The effect of the efflux pump inhibitor Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP) on the susceptibility to imipenem and cefepime in clinical strains of Acinetobacter baumannii.

INTRODUCTION: In the last years the rapid expansion of multidrug-resistant A. baumannii strains have become a major health problem. Efflux pumps are a group of transport proteins that contribute to the development of antibiotic resistance. The aim of this study was to evaluate the effect of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on the antimicrobial action of imipenem and cefepime on clinical strains of A. baumannii. MATERIALS AND METHODS: A total of 49 non-duplicate clinical samples were collected during January through December of 2018 from patients hospitalized in the Hospital Regional Docente de Cajamarca. Of the 49 samples obtained, the confirmatory identification of A. baumannii was performed on 47 samples by molecular methods. The amplification of the blaOXA-51-like gene was carried out by polymerase chain reaction (PCR). The determination of the minimum inhibitory concentration (MIC) was calculated using the microdilution method in culture broth. The susceptibility to both antibiotics (cefepime and imipenem) was evaluated in the presence and absence of the inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). RESULTS: A total of 47 strains of A. baumannii were isolated: 97.87% (46/47) were resistant to Imipenem, 2.13% (1/47) of them were classified as intermediate and none of these strains were susceptible. On the other hand, 51.06% (24/47) of isolates were resistant to cefepime; 19.15% (9/47) intermediate and 29.79% (14/47) susceptible. We considered a significant difference in antibiotic susceptibility if the MIC changed at least 4 dilutions, after the addition of the inhibitor. In the case of CCCP in addition to imipenem, 2.1% (1/47) had a significant change of 4 or more reductions in MIC, 59.6% (28/47) achieved a change equal or less than 3 dilutions and 17.0% (8/47) did not have any change. In the case of CCCP with cefepime the percentage of strains with the significant change of MIC was 8.5% (4/47). On the other hand, 53.2% (24/47) presented a reduction equal or less than 3 dilutions and 12.8% (6/47) did not show changes. CONCLUSION: In conclusion, our results demonstrate that the use of CCCP may improve the antibiotic effect of imipenem and cefepime on clinical strains of A. baumannii. The relevance of this study is that it provides evidence that this efflux pump inhibitor may be an alternative treatment against multidrug-resistant A. baumannii.

Defining the molecular mechanisms of the mitochondrial permeability transition through genetic manipulation of F-ATP synthase.

F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway.

Active Efflux Leads to Heterogeneous Dissipation of Proton Motive Force by Protonophores in Bacteria.

Various toxic compounds disrupt bacterial physiology. While bacteria harbor defense mechanisms to mitigate the toxicity, these mechanisms are often coupled to the physiological state of the cells and become ineffective when the physiology is severely disrupted. Here, we characterized such feedback by exposing Escherichia coli to protonophores. Protonophores dissipate the proton motive force (PMF), a fundamental force that drives physiological functions. We found that E. coli cells responded to protonophores heterogeneously, resulting in bimodal distributions of cell growth, substrate transport, and motility. Furthermore, we showed that this heterogeneous response required active efflux systems. The analysis of underlying interactions indicated the heterogeneous response results from efflux-mediated positive feedback between PMF and protonophores' action. Our studies have broad implications for bacterial adaptation to stress, including antibiotics. IMPORTANCE An electrochemical proton gradient across the cytoplasmic membrane, alternatively known as proton motive force, energizes vital cellular processes in bacteria, including ATP synthesis, nutrient uptake, and cell division. Therefore, a wide range of organisms produce the agents that collapse the proton motive force, protonophores, to gain a competitive advantage. Studies have shown that protonophores have significant effects on microbial competition, host-pathogen interaction, and antibiotic action and resistance. Furthermore, protonophores are extensively used in various laboratory studies to perturb bacterial physiology. Here, we have characterized cell growth, substrate transport, and motility of Escherichia coli cells exposed to protonophores. Our findings demonstrate heterogeneous effects of protonophores on cell physiology and the underlying mechanism.

SUMOylation of mitofusins: A potential mechanism for perinuclear mitochondrial congression in cells treated with mitochondrial stressors.

Depolarized/damaged mitochondria aggregate at the perinuclear region prior to mitophagy in cells treated with mitochondrial stressors. However, the cellular mechanism(s) by which damaged mitochondria are transported and remain aggregated at the perinuclear region is unknown. Here, we demonstrate that mitofusins (Mfn1/2) are post-translationally modified by SUMO2 (Small Ubiquitin-related Modifier 2) in Human embryonic kidney 293 (Hek293) cells treated with protonophore CCCP and proteasome inhibitor MG132, both known mitochondrial stressors. SUMOylation of Mfn1/2 is not for their proteasomal degradation but facilitate mitochondrial congression at the perinuclear region in CCCP- and MG132-treated cells. Additionally, congressed mitochondria (mito-aggresomes) colocalize with LC3, ubiquitin, and SUMO2 in CCCP-treated cells. Knowing that SUMO functions as a "molecular glue" to facilitate protein-protein interactions, we propose that SUMOylation of Mfn1/2 may congress, glues, and confines damaged mitochondria to the perinuclear region thereby, protectively quarantining them from the heathy mitochondrial network until their removal via mitophagy in cells.

Overcoming epithelial-mesenchymal transition-mediated drug resistance with monensin-based combined therapy in non-small cell lung cancer.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is a key process in tumor progression and metastasis and is also associated with drug resistance. Thus, controlling EMT status is a research of interest to conquer the malignant tumors. MATERIALS AND METHODS: A drug repositioning analysis of transcriptomic data from a public cell line database identified monensin, a widely used in veterinary medicine, as a candidate EMT inhibitor that suppresses the conversion of the EMT phenotype. Using TGF-ß-induced EMT cell line models, the effects of monensin on the EMT status and EMT-mediated drug resistance were assessed. RESULTS: TGF-ß treatment induced EMT in non-small cell lung cancer (NSCLC) cell lines and the EGFR-mutant NSCLC cell lines with TGF-ß-induced EMT acquired resistance to EGFR-tyrosine kinase inhibitor. The addition of monensin effectively suppressed the TGF-ß-induced-EMT conversion, and restored the growth inhibition and the induction of apoptosis by the EGFR-tyrosine kinase inhibitor. CONCLUSION: Our data suggested that combined therapy with monensin might be a useful strategy for preventing EMT-mediated acquired drug resistance.

AMPK activation induces mitophagy and promotes mitochondrial fission while activating TBK1 in a PINK1-Parkin independent manner.

Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.

Direct determination of anaerobe contributions to the energy metabolism of Trypanosoma cruzi by chip calorimetry.

We describe a chip calorimetric technique that allows the investigation of biological material under anoxic conditions in a micro-scale and in real time. Due to the fast oxygen exchange through the sample flow channel wall, the oxygen concentration inside the samples could be switched between atmospheric oxygen partial pressure to an oxygen concentration of 0.5% within less than 2 h. Using this technique, anaerobic processes in the energy metabolism of Trypanosoma cruzi could be studied directly. The comparison of the calorimetric and respirometric response of T. cruzi cells to the treatment with the mitochondrial inhibitors oligomycin and antimycin A and the uncoupler FCCP revealed that the respiration-related heat rate is superimposed by strong anaerobic contributions. Calorimetric measurements under anoxic conditions and with glycolytic inhibitors showed that anaerobic metabolic processes contribute from 30 to 40% to the overall heat production rate. Similar basal and antimycin A heat rates with cells under anoxic conditions indicated that the glycolytic rates are independent of the oxygen concentration which confirms the absence of the "Pasteur effect" in Trypanosomes. Graphical abstract.

Metabolic Inhibition Induces Transient Increase of L-type Ca2+ Current in Human and Rat Cardiac Myocytes.

Metabolic inhibition is a common condition observed during ischemic heart disease and heart failure. It is usually accompanied by a reduction in L-type Ca2+ channel (LTCC) activity. In this study, however, we show that metabolic inhibition results in a biphasic effect on LTCC current (ICaL) in human and rat cardiac myocytes: an initial increase of ICaL is observed in the early phase of metabolic inhibition which is followed by the more classical and strong inhibition. We studied the mechanism of the initial increase of ICaL in cardiac myocytes during ß-adrenergic stimulation by isoprenaline, a non-selective agonist of ß-adrenergic receptors. The whole-cell patch⁻clamp technique was used to record the ICaL in single cardiac myocytes. The initial increase of ICaL was induced by a wide range of metabolic inhibitors (FCCP, 2,4-DNP, rotenone, antimycin A). In rat cardiomyocytes, the initial increase of ICaL was eliminated when the cells were pre-treated with thapsigargin leading to the depletion of Ca2+ from the sarcoplasmic reticulum (SR). Similar results were obtained when Ca2+ release from the SR was blocked with ryanodine. These data suggest that the increase of ICaL in the early phase of metabolic inhibition is due to a reduced calcium dependent inactivation (CDI) of LTCCs. This was further confirmed in human atrial myocytes where FCCP failed to induce the initial stimulation of ICaL when Ca2+ was replaced by Ba2+, eliminating CDI of LTCCs. We conclude that the initial increase in ICaL observed during the metabolic inhibition in human and rat cardiomyocytes is a consequence of an acute reduction of Ca2+ release from SR resulting in reduced CDI of LTCCs.

Wnt signaling inhibition by monensin results in a period of Hippo pathway activation during intestinal adaptation in zebrafish.

Intestinal adaptation (IA) is a critical response to increase epithelial surface area after intestinal loss. Short bowel syndrome (SBS) may follow massive intestinal resection in human patients, particularly without adequate IA. We previously validated a model in zebrafish (ZF) that recapitulates key SBS pathophysiological features. Previous RNA sequencing in this model identified upregulation of genes in the Wnt and Hippo pathways. We therefore sought to identify the timeline of increasing cell proliferation and considered the signaling that might underpin the epithelial remodeling of IA in SBS. SBS was created in a ZF model as previously reported and compared with sham fish with and without exposure to monensin, an ionophore known to inhibit canonical Wnt signaling. Rescue of the monensin effects was attempted with a glycogen synthase kinase 3 inhibitor that activates wnt signaling, CHIR-99021. A timeline was constructed to identify peak cellular proliferation, and the Wnt and Hippo pathways were evaluated. Peak stem cell proliferation and morphological changes of adaptation were identified at 7 days. Wnt inhibition diminished IA at 2 wk and resulted in activation of genes of the Wnt/ß-catenin and Yes-associated protein (YAP)/Hippo pathway. Increased cytoplasmic YAP was observed in monensin-treated SBS fish. Genes of the WASP-interacting protein (WIP) pathway were elevated during Wnt blockade. In conclusion, cellular proliferation and morphological changes accompany SBS even in attempted Wnt blockade. Wnt/ß-catenin, YAP/Hippo pathway, and WIP pathway genes increase during early Wnt blockade. Further understanding of the effects of Wnt and YAP pathway signaling in proliferating stem cells might enrich our knowledge of targets to assist IA. NEW & NOTEWORTHY Intestinal adaptation is a critical response to increase epithelial surface area after large intestinal losses. Inhibition of Wnt/ß-catenin signaling impairs intestinal adaptation in a zebrafish model of short bowel syndrome. There is a subsequent upregulation in genes of the Yes-associated protein/Hippo and WIP pathway. These may be targets for future human therapies, as patients are salvaged by the compensation of increased intestinal epithelial surface area through successful intestinal adaptation.

Cleaved PGAM5 is released from mitochondria depending on proteasome-mediated rupture of the outer mitochondrial membrane during mitophagy.

PGAM5 is a unique type of protein phosphatase that exists in mitochondria. It has been shown to exist in the inner mitochondrial membrane through its transmembrane domain and to be cleaved within the transmembrane domain upon mitochondrial dysfunction. However, its submitochondrial localization remains controversial; many researchers claim that PGAM5 localizes to the outer mitochondrial membrane based on the findings that PGAM5 associates with many cytoplasmic proteins. Here, we found that cleaved PGAM5 was released from mitochondria during mitophagy, a selective form of autophagy specific for mitochondria, and that the release was inhibited by proteasome inhibitors in HeLa cells stably expressing the E3 ubiquitin ligase Parkin. However, treatment of parental HeLa cells lacking Parkin with mitophagy-inducing agents caused PGAM5 cleavage but did not cause its release from mitochondria. Thus, cleaved PGAM5 appears to be released from mitochondria depending on proteasome-mediated rupture of the outer membrane during mitophagy, which has been previously shown to precede autophagy-mediated degradation of whole mitochondria. This study suggests that PGAM5 senses mitochondrial dysfunction in the inner mitochondrial membrane and serves as a signalling intermediate that regulates the cellular response to mitochondrial stress upon its cleavage and release from mitochondria.

Mitochondrial membrane depolarization enhances TRAIL-induced cell death in adult human granulosa tumor cells, KGN, through inhibition of BIRC5.

BACKGROUND: Cellular metabolic changes that accompany malignant transformation have been heralded as hallmark features of cancer. However, metabolic signatures between neoplasms can be unique, allowing for distinctions in malignancy, invasion and chemoresistance between cancer types and subtypes. Mitochondria are central metabolic mediators, as cellular bioenergetics veers from oxidative phosphorylation to glycolysis. Herein, we evaluate the role of mitochondria in maintenance of cellular metabolism, proliferation, and survival in the adult granulosa tumor cell line, KGN, as well as three epithelial ovarian cancer cell lines to determine distinctions in specific features. RESULTS: Notably, KGN cells were susceptible to TRAIL- and cisplatin-induced death following pretreatment with the metabolic inhibitor FCCP, but not oligomycin A. Collapse of mitochondrial membrane potential was found concomitant with cell death via apoptosis, independent from extrinsic canonical apoptotic routes. Rather, treatment with FCCP resulted in elevated cytochrome c release from mitochondria and decreased responsiveness to BIRC5. Following knockdown of BIRC5, mitochondrial membrane depolarization further sensitized KGN cells to induction of apoptosis via TRAIL. CONCLUSIONS: These results indicate an essential role, distinct from metabolism, for mitochondrial membrane potential in KGN cells to sense and respond to external mediators of apoptotic induction.

Mitochondrial uncoupling reveals a novel therapeutic opportunity for p53-defective cancers.

There are considerable challenges in directly targeting the mutant p53 protein, given the large heterogeneity of p53 mutations in the clinic. An alternative approach is to exploit the altered fitness of cells imposed by loss-of-wild-type p53. Here we identify niclosamide through a HTS screen for compounds selectively killing p53-deficient cells. Niclosamide impairs the growth of p53-deficient cells and of p53 mutant patient-derived ovarian xenografts. Metabolome profiling reveals that niclosamide induces mitochondrial uncoupling, which renders mutant p53 cells susceptible to mitochondrial-dependent apoptosis through preferential accumulation of arachidonic acid (AA), and represents a first-in-class inhibitor of p53 mutant tumors. Wild-type p53 evades the cytotoxicity by promoting the transcriptional induction of two key lipid oxygenation genes, ALOX5 and ALOX12B, which catalyzes the dioxygenation and breakdown of AA. Therefore, we propose a new paradigm for targeting cancers defective in the p53 pathway, by exploiting their vulnerability to niclosamide-induced mitochondrial uncoupling.

Efflux pump inhibitor CCCP to rescue colistin susceptibility in mcr-1 plasmid-mediated colistin-resistant strains and Gram-negative bacteria.

Objectives: Efflux in bacteria is a ubiquitous mechanism associated with resistance to antimicrobials agents. Efflux pump inhibitors (EPIs) have been developed to inhibit efflux mechanisms and could be a good alternative to reverse colistin resistance, but only CCCP has shown good activity. The aim of our study was to identify CCCP activity in a collection of 93 Gram-negative bacteria with known and unknown colistin resistance mechanisms including isolates with mcr-1 plasmid-mediated colistin resistance. Methods: Colistin MIC was evaluated with and without CCCP and the fold decrease of colistin MIC was calculated for each strain. In order to evaluate the effect of this combination, a time-kill study was performed on five strains carrying different colistin resistance mechanisms. Results: Overall, CCCP was able to reverse colistin resistance for all strains tested. The effect of CCCP was significantly greater on intrinsically colistin-resistant bacteria (i.e. Proteus spp., Serratia marcescens, Morganella morganii and Providencia spp.) than on other Enterobacteriaceae (P < 0.0001). The same was true for bacteria with a heteroresistance mechanism compared to bacteria with other colistin resistance mechanisms (P < 0.0001). A time-kill study showed the combination was bacteriostatic on strains tested. Conclusions: These results suggest an efflux mechanism, especially on intrinsically resistant bacteria and Enterobacter spp., but further analysis is needed to identify the molecular support of this mechanism. EPIs could be an alternative for restoring colistin activity in Gram-negative bacteria. Further work is necessary to identify new EPIs that could be used in humans.

Unravelling mechanisms of nitrofurantoin resistance and epidemiological characteristics among Escherichia coli clinical isolates.

The aim of this study was to investigate mechanisms of nitrofurantoin resistance and epidemiological characteristics in Escherichia coli clinical isolates. From a total of 1444 E. coli clinical isolates collected from our hospital in 2015, 18 (1.2%) nitrofurantoin-resistant E. coli isolates were identified with nitrofurantoin minimum inhibitory concentrations (MICs) ranging from 128 µg/mL to ≥512 µg/mL. The prevalence of the nfsA gene in nitrofurantoin-resistant, -intermediate and -susceptible isolates was 88.9%, 88.9% and 100%, respectively, and the prevalence of the nfsB gene was 66.7%, 61.1% and 100%, respectively. Eight nitrofurantoin-resistant isolates and two nitrofurantoin-intermediate isolates possessed oqxAB genes. In nitrofurantoin-resistant isolates, mutations in NfsA (the majority of mutated sites were I117T and G187D, accounting for 38.9%) and/or NfsB were detected, whereas only NfsA mutations were found in intermediate isolates and no sequence changes were detected in susceptible isolates. A ≥4-fold decrease in MIC was observed in eight nitrofurantoin-resistant isolates following addition of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mean expression level of oqxB in nitrofurantoin-resistant isolates increased ca. 7-fold compared with intermediate isolates. Multilocus sequence typing (MLST) categorised the 18 nitrofurantoin-resistant isolates into 11 different sequence types. Pulsed-field gel electrophoresis (PFGE) analysis revealed that homology among the nitrofurantoin-resistant isolates was low and sporadic. In conclusion, mutations in nfsA and nfsB were the main mechanisms leading to nitrofurantoin resistance, and overexpression of the oqxAB gene might help to further increase the MIC of nitrofurantoin.

Short communication: Field study to investigate the associations between herd-level risk factors for milk fat depression and bulk tank milk fat percent in dairy herds feeding monensin.

Fat is the most variable milk component, and maintaining milk fat continues to be a challenge on commercial dairy farms. Our objectives were to establish associations between herd-level risk factors for milk fat depression and bulk tank milk fat content in commercial dairy herds feeding monensin. Seventy-nine Holstein commercial dairy herds in the northeast and Upper Midwest United States were enrolled in an observational study. Data were collected on herd characteristics, total mixed ration (TMR) samples, all component silage samples, and bulk tank milk samples. The unconditional univariable association of each explanatory variable and bulk tank milk fat percentage was evaluated using simple linear regression and multivariable regression models. Milk fat content of trans-10 C18:1 had an exponentially negative relationship to herd milk fat percentage. In general, milk fat content of fatty acids synthesized de novo in the mammary gland were positively related to herd milk fat, and the content of several trans-C18:1 fatty acids, which would be products of alternate pathways of ruminal biohydrogenation, were negatively related to herd milk fat. Variables related to TMR composition did not have univariable relationships with herd milk fat percentage. Herds that had >49.8% of the TMR particles on the middle screen of the Penn State particle separator had higher milk fat percentage than those with ≤49.8%, and herds with >54.0% of TMR particles in the bottom pan had lower milk fat percentage than herds with ≤54.0%. Dietary content of monounsaturated fatty acids (C16:1 and C18:1) had negative relationships with herd milk fat percentage; however, no single diet component accounted for more than 11% of the variation in herd-level milk fat percentage. Univariable monensin dose was not associated with herd milk fat percentage. The relative lack of significant univariate relationships with herd-level milk fat suggests many factors contribute to milk fat content, and herds experiencing low milk fat will need to examine many potential risk factors when working to troubleshoot this challenge.

Zn2+-induced disruption of neuronal mitochondrial function: Synergism with Ca2+, critical dependence upon cytosolic Zn2+ buffering, and contributions to neuronal injury.

Excitotoxic Zn2+ and Ca2+ accumulation contributes to neuronal injury after ischemia or prolonged seizures. Synaptically released Zn2+ can enter postsynaptic neurons via routes including voltage sensitive Ca2+ channels (VSCC), and, more rapidly, through Ca2+ permeable AMPA channels. There are also intracellular Zn2+ binding proteins which can either buffer neuronal Zn2+ influx or release bound Zn2+ into the cytosol during pathologic conditions. Studies in culture highlight mitochondria as possible targets of Zn2+; cytosolic Zn2+ can enter mitochondria and induce effects including loss of mitochondrial membrane potential (ΔΨm), mitochondrial swelling, and reactive oxygen species (ROS) generation. While brief (5 min) neuronal depolarization (to activate VSCC) in the presence of 300 µM Zn2+ causes substantial delayed neurodegeneration, it only mildly impacts acute mitochondrial function, raising questions as to contributions of Zn2+-induced mitochondrial dysfunction to neuronal injury. Using brief high (90 mM) K+/Zn2+ exposures to mimic neuronal depolarization and extracellular Zn2+ accumulation as may accompany ischemia in vivo, we examined effects of disrupted cytosolic Zn2+ buffering and/or the presence of Ca2+, and made several observations: 1. Mild disruption of cytosolic Zn2+ buffering-while having little effects alone-markedly enhanced mitochondrial Zn2+ accumulation and dysfunction (including loss of ∆Ψm, ROS generation, swelling and respiratory inhibition) caused by relatively low (10-50 µM) Zn2+ with high K+. 2. The presence of Ca2+ during the Zn2+ exposure decreased cytosolic and mitochondrial Zn2+ accumulation, but markedly exacerbated the consequent dysfunction. 3. Paralleling effects on mitochondria, disruption of buffering and presence of Ca2+ enhanced Zn2+-induced neurodegeneration. 4. Zn2+ chelation after the high K+/Zn2+ exposure attenuated both ROS production and neurodegeneration, supporting the potential utility of delayed interventions. Taken together, these data lend credence to the idea that in pathologic states that impair cytosolic Zn2+ buffering, slow uptake of Zn2+ along with Ca2+ into neurons via VSCC can disrupt the mitochondria and induce neurodegeneration.

Effect of supplementing a diet with monensin sodium and Saccharomyces Cerevisiae on reproductive performance of Ghezel ewes.

Effect of supplementing a diet, in an attempt to enhance reproduction, with monensin sodium and Saccharomyces cerevisiae yeast on reproductive performance was investigated during the breeding season using 44 Ghezel ewes (body weight 56.97±7.47kg, age 2-5 years and body condition score (BCS) 2.5) which were allocated randomly in equal numbers to the four dietary treatments as follows: 1) Basal diet plus supplemental feed (450g/ewe/d) plus monensin sodium (30mg/ewe/d) (MS); 2) Basal diet plus supplemental feed (450 g/ewe/d) plus Saccharomyces cerevisiae yeast (4×109CFU/ewe/d) (SC); 3) Basal diet plus supplemental feed (450g/ewe/d) (FG); 4) Basal diet (only grazing on pasture, Control; G). Estrous synchronization of all ewes was done using controlled internal drug release (CIDR) and all ewes were mated with purebred Ghezel rams after CIDR removal. The results indicated that MS and SC treatments with 15 lambs had greater number of lambs than ewes of the other two treatment groups. Ewes in MS group with 50% twining rate had the greatest value followed by the FG, SC and G treatment groups (P<0.05). The lambs from ewes in MS and SC groups were heavier in weight than those in FG and G treatments (P<0.01). Blood sample analysis provided evidence that ewes in MS and SC groups had greater concentrations of 17ß-estradiol (E2), progesterone (P4), blood urea nitrogen (P<0.05), insulin, glucose, cholesterol and total protein (P<0.01) than ewes of the other groups. These results indicated that using a diet for enhancing reproduction, including monensin sodium and Saccharomyces cerevisiae yeast in the breeding season could have beneficial effects on reproductive performance of Ghezel ewes.

Superoxide drives progression of Parkin/PINK1-dependent mitophagy following translocation of Parkin to mitochondria.

Reactive oxygen species (ROS) and mitophagy are profoundly implicated in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease (PD). Several studies have suggested that ROS are not involved in mitochondrial translocation of Parkin which primes mitochondria for autophagic elimination. However, whether ROS play a role in the execution of mitophagy is unknown. In the present study, we show that carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment induced both mitochondrial depolarization and generation of ROS that were needed for the mitophagy process. Cells failed to proceed to complete mitophagy if CCCP treatment was discontinued even after recruitment of Parkin and autophagy machinery to mitochondria. Notably, treatment of pro-oxidant was able to replace CCCP treatment to take mitophagy forward, while it alone was insufficient to induce translocation of Parkin to mitochondria or autophagic clearance of mitochondria. In addition, an SOD mimetic that attenuated the superoxide level suppressed mitophagy, while an SOD inhibitor accumulated cellular superoxide and promoted mitophagy. Furthermore, blockage of the p38 signaling pathway inhibited mitophagy induced by ROS, suggesting that it may contribute to the activation of ROS-mediated mitophagy. Together, our study sheds light on the link between ROS and mitophagy at a molecular level, and suggests the therapeutic potential of regulating mitophagy through the superoxide-p38-mitophagy axis.

Differential effects of mitochondrial inhibitors on porcine granulosa cells and oocytes.

Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP.

Cross talk between increased intracellular zinc (Zn2+) and accumulation of reactive oxygen species in chemical ischemia.

Both zinc (Zn2+) and reactive oxygen species (ROS) have been shown to accumulate during hypoxic-ischemic stress and play important roles in pathological processes. To understand the cross talk between the two of them, here we studied Zn2+ and ROS accumulation by employing fluorescent probes in HeLa cells to further the understanding of the cause and effect relationship of these two important cellular signaling systems during chemical-ischemia, stimulated by oxygen and glucose deprivation (OGD). We observed two Zn2+ rises that were divided into four phases in the course of 30 min of OGD. The first Zn2+ rise was a transient, which was followed by a latent phase during which Zn2+ levels recovered; however, levels remained above a basal level in most cells. The final phase was the second Zn2+ rise, which reached a sustained plateau called Zn2+ overload. Zn2+ rises were not observed when Zn2+ was removed by TPEN (a Zn2+ chelator) or thapsigargin (depleting Zn2+ from intracellular stores) treatment, indicating that Zn2+ was from intracellular storage. Damaging mitochondria with FCCP significantly reduced the second Zn2+ rise, indicating that the mitochondrial Zn2+ accumulation contributes to Zn2+ overload. We also detected two OGD-induced ROS rises. Two Zn2+ rises preceded two ROS rises. Removal of Zn2+ reduced or delayed OGD- and FCCP-induced ROS generation, indicating that Zn2+ contributes to mitochondrial ROS generation. There was a Zn2+-induced increase in the functional component of NADPH oxidase, p47phox, thus suggesting that NADPH oxidase may mediate Zn2+-induced ROS accumulation. We suggest a new mechanism of cross talk between Zn2+ and mitochondrial ROS through positive feedback processes that eventually causes excessive free Zn2+ and ROS accumulations during the course of ischemic stress.