This initiates feedforward, allosteric or Ca2+-mediated, T-sarcoplasmic reticular (SR) junctional, voltage sensor-Cav1.1/Cav1.2 and ryanodine receptor-RyR1/RyR2 interaction. We review recent structural, physiological and translational studies on possible feedback actions of the resulting SR Ca2+ release on Nav1.4/Nav1.5 function in native muscle. Finite-element modelling predicted potentially regulatory T-SR junctional [Ca2+]TSR domains. Nav1.4/Nav1.5, III-IV linker and C-terminal domain structures included Ca2+ and/or calmodulin-binding sites whose mutations corresponded to specific clinical conditions. Loose-patch-clamped native murine skeletal muscle fibres and cardiomyocytes showed reduced Na+ currents (INa) following SR Ca2+ release induced by the Epac and direct RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate and caffeine, abrogated by the RyR inhibitor dantrolene. Conversely, dantrolene and the Ca2+-ATPase inhibitor cyclopiazonic acid increased INa. Experimental, catecholaminergic polymorphic ventricular tachycardic RyR2-P2328S and metabolically deficient Pgc1β−/− cardiomyocytes also showed reduced INa accompanying [Ca2+]i abnormalities rescued by dantrolene- and flecainide-mediated RyR block. Finally, hydroxychloroquine challenge implicated action potential (AP) prolongation in slowing AP conduction through modifying Ca2+ transients. The corresponding tissue/organ preparations each showed pro-arrhythmic, slowed AP upstrokes and conduction velocities. We finally extend discussion of possible Ca2+-mediated effects to further, Ca2+, K+ and Cl−, channel types.Skeletal and cardiac muscle excitation–contraction coupling commences with NaThis article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’. Tubular Cav1.1 or Cav1.2 channels then sense the resulting voltage changes and transduce these into a ryanodine receptor (RyR1 or RyR2)-mediated sarcoplasmic reticular (SR) Ca2+ store release. This takes place through either reciprocal allosteric Cav1.1-RyR1 i, that activates troponin, in turn triggering the myofilament action that causes myocyte contraction. However, in addition to these feedforward processes, recent speculations have considered possible feedback modulation of this process for which one example might involve a long loop Ca2+-mediated feedback signalling influencing the Na+ channel itself. This action would be consistent with the function of intracellular Ca2+ in acting as a strategic second messenger known to regulate widespread protein activity through numerous cell types.Skeletal and cardiac muscle excitation–contraction coupling commences with Na2+ and Nav1.4/Nav1.5 structures required for Ca2+ feedback action. These features are next related to results of loose-patch clamp studies exploring for alterations in voltage-dependent Na+ current densities, INa, half-maximal activation and inactivation voltages V1/2, and steepness factors, k, following manipulations of intracellular [Ca2+] in native skeletal muscle fibres and cardiomyocytes. The latter employed the Exchange protein directly activated by cAMP (Epac) and RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate (8-CPT) and caffeine, using as controls the RyR1/RyR2 inhibitor dantrolene, and the RyR1/RyR2 and Ca2+-ATPase inhibitors dantrolene and cyclopiazonic acid (CPA). Genetic disease and other chronic clinical models similarly permitted correlations between reduced INa and [Ca2+]i changes and their specific pharmacological rescue. These included studies on murine gain of function, RyR2-P2328S, catecholaminergic polymorphic ventricular tachycardic (CPVT) and loss-of-function Pgc1β−/− paradigms for pro-arrhythmic metabolic cardiac disease. Findings were related to physiological effects bearing on the consequent velocity of propagation of cell excitability and its translational consequences in intact organs. We finally discuss reports describing hydroxychloroquine (HCQ) challenge and the possibility of a Ca2+ modulation arising from altered Ca2+ release transients consequent upon prolonged AP recovery. We conclude speculating on possible roles for Ca2+-mediated feedback regulation as a general phenomenon that might similarly affect other, Ca2+, K+ and Cl−, channel activity in vivo.We here review recent evidence for such a mechanism as applied to intact native functioning skeletal and cardiac muscle. We explore the possible anatomical and molecular background suggested by theoretical and experimental structural studies for such schemes, then examine evidence for its possible electrophysiological operation in native intact skeletal and cardiac muscle. These insights are translated into experimental and clinical human disease models. This involves first summarizing cell modelling and molecular background evidence bearing on the necessary diffusional and structural conditions of elevated Ca. 22+ release to T-SR triadic or dyadic junctions TSR to levels sufficient to mediate regulatory feedback effects.Structural descriptions of skeletal or cardiac muscle, sarcomeres and their surface, T-tubular and SR membranes localize the processes coupling T-tubular membrane potential changes and SR Caunctions –5. Theseantified a using fe fibres b 6]. T-. T-2+ re2+ indicator results were compatible with such [Ca2+]TSR microdomains with their small dimensions and dispersed nature [2+ sensors localized to T-SR gap regions. This involved Ca2+ sensor conjugation to FKBP12.6 [v1.1 γ1 auxiliary subunit [2+] with those existing in the bulk cardiac [2+ probes [2+] (100 nM) attributable to background SR Ca2+ leak [2+ probes was distributed throughout the muscle fibres with large Ca2+-dependent changes and obvious kinetic delays [2+] more rapidly reached distinct maximum values distinctly higher [2+]. These findings prompted preliminary modelling explorations of the functions of possible dedicated and promiscuous calmodulin pools, respectively, directly regulating channel activity and sensing local Ca2+ to trigger downstream Ca2+/CaM-dependent protein kinase II and calcineurin i, before similarly replicating their eventual first-order SERCA-mediated Ca2+ SR re-sequestration TSR ranging from approximately 0.3–0.4 to 17–20 µM at voltages corresponding to contraction threshold and full activation, respectively. These traversed the entire axial T-SR distance (c) with radial, greater than fivefold, [Ca2+] differences between T-SR junction centre and edge (di–iii). The Ca2+ microdomains robustly persisted through 104-fold variations in Ca2+ diffusion coefficient, and fivefold variations in T-SR distance and diameter, at constant Jinflux, and 10- and 100-fold Jinflux reductions below threshold levels of Ca2+ release. Finally, they also persisted giving T-SR centre [Ca2+] at μM levels at Jinflux corresponding to reported bulk cytosolic [Ca2+]i even in resting as opposed to activated muscle [2+] through typical reported resting bulk cytosolic [Ca2+] values (e). In such simulations, muscle activation by successively larger depolarizing steps recruited correspondingly greater proportions of T-tubular membrane that experienced successively higher, 0.1 to 10 µM, [Ca2+] (f).The resulting Cadistance c with raand edge di–iii. Td muscle –15. Sign] values e. In suc, [Ca2+] f.2+]TSR microdomains generated by RyR-mediated Ca2+ release could then access T-tubular Nav1.4 or Nav1.5 channels in the T-SR junction vicinity. The available evidence could prompt similar future studies extending to cardiac Nav1.5 as opposed to skeletal muscle Nav1.4 channels. Mammalian atrial and ventricular cardiomyocyte junctions between surface and SR membranes differ from those in skeletal muscle in involving peripheral couplings or dyadic rather than triadic junctions i increases from approximately 100 nM to 1–10 µM [+ channel function in studies using in vitro expression systems [2+ buffering (Kd = 4 µM) permitting [Ca2+]i approximately 10 µM to be reached; Nitr-photo-Ca2+ uncaging and activating co-expressed Cav1.2 gave concordant results. All reduced maximum INa in HEK293 cells expressing Nav1.4, or Nav1.5 chimeras with the Nav1.4 CTD, but not those expressing Nav1.5 or Nav1.4 chimeras with the Nav1.5 CTD [INa with [Ca2+]i elevation produced by patch electrode Ca2+-BAPTA or caffeine challenge and 24 h sustained culture medium [Ca2+] elevations, respectively. Inactivation V1/2s was unaffected through all these studies TSR could reach still higher levels, sufficient for regulatory Ca2+-CaM binding. Intact functioning native skeletal muscle fibres and cardiomyocytes both also demonstrated corresponding INa alterations during loose-patch clamp recording under conditions of perturbed Ca2+ homeostasis [2+]i homeostasis arising from the measurement procedures themselves. Altered [Ca2+]i homeostasis could accompany and affect results from other experimental studies, including conventional patch clamp, which further often employ Ca2+-chelating EGTA- and F−-containing pipette solutions. Furthermore, loose-patch clamping additionally permits sequential and multiple recordings to be made with the same standardized pipette before and after pharmacological challenge, and studies to be made in intact in situ skeletal muscle fibres and cardiomyocytes, as opposed to isolated or cultured cells. Features of their reported Na+ current, INa, concurred with those obtained on earlier occasions and with results from other voltage clamp methods applied to mammalian skeletal muscle fibres . Exchange protein directly activated by cAMP (Epac) causes a downstream RyR phosphorylation stimulating SR Ca2+ release (a). In murine skeletal muscle fibres, the Epac activator 8-CPT (1 µM) i) transients, increased amplitudes of [Ca2+]i transients evoked by AP excitation, and spectrofluometrically measurable spontaneous Ca2+ waves suggesting Ca2+ homeostatic changes (e) . F. FINa incordings .. 52+ homeostasis, potentially altering [Ca2+]TSR (a), yielded concordant results i in mammalian skeletal muscle fibres (b) [db) and increases in INa when 0.5 and 2 mM caffeine was added before but not following establishing the loose-patch clamp seal (c). Dantrolene (10 µM) abrogated these effects (e). Applied by itself it induced small INa increases suggesting that it relieved inhibitory effects of a persistent resting Ca2+ release  [Independent pharmacological manipulations of intracellular skeletal muscle fibre CaCa2+]TSR a, yielde results . The RyRe fibres b 61]. E. E2+ homamp seal c. Dantro effects e. Applie release da,ea [622+-ATPase inhibitor CPA (0.1 and 1 µM) has been similarly reported to increase bulk cytosolic [Ca2+]i through a contrasting mechanism inhibiting SR Ca2+ re-uptake (a). However, INa now increased by approximately 30% , with insignificant effects on V1/2 and k (fc), within 1–4 min of introducing CPA-containing but not control solution through a bespoke flow system in identical stable membrane patches in skeletal muscle fibres i microdomain. This could result from SR Ca2+ depletion and would transiently reduce local [Ca2+]TSR. These findings thus together implicate possible local T-SR domains in which [Ca2+]TSR was either increased or reduced in such Nav1.4 down- or upregulation [2+], caffeine and CPA, expected to elevate [Ca2+]i, also reduced mean peak inward INa in murine atrial preparations i could, respectively, increase or decrease at sustained high- or low-firing rates. This could modify Nav1.4/Nav1.5 function either directly or through Ca2+-CaM diminishing or enhancing Nav1.4/Nav1.5 availability for driving AP upstroke and propagation resulting in a form of Ca2+ memory. [Ca2+]i could also increase with compromised SR Ca2+-ATPase activity following ATP depletion. In skeletal muscle, this could result in physiologically adaptive reductions in cell excitability permitting recovery from fatiguing stimulation [2+]i could be physiologically important particularly under conditions of intense and prolonged muscle activation including tetanic stimulation and metabolic exhaustion. For example, tetanic stimulation in amphibian muscle was followed by significantly prolonged decays in [Ca2+]i during recovery, extending even beyond full muscle relaxation. These processes could elevate [Ca2+]i to even approximately 1 µM levels. Both these features were accentuated by increasing stimulation duration i [The presence of a Cautations . In skelhibition . One suctivation . A furthted V1/2 . Finally/2 [2+]i .. 7v1.5 function have been implicated in specific pathological pro-arrhythmic phenotypes. Thus, some cases of the pro-arrhythmic loss of Nav1.5 function Brugada Syndrome (BrS) show mutations in site B of the DIII-DIV linker i elevations (a) [RyR2-P2328S channels correspondingly exhibited enhanced channel activity at a cytoplasmic [Ca2+] of 1 µM as well as greater than 10-fold negative and greater than 1000-fold positive shifts in cytosolic Ca2+ dependences of activation and inactivation, respectively, but normal unit channel conductances [Cardiac utations  are assoutations . Experimevations a 77]. B. BRyR2, uctances .Figure INa potentially could contribute to the CPVT phenotype associated with the RyR2-P2328S variant. Loose-patch clamp measurements in superfused homozygotic RyR2-P2328S atria revealed markedly reduced peak INa to extents comparable to findings in Nav1.5-haploinsufficient Scn5a+/− atria . Activation and inactivation current–voltage relationships were unchanged i transients that in turn might exert feedback effects on Nav1.5  transients. This offers a potential mechanism for the observed downregulation of INa and CV invoking a Ca2+-mediated feedback on Nav1.5 function. Furthermore, (HCQ+AZM) accentuated all these effects of HCQ. It further disrupted AP propagation and induced alternans and torsades-like episodes during forced pacing (g) ..IKr and. 112+-mediated effects on Nav1.4 and Nav1.5 that might exert an in vivo feedback modulation of excitation–contraction coupling in intact native skeletal muscle fibres and cardiac myocytes. Such actions could form a subclass of a range of feedback mechanisms in which local-elevated microdomain [Ca2+] resulting from store Ca2+ release might modify surface membrane channel activity and reduce cell excitability   . They maain [2+]  and infli increases that could arise from RyR-induced Ca2+ release [in vitro adult and neonatal cardiomyocyte preparations and intact perfused hearts demonstrated increased SK2 expression and pharmacologically detectable effects upon AP recoveries paralleling ventricular hypertrophic changes induced by angiotensin II and p21-activated kinase type 1 downregulation [2+]i-mediated SK regulation could then modify membrane potential or excitability and consequent arrhythmogenicity.By contrast, small conductance (SK) K and SK3 ,125, sub release  but are  release ,128. Nor release . Its exa release  though n release . Neverth release . Further release ,134 obse release . Studiesgulation . These p2+]-sensitivity properties also occur in some anion channels. Thus, Ca2+-activated Cl− TMEM16A channels [Finally, [Cachannels , additiochannels , may alschannels  and skelchannels .


Ferroptosis induction through the suppression of glutathione peroxidase 4 (GPX4) and apoptosis-inducing factor mitochondria-associated 2 (AIFM2) has proven to be an effective approach in eliminating chemotherapy-resistant cells of various types. However, a comprehensive understanding of the roles of GPX4 and AIFM2 in acute myeloid leukemia (AML) has not yet been achieved. Using cBioPortal, DepMap, GEPIA, Metascape, and ONCOMINE, we compared the transcriptional expression, survival data, gene mutation, methylation, and network analyses of GPX4- and AIFM2-associated signaling pathways in AML. The results revealed that high expression levels of GPX4 and AIFM2 are associated with an adverse prognosis for AML patients. Overexpression of AIFM2 correlated with elevated mutation frequencies in NPM1 and DNMT3A. GPX4 upregulation modulated the following pathways: GO:0045333, cellular respiration; R-HSA-5389840, mitochondrial translation elongation; GO:0009060, aerobic respiration; R-HSA-9609507, protein localization; and R-HSA-8953854, metabolism of RNA. On the other hand, the overexpression of AIFM2 influenced the following processes: GO:0048704, embryonic skeletal system morphogenesis; GO:0021546, rhombomere development; GO:0009954, proximal/distal pattern formation; and GO:0048732, gland development. This study identifies the high expression of GPX4 and AIFM2 as novel biomarkers predicting a poor prognosis for AML patients. Furthermore, ferroptosis induction may improve the stratified treatment of AML. Acute myeloid leukemia (AML) comprises a group of heterogeneous hematologic malignancies characterized by an increased number of cytogenetic and molecular abnormalities, imposing a profound burden on affected individuals worldwide , 2. DespFerroptosis induction, an iron-dependent form of necrotic cell death triggered by excessive peroxidation of polyunsaturated fatty acids (PUFAs), is being explored as an alternative approach to eradicate apoptosis-resistant cancer cells , 6. FerrPhospholipid (PL) detoxification is typically regulated by the glutathione peroxidase (GPX) family . To date10). This compound neutralizes lipid peroxyl radicals, moderating PL peroxidation. AIFM2 catalyzes the production of CoQ10 via NAD(P)H. Therefore, the AIFM2-CoQ10-NAD(P)H pathway constitutes a parallel system that coordinates with GPX4 and glutathione to maintain the homeostasis of phospholipid peroxidation and ferroptosis [Beyond GPX4, ferroptosis suppressor protein 1 (FSP1), also known as apoptosis-inducing factor mitochondria-associated 2 (AIFM2), has recently been identified as another player in suppressing ferroptosis, particularly in the context of GPX4 knockout. AIFM2 protects against ferroptosis through a glutathione-independent mechanism mediated by ubiquinone  of AML patients .Given that gene mutations are prevalent in AML, we examined the correlation between GPX4 and AIFM2 transcriptional levels and gene mutations using the cBioPortal tool. Results demonstrated that AIFM2 overexpression correlated with a high mutation frequency of nucleophosmin (NPM1) and DNA methyltransferase 3A (DNMT3A) . The comWe examined genes coexpressed with GPX4 and AIFM2 in the TCGA-AML dataset using the cBioPortal tool. The expression of GPX4 exhibited a positive correlation with the upregulation of several genes, including POLR2E, ATP5F1D, NDUFS8, NAA10, PLEKHJ1, C19ORF24, CIAO2B, NDUFB7, SIRT6, MRPS12, UQCR11, C19ORF53, CCDC124, TBCB, and NDUFS6. Similarly, AIFM2 expression was positively correlated with elevated levels of genes such as ABHD11, LINC00899, HOXA10, HOXA6, HOXA3, HOXA7, H2AFY2, CPNE8, RINL, HOXA4, ITM2A, HOXA9, HOXA11, HOXA5, and SHISA4. Subsequently, two lists of the most frequently coexpressed genes, one list of genes related to GPX4 and another related to AIFM2, were compiled to show the results of the pathway enrichment assays using GO and KEGG tools in Metascape. The results illustrated that GPX4 affected the following processes: GO:0045333, cellular respiration; R-HSA-5389840, mitochondrial translation elongation; GO:0009060, aerobic respiration; R-HSA-9609507, protein localization; R-HSA-8953854, metabolism of RNA, GO:0043161, proteasome-mediated ubiquitin-dependent protein catabolic process; GO:0009127, purine nucleoside monophosphate biosynthetic process; GO:0034660, NcRNA metabolic process; GO:0006122, mitochondrial electron transport from ubiquinol to cytochrome c; CORUM:2914, respiratory chain complex I (beta subunit) mitochondria; GO:1990542, mitochondrial transmembrane transport; GO:0009451, RNA modification; GO:0006515, protein quality control for misfolded or incompletely synthesized protein; hsa01200, carbon metabolism; GO:0044743, protein transmembrane import into intracellular organelle; R-HSA-73894, DNA repair; R-HSA-70895, branched-chain amino acid catabolism; R-HSA-9609523, insertion of tail-anchored proteins into the endoplasmic reticulum membrane; GO:0032787, monocarboxylic acid metabolic process; and GO:0042147, retrograde transport from endosomes to Golgi –6C. AIFMCancer cells with dysregulated ROS production, altered iron levels, and increased lipid peroxidation are more susceptible to ferroptosis, a phenomenon that has garnered significant attention in recent decades. While studies across various cancers have highlighted the efficacy of ferroptosis induction, particularly by inhibiting GPX4 and AIFM2, in eliminating apoptosis-resistant cancer cells, the exploration of ferroptosis in the context of AML remains relatively limited. In this study, we conducted a bioinformatics analysis to assess ferroptosis inducibility in AML patients. The findings revealed that high expression levels of both GPX4 and AIFM2 are associated with an adverse prognosis, suggesting the potential therapeutic use of ferroptosis inducers in treating AML patients.Intracellular iron should be tightly controlled, and iron overload induces the overproduction of ROS through the “Fenton reaction” and “Haber-Weiss reaction” , 30. ROSExcessive production of extracellular ROS (mean > 10-fold change) was observed in >60% of primary AML blasts, and this change was not correlated with the molecular subtype of AML. These accumulated ROS promote the proliferation of AML cell lines and primary AML blasts but promote normal CD34+ cell growth to a lesser extent . InteresFLT3 mutation occurs in almost 30–35% of AML, and FLT3-ITD is considered closely related to increased ROS production in AML , 37. ForIDH1 and IDH2 mutations are identified in nearly 10-15% of AML cases , 43. TheDNMT3A mutations, found in approximately 20% of AML cases, particularly in M4-AML and M5-AML, result in the dysfunction of DNMT3A protein and alterations in DNA methylation patterns , 47. NotIron overload contributes to cellular dysfunction by upregulating ROS levels in AML cells. Interestingly, high levels of both GPX4 and AIFM2 protect AML cells from ferroptosis, underscoring the dependency of AML cells on GPX4 and AIFM2 for survival. Moreover, AML patients exhibiting elevated levels of either GPX4 or AIFM2 experienced worse overall survival (OS) compared to those with lower levels of either GPX4 or AIFM2.These findings provide valuable insights into the intricate interplay between genetic mutations, ROS regulation, and the roles of GPX4 and AIFM2 in AML pathogenesis and prognosis.c- [Metabolomics studies have revealed that AML cells exhibit an increased demand for glucose and glutamine, channeling these resources to sustain proliferation advantages compared to normal cells , 50. Addc- . Overallc- .c- inhibition, for instance, effectively suppresses tumor growth in experimental models of fibrosarcoma and diffuse large B-cell lymphoma. Depletion of GSH has been found to increase the survival of chronic lymphocytic leukemia model mice [c-, thereby inducing the ferroptosis of AML cells [Certain ferroptosis inducers (FIs) have entered clinical trials and have shown promise in hematological malignancies. System Xdel mice –58. In adel mice , 60. ThiML cells . In addiML cells . HoweverThese findings underscore the potential of targeting ferroptosis pathways and associated cofactors as a therapeutic strategy in AML, opening avenues for further exploration and development of novel treatments.In this study, the utilization of GO and KEGG online analysis tools allowed us to elucidate the intricate association patterns between GPX4, AIFM2, and their most frequently altered linked genes in AML initiation and prognosis. Several enriched pathways emerged as noteworthy for future investigations, including GO:0045333, cellular respiration, R-HSA-5389840, mitochondrial translation elongation; GO:0009060, aerobic respiration; R-HSA-9609507, protein localization; R-HSA-8953854, metabolism of RNA; GO:0048704, embryonic skeletal system morphogenesis; and R-HSA-5617472, activation of anterior HOX genes in hindbrain development during early embryogenesis. Among these frequently altered correlated genes, R-HSA-8953854, related to the metabolism of RNA, inspired us to discover the correlation between RNA stability and ferroptosis.The apparent paradox observed in the GEPIA and ONCOMINE studies may be attributed to the different acquisition sites of normal samples in the GTEx database and AML patients in the TCGA database. Healthy control samples in the GTEx database are derived from bone marrow, the body’s most abundant source of iron. This suggests that hematopoietic stem cells (HSCs) and differentiated cells in bone marrow must upregulate GPX4 and AIFM2 to protect themselves against ferroptosis under iron overload conditions, maintaining normal hematopoietic function. On the other hand, samples from AML patients in the TCGA dataset were obtained from peripheral blood, an environment with lower iron levels. This seeming paradox prompted consideration for using a uniform sample type to more accurately evaluate the inducibility of ferroptosis.The challenge of chemo-drug resistance in traditional AML treatment has prompted the exploration of novel cell death mechanisms. Ferroptosis, a recently observed form of cell death, holds promise for overcoming chemo-drug resistance and eliminating resilient cancer cells. This study delves into the correlation between the expression of key genes involved in ferroptosis and the occurrence and progression of AML. The elevated expression of GPX4 and AIFM2 emerges as a potential indicator, suggesting their utility as biomarkers for predicting poor prognosis in AML patients. These genes not only serve as prognostic biomarkers but also present themselves as potential targets for treatment in AML. The induction of ferroptosis, combined with traditional treatments, may pave the way for improved stratified therapy in AML. In conclusion, the exploration of ferroptosis in the context of AML opens avenues for innovative therapeutic strategies, offering hope for enhanced treatment outcomes and a step forward in overcoming drug resistance challenges.https://depmap.org/portal/) is a processor used to identify essential genes across cancer cell lines by genome-wide CRISPR and shRNA screening, DepMap has already been validated as a powerful tool useful for discovering the genetic vulnerabilities of cancer cells and identifying potential targets for candidate drug screening. For a candidate gene, the calculated “perturbation score” represents the degree to which its loss prohibits cell proliferation in a selected cell line. A lower score (<0) means that the candidate gene is more likely to be required in the selected cell line. A score = 0 is equivalent to a gene that is not essential whereas a score of −1 corresponds to the median of all common essential genes. We used the DepMap portal to assay the dependence of AML cell lines on GPX4 and AIFM2.The Cancer Dependency Map portal (DepMap) (http://metascape.org) provides an online assay tool, that covers more than 40 independent knowledge bases in an integrated database including functional enrichment, interactome analysis, gene annotation, and a membership search in combination with Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) tools within Metascape [The Metascape portal (etascape . Genes wGEPIA is a web-based tool that enables researchers to perform fast and customizable assays based on TCGA and Genotype-Tissue Expression (GTEx) data . Using GP value was defined as 0.05, the fold change was defined as all.The ONCOMINE database serves as an online analysis tool that enables users to assay the transcriptional level of genes of interest in different cancer types based on a microarray database . GPX4 anhttp://xena.ucsc.edu/) is regarded as a visual integration and exploration tool for use with public and private multiomics datasets. The Xena browser enables researchers to explore data across multiple Xena hubs using a variety of visualization types and analyses [http://www.cbioportal.org/) is designed for the interactive exploration of multidimensional cancer genomics databases. It supports and stores data that include mutations, copy number alterations, mRNA expression changes, and DNA methylation values, as well as clinical parameters. The online analyses tool in cBioPortal allows researchers to interrogate datasets across genes, samples, and data types, allowing them to examine several different biologically and/or clinically relevant hypotheses. Ferroptosis-related gene mRNA expression analyses in adult patients with the FAB subtype of AML and in AML cell lines were conducted by cBioPortal [https://www.ebi.ac.uk/gxa/home), and the expression values were log-transformed and a heat map was generated using GraphPad Prism 7.0.The UCSC Xena (analyses . We usedioPortal . Moreovehttps://www.cbioportal.org/, UCSC Xena at https://xenabrowser.net/.The data that support the findings of this study are available in open public database. These data were derived from the following resources available in the public domain: Cbioportal at Supplementary Figures


In this review, we (1) provide a comprehensive overview of the current state of research on the dynamics and regulation of nuclear Ca2+ signaling in cardiomyocytes, (2) address the role of nuclear Ca2+ in the development and progression of cardiac pathologies, such as heart failure and atrial fibrillation, and (3) discuss novel aspects of experimental methods to investigate nuclear Ca2+ handling and its downstream effects in the heart. Finally, we highlight current challenges and limitations and recommend future directions for addressing key open questions.The nucleus of a cardiomyocyte has been increasingly recognized as a morphologically distinct and partially independent calcium (Ca As the large majority of SERCA is located on the outer nuclear membrane, Ca2+ first needs to diffuse out of the nucleus through NPCs to be taken up again into the lumen of the NE [2+ levels—SERCA function is the single most important factor regulating diastolic [Ca2+]nuc  and [ATP] [2+]nuc increases to a similar extent as [Ca2+]cyto in response to faster pacing, diastolic [Ca2+]nuc rises to about twice the [Ca2+]cyto, suggesting that slower nucleoplasmic CaT kinetics provoke a build-up of Ca2+ when diastole is shortened [2+]nuc overload may lead to the activation of Ca2+-mediated hypertrophic pathways in the nucleus with important structural and functional consequences for the heart.Every cytosolic Calear CaT , left. TCa2+]nuc . Nuclearmplitude . This id nucleus ,27,28. Sf the NE ,29. In aCa2+]nuc . Althougnd [ATP] , as wellnd [ATP] . Due to hortened . If prol2+ overload, especially in conditions of physiological stress. Namely, Voglhuber and Holzer et al. found that in healthy cardiomyocytes, densely packed mitochondria in perinuclear spaces—small areas of cytoplasm adjacent to the nucleus in the longitudinal direction—shape nucleoplasmic CaTs by taking up significant amounts of Ca2+ in close proximity to the nucleus. In contrast, pharmacological inhibition of mitochondrial Ca2+ uptake led to a significant increase in Ca2+ levels in and around the nucleus nuc independent of [Ca2+]cyto [2+ content without influencing global [Ca2+] cyto ,34. Simil [Ca2+] . PLB, thdynamics ,35. Indec stress ,36, the 2+ from the cytoplasm or specialized microdomains such as dyads, [Ca2+]nuc can be additionally and independently regulated via the direct release of Ca2+ from the NE nuc is consistently shown to be mediated by Ca2+ release from perinuclear stores, precaution is needed when choosing an appropriate model to study IP3R signaling, since it is dependent on specific upstream stimuli, cardiomyocyte species and their maturation stage nuc is further sensed by CaN, which activates nuclear factor of activated T cells (NFAT)-dependent transcription  in cardiomyocytes. CaN, as CaMKII, is activated by Ca2+/calmodulin and thereby sensitive to elevated [Ca2+]. Several factors including differences in association rate with Ca2+/calmodulin, regional distribution of CaN, the presence of competing binding partners and endogenous inhibitors allow CaN to evade continuous activation following each contraction [2+ [2+]nuc remains to be addressed.Nuclear Cal stress ,70. The fibrosis ,72. Othefibrosis  and nuclfibrosis . Subedi fibrosis , while Sfibrosis . Similar cytosol . Still, traction . On the traction . Importation  have generally been accepted as drivers of pathological events in cardiomyocytes, studies reporting isolated changes in nucleoplasmic Ca2+ levels are gravely underrepresented in the literature. However, many arguments support the role of [Ca2+]nuc as an important regulator of pathological transcription. One strong argument is that functional and structural alterations of the nucleus precede the development of cardiac pathologies. These include changes in nuclear size and shape in the form of NE invaginations and blebbing, together with changes in expression of Ca2+ handling proteins, most importantly RyR2s, SERCA and IP3Rs cyto and [Ca2+]nuc kinetics. The changes in nucleoplasmic CaTs were more prominent than the cytoplasmic ones, and associated with accumulation of activated CaMKIIδC on the NE. This initial phenotype of preferentially sped up nuclear CaTs prevented the rise of diastolic [Ca2+]nuc and the activation of hypertrophic signaling pathways mediated by IL6R. However, such adaptative response was of limited duration only before transitioning into maladaptive changes, first in the nucleus and then in the cytoplasm as well nuc nuc through newly expressed IP3Rs would continue promoting a vicious cycle of CaN-NFAT pro-hypertrophic transcription nuc . Strikincription .2+ handling, gene expression, structure and function persist, they gradually start manifesting as cardiac dysfunction at organ level and eventually progress to the full-blown HF phenotype  rose, CaT amplitude decreased and the rate of [Ca2+] decline slowed down in both cytosol and nucleus  and slowed CaT kinetics in both subcellular compartments nuc in cardiomyocytes [2+ uptake compared with intrafibrillar mitochondria at baseline and under physiological stress protocol. This may partially explain a disproportionate rise in [Ca2+]nuc compared to [Ca2+]cyto in failing cardiomyocytes at increased stimulation frequencies. On the other hand, upregulation of the mitochondrial calcium uniporter (MCU) under stress conditions elicits protective effects against cardiomyocyte death from Ca2+ overload nuc as a clinically relevant parameter and potential therapeutic target for HF patients.The complex dysregulation of nuclear Ca2+ handling abnormalities, structural remodeling and autonomic neural dysfunction [2+ handling in AF and fail to consider [Ca2+]nuc and its downstream transcriptional targets as contributors to atrial arrhythmogenesis and contractile dysfunction.While ventricular and atrial cardiomyocytes share functional and structural commonalities in the form of ion handling and ECC, there are distinct tissue-specific differences in distribution of ion channel expression and thus electrophysiological properties. AF is the most common sustained cardiac arrhythmia with a lifetime risk of around 20%  and subsfunction . An incrfunction . Similar2+ alterations in atrial cardiomyocytes during AF was published only recently [2+]nuc in AF samples, mediated by slower nuclear [Ca2+] decay kinetics -derived cardiomyocytes under AF stimulation protocol nuc nuc .2+ handling was studied more extensively and it was documented in cardiomyocytes with mutations in sarcomeric and ion handling genes such as TNNT  and PLNroponin; ), while  cytosol . Indeed, cytosol ,35 and C cytosol ,101, it 2+ signals in cardiomyocytes is essential for assessing and understanding compartmentalized Ca2+ fluxes and their roles in ECC and ETC [2+ indicator and experimental model for acquiring signals in the subcellular compartment of interest.Reliable and accurate quantification of subcellular Ca and ETC . For suc2+] changes in different cellular spaces is directly linked to Ca2+ binding affinities of fluorescent Ca2+ dyes, reflected by the dissociation constant (Kd). Kd allows an estimate of the detectable [Ca2+] range and should generally be near the midpoint of the expected [Ca2+] fluctuations [2+ indicators  are typically used for the visualization of [Ca2+] changes in the SR or NE and high affinity Ca2+ indicators  for measuring changes in the cytosolic and nucleoplasmic free [Ca2+]  ,126,127.ell line . Cal-520ell line ; however2+ indicators have several technical shortcomings: (1) their Ca2+ binding affinities and fluorescent properties vary between cytosol and nucleus, (2) they may be differentially sequestered into intracellular organelles or (3) they may leak from the cytoplasm to the extracellular medium via sarcolemmal anion transporters [2+] by determining in situ calibration curves in different types of cardiomyocytes isolated from different experimental animal models. Such curves take into account the effects of a particular cellular environment on indicator properties and allow for a direct comparison between [Ca2+] in different subcellular spaces  have been successfully used in isolated cardiomyocytes in the past [2+ was specifically buffered using a nuclear-targeted, red fluorescent protein-tagged form of the neuronal Ca2+ binding protein calbindin, demonstrating that increased nuclear Ca2+ levels are required for the induction of atrial natriuretic factor expression [2+ in the perinuclear space and showed that buffering perinuclear Ca2+ completely inhibits isoprenaline-induced perinuclear CaN activation in both neonatal and adult myocytes [2+ regulatory proteins can be of great value for studying nucleus-restricted Ca2+ dynamics.To our knowledge, two genetically encoded Cathe past ,153. In myocytes . Further2+ handling—although not as extensively studied as an independent parameter of cardiac remodeling—significantly complements the knowledge on cytoplasmic Ca2+ regulation and alterations, and it augments the likelihood of mitigating pathological cardiac remodeling in the coming years. Future technological breakthroughs in the field of subcellular Ca2+ quantification, cardiac disease modeling and small molecule generation will surely help to better describe remodeling mechanisms and prevent or even halt cardiac disease progression. Understanding nuclear Ca2+ regulation will undoubtedly play an important role in these efforts, as Ca2+ cycling in the nucleus may be the “mastermind” behind the initiation and progression of various cardiac diseases [In this review, we demonstrate that nuclear Cadiseases .


Mutations in the gene encoding the transient receptor potential cation channel, subfamily V, member 6 (TRPV6) have been identified as causative mutations of transient neonatal hyperparathyroidism due to insufficient maternal–fetal Ca2+ transport in the placenta. In this study, we found two novel mutations in subjects that have transient neonatal hyperparathyroidism. TRPV6 carrying the mutation p.Arg390His that localizes to the outer edge of the first transmembrane domain (S1) showed impaired trafficking to the plasma membrane, whereas TRPV6 having the mutation p.Gly291Ser in the sixth ankyrin repeat (AR) domain had channel properties that were comparable those of WT channels, although the increases in steady-state intracellular Ca2+ concentration could have led to Ca2+ overload and subsequent death of cells expressing this mutant channel. These results indicate that the AR6 domain contributes to TRPV6-mediated maintenance of intracellular Ca2+ concentrations, and that this region could play a novel role in regulating the activity of TRPV6 Ca2+-selective channels.Maternal–fetal calcium (Ca Blood Ca2+ levels are finely maintained by the parathyroid hormone (PTH)–vitamin D axis that modulates bone resorption and intestinal Ca2+ absorption. In fetuses and neonates, additional Ca2+ transport occurs to maintain bone mineralization i-dependent inactivation over a longer time range leads to Ca2+ overload and cell death  or 30 Ca bath solution, likely because the Ca2+ concentration was nearly saturated. These results supported our hypothesis that the Gly291Ser mutation causes impaired [Ca2+]i-dependent inactivation across a longer time range that may be too long to allow detection in patch-clamp recordings.Thus, we hypothesized that trafficking of p.Arg390His proteins was impaired. Indeed, similar to the TRPV6 S2 transmembrane domain mutant p.Gly428Arg that was previously shown to have deficient trafficking, p.Arg390His did not localize to the plasma membrane, but was present in total cell lysates  is a member of the TRP channel family that has very high Caectivity . TRPV6 intestine , 14 and ntestine , as wellplacenta , suggest tissues , 17. Des2+ overload due to impaired [Ca2+]i-dependent inactivation i-dependent inactivation, in our patch-clamp recordings we found no significant difference between the mutant and WT upon repetitive application of calcium over 20–30 s. This outcome is a general indicator for the [Ca2+]i-dependent fast/intermediate inactivation. Meanwhile, the intracellular Ca2+ concentration was significantly higher in the mutant-expressing cells than in cells expressing WT. Although the reason for this difference is unclear, Ca2+-selective channels can be inhibited via multiple pathways, including those having fast and slow inactivation kinetics i homeostasis that is crucial for sustained, unidirectional Ca2+ transport.Mutations in the gene encoding the TRP channel TRPV6 cause transient neonatal hyperparathyroidism (TNHP). In this study, we report novel mutations in the TRPV6 S1 and AR6 domains that occurred outside of domains recognized as being mutation hotspots . Unexpectedly, we found that the mutation in AR6 was associated with increased intracellular CaAdditional file 1: Table S1. Oligonucleotide sequences of primers used for Sanger sequencing of the human TRPV6 gene.Additional file 2: Figure S1. Representative time trace of whole cell currents from HEK293T cells expressing WT or p.Arg390His-TRPV6 at a − 60 mV holding potential. Dashed lines indicate zero current level. Whole-cell patch-clamp recordings were carried out 22–24 hours after the transfection using standard bath solution , divalent-free (DVF) solution , NMDG solution , or 30 mM calcium solution , and pipette solution containing 100 mM Cs-aspartate, 40 mM CsCl, 1 mM MgCl2, 10 mM EGTA, 5 mM HEPES (pH 7.2 with CsOH). Osmolarity was confirmed to be ~290 mOSm/kg. Data were sampled using an Axopatch 200B amplifier and pCLAMP software . Membrane potential was clamped at − 60 mV.Additional file 3: Figure S2. Localization of TRPV6 proteins in the plasma membrane. Plasma membrane proteins of transfected HEK293 cells were biotinylated, collected with streptavidin beads. Myc-TRPV6 proteins were analyzed by Western blotting using anti c-myc antibody. The 80 kDa band was observed for cells expressing WT and p.Gly291Ser. Anti-Na+-K+-ATPase antibody was used for the loading controls. Plasma membrane protein biotinylation were carried out 22 hours after initiating transfection. Transfected cells were incubated twice with 0.5 mg/ml EZ-link-NHS-LC-biotin  at 37 ºC for 10 min. The biotinylation was stopped with quenching buffer  before the cells were from the dish by adding the lysis buffer . Biotinylated proteins were collected by the magnetic beads  under manufacture’s instruction. Western blotting was performed with 7.5 % TGX gel , and the signal was visualized by the Light capture system . Anti-Na+,K+-ATPase antibody with HRP  was used for the loading control with 1/1000 dilution.


The uncontrolled increase in intracellular calcium concentration ([Ca2+]i), leading to the development of Ca2+ dysregulation events and related excitable and synaptic dysfunctions, is a consolidated hallmark of AD onset and possibly other neurodegenerative diseases. Since RyRs contribute to increasing [Ca2+]i and are thought to be a promising target for AD treatment, the goal of this review is to summarize the current level of knowledge regarding the involvement of RyRs in governing neuronal function both in physiological conditions and during the onset of AD.Research into the early impacts of Alzheimer’s disease (AD) on synapse function is one of the most promising approaches to finding a treatment. In this context, we have recently demonstrated that the Abeta42 peptide, which builds up in the brain during the processing of the amyloid precursor protein (APP), targets the ryanodine receptors (RyRs) of mouse hippocampal neurons and potentiates calcium (Ca There is considerable evidence to indicate that Abeta oligomers affect Ca2+ entry through N-methyl-D-aspartate receptors (NMDARs) [2+]i increase involves the upregulation of ryanodine (RyRs) and inositol triphosphate receptors (IP3Rs) and increasing amounts of evidence suggest that they are impaired during AD onset. These two receptors are not equally distributed along the ER membrane and, while IP3Rs are more concentrated at the somatic region, RyRs prefer the portion of ER membrane that is in the vicinity of the synaptic sites [2+]i and directly responsible for governing synaptic function. Three different RyR isoforms are known and, while it was previously thought that RyR1 was principally expressed in skeletal muscle, RyR2 in the heart, and RyR3 in neurons [2 [3 [2+ channels and are characterized by high permeability [1 for calcium is 110 pS and that this can increase to over 700 pS for potassium [2+]i becomes evident. The Ca2+ released from RyRs can be triggered by Ca2+ entry through the Ca2+ channels located in the plasma membrane; a phenomenon called “Ca2+ induced Ca2+ release” (CICR) that is typically observed in cardiac myocytes where RyR2 is the principally expressed isoform. VGCCs i that are induced by Abeta42 and improves the excitable and synaptic properties of hippocampal neurons [2 adrenoreceptors [In AD mouse models, it has recently been shown that the accumulation of Abeta oligomers prevents the proper interaction between RyRs and the intracellular stabilizer proteins calstabins, preventing the complete closure of RyRs, which assume a leaky conformational state and generate continuous slow release and accumulation of calcium in the cytoplasm . It has for RyR2 . In our  neurons  (Figure  neurons , but its neurons . New cla neurons ,85,89, w neurons ,93. This neurons . On the Studies performed in recent years indicate that RyRs may play a role in the onset of AD and other neurodegenerative diseases . The dev


While important, other Ca2+ channels, particularly T-type could contribute to pressure regulation within defined voltage ranges. This study examined the role of one T-type Ca2+ channel (CaV3.1) using C57BL/6 wild type and CaV3.1−/− mice. Patch-clamp electrophysiology, pressure myography, blood pressure and Ca2+ imaging defined the CaV3.1−/− phenotype relative to C57BL/6. CaV3.1−/− mice had absent CaV3.1 expression and whole-cell current, coinciding with lower blood pressure and reduced mesenteric artery myogenic tone, particularly at lower pressures (20–60 mmHg) where membrane potential is hyperpolarized. This reduction coincided with diminished Ca2+ wave generation, asynchronous events of Ca2+ release from the sarcoplasmic reticulum, insensitive to L-type Ca2+ channel blockade . Proximity ligation assay (PLA) confirmed IP3R1/CaV3.1 close physical association. IP3R blockade  in nifedipine-treated C57BL/6 arteries rendered a CaV3.1−/− contractile phenotype. Findings indicate that Ca2+ influx through CaV3.1 contributes to myogenic tone at hyperpolarized voltages through Ca2+-induced Ca2+ release tied to the sarcoplasmic reticulum. This study helps establish CaV3.1 as a potential therapeutic target to control blood pressure.The arterial myogenic response to intraluminal pressure elicits constriction to maintain tissue perfusion. Smooth muscle [Ca This “myogenic response” was first described by Bayliss3 and is intimately tied to arterial depolarization, the activation of voltage-gated Ca2+ channels and the concomitant rise in cytosolic Ca2+ concentration ([Ca2+]i), which complexes with calmodulin driving myosin light chain phosphorylation4. Three subclasses of voltage-gated Ca2+ channels (CaV1–3), are encoded in the mammalian genome and each displays unique voltage-dependent properties5. In arterial smooth muscle, CaV1.2 (L-type) Ca2+ channels are principally responsible for extracellular Ca2+ entry and their blockade is notable for attenuating a range of constrictor responses, including those induced by pressure6.Smooth muscle cells in the arterial wall actively contract to intravascular pressure, maintaining organ blood flow under dynamic conditions2+ channels are classified as high-voltage-activated and dominate the setting of smooth muscle [Ca2+]i when intravascular pressure is elevated and arteries depolarized7. Their activity, however, markedly drops with hyperpolarization as pressure is reduced or when endothelial cell K+ channels are activated by selected agents8. As [Ca2+]i remains a determinant of tone, even in the hyperpolarized state, it follows that other Ca2+ channels, ones with a leftward voltage profile, should be expressed in vascular smooth muscle6. T-type Ca2+ channels display activation/inactivation properties decidedly more negative to their L-type counterparts10. Two subtypes (CaV3.1 and CaV3.2) are expressed in vascular smooth muscle, the latter linked to the activation of large-conductance Ca2+-activated K+ (BK) channels and a negative feedback response limiting arterial constriction10. This leaves CaV3.1 as to enabling myogenic tone at hyperpolarized voltages12, presumptively through a mechanism where Ca2+ influx directly contributes to the cytosolic Ca2+ pool or indirectly triggers sarcoplasmic reticulum Ca2+ release in the form of asynchronous Ca2+ waves15.L-type CaV3.1 channels enable myogenic tone development in mouse mesenteric arteries. This work entailed the use of C57BL/6 wild type and CaV3.1−/− mice, and the integrated use of cellular , tissue (pressure myography and rapid Ca2+ imaging) and whole animal (metabolic caging and blood pressure) techniques. Initial assays confirmed the absence of CaV3.1 in mesenteric arterial smooth muscle of knockout animals. This absence aligned with a drop in systemic blood pressure and reduced myogenic tone at lower pressures compared to controls. Subsequent experiments revealed that Ca2+ wave generation was attenuated in CaV3.1−/− arteries as this channel no longer resided near IP3R1 and that IP3R blockade in C57BL/6 arteries produced a CaV3.1−/− phenotype. These findings highlight a role for CaV3.1 in myogenic tone development and hemodynamic control through the triggering of sarcoplasmic reticulum Ca2+ waves. They additionally reveal the potential therapeutic value of CaV3.1 in the control of hypertension.This study explored whether and by what mechanisms CaV3.1−/− (in-house colony) mice (16–20 weeks of age) were humanely euthanized via CO2 asphyxiation. The mesentery was removed rapidly and placed in cold PBS solution (pH 7.4) containing (in mM): 138 NaCl, 3 KCl, 10 Na2HPO4, 2 NaH2PO4, 5 glucose, 0.1 CaCl2, and 0.1 MgSO4. Third and fourth-order mesenteric were isolated and cut into 2–3 mm segments and transferred to fresh cold PBS.Animal procedures were approved by the animal care committee at the University of Western Ontario ensuring compliance with federal and provincial standards and under consideration of the ARRIVE guidelines. Male C57BL/6  or CaV3.1−/− mice (n = 3) and DNA was extracted using the QIAamp Fast DNA Tissue Kit (QIAGEN). 200 ng of DNA was amplified via polymerase chain reaction (PCR) using previously published CaV3.1 primers: C57BL/6 F, 5ʹ-ATACGTGGTTCGAGCGAGTC-3ʹ; WT R, 5ʹ-CGAAGGCCTGACGTAGAAAG-3ʹ; CaV3.1−/− R, 5ʹ-CTGACTAGGGGAGGAGTAGAAG-3ʹ16. Gel electrophoresis was performed on PCR products at 95 V for 1 h on a 1.5% agarose gel. Gel was then imaged using the Bio-Rad ChemiDoc™ MP Imaging System and Image Lab 6.1 software (Bio-Rad).Ear tissue was collected from C57BL/6 and Ca2, 10 glucose and 10 HEPES with 1 mg/mL bovine serum albumin . Vessels were then exposed to a two-step digestion process that began with 14-min incubation (37 °C) in media containing 0.5 mg/mL papain and 1.5 mg/mL dithioerythritol, followed by 10-min incubation in media containing 100 μM Ca2+, and collagenases type H (0.7 mg/mL) and type F (0.4 mg/mL). Following incubation, tissues were washed repeatedly with ice-cold isolation medium and triturated with a fire-polished pipette. Liberated cells were stored in ice-cold isolation medium for use the same day.Third and fourth-order mesenteric arteries were placed in an isolation medium  containing (in mM): 60 NaCl, 80 Na-glutamate, 5 KCl, 2 MgClV3.1 expression was assessed in mesenteric arterial smooth muscle cells isolated from C57BL/6 and CaV3.1−/− mice. Briefly, isolated cells were fixed onto a microscope cover glass in PBS (pH 7.4) containing 4% paraformaldehyde and 0.2% Tween 20. Fixed cells were blocked  with a quench solution (PBS supplemented with 3% donkey serum and 0.2% Tween 20) and subsequently incubated overnight  with rabbit anti-CaV3.1 primary antibody diluted in quench solution (1:100). In the following morning, cells were washed 3× in PBS-0.2% Tween 20 and then incubated  in a PBS-0.2% Tween 20 buffer containing Alexa Fluor 488 donkey anti-rabbit IgG-secondary antibody (1:200). After further washing, isolated cells and whole-mount preparations were mounted with Prolong Diamond Antifade Mountant with DAPI. Immunofluorescence was detected through a 63× oil immersion lens coupled to a Leica-TCS SP8 confocal microscope equipped with the appropriate filter sets. Smooth muscle cells isolated from C57BL/6 cerebral arteries were used as CaV3.1 positive controls. Secondary antibody controls were performed and were negative for nonselective labelling.Ca2+ currents in smooth muscle cells isolated from mesenteric arteries. Cell averaged capacitance was 12–18 pF. Recording electrodes  were fashioned from borosilicate glass using a micropipette puller  and backfilled with pipette solution containing (in mM): 135 CsCl, 5Mg-ATP, 10 HEPES, and 10 EGTA (pH 7.2). To attain a whole-cell configuration, the pipette was lowered onto a cell while applying negative pressure to rupture the membrane and garner intracellular access. Cells were voltage clamped  and subjected to − 90 mV followed by 10 mV voltage steps (300 ms) starting from − 50 to 40 mV in a bath solution consisting of (mM): 110 NaCl, 1 CsCl, 1.2 MgCl2, 10 glucose, and 10 HEPES plus 10 BaCl2 (charge carrier). Delineation of vascular voltage-gated Ca2+ channels was performed by introducing 200 nM nifedipine to block L-type channels, followed by 50 µM Ni2+ to selectively block CaV3.2 channels without affecting CaV3.1. Currents were recorded using an Axopatch 200B patch-clamp amplifier  at room temperature (~ 22 °C). Data were filtered at 1 kHz, digitized at 5 kHz, and stored on a computer for offline analysis with Clampfit 10.3 software . Current/voltage relationships were plotted as peak current density (pA/pF) at the different voltage steps.Conventional patch-clamp electrophysiology was utilized to measure voltage-gated Ca2 consumption and CO2 production, the cumulative amount of food and water consumed, respiratory exchange rate, activity (number of infrared beam breaks), and sleep epochs were measured in C57BL/6 and CaV3.1−/− mice. Chambers were kept at 24 ± 1 °C with airflow of 0.5 L/min, and animals had ad libitum access to food and water. Metabolic parameters were recorded every 10 min for 48 h. Data from the same 12-h interval for each mouse was selected to standardize the data processing.Comprehensive Lab Animal Monitoring System (CLAMS) interface using Oxymax software  was utilized to measure the differences in OV3.1−/− mice using the non-invasive CODA tail-cuff system , as described, and following recommendations of the Subcommittee of Professional and Public Education of the American Heart Association Council on High Blood Pressure Research18. To minimize anxiety, the animals were properly acclimatized in advance, and a heating platform was used to maintain body temperature. Mice (n = 5) were subjected to 25-min recordings daily for one week, and the weekly averages were recorded.Blood pressure measurements were performed on awake C57BL/6 and Ca2, balance air) at 37 °C containing (in mM): 119 NaCl, 4.7 KCl, 1.7 KH2PO4, 1.2 MgSO4, 1.6 CaCl2, 10 glucose, and 20 NaHCO3. To limit the influence of endothelial receptors, air bubbles were passed through the vessel lumen (1 min). Arterial diameters were monitored using an automated edge detection system  and a 10× objective. Arteries were equilibrated at 15 mmHg, and contractile responsiveness was assessed by a brief (≈ 10 s) application of 60 mM KCl. After equilibration, intraluminal pressure was elevated from 20 to 100 mmHg in 20 mmHg increments for 10 min each, and arterial diameters were monitored in Ca2+ PSS (control), and in the presence of 0.3 µM nifedipine (L-type Ca2+ Channel blocker) alone or with 50 µM 2-APB or 3 µM xestospongin C (IP3R blockers) or 150 nM kurtoxin (CaV3.x blocker). A final passive diameter assessment was conducted in Ca2+-free + 2 mM EGTA. Arteries that did not respond to superfused KCl (60 mM) or were insensitive or hypersensitive to pressure were excluded from experimentation. Percentage of maximum tone and incremental distensibility were calculated as follows:Isolated mesenteric arteries were cannulated in an arteriograph and superfused with physiological salt solution  or presence of 0.3 µM nifedipine. Changes in diameter in response to each concentration were recorded and percentage of maximum constriction was calculated as follows:2+ PSS, and Endothelium-denuded mesenteric arteries were cannulated in a pressure myograph as explained above. Following the high potassium challenge, arteries were equilibrated at 60 mmHg, then subjected to the administration of phenylephrine (PE) into the bath solution. Increasing concentrations of PE (in M) 102+ indicator Fluo-8 and placed on the stage of a Nikon swept-field confocal microscope with enclosed Agilent 3B laser attached to Andor camera (iXon Ultra). Fluo-8 working solution (19.1 µM) was freshly prepared by dissolving 5 μL of stock solution (1.91 mM) in 5 μL pluronic acid plus 490 μL HBSS buffer consisting of (in mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2 10 HEPES, and 10 Glucose (pH 7.4). Incubation was done for 75 min at 37 °C in the dark. Vessels were then cannulated and equilibrated at an intraluminal pressure of 15 mmHg for 15 min in Ca2+ PSS solution. Intraluminal pressure was then raised to 60 mmHg, and Ca2+ waves were recorded in the presence and absence of 0.3 µM nifedipine (± 150 nM kurtoxin) or 50 µM 2-APB or 3 µM xestospongin C. Fluo-8-loaded arteries were excited at 488 nm and emission spectra at 510 nm viewed through a 60× water immersion objective (1.2 WI) and were monitored and analysed using Nikon NIS Elements software (AR 4.20.01). To limit laser-induced tissue injury, image acquisition was set to 45 s at 5 fps. A series of regions of interest (1 × 1 µm), created within the analysis software, was placed on 10 successive cells that were in sharp focus using the first visibly loaded smooth muscle cell as a starting point. A Ca2+ wave was defined as local fractional fluorescence , permeabilization  and blocking . Cells were then washed with PBS then incubated with primary antibodies  in Duolink antibody diluent solution at 4 °C overnight. Cells were then incubated with secondary Duolink PLA PLUS and MINUS probes for 1 h at 37 °C. If target proteins are within 40 nm of each other, synthetic oligonucleotides attached to the probes hybridize enabling their subsequent amplification and binding to complementary fluorescent oligonucleotide sequences, detected using Leica-TCS SP8 confocal microscope.To test the spatial proximity of Can indicates the number of cells, arteries, or animals. Power analysis was performed a priori to assess the sample size sufficient for obtaining statistical significance. No more than 1 experiment was performed on cells/tissues from any given animal. Where appropriate, paired/unpaired t-tests or two‐way analysis of variance (ANOVA) were performed to ascertain significant differences in mean values to a given condition/treatment. P values ≤ 0.05 were considered statistically significant.Data are expressed as means ± SD, and V3.1 and IP3R1 were purchased from NovusBio and Alomone Laboratories, respectively. PCR kit was obtained from Qiagen. Secondary antibody, Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L), and 2-APB were obtained from ThermoFisher. Duolink PLA detection kits, nifedipine, PE hydrochloride, DAPI, donkey serum kurtoxin, and all other chemicals were obtained from Sigma-Aldrich unless stated otherwise. In cases where DMSO was used as a solvent, the maximal DMSO concentration after application did not exceed 0.5%. Please see the Fluo-8 was acquired from Abcam. Primary antibodies against CaV3.1 channels was confirmed by PCR, immunohistochemical analysis and conventional whole-cell patch-clamp electrophysiology. In detail, PCR amplification of C57BL/6 and Cav3.1−/− mouse DNA with CaV3.1 primers resulted in different sized PCR products. C57BL/6 mice had a PCR product of 288 bp corresponding to the wild type allele, while the PCR product for CaV3.1−/− mice was seen at 385 bp . This analysis aligned with whole-cell electrophysiology, which noted dampened CaV3.1 activity in smooth muscle cells from CaV3.1−/− mice relative to C57BL/6 controls (P = 0.013). Note, CaV3.1 activity was measured by first monitoring the total inward Ba2+ current, the collective sum of CaV1.2, CaV3.1, and CaV3.2 currents20. Based on past studies, nifedipine and Ni2+ were then applied to abolish CaV1.2 (L-type) and CaV3.2 (T-type) activities, respectively, and the residual current was then assigned to CaV3.122. The current–voltage relationship of each Ca2+ channel is illustrated in Fig. V3.1 current in green), with peak current (at + 10 mV) summarized in Fig. V3.1−/− mice.Genetic deletion of Ca2 consumption, CO2 production, the respiratory exchange rate, along with cumulative food and water consumed during normal activity. No significant difference was observed among C57BL/6 and CaV3.1−/− mice  in physiological saline solutions, with Ca2+ and Ca2+-free + 2 mM EGTA. Traces and summative data are presented in Fig. V3.1−/− arteries displayed reduced myogenic tone compared to C57BL/6 controls, a trend that was statistically significant at lower intraluminal pressures . Arterial distensibility, a surrogate of vessel stiffness and defined as the percentage change in passive arteriolar diameter per change in intravascular pressure23, was comparable among the two groups of arteries  at a frequency of 9 waves/cell/min, each with a duration of 3–4 s  along with firing frequency (P < 0.0001) by 55% and 65%, respectively  and Ca2+ wave frequency (P = 0.0002) by 80% and 75%, respectively. Owing to the non-selective nature of 2-APB, and its reported inhibition of store-operated Ca2+ entry, the previous control experiments were repeated in the presence of xestospongin C, a selective IP3R blocker. Similar to 2-APB, xestospongin C attenuated the number of firing cells (P = 0.011) and Ca2+ wave frequency (P = 0.002)  ± 2-APB (50 µM). Of particular note, was the nifedipine-resistant tone that was present in C57BL/6 but not CaV3.1−/− arteries, particularly at lower intravascular pressures  consistent with IP3Rs and Ca2+ waves playing a role in its genesis  was used in place of 2-APB in C57BL/6 and it generated a contractile , and Ca2+ wave . Also note, whereas nifedipine had no impact on Ca2+ wave generation  and firing frequency (P = 0.002)).Given the preceding observation, a final set of functional experiments were performed to address the contributory role of IP3. This foundational response is now known to set basal tone in key organs and stabilizes organ perfusion as blood pressure fluctuates. Further, this response has been intimately tied to arterial depolarization and the rise in [Ca2+]i enabled by graded Ca2+ entry principally through L-type Ca2+ channels9. Vascular L-type Ca2+ channels are encoded by the CaV1.2 α1 pore-forming subunit whose steady-state voltage-dependent properties are shifted rightward to more depolarized potentials25. Recent work has revealed that L-type Ca2+ channels are not alone in vascular smooth muscle and that T-type Ca2+ channels are also expressed, with CaV3.1 being key to this investigation9. Its steady-state activation profile is hyperpolarized, and as such enables Ca2+ entry when L-type Ca2+ channels are deactivated. In theory, Ca2+ entry via T-type Ca2+ channels could impact tone development by directly contributing to the cytosolic Ca2+ pool or by acting locally and indirectly to trigger Ca2+ waves. Ca2+ waves are slow asynchronous events that spread from end to end and whose triggering is tied to the opening of sarcoplasmic reticulum IP3Rs by IP3 and Ca2+24. Using a CaV3.1−/− model, we tested whether Ca2+ entry through this particular T-type channel does indeed facilitate myogenic tone at hyperpolarized voltages and if this functional response is coupled to the governance of Ca2+ waves.Bayliss first described the intrinsic ability of resistance arteries to constrict to a rise in intravascular pressureV3.1 began with experiments to confirm the absence of CaV3.1 in mesenteric arterial smooth muscle cells from genetic deletion mice  modification in CaV3.1−/− mice. Second, protein analysis using immunohistochemistry showed that surface expression of CaV3.1 was notably lacking in CaV3.1−/− but not C57BL/6 cells. These observations aligned with the results from the third, functional approach (whole-cell electrophysiology), which revealed that the nifedipine/Ni2+ resistant Ba2+ current, previously ascribed to CaV3.122 was also absent in mesenteric arterial smooth muscle cells harvested from genetic deletion mice. The absence of this T-type Ca2+ channel coincided with a reduction in systolic and diastolic blood pressure, a finding consistent with a role in hemodynamic control. Past observations are limited, with one study reporting no difference in blood pressure, although values were unrealistically low for both CaV3.1−/− and C57BL/6 mice26. A second showed blood pressure trending lower in CaV3.1−/− mice, along with a more substantive reduction in blood pressure variability27. While the mechanism driving the blood pressure change is unclear, it’s reasonable to assert a role for diminished myogenic tone, an idea we tested in isolated mesenteric arteries across a full pressure range. Consistent with expectations, a reduction in myogenic tone was observed in CaV3.1−/− arteries, particularly at lower pressure , although without defining mechanism20. Finally, in using this genetic deletion model, acknowledgement of other cardiovascular effects is key, in particular bradycardia26 and impaired blood pressure regulation through impaired NO formation32.In contextualizing the preceding observations, one should recognize past inferential work linking T-type Ca2+ waves are slow-spreading, end-to-end events initiated by a stimulus that drives the release of Ca2+ from the sarcoplasmic reticulum34. The initiation and spread of these asynchronous events are tied to IP3R, Ca2+-permeable channels whose activation depends on IP3 and Ca2+ binding to cytosolic sites25. Past work in rat cerebral arteries has shown that Ca2+ waves are present at low intravascular pressure and that frequency rises as pressure is elevated into the lower physiological range24. Pharmacological attenuation of Ca2+ waves through IP3R blockade or impairment of store refilling results in diminished pressure-induced constriction particularly at low intravascular pressure when arteries are more hyperpolarized24. Respectful of these results, it follows that low threshold CaV3.1 channels provide the Ca2+ needed to trigger Ca2+ waves and foster myogenic tone when L-type Ca2+ channels are decidedly less active. This concept was tested three ways, the first examining Ca2+ wave generation in CaV3.1−/− arteries, the second ascertaining if CaV3.1 colocalized with IP3Rs, and the final determining if Ca2+ wave inhibition in C57BL/6 mice results in a CaV3.1−/− contractile phenotype. Findings in Fig. 2+ wave generation is robust in control mesenteric arteries as defined by the number of firing cells and the rate of Ca2+ waves per firing cell. Analogous to past work in rat cerebral arteries, nifedipine didn’t impact Ca2+ wave generation, consistent with L-type Ca2+ channels playing little role in initiating or maintaining these events24. Ca2+ waves were significantly reduced in CaV3.1−/− arteries and abolished in control arteries by 2-APB, and xestospongin C, IP3R inhibitors, findings consistent with this T-type Ca2+ channel driving sarcoplasmic reticulum dependent events. These intriguing findings aligned with results from the PLA that note a close spatial association between CaV3.1 and IP3R. In detail, this assay involves the binding of primary antibodies to two target proteins and then uses secondary antibodies with conjugated DNA strands which form a circular DNA template for amplification if proteins are < 40 nm apart10. The amplified product, detected as bright red puncta, is clearly visible in Fig. 2+ flux via CaV3.1 should be sufficient to open IP3Rs. In light of both results, final experiments assessed whether reduced Ca2+ wave production in C57BL/6 vessels generate a functional phenotype akin to CaV3.1−/− arteries. In this regard, we monitored myogenic tone in mesenteric arteries  at rest and following treatment with nifedipine alone or with 2-APB or xestospongin C  through IP3R blockade. This loss of tone parallels a similar loss in tone, and likewise Ca2+ waves in C57BL/6 mice when kurtoxin, a CaV3.x blocker was applied on top of nifedipine, an L-type Ca2+ channel blocker  2-APB treatment having no impact on global [Ca2+] while reducing myogenic tone35; (2) 2-APB only dilating arteries which prior to treatment were generating Ca2+ waves36; and (3) Ca2+ waves abrogation corelating with reduced myosin light chain phosphorylation particularly at lower pressure24.CaV3.1−/− and C57BL/6 arteries were evident at lower pressures (20–60 mmHg), the same trend was present at higher pressures, although without statistical significance. This finding is perhaps unsurprising as L-type Ca2+ channels rise to dominate [Ca2+]i as arteries depolarize with pressurization. Second, while our work noted Ca2+ wave insensitivity to L-type Ca2+ channel blockade, like the cerebral vasculature24, it lies in contrast to cremaster arterioles where nifedipine attenuated Ca2+ wave formation37. This discrepancy suggests there may be mechanistic uniqueness among vascular beds, which to date is unappreciated. Alternatively, one could potentially argue the higher concentration of nifedipine (1 μM) used on cremaster arteries may be blocking CaV3.1 and consequently the triggering of IP3R40. This perspective is consistent with electrophysiology observations noting that T-type Ca2+ channels are partially blocked by low micromolar nifedipine42.Two final points in this study require further consideration. First, while differences in myogenic tone between CaV3.1−/− mice have lower blood pressure, and mesenteric arteries display diminished myogenic tone compared to controls. Second, immunohistochemical analysis reveals that CaV3.1 lies within 40 nm of IP3R1, and when this arrangement is genetically disrupted, arteries generate fewer Ca2+ waves. Third, a pharmacological blockade of IP3Rs in C57BL/6 arteries produces a phenotype similar to CaV3.1−/− vessels, that being diminished myogenic tone at lower intravascular pressure. By establishing a clear sequential relationship between CaV3.1, Ca2+ waves and myogenic tone, this study advances the understanding of vascular contractility and highlights a new target for therapeutic control. In this regard, one could provocatively suggest that development of selective CaV3.1 blockers could be of value in the management of hypertension.This study presents three key findings, summarized in Fig. Supplementary Information.


The spleen is one of the most frequently affected organs in blunt abdominal trauma. Since Upadhyaya, the treatment of splenic trauma has undergone important changes. Currently, the consensus is that every splenic trauma presenting with hemodynamic stability should be initially treated nonoperatively, provided that the hospital has adequate structure and the patient does not present other conditions that indicate abdominal exploration. However, several topics regarding the nonoperative management (NOM) of splenic trauma are still controversial. Splenic angioembolization is a very useful tool for NOM, but there is no consensus on its precise indications. There is no definition in the literature as to how NOM should be conducted, neither about the periodicity of hematimetric control, the transfusion threshold that defines NOM failure, when to start venous thromboembolism prophylaxis, the need for control imaging, the duration of bed rest, and when it is safe to discharge the patient. The aim of this review is to make a critical analysis of the most recent literature on this topic, exposing the state of the art in the NOM of splenic trauma. The rate of failure of nonoperative management (NOM) is currently lower than 10%,,Although indications for NOM of splenic trauma have been extensively studied in the literature, there are few studies defining the evolution of the selected patients, and there are few widely used protocols for the conduction of NOMThe aim of this review is to critically analyze the evolution of NOM of splenic trauma, it’s predictors of failure, and the main controversies in the literature so far.Until the 1980s, splenic lesions were routinely treated with splenectomy,,The first reports of NOM for splenic injuries came from studies with children-It is currently agreed that every hemodynamically stable patient may initially undergo NOM if some resources are available in the hospitalAs NOM was increasingly instituted, cases of failure of NOM began to become more evident. In order to identify which patients were most at risk, several studies sought to determine the predictors of failure of NOM.,Most patients with low-grade splenic injuries (I to III according to AAST) are successfully submitted to NOM. However, even patients with high-grade lesions (IV-V AAST) may initially undergo NOM, provided they are hemodynamically stableSeveral factors have been studied in the literature, including age, degree of splenic injuryIn a study conducted by Olthof et al.In a more recent review, Olthof et al.15,37 that involved over 10,000 patients showed that 85% to 95% of failure of NOM cases occurred within the first 3 days of observation. The most recent study from 2008 also showed that monitoring for an additional 2 days made it possible to diagnose 1.5% more failure of NOM. Therefore, it is recommended that patient observation should be performed for 3 to 5 days, allowing identification of up to 97% of failure of NOM.Just as important as knowing the population at risk for failure of NOM is to know when the failure usually occurs. A 2005 study,Splenic embolization has been considered a tool to decrease failure of NOM cases. In a 1995 study,There is controversy when analyzing the meaning of tomographic blush in the main references on the subject. The presence of blush is of great importance in the Western Trauma Association guidelines, as it indicates angioembolization in grade IIIOnce decided by splenic embolization, the results regarding proximal (splenic artery trunk) or distal  embolization must be analyzed. Proximal embolization is usually used when bleeding is diffuse, when the patient’s hemodynamic condition is borderline, or when vascular anatomy is unfavorable. In cases where bleeding is focal, distal embolization is used. It is noteworthy that, although there are no prospective studies, proximal embolization is faster, and has lower failure and complication rates compared to distalThe main complications of splenic embolization are splenic infarction, abscess, hyperthermia and hyperalgesia without associated splenic infarction. Such complications may occur in up to 47% of casesA large national retrospective studyAlthough the spleen is one of the organs most commonly affected in blunt abdominal trauma, NOM is the method of choice for most patients, and angiography has a clear role in reducing the failure of NOM rates, there are still several controversies in the literature. While the indication of NOM is well established, and its main predictors of failure have been extensively studied, there are not many published norms related to the evolution of these patients. In other words, although there is consensus on which patients should undergo NOM, little is known about how to follow them once the nonoperative strategy has been established.How should hematimetric control be performed? How long should patients be kept at rest and when should they return to activities? When to start venous thromboembolism (VTE) prophylaxis? Is there a need for control imaging? What is the impact of splenic embolization on its immune function? These are some topics on which there is no consensus yet. Although extremely important for the proper follow-up of patients undergoing NOM, there is no strong evidence to suggest any specific recomendation. Thus, it is necessary to perform a critical analysis of the works already published.Most studies referring to hematimetric control discuss the frequency with which hemoglobin and hematocrit levels should be obtained. In fact, there is currently not enough evidence to guide a specific regimenThe relationship between early patient mobilization and failure of NOM is discussed as well. In a recent study, Teichman et al.,,,,,54 involving more than 36,000 blunt trauma patients undergoing NOM showed that early introduction (within 48h of the injury) of VTE chemoprophylaxis was associated with lower rates of deep vein thrombosis and pulmonary thromboembolism, without causing significant difference in the need for blood transfusion, the incidence of failure of NOM or mortality. Thus, despite being a controversial topic, the literature recommends early initiation of chemoprophylaxis in the first 48-72h, considering the magnitude of the splenic lesion and the risk of patient bleeding individually to decide the ideal moment to initiate VTE prophylaxis.Regarding the beginning of VTE prophylaxis, there is no consensus either. The incidence of thromboembolic complications in patients with solid organ injuries can reach 4.5%, resulting in morbidityAnother aspect discussed in the NOM of splenic lesions concerns the threshold of transfused blood units that would define failure of NOM and, therefore, the need for surgical intervention or embolization. There is no consensus in the literature about this value. Fodor et al. indicates that transfusion of 2 or more red blood cell concentrates should already be indicative of failure of NOM,,There is also discussion about the need for control imaging after the onset of NOM of splenic injury. It is currently accepted that vascular abnormalities may appear later than the first CT scan. One study showed that pseudoaneurysms can appear even in grade II and III,Asplenia is a condition associated with immune deficiency and can result in fulminant infections with encapsulated germs. Despite being a rare condition - occurs in only about 2% -, patients submitted to splenectomy are at risk of developing these infectionsNOM of blunt splenic injuries can be indicated in every hemodynamically stable patient, provided there are adequate resources in the hospital and there are no associated lesions that require surgical exploration. Once decided for NOM, it is imperative to identify the main predictors of failure. Although not contraindicating NOM, the presence of these factors should alert the physician, motivating him to closely monitor the patient. Unfortunately, there is no consensus on various aspects of follow-up of these patients, and protocols are not uniform in most services. Therefore, the treatment should be individualized according to the best available evidence.


Colletotrichum species, such as Colletotrichum gloeosporioides and Colletotrichum cuspidosporium. In recent years, Colletotrichum aenigma was reported as a causal agent of Grape anthracnose in China and South Korea. Peroxisome is an important organelle in eukaryotes, which plays a very important role in the growth, development, and pathogenicity of several plant-pathogenic fungal species i, but it has not been reported in C. aenigma. In this work, the peroxisome of C. aenigma was labeled with a fluorescent protein, using green fluorescent protein (GFP) and red fluorescent protein (DsRED and mCherry) as reporter genes. Via Agrobacterium tumefaciens-mediated transformation (AtMT), two fluorescent fusion vectors to mark the peroxisomes, with GFP and DsRED, respectively, were introduced into a wild-type strain of C. aenigma. In the transformants, bright dots of green or red fluorescence in hyphae and spores could be seen in the strains labeled peroxisome. The nuclei labeled by the same method showed bright round fluorescent spots. In addition, we also combined fluorescent protein labeling with chemical staining to show the localization more clearly. The ideal peroxisome and nuclear fluorescence-labeled C. aenigma strain was obtained, which provided a reference for the study of its growth, development, and pathogenicity.Anthracnose is one of the most widespread and destructive diseases in grapes. Grape anthracnose can be caused by various  China is the largest producer and consumer of fresh grapes in the world. There are reports indicating that after conducting field investigations in the main grape-producing areas of Xinjiang and Hebei, it was found that the grape-planting area of China has remained stable at over 10 million mu over the past five years. The production of grapes in China continued to increase, reaching 13.667 million tons in 2018 and becoming the world’s largest producer. The distribution of grape-planting areas in China is concentrated, with the main production areas located in historically advantageous planting areas, such as Xinjiang, Shanxi, Hebei, and Shandong. The planting areas of five provinces have reached 4.629 million mu, accounting for 42.6% of the total area.Colletotrichum species, such as Colletotrichum gloeosporioides and Colletotrichum cuspidosporium [Colletotrichum aenigma was also reported as a causal agent of Grape anthracnose in China and South Korea [Grape anthracnose, also known as late rot, is an important disease impacting grapes that is mainly caused by the fungus osporium . The disth Korea ,3,4,5.Magnaporthe oryzae, Botrytis cinerea, Fusarium graminearum, and so on [C. aenigma. The peroxisomes were found to be induced and greatly proliferated at the early stages of the conidial germination and infection of M. orzyae [C. aenigma.Peroxisome is a kind of monolayer-coated organelle with active metabolisms and highly dynamic changes in eukaryotes, which is mainly involved in the metabolism of fat, the production and scavenging of reactive oxygen species, etc. Additionally, it has been reported that peroxisome is essential for the pathogenicity of many phytopathogenic fungi, including nd so on . However. orzyae . TherefoAequorea victoria in 1962, has stable, intuitive, and convenient fluorescence properties and is the most widely used fluorescent protein at present. Red fluorescent protein (RFP) was isolated from Discosoma sp. by MATZ et al. in 1999. The different variants of red fluorescent protein show different spectral and physicochemical properties, emitting various colors of fluorescence. For example, mCherry emits 610 nm fluorescence under 587 nm excitation light and cherry-red under a fluorescence microscope. It has the advantages of rapid ripening and good monomer properties and has a wide range of applications. Calcofluor white (CFW) and DAPI are two kinds of dyes that display blue fluorescence under a fluorescent microscope [Magnaporthe oryzae [Fusarium oxysporum f. sp. niveum [Botrytis cinerea [C. aenigma.Fluorescent protein labeling is an effective method of studying promoter activity, gene expression dynamics, cellular and subcellular localization of proteins, and the growth and development of organisms . Green fcroscope . Wang J e oryzae . Fluores. niveum  and BotrC. aenigma via Agrobacterium tumefaciens-mediated fungal transformation (AtMT). Using the fluorescently labeled strains, the peroxisomal dynamics and nuclear distribution in C. aenigma were detected under a fluorescent microscope. In addition, we obtained a library of T-DNA insertion transformants for mutant selection. Consequently, we provide a tool and basic data for the further study of the organelle biogenesis, fungal growth, development, and pathogenicity of C. aenigma.In this present work, GFP-PTS1 , DsRED-PC. aenigma and its transformants were cultured on the complete medium (CM) [Colletotrichum aenigma (Genbank Taxonomy ID: 1215731) strain NH-8 was isolated from a grape vineyard in Ninghai country, Zhejiang province, China, identified by the morphology and the sequences of ITS, GAPDH, and tubulin, and kept in the fungal strain store in Zhejiang Academy of Agricultural Sciences (accession number ZN-05827).ium (CM)  in 28 °CC. aenigma by AtMT [A. tumefaciens strains were cultured in a YEB liquid medium containing 50 μg/mL ampicillin, 50 μg/mL rifampicin, and 50 μg/mL kanamycin in 28 °C darkness for 2 days with 180 r/min [C. aenigma spores which were cultured on CM medium at 28 °C for 5 days, we then prepared the spore suspension into 1 × 106/m L. Afterward, the spore liquid and A. tumefaciens were co-cultured on IM medium [PHMGA, pHMR1 , and pKD by AtMT  transfor80 r/min . ObtainiM medium  at 23 °CTaking CA-HMR1 transformants as an example to detect genetic stability is shown in C. aenigma wild strain was used as a negative control, and the corresponding plasmid DNA was used as a positive control. The PCR products were detected by electrophoresis with 1.0% agarose gel under 150V for 20 min. The primers used were listed in The transformants of each generation were detected by PCR to determine whether they contained the hygromycin resistance gene and the corresponding fluorescent protein gene. Genomic DNA was extracted by the CTAB  method. The transformants with strong fluorescence expression, clear localization, and stable subculture were selected, and a small number of hyphae and spores were selected to observe and record the fluorescence localization using a laser confocal microscope (ZEISS LSM780).The red fluorescence was observed at an excitation wavelength of 543 nm and an emission wavelength of 570–630 nm. The green fluorescence was observed at an excitation wavelength of 488 nm and an emission wavelength of 495–550 nm. In addition, the blue fluorescence was observed at an excitation wavelength of 405 nm and an emission wavelength of 410–480 nm.CFW and DAPI Staining: 10 μL 10 μg/mL CFW staining solution was added to avoid light staining for 5 min, and the blue fluorescence produced by the cell wall was observed . Using aC. aenigma with a hole punch, and finally transferred to the new CM medium plate with 3 repeats for each strain. After 3 days of dark culture at 28 °C, the colony diameter was measured and photographed, and the difference in growth rates was statistically analyzed. The growth rate was calculated with V = (D − d)/T; V is the growth rate (mm/d); D is the average diameter of the colony (mm); d is the diameter of the inoculated plaque (mm); and T is the growth time (d).The tested strain was inoculated on the CM medium plate for 3 days, and then the round mycelial blocks with a diameter of 0.5 cm were obtained at the colony edge of the transformant and strain To harvest the spores, 4 mL of sterilized distilled water was added to each CM culture plate. Then the colony surface was gently scraped with a disposable coating stick to wash the spores into the water. The spore suspension was filtered with three layers of sterilized lens wipes with 3 repeats for each treatment. The spore concentration was calculated on a Hemocytometer, and the spore generation of each strain was calculated and compared.C. aenigma were prepared in 106 mL−1. Afterward, 20 fresh grapes and a few leaves were wiped briefly with 75% alcohol, naturally dried, and acupunctured using sterile needles, then they were inoculated by dropping the suspension on the acupunctured sites with pipettes.The pathogenicity was tested by acupuncture inoculation (acupuncture 0.5 cm). The spore suspensions of the transformants and wild strain of The mycelial blocks were also used in inoculation by placing them on the acupuncture site of the leaves. The sterile water was used as the control, and a group of non-invasive inoculation groups was set up. The treatment was repeated 3 times in each group. After inoculation, the fruits were cultured in the dark at 28 °C and we added 10 mL of sterile water with sterilized cotton or filter paper to maintain humidity. The incidence of fruits and leaves was observed and recorded.The partial transformants of CA-HMGA A, CA-HMRC. aenigma transformants may be related to their multinucleated characteristics. The cells of transformants with T-DNA integrations in partial nuclei can emit red fluorescence. However, in the process of subculture, the untransformed nuclei occasionally replaced and occupied the cells, and resulted in the loss of fluorescence. Therefore, the stable strains can be obtained via successive subcultures and selection.In order to determine the genetic stability of transformants, five CA-HMR1 transformants (T0 generation) expressing high green fluorescence were randomly selected and subcultured for 7 generations, and the fluorescence of each generation was observed. The results showed that among the 25 transformants of T1 generation, 6 transformants lost fluorescence. In the T2 generation, 19 transformants could emit light, and 6 of 25 transformants disappeared after subculture to the T7 generation. However, for the transformants whose fluorescence disappeared, the fluorescence could not be recovered after 2 generations. The results showed that the subculture led to the loss of fluorescence of some of the transformants. The genetic instability of C. aenigma was obtained at the same time as the fluorescence localization. In order to test the effect of foreign gene introduction on the phenotype of transformants, 5 transformants of CA-HMGA, CA-HMR1, and CA-mCherry-H2B were randomly selected to observe the colony morphology (C. aenigma, and a few transformants showed slower growth rates. As a result, we concluded that the phenotypic variation of transformants caused by the random insertion of foreign genes is a low-probability event, and most fluorescent transformants can simulate the dynamics of organelles in the wild type. At the same time, specific phenotypic transformants can be obtained through screening, which lays a foundation for the identification and study of functional genes.Through transformation, a random insertion transformation library of rphology A, spore rphology C, growthrphology B, and paC. aenigma, CA-HMGA, and CA-mCherry-H2B were selected to test virulence. After inoculating healthy grape leaves for three days, the inoculation site began to turn black (C. aenigma disappear.Secondly, several strains of rn black A. Seven rn black B, and itrn black D and thern black C. At theThe successfully verified transformants were selected to observe the fluorescence expression and localization under confocal fluorescence microscopy. Under the action of the H3 promoter, mCherry-H2B fusion protein showed a high brightness of red fluorescence in both mycelium and spores. The red fluorescence was concentrated on the round spots inside the cells, and the location and size of the spots were consistent with the characteristics of nuclear localization. We also found that with the advance of spore germination, the nucleus in the spore gradually disappeared, while new nuclei appeared in the newly formed appressorium A. These MPG1 gene promoter, the green fluorescence of GFP-PTS1(C. aenigma. We can also clearly see the green fluorescence and red fluorescence localization in the appressorium. Peroxisome slowly appears in the germ tube over time, so we can observe the number and dynamic changes of peroxisome.Under a laser confocal microscope, the transformants of CA-HMGA, CA-HMR1, and CA-HMGA-PNMcherry were observed. Under the action of the  GFP-PTS1A, red fl GFP-PTS1B, or red GFP-PTS1C of DsREC. aenigma and the cell walls of the three transformants CA-HMGA, CA-HMR1, and CA-mCherry-H2B were stained with CFW for co-localization observation (In order to observe the location of different cell structures and organelles more clearly and intuitively, the appressorium of ervation . As obseNeurospora crassa, M. oryzae, and so on [M. oryzae and F. graminearum, there are fewer studies on the molecular mechanism of the C. aenigma infection and the labeling of organelles with fluorescent proteins. In this study, the peroxisome and nucleus of C. aenigma were successfully labeled by GFP and the red fluorescent proteins DsRED and mCherry.Using fluorescent protein labeling to track the growth, development, and pathogenicity of fungal strains is an effective method in plant and animal pathogenic fungi. At the same time, fluorescent proteins are also widely used to study the spatial-temporal expression of genes, and the cellular and subcellular localization of proteins. At present, many different fluorescent proteins have been applied in fungi, such as nd so on . HoweverThe number of nuclei and the dynamic processes of nuclear division, movement, and degradation are some of the important contents of fungal developmental biology .M. oryzae, there is a precise regulation of nuclear division and distribution to daughter cells. As a multinucleated fungus, the mechanism of nuclear division, separation, and regulation of C. aenigma may be more complex. The formation and biochemical metabolism of peroxisome are essential to the infection of M. oryzae and B. cinerea [C. aenigma, which provides a reference and source of material for studying the growth, development, pathogenic process, and molecular mechanism of the fungus.In the process of the appressorium formation of  cinerea . The fluH3 and MPG1 promoters from M. oryzae could be used to trigger the gene expression in C. aenigma. The fluorescent protein genes under the two promoters were abundantly expressed in the cells of C. aenigma in hyphae and conidia. The expression intensity was adequate for labeling the peroxisomes and the nuclei. Our results thus gave more options for effective fungal promoters regarding the study of fungal genes. In order to show the localization more clearly, we also combined applying the localization of fluorescent proteins with chemical fluorescent staining. We provided important data for the study of cell structure, gene function, and protein-protein interaction.The appropriate promoter to trigger the expression of the fluorescent proteins is the basis of fluorescent labeling. The results showed that C. aenigma is comparable to that of filamentous fungi, such as M. oryzae and F. graminearum, but the genetic stability of the transformants of C. aenigma is lower, which may be due to the multinucleation of C. aenigma. In addition, the colony morphology, growth rate, sporulation, and pathogenicity in most of the transformants were not significantly different from those of the wild type. The results indicated that the growth and development of most of the transformants were not affected by the transformation processes, or the expression of hygromycin and fluorescent proteins.The transformation efficiency of Therefore, most of the fluorescent strains would be capable of tracing the organelles in further cellular research. A very small portion of the transformants showed phenotypic variation, in which the T-DNA may have been inserted into some important gene loci. These transformants were important in identifying the functional genes in fungal molecular biology. Thus, our work provides a useful tool for the study of molecular and cellular biology in fungi.


Previous studies have shown reduced surgical site infections (SSIs) with β-lactam prophylaxis compared with non-β-lactam alternatives; however, variations in timing and dosing have been reported. Few studies have investigated the impact of preoperative antibiotic timing or dosing on reported SSI differences between regimens.A multi-center, retrospective cohort study was performed utilizing available data from elective colorectal surgeries in Michigan. Adult patients receiving surgical prophylaxis between July 2012 and June 2021 were included. Exclusions included emergent or urgent surgeries and missing follow-up and antibiotic information. The primary objective was to evaluate the incidence of SSIs between patients receiving surgical prophylaxis with β-lactam antibiotics with those who received non-β-lactam alternatives. A multivariable logistic regression model tested the association of SSIs and covariates including antibiotic dosing and timing.A total of 28,899 procedures were included. Of these, 25,875 received β-lactam prophylaxis and 3,024 received non-β-lactam alternatives. Patients in the β-lactam cohort received higher recommended dosing , timing , combined recommended dose and timing , and had a lower unadjusted incidence of SSIs . Multivariable logistic regression showed β-lactam prophylaxis was associated with a reduction in SSIs (adjusted odds ratio (aOR) 0.79; 95% CI 0.64 to 0.98; p =0.033) (Table 1). Recommended dosing  and timing  were not associated with any change in SSIs. Older age clean/contaminated procedures, and colectomies were associated with a decreased SSI risk. Higher body mass index, corticosteroid use, contaminated and dirty procedures, ASA scores 3-5, and open procedures were associated with an increased risk.The use of β-lactams for surgical prophylaxis in elective colorectal surgery procedures was associated with decreased SSIs. Significant differences in compliance with recommended prophylaxis dosing and timing between cohorts was not associated with changes in SSIs.Robert K. Cleary, MD, FASCRS, FACS, Intuitive Surgical Inc: Honoraria