Frequencies of insecticide resistance mutations detected by the amplicon sequencing in Plutella xylostella (Lepidoptera: Plutellidae) and Spodoptera exigua (Lepidoptera: Noctuidae) from China.

The globally prevalent pests, Diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) and Beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), pose significant threats to cruciferous vegetables. They have rapidly developed resistance to a wide range of insecticides, leading to significant yield losses and increased control expenses. In this study, we have established an efficient approach utilizing amplicon sequencing to detect the frequency of 15 target resistance mutant sites in 6 molecular targets, acetylcholinesterase 1 (ACE1), chitin synthase 1 (CHS1), the γ-aminobutyric acid receptor (GABAR), glutamate-gated chloride channel (GluCl), voltage-gated sodium channels (NaV), and ryanodine receptor (RyR) in P. xylostella and the frequency of 11 mutations in 5 molecular targets (except GluCl) in S. exigua in China. Our findings indicate that P. xylostella exhibits remarkably high frequency (over 88.67%) in pyrethroid resistance-related mutations T929I and L1014F of NaV. In S. exigua, the frequencies of L659F mutation were ranging from 41.92% to 74.89%. In addition, the organophosphorus resistance-related mutations A298S and G324A of ACE1 were detected at frequencies ranging from 34.29% to 75.66%, and these 2 mutations occurred simultaneously (from 29.22% to 65.79%) in P. xylostella. An interannual variation in mutation frequency from 2019 to 2021 was found for P. xylostella in HNCS. The frequency of A298S and G324A mutations steadily increased while the frequency of G4946E and I4790M mutations continuously decreased. These results unveil a worrisome scenario of multiple resistance sites in these 2 pests in China and provide valuable insights for the practical application of pesticides in the field.

Electrophysiological and pharmacological properties of the slowpoke channel in the diamondback moth, Plutella xylostella.

The slowpoke channel responds to the intracellular calcium concentration and the depolarization of the cell membrane. It plays an important role in maintaining the resting potential and regulating the homeostasis of neurons, but it can also regulate circadian rhythm, sperm capacitation, ethanol tolerance, and other physiological processes in insects. This renders it a potentially useful target for the development of pest control strategies. There are relatively few studies on the slowpoke channels in lepidopteran pests, and their pharmacological properties are still unclear. So, in this study, the slowpoke gene of Plutella xylostella (Pxslo) was heterologous expressed in HEK293T cells, and the I-V curve of the slowpoke channel was measured by whole cell patch clamp recordings. Results showed that the slowpoke channel could be activated at -20 mV with 150 µM Ca2+. The subsequent comparison of the electrophysiological characteristics of the alternative splicing site E and G deletions showed that the deletion of the E site enhances the response of the slowpoke channel to depolarization, while the deletion of the G site weakens the response of the slowpoke channel to depolarization. Meanwhile, the nonspecific inhibitors TEA and 4-AP of the Kv channels, and four pesticides were tested and all showed an inhibition effect on the PxSlo channel at 10 or 100 µM, suggesting that these pesticides also target the slowpoke channel. This study enriches our understanding of the slowpoke channel in Lepidopteran insects and can aid in the development of relevant pest management strategies.

Appressoria Formation in Phytopathogenic Fungi Suppressed by Antimicrobial Peptides and Hybrid Peptides from Black Soldier Flies.

Antimicrobial peptides (AMPs) from black solider flies (Hermetia illucens, BSF) exhibiting broad-spectrum antimicrobial activity are the most promising green substitutes for preventing the infection of phytopathogenic fungi; therefore, AMPs have been a focal topic of research. Recently, many studies have focused on the antibacterial activities of BSF AMPs against animal pathogens; however, currently, their antifungal activities against phytopathogenic fungi remain unclear. In this study, 7 AMPs selected from 34 predicted AMPs based on BSF metagenomics were artificially synthesized. When conidia from the hemibiotrophic phytopathogenic fungi Magnaporthe oryzae and Colletotrichum acutatum were treated with the selected AMPs, three selected AMPs-CAD1, CAD5, and CAD7-showed high appressorium formation inhibited by lengthened germ tubes. Additionally, the MIC50 concentrations of the inhibited appressorium formations were 40 µM, 43 µM, and 43 µM for M. oryzae, while 51 µM, 49 µM, and 44 µM were observed for C. acutatum, respectively. A tandem hybrid AMP named CAD-Con comprising CAD1, CAD5, and CAD7 significantly enhanced antifungal activities, and the MIC50 concentrations against M. oryzae and C. acutatum were 15 µM and 22 µM, respectively. In comparison with the wild type, they were both significantly reduced in terms of virulence when infection assays were performed using the treated conidia of M. oryzae or C. acutatum by CAD1, CAD5, CAD7, or CAD-Con. Meanwhile, their expression levels of CAD1, CAD5, and CAD7 could also be activated and significantly increased after the BSF larvae were treated with the conidia of M. oryzae or C. acutatum, respectively. To our knowledge, the antifungal activities of BSF AMPs against plant pathogenic fungi, which help us to seek potential AMPs with antifungal activities, provide proof of the effectiveness of green control strategies for crop production.

Exosomal microRNA­4516, microRNA­203 and SFRP1 are potential biomarkers of acute myocardial infarction.

Acute myocardial infarction (AMI) is a serious disease which threatens public health. Exosomes (exos) contain certain genetic information and are important communication vehicles between cells. In the present study, different exosomal microRNAs (miRs), which exhibit a notable association between expression levels in plasma and AMI were assessed to support the development of new diagnostic and clinical assessment markers of patients with AMI. In total, 93 individuals, including 31 healthy controls and 62 patients with AMI, were recruited for the present study. Data on age, blood pressure, glucose levels, lipid levels and coronary angiography images were collected from the enrolled individuals, and plasma samples were collected. Plasma exos were extracted and verified using ultracentrifugation, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting (WB). Exo­miR­4516 and exo­miR­203 in plasma exos were identified by exosomal miRNA sequencing analysis, reverse transcription­quantitative PCR was performed to detect the levels of exo­miR­4516 and exo­miR­203 in plasma exos, and ELISA was performed to detect the levels of secretory frizzled­related protein 1 (SFRP1) in samples. The correlation analysis between exo­miR­4516, exo­miR­203 and SFRP1 in plasma exos and AMI was presented as receiver operating characteristic curves (ROCs) of the SYNTAX score, cardiac troponin I (cTnI), low­density lipoprotein (LDL) and each indicator separately. Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed to predict relevant enrichment pathways. Exos were successfully isolated from plasma by ultracentrifugation, which was confirmed by TEM, NTA and WB. Exo­miR­4516, exo­miR­203 and SFRP1 levels in plasma were significantly higher in the AMI group compared with the healthy control group. ROCs demonstrated that exo­miR­4516, exo­miR­203 and SFRP1 levels had a high diagnostic efficiency in predicting AMI. Exo­miR­4516 was positively correlated with SYNTAX score, and plasma SFRP1 was positively correlated with plasma cTnI and LDL. In conclusion, the data demonstrated that exo­miR­4516, exo­miR­203 and SFRP1 levels could be used in combination to diagnose and assess the severity of AMI. The present study was retrospectively registered (TRN, NCT02123004).

Streptomyces longhuiensis sp. nov, a novel species isolated from soil of Lilium brownii in Hunan Province, PR China.

An actinobacterium strain, designated BH-MK-02T, was isolated from the soil of Lilium brownii. The taxonomic position was determined using a polyphasic approach. Strain BH-MK-02T grew well on International Streptomyces Project series media and formed well-developed, branched substrate hyphae and aerial mycelium that differentiated into straight spore chains with a wrinkled surface. The diagnostic diamino acid was ll-diaminopimelic acid. The major menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides, phosphatidylglycerol and unidentified lipid spots. The predominant fatty acids were anteiso-C15 : 0, iso-C16 : 0, C16 : 0 and C16 : 1 ω7c/C16 : 1 ω6c. The phenotypic characteristics of strain BH-MK-02T indicated that it belonged to the genus Streptomyces. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain BH-MK-02T was most closely related to Streptomyces aureus CGMCC 4.1833T (99.7 %). However, the average nucleotide identity and digital DNA-DNA hybridization values between the whole-genome sequences of strain BH-MK-02T and S. aureus CGMCC 4.1833T were 78.1 and 23.2 %, respectively, below the 96.7 and 70 % cut-off points respectively recommended for delineating Streptomyces species. Furthermore, the novel isolate could be distinguished from S. aureus CGMCC 4.1833T by morphological, physiological and biochemical characteristics. Based on all these data, strain BH-MK-02T (=MCCC 1K06237T=JCM 34789T) clearly represents a novel species within the genus Streptomyces, for which the name Streptomyces longhuiensis sp. nov. is proposed.

Pharmacological Properties and Function of the PxOctß3 Octopamine Receptor in Plutella xylostella (L.).

The diamondback moth (Plutella xylostella) is one of the most destructive lepidopteran pests of cruciferous vegetables, and insights into regulation of its physiological processes contribute towards the development of new pesticides against it. Thus, we investigated the regulatory functions of its ß-adrenergic-like octopamine receptor (PxOctß3). The open reading frame (ORF) of PxOctß3 was phylogenetically analyzed, and the levels of expression of the receptor mRNA were determined. This ORF was also cloned and expressed in HEK-293 cells. A series of octopamine receptor agonists and antagonists were tested against PxOctß3. We showed that the receptor is a member of the Octß3 protein family, and an analysis using quantitative PCR showed that it was expressed at all developmental stages of P. xylostella. Octopamine activated PxOctß3, resulting in increased levels of intracellular cAMP. Furthermore, the agonists naphazoline, clonidine, 2-phenethylamine, and amitraz activated the PxOctß3 receptor, and naphazoline was the most effective. Only metoclopramide and mianserin had significant antagonistic effects on PxOctß3, whereas yohimbine, phentolamine, and chlorpromazine lacked obvious antagonistic effects. The injection of double-stranded RNA in an RNA interference assay indicated that PxOctß3 regulates development in P. xylostella. This study demonstrated the pharmacological properties and functions of PxOctß3 in P. xylostella, thus, providing a theoretical basis for the design of pesticides that target octopamine receptors.

Prevalence and heart rate variability characteristics of premature ventricular contractions detected by 24-hour Holter among outpatients with palpitations in China: a cross-sectional study.

OBJECTIVE: To analyse the prevalence and heart rate variability (HRV) characteristics of premature ventricular contraction (PVC) detected by 24-hour Holter among Chinese outpatients with palpitations. DESIGN: A cross-sectional study. SETTING: This study was conducted in a tertiary hospital. PARTICIPANTS: A total of 4754 outpatients who received 24-hour Holter for palpitations. MAIN OUTCOME MEASURES: Prevalence, HRV time-domain and frequency-domain analyses of 24-hour Holter, and echocardiographic parameters were assessed. Propensity score matching (PSM) was applied to balance baseline variables (age, gender) to decrease the bias between comparison groups. RESULTS: The prevalence of PVC was 67.7% (3220/4754), and was higher in men than women (69.9% vs 66.0%, p=0.004); the prevalence of frequent PVCs (PVC burden≥5%) was 7.7% (368/4754). Older patients had the highest frequency of PVC among all patients. However, among 3220 patients with PVC, younger patients' PVC burden was much higher. Matched 1:1 by age and gender, the HRV time-domain parameters in patients with PVC were all lower than those in patients without PVC (all p<0.05); for the HRV frequency-domain parameters, the patients with frequent PVCs had a higher low frequency/high frequency (LF/HF) ratio (5.4 vs 2.8, p<0.001) than those with PVC burden less than 5%. CONCLUSIONS: The prevalence of PVC and frequent PVCs were 67.7% and 7.7%, respectively, detected by 24-hour Holter among Chinese outpatients with palpitations. Decreased HRV time-domain parameters suggested the occurrence of PVC, and increased LF/HF ratio represented the imbalance of autonomic nervous system in patients with frequent PVCs. Further studies are needed to understand the HRV indexes in PVC patients.

C/EBPα Regulates PxTreh1 and PxTreh2 Trehalase-Related Bt Resistance in Plutella xylostella (L.).

Trehalase regulates energy metabolism in insects by converting trehalose into two glucose molecules. High amounts of trehalase are critical for insect flight and larval stress resistance. However, whether trehalase participates in the development of pesticide resistance remains unclear. In this study, we explored this phenomenon and the mechanism that underlies the regulation of Trehalase transcription. We found that overexpression of PxTreh1 and PxTreh2 induced Bacillus thuringiensis (Bt) resistance in Plutella xylostella. The promoter sequences of PxTreh1 and PxTreh2 were also cloned and identified. The dual-luciferase reporter system and RNA interference technology revealed that the expression of PxTreh1 and PxTreh2 genes is possibly regulated by the CCAAT enhancer-binding protein (C/EBPα). A yeast one-hybrid experiment confirmed the interaction between C/EBPα and the PxTreh2 promoter. The findings of this study suggest that C/EBPα mediates the adaptability of P. xylostella to adverse environmental stressors by regulating the expression of trehalase.

Streptomyces liliifuscus sp. nov and an anti-ginger plague agent Streptomyces liliiviolaceus sp. nov, two novel species isolated from soil of Lilium lancifolium.

Two novel strains of actinobacteria, ZYC-3T and BH-SS-21T, were isolated from Hunan Province, PR China. The fermentation broth of BH-SS-21T inhibited the rapid spread of ginger blast, unlike that of ZYC-3T. The taxonomic characteristics of ZYC-3T and BH-SS-21T were defined using a polyphasic approach. The analysis of the full-length 16S rRNA gene sequence revealed that ZYC-3T and BH-SS-21T represented members of the genus Streptomyces. ZYC-3T had less than 98.7% sequence similarities to all species of the genus Streptomyces, while BH-SS-21T exhibited 99.97, 98.95, 98.83, 98.82, 98.75 and less than 98.7% sequence similarities to 'Streptomyces dioscori' A217, Streptomyces ederensis JCM 4958T, Streptomyces glomeroaurantiacus NBRC 15418T, Streptomyces aurantiacus NBRC 13017T, Streptomyces umbrinus JCM 4521T and other species with validly published names in the genus Streptomyces. However, the digital DNA-DNA relatedness and average nucleotide identity values between ZYC-3T, BH-SS-21T, and their closely related strains were significantly lower than the recommended threshold values. Also, phenotypic, chemotaxonomic and genetic features distinguished ZYC-3T and BH-SS-21T from their reference strains. On the basis of their genotypic and phenotypic characteristics, strains ZYC-3T and BH-SS-21T were classified as representing novel species of the genus Streptomyces under the names Streptomyces liliifuscus sp. nov. ZYC-3T (=CICC 25040T=JCM 34560T=MCCC 1K04978T) and Streptomyces liliiviolaceus sp. nov. BH-SS-21T (=MCCC 1K06236T=JCM 34767T), respectively.

microRNA-148a in Exosomes Derived from Bone Marrow Mesenchymal Stem Cells Alleviates Cardiomyocyte Apoptosis in Atrial Fibrillation by Inhibiting SMOC2.

Exosomes-related microRNAs (miRNAs) have been considered to be the significant biomarkers contributing to the development of atrial fibrillation (AF). We observed the implicit mechanism of exosomes-miR-148a derived from bone marrow mesenchymal stem cells (BMSCs) in AF. The AF cell and mice models were established firstly. QRT-PCR and Western blot analysis were applied to detect the expression of miR-148a, SPARC-associated modular calcium-binding protein 2 (SMOC2), Bcl-2, Bax, and caspase-3. BMSCs were separated from healthy mice and exosomes were obtained from BMSCs. BMSCs were transfected with mimics and inhibitor, and HL-1 cells were treated with mimics and pcDNA3.1. MTT assay were used to detect cell viability of cells. Flow cytometric analysis and TUNEL analysis were used for detecting cell apoptosis of cells. In our study, exosomes derived from BMSCs inhibited the development of AF, and miR-148a acted a vital role in this segment. SMOC2 was a target gene of miR-148a and promoted apoptosis of HL-1 cells. Additionally, miR-148a mimics decreased cellular apoptosis, eliminated SMOC2 expression, and elevated Bcl-2 expression in AF-treated cells. Collectively, miR-148a overexpressed in BMSC-exosomes restrained cardiomyocytes apoptosis by inhibiting SMOC2.

Identification of a novel missense mutation in the TPM1 gene via exome sequencing in a Chinese family with dilated cardiomyopathy: A case report and literature review.

RATIONALE: Dilated cardiomyopathy (DCM) is a cardiovascular disorder characterized by consecutive ventricular dilation and contractile dysfunction, often leading to congestive heart failure. DCM type 1Y (DCM1Y) is caused by a mutation in the TPM1 (tropomyosin 1) gene. To date, about thirty TPM1 gene mutations have been reported to be related to DCM1Y. However, mutational screening of the TPM1 gene is still far from being complete. Identification of TPM1 mutation is particularly important in the diagnosis of DCM1Y and will give more insights into the molecular pathogenesis of DCM1Y. PATIENT CONCERNS: A Chinese Han family with DCM phenotypes was examined. DIAGNOSIS: A novel missense mutation, c.340G > C in exon 3 of the TPM1 gene, was identified. INTERVENTIONS: Next-generation sequencing (NGS) of DNA samples was performed to detect the gene mutation in the proband, which was confirmed by Sanger sequencing. OUTCOMES: This novel heterozygous mutation results in the substitution of glutamic acid with glutamine (p.E114Q). Based on this finding and clinical manifestations, a final diagnosis of DCM1Y was made. LESSONS: We present evidence that p.E114Q mutation represents a novel TPM1 mutation in a Chinese Han family with DCM. Our data expand the mutation spectrum of the TPM1 gene and may facilitate the clinical diagnosis of DCM1Y.

Analysis of the Antifungal Properties of Bacillus velezensis B-4 Through a Bioassay and Complete-Genome Sequencing.

The strain B-4, isolated from a field in Changsha (China), presents strong antifungal activities, as identified by the Kirby-Bauer test, especially for pathogens that harm crops. Here, we obtained the complete genome sequence of the strain B-4 by Pacific Biosciences single-molecule real-time sequencing, making it well analyzed for understanding mechanisms and creating biological agents. Its 3,919-kb circular chromosome genome has 3,725 protein-coding genes [coding sequences (CDSs)] and 46.7% guanine-cytosine content. A comparative genome analysis of B-4 with other published strains (including Bacillus velezensis, Bacillus amyloliquefaciens, and Bacillus subtilis) revealed that the strain B-4 is a B. velezensis strain. These different strains have 2,889 CDSs in common, whereas 179 CDSs were found to be unique in the strain B-4, which is a far greater number than that in other strains. Regarding the antifungal activities of B-4, we were specifically concerned with the genes involved in the biosynthesis of secondary metabolites. In total, more than 19.56% of the genome was annotated to 12 gene clusters relating to synthesis of antimicrobial metabolites, which contained various enzyme-encoding operons for non-ribosomal peptide synthetases, polyketide synthases, and lantipeptide synthesis proteins. They were all considered to be related to the production of bacteriostatic substances or stimulation of induced systemic resistance by bacterial metabolites. These situations also present an advantage over those of other strains for biocontrol potential. We provide evidence that the biological control effect of the strain B-4, as demonstrated in antibacterial activity experiments and predicted from the complete genome sequence analysis, provides the basis for research promoting agricultural research on sustainable development, especially the contribution of biotechnology to agriculture.

Hepatocyte growth factor modification enhances the anti-arrhythmic properties of human bone marrow-derived mesenchymal stem cells.

BACKGROUND/AIMS: Chronic myocardial infarction (MI) results in the formation of arrhythmogenic substrates, causing lethal ventricular arrhythmia (VA). We aimed to determine whether mesenchymal stem cells (MSCs) carrying a hepatocyte growth factor (HGF) gene modification (HGF-MSCs) decrease the levels of arrhythmogenic substrates and reduce the susceptibility to developing VA compared with unmodified MSCs and PBS in a swine infarction model. METHODS: The left descending anterior artery was balloon-occluded to establish an MI model. Four weeks later, the randomly grouped pigs were administered MSCs, PBS or HGF-MSCs via thoracotomy. After an additional four weeks, dynamic electrocardiography was performed to assess heart rate variability, and programmed electrical stimulation was conducted to evaluate the risk for VA. Then, the pigs were euthanized for morphometric, immunofluorescence and western blot analyses. RESULTS: The HGF-MSC group displayed the highest vessel density and Cx43 expression levels, and the lowest levels of apoptosis, and tyrosine hydroxylase (TH) and growth associated protein 43 (GAP43) expression. Moreover, the HGF-MSC group exhibited a decrease in the number of sympathetic nerve fibers, substantial decreases in the low frequency and the low-/high- frequency ratio and increases in the root mean square of successive differences (rMSSD) and the percentage of successive normal sinus R-R intervals longer than 50 ms (pNN50), compared with the other two groups. Finally, the HGF-MSC group displayed the lowest susceptibility to developing VA. CONCLUSION: HGF-MSCs displayed potent antiarrhythmic effects, reducing the risk for VA.

Establishment and evaluation of a swine model of acute myocardial infarction and reperfusion-ventricular fibrillation-cardiac arrest using the interventional technique.

BACKGROUND: Ventricular fibrillation is the main cause of sudden cardiac death among patients with acute myocardial infarction (AMI). Substantial benefits could be obtained by both researchers and practitioners if an AMI reperfusion-ventricular fibrillation-cardiac arrest model were established. METHODS: Twenty swine were anesthetized and underwent occlusion of the left anterior descending branch for 90 minutes prior to blood reperfusion. Throughout this process, continuous 12-lead electrocardiography (ECG) was used to monitor heart rate, rhythm, and electrocardiogram alteration. Thereafter, AMI was confirmed by ECG and left ventricular angiography. Heart tissue was collected for pathological analysis, and for evaluation of the establishment of a model of AMI reperfusion. RESULTS: Seven swine died during the model establishment, and the 13 surviving swine were proven to have myocardial infarction; nine of those survivors had ventricular fibrillation-cardiac arrest after reperfusion based on the electrocardiograph and pathological examination. CONCLUSION: Blocking the left anterior descending branch by inflation of an over-the-wire coronary balloon catheter in swine can result in successful establishment of a swine model of AMI and reperfusion-ventricular fibrillation-cardiac arrest, with good reproducibility and a high survival rate.