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The worldwide HIV cases were 39.0 million (33.1–45.7 million) in 2022. Due to genetic variations, HIV-1 is more easily transmitted than HIV-2 and favours CD4 + T cells and macrophages, producing AIDS. Conventional HIV drug therapy has many drawbacks, including adherence issues leading to resistance, side effects that lower life quality, drug interactions, high costs limiting global access, inability to eliminate viral reservoirs, chronicity requiring lifelong treatment, emerging toxicities, and a focus on managing infections. Conventional dosage forms have bioavailability issues due to intestinal P-glycoprotein (P-gp) efflux, which can reduce anti-retroviral drug efficacy and lead to resistance. Use of phyto-constituents with P-gp regulating actions has great benefits for semi-synthetic modification to create formulations with greater bioavailability and reduced toxicity, which improves drug effectiveness. Lipid-based nanocarriers, solid lipid nanoparticles, nanostructured lipid carriers, polymer-based nanocarriers, and inorganic nanoparticles may inhibit P-gp efflux. Employing potent P-gp inhibitors within nanocarriers as a Trojan horse approach can enhance the intracellular accumulation of anti-retroviral drugs (ARDs), which are substrates for efflux transporters. This technique increases oral bioavailability and offers lower-dose options, boosting HIV patient compliance and lowering costs. Molecular docking of the inhibitor with P-gp may anticipate optimum binding and function, allowing drug efflux to be minimised. [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance transporter 2 (MRP2) are efflux transporters involved in the absorption, excretion, and distribution of drugs. Bidirectional cell assays are recognized models for evaluating the potential of new drugs as substrates or inhibitors of efflux transporters. However, the assays are complicated by a lack of selective substrates and/or inhibitors, as well simultaneous expression of several efflux transporters in cell lines used in efflux models. This project aims to evaluate an in vitro efflux cell assay employing model substrates and inhibitors of P-gp, BCRP and MRP2 with knockout (KO) cell lines. The efflux ratios (ER) of P-gp (digoxin, paclitaxel), BCRP (prazosin, rosuvastatin), MRP2 (etoposide, olmesartan) and mixed (methotrexate, mitoxantrone) substrates were determined in wild-type C2BBe1 and KO cells. For digoxin and paclitaxel, the ER decreased to less than 2 in the cell lines lacking P-gp expression. The ER decreased to less than 3 for prazosin and less than 2 for rosuvastatin in the cell lines lacking BCRP expression. For etoposide and olmesartan, the ER decreased to less than 2 in the cell lines lacking MRP2 expression. The ER of methotrexate and mitoxantrone decreased in single- and double-KO cells without BCRP and MRP2 expression. These results show that KO cell lines have the potential to better interpret complex drug-transporter interactions without depending upon multi-targeted inhibitors or overlapping substrates. For drugs that are substrates of multiple transporters, the single- and double-KO cells may be used to assess their affinities for the different transporters. [ABSTRACT FROM AUTHOR]

Antifungal resistance and antifungal tolerance are two distinct terms that describe different cellular responses to drugs. Antifungal resistance describes the ability of a fungus to grow above the minimal inhibitory concentration (MIC) of a drug. Antifungal tolerance describes the ability of drug susceptible strains to grow slowly at inhibitory drug concentrations. Recent studies indicate antifungal resistance and tolerance have distinct evolutionary trajectories. Superficial candidiasis bothers millions of people yearly. Miconazole has been used for topical treatment of yeast infections for over 40 years. Yet, fungal resistance to miconazole remains relatively low. Here we found different clinical isolates of Candida albicans had different profile of tolerance to miconazole, and the tolerance was modulated by physiological factors including temperature and medium composition. Exposure of non-tolerant strains with different genetic backgrounds to miconazole mainly induced development of tolerance, not resistance, and the tolerance was mainly due to whole chromosomal or segmental amplification of chromosome R. The efflux gene CDR1 was required for maintenance of tolerance in wild type strains but not required for gain of aneuploidy-mediated tolerance. Heat shock protein Hsp90 and calcineurin were essential for maintenance as well as gain of tolerance. Our study indicates development of aneuploidy-mediated tolerance, not resistance, is the predominant mechanism of rapid adaptation to miconazole in C. albicans, and the clinical relevance of tolerance deserves further investigations. [ABSTRACT FROM AUTHOR]

Antibiotic-resistant strains of Staphylococcus aureus are being viewed as a serious threat by various public health agencies. Identifying novel targets in this important pathogen is crucial to the development of new effective antibacterial formulations. We investigated the antibacterial effect of a colloidal nanosilver formulation, Silversol®, against an antibiotic-resistant strain of S. aureus using appropriate in vitro assays. Moreover, we deciphered the molecular mechanisms underlying this formulation's anti-S. aureus activity using whole transcriptome analysis. Lower concentrations of the test formulation exerted a bacteriostatic effect against this pathogen, and higher concentrations exerted a bactericidal effect. Silversol® at sub-lethal concentration was found to disturb multiple physiological traits of S. aureus such as growth, antibiotic susceptibility, membrane permeability, efflux, protein synthesis and export, biofilm and exopolysaccharide production, etc. Transcriptome data revealed that the genes coding for transcriptional regulators, efflux machinery, transferases, β-lactam resistance, oxidoreductases, metal homeostasis, virulence factors, and arginine biosynthesis are expressed differently under the influence of the test formulation. Genes (argG and argH) involved in arginine biosynthesis emerged among the major targets of Silversol®'s antibacterial activity against S. aureus. [ABSTRACT FROM AUTHOR]

L-alanine is one of the smallest chiral compounds and has been widely used in food, medicine, and daily chemical fields. Microbial production of L -alanine has problems such as long fermentation period and low productivity. In this study, metabolic engineering strategies such as strengthen precursors supply, promoter engineering and transporter engineering was used to construct an Escherichia coli cell factory with high-titer production of L-alanine. The production process of L -alanine was optimized by biochemical engineering strategies to improve the production performance of the Escherichia coli cell factory with production of L-alanine. Results: Overexpression of gapA (Glyceraldehyde 3-phosphate dehydrogenase gene) enhanced precursor supply, increased the titer of L-alanine and the conversion of glucose by 5.1% and 15.6%, respectively. Through the optimization of gapA expression by promoter engineering, the titer of L-alanine and the conversion of glucose reached 18.3 g/L and 0.55 g/g, respectively. Overexpression of L-alanine transporter (AlaE) enhanced L-alanine transport, the titer of L-alanine reached 20.4 g/L. The composition of culture medium was optimized by biochemical engineering strategy, the best carbon source was glucose 40 g/L and the best nitrogen source was (NH4)2SO4 25 g/L. The optimum fermentation conditions were as follows: inoculum size: 15%, fermentation mode: 10 h conversion to anaerobic fermentation and feeding mode: variable speed feed. After 36 h fermentation in a 5 L bioreactor, the constructed strain E. coli W-135 produced 127.2 g/L of L-alanine, with a yield of 0.83 g/g glucose and a productivity of 3.53 g/ L/h, which were 64.3%, 50.9% and 64.2% higher than those before optimization, respectively. In this study, a L-ala-nine high-titer strain with short fermentation period and simple fermentation process was constructed by using the strategy of system metabolic engineering and biochemical engineering, which provided a theoretical basis for the industrial production of L-alanine. [ABSTRACT FROM AUTHOR]

The dependence on metals and metalloids significantly impacts global heavy metal pollution and the fate of living organisms on this planet. Microorganisms like bacteria are no exception, and due to continuous exposure to metalliferous environments, many bacteria have evolved heavy metal resistance. Bacteria can also detoxify and subsequently remove the toxic metals using a variety of mechanisms. Efflux systems and associated transporter proteins are integral to heavy metal homoeostasis, tolerance, and detoxification in bacteria. Transport proteins are crucial for facilitating the transfer of hydrophilic substrates across hydrophobic membranes, constituting a varied array of proteins with distinctions in topology, substrate specificity, energy coupling mechanisms, and sequence similarity. This category of transporter proteins encompasses P-type ATPases, RND (resistance, nodulation, cell division), CDF (cation diffusion facilitator), ATP binding cassette (ABC) transporters, HoxN, CHR, MIT family proteins, and more. This article provides metal-specific insights into transporters, delineating their functions in bacterial resistance and homeostasis concerning potentially toxic metals and metalloids. The roles of assorted genes and operons in fulfilling this purpose and the physiological impact of different heavy metals on bacterial cells are also elaborated upon in this review. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

MDCK/Caco-2 assays serve as essential in vitro tools for evaluating membrane permeability and active transport, especially mediated by P-glycoprotein (P-gp). Despite their utility, challenges remain in quantifying active transport and using the efflux ratio (ER) to determine intrinsic values for active efflux. Such an intrinsic value for P-gp facilitated efflux necessitates knowing whether this transporter transports the neutral or ionic species of a compound. Utilising MDCK-MDR1 assays, we investigate a method for determining transporter substrate fraction preference by studying ER pH-dependence for basic, acidic and non-dissociating compounds. These results are compared with model fits based on various assumptions of transporter species preference. As an unexpected consequence of these assays, we also give evidence for an additional influx transporter at the basolateral membrane, and further extend our model to incorporate this transport. The combined influences of paracellular transport, the previously unaccounted for basolateral influx transporter, as well as potential pH effects on the transporter impedes the extraction of intrinsic values for active transport from the ER. Furthermore, we determined that using inhibitor affects the measurement of paracellular transport. While clear indications of transporter species preference remain elusive, this study enhances understanding of the MDCK system.

Cholesterol homeostasis is crucial for cellular and systemic function. The disorder of cholesterol metabolism not only accelerates the onset of cardiovascular disease (CVD) but is also the fundamental cause of other ailments. The regulation of cholesterol metabolism in the human is an extremely complex process. Due to the dynamic balance between cholesterol synthesis, intake, efflux and storage, cholesterol metabolism generally remains secure. Disruption of any of these links is likely to have adverse effects on the body. At present, increasing evidence suggests that abnormal cholesterol metabolism is closely related to various systemic diseases. However, the exact mechanism by which cholesterol metabolism contributes to disease pathogenesis remains unclear, and there are still unknown factors. In this review, we outline the metabolic process of cholesterol in the human body, especially reverse cholesterol transport (RCT). Then, we discuss separately the impact of abnormal cholesterol metabolism on common diseases and potential therapeutic targets for each disease, including CVD, tumors, neurological diseases, and immune system diseases. At the end of this review, we focus on the effect of cholesterol metabolism on eye diseases. In short, we hope to provide more new ideas for the pathogenesis and treatment of diseases from the perspective of cholesterol. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. The N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections. [ABSTRACT FROM AUTHOR]

Salicylic acids have been used in human and veterinary medicine for their anti-pyretic, anti-inflammatory, and analgesic properties for centuries. A key role of salicylic acid—immune modulation in response to microbial infection—was first recognized during studies of their botanical origin. The effects of salicylic acid on bacterial physiology are diverse. In many cases, they impose selective pressures leading to development of cross-resistance to antimicrobial compounds. Initial characterization of these interactions was in Escherichia coli, where salicylic acid activates the multiple antibiotic resistance (mar) operon, resulting in decreased antibiotic susceptibility. Studies suggest that stimulation of the mar phenotype presents similarly in closely related Enterobacteriaceae. Salicylic acids also affect virulence in many opportunistic pathogens by decreasing their ability to form biofilms and increasing persister cell populations. It is imperative to understand the effects of salicylic acid on bacteria of various origins to illuminate potential links between environmental microbes and their clinically relevant antimicrobial-resistant counterparts. This review provides an update on known effects of salicylic acid and key derivatives on a variety of bacterial pathogens, offers insights to possible potentiation of current treatment options, and highlights cellular regulatory networks that have been established during the study of this important class of medicines. [ABSTRACT FROM AUTHOR]

Background and Purpose: Blood–brain barrier (BBB) ABCB1, ABCG2 and ABCC5 transporters influence central therapeutic drug distribution. Transporter expression is regulated by the NR3C1, NR1I3 and NR1I2 nuclear receptors, but their precise roles in brain are poorly understood. We investigated the effects of selective ligand‐based activation of NR3C1, NR1I3, NR1I2 and NR2B1 in porcine brain endothelial cells (PBECs). Experimental Approach: Primary cultures of PBECs were exposed to NR3C1, NR1I3 and NR1I2 ligands and ABCB1, ABCG2 and ABCC5 transporter activities determined by measuring intracellular accumulation of fluorescent probes. Western blotting was used to determine the effects of receptor ligands on expression of ABCB1, ABCG2, ABCC5, NR1I2, NR1I3, NR3C1 and NR2B1. Fluorescent immunocytochemistry was employed to assess the effects of receptor ligands on the cellular localisation of NR1I2 and NR1I3. Key Results: The NR1I2 agonist rifampicin significantly up‐regulated ABCG2 activity, which is counteracted by co‐treatment with NR1I2 antagonist l‐sulforaphane. The NR1I3 agonist 6‐(4‐chlorophenyl)‐imidazo[2,1‐b]thiazole‐5‐carbaldehyde and inverse agonist meclizine significantly down‐regulated ABCB1, ABCG2 and ABCC5 activity. NR3C1 agonist dexamethasone significantly increased ABCB1, ABCG2 and ABCC5 activity and ABCG2 and ABCC5 protein expression, which was counteracted by co‐treatment with the NR3C1 antagonist mifepristone. This first study demonstrates that NR1I3 and NR3C1 regulate ABCC5 activity and protein expression in BBB endothelial cells. Conclusions and Implications: In PBECs, expression of key ATP‐binding cassette (ABC) transporters and nuclear receptors is differentially regulated by NR1I3, NR1I2, NR3C1 and NR2B1. This will help to better understand the response of the BBB to physiological and pharmacological activation of nuclear receptors. [ABSTRACT FROM AUTHOR]

Abstract Cholesterol homeostasis is crucial for cellular and systemic function. The disorder of cholesterol metabolism not only accelerates the onset of cardiovascular disease (CVD) but is also the fundamental cause of other ailments. The regulation of cholesterol metabolism in the human is an extremely complex process. Due to the dynamic balance between cholesterol synthesis, intake, efflux and storage, cholesterol metabolism generally remains secure. Disruption of any of these links is likely to have adverse effects on the body. At present, increasing evidence suggests that abnormal cholesterol metabolism is closely related to various systemic diseases. However, the exact mechanism by which cholesterol metabolism contributes to disease pathogenesis remains unclear, and there are still unknown factors. In this review, we outline the metabolic process of cholesterol in the human body, especially reverse cholesterol transport (RCT). Then, we discuss separately the impact of abnormal cholesterol metabolism on common diseases and potential therapeutic targets for each disease, including CVD, tumors, neurological diseases, and immune system diseases. At the end of this review, we focus on the effect of cholesterol metabolism on eye diseases. In short, we hope to provide more new ideas for the pathogenesis and treatment of diseases from the perspective of cholesterol.

The resistance–nodulation–division efflux machinery confers antimicrobial resistance to Gram-negative bacteria by actively pumping antibiotics out of the cell. The protein complex is powered by proton motive force; however, the proton transfer mechanism itself and indeed even its stoichiometry is still unclear. Here we review computational studies from the last decade that focus on elucidating the number of protons transferred per conformational cycle of the pump. Given the difficulties in studying proton movement using even state-of-the-art structural biology methods, the contributions from computational studies have been invaluable from a mechanistic perspective.

Background: Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic virulent bacterium with natural resistance to several antibiotics. It is an important causative agent in respiratory tract infections, surgical wound infections, burn wounds, and urinary tract infections. It has many virulent factors enhancing its pathogenesis e.g., tox A, type III secretion system that play a crucial role in tissue destruction. Pseudomonas aeruginosa is resistant to many antibiotics through different mechanisms including the availability of an efflux pump that extrudes antibiotics outside the bacteria. Numerous efflux genes are discovered e.g., MexAB-OprM, MexCD-OprJ, or MexXY (-OprA). Aim of work: To investigate some of the virulence genes and efflux pump genes in P. aeruginosa and to estimate their impact on antibiotic resistance. Methods: This current study was conducted on 250 different samples (sputum, urine, surgical wound, and burns) culture and sensitivity were done to the isolated bacteria, then molecular detection of efflux pump and some virulence genes by PCR. Results: Out of 250 samples, 46 yielded P. aeruginosa. These 46 samples were collected from 22 males and 24 females. Forty percent of the isolates were resistant to ceftazidime and cefepime followed by 37% resistance to ofloxacin and 36% resistance to gatifloxacin. Imipenem was the most susceptible antibiotic. Of all the isolated Pseudomonas 87% were multidrug-resistant. Exotoxin S (Exo S) gene was found predominantly in surgical wound samples 10 (21.7%), followed by burn samples, and sputum samples. Conclusion: There was a significant association between the availability of efflux genes and antibiotic resistance in Pseudomonas. Pseudomonas aeruginosa carries multiple mechanisms to destroy human cells and to compete with antibiotics, knowing these factors may be helpful to counteract them by targeting virulence or inhibiting efflux mechanisms.

Antibiotic-resistant strains of Staphylococcus aureus are being viewed as a serious threat by various public health agencies. Identifying novel targets in this important pathogen is crucial to the development of new effective antibacterial formulations. We investigated the antibacterial effect of a colloidal nanosilver formulation, Silversol®, against an antibiotic-resistant strain of S. aureus using appropriate in vitro assays. Moreover, we deciphered the molecular mechanisms underlying this formulation’s anti-S. aureus activity using whole transcriptome analysis. Lower concentrations of the test formulation exerted a bacteriostatic effect against this pathogen, and higher concentrations exerted a bactericidal effect. Silversol® at sub-lethal concentration was found to disturb multiple physiological traits of S. aureus such as growth, antibiotic susceptibility, membrane permeability, efflux, protein synthesis and export, biofilm and exopolysaccharide production, etc. Transcriptome data revealed that the genes coding for transcriptional regulators, efflux machinery, transferases, β-lactam resistance, oxidoreductases, metal homeostasis, virulence factors, and arginine biosynthesis are expressed differently under the influence of the test formulation. Genes (argG and argH) involved in arginine biosynthesis emerged among the major targets of Silversol®’s antibacterial activity against S. aureus.

The metabolism of a bacterial cell stretches beyond its boundaries, often connecting with the metabolism of other cells to form extended metabolic networks that stretch across communities, and even the globe. Among the least intuitive metabolic connections are those involving cross-feeding of canonically intracellular metabolites. How and why are these intracellular metabolites externalized? Are bacteria simply leaky? Here I consider what it means for a bacterium to be leaky, and I review mechanisms of metabolite externalization from the context of cross-feeding. Despite common claims, diffusion of most intracellular metabolites across a membrane is unlikely. Instead, passive and active transporters are likely involved, possibly purging excess metabolites as part of homeostasis. Re-acquisition of metabolites by a producer limits the opportunities for cross-feeding. However, a competitive recipient can stimulate metabolite externalization and initiate a positive-feedback loop of reciprocal cross-feeding. [ABSTRACT FROM AUTHOR]

Polar auxin transport is a quintessential feature of higher plant physiology and it has been known for many years that some of the primary drivers of polar auxin transport are the PIN-formed (PIN) auxin efflux proteins. Formative research established many key biochemical features of the transport system and discovered inhibitors such as 1-naphthylphthalamic acid (NPA), but the mechanism of action of PINs has remained elusive. This changed in 2022 with the publication of high-resolution structures of the membrane-spanning domains of three PIN proteins. The atomic structures and associated activity assays reveal that PINs use an elevator mechanism to transport auxin anions out of the cell. NPA was shown to be a competitive inhibitor that traps PINs in their inward-open conformation. The secrets of the hydrophilic cytoplasmic loop of PIN proteins remain to be discovered. [ABSTRACT FROM AUTHOR]

Background: Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic virulent bacterium with natural resistance to several antibiotics. It is an important causative agent in respiratory tract infections, surgical wound infections, burn wounds, and urinary tract infections. It has many virulent factors enhancing its pathogenesis e.g., tox A, type III secretion system that play a crucial role in tissue destruction. Pseudomonas aeruginosa is resistant to many antibiotics through different mechanisms including the availability of an efflux pump that extrudes antibiotics outside the bacteria. Numerous efflux genes are discovered e.g., MexABOprM, MexCD-OprJ, or MexXY (-OprA). Aim of work: To investigate some of the virulence genes and efflux pump genes in P. aeruginosa and to estimate their impact on antibiotic resistance. Methods: This current study was conducted on 250 different samples (sputum, urine, surgical wound, and burns) culture and sensitivity were done to the isolated bacteria, then molecular detection of efflux pump and some virulence genes by PCR. Results: Out of 250 samples, 46 yielded P. aeruginosa. These 46 samples were collected from 22 males and 24 females. Forty percent of the isolates were resistant to ceftazidime and cefepime followed by 37% resistance to ofloxacin and 36% resistance to gatifloxacin. Imipenem was the most susceptible antibiotic. Of all the isolated Pseudomonas 87% were multidrugresistant. Exotoxin S (Exo S) gene was found predominantly in surgical wound samples 10 (21.7%), followed by burn samples, and sputum samples. Conclusion: There was a significant association between the availability of efflux genes and antibiotic resistance in Pseudomonas. Pseudomonas aeruginosa carries multiple mechanisms to destroy human cells and to compete with antibiotics, knowing these factors may be helpful to counteract them by targeting virulence or inhibiting efflux mechanisms. [ABSTRACT FROM AUTHOR]

ABSTRACT To evaluate the resistance mechanisms among Pseudomonas aeruginosa clinical isolates exhibiting meropenem (MEM) MIC values higher than meropenem-vaborbactam (MEV). P. aeruginosa clinical isolates collected in US hospitals from 2014 to 2019 were susceptibility tested. Whole-genome and transcriptome sequencing were performed. Results were analyzed for strain typing, acquired β-lactamases, and mutations in chromosomal genes; gene expression was measured for known β-lactam resistance contributors. Results were compared to a control group of 10 P. aeruginosa isolates displaying MIC values at 8 mg/L for meropenem ± vaborbactam (MEM = MEV). Out of 88 isolates displaying MEM > MEV, 33 (37.5%) isolates had reproducibly lower MIC values for meropenem-vaborbactam compared to meropenem when retested. The expression of mexX, mexY, mexZ, and ampC was significantly greater among a higher percentage of the MEM > MEV isolates. Furthermore, the association of mexXY and ampC overexpression was detected in 17/33 MEM > MEV isolates and only 1/10 MEM = MEV isolate. In addition, the Pseudomonas-derived cephalosporinase amino acid substitution R79Q was detected among 33.3% of the isolates displaying MEM > MEV, and none of the isolates displayed MEM = MEV. Other resistance mechanisms were not observed or were equally observed in both groups. In rare cases, vaborbactam plays a role in lowering the meropenem MIC values in P. aeruginosa clinical isolates likely due to the inhibition of the AmpC gene that was overexpressed in the presence of upregulation of MexXY with or without alterations in the AmpC gene. IMPORTANCE Pseudomonas aeruginosa isolates are intrinsically resistant to multiple antimicrobial agents and meropenem is an important therapeutic option to treat infections caused by this organism. Meropenem-vaborbactam activity is similar to that of meropenem alone against P. aeruginosa isolates. Isolates belonging to this species that display lower meropenem-vaborbactam compared to meropenem are rare. We initiated this study to understand the resistance mechanisms that could lead to lower meropenem-vaborbactam MIC values when compared to meropenem alone. We documented that isolates displaying lower meropenem-vaborbactam exhibited overexpression of MexXY and AmpC. In addition, isolates displaying the R79Q PDC (AmpC) mutation were more likely to display lower meropenem-vaborbactam when compared to isolates displaying the same MIC values for these agents.

Synovial fluid (SF) from human knee joints with osteoarthritis (OA) has elevated levels of lysophosphatidylcholine (LPC) species, but their functional role is not well understood. This in vitro study was designed to test the hypothesis that various LPCs found elevated in OA SF and their metabolites, lysophosphatidic acids (LPAs), modulate the abundance of proteins and phospholipids (PLs) in human fibroblast-like synoviocytes (FLSs), with even minute chemical variations in lysophospholipids determining the extent of regulation. Cultured FLSs (n = 5–7) were treated with one of the LPC species, LPA species, IL-1β, or a vehicle. Tandem mass tag peptide labeling coupled with LC-MS/MS/MS was performed to quantify proteins. The expression of mRNA from regulated proteins was analyzed using RT-PCR. PL synthesis was determined via ESI-MS/MS, and the release of radiolabeled PLs was determined by means of liquid scintillation counting. In total, 3960 proteins were quantified using multiplexed MS, of which 119, 8, and 3 were significantly and reproducibly regulated by IL-1β, LPC 16:0, and LPC 18:0, respectively. LPC 16:0 significantly inhibited the release of PLs and the synthesis of phosphatidylcholine, LPC, and sphingomyelin. Neither LPC metabolite—LPA 16:0 nor LPA 18:0—had any reproducible effect on the levels of each protein. In conclusion, small chemical variations in LPC species can result in the significantly altered expression and secretion of proteins and PLs from FLSs. IL-1β influenced all proteins that were reproducibly regulated by LPC 16:0. LPC species are likely to modulate FLS protein expression only in more advanced OA stages with low IL-1β levels. None of the eight proteins being significantly regulated by LPC 16:0 have been previously reported in OA. However, our in vitro findings show that the CD81 antigen, calumenin, and B4E2C1 are promising candidates for further study, focusing in particular on their potential ability to modulate inflammatory and catabolic mechanisms. [ABSTRACT FROM AUTHOR]

Objective(s): This research was designed to study the prevalence of OqxAB efflux pump genes and also to investigate the relationship between efflux pump and resistance to antibiotics, especially to fluoroquinolones, evaluate the expression levels of OqxAB genes, and molecular typing of Klebsiella pneumoniae isolated from hospitalized patients in Hamadan hospitals, west of Iran.Materials and Methods: In a cross-sectional study, 100 clinical strains of K. pneumoniae were isolated from hospitalized patients in three major teaching hospitals from January to June 2021. The antibiotic susceptibility of isolates was evaluated by the disk-diffusion agar method. The frequency of genes encoding oqxA and oqxB of efflux pump genes was investigated by PCR, and the expression of the oqxA efflux pump gene was investigated by the Real-time PCR method. The genetic relationship of K. pneumoniae isolates was analyzed by the Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR technique.Results: According to our results, the multi-drug resistance phenotype (MDR) in 65% and high prevalence resistance to ciprofloxacin in 89% of K. pneumoniae isolates was detected. The higher prevalence of oqxA (95%) and oqxB (98%) was also detected. There was a significant relationship between ciprofloxacin resistance and the oqxB gene as well as between ceftriaxone and chloramphenicol resistance and the oqxA gene. The expression of the oqxA gene was higher in ciprofloxacin-resistant isolates.Conclusion: The results of this study suggest a potential reservoir for the spread of OqxAB genes among hospital-acquired bacteria. Infection control strategies should be used prudently to reduce the spread of resistant strains of K. pneumoniae in hospitals.

ATP-binding cassette (ABC) transporters are believed to protect aquatic organisms by pumping xenobiotics out, and recent investigation has suggested their involvement in the detoxification and efflux of nanoparticles (NPs), but their roles in fish embryos are poorly understood. In this regard, this paper summarizes the recent advances in research pertaining to the development of ABC transporter-mediated multi-xenobiotic resistance (MXR) mechanism in fish embryos and the potential interaction between ABC transporters and NPs. The paper focuses on: (1) Expression, function, and modulation mechanism of ABC proteins in fish embryos; (2) Potential interaction between ABC transporters and NPs in cell models and fish embryos. ABC transporters could be maternally transferred to fish embryos and thus play an important role in the detoxification of various chemical pollutants and NPs. There is a need to understand the specific mechanism to benefit the protection of aquatic resources.

Advanced age, accompanied by impaired glymphatic function, is a key risk factor for many neurodegenerative diseases. To study age-related differences in the human glymphatic system, we measured the influx and efflux activities of the glymphatic system via two non-invasive diffusion magnetic resonance imaging (MRI) methods, ultra-long echo time and low-b diffusion tensor imaging (DTIlow-b) measuring the subarachnoid space (SAS) flow along the middle cerebral artery and DTI analysis along the perivascular space (DTI-ALPS) along medullary veins in 22 healthy volunteers (aged 21-75 years). We first evaluated the circadian rhythm dependence of the glymphatic activity by repeating the MRI measurements at five time points from 8:00 to 23:00 and found no time-of-day dependence in the awake state under the current sensitivity of MRI measurements. Further test-retest analysis demonstrated high repeatability of both diffusion MRI measurements, suggesting their reliability. Additionally, the influx rate of the glymphatic system was significantly higher in participants aged >45 years than in participants aged 21-38, while the efflux rate was significantly lower in those aged >45 years. The mismatched influx and efflux activities in the glymphatic system might be due to age-related changes in arterial pulsation and aquaporin-4 polarization. [ABSTRACT FROM AUTHOR]

Resistance Nodulation Division (RND) efflux systems are ubiquitous transporters in gram-negative bacteria that provide protection against antimicrobial agents and thereby enhance survival in virtually all environments these prokaryotes inhabit. Vibrio cholerae is a dual lifestyle enteric pathogen that spends much of its existence in aquatic environments. An unwitting encounter with a human host can lead to V. cholerae intestinal colonization by strains that encode cholera toxin and toxin co-regulated pilus virulence factors leading to potentially fatal cholera diarrhea and dissemination in the environment. Adaptive response mechanisms to host factors encountered by these pathogens are therefore critical both to engage survival mechanisms such as RND-mediated transporters and to induce timely expression of virulence factors. Sensing of cues encountered in the host may therefore activate more than protective responses such as efflux systems, but also be coordinated to initiate expression of virulence factors. This review summarizes recent advances that contribute towards the understanding of RND efflux physiological functions and how the transport systems interface with the regulation of virulence factor production in V. cholerae. [ABSTRACT FROM AUTHOR]

Antipsychotic (AP)—induced adverse drug reactions (ADRs) are a current problem of biological and clinical psychiatry. Despite the development of new generations of APs, the problem of AP-induced ADRs has not been solved and continues to be actively studied. One of the important mechanisms for the development of AP-induced ADRs is a genetically-determined impairment of AP efflux across the blood-brain barrier (BBB). We present a narrative review of publications in databases (PubMed, Springer, Scopus, Web of Science E-Library) and online resources: The Human Protein Atlas; GeneCards: The Human Gene Database; US National Library of Medicine; SNPedia; OMIM Online Mendelian Inheritance in Man; The PharmGKB. The role of 15 transport proteins involved in the efflux of drugs and other xenobiotics across cell membranes (P-gp, TAP1, TAP2, MDR3, BSEP, MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, MRP7, MRP8, MRP9, BCRP) was analyzed. The important role of three transporter proteins (P-gp, BCRP, MRP1) in the efflux of APs through the BBB was shown, as well as the association of the functional activity and expression of these transport proteins with low-functional and non-functional single nucleotide variants (SNVs)/polymorphisms of the ABCB1, ABCG2, ABCC1 genes, encoding these transport proteins, respectively, in patients with schizophrenia spectrum disorders (SSDs). The authors propose a new pharmacogenetic panel "Transporter protein (PT)—Antipsychotic (AP) Pharmacogenetic test (PGx)" (PTAP-PGx), which allows the evaluation of the cumulative contribution of the studied genetic biomarkers of the impairment of AP efflux through the BBB. The authors also propose a riskometer for PTAP-PGx and a decision-making algorithm for psychiatrists. Conclusions: Understanding the role of the transportation of impaired APs across the BBB and the use of genetic biomarkers for its disruption may make it possible to reduce the frequency and severity of AP-induced ADRs, since this risk can be partially modified by the personalized selection of APs and their dosing rates, taking into account the genetic predisposition of the patient with SSD. [ABSTRACT FROM AUTHOR]

The main factors that determine the low effectiveness of chemotherapy are the low target bioavailability of antitumor drugs and the efflux process. In attempts to overcome this problem, several approaches are proposed here. Firstly, the development of polymeric micellar systems based on chitosan grafted by fatty acids (different types to optimize their properties), which, on the one hand, increase the solubility and bioavailability of cytostatics and, on the other hand, effectively interact with tumor cells due to the polycationic properties of chitosan, allowing for more effective penetration of cytostatic drugs into the cells. Secondly, the use of adjuvants—synergists of cytostatics (such as eugenol) included in the same micellar formulation—that selectively enhance the accumulation and retention of cytostatics in the tumor cells. pH- and temperature-sensitive polymeric micelles developed show high entrapment efficiency for both cytostatics and eugenol (EG) >60% and release the drug in a prolonged manner for 40 h in a weakly acidic medium corresponding to the microenvironment of tumors. In a slightly alkaline environment, the drug circulates longer (more than 60 h). The thermal sensitivity of micelles is realized due to an increase in the molecular mobility of chitosan, which undergoes a phase transition at 32–37 °C. The effect of the cytostatic drug doxorubicin (Dox) on cancerous A549 cells and model healthy cells of human embryonic renal epithelium (HEK293T) was studied by FTIR spectroscopy and fluorescence microscopy. Micellar Dox penetrates into cancer cells 2–3 times more efficiently when using EG adjuvant, which inhibits efflux, as demonstrated by a significant increase in the ratio of intra- and extracellular concentrations of the cytostatic. However, here it is worth remembering about healthy cells that they should not be damaged: according to changes in the FTIR and fluorescence spectra, the penetration of Dox into HEK293T when using micelles in combination with EG is reduced by 20–30% compared to a simple cytostatic. Thus, experimental developments of combined micellar cytostatic drugs have been proposed to increase the effectiveness of cancer treatment and overcome multiple drug resistance. [ABSTRACT FROM AUTHOR]

Simple Summary: Staphylococcus pseudintermedius is the main bacterial agent of skin and soft tissue infections in companion animals. The rising antimicrobial resistance in this species is a public health concern. Efflux activity is a resistance mechanism poorly characterized for this bacterium. This study aimed to evaluate efflux as contributor of biocide and fluoroquinolone resistance in S. pseudintermedius. Determination and application of cut-off values detected a non-wild type population against the biocide tetraphenylphosphonium bromide, linked to increased efflux activity. Further characterization of this efflux activity demonstrated that it is strain-specific and glucose-dependent. Fluoroquinolone resistance was mainly related to target mutations, which may be masking the contribution of efflux. This study highlights the relevance of efflux-mediated resistance in S. pseudintermedius, particularly to biocides, and provides a methodological basis for further studies on the efflux activity on this important veterinary pathogen. Staphylococcus pseudintermedius is the main bacterial cause of skin and soft tissue infections (SSTIs) in companion animals, particularly dogs. The emergence of methicillin-resistant S. pseudintermedius (MRSP) strains, frequently with multidrug resistance phenotypes is a public health concern. This study aimed to evaluate efflux, a resistance mechanism still poorly characterized in S. pseudintermedius, as a contributor to biocide and fluoroquinolone resistance. Susceptibility to the efflux pump substrates ethidium bromide (EtBr), tetraphenylphosphonium bromide (TPP) and ciprofloxacin (CIP) was evaluated by minimum inhibitory concentration (MIC) determination for 155 SSTIs-related S. pseudintermedius in companion animals. EtBr and TPP MIC distributions were analyzed to estimate cut-off (COWT) values. The effect of the efflux inhibitors (EIs) thioridazine and verapamil was assessed upon MICs and fluorometric EtBr accumulation assays, performed with/without glucose and/or EIs. This approach detected a non-wild type population towards TPP with increased efflux, showed to be strain-specific and glucose-dependent. Resistance to fluoroquinolones was mainly linked to target gene mutations, yet a contribution of efflux on CIP resistance levels could not be ruled out. In sum, this study highlights the relevance of efflux-mediated resistance in clinical S. pseudintermedius, particularly to biocides, and provides a methodological basis for further studies on the efflux activity on this important pathogen of companion animals. [ABSTRACT FROM AUTHOR]

The discovery void of antimicrobial development has occurred at a time when the world has seen a rapid emergence and spread of antimicrobial resistance, the 'perfect storm' as it has often been described. While the discovery and development of new antibiotics has continued in the research sphere, the pipeline to clinic has largely been fed by derivatives of existing classes of antibiotics, each prone to pre-existing resistance mechanisms. A novel approach to infection management has come from the ecological perspective whereby microbial networks and evolved communities already possess small molecular capabilities for pathogen control. The spatiotemporal nature of microbial interactions is such that mutualism and parasitism are often two ends of the same stick. Small molecule efflux inhibitors can directly target antibiotic efflux, a primary resistance mechanism adopted by many species of bacteria and fungi. However, a much broader anti-infective capability resides within the action of these inhibitors, borne from the role of efflux in key physiological and virulence processes, including biofilm formation, toxin efflux, and stress management. Understanding how these behaviors manifest within complex polymicrobial communities is key to unlocking the full potential of the advanced repertoires of efflux inhibitors. [ABSTRACT FROM AUTHOR]

LacY catalyzes accumulation of galactosides against a concentration gradient by coupling galactoside and H+ transport (i.e., symport). While alternating access of sugar- and H+-binding sites to either side of the membrane is driven by binding and dissociation of sugar, the electrochemical H+ gradient ([Formula: see text]) functions kinetically by decreasing the Km for influx 50- to 100-fold with no change in Kd The affinity of protonated LacY for sugar has an apparent pK (pKapp) of ∼10.5, due specifically to the pKa of Glu325, a residue that plays an irreplaceable role in coupling. In this study, rates of lactose/H+ efflux were measured from pH 5.0 to 9.0 in the absence or presence of a membrane potential (ΔΨ, interior positive), and the effect of the imposed ΔΨ on the kinetics of efflux was also studied in right-side-out membrane vesicles. The findings reveal that [Formula: see text] induces an asymmetry in the transport cycle based on the following observations: 1) the efflux rate of WT LacY exhibits a pKapp of ∼7.2 that is unaffected by the imposed ΔΨ; 2) ΔΨ increases the rate of efflux at all tested pH values, but enhancement is almost 2 orders of magnitude less than observed for influx; 3) mutant Glu325 - Ala does little or no efflux in the absence or presence of ΔΨ, and ambient pH has no effect; and 4) the effect of ΔΨ (interior positive) on the Km for efflux is almost insignificant relative to the 50- to 100-fold decrease in the Km for influx driven by ΔΨ (interior negative).

A small subpopulation of Vibrio splendidus AJ01 that was exposed to tetracycline at 10 times the minimal inhibitory concentration (MIC) still survived, named tetracycline-induced persister cells in our previous work. However, the formation mechanisms of persister is largely unknown. Here, we investigated tetracycline-induced AJ01 persister cells by transcriptome analysis and found that the purine metabolism pathway was significantly downregulated, which was consistent with lower levels of ATP, purine, and purine derivatives in our metabolome analysis. Inhibition of the purine metabolism pathway by 6-mercaptopurine (6-MP, inhibits ATP production), increased persister cell formation and accompanied with the decreasing intracellular ATP levels and increasing cells with protein aggresome. On the other hand, the persister cells had reduced intracellular tetracycline concentrations and higher membrane potential after 6-MP treatment. Inhibition of the membrane potential by carbonyl cyanide m-chlorophenyl hydrazone reversed 6-MP-induced persistence and resulted in higher levels of intracellular tetracycline accumulation. Meanwhile, cells with 6-MP treatment increased the membrane potential by dissipating the transmembrane proton pH gradient, which activated efflux to decrease the intracellular tetracycline concentration. Together, our findings show that reduction of purine metabolism regulates AJ01 persistence and is associated with protein aggresome formation and intracellular tetracycline efflux. [ABSTRACT FROM AUTHOR]

Aim To investigate the effect of adapting Klebsiella pneumoniae clinical isolates harboring ESBL genes to cetrimide (CT) in terms of subsequent cross-resistance to other biocides and antibiotics, and to investigate changes to virulence markers, such as biofilm formation and efflux activity. The changes between phenotypic extended spectrum β-lactamases (pESBL) expressing isolates and non-phenotypic ESBL (npESBL) isolates was compared. Methods and results Kl. pneumoniae isolates (14 pESBL and 17 npESBL) were adapted to increasing concentrations of CT, until 4 × MIC was reached. The MIC of the adapted isolates was tested against chloroxylenol (CX) and chlorhexidine. Disk diffusion techniques were used to determine the susceptibility of the isolates to different antibiotics. Biofilm formation was assessed for the isolates using the crystal violet method and efflux pump activity was studied using the ethidium bromide assay. After CT adaptation, 100% of npESBL isolates and 85.7% of pESBL isolates showed increase in CT MIC after CT adaptation. While 41.2% of npESBL and 57.1% of the pESBL isolates showed a cross-resistance with chlorhexidine. CT adaptation resulted in a significant decrease in the susceptibility of npESBL isolates to aztreonam and cefotaxime compared to pESBL isolates, which could be linked to the increase in efflux activity of npESBL compared to pESBL. Biofilm formation was significantly increased after CT adaptation regardless of the type of isolate. Conclusions The extensive use of biocides in the environment can induce cross-resistance to other biocides and antibiotics, and can increase the ability of bacteria to form biofilms. The response of bacteria to biocide adaptation differs between pESBL and npESBL isolates, although the effect is strain specific. [ABSTRACT FROM AUTHOR]

Objective(s): This research was designed to study the prevalence of OqxAB efflux pump genes and also to investigate the relationship between efflux pump and resistance to antibiotics, especially to fluoroquinolones, evaluate the expression levels of OqxAB genes, and molecular typing of Klebsiella pneumoniae isolated from hospitalized patients in Hamadan hospitals, west of Iran. Materials and Methods: In a cross-sectional study, 100 clinical strains of K. pneumoniae were isolated from hospitalized patients in three major teaching hospitals from January to June 2021. The antibiotic susceptibility of isolates was evaluated by the disk-diffusion agar method. The frequency of genes encoding oqxA and oqxB of efflux pump genes was investigated by PCR, and the expression of the oqxA efflux pump gene was investigated by the Real-time PCR method. The genetic relationship of K. pneumoniae isolates was analyzed by the Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR technique. Results: According to our results, the multi-drug resistance phenotype (MDR) in 65% and high prevalence resistance to ciprofloxacin in 89% of K. pneumoniae isolates was detected. The higher prevalence of oqxA (95%) and oqxB (98%) was also detected. There was a significant relationship between ciprofloxacin resistance and the oqxB gene as well as between ceftriaxone and chloramphenicol resistance and the oqxA gene. The expression of the oqxA gene was higher in ciprofloxacin-resistant isolates. Conclusion: The results of this study suggest a potential reservoir for the spread of OqxAB genes among hospital-acquired bacteria. Infection control strategies should be used prudently to reduce the spread of resistant strains of K. pneumoniae in hospitals. [ABSTRACT FROM AUTHOR]

α‐Aminoadipic acid (AAA) is a nonproteinogenic amino acid with potential applications in pharmaceutical, chemical and animal feed industries. Currently, AAA is produced by chemical synthesis, which suffers from high cost and low production efficiency. In this study, we engineered Escherichia coli for high‐level AAA production by coupling lysine biosynthesis and degradation pathways. First, the lysine‐α‐ketoglutarate reductase and saccharopine dehydrogenase from Saccharomyces cerevisiae and α‐aminoadipate‐δ‐semialdehyde dehydrogenase from Rhodococcus erythropolis were selected by in vitro enzyme assays for pathway assembly. Subsequently, lysine supply was enhanced by blocking its degradation pathway, overexpressing key pathway enzymes and improving nicotinamide adenine dineucleotide phosphate (NADPH) regeneration. Finally, a glutamate transporter from Corynebacterium glutamicum was introduced to elevate AAA efflux. The final strain produced 2.94 and 5.64 g/L AAA in shake flasks and bioreactors, respectively. This work provides an efficient and sustainable way for AAA production. [ABSTRACT FROM AUTHOR]

Resistance Nodulation Division (RND) efflux systems are ubiquitous transporters in gram-negative bacteria that provide protection against antimicrobial agents and thereby enhance survival in virtually all environments these prokaryotes inhabit. Vibrio cholerae is a dual lifestyle enteric pathogen that spends much of its existence in aquatic environments. An unwitting encounter with a human host can lead to V. cholerae intestinal colonization by strains that encode cholera toxin and toxin co-regulated pilus virulence factors leading to potentially fatal cholera diarrhea and dissemination in the environment. Adaptive response mechanisms to host factors encountered by these pathogens are therefore critical both to engage survival mechanisms such as RND-mediated transporters and to induce timely expression of virulence factors. Sensing of cues encountered in the host may therefore activate more than protective responses such as efflux systems, but also be coordinated to initiate expression of virulence factors. This review summarizes recent advances that contribute towards the understanding of RND efflux physiological functions and how the transport systems interface with the regulation of virulence factor production in V. cholerae.

PurposeImage-guided prostate biopsies are routinely acquired in the diagnosis and treatment monitoring of prostate cancer, yielding useful tissue for identifying metabolic biomarkers and therapeutic targets. We developed an optimized biopsy tissue culture protocol in combination with [1,6-13 C2 ]glucose labeling and quantitative high-resolution NMR to measure glycolysis and tricarboxcylic acid (TCA) cycle activity in freshly acquired living human prostate biopsies.MethodsWe acquired 34 MRI-ultrasound fusion-guided prostate biopsies in vials on ice from 22 previously untreated patients. Within 15 min, biopsies were transferred to rotary tissue culture in 37°C prostate medium containing [1,6-13 C2 ]glucose. Following 24 h of culture, tissue lactate and glutamate pool sizes and fractional enrichments were quantified using quantitative 1 H high resolution magic angle spinning Carr-Purcell-Meiboom-Gill (CPMG) spectroscopy at 1°C with and without 13 C decoupling. Lactate effluxed from the biopsy tissue was quantified in the culture medium using quantitative solution-state high-resolution NMR.ResultsLactate concentration in low-grade cancer (1.15 ± 0.78 nmol/mg) and benign (0.74 ± 0.15 nmol/mg) biopsies agreed with prior published measurements of snap-frozen biopsies. There was substantial fractional enrichment of [3-13 C]lactate (≈70%) and [4-13 C]glutamate (≈24%) in both low-grade cancer and benign biopsies. Although a significant difference in tissue [3-13 C]lactate fractional enrichment was not observed, lactate efflux was significantly higher (P < 0.05) in low-grade cancer biopsies (0.55 ± 0.14 nmol/min/mg) versus benign biopsies (0.31 ± 0.04 nmol/min/mg).ConclusionA protocol was developed for quantification of lactate production-efflux and TCA cycle activity in single living human prostate biopsies, allowing metabolic labeling on a wide spectrum of human tissues (e.g., metastatic, post-non-surgical therapy) from patients not receiving surgery.

ABSTRACT: Objectives: We evaluated the azole resistance mechanisms and epidemiology of fluconazole-resistant Candida glabrata from a global survey. Methods: A total of 2992 Candida spp. isolates collected during 2018–2019 were susceptibility tested by the broth microdilution reference method following CLSI guidelines. Fluconazole-resistant C. glabrata isolates were submitted to whole genome sequencing and gene expression assays using qRT-PCR. Results: Among 561 CGLA isolates tested, 34 (6.1%) were fluconazole resistant. These isolates were collected from 11 countries and mainly recovered from bloodstream infections (79.4%). All fluconazole-resistant C. glabrata isolates were non-wild type for voriconazole, 24/34 were non-wild type for posaconazole, but only 2/34 were non-wild type for itraconazole. Isavuconazole MIC values ranged from 0.25 to >4 mg/L. Fluconazole-resistant C. glabrata isolates belonged to 14 different sequence types (ST). None of the isolates exhibited alterations in ERG3 or ERG11, the target of azoles. All but two fluconazole-resistant isolates displayed overexpression of CgCDR1 (22/34; 64.7%) and/or CgCDR2 (26/34; 76.5%), while 16 isolates had both genes overexpressed. Overexpression of CgSNQ2 or ERG11 was not observed. Gain of function (GoF) alterations in the transcription factor CgPDR1 were noted in 14 isolates. Four (11.8%) isolates that were nonsusceptible to one or more echinocandins had FKS2 HS1 alterations (2 S663P and 2 F659Y/deletion). Conclusion: Fluconazole-resistant C. glabrata was driven by overexpression of CgCDR1 and/or CgCDR2. GoF alterations in PDR1 that have been associated with increased virulence were observed. Susceptibility results and surveillance data are needed to guide treatment for these isolates.

Jia-Ling Yang,1 Chia-Jui Yang,2,3 Yu-Chung Chuang,1 Wang-Huei Sheng,1 Yee-Chun Chen,1 Shan-Chwen Chang1 1Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of China; 2Department of Internal Medicine, Far Eastern Memorial Hospital, New Taipei City, Taiwan; 3School of Medicine, National Yang Ming Chiao Tung University, Taipei, TaiwanCorrespondence: Yu-Chung Chuang, Division of Infectious Diseases, Department of Internal Medicine, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Zhongzheng Dist, Taipei, 100225, Taiwan, Republic of China, Tel +886 2 2312 3456 ext 65054, Fax +886 2 2397 1412, Email weischuang@gmail.comPurpose: In this study, we evaluated the minocycline susceptibility rate in carbapenem-resistant Acinetobacter baumannii (CRAB) clinical strains, and the association between tetB carriage and minocycline susceptibility in CRAB.Patients and Methods: A total of 100 genetically unrelated CRAB clinical strains from bloodstream infection were randomly collected from a medical center in Taiwan. An argument for a new minocycline susceptibility breakpoint of 1 mg/L was suggested based on pharmacokinetic (PK) and pharmacodynamic (PD) studies. Strains with minocycline minimum inhibitory concentrations (MICs) of > 1 mg/L were classified as PK-PD non-susceptible. TetB carriage was detected by polymerase chain reaction (PCR).Results: Fifty-five (55%) CRAB strains were susceptible to minocycline according to the Clinical and Laboratory Standards Institute (CLSI) criteria, among which 98.2% (54/55) were PK-PD non-susceptible. The minocycline MIC50/90 was 4/16 mg/L. Ninety-seven (97%) strains carried tetB. All of the tetB-positive strains and 66.7% (2/3) of the tetB-negative strains were PK-PD non-susceptible. By statistical analysis, tetB carriage was significantly correlated with PK-PD non-susceptibility (P = 0.03) and a higher minocycline MIC (P = 0.02). The sensitivity and specificity of the tetB PCR for predicting PK-PD non-susceptibility were 98% and 100%, respectively.Conclusion: At our institute, most CRAB strains were PK-PD non-susceptible and most carried tetB gene. Recognizing the minocycline MIC and tetB status may be essential when using minocycline to treat CRAB-related infections.Keywords: CRAB, minocycline PK-PD non-susceptibility, multidrug resistance, efflux

Mycobacterium tuberculosis is a deadly pathogen, currently the leading cause of death worldwide from a single infectious agent through tuberculosis infections. If the End TB 2030 strategy is to be achieved, additional drugs need to be identified and made available to supplement the current treatment regimen. In addition, drug resistance is a growing issue, leading to significantly lower treatment success rates, necessitating further drug development. Vanoxerine (GBR12909), a dopamine re-uptake inhibitor, was recently identified as having anti-mycobacterial activity during a drug repurposing screening effort. However, its effects on mycobacteria were not well characterized. Herein, we report vanoxerine as a disruptor of the membrane electric potential, inhibiting mycobacterial efflux and growth. Vanoxerine had an undetectable level of resistance, highlighting the lack of a protein target. This study suggests a mechanism of action for vanoxerine, which will allow for its continued development or use as a tool compound.

HepaRG cells are highly‐differentiated human hepatoma cells, which are increasingly recognized as a convenient cellular model for in vitro evaluation of hepatic metabolism, transport, and/or toxicity of drugs. The present study was designed to evaluate whether HepaRG cells can also be useful for studying drug‐mediated inhibition of canalicular and/or sinusoidal hepatic efflux of bile acids, which constitutes a major mechanism of drug‐induced liver toxicity. For this purpose, HepaRG cells, initially loaded with the bile acid taurocholate (TC), were reincubated in TC‐free transport assay medium, in the presence or absence of calcium or drugs, before analysis of TC retention. This method allowed us to objectivize and quantitatively measure biliary and sinusoidal efflux of TC from HepaRG cells, through distinguishing cellular and canalicular compartments. In particular, time‐course analysis of the TC‐free reincubation period of HepaRG cells, that is, the efflux period, indicated that a 20 min‐efflux period allowed reaching biliary and sinusoidal excretion indexes for TC around 80% and 60%, respectively. Addition of the prototypical cholestatic drugs bosentan, cyclosporin A, glibenclamide, or troglitazone during the TC‐free efflux phase period was demonstrated to markedly inhibit canalicular and sinusoidal secretion of TC, whereas, by contrast, incubation with the noncholestatic compounds salicylic acid or flumazenil was without effect. Such data therefore support the use of human HepaRG cells for in vitro predicting drug‐induced liver toxicity (DILI) due to the inhibition of hepatic bile acid secretion, using a biphasic TC loading/efflux assay. [ABSTRACT FROM AUTHOR]

Cinnamaldehyde (CNA), the main component of cinnamon essential oil, is one of the most active plant compounds against nosocomial pathogen Pseudomonas aeruginosa. Exposure of wild-type strain PA14 (MIC 700 µg/mL) for 5 to 10 days to fixed (900 µg/mL) or increasing (from 900 to 1400 µg/mL) concentrations of this natural antibacterial resulted in emergence of resistant mutants CNA-A1 to A3, and CNA-B1 to B7, respectively. Genome sequencing experiments showed that each of CNA-A1 to A3 mutants differed from PA14 by one SNP, and a slight increase in CNA resistance level (from 700 to 900 µg/mL). By comparison, mutants B1 to B7 were more resistant (up to 1100 µg/mL); each of them harbored multiple SNPs (from 24 to 39) likely as a consequence of alteration of DNA mismatch repair gene mutS. Of the ten mutants selected, eight contained mutations in gene nalC, which indirectly downregulates expression of the operon that codes for multidrug efflux system MexAB-OprM, and showed increased resistance (up to 16-fold versus PA14) to antibiotic molecules exported by the pump, including ß-lactams and fluoroquinolones. Of the six mutants with the highest CNA resistance, five were no longer motile because of alteration of genes flgJ, fliE and/or pilJ genes. Altogether, our data show that P. aeruginosa is able to adapt to strong electrophilic molecules such as CNA by upregulating its intrinsic efflux pump MexAB-OprM, and through less well-characterized pleiotropic changes. Whether multidrug-resistant mutants can emerge in patients using cinnamon essential oil as self-medication needs to be assessed further. [ABSTRACT FROM AUTHOR]

Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate studies on resistance mechanisms towards QACs, we synthesized a fluorescent quaternary ammonium compound, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl] azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. As a proof-of-concept, NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC, and benzyl-dimethyl-dodecylammonium chloride (BAC12), a monoconstituent BAC with alkyl-chain length of 12 and high structural similarity to NBD-DDA. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBDDDA is similar to that of BAC. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized to the cell envelope of E. coli, which is a primary target of BAC and other QACs. Leveraging these findings and NBDDDA' s fluorescent properties, we show that reduced cellular accumulation is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC. Overall, NBD-DDA's antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study resistance mechanisms of QACs in bacteria and highlight its potential to gain detailed insights into its mode-of-action. [ABSTRACT FROM AUTHOR]

In addition to sphingomyelin and ceramide, sugar derivatives of ceramides, hexosylceramides (HexCer) are the major circulating sphingolipids. We have shown that silencing of ABCA1 transmembrane protein function for instance in cases of loss of function of ABCA1 gene results in low levels of HDL as well as a concomitant reduction in plasma HexCer levels. However, proteins involved in hepatic synthesis and egress of HexCer from cells is not well known although ABCA1 seems to be indirectly controlling the HexCer plasma levels by supporting HDL synthesis. In this study, we hypothesized that protein(s) other than ABCA1 are involved in the transport of HexCer to HDL. Using an unbiased knockdown approach, we found that ATP-binding cassette transporter protein C10 (ABCC10) participates in the synthesis of HexCer and thereby affects egress to HDL in human hepatoma Huh-7 cells. Furthermore, livers from ABCC10 deficient mice had significantly lower levels of HexCer compared to wild type livers. These studies suggest that ABCC10 partakes in modulating the synthesis and subsequent efflux of HexCer to HDL in liver cells. [ABSTRACT FROM AUTHOR]

Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate studies on resistance mechanisms towards QACs, we synthesized a fluorescent quaternary ammonium compound, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. As a proof-of-concept, NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC, and benzyl-dimethyl-dodecylammonium chloride (BAC12), a mono-constituent BAC with alkyl-chain length of 12 and high structural similarity to NBD-DDA. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBD-DDA is similar to that of BAC. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized to the cell envelope of E. coli, which is a primary target of BAC and other QACs. Leveraging these findings and NBD-DDA‘s fluorescent properties, we show that reduced cellular accumulation is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC. Overall, NBD-DDA’s antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study resistance mechanisms of QACs in bacteria and highlight its potential to gain detailed insights into its mode-of-action.

Uranium is a radioactive heavy metal and a significant public health concern; however, its associated underlying toxicological mechanisms remain largely unknown. In this work, the uptake and efflux processes of uranium in CHO-k1 cells were studied and the cytotoxicity effects were explored. It was found that both the uptake and efflux processes took place rapidly and half of the internalized uranium was expelled within 8 h. The uranium exposure caused a decrease of cell viability and adhesion ability in a dose-dependent manner and blocked the cell cycle at the G1 stage. In addition, gene expression analysis revealed relative changes in the transcription of metabolism related genes. Further studies revealed that the cytotoxicity of uranium could be alleviated by exposing cells to a lower temperature or by the addition of amantadine-HCl, an endocytosis inhibitor. Interestingly, after uranium exposure, needle-like precipitates were observed in both intracellular and extracellular regions. These findings collectively suggest that the cellular transport of uranium is a rapid process that disturbs cell metabolism and induces cytotoxicity, and this impact could be reduced by slowing down endocytic processes.

We report the emergence of imipenem-relebactam nonsusceptible Pseudomonas aeruginosa in 5 patients treated for nosocomial pneumonia for 10–28 days. Genome sequence analysis identified treatment-emergent mutations in MexAB-OprM and/or MexEF-OprN efflux operons that arose independently in each patient across distinct P. aeruginosa sequence types. Testing with efflux-inhibitor PAβN restored imipenem-relebactam susceptibility. [ABSTRACT FROM AUTHOR]