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Journal of Economic Entomology May 2024The globally prevalent pests, Diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) and Beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), pose...
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.
PubMed: 38748551
DOI: 10.1093/jee/toae109 -
Journal of Cachexia, Sarcopenia and... Jun 2024Autosomal-recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated...
Integrated multi-omics approach reveals the role of striated muscle preferentially expressed protein kinase in skeletal muscle including its relationship with myospryn complex.
BACKGROUND
Autosomal-recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated cardiomyopathy (CNM5). Loss of SPEG is associated with defective triad formation, abnormal excitation-contraction coupling, calcium mishandling and disruption of the focal adhesion complex in skeletal muscles. To elucidate the underlying molecular pathways, we have utilized multi-omics tools and analysis to obtain a comprehensive view of the complex biological processes and molecular functions.
METHODS
Skeletal muscles from 2-month-old SPEG-deficient (Speg-CKO) and wild-type (WT) mice were used for RNA sequencing (n = 4 per genotype) to profile transcriptomics and mass spectrometry (n = 4 for WT; n = 3 for Speg-CKO mice) to profile proteomics and phosphoproteomics. In addition, interactomics was performed using the SPEG antibody on pooled muscle lysates (quadriceps, gastrocnemius and triceps) from WT and Speg-CKO mice. Based on the multi-omics results, we performed quantitative real-time PCR, co-immunoprecipitation and immunoblot to verify the findings.
RESULTS
We identified that SPEG interacts with myospryn complex proteins CMYA5, FSD2 and RyR1, which are critical for triad formation, and that SPEG deficiency results in myospryn complex abnormalities (protein levels decreased to 22 ± 3% for CMYA5 [P < 0.05] and 18 ± 3% for FSD2 [P < 0.01]). Furthermore, SPEG phosphorylates RyR1 at S2902 (phosphorylation level decreased to 55 ± 15% at S2902 in Speg-CKO mice; P < 0.05), and its loss affects JPH2 phosphorylation at multiple sites (increased phosphorylation at T161 [1.90 ± 0.24-fold], S162 [1.61 ± 0.37-fold] and S165 [1.66 ± 0.13-fold]; decreased phosphorylation at S228 and S231 [39 ± 6%], S234 [50 ± 12%], S593 [48 ± 3%] and S613 [66 ± 10%]; P < 0.05 for S162 and P < 0.01 for other sites). On analysing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix-receptor interaction (P < 1e) and peroxisome proliferator-activated receptor signalling (P < 9e).
CONCLUSIONS
We have elucidated the critical role of SPEG in the triad as it works closely with myospryn complex proteins (CMYA5, FSD2 and RyR1), it regulates phosphorylation levels of various residues in JPH2 and S2902 in RyR1, and its deficiency is associated with dysregulation of several pathways. The study identifies unique SPEG-interacting proteins and their phosphorylation functions and emphasizes the importance of using a multi-omics approach to comprehensively evaluate the molecular function of proteins involved in various genetic disorders.
Topics: Animals; Mice; Muscle, Skeletal; Mice, Knockout; Muscle Proteins; Ryanodine Receptor Calcium Release Channel; Proteomics; Phosphorylation; Multiomics; Myosin-Light-Chain Kinase
PubMed: 38725372
DOI: 10.1002/jcsm.13470 -
Frontiers in Physiology 2024The loose-patch clamp technique was first developed and used in native amphibian skeletal muscle (SkM), offering useful features complementing conventional sharp...
Loose-patch clamp analysis applied to voltage-gated ionic currents following pharmacological ryanodine receptor modulation in murine hippocampal cornu ammonis-1 pyramidal neurons.
INTRODUCTION
The loose-patch clamp technique was first developed and used in native amphibian skeletal muscle (SkM), offering useful features complementing conventional sharp micro-electrode, gap, or conventional patch voltage clamping. It demonstrated the feedback effects of pharmacological modification of ryanodine receptor (RyR)-mediated Ca release on the Na channel (Nav1.4) currents, initiating excitation-contraction coupling in native murine SkM. The effects of the further RyR and Ca-ATPase (SERCA) antagonists, dantrolene and cyclopiazonic acid (CPA), additionally implicated background tubular-sarcoplasmic Ca domains in these actions.
MATERIALS AND METHODS
We extend the loose-patch clamp approach to ion current measurements in murine hippocampal brain slice cornu ammonis-1 (CA1) pyramidal neurons. We explored the effects on Na currents of pharmacologically manipulating RyR and SERCA-mediated intracellular store Ca release and reuptake. We adopted protocols previously applied to native skeletal muscle. These demonstrated Ca-mediated feedback effects on the Na channel function.
RESULTS
Experiments applying depolarizing 15 ms duration loose-patch clamp steps to test voltages ranging from -40 to 120 mV positive to the resting membrane potential demonstrated that 0.5 mM caffeine decreased inward current amplitudes, agreeing with the previous SkM findings. It also decreased transient but not prolonged outward current amplitudes. However, 2 mM caffeine affected neither inward nor transient outward but increased prolonged outward currents, in contrast to its increasing inward currents in SkM. Furthermore, similarly and in contrast to previous SkM findings, both dantrolene (10 μM) and CPA (1 μM) pre-administration left both inward and outward currents unchanged. Nevertheless, dantrolene pretreatment still abrogated the effects of subsequent 0.5- and 2-mM caffeine challenges on both inward and outward currents. Finally, CPA abrogated the effects of 0.5 mM caffeine on both inward and outward currents, but with 2 mM caffeine, inward and transient outward currents were unchanged, but sustained outward currents increased.
CONCLUSION
We, thus, extend loose-patch clamping to establish pharmacological properties of murine CA1 pyramidal neurons and their similarities and contrasts with SkM. Here, evoked though not background Ca-store release influenced Nav and Kv excitation, consistent with smaller contributions of background store Ca release to resting [Ca]. This potential non-canonical mechanism could modulate neuronal membrane excitability or cellular firing rates.
PubMed: 38720787
DOI: 10.3389/fphys.2024.1359560 -
The Journal of Physical Chemistry. B May 2024Ryanodine receptor type 1 (RyR1) is a Ca-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger...
Ryanodine receptor type 1 (RyR1) is a Ca-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger motif positioned within the cytoplasmic C-terminal domain (CTD). Yet, owing to limitations in cryo-EM resolution, RyR1 structures lack precision in detailing the metal coordination structure, prompting the need for an accurate model. In this study, we employed molecular dynamics (MD) simulations and the density functional theory (DFT) method to refine the binding characteristics of Zn in the zinc-finger site of the RyR1 channel. Our findings also highlight substantial conformational changes in simulations conducted in the absence of Zn. Notably, we observed a loss of contact at the interface between protein domains proximal to the zinc-finger site, indicating a crucial role of Zn in maintaining structural integrity and interdomain interactions within RyR1. Furthermore, this study provides valuable insights into the modulation of ATP, Ca, and caffeine binding, shedding light on the intricate relationship between Zn coordination and the dynamic behavior of RyR1. Our integrative approach combining MD simulations and DFT calculations enhances our understanding of the molecular mechanisms governing ligand binding in RyR1.
Topics: Ryanodine Receptor Calcium Release Channel; Molecular Dynamics Simulation; Zinc; Ligands; Calcium; Density Functional Theory; Binding Sites; Protein Binding; Zinc Fingers; Caffeine; Adenosine Triphosphate; Humans
PubMed: 38717304
DOI: 10.1021/acs.jpcb.4c01189 -
Advanced Materials (Deerfield Beach,... May 2024Tumor cells harness Ca to maintain cellular homeostasis and withstand external stresses from various treatments. Here, a dual-channel Ca nanomodulator (CAP-P-NO) is...
Tumor cells harness Ca to maintain cellular homeostasis and withstand external stresses from various treatments. Here, a dual-channel Ca nanomodulator (CAP-P-NO) is constructed that can induce irreversible intracellular Ca disorders via the redistribution of tumor-inherent Ca for disrupting cellular homeostasis and thus improving tumor radiosensitivity. Stimulated by tumor-overexpressed acid and glutathione, capsaicin and nitric oxide are successively escaped from CAP-P-NO to activate the transient receptor potential cation channel subfamily V member 1 and the ryanodine receptor for the influx of extracellular Ca and the release of Ca in the endoplasmic reticulum, respectively. The overwhelming level of Ca in tumor cells not only impairs the function of organelles but also induces widespread changes in the gene transcriptome, including the downregulation of a set of radioresistance-associated genes. Combining CAP-P-NO treatment with radiotherapy achieves a significant suppression against both pancreatic and patient-derived hepatic tumors with negligible side effects. Together, the study provides a feasible approach for inducing tumor-specific intracellular Ca overload via endogenous Ca redistribution and demonstrates the great potential of Ca disorder therapy in enhancing the sensitivity for tumor radiotherapy.
PubMed: 38690593
DOI: 10.1002/adma.202401222 -
Proceedings of the National Academy of... May 2024Type 1 voltage-activated calcium channels (CaV1) in the plasma membrane trigger calcium release from the sarcoplasmic reticulum (SR) by two mechanisms. In...
Type 1 voltage-activated calcium channels (CaV1) in the plasma membrane trigger calcium release from the sarcoplasmic reticulum (SR) by two mechanisms. In voltage-induced calcium release (VICR), CaV1 voltage sensing domains are directly coupled to ryanodine receptors (RYRs), an SR calcium channel. In calcium-induced calcium release (CICR), calcium ions flowing through activated CaV1 channels bind and activate RYR channels. VICR is thought to occur exclusively in vertebrate skeletal muscle while CICR occurs in all other muscles (including all invertebrate muscles). Here, we use calcium-activated SLO-2 potassium channels to analyze CaV1-SR coupling in body muscles. SLO-2 channels were activated by both VICR and external calcium. VICR-mediated SLO-2 activation requires two SR calcium channels (RYRs and IP3 Receptors), JPH-1/Junctophilin, a PDZ (PSD95, Dlg1, ZO-1 domain) binding domain (PBD) at EGL-19/CaV1's carboxy-terminus, and SHN-1/Shank (a scaffolding protein that binds EGL-19's PBD). Thus, VICR occurs in invertebrate muscles.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Calcium; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Muscles; Inositol 1,4,5-Trisphosphate Receptors; Large-Conductance Calcium-Activated Potassium Channels; Membrane Proteins; Calcium Signaling; Muscle Proteins; Calcium Channels; Membrane Transport Proteins
PubMed: 38687794
DOI: 10.1073/pnas.2317753121 -
Zhonghua Yi Xue Yi Chuan Xue Za Zhi =... May 2024To explore the clinical features and genetic etiology of a child with Central core disease (CCD).
OBJECTIVE
To explore the clinical features and genetic etiology of a child with Central core disease (CCD).
METHODS
A child with CCD who was treated at the Children's Hematology Department of the First Affiliated Hospital of Zhengzhou University in February 2022 was selected as the study subject. Muscle biopsy was performed. Peripheral blood samples were collected from the child and his parents for the extraction of genomic DNA. The child was subjected to whole exome sequencing (WES), and candidate variant was verified by Sanger sequencing.
RESULTS
The child, a 12-year-old boy, had manifested motor retardation, facial weakness, ptosis, pectus carinatum, scoliosis, etc. Muscle biopsy showed that the central nucleus muscle fibers and atrophic muscle fibers were mainly type I. WES revealed that the child has harbored c.10561G>A (p.G3521S) and c.3448T>C (p.C1150R) compound heterozygous variants of the RYR1 gene. Sanger sequencing confirmed that they were inherited from his mother and father, respectively. Based on the guidelines from the American College of Medical Genetics and Genomics, both variants were considered as likely pathogenic (PS4+PM1+PM2_Supporting+PP3;PM1+PM2_Supporting+PM3+PP3).
CONCLUSION
By combining his clinical manifestation and results of muscle pathology and genetic testing, the child was diagnosed with CCD, which may be attributed to the c.10561G>A (p.G3521S) and c.3448T>C (p.C1150R) compound heterozygous variants of the RYR1 gene.
Topics: Humans; Ryanodine Receptor Calcium Release Channel; Male; Child; Heterozygote; Myopathy, Central Core; Exome Sequencing; Mutation; Genetic Testing
PubMed: 38684305
DOI: 10.3760/cma.j.cn511374-20230418-00216 -
Archives of Cardiovascular Diseases 2024With the development of advanced sequencing techniques, genetic testing has emerged as a valuable tool for the work-up of non-ischaemic sudden cardiac arrest (SCA).
BACKGROUND
With the development of advanced sequencing techniques, genetic testing has emerged as a valuable tool for the work-up of non-ischaemic sudden cardiac arrest (SCA).
AIMS
To evaluate the effectiveness of genetic testing in patients with unexplained SCA, according to clinical phenotype.
METHODS
All patients who underwent molecular genetic testing for non-ischaemic SCA with no left ventricular cardiomyopathy between 2012 and 2021 in two French university hospitals were included.
RESULTS
Of 66 patients (mean age 36.7±11.9years, 54.5% men), 21 (31.8%; 95% confidence interval 22.4-45.3%) carried a genetic variant: eight (12.1%) had a pathogenic or likely pathogenic (P/LP) variant and 13 (19.7%) had a variant of uncertain significance (VUS). Among 37 patients (56.1%) with no phenotypic clues, genetic testing identified a P/LP variant in five (13.5%), mainly in RYR2 (n=3) and SCN5A (n=2), and a VUS in nine (24.3%). None of the nine patients with phenotypic evidence of channelopathies had P/LP variants, but two had VUS in RYR2 and NKX2.5. Among the 20 patients with suspected arrhythmogenic cardiomyopathy, three P/LP variants (15.0%) and two VUS (10.0%) were found in DSC2, PKP2, SCN5A and DSG2, TRPM4, respectively. Genetic testing was performed sooner after cardiac arrest (P<0.001) and results were obtained more rapidly (P=0.02) after versus before 2016.
CONCLUSION
This study highlights the utility of molecular genetic testing with a genetic variant of interest identified in one-third of patients with unexplained SCA. Genetic testing was beneficial even in patients without phenotypic clues, with one-fourth of patients carrying a P/LP variant that could have direct implications.
Topics: Humans; Male; Female; Death, Sudden, Cardiac; Phenotype; Adult; Genetic Testing; Middle Aged; Genetic Predisposition to Disease; Predictive Value of Tests; France; Young Adult; Risk Factors; Hospitals, University; Retrospective Studies; Mutation; Ryanodine Receptor Calcium Release Channel; NAV1.5 Voltage-Gated Sodium Channel; Genetic Markers
PubMed: 38670870
DOI: 10.1016/j.acvd.2024.02.005 -
Biosensors Apr 2024The homeostasis of cellular calcium is fundamental for many physiological processes, while the calcium levels remain inhomogeneous within cells. During the onset of...
The homeostasis of cellular calcium is fundamental for many physiological processes, while the calcium levels remain inhomogeneous within cells. During the onset of asthma, epithelial and inflammatory cells secrete platelet-derived growth factor (PDGF), inducing the proliferation and migration of airway smooth muscle (ASM) to the epidermal layer, narrowing the airway. The regulation of ASM cells by PDGF is closely related to the conduction of calcium signals. In this work, we generated subcellular-targeted FRET biosensors to investigate calcium regulation in the different compartments of ASM cells. A PDGF-induced cytoplasmic calcium [Ca] increase was attributed to both extracellular calcium influx and endoplasmic reticulum (ER) calcium [Ca] release, which was partially regulated by the PLC-IPR pathway. Interestingly, the removal of the extracellular calcium influx led to inhibited ER calcium release, likely through inhibitory effects on the calcium-dependent activation of the ER ryanodine receptor. The inhibition of the L-type calcium channel on the plasma membrane or the SERCA pump on the ER resulted in both reduced [Ca] and [Ca] from PDGF stimulation, while IPR channel inhibition led to reduced [Ca] only. The inhibited SERCA pump caused an immediate [Ca] increase and [Ca] decrease, indicating active calcium exchange between the cytosol and ER storage in resting cells. PDGF-induced calcium at the outer mitochondrial membrane sub-region showed a similar regulatory response to cytosolic calcium, not influenced by the inhibition of the mitochondrial calcium uniporter channel. Therefore, our work identifies calcium flow pathways among the extracellular medium, cell cytosol, and ER via regulatory calcium channels. Specifically, extracellular calcium flow has an essential function in fully activating ER calcium release.
Topics: Biosensing Techniques; Platelet-Derived Growth Factor; Calcium; Myocytes, Smooth Muscle; Fluorescence Resonance Energy Transfer; Humans; Endoplasmic Reticulum; Calcium Channels; Calcium Signaling
PubMed: 38667172
DOI: 10.3390/bios14040179 -
Nature Communications Apr 2024Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the...
Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the aging phenotype. Our data indicate that age-associated overexpression of BIN1 occurs alongside dysregulated endosomal recycling and disrupted trafficking of cardiac Ca1.2 and type 2 ryanodine receptors. These deficiencies affect channel function at rest and their upregulation during acute stress. In vivo echocardiography reveals reduced systolic function in old mice. BIN1 knockdown using an adeno-associated virus serotype 9 packaged shRNA-mBIN1 restores the nanoscale distribution and clustering plasticity of ryanodine receptors and recovers Ca transient amplitudes and cardiac systolic function toward youthful levels. Enhanced systolic function correlates with increased phosphorylation of the myofilament protein cardiac myosin binding protein-C. These results reveal BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium.
Topics: Animals; Adaptor Proteins, Signal Transducing; Male; Aging; Mice; Tumor Suppressor Proteins; Myocardium; Ryanodine Receptor Calcium Release Channel; Gene Knockdown Techniques; Endosomes; Calcium Channels, L-Type; Heart; Mice, Inbred C57BL; Humans; Myocytes, Cardiac; Nuclear Proteins; RNA, Small Interfering; Systole; Nerve Tissue Proteins
PubMed: 38664444
DOI: 10.1038/s41467-024-47847-8