Carbonyl Cyanide 3-chlorophenylhydrazone Research Articles

Mito-TEMPO Ameliorates Sodium Palmitate Induced Ferroptosis in MIN6 Cells through PINK1/Parkin-Mediated Mitophagy

ObjectiveMitochondrial reactive oxygen species (mtROS) could cause damage to pancreatic β-cells, rendering them susceptible to oxidative damage. Hence, investigating the potential of the mitochondriatargeted antioxidant (Mito-TEMPO) to protect pancreatic β-cells from ferroptosis by mitigating lipid peroxidation becomes crucial. MethodsMIN6 cells were cultured in vitro with 100 μmol/L sodium palmitate (SP) to simulate diabetes. FerroOrange was utilized for the detection of Fe2+ fluorescence staining, BODIPY581/591C11 for lipid reactive oxygen species, and MitoSox-Red for mtROS. Alterations in mitophagy levels were assessed through the co-localization of lysosomal and mitochondrial fluorescence. Western blotting was employed to quantify protein levels of Acsl4, GPX4, FSP1, FE, PINK1, Parkin, TOMM20, P62, and LC3. Subsequently, interventions were implemented using Mito-TEMPO and Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to observe changes in ferroptosis and mitophagy within MIN6 cells. ResultsWe found that SP induced a dose-dependent increase in Fe2+ and lipid ROS in MIN6 cells while decreasing the expression levels of GPX4 and FSP1 proteins. Through bioinformatics analysis, it has been uncovered that mitophagy assumes a crucial role within the ferroptosis pathway associated with diabetes. Additionally, SP decreased the expression of mitophagy-related proteins PINK1 and Parkin, leading to mtROS overproduction. Conversely, Mito-TEMPO effectively eliminated mtROS while activating the mitophagy pathways involving PINK1 and Parkin, thereby reducing the occurrence of ferroptosis in MIN6 cells. CCCP also demonstrated efficacy in reducing ferroptosis in MIN6 cells. ConclusionIn summary, Mito-TEMPO proved effective in attenuating mtROS production and initiating mitophagy pathways mediated by PINK1 and Parkin in MIN6 cells. Consequently, this decreased iron overload and lipid peroxidation, ultimately safeguarding the cells from ferroptosis.

Identification of nanaomycin A and its analogs by a newly established screening method for functional inhibitors of the type IX secretion system in Porphyromonas gingivalis.

Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, is a key pathogen in chronic periodontitis. P. gingivalis has a type IX secretion system (T9SS) that secretes highly hydrolytic proteinases called gingipains for obtaining peptides as an energy source. Although most T9SS-related proteins have been identified, no specific inhibitor of T9SS has been reported. To screen T9SS inhibitors, we focused on and characterized a minimal liquid medium called mC medium that contains milk casein as the sole protein source. We found that P. gingivalis wild-type strain ATCC 33277 caused cloudiness of mC medium without growth. In mC medium, an alkylating agent, iodoacetamide (IAM) that is an inhibitor of gingipains, and a protonophore, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) that dissipates the proton motive force required for T9SS-mediated secretion, clearly inhibited the increase in turbidity. Moreover, neither the gingipain-null mutant nor the T9SS-deficient mutant caused mC medium cloudiness, suggesting that mC medium cloudiness is dependent on gingipain activity and T9SS. These results indicated that mC medium can be used to assess P. gingivalis gingipain activity and its functional T9SS. Using an assay system with mC medium, we discovered that OM-173αA and OM-173βA in the Ōmura Natural Compound Library and nanaomycin A were probable T9SS inhibitors. The compounds need to be further investigated as tools for analyzing T9SS and as potential therapeutic agents for periodontal disease.

AdeABC, AdeFGH, and AdeIJK efflux pumps as key factors in tigecycline resistance of Acinetobacter baumannii: a study from Western Balkan hospitals.

The present study investigated the role of resistance-nodulation-cell division (RND) efflux pumps in tigecycline resistance of Acinetobacter baumannii clinical isolates recovered from three Western Balkan countries (Serbia, Bosnia and Herzegovina and Montenegro). A total of 37 A. baumannii isolates recovered from seven tertiary care hospitals in 2016 and 2022 were tested against tigecycline using broth microdilution method. Then, efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used to determine the involvement of efflux pumps in tigecycline resistance. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multiplex PCR-based determination of clonal lineage. Regulators of efflux pumps were analyzed for amino acid substitutions, while reverse transcription-quantitative PCR (RT-qPCR) enabled quantification of RND efflux pumps expression. All tested isolates were interpreted as resistant to tigecycline and showed reduced tigecycline minimum inhibitory concentration (MIC) values in the presence of CCCP. PFGE analysis showed significant diversity among isolates grouped in cluster I including IC2 (n = 32) and IC3 (n = 1) isolates, while cluster II was comprised of four IC1 isolates. The most prevalent substitutions in AdeR were V120I and A136V and in AdeS G186V and N268H (n = 33). The Q262R substitution was detected in AdeL proteins of IC1 isolates, whereas no alterations were observed within AdeN. The expression of the adeB, adeG, and adeJ genes in selected isolates was upregulated in five (1.16- to 3-fold), sixteen (1.35- to 2.82-fold), and twelve isolates (1.62- to 4-fold) compared to ATCC19606, respectively. This study revealed that overexpression of RND efflux pumps underlies tigecycline resistance in A. baumannii clinical isolates from the Western Balkans.

HSF1/HSP25 system protects mitochondria function from heat stress and assists steroidogenesis in MA-10 Leydig cells

Heat shock response is characterized by the induction of heat shock proteins (HSPs) or molecular chaperones that maintain protein homeostasis. Heat shock transcription factor 1 (HSF1) plays a central role in heat shock response in mammalian cells. To investigate the impact of the heat shock response mechanism on steroidogenesis, we generated MA-10 mouse Leydig tumor cells deficient in HSF1 using CRISPR-Cas9 genome editing. Under heat stress conditions, the levels of StAR protein, but not its mRNA, decreased more in HSF1-knockout cells than in wild-type cells, confirming that HSF1 stabilizes StAR protein. Simultaneously, HSP110, HSP70, and HSP25 were markedly upregulated in a manner dependent on HSF1. Mitochondrial membrane potential (MMP) and ATP synthesis were decreased in HSF1-knockout cells under heat stress conditions, and mitochondrial fragmentation was enhanced. Furthermore, treatment with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a disruptor of MMP, reduced the levels of StAR protein to a greater extent in HSF1-knockout cells than in wild-type cells, which was associated with decreased MMP and ATP synthesis. Unexpectedly, HSP25 expression was markedly increased in wild-type cells following CCCP treatment. HSP25 knockdown reduces MMP under heat stress conditions and decreases StAR protein levels and progesterone synthesis. HSP25 overexpression in HSF1KO cells restored StAR protein levels. These results show that the HSF1/HSP25 pathway protects mitochondrial function and maintains StAR synthesis.

Mitochondrial dysfunction matures Ras-induced early senescence to full senescence with a proinflammatory senescence-associated secretory phenotype in the fish cell line, EPC

Fish cell lines differ from most mammalian diploid cell lines by the fact that cellular senescence is not readily induced. Previously, we demonstrated that the absence of the p16 gene in the fish genome prevents cells from reaching full senescence even when Ras is activated. Drosophila also lacks p16; however, early senescence triggered by Ras activation progresses to full senescence and is accompanied by a proinflammatory senescence-associated secretory phenotype (SASP), due to mitochondrial deficiency. It is unclear whether mitochondrial deficiency can also induce the maturation of Ras-induced early senescence (RIS) to full senescence along with a proinflammatory SASP in fish cell lines. Here, we investigated whether mitochondrial dysfunction induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in concert with activated Ras results in full senescence and whether this is accompanied by a proinflammatory SASPs in the EPC fish cell line. We found that although EPC cells with mitochondrial dysfunction exhibited a proinflammatory SASP, this did not result in permanent cell proliferation arrest or the upregulation of endogenous Ras expression. These findings suggest that other factors must act in concert with mitochondrial dysfunction to induce full senescence. The proliferation of EPC cells overexpressing a constitutively active mutant of H-Ras (H-RasV12) was markedly reduced, irrespective of CCCP treatment. These findings suggest that there are similarities between the cellular senescence observed in fish and Drosophila cells lacking the p16 gene. However, it should be noted that fish cells differ from Drosophila cells in that mitochondrial dysfunction alone can induce proinflammatory SASP factors.

SUMOylation of MFF coordinates fission complexes to promote stress-induced mitochondrial fragmentation.

Mitochondria undergo fragmentation in response to bioenergetic stress, mediated by dynamin-related protein 1 (DRP1) recruitment to the mitochondria. The major pro-fission DRP1 receptor is mitochondrial fission factor (MFF), and mitochondrial dynamics proteins of 49 and 51 kilodaltons (MiD49/51), which can sequester inactive DRP1. Together, they form a trimeric DRP1-MiD-MFF complex. Adenosine monophosphate-activated protein kinase (AMPK)-mediated phosphorylation of MFF is necessary for mitochondrial fragmentation, but the molecular mechanisms are unclear. Here, we identify MFF as a target of small ubiquitin-like modifier (SUMO) at Lys151, MFF SUMOylation is enhanced following AMPK-mediated phosphorylation and that MFF SUMOylation regulates the level of MiD binding to MFF. The mitochondrial stressor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) promotes MFF SUMOylation and mitochondrial fragmentation. However, CCCP-induced fragmentation is impaired in MFF-knockout mouse embryonic fibroblasts expressing non-SUMOylatable MFF K151R. These data suggest that the AMPK-MFF SUMOylation axis dynamically controls stress-induced mitochondrial fragmentation by regulating the levels of MiD in trimeric fission complexes.

Forkhead box O-1 regulates the biological behavior of BMP-2-induced human bone mesenchymal stem cells through mitochondrial dynamics and autophagy.

This study explored the mechanism by which forkhead box O-1 (FoxO1) modulates the biological behaviors of bone mesenchymal stem cell (BMSC). Human BMSCs were cultured for 7 days in DMEM containing bone morphogenetic protein-2 (BMP-2) and treated with a short hairpin-FoxO1 plasmid. The study assessed cell proliferation, migration, apoptosis, adenosine triphosphate (ATP) levels, mitochondrial DNA levels, membrane potential, autophagy, and the levels of FoxO1, apoptosis-associated proteins, osteogenic differentiation-associated proteins, mitochondrial fusion and fission proteins and mitochondrial autophagy-related proteins. The cells were also treated with the mitochondrial fusion activator MASM7 and the mitochondrial autophagy activator carbonyl cyanide 3-chlorophenylhydrazone. The study evaluated whether mitochondrial dynamics and autophagy activation could rescue the FoxO1 knockdown-induced changes in BMSC biological behaviors, mitochondrial dynamics, and mitochondrial autophagy. BMP-2-induced BMSCs exhibited up-regulated FoxO1 expression, enhanced proliferation and migration, and induced osteogenic differentiation, while FoxO1 knockdown inhibited BMP-2-induced BMSC proliferation, migration and osteogenic differentiation, increased apoptosis, and affected mitochondrial dynamics and autophagy. Promoting mitochondrial fusion partially reversed the regulatory effects of FoxO1 downregulation on mitochondrial autophagy and the inhibitory effects of FoxO1 silencing on BMP-2-induced BMSC biological behaviors. Activated mitochondrial autophagy facilitated the homeostasis of mitochondrial dynamics and partially counteracted the inhibitory effects of FoxO1 knockdown on BMP-2-induced BMSC biological behaviors. In conclusion, FoxO1 regulates mitochondrial dynamics and autophagy to modulate the osteogenic differentiation of BMP-2-induced human BMSCs.

Automated Image-Based Fluorescence Screening of Mitochondrial Membrane Potential in Daphnia magna: An Advanced Ecotoxicological Testing Tool.

This study demonstrated the strengths of in vivo molecular staining coupled with automated imaging analysis in Daphnia magna. A multiwell plate protocol was developed to assess mitochondrial membrane potential using the JC-1 dye. The suitability of five common anesthetics was initially tested, and 5% ethanol performed best in terms of anesthetic effects and healthy recovery. The staining conditions were optimized to 30 min staining with 2 μM JC-1 for best J-aggregate formation. The protocol was validated with the model compound carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and used to measure the effect of four environmental contaminants, 2,4-dinitrophenol, triclosan, n-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), and ibuprofen, on mitochondrial health. Test organisms were imaged using an automated confocal microscope, and fluorescence intensities were automatically quantified. The effect concentrations for CCCP were lower by a factor of 30 compared with the traditional OECD 202 acute toxicity test. Mitochondrial effects were also detected at lower concentrations for all tested environmental contaminants compared to the OCED 202 test. For 2,4-dinitrophenol, mitochondria effects were detectable after 2 h exposure to environmentally relevant concentrations and predicted organism death was observed after 24 h. The high sensitivity and time efficiency of this novel automated imaging method make it a valuable tool for advancing ecotoxicological testing.

Mitochondrial elongation confers protection against doxorubicin-induced cardiotoxicity

Doxorubicin (DOX)-induced cardiac damage remains a leading cause of death amongst cancer survivors. DOX-induced cardiotoxicity (DIC) is mediated by disturbed mitochondrial dynamics, but it remains debated that the mechanisms by which DOX disrupted equilibrium between mitochondrial fission and fusion. In the present study, we observed that DOX induced mitochondrial elongation in multiple cardiovascular cell lines. Mechanically, DOX not only downregulated the mitochondrial fusion proteins including Mitofusin 1/2 (MFN1/2) and Optic atrophy 1 (OPA1), but also induced lower motility of dynamin-related protein 1(Drp1) and its phosphorylation on 637 serine, which could inhibit mitochondrial fission. Interestingly, DOX failed to induce mitochondrial elongation in cardiomyocytes co-treated with protein kinase A (PKA) inhibitor H89 or expressing phosphodeficient Drp1-S637A variants. Besides, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was able to blocked the mitochondrial elongation induced by DOX treatment, which could be phenocopied by OPA1 knockdown. Therefore, we speculated that DOX inhibited mitochondrial fission and fusion simultaneously, yet enabled mitochondrial fusion dominate the mitochondrial dynamics, resulting in mitochondrial elongation as the main manifestation. Notably, blocking mitochondrial elongation by inhibiting Drp1-S637 phosphorylation or OPA1 knockdown aggravated DOX-induced cardiomyocytes death. Based on these results, we propose a novel mechanistic model that DOX-induced mitochondrial elongation is attributed to the equilibrium disturbance of mitochondrial dynamics, which serves as an adaptive response and confers protection against DIC.

Inside-out, antimicrobial resistance mediated by efflux pumps in clinical strains of Acinetobacter baumannii isolated from burn wound infections.

Acinetobacter baumannii belongs to the ESKAPE group. It is classified as a critical priority group by the World Health Organization and a global concern on account of its capacity to acquire and develop resistance mechanisms to multiple antibiotics. Data from the United States indicates 500 deaths annually. Resistance mechanisms of this bacterium include enzymatic pathways such as ß-lactamases, carbapenemases, and aminoglycoside-modifying enzymes, decreased permeability, and overexpression of efflux pumps. A. baumannii has been demonstrated to possess efflux pumps, which are classified as members of the MATE family, RND and MFS superfamilies, and SMR transporters. The aim of our work was to assess the distribution of efflux pumps and their regulatory gene expression in clinical strains of A. baumannii isolated from burned patients. METHODS: From the Clinical Microbiology Laboratory at the Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra collection in Mexico, 199 strains were selected. Antibiotics susceptibilities were performed by broth microdilutions to determine minimal inhibitory concentrations. Phenotypic assays with efflux pump inhibitors were conducted using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and phenylalanine-arginine ß-naphthylamide (PAßN) in conjunction with amikacin, ceftazidime, imipenem, meropenem and levofloxacin. A search was conducted for structural genes that are linked to efflux pumps, and the relative expression of the adeR, adeS, and adeL genes was analyzed. RESULTS: Among a total of 199 strains, 186 exhibited multidrug resistance (MDR). Fluoroquinolones demonstrated the highest resistance rates, while minocycline and amikacin displayed comparatively reduced resistance rates (1.5 and 28.1, respectively). The efflux activity of fluorquinolones exhibited the highest phenotypic detection (from 85 to 100%), while IMP demonstrated the lowest activity of 27% with PAßN and 43.3% with CCCP. Overexpression was observed in adeS and adeL, with adeR exhibiting overexpression. Concluding that clinical strains of A. baumannii fromour institution exhibited efflux pumps as one of the resistance mechanisms.

Uptake and transport of antibiotic kasugamycin from leaves to roots in rice (Oryza sativa L.) seedlings

AbstractKasugamycin (KSM), an aminoglycoside antibiotic, has been widely used as a natural fungicide to control plant diseases, particularly for managing rice blast. However, its uptake mechanism and transport in rice remain to be explored. In this article, rice seedlings were treated by the foliar spraying method, and the content of KSM in rice leaves, stems and roots under different treatments was detected by high‐performance liquid chromatography tandem mass spectrometry (HPLC‐MS/MS), respectively. Results showed that leaf‐applied KSM could be transported to the phloem and migrate to roots and stems after uptake by leaves. Concentration, temperature and pH had significant effects on the uptake of KSM. Compared with the control, the competitive inhibitors d‐glucose and phlorizin both inhibited the uptake of KSM, demonstrating that sugar transporter proteins were involved in the uptake process. The energy inhibitors dinitrophenol (DNP) and carbonyl cyanide chlorophenylhydrazone (CCCP) also significantly inhibited the uptake of KSM, indicating that the uptake of KSM required energy consumption. Thus, the uptake of KSM by rice was an active process involving sugar transporter proteins, and it could migrate downward through the phloem. This study contributes to the promotion of the scientific application of antibiotics and the biological control of crop diseases. It will also provide a theoretical basis for the development of root‐targeted pesticides and transport pesticides with phloem mobility.

Differentiation activates mitochondrial OPA1 processing in myoblast cell lines

Mitochondrial optic atrophy-1 (OPA1) plays key roles in adapting mitochondrial structure to bioenergetic function. When transmembrane potential across the inner membrane (Δψm) is intact, long (L-OPA1) isoforms shape the inner membrane through membrane fusion and the formation of cristal junctions. When Δψm is lost, however, OPA1 is cleaved to short, inactive S-OPA1 isoforms by the OMA1 metalloprotease, disrupting mitochondrial structure and priming cellular stress responses such as apoptosis. Previously, we demonstrated that L-OPA1 of H9c2 cardiomyoblasts is insensitive to loss of Δψm via challenge with the protonophore carbonyl cyanide chlorophenyl hydrazone (CCCP), but that CCCP-induced OPA1 processing is activated upon differentiation in media with low serum supplemented with all-trans retinoic acid (ATRA). Here, we show that this developmental induction of OPA1 processing in H9c2 cells is independent of ATRA; moreover, pretreatment of undifferentiated H9c2s with chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, recapitulates the Δψm-sensitive OPA1 processing observed in differentiated H9c2s. L6.C11 and C2C12 myoblast lines display the same developmental and CAP-sensitive induction of OPA1 processing, demonstrating a general mechanism of OPA1 regulation in mammalian myoblast cell settings. Restoration of CCCP-induced OPA1 processing correlates with increased apoptotic sensitivity. Moreover, OPA1 knockdown indicates that intact OPA1 is necessary for effective myoblast differentiation. Taken together, our results indicate that a novel developmental mechanism acts to regulate OMA1-mediated OPA1 processing in myoblast cell lines, in which differentiation engages mitochondrial stress sensing.

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.

Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue

ABSTRACT The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and enzyme-linked immunosorbent assay. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens. Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ELISA: enzyme-linked immunosorbent assay; HEK293E cell: human embryonic kidney E cell; ICC: immunocytochemistry; IHC: immunohistochemistry: KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MEF: mouse embryonic fibroblast; MSD: Meso Scale Discovery; n.s.: non-significant; nonTg: non-transgenic; PBMC: peripheral blood mononuclear cell; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated Ub at serine 65; Ub: ubiquitin; WT: wild-type.

Unveiling the variability in cadmium accumulation and tolerance characteristics: a comparative study of Basma and Yunyan 87 tobacco varieties

ABSTRACT Tobacco (Nicotiana tabacum L.) shows promise for remediating Cd-contaminated soil due to its significant Cd accumulation capabilities. Although various tobacco varieties exhibit distinct Cd bioaccumulation capacities, a comprehensive understanding of the underlying mechanisms is lacking. This study, conducted using hydroponics, explores differences in Cd accumulation and tolerance mechanisms between two tobacco varieties, Basma and Yunyan 87. The results showed that Cd stress reduced the dry weight, tolerance index, and root morphology for both varieties. Basma exhibited a relatively smaller decline in these indices compared to Yunyan 87. Moreover, Basma demonstrated a higher Cd bioconcentration factor (BCF), concentration, and accumulated content, signifying its superior tolerance and bioaccumulation capacity to Cd compared to Yunyan 87. The Carbonyl Cyanide3-ChloroPhenylhydrazone (CCCP) addition resulted in reduced Cd accumulation and BCFs in both tobacco species. This effect was more pronounced in Basma, suggesting that Basma relies more on an active transport process than Yunyan 87. This could potentially explain its enhanced bioaccumulation ability. Subcellular Cd distribution analysis revealed Basma's preference for distributing Cd in soluble fractions, while Yunyan 87 favoured the cell wall fractions. Transmission electron microscope showed that Basma's organelles were less damaged than Yunyan 87's under Cd stress, possibly contributing to the superior tolerance of Basma. Therefore, these results provided a theoretical foundation for development of Cd-contaminated soil tobacco remediation technology.

Molecular epidemiology and resistance mechanisms of tigecycline-non-susceptible Acinetobacter baumannii isolated from a tertiary care hospital in Chongqing, China.

We studied 34 isolates of Tigecycline-Non-Susceptible A. baumannii (TNAB) obtained from clinical specimens at a large tertiary care hospital in Chongqing, China. These 34 strains belonged to 8 different clones including ST195 (35.3%) and ST208 (17.7%). EBURST analysis found that these 8 ST types belonged to the Clonal Complex 92. Tigecycline resistance-associated genes adeR, adeS, adeL, adeN, rrf, rpsJ, and trm were detected in most strains. The expression level of the resistance-nodulation-cell division (RND) efflux pumps in TNAB strains was higher than the reference strain ATCC19606. 58.8% of strains had a decrease in the tigecycline minimum inhibitory concentration (MIC) after the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The TNAB strains in our hospital have a high degree of affinity and antibiotic resistance. Regular surveillance should be conducted to prevent outbreaks of TNAB epidemics.

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

Aim: To evaluate the action of promethazine, fluoxetineand 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 bromideto 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.

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.

DDIT3/CHOP promotes LPS/ATP-induced pyroptosis in osteoblasts via mitophagy inhibition

Inflammatory environments can trigger endoplasmic reticulum (ER) stress and lead to pyroptosis in various tissues and cells, including liver, brain, and immune cells. As a key factor of ER stress, DNA damage-inducible transcript 3 (DDIT3)/CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is upregulated in osteoblasts during inflammatory stimulation. DDIT3/CHOP may therefore regulate osteoblast pyroptosis in inflammatory conditions. During this investigation, we found that lipopolysaccharides (LPS)/adenosine 5′-triphosphate (ATP) stimulation in vitro induced osteoblasts to undergo pyroptosis, and the expression of DDIT3/CHOP was increased during this process. The overexpression of DDIT3/CHOP further promoted osteoblast pyroptosis as evidenced by the increased expression of the inflammasome NLR family pyrin domain containing 3 (NLRP3) and ratios of caspase-1 p20/caspase-1 and cleaved gasdermin D (GSDMD)/GSDMD. To explore the specific mechanism of this effect, we found through fluorescence imaging and Western blot analysis that LPS/ATP stimulation promoted PTEN-induced kinase 1 (PINK1)/E3 ubiquitin-protein ligase parkin (Parkin)-mediated mitophagy in osteoblasts, and this alteration was suppressed by the DDIT3/CHOP overexpression, resulting in increased ratio of pyroptosis compared with the control groups. The impact of DDIT3/CHOP on pyroptosis in osteoblasts was reversed by the application of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a specific mitophagy agonist. Therefore, our data demonstrated that DDIT3/CHOP promotes osteoblast pyroptosis by inhibiting PINK1/Parkin-mediated mitophagy in an inflammatory environment.

The SCF-FBW7β E3 ligase mediates ubiquitination and degradation of the serine/threonine protein kinase PINK1

Understanding the mechanisms that govern the stability of functionally crucial proteins is essential for various cellular processes, development, and overall cell viability. Disturbances in protein homeostasis are linked to the pathogenesis of neurodegenerative diseases (NDDs). PINK1, a protein kinase, plays a significant role in mitochondrial quality control and cellular stress response, and its mutated forms lead to early-onset Parkinson's disease (PD). Despite its importance, the specific mechanisms regulating PINK1 protein stability have remained unclear. This study reveals a cytoplasmic interaction between PINK1 and FBW7β in mammalian cells. FBW7β, a component of the Skp1-Cullin-1-F-box protein (SCF) complex-type ubiquitin ligase, is instrumental in recognizing substrates. Our findings demonstrate that FBW7β regulates PINK1 stability through the SCF complex and the proteasome pathway. It facilitates the K48-linked polyubiquitination of PINK1, marking it for degradation. When FBW7 is absent, PINK1 accumulates, leading to heightened mitophagy triggered by carbonyl cyanide 3-chlorophenylhydrazone treatment. Moreover, exposure to the toxic compound staurosporine accelerates PINK1 degradation via FBW7β, correlating with increased cell death. This study unravels the intricate mechanisms controlling PINK1 protein stability and sheds light on the novel role of FBW7β. These findings deepen our understanding of PINK1-related pathologies and potentially pave the way for therapeutic interventions.