenes common to both sexes were verified using semi-quantitive RT-PCR; results showed expression levels of these 22 unigenes were relatively consistent with the transcriptome analysis. Using all unigenes as references, we identified 6 657 putative simple sequence repeats. The transcriptome sequences and gene expression profiles of female and male H. vigintioctopunctata help explore genetic mechanisms of sexual dimorphism.

miR-532-3p is a widely documented microRNA (miRNA) involved in multifaceted processes of cancer tumorigenesis and metastasis. However, the clinical significance and biological functions of miR-532-3p in bone metastasis of prostate cancer (PCa) remain largely unknown. Herein, we report that miR-532-3p was downregulated in PCa tissues with bone metastasis, and downexpression of miR-532-3p was significantly associated with Gleason grade and serum prostate-specific antigen (PSA) levels and predicted poor bone metastasis-free survival in PCa patients. Upregulating miR-532-3p inhibited invasion and migration abilities of PCa cells in vitro, while silencing miR-532-3p yielded an opposite effect on invasion and migration abilities of PCa cells. Importantly, upregulating miR-532-3p repressed bone metastasis of PCa cells in vivo. Our results further demonstrated that overexpression of miR-532-3p inhibited activation of nuclear facto κB (NF-κB) signaling via simultaneously targeting tumor necrosis factor receptor-associated factor 1 (TRAF1), TRAF2, and TRAF4, which further promoted invasion, migration, and bone metastasis of PCa cells. Therefore, our findings reveal a novel mechanism contributing to the sustained activity of NF-κB signaling underlying the bone metastasis of PCa.

The prime issue derived from prostate cancer (PCa) is its high prevalence to metastasize to bone. MicroRNA-204-5p (miR-204-5p) has been reported to be involved in the development and metastasis in a variety of cancers. However, the clinical significance and biological functions of miR-204-5p in bone metastasis of PCa are still not reported yet. In this study, we find that miR-204-5p expression is reduced in PCa tissues and serum sample with bone metastasis compared with that in PCa tissues and serum sample without bone metastasis, which is associated with advanced clinicopathological characteristics and poor bone metastasis-free survival in PCa patients. Moreover, upregulation of miR-204-5p inhibits the migration and invasion of PCa cells in vitro, and importantly, upregulating miR-204-5p represses bone metastasis of PCa cells in vivo. Our results further demonstrated that miR-204-5p suppresses invasion, migration, and bone metastasis of PCa cells via inactivating nuclear factor κB (NF-κB) signaling by simultaneously targeting TRAF1, TAB3, and MAP3K3. In clinical PCa samples, miR-204-5p expression negatively correlates with TRAF1, TAB3, and MAP3K3 expression and NF-κB signaling activity. Therefore, our findings reveal a new mechanism underpinning the bone metastasis of PCa, as well as provide evidence that miR-204-5p might serve as a novel serum biomarker in bone metastasis of PCa.This study identifies a novel functional role of miR-204-5p in bone metastasis of prostate cancer and supports the potential clinical value of miR-204-5p as a serum biomarker in bone metastasis of PCa.

Fluralaner, a systemic pesticide, was originally registered with the US Food and Drug Administration in 2014 under the trade name Bravecto for flea treatment for pets. As a GABA antagonist, the footprint of fluralaner has expended beyond medical and veterinary pests in recent years. In this study, we examined the acute toxicity of fluralaner against three pests of Henosepilachna vigintioctopunctata, Megalurothrips usitatus, and Phyllotreta striolata in the Solanaceae, Fabaceae, and Cruciferae families, respectively, and the sublethal impact of fluralaner on Propylaea japonica, a widely distributed predatory ladybeetle. Based on LC50, fluralaner was effective against H. vigintioctopunctata (0.098 mg a.i. L−1 for the second instar larvae), M. usitatus (0.134 mg a.i. L−1 for adult females), and P. striolata (0.595 mg a.i. L−1 for adults). For P. japonica, however, fluralaner was substantially less effective (1.177 mg a.i. L−1 for the third instar larvae). Furthermore, the LC10 and LC30 of P. japonica were also consistently higher than the LC50 of the three pests. In addition, we did not observe any significant impacts of fluralaner at LC10 and LC30 on the life history traits, including body weight, developmental time, pre-oviposition period, and fecundity of P. japonica. Based on our results from acute toxicities and sublethal impacts, fluralaner is effective against vegetable pests, while potentially friendly to P. japonica when employed as a biological control agent.

Abstract This paper considers the Cauchy problem for fast diffusion equation with nonlocal source ut=Δum+(∫Rnuq(x,t)dx)p−1qur+1 $u_{t}=\Delta u^{m}+ (\int_{\mathbb{R}^{n}}u^{q}(x,t)\,dx )^{\frac{p-1}{q}}u^{r+1}$, which was raised in [Galaktionov et al. in Nonlinear Anal. 34:1005–1027, 1998]. We give the critical Fujita exponent pc=m+2q−n(1−m)−nqrn(q−1) $p_{c}=m+\frac{2q-n(1-m)-nqr}{n(q-1)}$, namely, any solution of the problem blows up in finite time whenever 1pc $p>p_{c}$.

A number of studies have reported that aberrant expression of microRNAs (miRNAs) closely correlates with the bone metastasis of prostate cancer (PCa). However, clinical significance and functional roles of both strands of a single miRNA in bone metastasis of PCa remain undefined. Here, we reported that miR-582-3p and miR-582-5p expression were simultaneously reduced in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Downexpression of miR-582-3p and miR-582-5p strongly and positively correlated with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients. Upregulating miR-582-3p and miR-582-5p inhibited invasion and migration abilities of PCa cells in vitro, as well as repressed bone metastasis in vivo. Our results further revealed that miR-582-3p and miR-582-5p attenuated bone metastasis of PCa via inhibiting transforming growth factor β (TGF-β) signaling by simultaneously targeting several components of TGF-β signaling, including SMAD2, SMAD4, TGF-β receptor I (TGFBRI), and TGFBRII. Moreover, deletion contributes to miR-582-3p and miR-582-5p downexpression in PCa tissues. Finally, clinical negative correlations of miR-582-3p and miR-582-5p with SMAD2, SMAD4, TGFBRI, and TGFBRII were demonstrated in PCa tissues. Thus, our findings explore a novel tumor-suppressive miRNA with its both strands implicated in bone metastasis of PCa, suggesting its potential therapeutic value in treatment of PCa bone metastasis.

Background: Acute ischemic stroke (AIS) is the main cause of worldwide death and disability. Early neurological deterioration (END) can further increase the probability of death and disability in patients with ischemic stroke. Therefore, it is essential to find biomarkers to predict END early. Inflammatory response plays a crucial role in determining the course, outcome, and prognosis of END. Earlier studies focused on the relationship between routine hematological inflammatory markers and END, which limited the results. At present, relatively new and comprehensive markers of inflammatory response are relatively scarce. In this study, we investigate the predictive value of inflammatory markers in acute ischemic stroke cases for END which include systemic inflammatory response index (SIRI), platelet/lymphocyte ratio (PLR), lymphocyte/monocyte ratio (LMR), neutrophil/lymphocyte ratio (NLR), and then to establish a nomogram model. Methods: A total of 375 patients with AIS were analyzed who were admitted to the Second Affiliated Hospital of Harbin Medical University from September 2019 to June 2021. The associations between END and inflammatory markers were studied by employing the analysis of univariate. Following that, through regression models of the least absolute shrinkage and selection operator, the END risk model’s feature selection was optimized. The development of the model of prediction was carried out by applying the multivariable logistic regression analysis. The calibration, discrimination, and clinical efficacy of the prediction model were studied via calibration plot, C-index, and decision curve analysis (DCA). The bootstrapping validation method was used for the evaluation of internal validation. Results: We constructed a nomogram consisting of CRP, monocytes, NIHSS and SIRI. This model had desirable calibration and discrimination, with a C-index of 0.757 (95% confidence interval: 0.702–0.805). Interval validation could still achieve the higher C-index value of 0.747. When the risk threshold for END was greater than 13% but less than 84%, DCA proved to be clinically useful. Conclusions: Our research shows that SIRI can be used as a new predictor of END, as well as a monitor of treatment response. Compared with the traditional single inflammatory indicator, the integration of SIRI nomogram can predict the occurrence of END more objectively and reliably.

Cell death is a vital event in life. Infections and injuries cause lytic cell death, which gives rise to danger signals that can further induce cell death, inflammation, and tissue damage. The mevalonate (MVA) pathway is an essential, highly conserved and dynamic metabolic pathway. Here, we discover that farnesyl pyrophosphate (FPP), a metabolic intermediate of the MVA pathway, functions as a newly identified danger signal to trigger acute cell death leading to neuron loss in stroke. Harboring both a hydrophobic 15-carbon isoprenyl chain and a heavily charged pyrophosphate head, FPP leads to acute cell death independent of its downstream metabolic pathways. Mechanistically, extracellular calcium influx and the cation channel transient receptor potential melastatin 2 (TRPM2) exhibit essential roles in FPP-induced cell death. FPP activates TRPM2 opening for ion influx. Furthermore, in terms of a mouse model constructing by middle cerebral artery occlusion (MCAO), FPP accumulates in the brain, which indicates the function of the FPP and TRPM2 danger signal axis in ischemic injury. Overall, our data have revealed a novel function of the MVA pathway intermediate metabolite FPP as a danger signal via transient receptor potential cation channels.

Abstract This paper considers the following coupled chemotaxis system: { u t = ∇ ⋅ ( ϕ ( u ) ∇ u ) − χ 1 ∇ ⋅ ( u ∇ w ) + μ 1 u ( 1 − u − a 1 v ) , v t = ∇ ⋅ ( ψ ( v ) ∇ v ) − χ 2 ∇ ⋅ ( v ∇ w ) + μ 2 v ( 1 − a 2 u − v ) , w t = Δ w − γ w + α u + β v , $$\textstyle\begin{cases} u_{t}=\nabla\cdot(\phi(u)\nabla u)-\chi_{1} \nabla\cdot(u\nabla w)+\mu_{1} u(1-u-a_{1} v), \\ v_{t}=\nabla\cdot(\psi(v)\nabla v)-\chi_{2} \nabla\cdot(v\nabla w)+\mu_{2} v(1-a_{2}u-v), \\ w_{t}=\Delta w-\gamma w+\alpha u+\beta v, \end{cases} $$ with homogeneous Neumann boundary conditions in a bounded domain Ω ⊂ R N $\Omega\subset\mathbb{R}^{N}$ ( N ≥ 3 $N\ge3$ ) with smooth boundaries, where χ 1 $\chi_{1}$ , χ 2 $\chi_{2}$ , μ 1 $\mu_{1}$ , μ 2 $\mu_{2}$ , a 1 $a_{1}$ , a 2 $a_{2}$ , α, β and γ are positive. Based on the maximal Sobolev regularity, the existence of a unique global bounded classical solution of the problem is established under the assumption that both μ 1 $\mu_{1}$ and μ 2 $\mu_{2}$ are sufficiently large.

The gut bacteria of insects positively influence the physiology of their host, however, the dynamics of this complicated ecosystem are not fully clear. To improve our understanding, we characterized the gut prokaryotic of Henosepilachna vigintioctopunctata that fed on two host plants, Solanum melongena (referred to as QZ hereafter) and Solanum nigrum (referred to as LK hereafter), by sequencing the V3-V4 hypervariable region of the 16S rRNA gene using the Illumina MiSeq system. The results revealed that the gut bacterial composition varied between specimens that fed on different host plants. The unweighted pair group method with arithmetic mean analyses and principal coordinate analysis showed that the bacterial communities of the LK and QZ groups were distinct. Four phyla (Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria) were present in all H. vigintioctopunctata gut samples. It is noteworthy that bacteria of the phylum Cyanobacteria were only found in the LK group, with a low relative abundance. Proteobacteria and Enterobacteriaceae were the predominant phylum and family, respectively, in both the LK and QZ groups. Linear discriminant analysis effect size (LEfSe) analyses showed that the QZ group enriched the Bacilli class and Lactococcus genus; while the LK group enriched the Alphaproteobacteria class and Ochrobactrum genus. PICRUSt analysis showed that genes predicted to be involved in xenobiotic biodegradation and metabolism, metabolism of other amino acids, signaling molecules, and interaction were significantly higher in the QZ group. Genes predicted to be involved in the metabolism of cofactors and vitamins were significantly higher in the LK group. Furthermore, the complexity of the network structure and the modularity were higher in the LK group than in the QZ group. This is the first study to characterize the gut bacteria of H. vigintioctopunctat, our results demonstrate that the two host plants tested had a considerable impact on bacterial composition in the gut of H. vigintioctopunctata and that the bacterial communities were dominated by relatively few taxa.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying expression levels of targeted genes during various biological processes in numerous areas of clinical and biological research. Selection of appropriate reference genes for RT-qPCR normalization is an elementary prerequisite for reliable measurements of gene expression levels. Here, by analyzing datasets published between 2008 and 2017, we summarized the current trends in reference gene selection for insect gene expression studies that employed the most widely used SYBR Green method for RT-qPCR normalization. We curated 90 representative papers, mainly published in 2013–2017, in which a total of 78 insect species were investigated in 100 experiments. Furthermore, top five journals, top 10 frequently used reference genes, and top 10 experimental factors have been determined. The relationships between the numbers of the reference genes, experimental factors, analysis tools on the one hand and publication date (year) on the other hand was investigated by linear regression. We found that the more recently the paper was published, the more experimental factors it tended to explore, and more analysis tools it used. However, linear regression analysis did not reveal a significant correlation between the number of reference genes and the study publication date. Taken together, this meta-analysis will be of great help to researchers that plan gene expression studies in insects, especially the non-model ones, as it provides a summary of appropriate reference genes for expression studies, considers the optimal number of reference genes, and reviews the average number of experimental factors and analysis tools per study.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a momentous technique for quantifying expression levels of the targeted genes across various biological processes. Selection and validation of appropriate reference genes for RT-qPCR analysis are a pivotal precondition for reliable expression measurement. Henosepilachna vigintioctopunctata is one of the most serious insect pests that attack Solanaceae plants in Asian countries. Recently, the transcriptomes of H. vigintioctopunctata were sequenced, promoting gene functional studies of this insect pest. Unfortunately, the reference genes for H. vigintioctopunctata have not been selected and validated. Here, a total of 7 commonly used reference genes, namely, Actin, GAPDH, RPL13, RPL6, RPL32, RPS18, and ATPB, were selected and assessed for suitability under four experimental conditions, namely, developmental stage, tissue, temperature, and host plant, using RefFinder, which integrates four different analytical tools (Normfinder, geNorm, the ΔCt method, and BestKeeper). The results displayed that RPL13 and RPS18 were the best suitable reference genes for each experimental condition. The relative transcript levels of 2 target genes, lov and TBX1, varied greatly according to normalization with the two most- and least-suited reference genes. Our results will be helpful for improving the accuracy of the RT-qPCR analysis for future functional investigations of target gene expression in H. vigintioctopunctata.

Asian citrus psyllid Diaphorina citri Kuwayama is an important economic pest of citrus, as it transmits Candidatus Liberibacter asiaticus, the causative agent of huanglongbing. In this study, we used RNA-seq to identify novel genes and provide the first high-resolution view of the of D. citri transcriptome throughout development. The transcriptomes of D. citri during eight developmental stages, including the egg, five instars, and male and female adults were sequenced. In total, 115 million clean reads were obtained and assembled into 354,726 unigenes with an average length of 925.65 bp and an N50 length of 1733 bp. Clusters of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to functionally annotate the genes. Differential expression analysis highlighted developmental stage-specific expression patterns. Furthermore, two trehalase genes were characterized with lower expression in adults compared to that in the other stages. The RNA interference (RNAi)-mediated suppression of the two trehalase genes resulted in significantly high D. citri mortality. This study enriched the genomic information regarding D. citri. Importantly, these data represent the most comprehensive transcriptomic resource currently available for D. citri and will facilitate functional genomics studies of this notorious pest.

RNA interference (RNAi) techniques have emerged as powerful tools in the development of novel management strategies for the control of insect pests, such as Henosepilachna vigintioctopunctata, which is a major solanaceous pest in Asia. Our results showed that levels of HvSnf7 expression were greater in larval midguts than in other tissues. Silencing of HvSnf7 led to greater H. vigintioctopunctata mortality rates and appeared to be time- and partially dose-dependent. Bacterially expressed dsHvSnf7 that was applied to detached plant leaves caused 98, 88, and 60% mortality in 1st and 3rd instars, and adults after 10, 12, and 14 d, respectively; when applied to living plants, bacterially expressed dsHvSnf7 led to mortality in 1st and 3rd instars, with no effect on adults. Bacterially expressed dsHvSnf7 led to improved plant protection against H. vigintioctopunctata. Ultrastructural changes caused by HvSnf7-RNAi in larval midguts showed extensive loss of cellular contents that indicate loss of membrane integrity. This study indicate that HvSnf7 potentially can be used as RNAi target gene for controlling of H. vigintioctopunctata.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique commonly used in molecular biology to analyze RNA expression. The selection of suitable reference genes for data normalization is a precondition for credible measurements of gene expression levels using RT-qPCR. Propylea japonica is one of the most common pests of many crop systems throughout East Asia, and has often been used in the testing of non-target impacts during environmental risk assessments of genetically engineered plants. The present study assessed the suitability of nine frequently used reference genes for comparisons of P. japonica gene expression. Expression stability was compared across developmental stages, sex, a range of tissues, and following exposure to different temperatures. Data were analyzed using RefFinder, which integrated the results obtained using NormFinder, geNorm, BestKeeper, and the ΔCt method. This led to the identification of unique sets of reference genes for each experimental condition: ribosomal protein S18 (RPS18) and elongation factor 1 α (EF1A) for developmental stage comparisons, RPS18 and EF1A for sex comparisons, EF1A and ribosomal protein L4 for tissue comparisons, and RPS18 and EF1A for analyses of temperature-mediated effects. These reference genes will help to enhance the accuracy of RT-qPCR analyses of P. japonica gene expression. This work represents an initial move towards building a standardized system for RT-qPCR analysis of P. japonica, providing a basis for the ecological risk assessment of RNAi-based insect control products.

The development of genetically-engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is RT-qPCR, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata, a commonly-used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper, the ΔCt method, and RefFinder, were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended respectively for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata, our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants.

Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae) is a major pest affecting Solanaceae plants in Asian countries. In this study, we sequenced the ovary and testis transcriptomes of H. vigintioctopunctata to identify gonad-related genes. Comparison of the unigene sequences in ovary and testis libraries identified 1,421 and 5,315 ovary- and testis-specific genes, respectively. Among the ovary-specific genes, we selected the RC2-like and PSHS-like genes to investigate the effects of gene silencing on the mortality, percentage infertility, pre-oviposition period, fecundity, daily number of eggs laid, and hatching rate in female adults. Although the percentage mortality and infertility of females did not differ significantly among dsRNA treatments, fecundity was significantly reduced in the dsRC2-like and dsPSHS-like treatment groups. Moreover, the pre-oviposition period was markedly prolonged in response to dsPSHS-like treatment. This is the first reported RNA sequencing of H. vigintioctopunctata. The transcriptome sequences and gene expression profiles of the ovary and testis libraries will provide useful information for the identification of gonad-related genes in H. vigintioctopunctata and facilitate further research on the reproductive biology of this species. Moreover, the gonad-specific genes identified may represent candidate target genes for inhibiting the population growth of H. vigintioctopunctata.

BACKGROUND/OBJECTIVE:RAC1 gene could influence susceptibility to renal failure by altering the activity and expression of Rac1, which is a member of the Rho family of small GTP-binding proteins. In clinical practice, renal transplantation provides the optimal treatment for people with end-stage renal disease (ESRD). The objective of this present study was to determine whether the RAC1 gene polymorphisms were associated with primary ESRD susceptibility in Chinese renal recipients. METHODS:Six single nucleotide polymorphisms (SNPs) of RAC1 gene, including rs836488 T>C, rs702482 A>T, rs10951982 G>A, rs702483 A>G, rs6954996 G>A, and rs9374 G>A, were genotyped in 300 renal transplant recipients (cases) and 998 healthy Chinese subjects (controls) by using TaqMan SNP genotyping assay. Allele, genotype, and haplotype frequencies of the six SNPs were compared between cases and controls. Odds ratios (OR) and 95% confidence intervals (CI) were calculated in logistic regression models to evaluate the associations of the six SNPs with ESRD risk. RESULTS:The genotype distributions for the six SNPs in controls were consistent with Hardy-Weinberg equilibrium (P > 0.05). Association analysis revealed that three SNPs were significantly associated with ESRD risk. Positive associations with ESRD risk were found for the rs836488, rs702482, and rs702483 in the co-dominant model (minor allele homozygotes versus major allele homozygotes); specifically, the frequencies of the minor allele homozygotes and the minor allele for the three SNPs were higher in the cases than in the controls. In addition, these three SNPs also had associations with increased ESRD risk under the additive model (P < 0.05), and positive associations were also found for the rs836488 in the dominant model (P < 0.05) and for the rs702483 in the recessive model (P < 0.05). All these associations were independent of confounding factors. The other three SNPs (rs10951982, rs6954996, and rs9374), in all comparison models, were not associated with ESRD risk (P > 0.05). In haplotype analysis, carriers with "C-T-G-G-G-G" haplotype had a significantly higher risk of ESRD compared with the most common haplotype "T-A-G-A-G-G" (P = 0.011, OR = 1.46, 95% CI = 1.09-1.94). CONCLUSION:This study suggested that polymorphisms of RAC1 gene might influence the susceptibility to ESRD in Chinese Han population. Further studies are necessary to confirm our findings.

Neural stem cells are self-renewing, multipotent and undifferentiated precursors that retain the capacity for differentiation into both glial (astrocytes and oligodendrocytes) and neuronal lineages. Neural stem cells offer cell-based therapies for neurological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease and spinal cord injuries. However, their cellular behavior is poorly understood. MicroRNAs (miRNAs) are a class of small noncoding RNAs involved in cell development, proliferation and differentiation through regulating gene expression at post-transcriptional level. The role of miR-381 in the development of neural stem cells remains unknown. In this study, we showed that overexpression of miR-381 promoted neural stem cells proliferation. It induced the neural stem cells differentiation to neurons and inhibited their differentiation to astrocytes. Furthermore, we identified HES1 as a direct target of miR-381 in neural stem cells. Moreover, re-expression of HES1 impaired miR-381-induced promotion of neural stem cells proliferation and induce neural stem cells differentiation to neurons. In conclusion, miR-381 played important role in neural stem cells proliferation and differentiation.

Quantitative real-time PCR (qRT-PCR) is a powerful technique to quantify gene expression. To standardize gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to consistently expressed housekeeping genes (HKGs) is required. In this study, ten candidate HKGs including elongation factor 1 α (EF1A), ribosomal protein L11 (RPL11), ribosomal protein L14 (RPL14), ribosomal protein S8 (RPS8), ribosomal protein S23 (RPS23), NADH-ubiquinone oxidoreductase (NADH), vacuolar-type H+-ATPase (ATPase), heat shock protein 70 (HSP70), 18S ribosomal RNA (18S), and 12S ribosomal RNA (12S) from the cowpea aphid, Aphis craccivora Koch were selected. Four algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method were employed to evaluate the expression profiles of these HKGs as endogenous controls across different developmental stages and temperature regimes. Based on RefFinder, which integrates all four analytical algorithms to compare and rank the candidate HKGs, RPS8, RPL14, and RPL11 were the three most stable HKGs across different developmental stages and temperature conditions. This study is the first step to establish a standardized qRT-PCR analysis in A. craccivora following the MIQE guideline. Results from this study lay a foundation for the genomics and functional genomics research in this sap-sucking insect pest with substantial economic impact.

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring mRNA expression. To facilitate gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to stable housekeeping genes is mandatory. In this study, ten housekeeping genes, including beta-actin (Actin) , elongation factor 1 α (EF1A) , glyceralde hyde-3-phosphate dehydrogenase (GAPDH) , ribosomal protein L13 (RPL13) , ribosomal protein 49 (RP49) , α-tubulin (Tubulin) , vacuolar-type H+-ATPase (v-ATPase) , succinate dehydrogenase subunit A (SDHA) , 28S ribosomal RNA (28S) , and 18S ribosomal RNA (18S) from the two-spotted spider mite, Tetranychus urticae, were selected as the candidate reference genes. Four algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the performance of these candidates as endogenous controls across different developmental stages. In addition, RefFinder, which integrates the above-mentioned software tools, provided the overall ranking of the stability/suitability of these candidate reference genes. Among them, PRL13 and v-ATPase were the two most stable housekeeping genes across different developmental stages. This work is the first step toward establishing a standardized qRT-PCR analysis in T. urticae following the MIQE guideline. With the recent release of the T. urticae genome, results from this study provide a critical piece for the subsequent genomics and functional genomics research in this emerging model system.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for measuring and evaluating gene expression during variable biological processes. To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the main vector of tomato spotted wilt virus (TSWV), is a destructive invasive species. In this study, the expression profiles of 11 candidate reference genes from nonviruliferous and viruliferous F. occidentalis were investigated. Five distinct algorithms, geNorm, NormFinder, BestKeeper, the ΔCt method, and RefFinder, were used to determine the performance of these genes. geNorm, NormFinder, BestKeeper, and RefFinder identified heat shock protein 70 (HSP70), heat shock protein 60 (HSP60), elongation factor 1 α, and ribosomal protein l32 (RPL32) as the most stable reference genes, and the ΔCt method identified HSP60, HSP70, RPL32, and heat shock protein 90 as the most stable reference genes. Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis. This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions.