Carbonyl Cyanide M-chlorophenylhydrazone Research Articles

Investigation of human organoid retina with digital holographic transmission matrix measurements

Advanced manufacturing of retinal organoid samples from human induced pluripotent stem cells represents a promising way to study the development of retinal diseases. The retina is an epithelium composed of different cell layers with unique optical properties and detects light by photoreceptor neurons for visual function. There are still many challenges in detecting early and distinct cellular changes in retinal disease. In this paper, we study the capability of the optical transmission matrix, which fully describes the transition of a light field propagating through a scattering sample. Despite its rich information content, the transmission matrix is commonly just used for light delivery through scattering media. Digital holography is employed to measure the complex light-field information of the transmitted light. We demonstrate that singular value decomposition of the transmission matrix allows to discriminate phantom tissues with varying scattering coefficient. We apply these findings to retinal organoid tissues. Application of the protonophore carbonyl cyanide m-chloro-phenylhydrazone (CCCP), a known inducer of retinal damage in animals, caused cell death and structural changes in human retinal organoids, which resulted in distinct changes in the transmission matrix. Our data indicate that the analysis of the transmission matrix can distinguish pathologic changes of the retina towards the development of imaging-based biomarkers.

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.

IntroductionIn 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 methodsA 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).ResultsA 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.ConclusionIn 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.

Membrane potential-dependent regulation of mitochondrial complex II by oxaloacetate in interscapular brown adipose tissue.

Classically, mitochondrial respiration responds to decreased membrane potential (ΔΨ) by increasing respiration. However, we found that for succinate‐energized complex II respiration in skeletal muscle mitochondria (unencumbered by rotenone), low ΔΨ impairs respiration by a mechanism culminating in oxaloacetate (OAA) inhibition of succinate dehydrogenase (SDH). Here, we investigated whether this phenomenon extends to far different mitochondria of a tissue wherein ΔΨ is intrinsically low, i.e., interscapular brown adipose tissue (IBAT). Also, to advance our knowledge of the mechanism, we performed isotopomer studies of metabolite flux not done in our previous muscle studies. In additional novel work, we addressed possible ways ADP might affect the mechanism in IBAT mitochondria. UCP1 activity, and consequently ΔΨ, were perturbed both by GDP, a well‐recognized potent inhibitor of UCP1 and by the chemical uncoupler carbonyl cyanide m‐chlorophenyl hydrazone (FCCP). In succinate‐energized mitochondria, GDP increased ΔΨ but also increased rather than decreased (as classically predicted under low ΔΨ) O2 flux. In GDP‐treated mitochondria, FCCP reduced potential but also decreased respiration. Metabolite studies by NMR and flux analyses by LC‐MS support a mechanism, wherein ΔΨ effects on the production of reactive oxygen alters the NADH/NAD+ ratio affecting OAA accumulation and, hence, OAA inhibition of SDH. We also found that ADP‐altered complex II respiration in complex fashion probably involving decreased ΔΨ due to ATP synthesis, a GDP‐like nucleotide inhibition of UCP1, and allosteric enzyme action. In summary, complex II respiration in IBAT mitochondria is regulated by UCP1‐dependent ΔΨ altering substrate flow through OAA and OAA inhibition of SDH.

The utilization of photophosphorylation uncoupler to improve lipid production of Chlorella, a case study using transcriptome and functional gene expression analysis to reveal its mechanism

Uncoupling of photophosphorylation appears to have positive effect on lipid production of microalgae, however, underlying mechanisms involved in the effect are still unresolved. In this study, an uncoupler of photophosphorylation, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), was used for the promotion of microalgal lipid accumulation. The results indicated that addition of CCCP was able to enhance the lipid productions of all tested species of Chlorella, especially when cultivated under mixotrophic condition. Based on the transcriptome and functional gene expression analyses, the functional genes involving in photosynthetic electron transport (PET), fatty acids synthesis, tricarboxylic acid (TCA) cycle, glycolysis and pentose phosphate (PP) pathway were found to be up-regulated while the genes participating in photosystem II, ATP synthesis during photosynthesis and calvin cycle were to be down-regulated by the presence of CCCP. According to these observations, the mechanism of CCCP to the promotion of lipid synthesis probably through channeling the NAD(P)H metabolic fluxes from carbon fixation towards fatty acid biosynthesis and lipid production, which was accomplished by (1) down-regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase genes that involving in CO2 fixation via calvin cycle to reduce the NADPH consumption; (2) up-regulation of genes involved in TCA cycle and PP pathway to produce more NAD(P)H. The results of this study provide a new method to promote microalgal lipid production.

Cloning and sequencing of lsaE efflux pump gene from MDR Enterococci and its role in erythromycin resistance

Enterococci are opportunistic members of intestinal microbiota with notable ability to transmit antimicrobial resistance genes. Among the different resistance mechanisms, multidrug efflux is evolving as a huge problem in conferring multidrug resistance to bacterial cells because these pumps extrude a broad range of antimicrobials. Therefore, the aim of this work was to evaluate role of efflux pumps in the development of multi-drug resistance in Enterococci through studying the antimicrobial resistance profiles of Enterococci isolates, phenotypically and genotypically investigating the role of active efflux pumps in development of resistance, in addition to characterizing the most common efflux pump genes. The study involved the recovery of 149 Enterococci isolates from specimens of patients suffering infections in some hospitals in Egypt. Antimicrobial resistance profiles of isolates showed that only 1.3% of the isolates were resistant to each of linezolid, daptomycin, and fosfomycin. The highest resistance was to ampicillin (60.4%) while 47 of the isolates (31.54%) were found to be multidrug-resistant. Efflux pumps have shown to have a significant role in erythromycin resistance in 11 isolates (23.4% of MDR isolates) as indicated by an 8 or more fold decrease in minimum inhibitory concentration in the presence of the efflux pump inhibitor, carbonyl cyanide m- chlorophenylhydrazone (CCCP). End point PCR was used to detect efflux pump genes lsaE, msrC, and mefA in the 11 isolates at which efflux pumps were found to play a significant role in resistance. Nine out of the 11 isolates (81.8%) were found to carry lsaE gene. This gene was inserted into pUC21 vector and cloned into DH5α E. coli resulting in successful transformation and expression of erythromycin resistance in this host. Finally, sequencing of the lsaE gene was carried out. To the best of our knowledge, this is the first report on the cloning of lsaE gene from MDR Enterococcus isolates.

Decreased dynamin-related protein 1-related mitophagy induces myocardial apoptosis in the aging heart.

An increase in cardiomyocyte apoptosis is the main contributor to the observed high morbidity of cardiac disease during aging. Mitochondria play important roles in cardiac apoptosis, and dynamin-related protein 1 (Drp1) is the critical factor that participates in mitochondrial fission and induces mitophagy to maintain mitochondria quality. However, whether Drp1 is involved in the increase of apoptosis in aging heart remains unclear. The purpose of this study was to determine whether Drp1 participates in inducing the apoptosis through regulating mitophagy in aging myocardium. To explore the effect of mitophagy and apoptosis in aging heart, we detected the expression of COX IV and the co-localization of COX IV and LC3 II, which reflect mitophagy, and measured adenosine triphosphate and reactive oxygen species contents, which reflect mitochondrial injury. Cell apoptosis was detected by measuring the activity of caspase-3 and the expression of cleaved caspase-3 and further confirmed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The results showed an increase in apoptosis and a decrease in mitophagy in aging cardiomyocytes, and apoptosis was ameliorated after the induction of mitophagy by carbonyl cyanide m-chlorophenyl hydrazone (a mitophagy activator) in D-galactose (D-gal)-induced senescence H9c2 cells. To clarify the role of Drp1 in apoptosis, we knocked down Drp1 by transfecting si-Drp1, or overexpressed Drp1 in senescent cells, and then detected mitophagy, mitochondrial injury, and apoptosis. The data showed that downregulated Drp1 induces mitochondrial damage and apoptosis. In addition, to explore the regulatory relationship between Drp1 and phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy, we detected the expressions of PINK1 and Parkin after the overexpression of Drp1 in the D-gal group cells and found that Drp1-mediated mitophagy inhibited the PINK1/Parkin pathway in senescent cells. Our results demonstrated that insufficient Drp1 induces cardiomyocyte apoptosis by inhibiting mitophagy, and Drp1 affects the PINK1/Parkin pathway of mitophagy in the aging heart.

Expression of Mex AB-Opr M efflux pump system and meropenem resistance in Pseudomonas aeruginosa isolated from surgical intensive care unit

Background: Meropenem resistance of Pseudomonas aeruginosa (P. aeruginosa) is considered an increasing problem. Efflux pump is one of multiple mechanisms that are responsible for this resistance. This study aimed to phenotypically and genotypically detect prevalence of efflux pump mediated meropenem resistance among P. aeruginosa isolates. Methods: Pseudomonas aeruginosa was isolated from different clinical specimens and identified by conventional methods and confirmed by Vietek MS Malditof Mass Spectroscopy. Antibiotic susceptibility test was done by disc diffusion method then minimum inhibitory concentrations (MIC) for meropenem was detected twice by agar dilution method without and after addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Efflux pump genes were detected by polymerase chain reaction ( PCR). Results: Out of 265 specimens, 78 P. aeruginosa were isolated with an isolation rate (29.4%). By disc diffusion method and MIC by agar dilution methods, 35 (44.8%) isolates were meropenem resistant. There was a significant difference regarding distribution of efflux pump genes in meropenem resistant isolates as 23 isolates (65.7%) were positive for efflux pump genes and 12 (34.3%) were negative (p value= 0.13). The MICs of meropenem for P. aeruginosa isolates were significantly decreased after addition of CCCP where MIC of 21 (60%) meropenem resistant isolates had an efflux pump-overexpressing phenotype (p value =0.001). Conclusion: High prevalence of meropenem resistance in P. aeruginosa is mediated by efflux pump genes including, mex A, mex B and opr M.

CVB3-Mediated Mitophagy Plays an Important Role in Viral Replication via Abrogation of Interferon Pathways.

Coxsackievirus B3 (CVB3) is a common enterovirus that causes systemic inflammatory diseases, such as myocarditis, meningitis, and encephalitis. CVB3 has been demonstrated to subvert host cellular responses via autophagy to support viral replication in neural stem cells. Mitophagy, a specialized form of autophagy, contributes to mitochondrial quality control via degrading damaged mitochondria. Here, we show that CVB3 infection induces mitophagy in human neural progenitor cells, HeLa and H9C2 cardiomyocytes. In particular, CVB3 infection triggers mitochondrial fragmentation, loss of mitochondrial membrane potential, and Parkin/LC3 translocation to the mitochondria. Rapamycin or carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment led to increased CVB3 RNA copy number in a dose-dependent manner, suggesting enhanced viral replication via autophagy/mitophagy activation, whereas knockdown of PTEN-induced putative kinase protein 1(PINK1) led to impaired mitophagy and subsequent reduction in viral replication. Furthermore, CCCP treatment inhibits the interaction between mitochondrial antiviral signaling protein (MAVS) and TANK-binding kinase 1(TBK1), thus contributing to the abrogation of type I and III interferon (IFN) production, suggesting that mitophagy is essential for the inhibition of interferon signaling. Our findings suggest that CVB3-mediated mitophagy suppresses IFN pathways by promoting fragmentation and subsequent sequestration of mitochondria by autophagosomes.

Effects of Superoxide Dismutase Inhibitors and Glucose on Cell Death and Generation of Reactive Oxygen Species in Pea Leaves.

The effects of superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC), triethylenetetramine (trien), and their combination with glucose on cells of the epidermis from pea leaves of different age (rapidly growing young leaves and slowly growing old leaves) was investigated. DDC and trien caused death of the guard cells as determined by destruction of their nuclei. Glucose did not affect destruction of the nuclei induced by SOD inhibitors in the cells from old leaves, but intensified it in the cells from young leaves. 2-Deoxyglucose, an inhibitor of glycolysis, and propyl gallate, SOD-mimic and antioxidant, suppressed destruction of the nuclei that was caused by SOD inhibitors and glucose in cells of the epidermis from the young, but not from the old leaves. Glucose and trien stimulated, and propyl gallate reduced generation of reactive oxygen species (ROS) in the pea epidermis as determined by the fluorescence of 2',7'-dichlorofluorescein (DCF). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophoric uncoupler of oxidative and photosynthetic phosphorylation, suppressed the DCF fluorescence in the guard cells. Treatment of the cells with CCCP followed by its removal with washing increased destruction of the nuclei caused by SOD inhibitors and glucose. In young leaves, CCCP was less effective than in old ones. The findings demonstrate the effects of SOD inhibitors and glucose on the cell death and generation of ROS and could indicate glycolysis-dependent ROS production.

Parkinson\u2019s disease\u2013associated VPS35 mutant reduces mitochondrial membrane potential and impairs PINK1/Parkin-mediated mitophagy

BackgroundMitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson’s disease (PD), and several genes linked to familial PD, including PINK1 (encoding PTEN-induced putative kinase 1 [PINK1]) and PARK2 (encoding the E3 ubiquitin ligase Parkin), are directly involved in processes such as mitophagy that maintain mitochondrial health. The dominant p.D620N variant of vacuolar protein sorting 35 ortholog (VPS35) gene is also associated with familial PD but has not been functionally connected to PINK1 and PARK2.MethodsTo better mimic and study the patient situation, we used CRISPR-Cas9 to generate heterozygous human SH-SY5Y cells carrying the PD-associated D620N variant of VPS35. These cells were treated with a protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce the PINK1/Parkin-mediated mitophagy, which was assessed using biochemical and microscopy approaches.ResultsMitochondria in the VPS35-D620N cells exhibited reduced mitochondrial membrane potential and appeared to already be damaged at steady state. As a result, the mitochondria of these cells were desensitized to the CCCP-induced collapse in mitochondrial potential, as they displayed altered fragmentation and were unable to accumulate PINK1 at their surface upon this insult. Consequently, Parkin recruitment to the cell surface was inhibited and initiation of the PINK1/Parkin-dependent mitophagy was impaired.ConclusionOur findings extend the pool of evidence that the p.D620N mutation of VPS35 causes mitochondrial dysfunction and suggest a converging pathogenic mechanism among VPS35, PINK1 and Parkin in PD.

Protective effect of mitophagy against aluminum-induced MC3T3-E1 cells dysfunction

Aluminum (Al) is a ubiquitous environmental metal toxicant that causes osteoblast (OB) damage which leads to Al-related bone diseases. Mitochondrial damage plays a key role in Al-related bone diseases, and while mitophagy can clear damaged mitochondria and improve OB function, the relationship between mitophagy and Al-induced OB dysfunction is unknown. To explore the role of mitophagy in Al-induced OB dysfunction in vitro, we used 2 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 0.4 μM Cyclosporin A (CsA) to activate and inhibit mitophagy, respectively. MC3T3-E1 cells were treated with 0 mM AlCl3 (control group); 2 mM AlCl3 (Al group); 2 μM CCCP (CCCP group); 2 μM CCCP and 2 mM AlCl3 (CCCP + Al group); 0.4 μM CsA (CsA group); 0.4 μM CsA and 2 mM AlCl3 (CsA + Al group). The results showed that Al induced ultrastructural and functional impairment of MC3T3-E1 cells. Compared to the Al group, mitophagy activation caused mitochondrial membrane potentials to collapse, up-regulated PINK1, Parkin, and LC3 expression, down-regulated p62 expression, and increased mitophagosome numbers. Mitophagy activation also reduced Al-induced oxidative stress and MC3T3-E1 cell functional damage, as seen in improvement in cell viability, cellular calcium and phosphorus contents, and collagen I, osteocalcin, and bone alkaline phosphatase gene expression. Mitophagy inhibition had the opposite effects on activation. Overall, these results show that mitophagy can protect against Al-induced OB dysfunction.

Active-Targeting Polymeric Dual Nanosensor for Ratiometrically Measuring Proton and Oxygen Concentrations in Mitochondria

Dysfunction of mitochondria is closely related to neurodegenerative diseases, heart diseases, cancers, and so on. Because both proton and oxygen participate in vital biochemical reactions occurring in mitochondria such as adenosine triphosphate (ATP) generation, measuring proton and oxygen concentrations in mitochondria is therefore crucial for monitoring mitochondria activities and understanding cellular behavior. For this purpose, we developed a ratiometric fluorescent nanosensor for simultaneously sensing and imaging O2 and pH in mitochondria. The steps are as follows: (1) Styrene was copolymerized with 2-aminoethyl methacrylate hydrochloride to produce amino-functionalized polymer nanoparticles. (2) The reference dye rhodamine B isothiocyanate (RBITC) and oxygen-sensitive dye platinum(II) octaethylporphyrin (PtOEP) were encapsulated into a polymer sphere during polymerization, while the pH indicator fluorescein isothiocyanate (FITC) and mitochondrial-targeting molecule (3-carboxypropyl)triphenylphosphonium bromide (TPP) were further modified on the surface of the nanoparticles. The developed nanosensor shows a narrow distribution of particle size, high sensitivity toward O2 and pH, excellent stability, and low cytotoxicity. These remarkable features of the dual nanosensor render them capable of real-time sensing and imaging of O2 and pH in mitochondria with high spatial resolution. Applying the mitochondrial-targeted dual nanosensor in HeLa cells, we quantitatively measured and imaged mitochondrial proton and oxygen concentration variations after carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment.

Susceptibility to biocides and the prevalence of biocides resistance genes in clinical multidrug-resistant Pseudomonas aeruginosa isolates from Hamadan, Iran.

This study aimed to investigate the association between biocides' reduced susceptibility and the presence of efflux pump genes including cepA, qacEΔ1 and qacE in multidrug-resistant (MDR) Pseudomonas aeruginosa. The MDR P. aeruginosa isolates were collected and identified from different clinical samples. The minimum inhibitory concentrations (MIC) of four biocides (chlorhexidine gluconate 1%, benzalkonium chloride 1%, Kohrsolin® extra, and SEPTI-Turbo) were determined by microbroth dilution with and without carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Polymerase chain reaction (PCR) was performed for detecting the efflux pump genes. In total, 92 MDR P. aeruginosa isolates were collected. The reduced susceptibility (8-128µg/ml) was seen against chlorhexidine gluconate 1%, benzalkonium chloride 1%, Kohrsolin® extra, and SEPTI-Turbo in 63 (68.5%), 59 (64.1%), 64 (69.6%), and 65 (70.6%) isolates, respectively. The Kohrsolin® extra was the most effective biocide. The cepA, qacE, and qacEΔ1 were detected in 56 (60.9%), 1 (1.1%), and 34 (36.9%) isolates, respectively. There was a significant association between the presence of biocide resistance genes and reduced susceptibility to studied biocides (P = 0.00001). The CCCP had no effect on benzalkonium chloride 1% and Kohrsolin® extra, but reduced the MICs of chlorhexidine gluconate 1% and SEPTI- Turbo by 2 to 128 fold. The P. aeruginosa isolates exhibited varying degrees of tolerance to biocides. The cepA was the most prevalent gene. There was a significant connection between the occurrence of the efflux pump genes cepA and qacEΔ1 with reduced biocide susceptibility.

Effect of Titanium Dioxide Nanoparticles on the Expression of Efflux Pump and Quorum-Sensing Genes in MDR Pseudomonas aeruginosa Isolates.

Most of the infections caused by multi-drug resistant (MDR) P. aeruginosa strains are extremely difficult to be treated with conventional antibiotics. Biofilm formation and efflux pumps are recognized as the major antibiotic resistance mechanisms in MDR P. aeruginosa. Biofilm formation by P. aeruginosa depends mainly on the cell-to-cell communication quorum-sensing (QS) systems. Titanium dioxide nanoparticles (TDN) have been used as antimicrobial agents against several microorganisms but have not been reported as an anti-QS agent. This study aims to evaluate the impact of titanium dioxide nanoparticles (TDN) on QS and efflux pump genes expression in MDR P. aeruginosa isolates. The antimicrobial susceptibility of 25 P. aeruginosa isolates were performed by Kirby–Bauer disc diffusion. Titanium dioxide nanoparticles (TDN) were prepared by the sol gel method and characterized by different techniques (DLS, HR-TEM, XRD, and FTIR). The expression of efflux pumps in the MDR isolates was detected by the determination of MICs of different antibiotics in the presence and absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Biofilm formation and the antibiofilm activity of TDN were determined using the tissue culture plate method. The effects of TDN on the expression of QS genes and efflux pump genes were tested using real-time polymerase chain reaction (RT-PCR). The average size of the TDNs was 64.77 nm. It was found that TDN showed a significant reduction in biofilm formation (96%) and represented superior antibacterial activity against P. aeruginosa strains in comparison to titanium dioxide powder. In addition, the use of TDN alone or in combination with antibiotics resulted in significant downregulation of the efflux pump genes (MexY, MexB, MexA) and QS-regulated genes (lasR, lasI, rhll, rhlR, pqsA, pqsR) in comparison to the untreated isolate. TDN can increase the therapeutic efficacy of traditional antibiotics by affecting efflux pump expression and quorum-sensing genes controlling biofilm production.

Mitophagy Impairment Aggravates Cisplatin-Induced Ototoxicity.

Cisplatin is an efficacious anticancer agent, but its use is limited by ototoxicity and resultant irreversible sensorineural hearing loss. Cisplatin ototoxicity is associated with cochlear cell oxidative stress and mitochondrial damage. However, mitophagy is vital for maintaining mitochondrial quality and cellular metabolism. Accordingly, we investigated the role of mitophagy in regulating cisplatin-induced ototoxicity using the auditory cell line HEI-OC1. In this study, HEI-OC1 cells were treated with either cisplatin alone (10 μM, 0, 8, 16, and 24 h); cisplatin (10 μM, 24 h) post transfection with small-interfering (si)RNAs targeting mitophagy-associated mRNAs; cisplatin (10 μM, 24 h) succeeding pretreatment with the mitophagy suppressor, 3-methyladenine (3-MA; 5 or 10 mM, 6 h); or cisplatin (30 μM, 24 h) following pretreatment with the mitophagy promoter, carbonyl cyanide m-chlorophenylhydrazone (CCCP; 1 or 2 μM, 2 h). The viability of cells, expression of mitophagy marker, and mitochondrial functions were then assessed in these cells. Cell viability was determined by a water-soluble tetrazolium assay; expression of mitophagy-associated proteins PINK1, Parkin, BNIP3, FUNDC1, p62, and LC3B was analyzed by Western blotting, mitochondrial membrane potential by flow cytometry, intracellular ATP by spectrophotometry, and mitochondrial degradation by dual staining for mitochondria and autophagosomes or lysosomes. Our results showed that cisplatin gradually reduced the viable cell number over time, induced mitochondrial depolarization, decreased intracellular ATP concentration, and enhanced the expression of PINK1, Parkin, BNIP3, p62, and LC3B. In addition, Parkin and BNIP3 knockdown accelerated cisplatin-induced loss of cell viability, mitochondrial membrane potential, mitophagosome/lysosome formation, and reduction in intracellular ATP production. Pretreatment with 3-MA aggravated the cisplatin-induced cytotoxicity, while that with CCCP reversed this effect. Overall, our findings indicate that mitophagy protects HEI-OC1 cells against cisplatin-induced cell death. Consequently, we strongly believe that targeted promotion of mitophagy may confer protection against cisplatin-induced ototoxicity.

β-Naphthothiazolium-based ratiometric fluorescent probe with ideal pKa for pH imaging in mitochondria of living cells

The weakly alkaline microenvironment (pH ~8.0) in mitochondria plays a vital role in maintaining its morphology and function. Thus monitoring mitochondrial pH (pHmito) is of great significance. Herein, a ratiometric fluorescent probe (ENBT) for pHmito imaging in mitochondria of living cells is reported. pH variation closely correlates to intramolecular charge transfer (ICT) from naphthol to β-naphthothiazolium. ENBT exhibits a remarkable decrease on ratiometric fluorescence at λem1/λem2 = F595/F700 in response to pH variation within 6.30–9.29. In addition, ENBT has an ideal pKa value of 7.94 ± 0.08, which is advantageous in accurate sensing of pHmito. Moreover, ENBT has a Stokes shift of >150 nm, which effectively eliminates the potential interference from the excitation irradiation. ENBT shows excellent capability for specific staining of mitochondria with low cytotoxicity, which is most suitable for pHmito imaging in live cells. The probe was applied for monitoring pHmito variation in mitochondria of live cells caused by H2O2, NAC (N-Acetyl-l-cysteine), NH4Cl, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and lactate/pyruvate. The morphological alterations of mitochondria in living cells after treatment by CCCP were further evaluated.

CCCP‐induced mitochondrial dysfunction – characterization and analysis of integrated stress response to cellular signaling and homeostasis

Mitochondrial dysfunction mediated by CCCP (carbonyl cyanide m-chlorophenyl hydrazone), an inhibitor of mitochondrial oxidative phosphorylation, evokes the integrated stress response (ISR), which is analyzed here by eIF2α phosphorylation and expression profiles of ATF4 and CHOP proteins. Our findings suggest that the CCCP-induced ISR pathway is mediated by activation of HRI kinase, but not by GCN2, PERK, or PKR. Also, CCCP activates AMPK, a cellular energy sensor, and AKT, a regulator implicated in cell survival, and suppresses phosphorylation of mTORC1 substrates eIF4E-BP1 and S6K. CCCP also downregulates translation and promotes autophagy, leading to noncaspase-mediated cell death in HepG2 cells. All these events are neutralized by NAC, an anti-ROS, suggesting that CCCP-induced mitochondrial dysfunction promotes oxidative stress. ISRIB, an inhibitor of the ISR pathway, mitigates CCCP-induced expression of ATF4 and CHOP, activation of AKT, and autophagy, similar to NAC. However, it fails to reverse CCCP-induced AMPK activation, suggesting that CCCP-induced autophagy is dependent on ISR and independent of AMPK activation. ISRIB restores partly, inhibition in eIF4E-BP1 phosphorylation, promotes eIF2α phosphorylation, albeit slowly, and mitigates suppression of translation accordingly, in CCCP-treated cells. These findings are consistent with the idea that CCCP-induced oxidative stress leading to eIF2α phosphorylation and ATF4 expression, which is known to stimulate genes involved in autophagy, play a pro-survival role together with AKT activation and regulate mTOR-mediated eIF4E-BP1 phosphorylation.

Bacteria Make a Living Breathing the Nitroheterocyclic Insensitive Munitions Compound 3-Nitro-1,2,4-triazol-5-one (NTO).

The nitroheterocyclic 3-nitro-1,2,4-triazol-5-one (NTO) is an ingredient of insensitive explosives increasingly used by the military, becoming an emergent environmental pollutant. Cometabolic biotransformation of NTO occurs in mixed microbial cultures in soils and sludges with excess electron-donating substrates. Herein, we present the unusual energy-yielding metabolic process of NTO respiration, in which the NTO reduction to 3-amino-1,2,4-triazol-5-one (ATO) is linked to the anoxic acetate oxidation to CO2 by a culture enriched from municipal anaerobic digester sludge. Cell growth was observed simultaneously with NTO reduction, whereas the culture was unable to grow in the presence of acetate only. Extremely low concentrations (0.06 mg L-1) of the uncoupler carbonyl cyanide m-chlorophenyl hydrazone inhibited NTO reduction, indicating that the process was linked to respiration. The ultimate evidence of NTO respiration was adenosine triphosphate production due to simultaneous exposure to NTO and acetate. Metagenome sequencing revealed that the main microorganisms (and relative abundances) were Geobacter anodireducens (89.3%) and Thauera sp. (5.5%). This study is the first description of a nitroheterocyclic compound being reduced by anaerobic respiration, shedding light on creative microbial processes that enable bacteria to make a living reducing NTO.

Effect of metabolic uncouplers on the performance of toluene-degrading biotrickling filter.

The biomass control potential of three metabolic uncouplers (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), carbonyl cyanide m-chlorophenylhydrazone (CCCP), and m-chlorophenol (m-CP)) was tested in biotrickling filters (BTFs) degrading toluene. The experiments employed two types of reactors: a traditional column design and a novel differential BTF (DBTF) reactor developed by De Vela and Gostomski (J Environ Eng 147:04020159, 2021). Uncouplers caused the toluene elimination capacity (EC) (~33 g/m3h for column reactors and ~600 g/m3h for DBTF) to decrease by 15-97% in a dose-dependent fashion. The EC completely recovered in the column reactor in 3 to 13 days, while only partial recovery happened in the DBTF. Short-term (1 to 3 days) true uncoupling was indicated by the 20-160% increase in %CO2 recovery, depending on concentration. FCCP and CCCP increased the pressure drop due to increased extracellular polymeric substances (EPS) production for protection against the uncouplers. The 4.0-mM m-CP weakened the biofilm in the BTF bed, as evidenced by the 130-500% increase in the total organic carbon in the liquid sump of the column and DBTF reactors. Moreover, a microbial shift led to the proliferation of genera that degrade uncouplers, further demonstrating that the uncouplers tested were not a sustainable biomass control strategy in BTFs.

Effect of membrane potential on entry of lactoferricin B-derived 6-residue antimicrobial peptide into single Escherichia coli cells and lipid vesicles.

The antimicrobial peptide (AMP) derived from lactoferricin B, LfcinB (4-9) (RRWQWR), and lissamine rhodamine B red-labeled peptide (Rh-LfcinB (4-9)) exhibit strong antimicrobial activities, and they can enter Escherichia coli cells without damaging the cell membranes. Thus, these peptides are cell-penetrating peptide (CPP) -type AMPs. In this study, to elucidate the effect of the membrane potential (Δφ) on the action of the CPP-type AMP, Rh-LfcinB (4-9), we investigated the interactions of Rh-LfcinB (4-9) with single E. coli cells and spheroplasts containing calcein in the cytosol using confocal laser scanning microscopy. At low peptide concentrations, Rh-LfcinB (4-9) entered the cytosol of single E. coli cells and spheroplasts without damaging the cell membranes, and the H+-ionophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) suppressed its entry. The studies using the time-kill method indicate that these low concentrations of peptide exhibit antimicrobial activity but CCCP inhibits this activity. Next, we investigated the effect of Δφ on the interaction of Rh-LfcinB (4-9) with single giant unilamellar vesicles (GUVs) comprising E. coli polar lipid extracts and containing a fluorescent probe, Alexa Fluor 647 hydrazide. At low concentrations (0.2-0.5 μM), Rh-LfcinB (4-9) showed significant entry to the single GUV lumen without pore formation in the presence of Δφ. The fraction of entry of peptide increased with increasing negative membrane potential, indicating that the rate of peptide entry into the GUV lumen increased with increasing negative membrane potential. These results indicate that Δφ enhances the entry of Rh-LfcinB (4-9) into single E. coli cells, spheroplasts, and GUVs and its antimicrobial activity.IMPORTANCE: Bacterial cells have a membrane potential (Δφ), but the effect of Δφ on action of cell-penetrating peptide-type antimicrobial peptides (AMPs) is not clear. Here, we investigated the effect of Δφ on the action of fluorescent probe-labeled AMP derived from lactoferricin B, Rh-LfcinB (4-9). At low peptide concentrations, Rh-LfcinB (4-9) enters the cytosol of Escherichia coli cells and spheroplasts without damaging their cell membrane, but a protonophore suppresses this entry and its antimicrobial activity. The rate of entry of Rh-LfcinB (4-9) into the giant unilamellar vesicles (GUVs) comprising E. coli lipids without pore formation increases with increasing Δφ. These results indicate that Δφ enhances the antimicrobial activity of Rh-LfcinB (4-9) and hence LfcinB (4-9) by increasing the rate of their entry into the cytosol.