The construction of a nomogram to predict the prognosis and recurrence risks of UPJO.

Objective: This study was conducted to explore the risk factors for the prognosis and recurrence of ureteropelvic junction obstruction (UPJO). Methods: The correlation of these variables with the prognosis and recurrence risks was analyzed by binary and multivariate logistic regression. Besides, a nomogram was constructed based on the multivariate logistic regression calculation. After the model was verified by the C-statistic, the ROC curve was plotted to evaluate the sensitivity of the model. Finally, the decision curve analysis (DCA) was conducted to estimate the clinical benefits and losses of intervention measures under a series of risk thresholds. Results: Preoperative automated peritoneal dialysis (APD), preoperative urinary tract infection (UTI), preoperative renal parenchymal thickness (RPT), Mayo adhesive probability (MAP) score, and surgeon proficiency were the high-risk factors for the prognosis and recurrence of UPJO. In addition, a nomogram was constructed based on the above 5 variables. The area under the curve (AUC) was 0.8831 after self cross-validation, which validated that the specificity of the model was favorable. Conclusion: The column chart constructed by five factors has good predictive ability for the prognosis and recurrence of UPJO, which may provide more reasonable guidance for the clinical diagnosis and treatment of this disease.

Identification and analysis of MSC-Exo-derived LncRNAs related to the regulation of EMT in hypospadias.

OBJECTIVE: This study aims to screen the differentially expressed long non-coding RNAs (DELncRNAs) related to the regulation of epithelial-mesenchymal transition (EMT) in hypospadias in mesenchymal stem cell-derived exosomes (MSC-Exons) and explore the potential mechanism of these lncRNAs for the EMT in hypospadias. METHODS: In this study, the microarray data related to MSC-Exos and hypospadias were downloaded from Gene Expression Omnibus (GEO). Besides, the lncRNAs highly expressed in MSC-Exos and the differentially expressed mRNAs and lncRNAs in children with hypospadias were screened, respectively. In addition, the lncRNAs enriched in MSC-Exos and differentially expressed lncRNAs in hypospadias were intersected to obtain the final DElncRNAs. Moreover, the co-expression interaction pairs of differentially expressed lncRNAs and mRNAs were analyzed to construct a Competing Endogenous RNA (ceRNA) network. Finally, the candidate lncRNAs in exosomes were subjected to in vitro cell function verification. RESULTS: In this study, a total of 4 lncRNAs were obtained from the microarray data analysis. Further, a ceRNA regulatory network of MSC-Exo-derived lncRNAs related to the regulation of EMT in hypospadias was constructed, including 4 lncRNAs, 2 mRNAs, and 6 miRNAs. The cell function verification results indicated that the exosomes secreted by MSCs may transport HLA complex group 18 (HCG18) into target cells, which promoted the proliferation, migration, and EMT of these cells. CONCLUSION: MSC-Exo-derived lncRNA HCG18 can enter target cells, and it may be involved in the regulation of EMT in hypospadias through the ceRNA network.

Nephroblastoma-specific dysregulated gene SNHG15 with prognostic significance: scRNA-Seq with bulk RNA-Seq data and experimental validation.

Wilms tumor (WT) is the most common malignancy of the genitourinary system in children. Currently, the Integration of single-cell RNA sequencing (scRNA-Seq) and Bulk RNA sequencing (RNA-Seq) analysis of heterogeneity between different cell types in pediatric WT tissues could more accurately find prognostic markers, but this is lacking. RNA-Seq and clinical data related to WT were downloaded from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. Small nucleolar RNA host gene 15 (SNHG15) was identified as a risk signature from the TARGET dataset by using weighted gene co-expression network analysis, differentially expressed analysis and univariate Cox analysis. After that, the functional mechanisms, immunological and molecular characterization of SNHG15 were investigated at the scRNA-seq, pan-cancer, and RNA-seq levels using Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), ESTIMATE, and CIBERSORT. Based on scRNA-seq data, we identified 20 clusters in WT and annotated 10 cell types. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing M2 macrophages as hubs for intercellular communication. In addition, in vitro cellular experiments showed that siRNAs interfering with SNHG15 significantly inhibited the proliferation and migration of G401 cells and promoted the apoptosis of G401 cells compared with the control group. The effect of siRNAs interfering with SNHG15 on EMT-related protein expression was verified by Western blotting assay. Thus, our findings will improve our current understanding of the pathogenesis of WT, and they are potentially valuable in providing novel prognosis markers for the treatment of WT.

Analysis of factors influencing the efficacy of vagus nerve stimulation for the treatment of drug-resistant epilepsy in children and prediction model for efficacy evaluation.

Objective: Vagus nerve stimulation (VNS) has been widely used in the treatment of drug-resistant epilepsy (DRE) in children. We aimed to explore the efficacy and safety of VNS, focusing on factors that can influence the efficacy of VNS, and construct a prediction model for the efficacy of VNS in the treatment of DRE children. Methods: Retrospectively analyzed 45 DRE children who underwent VNS at Qilu Hospital of Shandong University from June 2016 to November 2022. A ≥50% reduction in seizure frequency was defined as responder, logistic regression analyses were performed to analyze factors affecting the efficacy of VNS, and a predictive model was constructed. The predictive model was evaluated by receiver operating characteristic curve (ROC), calibration curves, and decision curve analyses (DCA). Results: A total of 45 DRE children were included in this study, and the frequency of seizures was significantly reduced after VNS treatment, with 25 responders (55.6%), of whom 6 (13.3%) achieved seizure freedom. There was a significant improvement in the Quality of Life in Childhood Epilepsy Questionnaire (15.5%) and Seizure Severity Score (46.2%). 16 potential factors affecting the efficacy of VNS were included, and three statistically significant positive predictors were ultimately screened: shorter seizure duration, focal seizure, and absence of intellectual disability. We developed a nomogram for predicting the efficacy of VNS in the treatment of DRE children. The ROC curve confirmed that the predictive model has good diagnostic performance (AUC = 0.864, P < 0.05), and the nomogram can be further validated by bootstrapping for 1,000 repetitions, with a C-index of 0.837. Besides, this model showed good fitting and calibration and positive net benefits in decision curve analysis. Conclusion: VNS is a safe and effective treatment for DRE children. We developed a predictive nomogram for the efficacy of VNS, which provides a basis for more accurate selection of VNS patients.

Impact of capital market volatility on economic growth - An analysis based on stochastic volatility model.

This paper delves into the relationship between the volatility of the capital market and economic growth within the broader framework of the macro capital market. By employing the Heston stochastic volatility model in tandem with macroeconomic theory, we aim to analyze the stochastic control problem between the allocation trajectory of macro-capital and economic fluctuations. Our mathematical analysis reveals that the influence of capital shifts on economic growth's volatility varies across different capital markets due to diverse risk levels inherent within the macro-capital market. To validate these mathematical findings, we embark on an empirical econometric analysis tailored to the nuances of China's capital market and its macroeconomic operations. This econometric exploration yields two primary insights: 1. Distinct components of China's capital market have varying influences on macroeconomic growth. 2. The structure of China's capital market, especially in its impact on macroeconomic development, exhibits imbalances and lacks optimal configuration.

The accumulation of modular serine protease mediated by a novel circRNA sponging miRNA increases Aedes aegypti immunity to fungus.

BACKGROUND: Mosquitoes transmit many infectious diseases that affect human health. The fungus Beauveria bassiana is a biological pesticide that is pathogenic to mosquitoes but harmless to the environment. RESULTS: We found a microRNA (miRNA) that can modulate the antifungal immunity of Aedes aegypti by inhibiting its cognate serine protease. Fungal infection can induce the expression of modular serine protease (ModSP), and ModSP knockdown mosquitoes were more sensitive to B. bassiana infection. The novel miRNA-novel-53 is linked to antifungal immune response and was greatly diminished in infected mosquitoes. The miRNA-novel-53 could bind to the coding sequences of ModSP and impede its expression. Double fluorescence in situ hybridization (FISH) showed that this inhibition occurred in the cytoplasm. The amount of miRNA-novel-53 increased after miRNA agomir injection. This resulted in a significant decrease in ModSP transcript and a significant increase in mortality after fungal infection. An opposite effect was produced after antagomir injection. The miRNA-novel-53 was also knocked out using CRISPR-Cas9, which increased mosquito resistance to the fungus B. bassiana. Moreover, mosquito novel-circ-930 can affect ModSP mRNA by interacting with miRNA-novel-53 during transfection with siRNA or overexpression plasmid. CONCLUSIONS: Novel-circ-930 affects the expression level of ModSP by a novel-circ-930/miRNA-novel-53/ModSP mechanism to modulate antifungal immunity, revealing new information on innate immunity in insects.

Identification of the expression patterns and potential prognostic role of m6A-RNA methylation regulators in Wilms Tumor.

BACKGROUND: To explore the potential role of m6A methylation modification in Wilms Tumor (WT) by m6A-RNA Methylation (m6A) regulators. METHODOLOGY: The association of m6A modification patterns with immune and prognostic characteristics of tumors was systematically evaluated using 19 m6A regulators extracted from Wilms Tumor's samples in public databases. A comprehensive model of "m6Ascore" was constructed using principal component analysis, and its prognostic value was evaluated. RESULTS: Almost all m6A regulators were differentially expressed between WT and normal tissues. Unsupervised clustering identified three distinct m6A clusters that differed in both immune cell infiltration and biological pathways. The m6Ascore was constructed to quantify m6A modifications in individual patients. Our analysis suggests that m6Ascore is an independent prognostic factor for WT and can be used as a novel predictor of WT prognosis. CONCLUSIONS: This study comprehensively explored and systematically characterized m6A modifications in WT. m6A modification patterns play a critical role in the tumor immune microenvironment (TIME) and WT prognosis. m6Ascore provides a more comprehensive understanding of m6A modifications in WT and offers a practical tool for predicting WT prognosis. This study will help clinicians to identify valid indicators of WT to improve the poor prognosis of this disease. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://www.aliyundrive.com/drive/folder/64be739cd6956a741fb24670baeea53422be6024 .

A qualitative study of ICU nurses assisting in Wuhan who suffered from workplace violence during the COVID-19 outbreak.

AIM: To explore the psychological experience and coping methods of nurses exposed to workplace violence and to propose measures to prevent and control workplace violence and provide psychological assistance for health workers. DESIGN: We adopted a phenomenological qualitative design. Twelve nurses in intensive care units assisting in Wuhan who experienced workplace violence during the COVID-19 outbreak were selected using purposeful sampling. Data were collected through semi-structured individual telephone interviews and analysed using Colaizzi's 7-step method. RESULTS: Analysis revealed three main categories including "Full of negative emotions", "Facing challenges and danger" and "Coping methods". The subjects experienced stress, fear, anger, helplessness, disappointment, sympathy and job burnout after suffering from workplace violence. The coping methods for workplace violence mainly included seeking support and help, escaping, making explanations, exercising tolerance and confronting the issue. PATIENT OR PUBLIC CONTRIBUTION: No patient or public contribution since nurses' experiences were explored.

OTU7B Modulates the Mosquito Immune Response to Beauveria bassiana Infection via Deubiquitination of the Toll Adaptor TRAF4.

The Aedes aegypti mosquito transmits devastating flaviviruses, such as Zika, dengue, and yellow fever viruses. For more effective control of the vector, the pathogenicity of Beauveria bassiana, a fungus commonly used for biological control of pest insects, may be enhanced based on in-depth knowledge of molecular interactions between the pathogen and its host. Here, we identified a mechanism employed by B. bassiana, which efficiently blocks the Ae. aegypti antifungal immune response by a protease that contains an ovarian tumor (OTU) domain. RNA-sequencing analysis showed that the depletion of OTU7B significantly upregulates the mRNA level of immunity-related genes after a challenge of the fungus. CRISPR-Cas9 knockout of OTU7B conferred a higher resistance of mosquitoes to the fungus B. bassiana. OTU7B suppressed activation of the immune response by preventing nuclear translocation of the NF-κB transcription factor Rel1, a mosquito orthologue of Drosophila Dorsal. Further studies identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as an interacting protein of OTU7B. TRAF4-deficient mosquitoes were more sensitive to fungal infection, indicating TRAF4 to be the adaptor protein that activates the Toll pathway. TRAF4 is K63-link polyubiquitinated at K338 residue upon immune challenge. However, OTU7B inhibited the immune signaling by enzymatically removing the polyubiquitin chains of mosquito TRAF4. Thus, this study has uncovered a novel mechanism of fungal action against the host innate immunity, providing a platform for further improvement of fungal pathogen effectiveness. IMPORTANCE Insects use innate immunity to defend against microbial infection. The Toll pathway is a major immune signaling pathway that is associated with the antifungal immune response in mosquitoes. Our study identified a fungal-induced deubiquitinase, OTU7B, which, when knocked out, promotes the translocation of the NF-κB factor Rel1 into the nucleus and confers enhanced resistance to fungal infection. We further found the counterpart of OTU7B, TRAF4, which is a component of the Toll pathway and acts as an adaptor protein. OTU7B enzymatically removes K63-linked polyubiquitin chains from TRAF4. The immune response is suppressed, and mosquitoes become much more sensitive to the Beauveria bassiana infection. Our findings reveal a novel mechanism of fungal action against the host innate immunity.

Mechanism of PKCε regulation of cardiac GIRK channel gating.

G-protein-gated inwardly rectifying potassium (GIRK) channel activity is regulated by the membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PI 4,5P2). Constitutive activity of cardiac GIRK channels in atrial myocytes, that is implicated in atrial fibrillation (AF), is mediated via a protein kinase C-ε (PKCε)-dependent mechanism. The novel PKC isoform, PKCε, is reported to enhance the activity of cardiac GIRK channels. Here, we report that PKCε stimulation leads to activation of GIRK channels in mouse atria and in human stem cell-derived atrial cardiomyocytes (iPSCs). We identified residue GIRK4(S418) which when mutated to Ala abolished, or to Glu, mimicked the effects of PKCε on GIRK currents. PKCε strengthened the interactions of the cardiac GIRK isoforms, GIRK4 and GIRK1/4 with PIP2, an effect that was reversed in the GIRK4(S418A) mutant. This mechanistic insight into the PKCε-mediated increase in channel activity because of GIRK4(S418) phosphorylation, provides a precise druggable target to reverse AF-related pathologies due to GIRK overactivity.

Bioengineered peptibodies as blockers of ion channels.

We engineered and produced an ion channel blocking peptibody, that targets the acetylcholine-activated inwardly rectifying potassium current (IKACh). Peptibodies are chimeric proteins generated by fusing a biologically active peptide with the fragment crystallizable (Fc) region of the human immunoglobulin G (IgG). The IKACh blocking peptibody was engineered as a fusion between the human IgG1 Fc fragment and the IKACh inhibitor tertiapinQ (TP), a 21-amino acid synthetic peptidotoxin, originally isolated from the European honey bee venom. The peptibody was purified from the culture supernatant of human embryonic kidney (HEK) cells transfected with the peptibody construct. We tested the hypothesis that the bioengineered peptibody is bioactive and a potent blocker of IKACh. In HEK cells transfected with Kir3.1 and Kir3.4, the molecular correlates of IKACh, patch clamp showed that the peptibody was ~300-fold more potent than TP. Molecular dynamics simulations suggested that the increased potency could be due to an increased stabilization of the complex formed by peptibody-Kir3.1/3.4 channels compared to tertiapin-Kir3.1/3.4 channels. In isolated mouse myocytes, the peptibody blocked carbachol (Cch)-activated IKACh in atrial cells but did not affect the potassium inwardly rectifying background current in ventricular myocytes. In anesthetized mice, the peptibody abrogated the bradycardic effects of intraperitoneal Cch injection. Moreover, in aged mice, the peptibody reduced the inducibility of atrial fibrillation, likely via blocking constitutively active IKACh. Bioengineered anti-ion channel peptibodies can be powerful and highly potent ion channel blockers, with the potential to guide the development of modulators of ion channels or antiarrhythmic modalities.

An exploration into the application of specialty-orientated CBL pedagogy in undergraduate teaching in pediatric surgery.

Objective: This study aims to identify whether the specialty-oriented case-based learning (CBL) pedagogy contributes to the teaching of basic theory and practical operation in undergraduate clinical teaching in pediatric surgery, and to assess the satisfaction of undergraduates. Methods: A total of 72 undergraduates in Grade 2016 who interned at Qilu Hospital of Shandong University were enrolled in this study. All these undergraduates voluntarily participated in this experimental study. They were randomly divided into the experimental group (the CBL group, n = 36) and the control group [the traditional lecture-based learning (LBL) group, n = 36] with the assistance of random number tables. In the control group, a traditional pedagogy was adopted and the knowledge in the textbook was explained according to the syllabus. In the experimental group, a specialty-oriented CBL pedagogy was adopted under the guidance of clinical instructors. After the teaching, a comparison was drawn between both groups in respect of the theoretical exam and practical exam scores. In addition, the teaching results were evaluated by a questionnaire survey. Results: The average theoretical exam scores and comprehensive scores of undergraduates in the CBL group were higher than those in the LBL group (P < 0.05). There was no significant difference in the practical exam scores between the CBL group and the LBL group (P > 0.05). However, those undergraduates in the CBL group attained higher scores in doctor-patient communication and perioperative diagnosis and treatment (P < 0.05). According to the questionnaire survey, the undergraduates in the CBL group had higher satisfaction than those in the LBL group. Besides, this specialty-oriented CBL pedagogy had higher performance in improving their ability to solve problems independently and cultivating and expanding their knowledge compared with the traditional pedagogy. Meanwhile, this specialty-oriented CBL pedagogy can cultivate the critical thinking of undergraduates, which could increase their learning efficiency and improve their interest in learning. Conclusion: This specialty-oriented CBL pedagogy could improve the mastery of professional knowledge, course satisfaction, doctor-patient communication ability in clinical practice, and perioperative diagnosis and treatment ability of these undergraduates. Therefore, it is worthwhile to recommend and popularize this pedagogy in undergraduate clinical teaching in pediatric surgery.

IKACh is constitutively active via PKC epsilon in aging mediated atrial fibrillation.

Atrial fibrillation (AF), the most common abnormal heart rhythm, is a major cause for stroke. Aging is a significant risk factor for AF; however, specific ionic pathways that can elucidate how aging leads to AF remain elusive. We used young and old wild-type and PKC epsilon- (PKCϵ) knockout mice, whole animal, and cellular electrophysiology, as well as whole heart, and cellular imaging to investigate how aging leads to the aberrant functioning of a potassium current, and consequently to AF facilitation. Our experiments showed that knocking out PKCϵ abrogates the effects of aging on AF by preventing the development of a constitutively active acetylcholine sensitive inward rectifier potassium current (IKACh). Moreover, blocking this abnormal current in the old heart reduces AF inducibility. Our studies demonstrate that in the aging heart, IKACh is constitutively active in a PKCϵ-dependent manner, contributing to the perpetuation of AF.

Ecdysone signaling mediates the trade-off between immunity and reproduction via suppression of amyloids in the mosquito Aedes aegypti.

The balance between immunity and reproduction is essential for many key physiological functions. We report that to maintain an optimal fertility, 20-hydroxyecdysone (20E) and the ecdysone receptor (EcR) downregulate the immune deficiency (IMD) pathway during the post blood meal phase (PBM) of the Aedes aegypti reproductive cycle. RNA interference-mediated depletion of EcR elicited an increased expression of the IMD pathway components, and these mosquitoes were more resistant to infection by Gram-negative bacteria. Moreover, 20E and EcR recruit Pirk-like, the mosquito ortholog of Drosophila melanogaster Pirk. CRISPR-Cas9 knockout of Pirk-like has shown that it represses the IMD pathway by interfering with IMD-mediated formation of amyloid aggregates. 20E and EcR disruption of the amyloid formation is pivotal for maintaining normal yolk protein production and fertility. Additionally, 20E and its receptor EcR directly induce Pirk-like to interfere with cRHIM-mediated formation of amyloid. Our study highlights the vital role of 20E in governing the trade-off between immunity and reproduction. Pirk-like might be a potential target for new methods to control mosquito reproduction and pathogen transmission.

The Arf6/PIP5K pathway activates IKACh in cigarette smoke mediated atrial fibrillation.

Cigarette smoking (CS) is a major cause of cardiovascular diseases. Smokers are at a significantly higher risk for developing atrial fibrillation (AF), a dangerous and abnormal heart rhythm. In the US, 15.5% of adults are current smokers, and it is becoming clear that CS is an independent risk factor for AF, but a detailed mechanistic understanding of how CS contributes to the molecular patho-electrophysiology of AF remains elusive. We investigated if CS related AF is in part mediated through a mechanism that depends on the cardiac acetylcholine activated inward rectifier potassium current (IKACh). We tested the hypothesis that CS increases IKACh via phosphatidylinositol 4-phosphate 5-kinase alpha (PIP5K) and ADP ribosylation factor 6 (Arf6) signaling, leading to AF perpetuation. In vivo inducibility of AF was assessed in mice exposed to CS for 8 weeks. AF duration was increased in CS exposed mice, and TertiapinQ, an IKACh blocker prevented AF development in CS exposed mice. In HEK293 cells stably transfected with Kir3.1 and Kir3.4, the molecular correlates of IKACh, CS exposure increased the expression of the Kir3.1 and Kir3.4 proteins at the cell surface, activated Arf6 and increased the IKACh current. Inhibition of PIP5K, or of Kir3.1/Kir3.4 trafficking via Arf6 abrogated the CS effects on IKACh. Cigarette smoke modifies the atrial electrophysiological substrate, leading to arrhythmogenesis, in part, through IKACh activation via an Arf6/PIP5K dependent pathway.

[Advances in application of molecularly imprinted polymers to the detection of polar pesticide residues].

Polar pesticides can be primarily classified as fungicides, herbicides, and insecticides; their rich variety and low cost have led to their extensive utilization in agriculture. However, the overuse of polar pesticides can lead to environmental contamination, such as water or soil pollution, which can also increase the risk of pesticide exposure among human life directly, or indirectly through contact with animal and plant-derived food. There are considerable differences in the physical and chemical properties of polar pesticides, as well as their trace amounts in complex food and environmental samples, posing immense challenges to their accurate detection. As a kind of artificially prepared selective adsorbent, molecularly imprinted polymers (MIPs) possess specific recognition sites complementary to template molecules in terms of the spatial structure, size, and chemical functional groups. With many advantages such as easy preparation, low cost, as well as good chemical and mechanical stability, MIPs have been widely applied in sample pretreatment and the analysis of polar pesticide residues. MIPs are typically used as adsorption materials in solid phase extraction (SPE) methods, including magnetic solid phase extraction (MSPE), dispersed solid phase extraction (DSPE), and stir bar sorptive extraction (SBSE). To rapidly detect polar pesticide residues with high sensitivity, MIPs are also used in the preparation of fluorescent sensors and electrochemical sensors. Furthermore, MIPs can be employed as the substrate in surface-enhanced Raman spectroscopy and as the substrate for the ion source in mass spectrometry for polar pesticide residue analysis. Thus far, various molecularly imprinted materials have been reported for the efficient separation and analysis of polar pesticide residues in various complex matrices. However, there is no review that summarizes the recent advances in MIPs for the determination of polar pesticides. This review introduces imprinting strategies and polymerization methods for MIPs, and briefly summarizes some new molecular imprinting strategies and preparation technologies. The application of MIPs in recent years (particularly the last five years) to the detection of polar pesticide residues including neonicotinoids, organophosphorus, triazines, azoles, and urea is then systematically summarized. Finally, the future development direction and trends for MIPs are proposed considering existing challenges, with the aim of providing reference to guide future research on MIPs in the field of polar pesticide residue detection.

Facile Synthesis of Antibody-Coupled Polydopamine-Coated Magnetic Graphene Oxide Composites for Efficient Immunopurification and Metabolomics Analysis of Mitochondria.

As a vital hub, a mitochondrion houses metabolic pathways that play important roles in cellular physiology. Aberrant metabolites occurring in mitochondria are closely associated with the emergence and progression of various mitochondria-related diseases. Therefore, a simple and versatile approach to efficiently purify intact mitochondria is urgently needed to precisely and comprehensively characterize the composition and abundance of the mitochondrial metabolome in different physiological and pathological states. In this work, novel immunoaffinitive magnetic composites MagG@PD@Avidin@TOM20 were prepared to achieve highly selective isolation of intact mitochondria from three different hepatocytes (LO2, HepG2, and Huh7). The prepared composites inherit combined merits, including strong magnetic responsiveness, excellent stability, and specific and high affinity between antibody TOM20 and mitochondrial outer membrane protein. These mitochondria attached on MagG@PD@Avidin@TOM20 were characterized by the western blot and fluorescence microscopy to confirm their purity and integrity, which are vital for reliable mitochondrial metabolic analysis. Subsequently, ultrahigh-performance liquid chromatography-high-resolution mass spectrometry-based untargeted metabolomics analysis was conducted to characterize the metabolomes in the immunopurified mitochondria and whole cells. Notably, the metabolite profiles of whole cells and mitochondria including itaconic acid, acetylcarnitine, malic acid, etc., were significantly different. These data underscore the importance of determining metabolites at the mitochondrial level, which would supplement us new knowledge at the subcellular level.

Juvenile Hormone-Sensitive Ribosomal Activity Enhances Viral Replication in Aedes aegypti.

Zika virus (ZIKV; Flaviviridae) is a devastating virus transmitted to humans by the mosquito Aedes aegypti. The interaction of the virus with the mosquito vector is poorly known. The double-stranded RNA (dsRNA)-mediated interruption or activation of immunity-related genes in the Toll, IMD, JAK-STAT, and short interfering RNA (siRNA) pathways did not affect ZIKV infection in A. aegypti. Transcriptome-based analysis indicated that most immunity-related genes were upregulated in response to ZIKV infection, including leucine-rich immune protein (LRIM) genes. Further, there was a significant increment in the ZIKV load in LRIM9-, LRIM10A-, and LIRM10B-silenced A. aegypti, suggesting their function in modulating viral infection. Further, gene function enrichment analysis revealed that viral infection increased global ribosomal activity. Silencing of RpL23 and RpL27, two ribosomal large subunit genes, increased mosquito resistance to ZIKV infection. In vitro fat body culture assay revealed that the expression of RpL23 and RpL27 was responsive to the Juvenile hormone (JH) signaling pathway. These two genes were transcriptionally regulated by JH and its receptor methoprene-tolerant (Met) complex. Silencing of Met also inhibited ZIKV infection in A. aegypti. This suggests that ZIKV enhances ribosomal activity through JH regulation to promote infection in mosquitoes. Together, these data reveal A. aegypti immune responses to ZIKV and suggest a control strategy that reduces ZIKV transmission by modulating host factors. IMPORTANCE Most flaviviruses are transmitted between hosts by arthropod vectors such as mosquitoes. Since therapeutics or vaccines are lacking for most mosquito-borne diseases, reducing the mosquito vector competence is an effective way to decrease disease burden. We used high-throughput sequencing technology to study the interaction between mosquito Aedes aegypti and ZIKV. Leucine-rich immune protein (LRIM) genes were involved in the defense in response to viral infection. In addition, RNA interference (RNAi) silencing of RpL23 and RpL27, two JH-regulated ribosomal large subunit genes, suppressed ZIKV infection in A. aegypti. These results suggest a novel control strategy that could block the transmission of ZIKV.

A benzopyran with antiarrhythmic activity is an inhibitor of Kir3.1-containing potassium channels.

Atrial fibrillation (AF) is the most commonly diagnosed cardiac arrhythmia and is associated with increased morbidity and mortality. Currently approved AF antiarrhythmic drugs have limited efficacy and/or carry the risk of ventricular proarrhythmia. The cardiac acetylcholine activated inwardly rectifying K+ current (IKACh), composed of Kir3.1/Kir3.4 heterotetrameric and Kir3.4 homotetrameric channel subunits, is one of the best validated atrial-specific ion channels. Previous research pointed to a series of benzopyran derivatives with potential for treatment of arrhythmias, but their mechanism of action was not defined. Here, we characterize one of these compounds termed Benzopyran-G1 (BP-G1) and report that it selectively inhibits the Kir3.1 (GIRK1 or G1) subunit of the KACh channel. Homology modeling, molecular docking, and molecular dynamics simulations predicted that BP-G1 inhibits the IKACh channel by blocking the central cavity pore. We identified the unique F137 residue of Kir3.1 as the critical determinant for the IKACh-selective response to BP-G1. The compound interacts with Kir3.1 residues E141 and D173 through hydrogen bonds that proved critical for its inhibitory activity. BP-G1 effectively blocked the IKACh channel response to carbachol in an in vivo rodent model and displayed good selectivity and pharmacokinetic properties. Thus, BP-G1 is a potent and selective small-molecule inhibitor targeting Kir3.1-containing channels and is a useful tool for investigating the role of Kir3.1 heteromeric channels in vivo. The mechanism reported here could provide the molecular basis for future discovery of novel, selective IKACh channel blockers to treat atrial fibrillation with minimal side effects.