The occurrence of gene mutation is a major contributor to the initiation and propagation of acute myeloid leukemia (AML). Accumulating evidence suggests that genes encoding cohesin subunits have a high prevalence of mutations in AML, especially in the t subtype. Therefore, it is important to understand how cohesin mutations contribute to leukemogenesis. However, the fundamental understanding of cohesin mutation in clonal expansion and myeloid transformation in hematopoietic cells remains ambiguous. Previous studies briefly introduced the cohesin mutation in AML; however, an in-depth summary of mutations in AML was not provided, and the correlation between cohesin and AML1-ETO in t  AML was also not analyzed. By summarizing the major findings regarding the cohesin mutation in AML, this review aims to define the characteristics of the cohesin complex mutation, identify its relationships with co-occurring gene mutations, assess its roles in clonal evolution, and discuss its potential for the prognosis of AML. In particular, we focus on the function of cohesin mutations in RUNX1-RUNX1T1 fusion. It has become apparent that acute myeloid leukemia (AML) is induced by the cooperative action of deregulated genes that alter cell proliferation and differentiation. As compared with other AML cytogenetic groups, patients with t8; 21) AML had a relatively favorable prognosis, and most of them (>85%) achieved complete remission (CR) with high-dose cytosine arabinoside (Ara-C). However, approximately 30% of the t AML patients relapsed within 1 year, and the overall survival at 5 years was close to 51%   can trigger the generation of the aberrant oncogenic fusion protein AML1-ETO , 4. Howeconserved regulatory elements (CREs) interactions by “looping out” the intervening chromatin segment to activate or inhibit the gene expression , RAD cohesin complex component (RAD21), and cohesin subunit SA (STAG1/STAG2) –17. Therpression , 30. Furpression . Mutatiopression , therefopression , 12, 34,The data of cohesin subunits mutations were collected from 16 articles. The percentage of cohesin mutations with different characteristics is displayed in As shown in de novo AML  AML. STAG2 was the most commonly mutated gene in adult AML. Besides, in agreement with our results, recent studies by Metzeler et al., demonstrated that STAG2 also tended to be more frequent in secondary AML (sAML) compared with novo AML . For difnovo AML . Intrigu), suggesting that cohesin mutations resulted in a decrease or loss of the cohesin function. Almost all SMC1A and SMC3 mutations were found to be missense, which was consistent with previous studies  AML, so we next asked whether there were specific mutant form and hotspot in the mutated cohesin gene. We found that the majority of STAG2 and RAD21 mutations were nonsense and frameshift in all the AML subtypes  AML patients . RAD21 iThe C-terminal plays an important role in the functions of STAG2 protein, as it is the location of many phosphorylation sites and potentially the nuclear localization signal , 41. TheMany leukemia genes are found to be infrequently mutated, and AML patients typically have more than one driver mutation , 43. FurSeveral studies have shown that mutations in the cohesin complex genes are often found in NPM1 mutation and CEBPAbi AML cases , 49, 50.Contradictory results have been reported regarding the correlation between genes in the cohesin complex and other mutations. Mutations in Ras-family oncogenes including ASXL1 or BCOR, and FLT3-ITD were enriched in leukemia patients carrying the cohesin mutation , 37. Howde novo AML  and disease-free survival (DFS) in novo AML . Regardlnovo AML . Howevernovo AML , 54, 55.novo AML , 56. In novo AML . Howevernovo AML . Thus thet al. found that recurrent cohesin mutations did not affect the prognostic results of t AML in 10 pediatric and 6 adult patients  AML, Faber patients . Besidespatients . Howeverpatients , 46. Amopatients . This reThese findings all emphasize that cohesin have different functions under various clinical features in the biological mechanism. Taken together, the AML subtype, karyotype, age, and co-mutated genes should be considered together with cohesin mutation as prognostic factors. In particular, it is worth evaluating the prognostic significance of cohesin mutations in t AML.de novo AML  was enhanced, and differentiation of HSPCs was impaired as a result of cohesin mutation or deletion , 66–68. Stag2 and Runx1 play an important role in regulating chromatin looping and transcription in hematopoiesis, and cohesin-Runx1 deficiency can induce myeloid-skewed expansion of HPSCs and myelodysplastic syndromes (MDS)  and P2  promoters of Runx1 (conserved regulatory elements (CRE)/promoter of Runx1 in the human leukemia K562 cell line  . In mousof Runx1 , 77, 78.ell line . It playh embryo , 79. Monsh model . Neverthsh model . Moreovepatients . These eCohesin mutations were prevalent in AML with RUNX1 abnormality , especiaIn this review, we summarized and analyzed cohesin mutations in AML, especially in t subtype, from previous studies. The conclusion and outlook are as follows:We found each subunit of cohesin mutated in different characteristics with different percentage. RAD21, SMC1A, and SMC3 mutations were remarkably higher in t than the other AML subtypes. STAG2 mutation occurred chiefly on adult AML and sAML. For FAB subtype, RAD21 and SMC3 are mainly found in FAB M2. SMC1A was common found in male. Such differences arise because each individual member of the cohesin complex has different influence and function in different characteristics of AML by different ways and mechanisms.Most of the current studies confirmed that cohesin can act as a growth regulator of HSCs. Whether cohesin gene mutation leads to protein functional activation or inactivation is unclear. Although mutated cohesin perturbed the balance between self-renewal and differentiation, no evidence demonstrated that the impaired cohesin function could independently lead to AML, suggesting that cooperative genetic/epigenetic alterations are involved in AML development. So far, co-mutations of cohesin with other genes are still controversial and the biological functions of cohesin are not yet fully understood in AML.The clinical outcomes of cohesin mutations are associated with the AML type, karyotype, and co-mutated gene. No specific therapeutic strategies targeting cohesin mutated AML have been described. In addition, most of the studies indicated that cohesin mutations are pre-leukemic and early genetic events in human leukemogenesis. Considering the four core subunits of cohesin associated with different prognosis and mutation frequencies, their specific functions in AML should be also investigated in depth.The mechanism with respect to cohesion subunits mutation is still unestablished in AML, especially in the t AML. How cohesin subunits mutation cooperates with AML1-ETO, thereby promoting leukemic transformation should be illustrated in future studies. Further understanding of the relationship between cohesin and AML1-ETO may provide unprecedented insights into the pathogenesis of AML.Conducting research on above questions would hopefully provide significant progress in diagnosis classification, prognostic stratification, and therapeutic strategies to determine AML risks in the near future.CH wrote the manuscript. XG and YL revised the manuscript critically. JZ, EY, and LZ prepared the figures and revisions. LY contributed to study design, writing, and editing. All authors contributed to the article and approved the submitted version.This work was supported by “Major New Drug Development Project” from Ministry of Science and Technology of China (2019ZX09201002003), Natural Science Foundation of Shenzhen University General Hospital (SUGH2020QD008), State Key Program of National Natural Science of China (82030076), National Natural Science Foundation of China , Beijing Natural Science Foundation (7202186), Shenzhen Science and Technology investigation project (JCYJ 20190808123005569 and JCYJ 20190808163601776), China Postdoctoral Science Foundation (2018M640824), and Science and Technology Foundation of Shenzhen (JCYJ20200109113810154).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RyRs are responsible for the release of Ca2+ from the SR during excitation–contraction coupling in striated muscle cells. Recent revolutionary advancements in cryo-electron microscopy have provided a number of near-atomic structures of RyRs, which have enabled us to better understand the architecture of RyRs. Thus, we are now in a new era understanding the gating, regulatory and disease-causing mechanisms of RyRs. Here we review recent advances in the elucidation of the structures of RyRs, especially RyR1 in skeletal muscle, and their mechanisms of regulation by small molecules, associated proteins and disease-causing mutations.Ryanodine receptors (RyRs) are huge homotetrameric Ca In striated muscle cells, when cells are stimulated, Ca2+ is rapidly released from intracellular Ca2+ stores of the sarcoplasmic reticulum (SR), a process known as excitation–contraction coupling (E–C coupling) and concentration of total Ca2+ released in the cytoplasm immediately reaches > 0.1 mM , Ca2+ release channels in the SR membrane  , which is gated via physical association with L-type voltage-dependent Ca2+ channels   Endo . All RyR2+, Mg2+, ATP and caffeine) and proteins   provides a number of near-atomic protein structures. Although the achievable resolution by cryo-EM depends on the molecular weight of the sample, there are several reported structures with resolution better than 2 Å , the huge mushroom-like structure rotates according to the arrow shown in the Fig. 2+. Thus, resolving the Ca2+-induced conformational changes that occur in CICR is currently difficult based on these structures. A structure in the open state with only Ca2+ bound will be necessary for a complete understanding of the gating mechanism of CICR, although Ca2+ alone only partially activates the channel under physiological condition.Figure 2+, ATP and caffeine) have been identified in RyR1  in Central domain and one carbonyl oxygen atom (T5001O) in CTD  exhibited similar Ca2+ sensitivity to the WT, while RyR2-E3847D (RyR1-E3893D) showed only minimal binding at higher Ca2+ concentrations. This suggests that RyR1-E3967 can more flexibly coordinate with Ca2+ than RyR1-E3893. This is supported by the structure, showing that the side chain of RyR1-E3893 has an extremely bent structure towards the bound Ca2+  and seems to be unsuitable for the coordination of Ca2+ binding. Nevertheless, RyR1-Q3970 and RyR2-Q3925 are very important residues, since RyR1-Q3970K causes central core disease  have reduced Ca2+ sensitivity  are involved in Ca2+-dependent inactivation, through direct or indirect mechanisms . In the tryptophan mutant, the interaction with isoleucine is lost, resulting in an increase in Ca2+ sensitivity -C4193W and RyR2(human)-A4607P) near the caffeine-binding sites greatly increase Ca2+ sensitivity by altering the hydrophobic interaction  and as an inhibitor at micromolar concentrations of Ca2+ (Ca2+-CaM)   is pulled out from inside RyR1 and rotates approximately 60 degrees  is a member of the EF-hand CaAlthough structures of RyR2 in complex with CaM have been determined at near-atomic resolution, high resolution structures of RyR1 in complex with CaM have not been reported. Thus, the mechanism of the activating effect of apo-CaM on RyR1 still remain unknown. High-resolution structures of RyR1 with bound CaM are expected to be generated in future research.2+ sensitivity for activation; Ca2+ sensitivity for inactivation; attainable maximum activity, i.e., gain) using live-cell Ca2+ imaging and [3H]ryanodine binding assays revealed qualitative and quantitative differences in alterations between mutations. Then, they observed tertiary structure of RyR1 determined by cryo-EM and the interactions that are important for channel regulation were predicted. Van Petegem et al. used X-ray crystallography of mutant RyR1 channels  simulation is a powerful tool to test how mutations affect the structure. Zheng et al. performed MD simulations of RyR1 or RyR2 mutants using the cryo-EM-determined structure of the NTD tetramer  have been determined in two different states (apo-CaM and Ca2+-CaM), and the molecular mechanism of Ca2+-CaM binding to RyR2 has also been clarified. However, this is just the first step in understanding the structure–function relationship of RyRs. In fact, the fundamental mechanisms of RyR, neither CICR nor DICR is still unclear just from the recent structures. It should be noted that the current models of RyRs by cryo-EM have major problems, in that 30% (1500 residues) of the regions of the receptor have no model building completed or amino acid residues identified, especially in the cytoplasmic region (Clarke and Hendrickson Since the first three reports of the near-atomic structures of RyR1 in 2015, there have been great advancements in our understanding of the structure–function relationship of RyRs. The binding sites of regulatory small molecules such as Ca


These changes may form a vicious cycle of positive feedback to promote cardiac dysfunction and arrhythmias. In HF rabbit cardiomyocytes investigated in this study, the inhibition of CaMKII, late Na+ current (INaL), and leaky ryanodine receptors (RyRs) all attenuated the prolongation and increased short-term variability (STV) of action potential duration (APD), but in age-matched controls these inhibitors had no or minimal effects. In control cardiomyocytes, we enhanced RyR leak (by low [caffeine] plus isoproterenol mimicking CPVT) which markedly increased STV and delayed afterdepolarizations (DADs). These proarrhythmic changes were significantly attenuated by both CaMKII inhibition and mitochondrial ROS scavenging, with a slight synergy with INaL inhibition. Inducing LQT by elevating INaL  caused markedly prolonged APD, increased STV, and early afterdepolarizations (EADs). Those proarrhythmic ATX-II effects were largely attenuated by mitochondrial ROS scavenging, and partially reduced by inhibition of CaMKII and pathological leaky RyRs using dantrolene. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) bearing LQT3 mutation SCN5A N406K, dantrolene significantly attenuated cell arrhythmias and APD prolongation. Targeting critical components of the Na+–Ca2+–CaMKII–ROS–INaL arrhythmogenic vicious cycle may exhibit important on-target and also trans-target effects . Incorporating this vicious cycle into therapeutic strategies provides novel integrated insight for treating cardiac arrhythmias and diseases.Cardiomyocyte Na Mordiseases , 20, 61,ses i)  and RyR [Ca2+]i) . Thus, t[Ca2+]i) , 45, 68 on-target effect but also trans-target effects in the loop as it may reduce the feedback activation of the vicious cycle. The on-target drug effects have been the focus of research in recent decades and showed benefits in HF. (1) Selective Na+ channel inhibitors  were shown to reverse the increased INaL and the prolongation of the APD in HF i–[Ca2+]i–ROS–CaMKII–RyR leak feedback interactions to proarrhythmic electrophysiological changes in HF rabbits  from 0.125 to 0.25 then 0.5 mmol/L. Finally, ventricular myocytes were kept in Tyrode’s solution at room temperature until use.Enzymatic isolation of left ventricular cardiomyocytes from New Zealand White rabbits  was performed as previously described . BrieflyHF was induced in New Zealand White rabbits  by aortic insufficiency and 4 weeks later by aortic constriction as previously described . Data he+ current dependent action potentials , where APDn and APDn+1 indicate the durations of the nth and (n + 1)th APs, and nbeats denotes the total number of consecutive beats analysed. APD alternans magnitude was calculated as the difference between the average APD90 of odd and even numbered beats during 50 consecutive APs recorded. Diastolic arrhythmogenic activities were elicited by cessation of 1-min tachypacing, and membrane potential was recorded for additional 1 min. Delayed afterdepolarizations (DADs) were defined as > 1 mV depolarization within 0.5 s. Spontaneous APs (sAPs) were defined as depolarizations showing overshoot with a fast upstroke phase. Early afterdepolarizations (EADs) were assessed at 0.2 Hz pacing, and EADs were defined as > 3 mV depolarization during AP repolarization.Action potentials (APs) were evoked by 2-ms-long supra-threshold depolarizing pulses delivered via the patch pipette. 50 consecutive APs were recorded to examine the average behaviour, and APD at 90% repolarization (APDNaL as previously described SR), freshly isolated rabbit cardiomyocytes were loaded with 8 μmol/L Mag-Fluo-4-AM  with 0.2% Pluronic F-127  for 2 h at room temperature. Subsequently, cells were washed twice in fresh Tyrode’s solution for 30 min to allow de-esterification to occur. Then, cardiomyocytes were placed in a narrow bath chamber with embedded field stimulation electrodes  and stimulated at 0.5 Hz frequency in Tyrode’s solution at room temperature (22 ± 1 °C). Mag-Fluo-4 was excited at 480 nm wavelength using an Optoscan monochromator  and fluorescence emission was collected at 535 ± 15 nm.To measure SR Ca2+]i, cardiomyocytes were loaded with 10 μmol/L Rhod2-AM  for 10 min at room temperature and subsequently left to de-esterify in fresh Tyrode’s solution for a minimum of 30 min. Then, cardiomyocytes were placed in a RC-27NE2 recording chamber and stimulated at 0.5 Hz frequency in Tyrode’s solution at room temperature. Rhod2 was excited at 561 nm wavelength using an Optoscan monochromator, and fluorescence was collected at 530 ± 20 nm. Fluorescence signals were recorded after steady state was reached in the cell during pacing.To measure  and ISO to investigate the role of the vicious cycle in proarrhythmic AP changes.To separate RyR leak from the complex HF phenotype, in terms of the arrhythmogenic feedback signalling network , we induced RyR leak by low [caffeine] (200 μmol/L) and isoproterenol  in healthy rabbit ventricular myocytes. Caffeine 3 min) slightly prolonged APD min slig90 did not change  significantly shortened APD90 following caffeine + ISO and attenuated the increase in STV  events between paced beats in myocytes loaded with the intra-SR [Ca2+] ([Ca2+]SR) fluorescent indicator, Mag-Fluo-4 , we tested the contribution of enhanced Io-4 Fig. a, b. In o-4 Fig. c–e. Follo-4 Fig. b and DADo-4 Fig. d was unco-4 Fig. e, especio-4 Fig. f. Importo-4 Fig. d. MitoTEo-4 Fig. g. Moreovo-4 Fig. d.Fig. 5R2+ release system following cessation of pacing. Caffeine + ISO induced spontaneous SR Ca2+ release events, which were attenuated in mitoTEMPOL-treated cells  significantly enhanced INaL during AP-clamp, and the net charge carried by INaL increased by 3.9-fold  and markedly increased STV  and prolonged the [Ca2+]i transient  and a decreased peak INa (due to reduced surface expression of Na+ channels) as loss-of-function (Brugada syndrome). Importantly, these changes in Na+ channel function are similar to the CaMKII-mediated effects i was found to be 3 mmol/L higher than in control i is required to trigger a spontaneous AP in failing cardiomyocytes  in HF  [2+] may increase ROS production i and CaMKII [2+]i has been previously shown to abolish EADs induced by ATX-II i and mitochondrial ROS production at a given [Ca2+]i. Incorporating these data may help to constrain and improve computational models in the future, which then would allow more controlled analysis of different branches of the vicious cycle.Here, we aimed to preserve physiological regulation in our cellular experiments to uncover interactions within the feedback loops. However, this approach has limitations on quantifying the exact role that each component plays in the vicious cycle. Full inhibition of one key component may break the whole loop and have a marked arrhythmia reducing effect, like that seen in HF Fig. . HoweverNaL Fig.  and RyR NaL Fig.  clearly ecainide  and ranoecainide . Future,+]i–[Ca2+]i–ROS-CaMKII-RyR leak vicious cycle signalling have already been shown; however, the strength of feedback interactions have not been previously investigated. Our conceptual novelty here is the identification of important trans-target effects beyond the on-target effects of the otherwise selective inhibitors. It may have important clinical implications suggesting that potentially a combination therapy targeting the major components of the arrhythmogenic vicious cycle described here can be synergistic and may provide substantial benefits in heart diseases by reducing cellular proarrhythmia. The use of combination therapy may also be advantageous by reducing the effective dose of each drug, thus reducing their adverse effects. Moreover, our data show that the most favourable drug target(s) may vary among heart diseases, and thus, personalized medicine approaches are required to identify the optimal drug combinations.As discussed above, many components of the [Na


Mycobacterium tuberculosis. BlaC was the major inactivator of the cephalosporin subclass of β-lactams. However, the PBPs PonA1 and PonA2 inactivated penicillins and carbapenems more effectively than BlaC. These findings demonstrate that select M. tuberculosis PBPs are effective at inactivating several β-lactams. Lesser-known PBPs, DacB, DacB1, DacB2, and Rv2864c, a putative PBP, were comparably more resistant to inhibition by all β-lactam subclasses. Additionally, Rv1730c exhibited low affinity to most β-lactams. Based on these findings, we conclude that in M. tuberculosis, BlaC is not the only source of inactivation of β-lactams. Therefore, the historical paradigm does not accurately describe the relationship between β-lactams and M. tuberculosis.Penicillin binding proteins (PBPs) have been extensively studied due to their importance to the physiology of bacterial cell wall peptidoglycan and as targets of the most widely used class of antibiotics, the β-lactams. The existing paradigm asserts that PBPs catalyze the final step of peptidoglycan biosynthesis, and β-lactams inhibit their activities. According to this paradigm, a distinct enzyme class, β-lactamases, exists to inactivate β-lactams. This paradigm has been the basis for how bacterial diseases are treated with β-lactams. We tested whether this historical view accurately reflects the relationship between β-lactams and the PBPs and the β-lactamase, BlaC, of IMPORTANCEM. tuberculosis, the causative agent of tuberculosis, kills more humans than any other bacterium. β-lactams are the most widely used class of antibiotics to treat bacterial infections. Unlike in the historical model that describes the relationship between β-lactams and M. tuberculosis, we find that M. tuberculosis penicillin binding proteins are able to inactivate select β-lactams with high efficiency. Staphylococcus aureus, Bacillus subtilis, and Escherichia coli. Since similar observations were made in these organisms, a paradigm that describes the mechanistic basis of β-lactam activity was developed and assumed to be generally applicable to a wide range of bacteria  are a class of enzymes that are present in virtually all bacteria , 2. Thei3–bacteria –14. Accopical PG , 16.M. tuberculosis, the causative agent of tuberculosis, kills more humans than any other bacterium . Download Table S2, TIF file, 1.6 MB.Amount of each β-lactam hydrolyzed by Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msphere.00039-22.5FIG S1M. tuberculosis PBPs. The amount of each β-lactam hydrolyzed by a PBP was measured for 120 min. Reaction conditions for all PBP-β-lactam pairs were identical. Each assay was performed in triplicate under identical reaction conditions. The mean and standard error are reported. Download FIG S1, TIF file, 1.3 MB.Hydrolysis of β-lactams by Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msphere.00039-22.6FIG S2M. tuberculosis PBPs in the presence of a β-lactam. Each panel shows nitrocefin hydrolyzed by a specific M. tuberculosis protein. Each M. tuberculosis PBP or BlaC was incubated with a β-lactam for 30 min, following which, nitrocefin was added. Hydrolysis of nitrocefin was monitored for 120 min. Each assay was performed in triplicate under identical reaction conditions. The mean and standard error are reported. Download FIG S2, TIF file, 1.1 MB.Hydrolysis of nitrocefin by Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the PonA2 exhibited a dominant inactivation profile against penicillins, as 24% of total hydrolytic activity against this β-lactam subclass was exhibited by this protein. PonA1, with 12% of the total hydrolytic activity against penicillins, was also more potent than BlaC (10%) , Table 1BlaC exhibited the most potent hydrolyzing activity (25%) against the cephalosporins. The levels of inactivation of cephalosporins by all other proteins were similar to each other with less than 10% each, while PonA1 and PonA2 were the least effective at inactivating this β-lactam subclass. The less-well-known putative PBPs Rv1367, Rv1730, and Rv2864 hydrolyzed ceftriaxone at a higher rate than other proteins, including BlaC .We extended these data to determine the overall susceptibility of each β-lactam subclass to the PBPs , Fig. S1M. tuberculosis PBPs.Inactivation of a β-lactam reporter by  In the above-described assay, one of the surprising findings was that PonA1 and PonA2 were more effective than BlaC at inactivating most penicillins and carbapenems. Based on these findings, we hypothesized the following: exposure of a protein to a β-lactam that it hydrolyzed at a high rate will not inhibit its β-lactamase activity. To test this hypothesis, we used on orthogonal approach based on determination of the amount of nitrocefin hydrolyzed by each PBP in the presence of each β-lactam. Nitrocefin is a commonly used chromogenic reporter for monitoring PBP hydrolytic activity , is then determined  with BlaC, Rv1367c, and Rv2864c could not be detected. Download FIG S3, TIF file, 0.4 MB.Verification of BOCILLIN FL binding to Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msphere.00039-22.4TABLE S4M. tuberculosis PBP gene locus IDs and sequences of DNA oligomers used to amplify DNA of each gene. Download Table S4, TIF file, 0.6 MB.List of Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 50 of each β-lactam and PBP pair is shown in 50 of most penicillins were among the highest observed. This outcome is expected as BOCILLIN belongs to the penicillin subclass, and therefore, it is unlikely that another penicillin is preferably bound by the proteins. However, an important exception was observed. The FIC50 of all β-lactams (except for faropenem) was consistently lowest for PbpB compared to other PBPs, indicating that this PBP has the highest binding affinity for penicillins, cephalosporins, and carbapenems. These data are in full accord with the inactivation results  was used for protein overexpression. Luria-Bertani broth, Tris-Cl, sodium chloride salts, and imidazole salts were purchased from Sigma-Aldrich. Powder forms of all β-lactams . The amino acid sequence of each protein was analyzed using TMHMM (E. coli BL21(DE3) to generate expression clones. Each protein was overexpressed by inducing BL21(DE3) culture in LB broth with 0.25 mM IPTG  overnight at 16°C with constant shaking at 150 rpm (6 tag were purified by Ni-nitrilotriacetic acid (NTA)-based affinity chromatography with minor protein-specific optimizations as necessary , PbpB(Rv2163c), PonA1(Rv0050), PonA2Rv3682), DacB(Rv3627c), DacB1(Rv3330), DacB2(Rv2911), Rv1367c, Rv1730c, Rv2864c, and BlaC(Rv2068c) were PCR amplified using genomic DNA (gDNA) of 2, DacB(Rng TMHMM  to identng TMHMM  and clonng TMHMM . All resecessary  70–72).–72.M. tu10.1128/msphere.00039-22.8FIG S4M. tuberculosis genes encoding PBPs and BlaC were cloned, overexpressed, and purified from E. coli. This SDS-PAGE shows the purified proteins. Download FIG S4, TIF file, 0.1 MB.Proteins used in the study. Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Each β-lactam was used at a final concentration of 1 mM, which was subsequently mixed with each PBP or BlaC each at 10 to 15 μM for a 100-μL final reaction volume in 10 mM Tris-Cl buffer, pH 7.4. The β-lactam ring opening reaction was monitored for 120 min, 25°C, using a SpectraMax 250 spectrophotometer. Each assay was performed in triplicate under identical reaction conditions. For each protein and β-lactam combination, the rate of β-lactam hydrolysis was determined, and the composite hydrolysis was computed for each β-lactam class by each PBP and BlaC , Fig. S1Nitrocefin  is a chromogenic β-lactam whose hydrolysis is quantified by measuring the transition from the substrate form (390 nm) to the product (496 nm) . Nitrocemax = 504 nm)  is a fluorescent penicillin (λ 504 nm) . BOCILLI 504 nm) . Each pr


Despite that, the molecular identity of the channel that mediates Ca2+ ion transport into mitochondria remained elusive for several years. Only in the last decade, the genes and pathways responsible for the mitochondrial uptake of Ca2+ began to be cloned and characterized. The gene coding for the pore-forming unit of the mitochondrial channel was discovered exactly 10 years ago, and its product was called mitochondrial Ca2+ uniporter or MCU. Before that, only one of its regulators, the mitochondria Ca2+ uptake regulator 1, MICU1, has been described in 2010. However, in the following years, the scientific interest in mitochondrial Ca2+ signaling regulation and physiological role has increased. This shortly led to the identification of many of its components, to the description of their 3D structure, and the characterization of the uniporter contribution to tissue physiology and pathology. In this review, we will summarize the most relevant achievements in the history of mitochondrial Ca2+ studies, presenting a chronological overview of the most relevant and landmarking discoveries. Finally, we will explore the impact of mitochondrial Ca2+ signaling in the context of muscle physiology, highlighting the recent advances in understanding the role of the MCU complex in the control of muscle trophism and metabolism.The notion of mitochondria being involved in the decoding and shaping of intracellular Ca Both intercellular and extracellular communication play fundamental roles in shaping cell behavior and driving cell fate decisions. Cell-to-cell and environmental signals are normally conveyed by distinct extracellular mediators  that are normally perceived by cells through surface receptors. These receptors convey them into a limited number of intracellular molecules, which are referred to as ‘second messengers’, which, in turn, forward the message to intracellular effectors finally activating the ultimate cellular responses. Despite the plethora of different signals and stimuli that cells may receive, only a few molecules to date have been described as second messengers of intracellular communication. Among them, the most common and, definitively, the most extensively studied is Ca2+ ions participate in the decoding of a vast range of stimuli and the variety of cellular components involved in the Ca2+ signal transduction is extremely wide, including basically all kinds of components, organelles, and molecules  conditions [2+-dependent assembly conformations of the beetle and human MCU holocomplexes with human MICUs have also been detailed [2+, the multiprotein complex shows a two-fold symmetry and consists of two V-shaped MCU-EMRE tetrameric subcomplexes and two MICU1-MICU2 heterodimers that bridge the tops of the subcomplexes , instead, the knockdown of MCU has shown crucial alterations not only during the early step of gastrulation, where the blastomere convergence and extension are altered, but also at later stages of development as the maturation of the notochord and anteroposterior axis formation were strongly impaired in morphant fish [MCU gene in Danio rerio inhibits mitochondrial Ca2+ influx, reduces oxidative phosphorylation, and induces lipid accumulation, a phenotype that was also observed in the hepatic tissue of liver-specific KO mouse [MCU has shown to be protective in neurons of Parkinson Disease zebrafish genetic model (namely in −/−pink fish), suggesting the crucial role of the MCU-dependent mitochondrial Ca2+ load on neuronal fitness [In 2013, three independent groups showed that the o models ,63,64. IKO mouse . Notably fitness .–/– mouse [−/− mice in a pure C57BL/6 background were not viable, displaying embryonic lethality at around E 11.5–E 13.5, thus suggesting a major involvement of mitochondrial Ca2+ uptake in organ metabolism and organism development that was compensated in a mixed genetic background [As for mammals, the MCU/– mouse  developsckground .2+ uptake affects Drosophila melanogaster development. In particular, the inhibition of the uniporter activity has been shown to be detrimental for memory establishment during the papulation stage. Indeed, during the development of adult flies, alterations in the structural and functional neuronal substrates, crucial for memory formation, occur in the MCU deficient fly [Of note, some years later, it has been reported that the block of MCU-dependent Caient fly .C. elegans model also provided additional interesting notions on its role in organism physiology. Indeed, MCU−/−C. elegans is viable and grossly normal, mirroring what was found in the first MCU−/− mouse model described [C. elegans deficient of a functional MCU [2+ transfer during high-intensity stimulation of the worm pharynx muscle. However, a lot still remains to be explored on the role of MCU in this model and most of the knowledge on mitochondrial Ca2+ regulation of muscle physiology has been achieved using the mammalian mouse model where MCU expression was genetically targeted, which we will briefly review in the following paragraphs.The genetic manipulation of MCU in escribed , even thonal MCU  suggeste2+ uniporter, since an alternative MCU isoform exists, named MCUb, which crosses the IMM and associates to MCU to form the calcium channel [CCDC109a paralog gene CCDC109b. Interestingly, this gene is found in vertebrates, but it is not present in other organisms in which MCU is expressed, such as plants, Nematoda, and Arthropoda. Despite the high structural similarity with MCU, MCUb sequence presents two critical aminoacidic substitutions in the loop region and in the TM1 domain, which explains its inability to transport Ca2+. Indeed, MCUb acts as a negative regulator of MCU activity, drastically reducing mitochondrial Ca2+ currents in vitro in planar lipid bilayer experiments and also when overexpressed in mammalian cells [2+ uptake [In 2013, Raffaello and co-authors discovered that MCU is not the only pore-forming subunit of the mitochondrial Ca channel  , which does not take part to the pore-forming domain of the channel, but it strongly regulates its activity in a Ca2+-dependent way [N-terminal mitochondrial targeting sequence, MICU1 shows two canonical EF-hand Ca2+ binding domains that confers the Ca2+-sensitivity  and electron microscopy studies of Gottschalk and collaborators . Accordi emerged . MICU1 wdiseases .MICU1 mRNA [2+ uptake upon stimulation in HeLa cells and skeletal muscle in vivo, and this increase is even higher than that observed after expression of the conventional MICU1 isoform [2+ more efficiently than the canonical MICU1-MICU2 pair, thus activating the uniporter at lower Ca2+ concentrations [2+ uptake required for the matching of ATP production to the energy expenditure of muscle contractile activity [2+ uptake, opening the path for future investigations on other tissue-specific isoforms and mechanisms controlling mitochondrial Ca2+ uptake.A variant of MICU1, named MICU1.1, has been discovered by our group as an alternative splicing product of the CU1 mRNA   and MCU channels [Although the functional role of MICU2 is still controversial, recent findings clarify some key aspects of uniporter modulation by this regulator. In mammalian cells, for example, it has been shown that MICU2 positively regulates MCU activation by controlling the cytosolic [Cachannels .−/− mouse model highlighted other important properties of this regulator [2+ uptake due to loss of the gatekeeping activity and overall loss of MCU-dependent Ca2+ influx due to the destabilization of the entire uniporter complex. These findings lead to the conclusion that the amount of MCU and MICUs proteins is crucial to maintain the stability of the whole complex. Moreover, some phenotypic features of the MICU2−/− mice are in common with models of cardiac pathologies, suggesting that MICU2 may act as a possible cardioprotective factor.Subsequently, the generation and characterization of the MICU2egulator . MICU2 gMICU3 is an evolutionarily conserved gene since it is present in plants and in vertebrates, with a peculiar tissue-specific distribution: indeed, it is mainly expressed in the brain, much less expressed in skeletal muscle, and virtually absent in other tissues [MICU3 gene encodes a 55 kDa protein that shares 34% and 47% protein sequence similarity with MICU1 and MICU2, respectively [N-terminal mitochondrial targeting sequence and binds Ca2+ thanks to the presence of EF-hand domains. The crystal structure of human MICU3 has been recently characterized in both Ca2+-free and Ca2+-bound conditions [2+ levels. Upon cytosolic Ca2+ increase, MICU heterodimers, including those containing MICU3, undergo a conformational change that releases the latch formed upon the uniporter mouth, thus allowing Ca2+ flux through the MCU pore [MICU3, previously known as EFHA2, is another MICU1 paralog, originally identified by the same genetic sequence analysis that described MICU2  Figure . This fi tissues ,89. The ectively . Like thnditions . This crMCU pore   is an additional constituent of the uniporter, discovered by quantitative mass spectrometry analysis of affinity-purified MCU complex components  Figure . EMRE isterminus . Moreoveterminus ,92. IndeLa cells . The folugmented . Accordiate tail . In the N-terminus facing the mitochondrial matrix. Secondly, it proposed that EMRE interaction with MCU occurs along with three major contact points of EMRE N-terminus, thus allowing Ca2+ ions to exit the channel vestibule. Finally, EMRE seems also to be crucial for MCU dimerization since, in its absence, the MCU channel is found as a monomer [Very recently, the cryo-EM structure of the human MCU complex added new insights into the interaction of EMRE within the complex and defined the structural elements by which EMRE exerts its action on the channel . In part monomer .2+ signals, cell metabolism, and cell survival.These new findings added important notions on the mechanisms of action and regulation of the MCU complex and highlighted the need for a precise orchestration of the holocomplex assembly to ensure its fundamental role in regulating cellular CaCCDC90A gene, resulted from an RNAi screening searching for mitochondrial membrane components involved in mitochondrial Ca2+ uptake regulation [The mitochondrial calcium uniporter regulator 1 or MCUR1 has been firstly described as an IMM-resident protein of 35 kDa, encoded by the gulation   model. In this context, MCUR1-dependent mitochondrial Careatment .The fact that skeletal muscle comprises around 40% of human body mass , is prim+/K+ ATPase and Ca2+ ATPase in the sarcolemma, sarcoplasmic reticulum Ca2+ ATPase in the ER). In addition, muscle is an extremely plastic tissue; muscle fibers can adapt and promptly respond to different workload demands shaping both their metabolic output and gene expression profile to match with changes in energy requirements of different types of exercise as well as during different phases of a single bout of exercise. For this reason, muscle metabolism needs to be highly efficient but flexible and rapidly adaptable. The main sources of ATP in muscles are the catabolism of glucose  and the oxidation of fatty acids . Other metabolic routes participate in the overall tissue energetics to a much-reduced extent [Of most relevance, muscles can contract to generate force, and, importantly, relax afterward to return to their resting length and condition. Obviously, contraction activity is a highly demanding process that massively consumes the cellular energy stored in the form of ATP molecules. Myosin, the major sarcomeric filament and the effective motor core of the myofiber, is the most abundant protein in muscle, comprising 25% of the total tissue protein  and it pd extent .2+ signaling on these two processes. The start of muscle contraction occurs at the membrane of myofibers, where the motor neuron-released neurotransmitter triggers sarcolemma depolarization and the consequent opening of voltage-gated ion channels (the DHPR). DHPR are structurally and functionally coupled to the Ca2+ release channels of the endo-sarcoplasmic reticulum (SR), the Ryanodine receptor (RyR), so that RyR also opens [2+ from the SR with the subsequent intracellular Ca2+ elevation and activation of troponins which, in turn, allow the actomyosin sliding and promote sarcomere contraction. This process is commonly defined as excitation-contraction coupling or ECC [2+ in skeletal muscle is, in the first place, associated with actomyosin contraction and ECC, the cation exerts at least two other crucial functions in muscle physiology. On the one hand, the elevation of intracellular Ca2+ regulates a plethora of Ca2+-sensitive effectors, such as nuclear transcription factors, calmodulins, and kinases which sequentially activate gene expression and downstream signaling cascades [2+ increase leads to mitochondrial Ca2+ uptake and the consequent stimulation of muscle aerobic metabolism and oxidative respiration [2+ rise induced in the myofiber by nerve activity leads to Ca2+ binding to calmodulin and consequent activation of the Ca2+-calmodulin-dependent phosphatase Calcineurin. Calcineurin dephosphorylates its target, the Nuclear Factor of Activated T-cells or NFAT [2+-dependent, Calcineurin-NFAT-mediated regulation of gene transcription was shown to be crucial for the expression of muscle-specific structural genes that characterize the different fiber-type populations [Finally, we will briefly describe the key molecular events leading to muscle contraction and muscle metabolic adaptation, with particular emphasis on the role played by Caso opens . This ing or ECC . Despitecascades . Secondlpiration ,117,118. or NFAT  that, on or NFAT ,121. Thiulations ,121.2+ mobilization induces Ca2+ uptake by myofiber mitochondria and enhances mitochondrial metabolism through the TCA cycle and respiratory chain [2+ and in particular of mitochondrial Ca2+ are necessarily associated with the regulation of skeletal muscle metabolism. This concept has been recently explored and we will report here some of the latest advances in the study of mitochondrial Ca2+ signaling and its influence on muscle metabolism and adaptation to both physiologic workload demands and pathologic conditions.On the other hand, the activity-dependent intracellular Cary chain ,117,118,2+ channel components [2+ uptake in vivo, including in skeletal muscle. Despite the first report of MCU gene deletion in the total knock out mouse model showed an unexpectedly mild phenotype, the MCU−/− mice display a reduction of both resting mitochondrial Ca2+ concentration and mitochondrial Ca2+ uptake after stimulation at the cellular level, and increased serum lactate at the systemic level [2 consumption in MCU−/− and wt MEF cells did not reveal significant differences [−/− mice, by the presence of an elevated amount of the phosphorylated inactive form of PDH, which reduces TCA cycle activity, and by a reduced exercise performance and muscle force of the KO animals [The molecular identification of the mitochondrial Camponents ,96,122 , and significant variability in myofiber size are found in the muscles of the analyzed patients. Despite that, the overall fiber-type distribution is maintained. However, the characterization of dermal fibroblast cells from MICU1-deficient patients together with that of muscle fibers from skeletal muscle-specific MICU1 KO mouse model (skmMicu1−/−) revealed a complex cellular mechanism underlying skeletal muscle dysfunction in the absence of this crucial regulator of mitochondrial Ca2+ entry [2+ dynamics of patient fibroblasts and skmMicu1−/− mouse myofibers showed the loss of both MCU gatekeeper activity and cooperative MCU activation [2+ resting level and an enhanced rate of mitochondrial Ca2+ uptake [2+ peak in stimulated MICU1 KO cells are not different from those of wt cells [2+ transients after cell stimulation are reduced in MICU1-deficient cells as a consequence of the enhanced mitochondrial buffering capacity [MICU1 deletion, as shown by an O2 consumption rate of patient cells [−/− mice [−/− mice showed enhanced lactate and creatine kinase levels, accompanied by reduced running time and overall decreased performance when assessed after treadmill running until exhaustion. The mechanism underlying muscle failure was unveiled by Evan’s blue dye analysis of muscle fiber integrity, which revealed clear signs of sarcolemma damage in skmMicu1−/− fibers, not found in wt fibers [2+ in myofiber plasma membrane recovery after exercise-induced damage in in vivo mouse skeletal muscle, but also in human fibroblasts of patients with MICU1 mutation after laser-induced injury [2+ signaling causes defects in the plasma membrane repair machinery in the C2C12 muscle cell model. Interestingly, the mitochondrial Ca2+-mediated healing of membrane damage has been shown to rely mainly on the activity of the small GTPase RhoA and the dynamics of actin filaments, while it appears substantially independent from the mitochondrial ATP and energy production [2+ signaling to the repair of fiber sarcolemma brings an additional level of complexity to the regulation of muscle fiber homeostasis by mitochondrial Ca2+ providing new opportunities for future investigations and, possibly, offering new targetable pathways for therapeutic interventions against neuromuscular disorders.In line with the high relevance of the MCU complex activity in the maintenance of muscle function demonstrated in animal and in vitro experimental models, disorder . From a disorder . The who2+ entry . The anativation . Indeed,+ uptake . Neverthwt cells . In addicapacity . The celnt cells  and a bl−/− mice  similar t fibers . MICU1 ad injury . These foduction . The con2+ signaling. We presented a synopsis of the discoveries that, in the last decade, led to the identification of most of the components that constitute the mitochondrial Ca2+ uptake machinery  [2+ uptake dictates the cell decision either to survive or to perish is still debated. Studies characterizing the heart of MCU-constitutive KO mice [MCU-deleted animals [2+-mediated activation of the mitochondrial Permeability Transition Pore (mtPTP) [2+-induced mtPTP opening together with a reduced cardiomyocyte death after ischemia-reperfusion [Another aspect of the biology of mitochondrial Caediated)  is a wid KO mice  and card animals ,57 showe (mtPTP) . On the erfusion ,57.The different results obtained from constitutive versus inducible tissue-specific MCU KO mouse models suggest the possibility that the MCU deletion may lead to long-term changes in the gene expression program. The characterization of these changes and the mechanisms by which they take place are yet to be defined.Finally, although the quantitative proteomic data provided by the group of Mootha clearly indicate that all the components of the MCU holocomplex (uniplex) and associated regulators have been already identified , the pos2+ levels and Ca2+ responses to match their metabolic requirement and physiological function. Indeed, timely and tissue-specific regulation of the mitochondrial Ca2+ uniporter activity is ensured by the different proportions of the MCU holocomplex components, as depicted in These considerations support the concept that the mitochondria of different cell types might have different Ca2+ role and regulation has grown massively in the last ten days; however, additional investigations are still needed to unravel the unresolved issues. Hopefully, scientists will take up this challenge and their work will provide further knowledge on the signaling and regulation of mitochondrial Ca2+ to better understand its role in cells and organs in physiology as well as to possibly develop new routes of intervention in pathological conditions.Concluding, our understanding of mitochondrial Ca


We simulated the changes in [Ca2+] through the sarcomere of these four fiber types, considering classical  and new  Ca2+ binding sites, during single and tetanic stimulation. We found that during a single twitch, the sarcoplasmic peak [Ca2+] for fibers type IIB and IIX was around 16 µM, and for fibers type I and IIA reached 10–13 µM. The release rate in fibers type I, IIA, IIX, and IIB was 64.8, 153.6, 238.8, and 244.5 µM ms−1, respectively. Both the pattern of change and the peak concentrations of the Ca2+-bound species in the sarcoplasm , the sarcolemma , and the SR (SERCA) showed the order IIB ≥ IIX > IIA > I. The capacity of the NCX was 2.5, 1.3, 0.9, and 0.8% of the capacity of SERCA, for fibers type I, IIA, IIX, and IIB, respectively. MITO peak [Ca2+] ranged from 0.93 to 0.23 µM, in fibers type I and IIB, respectively, while intermediate values were obtained in fibers IIA and IIX. The latter numbers doubled during tetanic stimulation. In conclusion, we presented a comprehensive mathematical model of the excitation–contraction coupling that integrated most classical and novel Ca2+ handling mechanisms, overcoming the limitations of the fast- vs. slow-fibers dichotomy and the use of slow dyes.Mag-Fluo-4 has revealed differences in the kinetics of the Ca The total [Ca2+] in MITO, considering both forms, bound to B and free, achieves a peak value of 0.26–1.12 µM after a single AP and 0.72–2.8 µM after 5 AP. Regarding the SR compartment, our calculations show that the available Ca2+ is reduced up to ~63%  for single transients, but to almost 53%  after a train of five shocks  both the pattern of change and the peak concentrations of the Ca2+-bound species in the sarcomere, the sarcolemma, and inside the SR showed the order IIB ≥ IIX > IIA > I, (iii) the mitochondrial peak [Ca2+] and the MITO buffers saturation showed the pattern I >> IIA >> IIX ≥ IIB.The main findings of the present work were: (i) during a single twitch, the sarcoplasmic peak . Our results with Mag-Fluo-4 . The numbers for type I and IIA fibers also agree with a study reporting peak Ca2+ for soleus slow fibers [2+] for fibers types I and IIA is between 6 and 12 µM [2+ flux during SOCE or excitation-coupled Ca2+ entry (ECCE) activations [2+ [2+] is raised. Trustable peak sarcoplasmic [Ca2+] also permits a better quantitation of the chemical species involved in ECC , including peak [Ca2+] inside compartments such as MITO.The very low affinity of the dye and the lack of saturation confer Mag-Fluo-4 the ability to determine a trustable peak sarcoplasmic  has ranged from 80 µM  to aroun–22 °C, , being ~of 85 µM  and ~110of 85 µM , and in 2+ release. Slow fibers are devoid of PV [The peak concentration of [CaPV] in fast fibers has ranged from 90 µM  to 120 µid of PV , or veryid of PV ,54, justThe peak [CaATP] we found for fibers IIX and IIB coincides with the values reported previously for fast fibers, i.e., 60 µM  and ~70 2+ release. Variations depend on differences in loading conditions used by the researchers in different laboratories. The value found for type I fibers in the present study was 3 µM. The order resembled that of the peak [Ca2+] in the sarcoplasm and the Ca2+-ATP reaction.The [CaDye] concentration reached a peak at around 22 µM  or 5 µM 2+] inside MITO was reported to be 0.35 µM for a single twitch in fast fibers in Marcucci et al. (2018) [2+] was ~0.25 µM in our results for fibers type IIX and IIB, about 25 ms after Ca2+ release. This concentration is similar to that obtained experimentally in fast mouse fibers [2+] peaked inside MITO in type I fibers, reaching a value of 0.9 µM. This delay likely reflects Ca2+ diffusion into MITO, as measured in mouse using genetically encoded sensors [2+] was far higher in I than in II, likely as the MCU of slow fibers has more capacity than that of the fast fibers  and [Ca2+] inside MITO in skeletal muscle fibers.Previous works have estimated the total concentration of MITO buffers [B]. From the simulated analysis of the total [B] in cardiac muscle fibers performed in , a value blunted ,47. Howe2+ pumped by the SERCA was estimated to be 1.5–3.5 µM in fast fibers , spatially refined models for fast fibers have shown up to a 20-fold gradient in the sarcoplasmic [Ca2+], depending on the distance of a subcellular region from the Ca2+ release units  and in the [Ca2+] bound to the binding sites. In the present study, the SOCE flux is associated with changes in [Ca2+] in the SR compartment. However, Ca2+ gradients in the SR have been measured during ECC and the Ca2+ levels decrease more rapidly in the terminal cisternae than in other regions of the SR  was performed according to the calibration method and the value parameters presented in [2+] was calculated using the expression:maxF and minF are the maximum and minimum fluorescences (150.9 A.U and 0.14 A.U) respectively, dK is the in situ dissociation constant of Mag-Fluo-4 (1.652 × 105 µM2), and [D]T is the total dye concentration (229.1 µM), at 20 °C. As the experimental Ca2+ transients that feed the model were acquired between 21–23 °C, no temperature corrections were performed in the calibration parameters.Typical fluorescence experimental recordings  with a system of differential equations. The rate of change of [Ca2+] with respect to time in the SR was determined byJRel is the release rate flux, JSERCA is the SERCA flux, and F the reaction rate of Ca2+ in the SR with calsequestrin (CSQ). The rate of change of sarcoplasmic [Ca2+] was expressed as:JNCX is the NCX flux, [Ca2+]MITO the mitochondrial [Ca2+], JSOCE is the SOCE flux, and F the reaction rate with the sarcoplasmic Ca2+ binding sites S. The change in time of [Ca2+] in the MITO was given byJMCU was the inflow through the MCU, JNCE outflow through the NCE, and F the reaction rate with B. The B was 20 µM of binding sites, which react with Ca2+  and [TNS] in the sarcoplasm was rescaled by the occupation of the MS volume in the sarcoplasm  . As eachThe [PV] in the fastest fibers can achieve large concentrations, 1.5–1.9 mM ,27. We a2+ by SERCA to the SR is described by the Michaelis–Menten kinetics and expressed as:VSERCA is the maximum flux rate, [Ca2+] is the Ca2+ concentration in the sarcoplasm, KSERCA is the dissociation constant and hSERCA the Hill coefficient. The SERCA isoform predominantly expressed in fast-twitch muscle fibers is the 1a, whereas in slow-twitch is the 2a MITO:VMCU is the maximum flux rate, [Ca2+] is the Ca2+ concentration in the sarcoplasm, and KMCU is the [Ca2+] where the transport rate is half-maximum. VMCU values were taken as 0.012 μM ms−1 in fast and 0.049 μM ms−1 in slow fibers at 16 °C mito decay phase, completed in a period of about 100 ms and measured during a single twitch, as in Rudolf et al. (2004)  in the SR, which can be described by the function:KSOCE and hSOCE are the Ca2+ dissociation constant of STIM1 (0.35 mM) and the Hill coefficient (4.7), respectively  is one order of magnitude higher than reported with slow dyes. The magnitudes of change of the Ca2+-bound forms of the Ca2+ buffers studied follow the order IIB ≥ IIX > IIA > I, except for mitochondrial peak [Ca2+] which showed the pattern I >> IIA >> IIX ≥ IIB. The kinetics for fibers IIA and IIX proved to be intermediate between I and IIB fibers, supporting dynamic data. The results may help better quantitate SOCE fluxes and thermal changes in mammalian fiber types in the future and support the use of fast Ca2+ dyes for most experimental approaches in skeletal muscle.Our mathematical, comprehensive model allows us to gain insight into the kinetics of the Ca


RAB27B is a member of Ras-like small GTPases that plays a role in endocytosis, exocytosis, and vesicle trafficking. We made an attempt to study the impacts of RAB27B on the proliferation and apoptosis of acute myeloid leukemia (AML) cells. The silencing of RAB27B was induced by siRNA for the detection of proliferation, cell cycle, and apoptosis, respectively by Cell Counting Kit-8 (CCK8), flow cytometry, and TUNEL. Related markers were also evaluated by Western blot analysis. The interaction between RAB27B and BDH2 was predicted by bioinformatics analysis and determined by immunoprecipitation. The gain of function of BDH2 was also detected by these functional assays. RAB27B exhibited high levels in AML cells, and RAB27B silencing led to reduced proliferation, increased cell cycle arrest and apoptosis levels. Then, the interaction between RAB27B and BDH2 was confirmed. Moreover, the effects of RAB27B inhibition on the proliferation, cell cycle arrest, and cell apoptosis were abolished after BDH2 overexpression. RAB27B inhibits proliferation and promotes apoptosis of leukemic cells by interacting with BDH2. Targeting RAB27B might be an effective method for the treatment of AML.  Acute myeloid leukemia (AML) is a genetically heterogeneous disease featured by the significantly rapid growth of abnormal myeloid progenitor cells in the bone marrow and blood that can interfere the function of normal blood cells ,2. As foThe Rab family is a type of Ras-like small GTPases that plays a role in endocytosis, exocytosis, and vesicle trafficking ,10. RAB2BDH2, originally named DHRS6, is recognized as a member of the short-chain dehydrogenase/reductase family . Its expWe assume that there exists an association between RAB27B and BDH2, which is involved in the progression of AML. In the present study, we explored the pivotal role of RAB27B in AML and discussed the potential association between RAB27B and BDH2. Moreover, we conducted a series of tests for the validation of our hypothesis that RAB27B might modulate the proliferation and apoptosis of leukemic cells via targeting BDH2.2 at 37°C. HS-5 cells were placed in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific) added with 10% FBS and 1% penicillin/streptomycin under the same condition.AML cell lines KG-1 and human bone marrow stromal cell line (HS-5) were purchased from American Type Culture Collection . AML-193 cells were obtained from Procell Life Science&Technology Co., Ltd , while K-652 cells from Kunming Cell Bank . AML cells were cultured in Iscove’s Modified Dulbecco’s Medium  with 10% FBS  and 1% penicillin/streptomycin  in an atmosphere with 5% COTotal protein was isolated from AML cells, and then a BCA Protein Assay Kit (Beyotime Biotechnology) was used to measure protein concentration. The samples were separated by electrophoresing on 10% SDS-PAGE and then transferred to PVDF membranes. Possible nonspecific binding was sealed by 5% nonfat milk and the proteins were then incubated overnight at 4°C with specific primary antibodies . After rinsing with TBST for three times, these membranes were incubated with secondary antibody . The bands were visualized using an enhanced chemiluminescence detection system . GAPDH served as the protein-loading control.−ΔΔCq method [Total RNA was isolated using TRIzol® reagent  based on the manufacturer’s protocol. RNA was synthesized into cDNA by the SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific) under the following conditions: 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min. cDNA was used for real-time PCR conducted by the Power SYBR Green Master Mix  on a 7300 Real-Time PCR System (Applied Biosystems). All values were normalized to GAPDH, and the calculation of the fold-change was conducted by 2q method . The priSiRNA targeting RAB27B or siRNA-NC , BDH2 overexpressing plasmids or empty plasmid (PcDNA3.1) were constructed and purchased from Genomeditech  transfected into AML cell using LipofectamineTM2000 with the manufacturer’s instructions . After 24 h, cells were used for further experiment.CCK-8 assayFor the detection of cell viability, the cells were seeded into 96-well plates at a concentration of 7500 cells/well and 10 μL CCK-8 reagent was added to each well at indicated time points. After that, the cells were incubated with normal cell culture medium for 2 h, and the absorbance was detected at 450 nm by a microplate reader.The transfected cells were harvested at 72 h and treated with 0.25% trypsin. Then, the cells were centrifuged in order to dump the supernatant. After being fixed by pre-cooled 70% ethanol solution and incubated at 4°C overnight, cells were washed for three times by PBS on the following day. Subsequently, cells were double-stained with Annexin VFITC and propidium iodide (PI), and then the cell cycle was determined. Data analyses were performed by FlowJo software .Terminal DNA transferase-mediated dUTP nick end labeling (TUNEL) assayTUNEL was conducted using in situ cell death detection kits TUNEL  according to the manufacturer’s protocol. DAPI  was used for the counterstaining of cell nuclei. The number of TUNEL-positive cells was calculated randomly for three visions under an inverted microscope.The cells were lysed by immunoprecipitation lysis buffer (Thermo Fisher Scientific) containing protease inhibitors and phosphatase inhibitors cocktail (Sigma-Aldrich). Cell lysates were then treated with anti-RAB27B or anti-BDH2 antibody and protein A/G beads (Thermo Fisher Scientific) at 4°C for 6 h. After being washed by immunoprecipitation lysis buffer, the beads were boiled for 5 min with 2× SDS loading buffer. Proteins were then analyzed by Western blot.GraphPad Prism software  was applied for data analysis. Data were expressed as the mean ± standard deviation (SD). The comparisons between the two independent groups were conducted by Student’s t-test, while the data among multiple groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered to indicate a statistically significant difference.http://gepia.cancer-pku.cn/) [To analyze the role of RAB27B in AML and discover the potential mechanism, we firstly hunted for the expression of RAB27B in AML by GEPIA database (pku.cn/) , where wAs RAB27B was high expressed in AML, we next silenced RAB27B to see if the malignant behaviors of AML cells were transformed. Results in RAB27B has been reported to regulate apoptosis in some cancers ,19,20. WThe anti-apoptotic factor BDH2 was shown to express at high level in AML, and there is a link between BDH2 and poor prognosis of AML patients. Thus, we attempted to get a clue regarding the role of BDH2 in AML cells. Prediction from BIOGRID tool revealed that RAB27B could bind to BDH2. To further analyze the underlying mechanism, we conducted RT-qPCR and found high expression of BDH2 in AML cells . FurtherTo investigate the potential mechanism by which RAB27B suppressed the proliferation of AML-193 cells, we overexpressed the expression of BDH2. Successful transfection was observed in Suppression of RAB27B promoted the cell cycle arrest at G0/G1 phase and the cell apoptosis by binding to BDH2Then, we analyzed whether RAB27B silencing did effects on the cell cycle and apoptosis of AML-193 cells by binding to BDH2. RAB27B knockdown induced an obvious increase in the cells of G0/G1 and a decrease in the G2/M population, which was reversed by BDH2 overexpression . AdditioIn this paper, the prediction from GEPIA tool indicated that the abnormally high expression of RAB27B was possibly correlated with the poor prognosis of AML patients. Concurrently, we determined the expression of RAB27B in AML cells, finding the elevated expression of RAB27B in various AML cells. Therefore, RAB27B may be an independent diagnostic and prognostic marker of AML.RAB27B, primarily recognized within platelets, was implicated in exosome secretion . Recent Since AML is a heterogeneous malignancy characterized by immature clonal myeloid cell proliferation and abnormal differentiation, many studies have identified cell proliferation inhibition targets for AML therapy . LncRNA BDH2 was found to play a key role in the pathogenesis of various cancers . CongrueOn the whole, we provide the first evidence that that RAB27B inhibits proliferation and promotes apoptosis of leukemic cells via interacting with BDH2, revealing that the RAB27B can functionally act as a prognostic marker of AML and targeting RAB27B may be conducive to the management of AML.


The model proposed is based on principles of charge attractivity and charge neutralization within the pore forming region of these receptors; it accurately predicts and reconciles experimental data across various platforms including Ca2+ permeability of GluA2-lacking AMPARs and ion selectivity within GluN3-containing di- and tri-heteromeric NMDARs. Additionally, the model provides insights into biophysical mechanisms regulating cation selectivity and permeability of these receptors and the role of various subunits in these processes.Glutamatergic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-D-aspartate) receptors are implicated in diverse functions ranging from synaptic plasticity to cell death. They are heterotetrameric proteins whose subunits are derived from multiple distinct gene families. The subunit composition of these receptors determines their permeability to monovalent and/or divalent cations, but it is not entirely clear how this selectivity arises in native and recombinantly-expressed receptor populations. By analyzing the sequence of amino acids lining the selectivity filters within the pore forming membrane helices (M2) of these subunits and by correlating subunit stoichiometry of these receptors with their ability to permeate Na While Ca2+ influx through NMDARs can bring about synaptic plasticity, the basis of learning and memory, or cell death from excitotoxicity  that are selective for and permeable to both Na+ and Ca2+. When assembled with glycine-binding GluN3 (GluN3A or GluN3B) subunits , d-NMDARs are no longer activatable by glutamate and are no longer permeable to Ca2+. Triheteromeric NMDARs, on the other hand, contain three different types of subunits , are selective for Ca2+ over Na+ for permeation and inward current  subunits of the same type  give rise to diheteromeric NMDARs ; Q00959, Grin2a (GluN2A); Q00960, Grin2b (GluN2B); Q00961, Grin2c (GluN2C); Q62645, Grin2d (GluN2D); Q9R1M7, Grin3a (GluN3A); Q8VHN2, Grin3b (GluN3B); P19490, Gria1 (GluA1); P19491, Gria2 (GluA2); P19492, Gria3 (GluA3); P19493, Gria4 (GluA4). The sequences were aligned using the Clustal Omega program in UniProt.1The amino acid sequences near the M2 segments of NMDA and AMPA receptor subunits were obtained from the UniProtKB protein knowledgebase for Rattus norvegicus  using accession numbers: + to Ca2+ concentration ratio is roughly a 102 to 1 and yet, influx of ions through a calcium channel is mediated predominantly by Ca2+, requiring severe selection against Na+. In contrast, K+ to Ca2+ concentration within the intracellular compartment of a spine head is roughly a 106 to 1 and so, discrimination against K+ is low  thereby affecting reversal potential which would be dominated by intracellular potassium rather than calcium concentration  with regions of low cation selectivity (the intracellular space within the spine head where the postsynaptic density is located) . At the ntration . Higher ntration . This tae inset, , suggestd-NMDARs  . Together, these residues constitute the putative selectivity filter that enables permeation of both Na+ and Ca2+ ions through these receptor channels (Site directed mutagenesis of amino acid residues within the pore-forming M2 domains of GluN1 and GluN2 subunits of heterologously expressed d-NMDARs  shows th1 (N598) , the man1 (N598) , and thi1 (N598) . The coachannels .d-NMDARs disrupts the ring of partial negativity observed in GluN2-containing d-NMDARs and diminishes the total negative ring charge by 2 , unlike GluN2, have non-polar glycine instead of asparagine at homologous positions within their M2 domains  and exprrq = –2; . Expresslutamate  and relaermeable  cf.  or divalent  cations  is diminished or lost and this situation is altered with the incorporation of GluN3 to make t-NMDARs, which acquire selectivity for Ca2+ over Na+ (Swapping a glycine-binding GluN1 subunit for a glycine-binding GluN3 subunit (3A or 3B) in GluN2-containing t-NMDARs  with a drq = –3; . The inc oocytes  which ishe brain ,b. Togetover Na+ .t-NMDARs is possible through ion selectivity  through a d- or t-NMDAR channel is assumed to be determined by the yes/no piecewise function (1), where A, defined as charge attractivity is multiplied by B, defined as inward drive. Both A (2) and B (3) are themselves binary  piecewise functions of iq, rq  and/or rq (charge of ion occupying the pore).The permeability of a cation with charge charge permeability Equation (1) above is based on the premise that following receptor activation, iq should first be attracted to the selectivity filter based on the magnitude of its charge and the total ring charge of the selectivity filter rq (note that like charges repel and unlike charges attract). Once attractivity is established, iq experiences an inward drive depending on (1) charge of the ion occupying the pore, if any; (2) neutralization of ring charge in the selectivity filter on account of pore occupancy and (3) the net electromotive force exerted by iq on rq to displace it inwards. The model assumes that ion selectivity precedes permeability (increased selectivity is correlated with increased permeability) and associates a distinct, albeit unquantified, dwell time for ions within the pore of the receptor channels where they are temporarily trapped and held in place to enable electrostatic interactions with other ions. Note that this trap and hold scenario is distinct from the binding of ions to designated sites within the channel pore. The model does not consider concentration gradient, which would tend to move iq from the synapse into the cell through the receptor and affect the rate of permeation, affinity of ions for specific binding sites within the pore of the channel in such a manner as to block it  or membrane potential . It instead assumes that the receptor is fully activated following binding of the neurotransmitter and devoid of any Mg2+ blockade or desensitization such that cations have free access to its selectivity filter through the pore. Further modeling of receptor dynamics would account for (i) the nature of Mg2+ blockade and (ii) temporal changes in receptor channel conductance (binary (1-permeable / 0-impermeable) and this information can be used as a prelude to the quantification of ion permeability across receptor channels (graded) using the Goldman-Hodgkin-Katz equations based on induced shifts in reversal potential  empty, (b) occupied by Na+ and (c) occupied by Ca2+  and B (R5C1) in determining iq’s permeability through the respective receptor channels . The model predicts that both Na+ and Ca2+ can permeate GluN2-containing d-NMDARs and that these receptors are selective for both cations by virtue of their selectivity filter properties. This is a departure from the commonly held perspective that these receptors are non-selective for monovalent and divalent cations. Note that while both Na+ and Ca2+ can displace themselves or each other from within the pore of the d-NMDAR channel  predicts selectivity for and permeability to Na+ when the pore is either empty or occupied by Na+ but not when occupied by Ca2+ .We assayed using the charge permeability Equation (1), the permeabilities of GluN2-containing rq = –4;  and GluNrq = –3;  to Na+ aCa2+ see . The sch channel , Na+ canharge rq . Ca2+, ot-NMDARs . This sufor Ca2+ . A furth–3 to –2  alters t by Ca2+ . Furthery A = 0; , suggest2+ permeability of AMPARs (2+ permeable unlike those assembled with it (rq from –4 to –2 (rq of –2 renders receptors selective for and permeable to Na+ but not Ca2+ (top panel). However, AMPARs assembled from any subunits other than GluA2 yield receptors with selectivity filters that have a rq of –4 (bottom panel) that are known to be selective for and permeable to both Na+ and Ca2+ , as predicted by the charge permeability equation (1), as a function of total ring charge of their respective selectivity filters  or empty . This may be the reason why d-NMDARs (rq = –4) are selective for and permeable to Na+ and Ca2+. For all other combinations (–3 ≤ rq ≤ –1), the receptors are either permeable to Na+ or Ca2+, but not both. This selective permeability arises in part from the attractivity of the selectivity filters for various ions and the interactions ions occupying the pore of the channel have with its ring charge. For example, at rq = –3, Na+ is permeable through the receptor channel until it is displaced from the pore and occupied by Ca2+. Once occupied by Ca2+, the receptor becomes permeable only to Ca2+ because a calcium ion alone can displace it from the pore of the channel. This may be the reason why GluN3-containing t-NMDARs (rq = –3) are highly Ca2+ permeable. At rq = –2, the attractivity to Ca2+ is zero and as Na+ can only displace Na+ but not Ca2+ from the pore of the channel, the receptor is rendered permeable only to Na+. This may be the reason why GluN2-containing AMPA and GluN3-containing d-NMDA receptors (rq = –2) are permeable to Na+ but not Ca2+. At rq = –1, the attractivity to Na+ is reduced to zero, and as Ca2+ can displace either Na+ or Ca2+ from the pore of the channel, these receptors would be rendered permeable only to Ca2+. However, we know of no known AMPA or NMDARs with selectivity filters whose rq = –1.To fully characterize the permeabilities of Na filters . Note thlters  that occupy fixed locations within the pore-forming M2 domains of each of the four subunits that compose these receptors. These amino acids are conserved across all members of a subunit family and their site directed mutagenesis has been shown to disrupt ion permeability in these receptors implying that they constitute a putative selectivity filter that enables them to screen for specific ions. Attractivity of cations by the selectivity filter ring charge, however, is not the sole determinant of their permeability. For example, Na+ is attracted to a receptor channel occupied by Ca2+ in both d-NMDARs  arranged along the outer periphery of a rotary dial marked with a receptor-selector , and three orthogonally oriented indicators  for readout of K+, Na+ and Ca2+ selectivity now incorporates a ring charge indicator  that enables correlation between the ring charge of the selectivity filter and permeability of the chosen receptors to Na+ and/or Ca2+. Cation selectivity levels are graded, color-coded (white/non-colored regions indicate non-selective and/or impermeant), and strategically positioned around the dial to capture the dynamics of change in ion selectivity as the knob is rotated to select for a particular receptor type and/or ring charge (bottom panels). Thus, it can be inferred that loss of K+ selectivity is associated with the emergence of Ca2+ permeability  receptors. However, the mechanisms proposed are all either structurally and/or functionally distinct from those indicated for AMPA and NMDARs in this study or are not fully understood. Neuronal nAChRs are pentameric structures whose subunits are classified into two families, alpha  and beta  2(β2)3 nAChR subtypes allow both Na+ and Ca2+ ((i) the presence of a ring of negatively charged glutamate residues at the narrow intracellular end of the receptor ((ii) differential effects of the hydrophobic constriction on water coordination by sodium and chloride ions; (iii) overall negative electrostatic environment in the M2 region and to some degree, the dynamics of the pore-lining residues and its crucial influence on the pore radius and ion hydration (3 receptors is under the control of charged rings at either end of the pore and dominated by the ring of negatively charged residues (glutamate) at the intracellular side of the channel. Changing the charge at this position has been shown to change ionic selectivity even in anion-selective receptors suggesting that electrostatic factors alone control selectivity in this family of receptors which also include nAChRs (3 channels may be homo (5-HT3A)- or heteropentameric (5-HT(3A)1(3B–3E)4) and are permeable to Na+, K+, and/or Ca2+ (3 receptors) also apply to AMPA and NMDARs, we increased the total negative ring charge of the selectivity filter rq to –5 and above , while querying for changes in ion selectivity and permeability using the charge permeability equation (1). Our model predicts that increasing the magnitude of the ring charge beyond –4 (rq < –4) makes the receptors ineffective in screening for cations, enabling both Na+ and Ca2+ to permeate the channel. This built-in redundancy may be one reason why AMPA and NMDARs assemble as tetramers and not pentamers. Given that the ability to screen for ions is also lost when the ring charge is completely ablated (rq = 0), suggests that cation selectivity and permeability in these receptors is also dominated by electrostatic mechanisms despite differences in the structural determinants of their selectivity filters.Subunit-dependent ion selectivity and permeability, based on charge attractivity principles and negative electrostatic environment in the M2 region, has also been suggested for other ligand-gated ionotropic receptors including nicotinic acetylcholine receptors (nAChRs) and serotonin  . Homo-oland Ca2+ . The undreceptor ; (ii) diydration . Ion sele nAChRs . Functio/or Ca2+ . To dete+/Ca2+) in AMPA and NMDARs, we asked how subunit-driven alterations in ring charge of their selectivity filters affect their selectivity and permeability to anions (Cl–). The model predicts that full permeability to Cl– alone does not arise until rq = –6 (see – impermeable. While attractivity is maintained throughout (A = 1), the inward drive (B) varies depending on the ring charge and the ions occupying the pore such that in the presence of Na+ and Ca2+, AMPA and NMDARs are rendered impermeable to Cl–, which is consistent with what has been reported in the literature for d-NMDARs  subunits are cation selective whereas those assembled from the R-form (rq = 0) subunits are permeant to anions and cations suggesting that differences in the anion vs. cation selectivity, in Ca2+ permeability and in channel conductance are likely to be determined by the difference in charge density of the channel , formulated primarily for assessing permeability of cations  is based, namely subunit-specific attractivity of ions and charge interactions within the pore of the receptor channels. Substitution of asparagine with glutamine at location 614 (N614Q) in the ε1 subunit has no effect on Ca2+ permeability but strongly reduces the affinity of Mg2+ for its binding site due to an increase in the rate of unbinding. The same mutation in the ζ1 subunit, on the other hand, reduces both the permeability of the channel for Ca2+ ions and its block by Mg2+ (2+ within the channel pores block the flux of sodium and other ions highlighting the emergence of ion selectivity and as a single mutation perturbs two distinct binding sites for Mg2+ and Ca2+ in opposite ways, the binding of divalent ions at the two sites interact with one another (Of note regarding selectivity and permeability of divalent cations through NMDARs are the differences in behavior of Cafor Ca2+ . Althoug by Mg2+ . The pre another . In summThe original contributions presented in the study are included in the article/SK helped in conceptualization, data analysis, and mathematical modeling. SSK helped in conceptualization, mathematical modeling, and writing of the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Parkin-mutant human fibroblasts, with defective oxidative phosphorylation activity, showed high basal cAMP level likely ascribed to increased activity/expression of soluble adenylyl cyclase and/or low expression/activity of the phosphodiesterase isoform 4 and to a higher Ca2+ level. Overall, these findings support the existence, in parkin-mutant fibroblasts, of an abnormal Ca2+ and cAMP homeostasis in mitochondria. In our previous studies resveratrol treatment of parkin-mutant fibroblasts induced a partial rescue of mitochondrial functions associated with stimulation of the AMPK/SIRT1/PGC-1α pathway. In this study we provide additional evidence of the potential beneficial effects of resveratrol inducing an increase in the pre-existing high Ca2+ level and remodulation of the cAMP homeostasis in parkin-mutant fibroblasts. Consistently, we report in these fibroblasts higher expression of proteins implicated in the tethering of ER and mitochondrial contact sites along with their renormalization after resveratrol treatment. On this basis we hypothesize that resveratrol-mediated enhancement of the Ca2+ level, fine-tuned by the ER–mitochondria Ca2+ crosstalk, might modulate the pAMPK/AMPK pathway in parkin-mutant fibroblasts.Parkin plays an important role in ensuring efficient mitochondrial function and calcium homeostasis.  PARK2 and PARK6, which encode for parkin and PINK1 respectively, are genes responsible of the onset of familial Parkinson’s disease (PD) a progressive degenerative disorder of the central nervous system characterized by dopaminergic neurodegeneration in the substantia nigra pars compacta. Several studies in PD models proved hallmarked dysfunctions of mitochondria, in particular, defect of the respiratory chain complex I, which plays a central role in mitochondrial oxidative phosphorylation (OXPHOS) efficiency and capacity . The re2+ ATPase (SERCA) is involved in maintaining the low resting Ca2+ concentration in cytosolic compartment. In both control and parkin-mutant fibroblasts, the co-treatment with resveratrol and thapsigargin (TG+RSV) did not further increase the high cytosolic level of Ca2+ elicited by TG alone. This leads to assume a limited inhibition of SERCA by resveratrol as already described [2+ level as compared to the TG-treated cells. This result is in agreement with the role of mitochondria in providing a local source of Ca2+ for ER refilling in Ca2+-depleted ER [parkin-mutant fibroblasts, the co-treatment TG+RSV induced an increase of mitochondrial Ca2+ level, as compared with TG-treated cells, showing a level of Ca2+ comparable to that measured in the presence of resveratrol alone. In parkin-mutant cells the TG+RSV co-treatment seems to prevent or, in any case, not to allow the mitochondrial Ca2+ ER refilling in Ca2+-depleted ER cells. The endoplasmic reticular Caescribed . Furtherleted ER . On the 2+ transfer from ER to mitochondria takes place [2+ level in the cytosolic compartment, in both control and parkin-mutant fibroblasts but a marked decrease of the mitochondrial Ca2+ only in control cells, as compared with Dan-treated cells. Under these conditions, in control cells, resveratrol could induce a partial inhibition of SERCA, causing an increase in cytosolic Ca2+ level and, as described for the TG-treatment, this could induce the ER refilling by the mitochondria. In parkin-mutant fibroblasts this process, even in this case, does not appear to occur and upon co-treatment (Dan+RSV) no significant changes of mitochondrial Ca2+ level were observed, as compared with Dan-treated cells. Previous studies clearly established in MAMs a functional and structural communication between mitochondria and ER ,109,110,es place . Co-incuparkin-mutant fibroblasts, the increased resveratrol-dependent Ca2+ level could be responsible for the enhanced activity of intramitochondrial Ca2+-sensitive dehydrogenases. This leads to an increased supply of reducing equivalents for the respiratory chain activity and consequent increase of ATP synthesis [parkin-mutant fibroblasts, the basal high level of mitochondrial Ca2+ is related to dysfunctional mitochondria, mainly derived by the failed auto(mito)phagic process. In this context, resveratrol-induced Ca2+ increase could lead to an improvement of the oxidative phosphorylation system and oxidative stress condition in new functional mitochondria derived from a rebalanced mitochondrial biogenesis vs. mitophagy, resulting from the Ca2+-dependent AMPK/SIRT1/PGC1α activation. In ynthesis ,111. We 2+ homeostasis, in parkin-mutant cells, is object of debate. It is reported that parkin-null cells and fibroblasts expressing mutant parkin showed reduced ER–mitochondria contact sites associated with a decrease in mitochondrial Ca2+ [2+ overload associated with a lower threshold of mPTP-opening, making neurons vulnerable to apoptosis [2+ stimulates the mitochondrial respiratory chain, an excessive Ca2+ load is dangerous for mitochondria by opening mPTP, which results in mitochondrial membrane potential dissipation and respiratory chain uncoupling, associated with a decrease of mitochondrial ATP synthesis [2+ uptake causes a reduction of mPTP opening making the cells resistant to apoptosis . Howeveoduction , we are oduction ,115. ThuR stress , resvera2+ transfer between ER and mitochondria, we studied the effect of 24 h resveratrol-treatment on the expression level of GRP75, Mfn2, and Miro2, three proteins involved in MAMs. Noteworthy, we observed a higher expression level of GRP75 in parkin-mutant fibroblasts, as compared with control cells. GRP75 is part of a multiprotein complex gathering IP3R and VDAC1, functionally coupling ER and mitochondria and promoting Ca2+ exchanges [2+ level [parkin-mutant fibroblasts could contribute to the higher basal Ca2+ level measured in the mitochondrial compartment. In keeping the importance of preserving a proper Caxchanges . In mous2+ level ,117. Theparkin-mutant fibroblasts showed a higher expression of Mfn2 as compared with control cells, which was decreased by resveratrol-treatment. Mfn2 is mainly localized at the MAM-related contact sites [parkin-mutant fibroblasts. In primary fibroblasts from parkin knockout mice and from parkin-mutant fibroblasts, a recent study showed an augmented ER–mitochondria tethering and ER-to-mitochondria Ca2+ transfer, likely due to increased Mfn2 level in MAMs [2+ transfer between the two organelles [parkin-mutant fibroblasts, the high expression level of Mfn2, likely due to the lack of its ubiquitination, could be involved in the increased steady-state Ca2+ level. Moreover, ct sites ,120,121,ct sites ,122. It ct sites ,124 for  in MAMs . Furtherganelles . In the 2+-sensing EF hand domains, in the ER–mitochondria contact sites regulation [2+ level in PINK1 mutant dopaminergic neurons [parkin-mutant fibroblasts, which could be responsible of the high Ca2+ level therein. Furthermore, it is worth mentioning that the ER stress upregulates GRP75 [2+ overload, exactly as observed by our group in parkin-mutant cells. Rho GTPases Miro1/2, localized in the mitochondrial outer membrane, are components of a complex that anchors mitochondria to motor proteins. Their ubiquitination by parkin leads to mitochondrial arrest that further facilitates the elimination of impaired mitochondria by mitophagy ,126. Recgulation ,69. Furtgulation . Consist neurons . As preves GRP75  and Mfn2es GRP75  expressiparkin-mutant fibroblasts. Thus, the resveratrol treatment, by modulating specific signaling pathways such as AMPK//SIRT1/PGC1α [2+ homeostasis; indeed, in colon cancer cells it has been shown that resveratrol induces a metabolic reprogramming, increasing oxidative capacities, pyruvate dehydrogenase activity, and ATP production. These effects were abrogated by Ca2+ chelation or the blockade of the mitochondrial Ca2+ uniporter as well as by the inhibition of AMPK pathway [Parkin plays a central role in the mitochondrial quality-control processes  in whichT1/PGC1α , might lT1/PGC1α ,131), dr pathway . 2+ homeostasis in human skin parkin-mutant fibroblasts, a parkin-null cellular model. The OXPHOS efficiency improvement by resveratrol, via the AMPK/SIRT1/PGC1α pathway, which we reported in parkin-mutant fibroblasts in our previous study [2+ levels and modulation of protein expression at the ER–mitochondria contact sites. In this study we showed that resveratrol induces a significant increase of cytosolic and mitochondrial Ca2+ level in parkin-mutant fibroblasts, resulting in a remodulation of the cAMP level. Moreover, resveratrol induces a significant downregulation of the expression level of Miro2 and Mfn2, proteins involved in the ERMCSs, highly expressed in parkin-mutant fibroblasts, likely regulating the Ca2+ traffic between ER and mitochondria. These findings might shed new light in identifying novel molecular targets for PD treatment.The aim of the present study was to investigate the effect of resveratrol on deregulated cAMP and Caus study , can be


Ci, on the orientation of the reinforcement direction α relative to the delamination front was investigated for two laminates of different interfacial ply arrangements. In the case of the first laminate, the delamination was located at the interface of the layers reinforced with symmetric fabric and unidirectional fabric. In the case of the second laminate, the delamination was located at the interface of layers reinforced with symmetric fabric. In both laminates, the orientation of fibers in the layers separated by the delamination differed by 45° regarding the warp directions. The investigations were carried out for Mode I, Mode II, and Mixed-Mode I/II (GII/GI = 1 and GII/GI = 1.7) loadings using hybrid beam specimens. The major problem appearing in the intended tests was the inevitable lack of symmetry in the xz and xy planes of the specimens and the resulting deformation and stress–strain couplings, causing undesired loading modes. To decrease these couplings, especially designed hybrid beam specimens were used. An auxiliary finite element analysis was performed to assess the remaining effects of the reduced couplings. To ascertain whether statistically significant differences between Gci values for different α occurred, the one-way analysis of variance supplemented by Levene’s test was carried out. The dependence of Gci on α was found out for both laminates. However, it was not equally strong, and it turned out that the loading mode and the interfacial ply were arrangement sensitive.Dependence of the initiation values of the Strain Energy Release Rate, G C on θ was broadly investigated, but mainly for laminates reinforced with unidirectional (UD) tapes. In ,,C on θ wapes. In  for 0°//apes. In , resistae.g., in ,16,17,18e.g., in ,20,21,221+ θ2), as shown in 1 could be considered as an angle of reinforcement below and θ2 above the delamination plane, as shown in The vast number of published papers focused on a particular delamination process in which a bisector of θ, θ = θ+ θ2, as ci(α) relationship.Due to economic reasons, an application of fabric reinforcement is equally common as UD tapes because the former allows for a less laborious layup. A typical fabric reinforced laminate comprises a certain number of UD and symmetric fabrics or symmetric fabrics only, which are rotated relative to each other by θ = 45°.  This research concerns resistance against delamination growth of such laminates and focuses on the Gci(α) relationship and (b) propagation rate da/dn , which can be described by the Paris law. The presented research concerns the former because it is crucial and must be obtained in the first place. Furthermore, despite this need, there are few papers with public access concerning the Gci(α) relationship.The reason for undertaking this research was the following. Very often, damage tolerance design must be supported by various numerical analyses. If a delamination growth is of interest, two pieces of information are needed. These are (a) initiation value of the Strain Energy Release Rate Gci(α) relationship for Mode I, Mode II, and Mixed-Mode I/II loadings only. This relationship was investigated experimentally, taking to a large extent advantage of the concepts presented in ASTM D5528, D7905, and D6671 standards 1 = 30°, orted in , resultsosed. In , resultsic with α would require very extensive fractographic studies, being a problem all by itself. In the frame of the presented research, it was possible to perform only very limited fractographic investigations. The results showed high complexity of the delamination surface morphology, as shown in In-depth investigation of the reasons for such a variation of GAt higher magnification, one could notice that the penetration of the strands by the propagating delamination was limited to the most external fibers of strands and roughly followed the strands’ envelope, as shown in ic with α. Unfortunately, very extensive fractographic investigations would be necessary to verify this hypothesis, as was mentioned. Such studies were out of the scope of this research; nevertheless, they shall be a topic of future investigations.The dependence of Gci on α was investigated under Mode I, Mode II, and Mixed-Mode I/II loadings for two different interfacial fiber arrangements. Delaminations were located at the interface of UD and symmetric fabrics (Laminate A) and at the interface of symmetric fabrics (Laminate B). Occurrence of the mentioned dependence was found out for both interfacial fiber arrangements; however, it was not equally strong. It was affected by the loading modes and the interfacial fiber arrangement.