Elevated Membrane Potential as a Tetracycline Resistance Mechanism in Escherichia coli.

The metabolic environment is responsible for antibiotic resistance, which highlights the way in which the antibiotic resistance mechanism works. Here, GC-MS-based metabolomics with iTRAQ-based proteomics was used to characterize a metabolic state in tetracycline-resistant Escherichia coli K12 (E. coli-RTET) compared with tetracycline-sensitive E. coli K12. The repressed pyruvate cycle against the elevation of the proton motive force (PMF) and ATP constructed the most characteristic feature as a consequence of tetracycline resistance. To understand the role of the elevated PMF in tetracycline resistance, PMF inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the pH gradient were used to investigate how the elevation influences bacterial viability and intracellular antibiotic concentration. A strong synergy was detected between CCCP and tetracycline to the viability, which was consistent with increasing intracellular drug and decreasing external pH. Furthermore, E. coli-RTET and E. coli-RGEN with high and low PMF concentrations were susceptible to gentamicin and tetracycline, respectively. The elevated PMF in E. coli-RTET was attributed to the activation of other metabolic pathways, except for the pyruvate cycle, including a malate-oxaloacetate-phosphoenolpyruvate-pyruvate-malate cycle. These results not only revealed a PMF-dependent mechanism for tetracycline resistance but also provided a solution to tetracycline-resistant pathogens by aminoglycosides and aminoglycoside-resistant bacteria by tetracyclines.

Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation.

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KEY POINTS: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage.

Combination of bone marrow mesenchymal stem cells and moxibustion restores cyclophosphamide-induced premature ovarian insufficiency by improving mitochondrial function and regulating mitophagy.

BACKGROUND: Premature ovarian insufficiency (POI) is a major cause of infertility. In this study, we aimed to investigate the effects of the combination of bone marrow mesenchymal stem cells (BMSCs) and moxibustion (BMSCs-MOX) on POI and evaluate the underlying mechanisms. METHODS: A POI rat model was established by injecting different doses of cyclophosphamide (Cy). The modeling of POI and the effects of the treatments were assessed by evaluating estrous cycle, serum hormone levels, ovarian weight, ovarian index, and ovarian histopathological analysis. The effects of moxibustion on BMSCs migration were evaluated by tracking DiR-labeled BMSCs and analyzing the expression of chemokines stromal cell-derived factor 1 (Sdf1) and chemokine receptor type 4 (Cxcr4). Mitochondrial function and mitophagy were assessed by measuring the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, and the mitophagy markers (Drp1, Pink1, and Parkin). Furthermore, the mitophagy inhibitor Mdivi-1 and the mitophagy activator CCCP were used to confirm the role of mitophagy in Cy-induced ovarian injury and the underlying mechanism of combination therapy. RESULTS: A suitable rat model of POI was established using Cy injection. Compared to moxibustion or BMSCs transplantation alone, BMSCs-MOX showed improved outcomes, such as reduced estrous cycle disorders, improved ovarian weight and index, normalized serum hormone levels, increased ovarian reserve, and reduced follicle atresia. Moxibustion enhanced Sdf1 and Cxcr4 expression, promoting BMSCs migration. BMSCs-MOX reduced ROS levels; upregulated MMP and ATP levels in ovarian granulosa cells (GCs); and downregulated Drp1, Pink1, and Parkin expression in ovarian tissues. Mdivi-1 significantly mitigated mitochondrial dysfunction in ovarian GCs and improved ovarian function. CCCP inhibited the ability of BMSCs-MOX treatment to regulate mitophagy and ameliorate Cy-induced ovarian injury. CONCLUSIONS: Moxibustion enhanced the migration and homing of BMSCs following transplantation and improves their ability to repair ovarian damage. The combination of BMSCs and moxibustion effectively reduced the excessive activation of mitophagy, which helped prevent mitochondrial damage, ultimately improving ovarian function. These findings provide a novel approach for the treatment of pathological ovarian aging and offer new insights into enhancing the efficacy of stem cell therapy for POI patients.

Efflux pump effects on levofloxacin resistance in Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.

Repurposing approved drugs as potential efflux pump inhibitors in multidrug-resistant Pseudomonas aeruginosa.

Aim: To evaluate the action of promethazine, fluoxetine and carbonyl cyanide 3-chlorophenylhydrazone as efflux pump inhibitors (EPIs) against multidrug-resistant Pseudomonas aeruginosa. Methods: The effect of the compounds was evaluated in planktonic cells and bacterial biofilms. Accumulation tests were performed with ethidium bromide to prove their action as EPIs. Then, they were associated with antimicrobials. Results: Effect on planktonic cells and biofilms was found. Assays with ethidium bromide indicate their action as EPIs. Significant reductions in the metabolic activity of biofilms were observed after the association with the antimicrobials, especially for meropenem. Conclusion: It is possible to prove the action of these compounds as EPIs for P. aeruginosa and demonstrate the relevance of efflux pumps in antimicrobial resistance.

[Box: see text].

Enhancing bactericidal activities of ciprofloxacin by targeting the trans-translation system that is involved in stress responses in Klebsiella pneumoniae.

Although the trans-translation system is a promising target for antcibiotic development, its antibacterial mechanism in Klebsiella pneumoniae (KP) is unclear. Considering that tmRNA was the core component of trans-translation, this study firstly investigated phenotypic changes caused by various environmental stresses in KP lacking trans-translation activities (tmRNA-deleted), and then aimed to evaluate antibacterial activities of the trans-translation-targeting antibiotic combination (tobramycin/ciprofloxacin) in clinical KP isolates based on inhibition activities of aminoglycosides against trans-translation. We found that the tmRNA-deleted strain P4325/ΔssrA was significantly more susceptible than the wild-type KP strain P4325 under environments with hypertonicity (0.5 and 1 M NaCl), hydrogen peroxide (40 mM), and UV irradiation. No significant differences in biofilm formation and survivals under human serum were observed between P4325/ΔssrA and P4325. tmRNA deletion caused twofold lower MIC values for aminoglycosides. As for the membrane permeability, tmRNA deletion increased ethidium bromide (EtBr) uptake of KP in the presence or absence of verapamil and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), decreased EtBr uptake in presence of reserpine in P4325/ΔssrA, and reduced EtBr efflux in P4325/ΔssrA in the presence of CCCP. The time-kill curve and in vitro experiments revealed significant bactericidal activities of the tmRNA-targeting aminoglycoside-based antibiotic combination (tobramycin/ciprofloxacin). Thus, the corresponding tmRNA-targeting antibiotic combinations (aminoglycoside-based) might be effective and promising treatment options against multi-drug resistant KP.

CCCP inhibits DPV infection in DEF cells by attenuating DPV manipulated ROS, apoptosis, and mitochondrial stability.

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.

Divergent Roles of Escherichia Coli Encoded Lon Protease in Imparting Resistance to Uncouplers of Oxidative Phosphorylation: Roles of marA, rob, soxS and acrB.

Uncouplers of oxidative phosphorylation dissipate the proton gradient, causing lower ATP production. Bacteria encounter several non-classical uncouplers in the environment, leading to stress-induced adaptations. Here, we addressed the molecular mechanisms responsible for the effects of uncouplers in Escherichia coli. The expression and functions of genes involved in phenotypic antibiotic resistance were studied using three compounds: two strong uncouplers, i.e., Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-Dinitrophenol (DNP), and one moderate uncoupler, i.e., Sodium salicylate (NaSal). Quantitative expression studies demonstrated induction of transcripts encoding marA, soxS and acrB with NaSal and DNP, but not CCCP. Since MarA and SoxS are degraded by the Lon protease, we investigated the roles of Lon using a lon-deficient strain (Δlon). Compared to the wild-type strain, Δlon shows compromised growth upon exposure to NaSal or 2, 4-DNP. This sensitivity is dependent on marA but not rob and soxS. On the other hand, the Δlon strain shows enhanced growth in the presence of CCCP, which is dependent on acrB. Interestingly, NaSal and 2,4-DNP, but not CCCP, induce resistance to antibiotics, such as ciprofloxacin and tetracycline. This study addresses the effects of uncouplers and the roles of genes involved during bacterial growth and phenotypic antibiotic resistance. Strong uncouplers are often used to treat wastewater, and these results shed light on the possible mechanisms by which bacteria respond to uncouplers. Also, the rampant usage of some uncouplers to treat wastewater may lead to the development of antibiotic resistance.

Knockout of adenylosuccinate synthase purA increases susceptibility to colistin in Escherichia coli.

Colistin is a cationic cyclic antimicrobial peptide used as a last resort against multidrug-resistant gram-negative bacteria. To understand the factors involved in colistin susceptibility, we screened colistin-sensitive mutants from an E. coli gene-knockout library (Keio collection). The knockout of purA, whose product catalyzes the synthesis of adenylosuccinate from IMP in the de novo purine synthesis pathway, resulted in increased sensitivity to colistin. Adenylosuccinate is subsequently converted to AMP, which is phosphorylated to produce ADP, a substrate for ATP synthesis. The amount of ATP was lower in the purA-knockout mutant than that in the wild-type strain. ATP synthesis is coupled with proton transfer, and it contributes to the membrane potential. Using the membrane potential probe, 3,3'-diethyloxacarbocyanine iodide [DiOC2(3)], we found that the membrane was hyperpolarized in the purA-knockout mutant compared to that in the wild-type strain. Treatment with the proton uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), abolished the hyperpolarization and colistin sensitivity in the mutant. The purA-knockout mutant exhibited increased sensitivity to aminoglycosides, kanamycin, and gentamicin; their uptake requires a membrane potential. Therefore, the knockout of purA, an adenylosuccinate synthase, decreases ATP synthesis concurrently with membrane hyperpolarization, resulting in increased sensitivity to colistin.

Cultured Rat Hippocampal Neurons Exposed to the Mitochondrial Uncoupler Carbonyl Cyanide Chlorophenylhydrazone Undergo a Rapid, Presenilin-Dependent Change in Neuronal Properties.

Presenilin 1 (PS1) is a transmembrane proteolytic subunit of γ-secretase that cleaves amyloid precursor proteins. Mutations in PS1 (mPS1) are associated with early-onset familial Alzheimer's disease (AD). The link between mutated PS1, mitochondrial calcium regulation, and AD has been studied extensively in different test systems. Despite the wide-ranging role of mPS1 in AD, there is a paucity of information on the link between PS1 and neuronal cell death, a hallmark of AD. In the present study, we employed the selective mitochondrial uncoupler carbonyl cyanide chlorophenylhydrazone (CCCP) and compared the reactivity of mPS1-transfected cultured rat hippocampal neurons with PS1 and control neurons in a situation of impaired mitochondrial functions. CCCP causes a slow rise in cytosolic and mitochondrial calcium in all three groups of neurons, with the mPS1 neurons demonstrating a faster rise. Consequently, mPS1 neurons were depolarized by CCCP and measured with TMRM, a mitochondrial voltage indicator, more than the other two groups. Morphologically, CCCP produced more filopodia in mPS1 neurons than in the other two groups, which were similarly affected by the drug. Finally, mPS1 transfected neurons tended to die from prolonged exposure to CCCP sooner than the other groups, indicating an increase in vulnerability associated with a lower ability to regulate excess cytosolic calcium.

Chaihu Shugan Powder inhibits interstitial cells of cajal mitophagy through USP30 in the treatment of functional dyspepsia.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaihu Shugan Powder (CHSGP) has significant clinical efficacy in the treatment of functional dyspepsia (FD), but the specific mechanism requires further study. AIM OF STUDY: The aim of this study was to investigate the therapeutic effect of CHSGP on FD rats and the underlying mechanism of the effect on interstitial cells of cajal (ICC) mitophagy. MATERIALS AND METHODS: The tail-clamping stimulation method was utilized to establish an FD rat model in vivo. Gastric emptying rate and small intestinal propulsion rate test, H&E staining, and Immunohistochemistry were conducted to evaluate the therapeutic effects of CHSGP on FD rats. In vitro, the regulatory effect of CHSGP on CCCP-mediated ICC mitophagy was further investigated by CCK8, Transmission electron microscope, immunofluorescence co-staining, Quantitative polymerase chain reaction and Western blot to reveal the potential mechanisms of CHSGP inhibited ICC mitophagy. RESULTS: Animal experiments provided evidence that CHSGP promoted gastric motility, increased ICC numbers, reduced Parkin expression, and elevated USP30 expression in FD rats. In vitro, further mechanism research demonstrated that CHSGP decreased LC3Ⅱ/LC3Ⅰ、PINK1、Parkin、PHB2 protein expression and increased USP30 protein expression. Furthermore, CHSGP increased Mfn2 protein expression by suppressing activation of the PINK1/Parkin pathway when USP30 is knocked down, consequently reducing CCCP-induced ICC mitophagy. CONCLUSIONS: These results suggest that CHSGP may treat FD against CCCP-induced ICC mitophagy by the up-regulation of via PINK1/Parkin pathway.

Varicella zoster virus glycoprotein E facilitates PINK1/Parkin-mediated mitophagy to evade STING and MAVS-mediated antiviral innate immunity.

Viruses have evolved to control mitochondrial quality and content to facilitate viral replication. Mitophagy is a selective autophagy, in which the damaged or unnecessary mitochondria are removed, and thus considered an essential mechanism for mitochondrial quality control. Although mitophagy manipulation by several RNA viruses has recently been reported, the effect of mitophagy regulation by varicella zoster virus (VZV) remains to be fully determined. In this study, we showed that dynamin-related protein-1 (DRP1)-mediated mitochondrial fission and subsequent PINK1/Parkin-dependent mitophagy were triggered during VZV infection, facilitating VZV replication. In addition, VZV glycoprotein E (gE) promoted PINK1/Parkin-mediated mitophagy by interacting with LC3 and upregulating mitochondrial reactive oxygen species. Importantly, VZV gE inhibited MAVS oligomerization and STING translocation to disrupt MAVS- and STING-mediated interferon (IFN) responses, and PINK1/Parkin-mediated mitophagy was required for VZV gE-mediated inhibition of IFN production. Similarly, carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-mediated mitophagy induction led to increased VZV replication but attenuated IFN production in a three-dimensional human skin organ culture model. Our results provide new insights into the immune evasion mechanism of VZV gE via PINK1/Parkin-dependent mitophagy.

USP30 inhibition induces mitophagy and reduces oxidative stress in parkin-deficient human neurons.

Ubiquitination of mitochondrial proteins plays an important role in the cellular regulation of mitophagy. The E3 ubiquitin ligase parkin (encoded by PARK2) and the ubiquitin-specific protease 30 (USP30) have both been reported to regulate the ubiquitination of outer mitochondrial proteins and thereby mitophagy. Loss of E3 ligase activity is thought to be pathogenic in both sporadic and inherited Parkinson's disease (PD), with loss-of-function mutations in PARK2 being the most frequent cause of autosomal recessive PD. The aim of the present study was to evaluate whether mitophagy induced by USP30 inhibition provides a functional rescue in isogenic human induced pluripotent stem cell-derived dopaminergic neurons with and without PARK2 knockout (KO). Our data show that healthy neurons responded to CCCP-induced mitochondrial damage by clearing the impaired mitochondria and that this process was accelerated by USP30 inhibition. Parkin-deficient neurons showed an impaired mitophagic response to the CCCP challenge, although mitochondrial ubiquitination was enhanced. USP30 inhibition promoted mitophagy in PARK2 KO neurons, independently of whether left in basal conditions or treated with CCCP. In PARK2 KO, as in control neurons, USP30 inhibition balanced oxidative stress levels by reducing excessive production of reactive oxygen species. Interestingly, non-dopaminergic neurons were the main driver of the beneficial effects of USP30 inhibition. Our findings demonstrate that USP30 inhibition is a promising approach to boost mitophagy and improve cellular health, also in parkin-deficient cells, and support the potential relevance of USP30 inhibitors as a novel therapeutic approach in diseases with a need to combat neuronal stress mediated by impaired mitochondria.

Investigation of Mitochondrial Homeostasis Changes in Lens Epithelium of High-Myopic Cataract.

PURPOSE: High myopia is demonstrated as a pathogenic factor for nuclear cataract. The main mechanism of high-myopia cataracts (HMC) is oxidative damage, which causes mitochondrial homeostasis imbalance. This study aimed to explore the mitochondrial homeostasis alterations in lens epithelial cells (LECs) of HMC. METHODS: The lens epithelium tissues of 20 patients with HMC and 20 control subjects with age-related cataracts (ARC) were collected. The real-time quantitative PCR and western blot assays were performed for gene expressions. Immunofluorescence (IF) assays were performed for mitochondrial marker TOM20, DNA damage marker 15A3, and autophagosome marker LC3. Transmission electron microscopy (TEM) was used to observe the changes in mitochondria morphology. Mitochondrial ROS, and mitochondrial membrane potential were detected by MitoSOX fluorescence, and JC-1 MitoMP staining, respectively. Rat lenses cultured in vitro were pretreated with CCCP and H2O2 (10 and 400 µM) for 24 h. RESULTS: The copy number of mtDNA was decreased in HMC patients compared to the ARC patients. Increased mitochondrial-oriented oxidative stress response was detected in LECs of HMC compared to that of ARC. Altered expressions of mitochondrial homeostasis and mitophagy markers, including TFAM, PGC1α, MFN1, MFN2, Drp1, PINK1, Parkin and LC3, were found in HMC patients. Reciprocally, no significant differences in the expression of BNIP3 and FUNDC1 were found between HMC and ARC patients. Importantly, TEM revealed that the obvious mitochondrial fission and mitophagy phenomena occur in the LECs of HMC patients compared to the ARC patients. Moreover, CCCP aggreated the mitoROS production and depolarized mitochondrial membrane potential in the H2O2-treated human lens epithelial cells line (SRA01/04); Most important, rat lens organ culture experiments indicated a significant increase in H2O2-induced lens opacity following mitochondrial uncoupling CCCP treatment. CONCLUSION: This study has identified for the first time the abnormal mitochondrial homeostasis in HMC, and provide a new perspective on the potential mechanisms of HMC, which occurs earlier and at a higher incidence rate than ARC.

Where Are the Children? A Thematic Analysis of State, Territory, and Tribal Organization Comprehensive Cancer Control Plans.

BACKGROUND: The Center for Disease Control's Comprehensive Cancer Control Program (CCCP) funds initiatives in fifty states, the District of Columbia, seven U.S. territories, and seven tribal organizations to prevent and control cancer. These initiatives influence policy, care, research, and advocacy for cancer treatment. We performed an analysis of CCCP plans for states, U.S. territories, and tribal organizations to understand the extent of inclusion of pediatric cancer care. METHODS: We conducted a thematic and quantitative analysis of CCCP plans for states, U.S. territories, and tribal organizations. Plans were assessed by two reviewers and scored for discussion of cancer prevention, risk factors, early detection and screening, treatment and innovation, access, barriers to care, and survivorship in childhood cancer. RESULTS: Plans from fifty states, the District of Columbia, seven territories, seven tribal organizations, and one Pacific Regional (USAPI) plan were reviewed, for a total of sixty-six plans. Up-to-date CCCP plans were available through the CDC or state websites for 74% of states, 57% of territories, and 71% of tribal organizations; older plans were available for all groups without up-to-date CCCP plans. While all plans referenced children, most did so in the context of childhood exposures influencing adult cancer risks (e.g., sun, tobacco, HPV). Few plans contained a section dedicated to childhood cancer (30% states, 14.3% territories, 14.3% tribes). A minority of plans specifically discussed early detection and screening (14% states, 0% territories, 14.3% tribes), treatment and innovation (32% states, 0% territories, 28.6% tribes), access to cancer care (38% states, 28.6% territories, 28.6% tribes), reducing barriers to cancer care (28% states, 42.9% territories, 28.6% tribes), and pediatric cancer survivorship (42% states, 0% territories, 28.6% tribes). CONCLUSIONS: Promoting inclusion of pediatric cancer in CCPs will help to standardize pediatric cancer care, eliminate treatment disparities across state lines, and allow for comprehensive understanding of pediatric oncology. LEVEL OF EVIDENCE: Level IV.

TRIM28 promotes porcine epidemic diarrhea virus replication by mitophagy-mediated inhibition of the JAK-STAT1 pathway.

Porcine epidemic diarrhea virus (PEDV) infection causes immunosuppression and clinical symptoms such as vomiting, watery diarrhea, dehydration, and even death in piglets. TRIM28, an E3 ubiquitin ligase, is involved in the regulation of autophagy. However, the role of TRIM28 in PEDV infection is unknown. This study aimed to determine whether TRIM28 acts as a host factor for PEDV immune escape. We found that depletion of TRIM28 inhibited PEDV replication, whereas overexpression of TRIM28 promoted the viral replication in host cells. Furthermore, knockdown of TRIM28 reversed PEDV-induced downregulation of the JAK/STAT1 pathway. Treatment with the mitophagic activator carbonyl cyanide 3-chlorophenylhydrazone (CCCP) attenuated the activating effect of TRIM28 depletion on the expression of the STAT1 pathway-related proteins. Treatment with CCCP also reduced the nuclear translocation of pSTAT1. Moreover, TRIM28, via its RING domain, interacted with PEDV N. Overexpression of TRIM28 induced mitophagy, which could be enhanced by co-expression with PEDV N. The results indicate that PEDV infection upregulates the expression of TRIM28, which induces mitophagy, leading to inhibition of the JAK-STAT1 pathway. This research unveils a new mechanism by which PEDV can hijack host cellular TRIM28 to promote its own replication.

Detection of Aminoglycoside Modifying Enzyme (AME) genes in Acinetobacter baumannii isolates and the inhibitory effect of efflux pump activity on drug susceptibility pattern.

INTRODUCTION: The development of aminoglycoside modifying enzymes (AMEs) and increased efflux activity are considered important aminoglycosides resistance mechanisms. AIM: This study is focused on the detection of the AMEs gene and assessing the effect of efflux pump inhibitor on the reversal of A. baumannii drug susceptibility. METHODOLOGY: Bacterial DNA was amplified using AMEs gene-specific primers. Isolates were also investigated for efflux pump activity using efflux pump inhibitor (EPI) i.e. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and the impact of both mechanisms was analyzed. RESULTS: Among A. baumannii isolates, 55% isolates (n â€‹= â€‹22/40) were identified to have aminoglycoside modifying enzymes genes; ant(3')-I gene (50%, 11/22), aac(6')-Ib gene (45.4%, 10/22), aph(3')-I gene (18.1%, 4/22) and aac(3)-I (9.1%, 2/22). Total 70% isolates have shown MIC alteration in different classes of drugs in response to EPI-CCCP. Such alteration was found in 100% amikacin sensitive and 58.6% amikacin resistant, 93.7% and 57.1% gentamicin sensitive and resistant isolates respectively. CONCLUSION: The presence of aminoglycosides modifying enzymes was frequent among aminoglycosides resistant A. baumannii isolates and the coexistence of efflux pumps activity also plays an important role to increase drug resistance. REPOSITORIES: Genbank and their accession numbers are MT903331[aac(3)-I], MT903332 MT903333 [ant(3')-I], MT903334, MT903335 [aph(3')-I)] and MT903336, MT940242 [ aac(6')-Ib].

Reducing Cancer Risk Through Primary Prevention Activities Among Children: A Demonstration Project.

CONTEXT: Opportunities to reduce the risk of cancer, including cervical, liver, and skin cancer, start early in life. To encourage adoption of primary prevention activities in childhood to reduce cancer risk later in life, Centers for Disease Control and Prevention conducted a demonstration project with 3 National Comprehensive Cancer Control Program (NCCCP) recipients. PROGRAM: Iowa, Northwest Portland Area Indian Health Board (NPAIHB), and Pennsylvania NCCCP recipients implemented evidence-based primary prevention activities for cervical, liver, and skin cancer among children using health care provider education, patient education, and policy development. IMPLEMENTATION: Iowa implemented an announcement approach to improve provider education on human papillomavirus (HPV) vaccination. Pennsylvania focused on patient education for reducing skin cancer risk and both provider and patient education for liver cancer prevention. NPAIHB created a sun safety intervention for tribal organizations, including a policy guide, media materials, and patient education. RESULTS: In Iowa, health care providers taking the announcement approach reported significantly higher mean scores on a posttest compared with a pretest regarding perceptions about HPV vaccination, self-efficacy, and behavioral intentions related to vaccination. Pennsylvania integrated sun safety education and sunscreen dispenser programs as a health and wellness initiative in 8 state parks and the Pennsylvania Department of Conservation and Natural Resources incorporated the program in its Pennsylvania Outdoor Recreation Plan. Pennsylvania also implemented health care provider education on the primary prevention of liver cancer through hepatitis B and hepatitis C screening and hepatitis B vaccination. The NPAIHB skin cancer policy guide was created and distributed for use to all 43 federally recognized tribes of Oregon, Washington, and Idaho served by NPAIHB. DISCUSSION: The identification, dissemination, and implementation of these efforts can serve as best practices for future childhood primary prevention programs. NCCCP recipients and public health professionals can use health care provider education, patient education, and policy development to reduce future risk for cervical, liver, and skin cancer among children.

AMP-activated protein kinase (AMPK) suppresses Ibaraki virus propagation.

The Ibaraki virus (IBAV) causes Ibaraki disease in cattle. Our previous studies have shown that IBAV uses macropinocytosis to enter the host cell and exit from the endosome to the cytosol in response to endosomal acidification. To further explore the mechanism of IBAV infection and replication, we examined the effect of inhibitors of mitochondrial oxidative phosphorylation, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and antimycin A, on IBAV propagation. These inhibitors significantly suppressed IBAV propagation, with reduced cellular ATP levels resulting from suppression of ATP synthesis. Furthermore, we identified AMP-activated protein kinase (AMPK), which is activated by CCCP or antimycin A, as a key signaling molecule in IBAV suppression. We also observed that IBAV infection induces ATP depletion and increases AMPK activity. Our findings suggest that AMPK is a potential target in Ibaraki disease.

Cristae dynamics is modulated in bioenergetically compromised mitochondria.

Cristae membranes have been recently shown to undergo intramitochondrial merging and splitting events. Yet, the metabolic and bioenergetic factors regulating them are unclear. Here, we investigated whether and how cristae morphology and dynamics are dependent on oxidative phosphorylation (OXPHOS) complexes, the mitochondrial membrane potential (ΔΨm), and the ADP/ATP nucleotide translocator. Advanced live-cell STED nanoscopy combined with in-depth quantification were employed to analyse cristae morphology and dynamics after treatment of mammalian cells with rotenone, antimycin A, oligomycin A, and CCCP. This led to formation of enlarged mitochondria along with reduced cristae density but did not impair cristae dynamics. CCCP treatment leading to ΔΨm abrogation even enhanced cristae dynamics showing its ΔΨm-independent nature. Inhibition of OXPHOS complexes was accompanied by reduced ATP levels but did not affect cristae dynamics. However, inhibition of ADP/ATP exchange led to aberrant cristae morphology and impaired cristae dynamics in a mitochondrial subset. In sum, we provide quantitative data of cristae membrane remodelling under different conditions supporting an important interplay between OXPHOS, metabolite exchange, and cristae membrane dynamics.