Duck plague virus (DPV) is extremely infectious and lethal, so antiviral drugs are urgently needed. Our previous study shows that DPV infection with duck embryo fibroblast (DEF) induces reactive oxygen species (ROS) changes and promotes apoptosis. In this study, we tested the antiviral effect of the carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a common mitochondrial autophagy inducer. Our results demonstrated a dose-dependent anti-DPV effect of CCCP, CCCP-treatment blocked the intercellular transmission of DPV after infection, and we also proved that CCCP could have an antiviral effect up to 48 hpi. The addition of CCCP reversed the DPV-induced ROS changes, CCCP can inhibit virus-induced apoptosis; meanwhile, CCCP can affect mitochondrial fusion and activate mitophagy to inhibit DPV. In conclusion, CCCP can be an effective antiviral candidate against DPV.

Improving the efficacy of existing antibiotics is significant for combatting antibiotic resistance that poses a major threat to human health. Carbonyl cyanide m-chlorophenylhydrazine (CCCP), a well-known protonophore for dissipating proton motive force (PMF), has been widely used to block the PMF-dependent uptake of aminoglycoside antibiotics and thus suppress aminoglycoside lethality. Here, we report that CCCP and its functional analog FCCP, but not other types of protonophores, unprecedently potentiate aminoglycosides (e.g., tobramycin and gentamicin) by 3-4 orders of magnitude killing of Escherichia coli, Staphylococcus aureus, Shigella flexneri, and Vibrio alginolyticus cells in stationary phase but not these cells in exponential phase nor other 12 bacterial species we examined. Overall, the effect of CCCP on aminoglycoside lethality undergoes a gradual transition from suppression against E. coli exponential-phase cells to potentiation against late stationary-phase cells, with the cell growth status and culture medium being crucial. Consistently, disturbance of the PMF by changing transmembrane proton gradient (ΔpH) or electric potential (ΔΨ) also potentiates tobramycin. Nevertheless, CCCP neither increases the intracellular concentration of tobramycin nor decreases the MIC of the antibiotic, thus excluding that CCCP acts as an efflux pump inhibitor to potentiate aminoglycosides. Rather, we show that the combined treatment dramatically enhances the cellular level of hydroxyl radical under both aerobic and anaerobic culturing conditions, under which the antioxidant N-acetyl cysteine fully suppresses both hydroxyl radical accumulation and cell death. Together, these findings open a new avenue to develop certain protonophores as aminoglycoside adjuvants against pathogens in stationary phase and also illustrate an essential role of hydroxyl radical in aminoglycoside lethality regardless of aerobic respiration.

Mitochondrial selective autophagy, known as mitophagy, surveils the mitochondrial population by eliminating superfluous and/or impaired organelles to mediate cellular survival and viability in response to injury/trauma and infection. In this study, the components of the mitophagy pathway in the Pacific oyster Crassostrea gigas were screened from NCBI with reference to the protein sequences of the human mitophagy process. A total of 10 mitophagy process-related genes were identified from C. gigas, including NIX, FUNDC1, PHB2, Cardiolipin, P62, VDAC2, MFN2, PARL, MPP, and OPTN. They shared high similarities with their homologs in the human mitophagy pathway and were expressed in various tissues of C. gigas. After CCCP exposure, the fluorescence intensity of the mitochondrial probe JC-1 monomers increased significantly in hemocytes, while the fluorescence intensity of JC-1 aggregates decreased significantly. Meanwhile, the fluorescence of lysosomes was found to be co-localized with that of CgLC3 and mitochondria in CCCP-treated hemocytes. Double- and single-membrane-bound vacuoles resembling autophagic structures were observed in the hemocytes after CCCP exposure. The fluorescence intensity of JC-1 monomers and the abundance of CgLC3Ⅱ in hemocytes both increased after Vibrio splendidus exposure. At the same time, the green signals of CgLC3 were co-localized with red signals of the mitochondria, and the fluorescence intensity of autophagy increased significantly in hemocytes after V. splendidus exposure. The results confirmed the existence of a complete mitophagy pathway in mollusks for the first time, which was helpful for further study on the function of mitochondrial autophagy in mollusks.

The retinal mitochondrial injury model in rat has been developed using the mitochondrial oxidative phosphorylation uncoupler, carbonylcyanide m-chlorophenyl hydrazine (CCCP). However, the CCCP-induced murine retinal mitochondrial injury model has not been reported. Here, the optimized conditions for the murine retinal mitochondrial injury model were established by intravitreal injection of different doses of CCCP (0, 2.5, 5, 7.5, 10, 12.5, 15 μg). Indeed, it has been reported that CCCP induces Opa1 cleavage and phosphorylation of ERK in cultured cells and rat retinas. Thus, we measured phosphorylated (p) -Erk and L/S-Opa1 following CCCP-induced retinal injury. Meanwhile, KW6002 (A2A receptor antagonist) pretreatment inhibited retinal injury induced by CCCP at 10 and 15 μg doses differently. Intravitreal injection of 10 μg doses of CCCP can induce apoptosis of retinal ganglion cells and decrease of retinal thickness, but intravitreal injection of 15 μg doses of CCCP is the appropriate dose to study the protective effect of A2A receptor. (1) Dose dependent effects of intravitreal injection of CCCP on the levels of L/S-Opa1 and p-Erk; (2) A2A receptor antagonist (KW6002) only inhibited the apoptosis of ganglion cells, but did not affect the thickness of retina with 10µg dosage of CCCP intravitreal injection; (3) A2A receptor antagonist (KW6002) inhibited the apoptosis of ganglion cells and increased the thickness of retina with 15µg dosage of CCCP intravitreal injection.

UNASSIGNED: Colistin is one of the few options for treating carbapenem-resistant Enterobacterales (CREs). There is little available information about the epidemic status of colistin-resistant CREs in Southern Sichuan, China. This study mainly investigated the genomic and phenotypic characteristics of an extensively drug resistant E. coli LZ00114 isolated from Luzhou, China.
UNASSIGNED: In 2020, LZ00114 was isolated from the urine of a patient with hydronephrosis and urinary tract infection in Luzhou, China. We assessed the resistance profile of LZ00114 in the presence and absence of the protonophore carbonyl cyano-m-chlorophenylhydrazine (CCCP) and 1-(1-naphthylmethyl)-piperazine (NMP) by antimicrobial susceptibility testing. The growth kinetics, motility, and pathogenicity of LZ00114 were determined to evaluate its microbial characteristics. In combination with whole genome sequencing (WGS) and real-time reverse transcription PCR (RT-PCR), we comprehensively analyzed the resistance mechanisms of LZ00114.
UNASSIGNED: LZ00114 was resistant to various antimicrobial agents, including meropenem, tetracycline, ciprofloxacin, gentamicin, fosfomycin, and polymyxin B. Notably, CCCP reversed the resistance of LZ00114 to polymyxin B. LZ00114 displayed high pathogenicity in the infection model (P<0.01) compared with the Lab-WT strain, and its growth rate and motility were not significantly different from the Lab-WT strain. WGS and conjugation revealed that LZ00114 belonged to ST410 and carried a bla NDM-5-harboring self-transmissible IncX3 plasmid and a multi-replicon IncFII/FIA/FIB plasmid carrying bla OXA-1-bla CTX-M-55-tet(B)-aac(6\')-Ib-cr-dfrA17-sul1-fosA3. Comparative genomics revealed genetic relatedness between LZ00114 and strains isolated from other regions. Furthermore, there were point mutations in pmrA (S29G, G144S), pmrB (D283G, Y358N), marR (G103S, Y137H), emrA (I219V), and emrD (G323D) of LZ00114. RT-PCR confirmed the overexpression of efflux pumps and PmrABC in LZ00114.
UNASSIGNED: This study provides valuable information for the surveillance of antimicrobial resistance and a theoretical basis for the prevention and control of colistin-resistant E. coli. There is still a need to be vigilant about the clone spread of the high-risk clone group E. coli ST410.

Blockade of adenosine A2A receptors (A2ARs) protects against neuronal damage caused by various brain insults including mitochondrial toxicity, but the precise neuroprotective mechanisms are unclear. Here, we studied the effects of the A2AR antagonist KW6002 on retinal injury induced by the mitochondrial oxidative phosphorylation uncoupler, carbonylcyanide m-chlorophenyl hydrazine (CCCP) and alterations in competing endogenous RNA (ceRNA) network. We found that KW6002 treatment partially reversed CCCP-induced reduction in retinal thickness and retinal ganglia cell number by increasing mitochondrial content and reducing retinal ganglia cells apoptosis. Furthermore, we employed whole-transcriptome sequencing to explore ceRNA network changes associated with CCCP-induced retinal injury and its reversal by KW6002. This analysis revealed that A2AR blockade reduced the number of CCCP-induced microRNAs by ∼60%, but increased the number of CCCP-induced circular RNAs by ∼50%. Among CeRNA network changes, CCCP-induced retinal injury was associated with a possible enrichment of the tumor necrosis factor signaling pathway and its related 126 microRNAs, 237 long non-coding RNAs, 58 circular RNAs competing. Moreover, the A2AR antagonist-mediated protection against CCCP-induced retinal injury was possibly associated with the up-regulation of mature brain-derived neurotrophic factor and its related 4 microRNAs competed by 43 long non-coding RNAs and 9 circular RNAs competing. These ceRNA network alterations by CCCP treatment and its reversal by A2AR antagonist may contribute to understanding the transcriptome mechanism for protection against CCCP-induced retinal injury by A2AR antagonists.

Hyperglycemia increases the risk of corneal endothelial dysfunction, resulting in damage to corneal endothelial structure and function. However, the effect and mechanism of hyperglycemia-induced corneal endothelial damage remain elusive. In this study, we demonstrated that hyperglycemia reduced the expression of pump-related protein Na+/K+ ATPase and barrier-related protein ZO-1. Moreover, we found hyperglycemia caused abnormal changes of morphological mitochondria and dynamics in vitro. In addition, the decreased levels of mitophagy were further confirmed Western blotting and LC3B-Mitotracker Immunofluorescence. Exogenous application of mitophagy agonist carbonyl cyanide m-chlorophenyl hydrazine (CCCP) increases the expression of Na+/K+ ATPase and ZO-1 in corneal endothelial cells through up-regulated mitophagy in vitro. In addition, CCCP effectively reverses the phenomenon of corneal opacity and increased corneal thickness in diabetic mice. Therefore, our demonstrated the novel function of mitophagy in the pathogenesis of diabetic cornea endothelial dysfunction, and provide potential approach for treating diabetic corneal endothelial dysfunction.

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This study aimed to identify the role and relationship with efflux pump of biofilm formation in Klebsiella pneumoniae. Sixty-one K. pneumoniae clinical isolates were collected between January and June of 2017 from the affiliated hospital of southwest medical university in Luzhou, China. The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) were determined using broth microdilution method. Crystal violet (CV) staining and confocal laser scanning microscope (CLSM) were used to monitor biofilm formation. Efflux pump expression was investigated qualitatively and quantitatively by polymerase chain reaction (PCR) and reverse transcriptase quantitative PCR (RT-qPCR). Crystal violet staining was performed to evaluate the effect of efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazine (CCCP) on K. pneumoniae biofilms. Our results showed that crystal violet staining and CLSM had good consistency in biofilm detection. Biofilm formation was an independent biological behavior of the strain and measured at 24 h was reasonable. Biofilms up-regulated antimicrobial resistance and expression of efflux pump gene acrA, emrB, oqxA, and qacEΔ1 in K. pneumoniae. CCCP inhibited biofilms but dose-dependent effect was obvious. Altogether, our data demonstrates that biofilm formation, as well as its interaction with efflux pump, promotes antimicrobial resistance in K. pneumoniae.

Recent studies show that mitochondria and actin filaments work together in two contexts: (1) increased cytoplasmic calcium induces cytoplasmic actin polymerization that stimulates mitochondrial fission and (2) mitochondrial depolarization causes actin assembly around mitochondria, with roles in mitophagy. It is unclear whether these two processes utilize similar actin assembly mechanisms. Here, we show that these are distinct actin assembly mechanisms in the acute phase after treatment (<10 min). Calcium-induced actin assembly is INF2 dependent and Arp2/3 complex independent, whereas depolarization-induced actin assembly is Arp2/3 complex dependent and INF2 independent. The two types of actin polymerization are morphologically distinct, with calcium-induced filaments throughout the cytosol and depolarization-induced filaments as \'clouds\' around depolarized mitochondria. We have previously shown that calcium-induced actin stimulates increases in both mitochondrial calcium and recruitment of the dynamin GTPase Drp1 (also known as DNM1L). In contrast, depolarization-induced actin is temporally associated with extensive mitochondrial dynamics that do not result in mitochondrial fission, but in circularization of the inner mitochondrial membrane (IMM). These dynamics are dependent on the protease OMA1 and independent of Drp1. Actin cloud inhibition causes increased IMM circularization, suggesting that actin clouds limit these dynamics.This article has an associated First Person interview with the first author of the paper.