We have reported that Kv4.3 proteins play an important role in delicate regulation of the membrane-associated CaMKII activity in ventricular myocytes. Here, we investigated the effect of in vivo Kv4.3 expression on ICa in HF left ventricular (LV) myocytes.CaCa density in subepicardial (SEP) and subendocardial (SEN) myocytes was proportionately reduced, without changing the transmural gradient. While the time course of ICa decay was hastened, the voltage-dependence of ICa activation and inactivation, however, remained unchanged. ICa recovery from inactivation was significantly accelerated. In line with the partial inhibition of CaMKII, the frequency-dependent Ca2+-induced ICa facilitation was recovered in the HF myocytes transfected with Kv4.3.Kv4.3 expression reduced overall CaMKII autophosphorylation with much greater reduction in the membrane compartmentalized CaMKII activity. ICa was recorded in dissociated SEP and SEN myocytes using whole-cell patch clamp method.Pressure-overload HF was induced by thoracic aortic banding. Kv4.3 expression was achieved by Ad-Kv4.3 injection in the LV myocardium. ICa remodeling via inhibition of the membrane-associated CaMKII, pointing to Kv4.3 restoration as a potential therapeutic approach for the disordered calcium regulation in HF.Kv4.3 expression in HF ventricle can effectively reverse I Ca is the key mediator for the excitation-contraction (E-C) coupling in cardiomyocytes. Ca2+ entry through L-type calcium channel (LTCC) triggers a release of Ca2+ from sarcoplasmic reticulum (SR), initiating myofilament contraction. The amplitude and the kinetics of ICa determine the amount of Ca2+ entry and consequently the contractile force and relaxation. In addition, Ca2+ entry and SR Ca2+ release activates a number of Ca2+-sensitive signaling cascades, most important among them is Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII plays a key role in regulation of E-C coupling by phosphorylation of a number of proteins involved in Ca2+ handling, such as LTCC, ryanodine receptor (RyR2), sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLB) i. This finding revealed that the CaMKII-Kv4.3 units are important intrinsic CaMKII inhibitor for the delicate regulation of CaMKII activity in ventricular myocytes. However, a growing body of evidence has elucidated that a reduction of Ito is a well-known feature of ventricular myocytes from HF animal models and patients, and this remodeling occurs at the early developing stage , was recovered by Kv4.3 expression. Altogether, these results suggest that the expression of Kv4.3 can successfully reverse ICa remodeling in ventricular myocytes of HF.In this study, we report a significant reduction in Imyocytes . In addif CaMKII , a commoin vivo expression of Kv4.3 in HF ventricular myocytes. Due to the membrane distribution i, which decreases the overall intracellular CaMKII activity. This is exactly what we observed in this study. However, since the LTCC is a membrane protein, the membrane-localized CaMKII activity is accordingly much more important for the regulation of ICa than the bulk CaMKII activity. In this study, we have demonstrated the key role of Kv4.3 expression in the regression of ICa remodeling in HF ventricular myocytes. We cannot, however, dissect the contribution of the overall CaMKII activation which is associated with the APD prolongation. This portion of contribution (if any) would be minor due to the fact of small change in the bulk CaMKII activity which is also away from the LTCC location. As an advantage, we recorded ICa in the myocytes with and without successful Kv4.3 expression in the same heart, which avoided the influence of heart condition and the variations of the dissociation-related cell quality.As an endogenous CaMKII inhibitor, Kv4.3 mainly affects CaMKII activity in the sarcolemmal compartment due to their membrane localization in myocytes. Meanwhile, Kv4.3 expression also leads to APD shortening and a consequent reduction in , we have separated the subepicardial and subendocardial myocytes for data acquisition and analysis.Currents were recorded in the LV myocytes isolated from HF mice 7 days after Ca and outward K+ current were recorded by whole-cell voltage clamp technique, with pipette resistances of 2-3 M when filled with internal solution. ICa was recorded at room temperature. Cells were depolarized every 10 s from a holding potential of -50 mV to test potentials between -40 to +60 mV (10mV steps) for 300 ms. To minimize the impact of ICa run-down, we added 5 mmol/L Mg-ATP to the pipette solution and conducted data acquisition after 5 - 10 minutes of equilibration between pipette solution and intracellular contents  and immunoblotted for Kv4.3, CaMKII, phospho-CaMKII (p-CaMKII), p-PLB-Ser16, Phospho-Thr17-PLB, PLB, and GAPDH, respectively. Primary antibodies used in this study include rabbit polyclonal and mouse monoclonal anti-CaMKII antibodies (M-176 and G-1) , polyclonal anti-phospho-CaMKII (Thr287) antibody , goat polyclonal anti-PLB antibody (Santa Cruz Biotechnology), rabbit polyclonal anti-P-Ser16PLB antibodies , rabbit polyclonal anti-P-Thr17PLB antibodies , rabbit polyclonal antibody against Kv4.3  and mouse monoclonal anti-GAPDH antibody (Millipore Corporation).Myocytes were homogenized in 1% TX-100 buffer [50 mM Tris-HCl, pH 7.4, 4% glycerol, 1 mM DTT, 1% Triton X-100, 1 mM EDTA, Mini-CompleteCa amplitude was evaluated as the peak inward current. Biexponential functions were used to model current decay kinetics. Peak outward K+ current amplitude was measured as the difference between the peak current and the sustained current remaining at the end of a 5000 ms pulse. In order to correct for variability in cell size, current were expressed as current density by dividing the absolute current amplitude by cell capacitance (pA/pF). Results are expressed as mean ± SEM. Statistical analysis was performed using Sigmastat for Windows (Version 3.5).). The paired and unpaired t-test was used for the comparisons and p<0.05 was regarded as significant.I


However, little is known about its spatiotemporal features in the context of osteoarthritis (OA). Herein, by examining the cartilage samples collected from patients undergoing knee arthroscopic surgery, we investigated the spatiotemporal features of spontaneous [Ca2+]i signaling in in situ chondrocytes at different OA stages. Our data showed zonal dependent spontaneous [Ca2+]i signaling in healthy cartilage samples under 4 mM calcium environment. This signal was significantly attenuated in healthy cartilage samples but increased in early-degenerated cartilage when cultured in 0 mM calcium environment. No significant difference was found in [Ca2+]i intensity oscillation in chondrocytes located in middle zones among ICRS 1–3 samples under both 4 and 0 mM calcium environments. However, the correlation was found in deep zone chondrocytes incubated in 4 mM calcium environment. In addition, increased protein abundance of Cav3.3 T-type voltage dependent calcium channel and Nfatc2 activity were observed in early-degenerated cartilage samples. The present study exhibited OA severity dependent spatiotemporal features of spontaneous [Ca2+]i oscillations of in situ chondrocytes, which might reflect the zonal specific role of chondrocytes during OA progression and provide new insight in articular cartilage degradation during OA progression.Intracellular calcium ([Ca Chondrocytes, as the highly specialized, metabolically active cells, regulate the development, maintenance, and repair of extracellular matrix (ECM) in articular cartilage2. Unlike its low cellular activity and matrix turnover ability in healthy articular cartilage4, chondrocytes play a pivotal role in OA initiation and progression by upregulating expression of matrix metalloproteinases , aggrecanase (ADAMTS-5) and inflammatory cytokines 5.Osteoarthritis (OA), which involves the dysfunction of adult articular cartilage, is the most common form of joint disease with manifestations of damaged articular cartilage, and may result in arthralgia, joint deformation, and limited mobility in patients. OA is the second leading cause of long-term disability in adults. Several risk factors of OA (2+]i) signaling is an essential universal secondary messenger that mediates the cellular metabolic activity in chondrocytes10. Based on its spatiotemporal parameters , encoded information is deciphered by various downstream transcription factors (e.g. nuclear factor of activated T cells (Nfat), nuclear factor-κB (NF-κB), c-Jun N-terminal kinase 1 (JNK1), myocyte enhancer factor-2 (MEF2), and cAMP response element-binding protein (CREB)), and ultimately leads to a range of metabolic and signaling processes11. Due to its unique role in extracellular-intracellular signal transduction, [Ca2+]i signaling has been widely studied in articular cartilage. It has been shown that chondrocytes not only respond to mechanical and chemical stimuli with [Ca2+]i signaling via modulating extracellular calcium influx and intracellular calcium release10, but also reflect its peri-cellular environment  with repetitive spontaneous [Ca2+]i signaling when statically cultured12, which plays a crucial role in regulating ECM metabolic activity12. With abnormal joint physiology such as increased mechanical loading and surrounding inflammatory activity, response of chondrocytes to their peri-cellular environment may be significantly altered, potentially contributing to the onset or progression of OA.Intracellular calcium  that affected chondrocyte mechanics19.Articular cartilage is a highly organized tissue, its zonal differences in biochemical content and bioelectrical properties are directly associated with depth-dependent changes in mechanical properties2+]i signaling of human articular chondrocytes located in its native PCM and ECM, and compared the zonal differences in [Ca2+]i signaling of in situ human chondrocytes. By comparing the spatiotemporal features of spontaneous [Ca2+]i signaling in in situ chondrocytes at different OA stages, the correlation between spontaneous [Ca2+]i signaling and OA severity was investigated. In addition, to address the possible mechanism of OA chondrocytes [Ca2+]i signaling alternation and downstream regulation of [Ca2+]i signaling, the protein level of T-type voltage-dependent calcium channels (VDCCs) and Nfatc2 activity were analyzed using total knee arthroplasty (TKA) samples.In the present study, we aimed to investigate the spontaneous [Ca2+]i signaling analyzed from each sample were classified into ICRS grades. Representative hematoxylin and eosin (H&E), alcian blue, and sirius red staining images for ICRS 0–3 grades were shown in Fig. Based on the International Cartilage Research Society (ICRS) grading system, the severity of each collected human articular cartilage sample was verified by histomorphometry approaches. Later on, the spatiotemporal features of spontaneous [Ca2+]i oscillations in ICRS 0 cartilage sample were shown in Fig. 2+]i oscillations were observed in a large portion of chondrons in middle and deep zones of both normal and OA cartilages. As shown in Fig. 2+]i oscillations propagated from cell number 2 to neighboring cells , and from cell number 6 to cell number 7. The time lag of [Ca2+]i peaks between each propagation of cell 1–7 also suggested this phenomenon.Spontaneous calcium signaling was observed in chondrocytes within both normal (ICRS 0) and OA (ICRS 1–3) articular cartilages without extraneous stimuli. Typical [Ca2+]i signaling in in situ chondrocytes were zonal, [Ca2+]e concentration, and ICRS grade dependent. Typical [Ca2+]i oscillations for both normal (ICRS 0) and early (ICRS 1) OA cartilages under 4 mM [Ca2+]e environment were shown in Fig. 2+]i oscillations increased with the depth of cartilage   located in single chondrons of deep zone demonstrated repetitive  and stronger [Ca2+]i peaks  during the same recording period  cells showed valid [Ca2+]i peak, with 0.41 ± 0.07 peaks (P > 0.05) and calcium peak magnitude of 1.91 ± 0.09 fold (P < 0.01) during the same recording period.Spatiotemporal features of e environment, the difference in each parameter of [Ca2+]i oscillations between middle and deep zones was abolished (P > 0.05), due to decreased [Ca2+]i oscillations of chondrocytes in deep zone when cultured in 0 mM [Ca2+]e environment . Furthermore, [Ca2+]i oscillations in chondrocytes in the superficial zone were completely diminished when cartilage was incubated in 0 mM [Ca2+]e environment.In 0 mM [Ca2+]i oscillations of chondrocytes between middle and deep zones was attenuated under 4 mM [Ca2+]e environment  . Furthermore, compared with 4 mM [Ca2+]e culture, chondrocytes in ICRS 1 deep zone displayed stronger [Ca2+]i when incubated in 0 mM [Ca2+]e environment .In early OA (ICRS 1) cartilage, zonal difference in [Ca05) Fig. . However2+]i signaling was not performed in superficial zones since this region was only presented in ICRS 0 samples. No zonal or [Ca2+]e concentration-dependent difference was found in the middle zones of all ICRS grades  and ICRS 3  samples demonstrated significantly weaker [Ca2+]i oscillations when incubated in 4 mM [Ca2+]e environment. Also, chondrocytes in ICRS 3 samples showed even weaker [Ca2+]i oscillations than that displayed by cells in ICRS 2 samples . However, this trend was not found in ICRS 1 samples. Chondrocytes located in the deep zone of ICRS 1 samples showed comparable [Ca2+]i oscillations of ICRS 0 chondrocytes when incubated in 4 mM [Ca2+]e environment . Furthermore, compared with samples within each ICRS grade in 4 mM [Ca2+]e environment, no significant difference was found in each ICRS 0, 2, 3 samples; while in ICRS 1 samples, significantly stronger [Ca2+]i oscillations were found in 0 mM [Ca2+]e environment as described above.In 0 mM [Ca2+]i signaling parameters of ICRS 0–3 samples to ICRS grading system under 4 mM and 0 mM [Ca2+]e environment, respectively (Table P < 0.001) and responsive rate  were moderately correlated with ICRS grade when cartilage samples were incubated in 4 mM [Ca2+]e environment. In contrast, when cartilage samples were incubated in 0 mM [Ca2+]e environment, the correlation between parameters of [Ca2+]i signaling and ICRS grading system was very weak.To analyze the possible correlation between spontaneous calcium signaling and OA severity, Spearman’s rank correlation coefficient method was used to analyze the relationship of [Caly Table . Results2+]i signaling in ICRS 1 samples, non-degenerated  and early-degenerated (OA) regions of TKA cartilage samples were used for RT-qPCR and Western boltting analyses. Compared with non-degenerated cartilage, chondrocytes in early-degenerated cartilage had significantly higher level of MMP-1, MMP-13, and COL1A1; and lower level of SOX-9 and SOX-11 22, mechanical sensitive calcium channels (MSCCs)18, transient receptor potential vanilloid (TRPV)7, and Ca2+ release-activated Ca2+ (CRAC) channel23. These calcium channels synergistically regulate [Ca2+]i homeostasis by modulating extracellular calcium influx24 and intracellular calcium release from endoplasmic reticulum25. In the present study, total abolishment and significant attenuation of spontaneous [Ca2+]i signaling in chondrocytes located in superficial and deep zones were noticed when incubated in 0 mM [Ca2+]e environment. Since cells in these zones were located in the peripheral area of cartilage samples and more susceptible to extracellular changes of environmental calcium concentration than cells in the middle zones, our results suggested that similar to MSCs26, spontaneous [Ca2+]i signaling in chondrocytes was dependent on extracellular calcium influx. However, unlike the results presented by Kono et al., our observation suggested that [Ca2+]i signal propagation existed in most chondrons of both healthy and OA cartilage. This contradiction could be explained by variation in the area where the cartilage sample was examined. In our observation, propagation of [Ca2+]i signal was only observed in middle and deep zones, where 5–10 chondrocytes were closely located in one chondron. Since articular cartilage is an avascular and an eurogenic connective tissue, in which a chondrocyte is isolated by its native PCM and lacks direct cell-cell connection27; [Ca2+]i signaling at a distance12 is possibly through extracellular messengers such as nitric oxide (NO) and nucleotides and nucleosides, ATP, uridine triphosphate (UTP), or adenosine diphosphate (ADP)28. Propagated [Ca2+]i signal in in situ chondrocytes may serve as an efficient intercellular communication pathway6.It is recently discovered that both isolated and 2+]i response, spontaneous [Ca2+]i signaling has been shown to regulate ECM maintenance in cartilage. Zhou et al. recently suggested that sustained spontaneous [Ca2+]i signaling enhanced mechanical properties of cartilage samples during long-term ex-vivo culture12. By analyzing the spatiotemporal features of spontaneous [Ca2+]i signaling in in situ chondrocytes of calf articular cartilage, they showed a high correlation between responsive rate and mechanical property of cultured explants. This observation was confirmed in the present study, in which we showed an increased responsive rate of spontaneous [Ca2+]i signaling from superficial to deep zone in normal cartilage. This phenomenon might reflect the distinct function of chondrocytes located in different zones revealed by microarray analysis29, which demonstrated that chondrocytes isolated from deep zones synthesized more proteoglycans than cells from the superficial zone30. In addition, Spearman’s rank correlation coefficient method also revealed that the responsive rate of spontaneous [Ca2+]i signaling was most correlated with the severity of cartilage degradation when incubated in 4 mM [Ca2+]e environment, suggesting that the spontaneous [Ca2+]i signaling could serve as a useful indicator of OA progression.Similar to mechanically induced [Ca2+]i signaling of in situ chondrocytes was compared among different ICRS grade samples, no significant difference was seen in middle zone chondrocytes, indicating that chondrocytes in this zone were relatively inactive during the whole process of OA. In contrast, decreased intensity of [Ca2+]i signaling in deep zones of late-stage OA cartilages was found among grades. Decreased calcium channel activity caused by alternation in channel structure of OA chondrocytes could be one of the reasons22. Animal model using combination of orally administered monosodium iodoacetate (MIA) and treadmill over-loading showed decreased protein level of TRPV5, a voltage-independent cation channel in OA chondrocytes31. A recent study using OA animal model and human sample also revealed decreased TRPV6 expression in degraded cartilages32. In addition, by using RNA-seq analysis, Dunn et al. compared gene expression changes in human OA cartilage samples and found that α2/δ, a subunit of voltage-dependent calcium channels regulating channel properties and increasing the functional channel33, was significantly down-regulated34. By using the larger volume of cartilage sample acquired during total knee arthroplasty (TKA) surgery, we showed that chondrocytes in the early-degenerated area had significantly increased expression level of MMP-1, MMP-13, COL1A1; and decreased expression level of SOX-9 and SOX-11. In addition, increased protein abundance of Cav3.3, a α1 subunit of T-type VDCC in the early-degenerated area was noticed. The transiently active Cav3.3 T-type VDCC requires weak membrane depolarization for activation. Increased Cav3.3 protein abundance might decrease the membrane potential35 and pronounce [Ca2+]i signaling of chondrocytes in deep zone of early OA sample when few calcium irons were presented; and ultimately activated Nfatc2, a transcription factor has been shown to be crucial in chondrogenesis in tracheal cartilage36, and function regulation of adult chondrocytes37. Despite the fact that early OA was most pronounced in the superficial and middle zones38, our data suggested a unique role of deep zone chondrocytes in the initiation of OA. In early OA, the increased Nfatc2 activity might serve as a compensatory mechanism to withstand the established damage in articular cartilage. There is a growing consensus that OA is a whole-joint disease39. Increased turnover of subchondral bone seen in OA patients40 and animal models41 indicated an important role of subchondral bone-deep zone cartilage crosstalk during the onset of OA. Our previous studies using human OA samples and destabilization of medial meniscus (DMM) model showed decreased thickness in calcified cartilage zone (CCZ) at early stage40, accompanied by increased invading vessels density in this region41. These observations suggested elevated paracrine transport of inflammatory factors and cytokines released from subchondral bone could influence the calcium channel activities of deep zone chondrocytes, ultimately altering the ECM components such as proteoglycan concentration and composition of collagen observed in the present study.When spontaneous [Ca2+]i signaling in OA progression remains unclear. More stable sample source such as cartilage collected from the large-animal model is needed for performing [Ca2+]i dependent downstream pathway analysis.The present study has several limitations. Firstly, although human biopsy samples at different OA stages represent the real progression of OA and retain the native PCM and ECM, they also suffer from disadvantages such as small sample volume, limited sample quantity, and restricted location for sample collection. In the current study, only a small volume of cartilage sample was acquired from each patient to prevent further damage to articular cartilage. To exclude the individual variation between patients, larger samples size and paired control sample representing non-degenerated cartilage from the same individual seems necessary for mechanism study. Secondly, the role of elevated spontaneous [Cain situ chondrocytes resided in both healthy and OA human articular cartilage displayed spontaneous [Ca2+]i signaling, and indicated that the spatiotemporal features of the [Ca2+]i oscillations were both zonal and OA severity dependent. While the exact mechanism needs further investigation, the changes of [Ca2+]i signaling might reflect the zonal specific role of chondrocytes during OA progression and provide new insight into molecular mechanism of articular cartilage degradation during OA progression. Also, our observation here also suggested that spontaneous [Ca2+]i signaling of in situ chondrocytes could serve as a potential biomarker for clinical diagnosis of early OA.Despite the limitations, the current study presented the direct evidence that Articular cartilage samples for calcium imaging and histology evaluation were collected from patients receiving knee arthroscopy by one senior surgeon in Center for Joint Surgery, Southwest Hospital, Third Military Medical University, Chongqing, China. Informed consent was obtained from participant patients. During the surgical process, a single 2 mm in diameter cylindrical osteochondral sample was collected using a custom-designed biopsy needle that fits the arthroscopic system. For ICRS 0 group, healthy cartilage samples were collected in the non-load bearing area in knee joints with no sign of cartilage degeneration. For ICRS 1–3 groups, samples representing the most severe wear in each knee were collected from the femoral condyle before chondroplasty. The severity of cartilage degradation in their knee joints was evaluated during surgery based on ICRS Visual Assessment Scale, and confirmed by histomorphometry approaches afterward. Cartilage samples containing defects caused by trauma, tumor, or rheumatoid arthritis (RA) were excluded from this study. 3–4 cartilage samples for each ICRS grade were collected from 15 patients  and fluorescence microscopy . As shown in Fig. 2PO4, 0.33; KCl, 4.0; MgCl2, 0.53; glucose, 5.5; HEPES, 5.0; CaCl2, 0 or 4 mM (the pH was adjusted to 7.4 with NaOH) at 37 °C for 15 min to remove any agitation during staining process26. Fluorescent images of chondrocytes were then recorded using 4 x objectives at room temperature for 15 min while the sample was undisturbed.Harvested samples containing full-thickness cartilage and subchondral bone were immediately transferred to laboratory in PBS . Samples were carefully cut into 2 half-cylinders using a scalpel. [Ca2+]i signaling, recorded image sequences were analyzed using Image J (Version 1.44p). The cartilage samples were separated into superficial, middle and deep zones in the image software according to chondrocyte/chondron size and arrangement. In detail, parallelly-arranged cells in cartilage surface were considered as superficial zone chondrocytes; cells in middle range of cartilage samples, and with the formation of medium-size chondrons in random arrangement were considered as middle zone chondrocytes; hypertrophy cells in deep range, and with the formation of large-size chondrons in vertical arrangement were considered as deep zone chondrocytes. For the superficial zone, 5–10 individual cells were randomly chosen and analyzed in each sample. For the middle and deep zone, cells in 5–10 randomly chosen chondrons were analyzed in each sample. Fluorescence intensity in individual cells was normalized with the mean background intensity obtained in three randomly chosen blank areas42. The calcium peak magnitude was reported as the mean fold increment of the peak intensity over the mean baseline fluorescence intensity in all tested chondrocytes. The responsive rate was determined by the number of cells whose calcium peak magnitude was over 2-fold of baseline divided by the number of total cells in the superficial zone or each chondron in middle and deep zone. For analyzing the number of peaks during the 15-min recording period, peaks with increment larger than two were counted as one peak, and the peak number was presented as the total number of peaks during the 15-min recording period.To analyze the i signaling, the statistical significance of the normalized fold increment, the number of peaks, and the responsive rate among all groups were determined by two-way ANOVA with Bonferroni’s post hoc test. Spearman’s rank correlation coefficient method was used to analyze the relationship between each parameter of [Ca2+]i signaling to ICRS grading system. For RT-qPCR and Western boltting data, the data from OA groups were compared with a hypothetical value of 1.0  by using the student’s t-test. Statistical significance was defined with p < 0.05.Data were presented as mean ± SEM. For demographic data of patients, the statistical significance of age and BMI was determined by one-way ANOVA with Bonferroni’s post hoc test. For spatiotemporal features of spontaneous [CaThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Table S1Supplementary Figure S1


Editorial on the Research TopicInter-Organelle Calcium Communication in Cancer2+ transfer, and its impact on cancer cell fate.Cancer is a leading cause of death worldwide, accounting for 8.2 million deaths  in 2012, with this number expected to rise to over 11 million by 2030 , suggest2+ store, interact with each other in response to changes in cellular metabolism , and Nogo-B/reticulon-4B as oncogenes or oncosuppressors. As mentioned, MAMs actively participate in the regulation of proteostasis, tuning the ER stress response to cope with metabolic demand under various stress stimuli. Carreras-Sureda et al. immerses us in the unfold protein response, its relation with MAMs, and the role this plays in cancer.It has been known since the 1950 that mitochondria and the ER, the main intracellular Catabolism . The ER–tabolism  as mitoc and PML , 11. Ped2+ is a ubiquitous signaling messenger that regulates various cellular processes ranging from muscle contraction to synaptic transmission and from cellular proliferation to cell death , generating specific spatiotemporal Ca2+ signals  receptor (IP3R), in the ER, which proves to be necessary to maintain the bioenergetic homeostasis of these cells , a highly Ca2+ permeable channel that controls metabolism and bioenergetics by modulating the access of Ca2+ as well as pyruvate, malate, succinate, and NADH to the inner mitochondrial membrane. Shoshan-Barmatz et al. review the role of VDAC1 in cancer with special emphasis on its role in apoptosis induced by [Ca2+]-increasing agents, which upregulate VDAC1 expression and subsequently results in VDAC1 oligomerization, the release of cytochrome C & Smac/Diablo, and eventually apoptosis. Once present in the mitochondrial intermembrane space, Ca2+ is taken up into the mitochondrial matrix through the MCU complex, whose structure, activity, and regulation has been explained by Mammucari et al. In addition, the authors highlighted the divergent roles of MCU in cancer, revealing both anti- and pro-tumorigenic roles dependent on the cancer type and cancer stage. Finally, Chen and Boehning assess the role of lipidation in the regulation of different signaling pathways that impact Ca2+ homeostasis, such as Ras in apoptosis and tumor progression.Call death , 13. Thi signals . Alteratgression , 16. In se cells , as delixidation . Ca2+ crPendin et al. These alterations enable cancer cells to avoid apoptosis and to cope with the sustained energy demand, particularly during migration, invasion, and metastasis. White concisely reviews the key steps controlled by Ca2+ during the invasion-metastasis cascade and reveals how changes in the Ca2+-handling components that reside in the MAMs might facilitate invasion and metastasis by impacting bioenergetics and reactive oxygen species generation. Along these lines, Hegedüs et al. provide experimental evidence that enhanced clearance of Ca2+ by increased expression of the plasma membrane Ca2+ ATPase 4b induced by histone deacetylase inhibitors, and reduces migration in a melanoma cell line.Mitochondria are highly dynamic organelles undergoing constant fusion and fission governed in part by the metabolic status of the cell . Cancer 2+ ions, impacts a plethora of cancer hallmarks and plays a critical role in oncogenesis. The emerging insights at the molecular level in the dependence of cancer cells on ER-mitochondrial Ca2+ signaling for their survival and migration will pave the path for new therapeutic strategies and tools (Cancer has a multifactorial nature. Over the last decade, it has become increasingly clear that ER-to-mitochondria communication, in particular, through the flux of Cand tools , hopefulCC drafted the manuscript that was reviewed and edited by PP and GB. All authors co-edited the Research Topic.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.


Firstly, data showed that Pb triggers endoplasmic reticulum (ER) stress response in rPT cells, as evidenced by the elevations of ER stress markers. Moreover, pharmacological modulation of Ca2+ mobilization in ER and cytoplasm with three chemicals (2-APB or TG or BAPTA-AM) can effectively increase or decrease the protein expression of ER stress markers in Pb-exposed rPT cells, demonstrating that Pb-induced ER stress is Ca2+-dependent. We found that Pb stimulates phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) to activate its activity. Meanwhile, inhibition of CaMKII with KN93 or KN62 attenuated Pb-activated caspase-12 and CCAAT/enhancer-binding protein homologous protein (CHOP) in rPT cells, demonstrating that CaMKII activation promoted ER stress in rPT cells. Likewise, Pb-induced apoptosis can be effectively inhibited by CaMKII inhibitor KN93 or KN62. Furthermore, co-treatment with KN93 or KN62 significantly reversed Pb-induced ER Ca2+ release and concomitant intracellular Ca2+ overload in rPT cells. In summary, these results expound the mechanisms involving in ER stress, Ca2+ dyshomeostasis and activated CaMKII, which all contribute to Pb-induced apoptosis. CaMKII acts as a critical mediator of ER stress and associated apoptosis via regulating intracellular Ca2+ mobilization from ER to cytoplasm.Lead (Pb) is a known nephrotoxic element. Recently we have proved that subcellular Ca Lead (Pb) is a nonessential toxic heavy metal and one of the most widely used metals in industries. Globally, Pb exposure is ubiquitous and routes of its exposure to animals and human beings include inhalation of Pb-contaminated dust particles or aerosols, and ingestion of Pb-contaminated food or water . The per2+ redistribution played a chief role in low-dose (0–1.0 µM) Pb-induced nephrotoxicity in rPT cells c and [Ca2+]ER in Pb-exposed rPT cells. 2-APB is a specific inhibitor of inositol 1, 4, 5-trisphosphate receptor (IP3R) that functions to release Ca2+ from ER stores [2+-ATPase [2+ chelator to attenuate the elevation of [Ca2+]c [2+]c elevation, respectively; 2-APB or BAPTA-AM treatment alone has no obvious effect on [Ca2+]c in rPT cells. However, treatment with TG further aggravated Pb-mediated cytosolic Ca2+ overload and TG treatment only caused significant cytosolic Ca2+ elevation in rPT cells. As shown in Figure 2+]ER depletion while TG treatment further aggravated Pb-mediated ER Ca2+ release. Treatment with BAPTA-AM has no effect on Pb-induced [Ca2+]ER. Neither 2-APB nor BAPTA-AM treatment only has no obvious effect on [Ca2+]ER in rPT cells, and TG treatment alone caused significant ER Ca2+ release in rPT cells. Data in Figure 2+]c elevation and [Ca2+]ER depletion.Next, we aim to confirm the effects of three CaR stores . TG rais+-ATPase , while B Ca2+]c . Data in+c . Data2+ modulators to explore the role of Ca2+ signaling in Pb-induced ER stress in rPT cells. As shown in Figure 2+ release with 2-APB markedly diminished the protein levels of GRP78 (A), GRP94 (B), CRT (C), CHOP (D) and active caspaes-12 (E) in Pb-exposed rPT cells. In contrast, induction of ER Ca2+ release with TG significantly aggravated the protein levels of GRP78 (A), GRP94 (B), CRT (C), CHOP (D) and active caspaes-12 (E) in Pb-exposed rPT cells  in rPT cells.Since activated CaMKII promotes apoptosis partly through regulation of ER Ca+ levels , we next2+ release and cytosolic Ca2+ overload c and [Ca2+]ER in this study, respectively. As expected, three modulators of Ca2+ signaling can potently regulate the oscillations of [Ca2+]c and [Ca2+]ER in Pb-treated rPT cells , DMEM-F12 (1:1), Hoechst 33258 solution and all other chemicals were purchased from Sigma-Aldrich. A phosphatase inhibitor (04906845001) was obtained from Roche Applied Science. Pluronic F-127 and Fluo-3-AM were obtained from Dojindo Laboratories . Mag-Fluo-4-AM and N, N, N′, N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) were purchased from Molecular Probes . KN62 and KN93 were purchased from Selleck . 2-Aminoethoxydiphenyl borate (2-APB) and 1, 2-Bis (2-aminophenoxy) ethane-N, N, N’, N’-tetraacetic acid acetoxymethyl ester (BAPTA-AM) were from Tocris Bioscience . Annexin V-FITC apoptosis detection kit was from Pharmingen . BCA protein assay kit and enhanced chemiluminescence (ECL) kit were obtained from Thermo Fisher Scientific Pierce. The following primary antibodies were used: GRP78 (cat No. 3183), GRP94 (cat No. 2104), Calreticulin , phospho-CaMKII , β-actin (cat No. 3700) and GAPDH (cat No. 5174) were purchased from Cell Signaling Technology. CHOP (cat No. ab11419), Caspase-12 (cat No. ab62484) and total-CaMKII  were obtained from Abcam. Secondary antibodies were conjugated to horseradish peroxidase .2+ signaling and cell viability c in this study. rPT cells were pretreated with 10 µM BAPTA-AM for 30 min, followed by incubation with 0.5 µM Pb for another 12 h. Also, cells were co-incubated with 10 µM 2-APB and 2.5 µM Cd or 1 µM TG and 2.5 µM Cd for 12 h. After the treatment, harvested cells were incubated with 0.5 mM of TPEN at 37°C for 10 min, loaded with 1 µM Fluo-3-AM (containing 0.02% Pluronic F-127) for 30 min in dark at 37°C, and then washed with D-Hank’s solution. Intracellular Ca2+ levels were represented with fluorescent intensity  of 10,000 cells on flow cytometer. This assay was replicated six different times using different batches of cells.Three chemicals, i.e., BAPTA-AM  of 10,000 cells on flow cytometer.It has been verified that Mag-Fluo-4-AM was selectively labelled on ER, making its specificity for measuring [Ca2+]ER . After cCells subjected to desired treatments were lysed in ice-cold RIPA buffer supplemented with protease inhibitor (Merck Millipore) and phosphatase inhibitor (Roche) to prepare the total cell lysates. After protein quantification with BCA method, samples were subjected to SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5% skim milk for 1 h at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies: GRP78 (diluted 1:1000), GRP94 (diluted 1:1000), CRT (diluted 1:1000), CHOP (diluted 1:1000), Caspase-12 (diluted 1:1000), t-CaMKII (diluted 1:1250), p-CaMKII (diluted 1:1000), β-actin (diluted 1:1000) and GAPDH (diluted 1:1000). After several washes with TBST, the membranes were incubated with appropriate secondary antibodies (1:5000 dilution) for 50 min at room temperature. Finally, each protein was detected on a Chemidoc XRS  by using the ECL kit. Proteins levels were determined by computer-assisted densitometric analysis . The density of each band was normalized to its respective loading control (β-actin or GAPDH). Data obtained were expressed as the ratio of intensity of the protein in chemical-treated cells to that of the corresponding protein in control cells. Each test was performed in four experiments with different batches of cells.Cells were pre-incubated with 10 µM KN62 or 10 µM KN93 for 2 h, then treated with 0.5 µM Pb for another 12 h to assess its effect on apoptosis, respectively. Apoptosis is characterized morphologically by condensation and fragmentation of nuclei. Thus, Hoechst 33258 staining was firstly applied to assess the morphological changes of treated cells, and 200 cells were randomly selected to count those apoptotic cells within every batch of experiment, each one performed in triplicate. Another concern is the quantitative analysis of apoptosis by flow cytometry. Both of these two methods have been extensively described in our previous study .2+]c and [Ca2+]ER in Pb-exposed rPT cells, respectively. Cells grown on coverslips were pre-incubated with 10 µM KN62 or 10 µM KN93 for 2 h, then exposed to 0.5 µM Pb for another 12 h. After incubated with 0.5 mM of TPEN (Pb chelator) at 37°C for 10 min, one part of slides were loaded with 2.5 µM Fluo-3-AM (containing 0.02% Pluronic F-127) in dark at 37°C for 30 min, and the other part of slides were incubated with 2.5 µM Mag-Fluo-4-AM (containing 0.02% Pluronic F-127) in dark at 37°C for 30 min. After washing with D-Hank’s solution, cells labeled with Fluo-3-AM or Mag-Fluo-4-AM were visualized by the confocal microscope  to assess the changes of [Ca2+]c and [Ca2+]ER, respectively. Representative confocal images were captured. [Ca2+]c and [Ca2+]ER were quantified using the ImageJ software under identical threshold conditions.Two CaMKII inhibitors, i.e., KN62 and KN93, were applied to assess its effect on the changes of [CaF test) after ascertaining the homogeneity of variance between the treatments, and P < 0.05 was regarded as significant.Experiments were performed at least three times with similar results. Data are presented as the mean ± SEM of the indicated number of replicates. Statistical comparisons were made using one-way analysis of variance (ANOVA) (Scheffe’s


Therefore, more than any other cell types, neurons are critically dependent on spatially and temporally controlled Ca2+ dynamics. This is achieved through an exquisite level of compartmentalization of Ca2+ storage and release from various organelles. The function of these organelles in the regulation of Ca2+ dynamics has been studied for decades using electrophysiological and optical methods combined with pharmacological and genetic alterations. Mitochondria and the endoplasmic reticulum (ER) are among the organelles playing the most critical roles in Ca2+ dynamics in neurons. At presynaptic boutons, Ca2+ triggers neurotransmitter release and synaptic plasticity, and postsynaptically, Ca2+ mobilization mediates long-term synaptic plasticity. To explore Ca2+ dynamics in live cells and intact animals, various synthetic and genetically encoded fluorescent Ca2+ sensors were developed, and recently, many groups actively increased the sensitivity and diversity of genetically encoded Ca2+ indicators (GECIs). Following conjugation with various signal peptides, these improved GECIs can be targeted to specific subcellular compartments, allowing monitoring of organelle-specific Ca2+ dynamics. Here, we review recent findings unraveling novel roles for mitochondria- and ER-dependent Ca2+ dynamics in neurons and at synapses.Calcium (Ca Therefore, regulation of intracellular Ca2+ homeostasis is central to the proper function of neuronal circuits. The maintenance of baseline levels of intracellular Ca2+ levels is regulated in part through exchangers and pumps such as the plasma membrane Ca2+-ATPase (PMCA pump), the Na+/Ca2+ exchanger (NCX), and the Na+/Ca2+-K+ exchanger (NCKX) which extrude Ca2+ through the plasma membrane into the extracellular space. In addition to these mechanisms, intracellular organelles, such as mitochondria and endoplasmic reticulum (ER), are able to regulate cytoplasmic Ca2+ ([Ca2+]c) through mitochondrial calcium uniporter (MCU) and smooth endoplasmic reticulum Ca2+-ATPase (SERCA), respectively.Calcium  were developed and applied both in vitro and in vivo. Also, GECIs tagged with target peptide sequences have allowed imaging of Ca2+ dynamics in specific organelles  followed by Ca2+ binding to sensors like synaptotagmins  plays a role in regulating presynaptic [Ca2+]c. This has been characterized in various species, neuronal cell types and circuits , the GTPase dMiro mutant lacks presynaptic mitochondria through impaired axonal transport  was not altered, but the evoked synaptic transmission was impaired during high frequency stimulation, and this defect was partially rescued by ATP  and increased asynchronous release , and insulin-like growth factor-1 receptor (IGF-1R) signaling regulates resting mitochondrial Ca2+ level and spontaneous transmission  fusion with the plasma membrane occurs following increase of [Cas Figure . At the 2+]c clearance, Ca2+ release from mitochondria plays important roles at presynaptic sites  was tagged to GECIs  and its binding peptide  revealed mitochondrial Ca2+ import simultaneously with cytosolic Ca2+  can be changed by Ca2+ influx  depending on cell types and stimulation protocol  sensors can be easily saturated by high [Ca2+]m and low affinity (high Kd) sensors may not be sensitive enough to detect small [Ca2+]m changes. Several studies reported low affinity mitochondrial Ca2+ probes for avoiding saturation  through SERCA and store-operated Ca2+ entry (SOCE) mechanism  and IP3-induced Ca2+ release  and voltage-gated Ca2+ channels (VGCCs), whereas IICR is triggered by IP3, which is generated via activation of phospholipase C (PLC) depending on metabotropic glutamate receptors (mGluRs) or other receptors like receptor tyrosine kinases , which mediate IICR, consist of three isoforms, IP3R1, IP3R2, and IP3R3, but IP3R1 is the dominant form in the brain  are involved in CICR, and they have three major subtypes; RyR1, RyR2, and RyR3. All of these isoforms are detected in the brain, and show region-specific expression , calcineurin (a Ca2+-dependent phosphatase), protein phosphatase 1 (PP1) and protein kinase C  stimulation and this mediates long-term depression (LTD) of PF-Purkinje cell pathway , has been the first applied for neuronal ER Ca2+ measurement, and after loading this dye in the cytoplasm and organelles, cytosolic dye was removed by perfusion with dye-free pipette solution  neurons -based Ca2+ sensor, D1ER, showed altered ER Ca2+ leak function in hippocampal neurons of presenilin double knockout and Alzheimer’s disease model mice  and GCaMPer (10.19), characterized ER Ca2+ uptake and release in cortical neurons and cerebellar Purkinje cells  will lead to the identification of synapse- and circuit-specific roles of mitochondria and ER Ca2+ in neurons.Recent studies characterized the roles of presynaptic mitochondria and circuit-specific ER Ca2+ uptake upon changes in cytosolic Ca2+ dynamics , Japan society for the promotion of science fellowship for research abroad and The Uehara Memorial Foundation (YH), and a grant from the Human Frontier Science Program long-term fellowship (S-KK).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.


Acinetobacter baumannii (MDRAB). We sought to determine the time effect of antibiotic exposure on multi-drug resistant Acinetobacter baumannii (MDRAB) acquisition and clearance.Information is limited about the effect of restricted carbapenem use on clearance of multi-drug resistant We conducted a retrospective observational study at the intensive care units of a tertiary medical center. Forty-two of a cohort of previously healthy young adults who were concurrently burned by a dust explosion was included. Cases consisted of those from whom MDRAB was isolated during hospitalization. Controls consisted of patients from whom MDRAB was not isolated in the same period. Use of antimicrobial agents was compared based on days of therapy per 1,000 patient-days . A 2-state Markov multi-state model was used to estimate the risk of acquisition and clearance of MDRAB.P < 0.001) before MDRAB isolation. For the cases, clearance of MDRAB was associated with lower use of carbapenem  and higher use of non-carbapenem beta-lactam . In multi-state model, each additional DOT of carbapenem increased the hazard of acquiring MDRAB (hazard ratio (HR), 1.08; 95% confidence interval (CI) 1.01–1.16) and each additional DOT of non-carbapenem beta-lactam increased the protection of clearing MDRAB .MDRAB was discovered in 9/42 (21.4%) individuals. The cases had significantly higher use of carbapenem (652 DOT/1,000PD vs. 385 DOT/1,000PD, Both acquisition and clearance of MDRAB were related to antibiotic exposure in a homogeneous population. Our findings suggest that early discontinuation of carbapenem could be an effective measure in antibiotic stewardship for the control of MDRAB spreading. Acinetobacter baumannii is an important pathogen, causing healthcare-associated infections such as pneumonia, urinary tract infection, soft tissue infection, and bloodstream infection [A. baumannii contributes to this organism’s survival in the environment, including persistence for extended periods of time on dry surfaces [A. baumannii ranks among the most important nosocomial pathogens [Acinetobacter baumannii (MDRAB) with decreased susceptibility to carbapenem poses a great threat to the health care system; alternative therapeutic options are limited and outcome is poor in patients infected by beta-lactam-resistant A. baumannii [nfection , 2. The surfaces . Given tathogens . The incaumannii , 6. An iaumannii –10.Studies have shown that antimicrobial use is associated with the emergence of drug-resistant pathogens; the implementation of an ASP may be effective in reducing resistance rates –17. WhilOn June 27, 2015, a dust explosion occurred in the late evening at the Formosa Fun Coast water park in northern Taiwan, creating over 400 burn victims and representing a major challenge to the health care system . Among tThis case-control study was conducted at the Chang Gung Memorial Hospital-Linkou Branch, a 3700-bed tertiary-care medical center with 308 ICU beds for critical care in northern Taiwan. The facility has two ICUs (48 beds) which were devoted exclusively to care of critically ill burn patients. The present investigation included 42 previously healthy young adults who had been admitted to the ICUs between June 27, 2015 and July 1, 2015 due to the dust burn occurred on June 27, 2015 in Taiwan. This analysis included all inpatient data until discharge or death. The study was approved by the Institutional Review Board of Chang Gung Memorial Hospital (104-9356B) and informed consent was waived due to the study’s retrospective nature.The key managements for the flame burn were accomplished as previously described . In addiInitially we tried to distinguish between infection and colonization status of the patients. However, after reviewing the charts, we found that we could not clearly define infection or colonization status regarding isolation of MDRAB based on objective data. The patients shared similar clinical findings, laboratory data, and isolates that were collected intraoperatively. Thus, we defined the condition of the patients as MDRAB infection/colonization.Demographics, underlying diseases, ventilator use, inhalation injury, and catheter use were reviewed. For evaluation of disease severity, an Acute Physiology And Chronic Health Evaluation (APACHE) II score was determined on the first day of admission. To quantify antimicrobial use, the prescriptions of each patient was reviewed and converted into days of therapy (DOTs). We defined the DOT as previously described . In shorFor the cases with MDRAB, the time-at-risk was calculated as the number of days from the date of admission to the date of MDRAB detection. For the controls without MDRAB isolation during hospitalization, the time-at-risk was calculated as for the cases starting on the date of admission. For the evaluation of clearance of MDRAB, the time-at-risk was the number of days between MDRAB isolation and MDRAB clearance. The adjusted rate of antimicrobial use (DOT/1000PD) was then calculated and compared between 1) the cases and the controls, and 2) before and after MDRAB isolation.A. baumannii was performed by Matrix-Assisted Laser Desorption-Time Of Flight (MALDI-TOF) mass spectrometry (MS). Susceptibility to all tested antibiotics except tigecycline was determined according to the Clinical and Laboratory Standards Institute (CLSI) interpretive criteria for disk diffusion method. Susceptibility to tigecycline was determined using the disk diffusion method with Mueller-Hinton agar , with breakpoints at ≧ 16 mm and ≦ 12 mm. MDRAB was defined as A. baumannii with full or intermediate resistance to amikacin, gentamicin, cefepime, ceftazidime, piperacillin, piperacillin-tazobactam, aztreonam, ciprofloxacin, and carbapenems [Identification of bapenems .p value <0.05 was considered statistically significant in all tests. All statistical analyses were performed with SAS software .We used a multistate model analyzing the process of acquisition and clearance of the MDRAB , 26. TheP < 0.001), lower use of non-carbapenem beta-lactam . There was no difference of overall antibiotic use between the case group and the control group . Measurement of DOT was comparable with that of DDD (correlation coefficient = 0.94) (Table MDRAB occurred in nine of forty-two patients during the study period 21.4%). In most (8 of 9) of these instances, MDRAB was detected from wounds. In only one instance was MDRAB isolated from the tip of a removed central venous catheter (CVC). Compared to the control group, the case group had similar demographics, total body surface area (TBSA) affected by burn, and APACHE II score on admission. No comorbid illness was reported in either group of patients. The median day to MDRAB occurrence was 16 days. Acquisition of MDRAB was associated with higher use of carbapenem . Characteristics, outcomes, as well as detailed antimicrobial use before and after the isolation of MDRAB in the cases (N = 9) were compared. Four of whom have stopped carbapenem and received ceftazidime after the MDRAB occurrence. Three of whom have continued carbapenem use. Two of whom have continued non-carbapenem beta-lactam use  (Table P = 0.024) (Table P = 0.020) as did non-carbapenem beta-lactam on MDRAB clearance   was 0.005, while the rate of transition from MDRAB positive to MDRAB negative (MDRAB clearance) was 0.01. The risk of acquiring MDRAB sharply increased 3 days after admission Fig. . In univ7) Table . GlycopeWe conducted this study to examine the association between carbapenem and MDRAB in an extremely homogeneous group of previously healthy young burn patients. Our results show that antibiotic exposure was related to both the acquisition and clearance of MDRAB. In particularly, MDRAB clearance was associated with restricted use of carbapenem. Each additional DOT of carbapenem increased the risk of MDRAB acquisition, and each additional DOT of non-carbapenem beta-lactam increased the chance of MDRAB clearance. Our findings highlight the impact of antimicrobial use on the occurrence of drug-resistant pathogens, suggesting that the early discontinuation of carbapenem use may help to control the emergence of MDRAB.A. baumannii colonization  by 5.1%. In the group of previously healthy young burn patients, we demonstrated that carbapenem was a time-dependent variable for MDRAB acquisition, and every additional DOT increased the risk of MDRAB acquisition by 8%. Both the studies explored the effect of carbapenem exposure as a continuous variable to further understand the association between time-dependent antibiotic exposure and acquisition of MDRAB. However, our cohort is uniquely positioned to evaluate the impact of antibiotic exposure on the occurrence of MDRAB infection/colonization, given that our patients were free of underlying conditions and were admitted at the same time due to the dust explosion; the results of these experiments provided additional data to facilitate the optimization of antibiotic prescription by clinicians.Munoz-Price et al.  reportedA. baumannii colonization lies in the high predictive value with regard to the subsequent development of carbapenem-resistant A. baumannii infection [A. baumannii is associated with a mortality rate of approximately 52%, compared with a 19% mortality rate observed following infection with carbapenem-susceptible A. baumannii [A. baumannii in the microbial ecology may solely reflect this organism’s resistance to antimicrobial agents under selective pressure.The importance of detection of carbapenem-resistant nfection . Notablyaumannii . Our finaumannii . Carbapeaumannii . HaltingThere has been a report that beta-lactam was a risk factor for acquisition of MDRAB . FurtherBoth DDD and DOT are standardized methods for measurement of antibiotic use . We chosOur study does have some limitations. Due to this study’s retrospective nature and single-center design, our results may be not applicable in other settings or hospitals. There were more than 400 injuries due to the dust explosion and only 42 of the them who were admitted to the ICUs of the hospital were included. This has implications for possible selection bias. However, our study population consisted of young adults who were previously healthy and admitted as a concurrent group following a single event (the dust explosion). The patients were admitted to the hospital at the same time between June 27, 2015 and July 1, 2015. We acknowledged that medical procedures and environmental factors played important roles in the process of MDRAB colonization. The integrated and intensive care provided by the hospital at that time maximally eliminated the confounding factors that were measurable . Hence, In conclusion, our results suggest that antibiotic exposure is associated with both acquisition and clearance of MDRAB in this homogeneous population of previously healthy, critically ill young burn patients. Our findings reinforce the utility of an ASP in reducing the occurrence of multidrug-resistant pathogens, and highlight the appropriateness of early discontinuation of carbapenem when the use of carbapenem is inevitable.


To propel a translational research for development of bronchodilator therapy, synergistic effects between SABAs with LAMAs were examined focused on Ca2+ signaling using simultaneous records of isometric tension and F340/F380 in fura-2-loaded tracheal smooth muscle. Glycopyrronium (3 nM), a LAMA, modestly reduced methacholine (1 μM)-induced contraction. When procaterol, salbutamol and SABAs were applied in the presence of glycopyrronium, relaxant effects of these SABAs are markedly enhanced, and percent inhibition of tension was much greater than the sum of those for each agent and those expected from the BI theory. In contrast, percent inhibition of F340/F380 was not greater than those values. Bisindolylmaleimide, an inhibitor of protein kinase C (PKC), significantly increased the relaxant effect of LAMA without reducing F340/F380. Iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ (KCa) channels, significantly suppressed the effects of these combined agents with reducing F340/F380. In conclusion, combination of SABAs with LAMAs synergistically enhances inhibition of muscarinic contraction via decreasing both Ca2+ sensitization mediated by PKC and Ca2+ dynamics mediated by KCa channels. PKC and KCa channels may be molecular targets for cross talk between β2-adrenoceptors and muscarinic receptors.Long-acting muscarinic antagonists (LAMAs) and short-acting β As a 2-adrenoceptor agonists .,24.2+-ac2+ dynamics and Ca2+ sensitization. The Ca2+ dynamics is a mechanism of airway smooth muscle contraction via activation of a Ca2+/calmodulin-dependent myosin light chain kinase and increase intracellular Ca2+ concentration via a Ca2+ influx through Ca2+ channels (Ca2+-dependent contraction). In contrast, since Phosphorylation (inactivation) of myosin phosphatase (MP) causes contraction as intracellular Ca2+ level unchanged, MP inhibition contributes to Ca2+ sensitization (Ca2+-independent contraction) [2+ signaling in airway smooth muscle plays an essential role in the pathophysiology of asthma and COPD [Various contractile agents such as acetylcholine cause smooth muscle contraction via GPCRs. Airway smooth muscle tone is regulated by Caraction) . MP is praction) ,27,28 anraction) ,30,31. Cand COPD ,35,36,372-adrenoceptor agonists (SABAs) is useful for symptom relief in patients with COPD. Because of the synergistic effect of these two agents on bronchodilation, pre-administration of LAMAs may be effective for as-needed use of SABAs, such as salbutamol and procaterol. Therefore, there is a need for the development of pharmacological treatments for COPD that exploit the synergistic effects between LAMAs and SABAs. This study was designed to determine whether procaterol and salbutamol (SABAs) and glycopyrronium (a LAMA) act synergistically to relax contracted airway smooth muscle. We promoted this study on the assumption of involvement of the major pathway in Ca2+ dynamics and Ca2+ sensitization, which play a key role of contraction of airway smooth muscle. In particular, mechanisms underlying this phenomenon were examined with a focus on Ca2+ signaling, specifically Ca2+ dynamics mediated by KCa channels and Ca2+ sensitization mediated by Rho-kinase and PKC in airway smooth muscle. The functional antagonism between β2-adrenergic and muscarinic action converges on KCa channel currents, and Rho-kinase and PKC play key role in muscarinic contraction of airway smooth muscle.Inhalation of rapid- and short-acting bronchodilators such as short-acting βn = 5, p < 0.05), and the reduction in tension was significantly greater than the reduction in F340/F380 at each concentration ≥1 nM (procaterol) and ≥10 nM  , but the reduction in F340/F380 was significantly greater than that in tension at each concentration (0.1–3 nM) (When procaterol (0.1–10 nM) and salbutamol (1–100 nM) were cumulatively applied to strips of tracheal smooth muscle pre-contracted with MCh , both inhibited MCh-induced contraction and reduced F340/F380 in a concentration-dependent manner. However, when almost complete relaxation was achieved at the maximum concentrations tested , F340/F380 was still higher than at the basal level a,b. The butamol) c,d. Howe.1–3 nM) .n = 5), respectively; procaterol (1 nM) reduced them by 37.0% ± 9.1% and 32.9% ± 5.4% (n = 5), respectively. When procaterol (1 nM) was applied to the MCh-precontracted strips in the presence of glycopyrronium (3 nM), inhibition of contraction and decrease in F340/380 were markedly enhanced, to 73.9% ± 8.2% and 55.3% ± 1.5% (n = 5), respectively . However, in the presence of glycopyrronium (3 nM), those values were augmented to 31.2% ± 13.8% and 26.9% ± 8.1% (n = 5), respectively , respectively. When procaterol (0.3 nM) was applied in the presence of glycopyrronium (3 nM), those values were augmented to 46.0% ± 7.7% and 38.5% ± 9.7% (n = 5), respectively  reduced MCh-induced tension and F340/F380 by 7.2% ± 4.5% and 23.6% ± 4.0% , respectively. When salbutamol (10 nM) was applied in the presence of glycopyrronium (3 nM), contraction was inhibited by 85.4% ± 6.2% and F340/F380 was reduced by 60.4% ± 8.4% , respectively. When salbutamol (3 nM) was applied in the presence of glycopyrronium (3 nM), contraction was inhibited by 56.7% ± 15.3% and F340/F380 was reduced by 45.7% ± 10.9%  inhibited MCh-induced contraction and reduced F340/F380 by 60.8% ± 10.6% and 59.8% ± 8.0% , respectively. When procaterol (1 nM) was applied to the MCh-precontructed strips in the presence of tiotropium (1 nM), inhibition of contruction and decrease in F340/F380 were markedly enhanced, to 55.6% ± 7.1% and 35.5% ± 5.5% (n = 5), respectively .Tiotropium (1 nM) reduced MCh-induced tension and F340/F380 by 2.9% ± 3.1% and 11.3% ± 3.6%  and F340/F380 . In contrast, the inhibitory effect of glycopyrronium on F340/F380 was not increased by bisindolylmaleimide, and was not greater in combination than the sum of the values for each agent , a potent inhibitor of PKC, caused modest decreases in MCh-induced tension  and glycopyrronium (3 nM) were applied together in the presence of bisindolylmaleimide (10 µM), percent inhibition of tension was almost the same as in the absence of bisindolylmaleimide c. Howeven = 5) and F340/F380  and F340/F380 by 26.5% ± 8.9% , a selective inhibitor of Rho-kinase, caused small decreases in MCh-induced tension  and 32.2% ± 10.8% (p < 0.01), respectively, compared to the values in the absence iberiotoxin (p < 0.001) and 36.2% ± 19.6% (p < 0.05), respectively, compared to those values in the absence of iberiotoxin . Recent reports have demonstrated that indacaterol, a LABA, synergistically augments the inhibitory effects of glycopyrronium against muscarinic contraction in airway smooth muscle [2-adrenoceptor agonists and muscarinic receptor antagonists.In the present study, GB (a LAMA) synergistically enhanced the inhibitory effect of procaterol and salbutamol (SABAs) on muscarinic contraction in airway smooth muscle via Cah muscle ,10. Moreh muscle . Therefo2+ signaling, mediated by Ca2+ dynamics via various channels and by Ca2+ sensitization due to the inactivation of MP via RhoA/Rho-kinase and PKC/CPI-17, contributes to airway smooth muscle tone induced by contractile agents via GPCRs, and other receptors [2+ concentration; however, the reduction in contraction is greater than the reduction in intracellular Ca2+ concentration  in the inhibition of muscarinic contraction is not identical between β2-adrenoceptor activation and muscarinic receptor suppression  and salbutamol (1–10 nM) were applied in the presence of glycopyrronium, the relaxant effect was greater than the sum of the effects of the equivalent concentrations of each agent  CPI-17 in endothelial cells [2-adrenergic relaxation is mediated by an inhibition of PKC-induced Ca2+ sensitization. Hence, PKC could be a target protein for the cross talk based on Ca2+ sensitization between β2-adrenoceptors and muscarinic receptors. From results of our study, SABA may attenuate PKC-induced Ca2+-sensitization via activation of PKA. To clarify this phenomenon, further investigation will be needed.As described above, given that contractile agonists, which stimulate GPCRs, activate RhoA and PKC, this indicates that Catagonist . These rrocesses . Becaused by PKA . Althougal cells . This fi2+ dynamics and Ca2+ sensitization [2+ dynamics (reducing intracellular Ca2+ concentration), not by affecting Ca2+ sensitization  [2-adrenoceptor agonists (β2-adrenoceptor desensitization) [Ca channel inhibitor, the relaxant effects of the SABA/glycopyrronium combination were markedly attenuated with an increase in F340/F380 , such as L-type voltage-dependent Ca2+ channels [i/KCa channel inhibitory linkage and an augmentation in the Gs/KCa channel stimulatory linkage are thought to be another functional intracellular mechanism of this synergistically relaxant effect of combination of β2-adrenoceptor agonists with muscarinic receptor antagonists. KCa channels also could be a target protein for the cross talk based on Ca2+ dynamics between these two GPCRs.Given that Keceptors , intraceeceptors ,20,22,50iveness)  and a deization) ,52. We e340/F380 . As showeceptors , consistchannels ,10,23  bubbled with a mixture of 99% O2 and 1% CO2 (pH 7.4) was perfused at a constant flow of 4 mL/min. Organ bath temperature was maintained at 37 °C. The animal protocols used in this work were evaluated and approved by the Research Center for Animal Life Science Committee, Shiga University of Medical Science .Tracheae were excised from male Hartley guinea pigs (300–350 g) after intraperitoneal injection of pentobarbital (150 mg/kg). The tracheal rings were opened by cutting longitudinally at the cartilaginous region, and the epithelium was peeled off carefully. Normal bathing solution  was added to increase the solubility of fura-2/AM. After the fura-2/AM had penetrated the tissue, the experimental chamber was filled with normal bathing solution. Isometric tension and fura-2 fluorescence of muscle strips were measured simultaneously, using a displacement transducer and a spectrofluorometer . The mucosal side of the smooth muscle was exposed to excitation light, and the intensities of excitation fluorescence at 340 nm and 380 nm were measured after background subtraction. Because the dissociation constant of fura-2 for Ca2+ in smooth muscle cytoplasm was shown to be different from that measured in vitro, the absolute amount of intracellular Ca2+ concentration was not calculated [2+ concentration. Methacholine  was administered for 5 min at 10 min intervals until the control response to 1 µM MCh was established, and then the experiment was started. Muscle tension and F340/F380 in the resting state were taken as 0%, and the value of percent inhibition of tension and F340/F380 were measured taking the control response to MCh (1 µM) as 100%. Indomethacin (2 µM) was administered throughout the experiments to abolish resting tone.This was conducted as described previously ,43,53. Ilculated . Therefo2+ concentration in the presence of β2-adrenoceptor agonists or muscarinic receptor antagonists, procaterol (0.1–10 nM), salbutamol (1–100 nM) or glycopyrronium (0.3–10 nM) were each administered cumulatively to tissue precontracted with MCh (1 µM throughout the study). To examine this relationship in the presence of a combination of β2-adrenoceptor agonists and muscarinic receptor antagonists, procaterol (0.1–1 nM) and salbutamol (1–10 nM) were added to MCh-precontracted tissue in the presence of glycopyrronium (3 nM), and procaterol (0.3–1 nM) was added to MCh-precontracted tissue in the presense of tiotropium (1 nM). To investigate the role of Ca2+ dynamics due to KCa channel activity in the synergistic effects of β2-adrenoceptor agonists and muscarinic receptor antagonists, procaterol and glycopyrronium were added together to MCh-precontracted tissue, in the presence of iberiotoxin (30 nM), a selective inhibitor of KCa channels, which are densely distributed on airway smooth muscle cell membrane [2+ sensitization due to Rho-kinase and PKC in the synergistic effects of β2-adrenoceptor agonists and muscarinic receptor antagonists, procaterol and glycopyrronium were both added to MCh-precontracted tissue in the presence of Y-27632 (1 µM), a selective inhibitor of Rho-kinase, and bisindolylmaleimide (10 µM), a potent inhibitor of PKC. Each reagent at each concentration was administered for 15 min (except MCh). Time-matched control tissues were treated similarly to the test tissues, but exposed continuously to the normal bathing solution (sham incubation) instead of procaterol, salbutamol, iberiotoxin, Y-27632, or bisindolylmaleimide.To examine the relationship between muscle tone and intracellular Camembrane . Moreove2+ concentration was evaluated using the BI theory. This model assumes that two or more agents act independently, with different modes and sites of action [x and y are the doses of two compounds in a combination experiment. If the combined experimental value is higher than the expected value, the interaction is synergistic. If it is lower, the interaction is antagonistic [The synergistic effect of glycopyrronium and procaterol or salbutamol and that of tiotropium and procaterol on contracted airway smooth muscle and change in intracellular Caf action , and is gonistic ,47.MCh, indomethacin, procaterol, salbutamol, and glycopyrronium were obtained from Wako pure Chemical Industries . iberiotoxin was obtained from Peptide Institute . Tiotropium, bisindolylmaleimide and Y-27632 were obtained from Sigma . Fura 2-AM was obtained from Dojin Laboratories .t-test or Student’s unpaired t-test and one-way analysis of variance. p < 0.05 was considered significant. Statistical analyses were performed using JMP version 11 .All data are expressed as the mean ± standard deviation (SD). Statistical significance was assessed by 2-adrenoceptor agonists with muscarinic receptor antagonists causes a synergistic inhibition of muscarinic contraction in airway smooth muscle. Ca2+ signaling via PKC-induced Ca2+ sensitization and KCa channel-induced Ca2+ dynamics play a functional role in this synergistic effect (Ca channels and PKC are key protein for cross talk between β2-adrenoceptors and muscarinic receptors (2-adrenoceptor agents and anti-cholinergic agents are a promising advancement in bronchodilator therapy for COPD and asthma, and that PKC and KCa channels may be novel targets for bronchodilator research and development.The combination of βc effect . KCa chaeceptors . Since ieceptors . Our res


For instance, the mitochondrial oxidative phosphorylation (OXPHOS) ensures a supply of adenosine triphosphate (ATP), but is also the main source of potentially harmful levels of reactive oxygen species (ROS). Moreover, mitochondrial function is strongly linked to mitochondrial Ca2+ homeostasis and mitochondrial shape, which undergo various alterations during aging. Since mitochondria play such a critical role in an organism’s process of aging, they also offer promising targets for manipulation of senescent cellular functions. Accordingly, interventions delaying the onset of age-associated disorders involve the manipulation of mitochondrial function, including caloric restriction (CR) or exercise, as well as drugs, such as metformin, aspirin, and polyphenols. In this review, we discuss mitochondria’s role in and impact on cellular aging and their potential to serve as a target for therapeutic interventions against age-related cellular dysfunction.Senescence is related to the loss of cellular homeostasis and functions, which leads to a progressive decline in physiological ability and to aging-associated diseases. Since mitochondria are essential to energy supply, cell differentiation, cell cycle control, intracellular signaling and Ca In a rapidly aging society, new treatment options for age-related disorders and diseases will be increasingly important ,2. Conse2+ homeostasis, as well as in endoplasmic reticulum (ER)-mitochondria crosstalk, could reduce the adaptive capacity of cells to withstand stress and increase their vulnerability to age-related diseases [As adenosine triphosphate (ATP)-producing power plants of the cell, mitochondria are in a unique position to influence an organism’s aging process. Recent reports suggest that mitochondrial function is linked to age-associated biphasic alterations in metabolic activity, including an increase and afterwards progressive decrease in mitochondrial function . In addidiseases . Consequdiseases  or exercdiseases , as welldiseases ,6, targediseases . Neverthdiseases , the rigNotably, impaired mitochondrial functions were reported to cause accelerated aging that affects primarily organs with high levels of energy demand, such as the brain, the heart, the skeletal muscle, as well as liver and kidney . The criThis review provides a summary of present knowledge about mitochondria’s unique role in the process of aging and the development of age-related disorders and diseases. Furthermore, the potential of mitochondria to serve as targets for therapeutic interventions against age-related diseases is highlighted.As descendants of α-proteobacteria, mitochondria are double-membraned and equipped with their own circular genome. After 1.5 billion years of incorporation into the eukaryotic cell, mitochondria are well-integrated and irreplaceable cellular compartments . While tThe protein complexes of the mitochondrial respiration chain are located at the cristae . These i2+ uptake from the IMS to the mitochondrial matrix, a complex machinery of proteins is located at the IMM [2+ uptake is linked to Ca2+ mobilization from the biggest internal Ca2+ store, the ER [2+ [2+ entry (SOCE). In this process low Ca2+ levels in the ER cause oligomerization and, finally, a conformational change of the Ca2+ sensing stromal interaction molecule-1 (STIM1), which then activates the plasma membrane Ca2+ channel (ORAI1) through protein-protein interactions, causing Ca2+ entry [2+ elevations are sensed by mitochondria, which take up Ca2+ to buffer cytosolic Ca2+ levels in distinct regions of the cytosol, so-called microdomains, and thereby control the activity of Ca2+-dependent enzymes [2+ to the ER [+/Ca2+ exchanger (NCX) and the plasma membrane Ca2+ ATPase (PMCA) also regulate cytosolic Ca2+ levels by extrusion of Ca2+ [To ensure and control mitochondrial Ca the IMM . Mitocho, the ER , or the e ER [2+ . The clo2+ entry . Cytosol enzymes  and ion  enzymes . Mitochoo the ER . Moreove of Ca2+ . 2+ uptake, including the pore-forming mitochondrial calcium uniporter (MCU) [2+-sensing proteins mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial Ca2+ uptake 2 (MICU2), which control mitochondrial Ca2+ homeostasis, to MCU [2+ uptake, including mitochondrial calcium uniporter regulator 1 (MCUR1) [2+ uptake [A finely-tuned complex of proteins ensures and controls the mitochondrial Caer (MCU) ,45, whicer (MCU) . Moreoveer (MCU)  links th, to MCU . In addi (MCUR1) , the EF- (MCUR1)  as well  (MCUR1) . Recentl+ uptake .2+ ions control the activity of the tricarboxylic acid (TCA) cycle by Ca2+-dependent dehydrogenases [2), donate electrons to the electron transport chain (ECT). Electrons are accepted at the NADH dehydrogenase (complex I) and cytochrome bc1 complex (complex III) and are shuttled via succinate dehydrogenase (complex II), ubiquinone, cytochrome bc1 complex (complex III), and cytochrome c to cytochrome c oxidase (complex IV), where O2 is reduced to H2O. The electron transport through complex I to complex IV is coupled to proton pumping from the mitochondrial matrix to the IMS. The electrochemical gradient produced is used for the proton transport from the IMS into the matrix through ATP synthase (complex V), causing a release of energy and phosphorylation of adenosine diphosphate (ADP) to ATP [2O2 [2O2 may diffuse from the mitochondria to the cytosol, in the matrix of mitochondria several enzymes, like peroxiredoxins (PRDX) 3 and 5, catalase, and glutathione peroxidases 1 and 4 reduce H2O2 to H2O [2O2 is also linked to the NAD and NADP systems and is a key signaling molecule [2O2 by overexpression of PRDX3 improved glucose tolerance in mice, potentially via inhibition of glycogen synthase kinase-3. Moreover, cells from animals overexpressing PRDX3 had increased resistance to stress-induced cell death and apoptosis [Mclk1+/− mutant mice that produce higher levels of ROS than wild-type mice, had an enhanced immune reactivity and slowed aging progression [Caenorhabditis elegans (C. elegans) [In the mitochondrial matrix, Caogenases ,54. The ) to ATP . If elec) to ATP . To neutATP [2O2 . While a2 to H2O . Notablymolecule . Hence, poptosis . High lepoptosis . In contpoptosis . Interesgression . Moreoveelegans) .c into the cytosol through a mechanism yet to be described [c binds to apoptotic protease activating factor 1 (APAF1) and ATP, forming the apoptosome complex, which cleaves pro-caspase 9 into its active form caspase-9, and thereby activates effector caspases [While mitochondria ensure cell viability by ATP production, they also execute programmed cell death . In respescribed . By deaccaspases . During caspases . Since all of these mitochondrial tasks are highly complex and need to be well-balanced and controlled, numerous targets may be affected by aging or impacted by dysregulation of the process of aging.2+ uptake and homeostasis undergoes remarkable changes. For instance, the MCU channel is oxidized by ROS, resulting in increased MCU channel activity, which, in turn, further elevates mitochondrial ROS levels and increases the risk for mitochondrial Ca2+ overload-induced cell death [2+ levels, while store-operated Ca2+ entry was inhibited via downregulation of STIM1 and ORAI1 in these aging neurons [2+ accumulation by inositol 1,4,5-trisphosphate receptor type 2 (ITPR2)-triggered Ca2+ release from the ER and mitochondrial Ca2+ uptake via MCU in human mammary epithelial cells (HMEC). In turn, loss of ITPR2 and MCU counteracted the development of senescence [2+ overload, as well as high levels of ROS promotes cell death by collapse of the mitochondrial membrane potential and opening of the mitochondrial permeability transition pore (mPTP) [2+, NAD+, and glutathione into the cytosol was shown to disrupt cellular homeostasis and to increase oxidative damage [2+ flux in response to cytosolic Ca2+ was reported to decrease progressively in C. elegans during aging [2+ uptake was demonstrated in mice lacking Fus1, a small mitochondrial protein regulating mitochondrial Ca2+ homeostasis. Loss of Fus1 caused inefficient accumulation of Ca2+ in mitochondria and decreased respiratory reserve capacity, resulting in the decreased lifespan of mice with knockout of Fus1 [2+ homeostasis during aging is a tightrope walk between meeting mitochondria’s demand for Ca2+ [2+ overload [During the process of aging, the protein machinery controlling mitochondrial Call death . Moreove neurons . In linenescence . Mitochoe (mPTP) . Moreovee (mPTP)  might fue (mPTP) . Releasee damage  and possng aging . The imp of Fus1 . These pfor Ca2+  and trigoverload .+/NADH ratio at middle-age (15–16 years) and elevation in increased age (28–32 years) [2+ levels mirrored those in energy production—initially elevated [2+ overload-induced damage [1FO ATP synthase activity and defective F1FO complex coupling, which may be caused by the mitochondrial protein cyclophilin D [Aging was typically associated with decreased mitochondrial metabolism . In appa2 years) . Moreoveelevated ,69 beford damage —it seemsd age 28–2 years [d damage . In thisd damage . Anotherd damage . Interesd damage . It seemd damage . In contd damage . Consistphilin D . Moreovephilin D . In summmt) was suggested as a possible mechanism to tolerate mtDNA deletions to a certain extent [mt is a protective transcriptional program from the cells to sense and respond to mitochondrial dysfunction by inducing mitochondrial protein homeostasis and regeneration of defective mitochondria [C. elegans strain, containing wild-type mtDNA and mtDNA lacking four essential genes, UPRmt was constitutively activated by dysfunctional OXPHOS. Moreover, even deleterious mtDNA was maintained by UPRmt, possibly in an attempt to recover OXPHOS activity by promoting mitochondrial biogenesis [C. elegans that OXPHOS dysfunction induces the activating transcription factor associated with stress (ATFS1), which binds directly to the OXPHOS gene promotors in the nuclear and mitochondrial genomes and thereby promotes OXPHOS recovery during the UPRmt [C. elegans and even rescued UPRmt signaling in ATFS1 deficient worms [mt on the process of aging was demonstrated in worms, in which UPRmt delayed aging and promoted life span via ATFS1 [mt was also shown to prolong the lifespan of Drosophila melanogaster by activation of the forkhead protein O (FOXO), which induces a set of chaperones implicated in lifespan extension [Mutations of mitochondrial DNA (mtDNA) are estimated to be 10- to 17-fold higher than in the nuclear genome . In linen extent . UPRmt ichondria . In a heogenesis . It was he UPRmt . In mammnt worms . Furtheria ATFS1 . Recentlxtension .mt in nematodes, ATFS1, controls mitochondrial biogenesis to compensate for loss of OXPHOS activity [mt activation during aging [C. elegans and linked to age-related decline in so-called mitophagy [mitochondria-on-a-string (MOAS), characterized by thin segments of mitochondria mixed with large ones, a phenotype already described for neurodegenerative diseases, was found and linked to incomplete mitochondrial fission due to dysregulation of the fission machinery [D. melanogaster. A shift towards accumulation of dysfunctional elongated mitochondria was thereby prevented and health, as well as lifespan, improved significantly [D. melanogaster (10–12 weeks), mitochondria showed cristae vacuolization and the distribution of functional proteins such as ATP synthase was hampered [D. melanogaster, characterized by the lack of well-developed cristae and cristae junctions and positively correlating with the rate of mitochondrial respiration [The main transcription factor of UPRactivity . Consequng aging . Indeed,itophagy . After mitophagy . For effitophagy . Notablyachinery . Short-tficantly . Notablyficantly . Neverthficantly . Consistficantly . Howeverficantly . Moreoveficantly . Acute dficantly . In longhampered . Anotherpiration . In contpiration . In cardpiration . In yeaspiration . In summC. elegans [2+ retention capacity was enhanced strongly by CR in comparison to ad libitum diet, offering protection from ischemia/reperfusion damage, a condition frequently observed in age [2+ and enhanced tolerance against excitotoxicity in rat brain mitochondria [+ levels [2 ratio at baseline benefited from CR in regard to muscle mitochondrial ATP synthesis rates and coupling [Caloric restriction: From  elegans  to prima elegans , caloric elegans ,115. The elegans . While r elegans . CR also elegans . Consist elegans . The effd in age . Additiochondria . In yeaschondria . Moreovechondria . AMPK wa+ levels  and to i+ levels . Indeed,+ levels . Neverthcoupling . These c2+ homeostasis has also been shown to be affected during aging, revealing downregulation of MCU in aged mice [2+ uptake and in mitochondrial shape by nine weeks of physical training or neuromuscular electrical stimulation. Improved muscle function and structure by both types of training were linked to increased protein levels of MCU. Interestingly, electron microscopy revealed remodeling of mitochondrial shape in muscles trained by electrical stimulation, which was potentially mediated by an increased expression of cristae structure-shaping protein OPA1 [Exercise: In a number of species, age-related muscle loss is linked to impaired mitochondrial biogenesis and function, as well as to decreased metabolic capacity . Prolifeged mice . Analysiein OPA1 . Interesein OPA1 .Polyphenols: Polyphenols, such as resveratrol (RSV) or green tea polyphenols, were shown to extend lifespan in Saccharomyces cerevisiae [C. elegans [D. melanogaster [French paradox, was hypothesized to be due to frequent consumption of RSV-rich red wine [French paradox has to be reevaluated.revisiae , C. eleg elegans , D. melanogaster , fish [1nogaster , and micnogaster . Moreovered wine . Clinicared wine . The lowred wine , as wellred wine . Howeverred wine . Thus, t2+ levels and activation of the CaMKKβ-AMPK pathway [+ levels [2O2 production, whereas the ubiquinone derivative, MitoQ, an electron scavenger preventing mitochondrial ROS formation [2O2 levels under this condition [Nevertheless, RSV was found to mimic features of CR, such as glucagon and catecholamine release, boosting cyclic adenosine monophosphate production, which, in turn, activates cell-type dependent effectors and leads to elevated Ca pathway . Enhance+ levels . Other r+ levels ,141, cau+ levels  and alte+ levels . Moreove+ levels . Low lev+ levels  as well + levels . Neverth+ levels . For insormation , could nondition .Antioxidants: In addition to MitoQ, the Murphy lab has synthesized several other mitochondria-targeted antioxidants by covalently attaching lipophilic triphenylphosphonium cations to endogenous antioxidants, including α-tocopherol (MitoVitE) and the synthetic spin trap compound α-phenyl-tert-butylnitrone (MitoPBN) [MitoPBN) . FurtherMitoPBN) . Due to MitoPBN) . In rat MitoPBN) . FurtherMitoPBN) . In addiMitoPBN) . MitoQ wMitoPBN) . MoreoveMitoPBN) , was sucMitoPBN)  and is cMitoPBN) . All of C. elegans [Nevertheless, the relevance of antioxidants in preventing age-related cellular dysfunction by the reduction of oxidative stress was challenged by the finding that small levels of ROS provoke a vaccination-like stimulus, thus causing development of antioxidant defense mechanisms and a positive impact on lifespan . Consequ elegans . The dif elegans . In anim elegans . In cont elegans . In the  elegans . Aspirin: Long-term usage of acetylsalicylic acid (Aspirin®), a nonsteroidal anti-inflammatory drug, was shown to slightly increase the lifespan of worms [C. elegans, aspirin treatment increased the expression of lipid hydrolysis and fatty acid β-oxidation-related genes, which activated DAF-12 and DAF-16, the worm orthologue to FOXO, and extended lifespan [of worms , flies [of worms , and mamof worms . After oof worms . This meof worms . Additiolifespan . Neverthlifespan  and potelifespan ,166, thelifespan .Omega-3 fatty acids: Treatment with ω-3 fatty acid α-linolenic acid (ALA) increased lifespan of C. elegans via the activation of the transcription factors NHR-49, the worm orthologue of human PPARα, and SKN1, the worm orthologue of human NRF2. NHR-49-activated genes are involved in the β-oxidation of free fatty acids, promoting the generation of energy via mitochondrial respiration and NRF2-induced mitochondrial biogenesis [ogenesis . In the ogenesis . Consistogenesis . NeverthMetformin: Metformin, a biguanide used for diabetes prevention and management for 60 years, was reported to display greatly beneficial effects on aging in cellular and animal models, as well as in pilot human studies, far beyond that expected from improving glycemic control [C. elegans [ control . Despite control , possibl control . That me control . Moreove elegans . As a ca elegans  and inhi elegans . Interes elegans . Consequ elegans , causingSenolytics: Senolytic drugs were reported to induce apoptosis of senescent cells, thus preventing the local and systemic pathogenic effects of senescent cells conveyed by the release of proinflammatory cytokines and chemokines, tissue-damaging proteases and inhibition of stem and progenitor cell function [function . By hostfunction . Accordifunction . The canfunction  and to ifunction . Navitocfunction . Moreovefunction . Therefo2+ handling [The process of aging evokes various alterations in mitochondrial Cahandling , mitochohandling , mitochohandling , as wellhandling , which ahandling . Initialhandling , exercishandling , and druhandling , antioxihandling , metformhandling , ω-3 fathandling , aspirinhandling , and senhandling , specifihandling . Consequently, it is of major importance to further investigate the molecular processes behind the role of mitochondria in aging, as well as their potential to serve as targets for therapeutic interventions.