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.
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).
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.
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.

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.

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.

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.

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.
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-β-induced EMT cell line models, the effects of monensin on the EMT status and EMT-mediated drug resistance were assessed.
TGF-β treatment induced EMT in non-small cell lung cancer (NSCLC) cell lines and the EGFR-mutant NSCLC cell lines with TGF-β-induced EMT acquired resistance to EGFR-tyrosine kinase inhibitor. The addition of monensin effectively suppressed the TGF-β-induced-EMT conversion, and restored the growth inhibition and the induction of apoptosis by the EGFR-tyrosine kinase inhibitor.
Our data suggested that combined therapy with monensin might be a useful strategy for preventing EMT-mediated acquired drug resistance.

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.

Nonalcoholic fatty liver disease (NAFLD) is estimated to affect up to one-third of the general population, and new therapies are urgently required. Our laboratory previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides. Although we previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, hepatic inflammation, and fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess its safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of longer-term CRMP treatment on hepatic mitochondrial oxidation and on the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in high-fat, fructose-fed cynomolgus macaques (n = 6) and spontaneously obese dysmetabolic rhesus macaques (n = 12). Using positional isotopomer nuclear magnetic resonance tracer analysis (PINTA), we demonstrated that acute CRMP treatment (single dose, 5 mg/kg) increased rates of hepatic mitochondrial fat oxidation by 40%. Six weeks of CRMP treatment reduced hepatic triglycerides in both nonhuman primate models independently of changes in body weight, food intake, body temperature, or adverse reactions. CRMP treatment was also associated with a 20 to 30% reduction in fasting plasma triglycerides and low-density lipoprotein (LDL)-cholesterol in dysmetabolic nonhuman primates. Oral administration of CRMP reduced endogenous glucose production by 18%, attributable to a 20% reduction in hepatic acetyl-coenzyme A (CoA) content [as assessed by whole-body β-hydroxybutyrate (β-OHB) turnover] and pyruvate carboxylase flux. Collectively, these studies provide proof-of-concept data to support the development of liver-targeted mitochondrial uncouplers for the treatment of metabolic syndrome in humans.

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 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 β-adrenergic stimulation by isoprenaline, a non-selective agonist of β-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.

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/β-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/β-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/β-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.