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Improving the nitrogen use efficiency is an effective way to increase the yield of rice, and maintaining carbon-nitrogen balance is essential for the normal growth and development of rice. To investigate the impact of the sucrose transporter protein OsSTP1 on nitrogen absorption in rice, in this study, we constructed transgenic lines overexpressing the sucrose transporter gene OsSTP1 ( OsSTP1-OE1 , OsSTP1-OE2 ) and mutant transgenic lines ( osstp1-1 , osstp1-2 ) from the wild type TB309. Further, we conducted a hydroponic experiment with four nitrogen supply levels of free nitrogen (FN, 0 mg/L), low nitrogen (LN, 10 mg/L), normal nitrogen (NN, 40 mg/L), and high nitrogen (HN, 80 mg/L) to study the responses of each line to different nitrogen supply levels during the seedling stage. The results showed that compared with the wild type and mutant lines in the LN group, the OsSTP1 -overexpressing lines exhibited significantly increased biomass, root length, and plant height, decreased soluble sugar content in the leaves, and increased soluble sugar content in the roots. The results indicate that the soluble sugars produced by leaf photosynthesis are transported to the roots through the phloem to promote root growth and nitrogen uptake, thus increasing the aboveground biomass. This study has identified that OsSTP1 can affect the long-distance transport of carbohydrates from source to sink to promote root growth, ultimately influencing rice's absorption and accumulation of nitrogen, improving nitrogen use efficiency and providing reference for reducing nitrogen fertilizer application.

Main Conclusion: In the present review, we discussed the detailed signaling cascades via membrane transporters that confer plant tolerance to abiotic stresses and possible significant use in plant development for climate-resilient crops. Plant transporters play significant roles in nutrient uptake, cellular balance, and stress responses. They facilitate the exchange of chemicals and signals across the plant's membrane by signal transduction, osmotic adjustment, and ion homeostasis. Therefore, research into plant transporters is crucial for understanding the mechanics of plant stress tolerance. Transporters have potential applications in crop breeding for increased stress resistance. We discuss new results about various transporter families (ABC, MATE, NRAMP, NRT, PHT, ZIP), including their functions in abiotic stress tolerance and plant development. Furthermore, we emphasize the importance of transporters in plant responses to abiotic stresses such as drought, cold, salt, and heavy metal toxicity, low light, flooding, and nutrient deficiencies. We discuss the transporter pathways and processes involved in diverse plant stress responses. This review discusses recent advances in the role of membrane transporters in abiotic stress tolerance in Arabidopsis and other crops. The review contains the genes discovered in recent years and associated molecular mechanisms that improve plants' ability to survive abiotic stress and their possible future applications by integrating membrane transporters with other technologies., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of Mycobacterium smegmatis , we identify a connection between wag31 and trehalose monomycolate (TMM) transporter mmpl3 in a suppressor screen and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 is responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation and in which the connection between Wag31, PlrA, and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate the synthesis of the different layers of the mycobacterial cell wall in changing conditions., Importance: This study is performed in Mycobacterium smegmatis , which is used as a model to understand the basic physiology of pathogenic mycobacteria such as Mycobacterium tuberculosis . In this work, we examine the function and regulation of three proteins involved in regulating cell wall elongation in mycobacterial cells, which occurs at the cell tips or poles. We find that Wag31, a regulator of polar elongation, works partly through the regulation of MmpL3, a transporter of cell wall constituents and an important drug target. Our work suggests that, beyond its transport function, MmpL3 has another function in controlling cell wall synthesis broadly in response to stress., Competing Interests: The authors declare no conflict of interest.

Small multidrug resistance (SMR) transporters are key players in the defense of multidrug-resistant pathogens to toxins and other homeostasis-perturbing compounds. However, recent evidence demonstrates that EmrE, an SMR from Escherichia coli and a model for understanding transport, can also induce susceptibility to some compounds by drug-gated proton leak. This runs down the ∆pH component of the proton-motive force (PMF), reducing the viability of the affected bacteria. Proton leak may provide an unexplored drug target distinct from the targets of most known antibiotics. Activating proton leak requires an SMR to be merely present, rather than be the primary resistance mechanism, and dissipates the energy source for many other efflux pumps. PAsmr, an EmrE homolog from Pseudomonas aeruginosa , transports many EmrE substrates in cells and purified systems. We hypothesized that PAsmr, like EmrE, may confer susceptibility to some compounds via drug-gated proton leak. Growth assays of E. coli expressing PAsmr displayed substrate-dependent resistance and susceptibility phenotypes, and in vitro solid-supported membrane electrophysiology experiments revealed that PAsmr performs both antiport and substrate-gated proton uniport, demonstrating the same functional promiscuity observed in EmrE. Growth assays of P. aeruginosa strain PA14 demonstrated that PAsmr contributes resistance to some antimicrobial compounds, but no growth defect is observed with susceptibility substrates, suggesting P. aeruginosa can compensate for the proton leak occurring through PAsmr. These phenotypic differences between P. aeruginosa and E. coli advance our understanding of the underlying resistance mechanisms in P. aeruginosa and prompt further investigation into the role that SMRs play in antibiotic resistance in pathogens., Importance: Small multidrug resistance (SMR) transporters are a class of efflux pumps found in many pathogens, although their contributions to antibiotic resistance are not fully understood. We hypothesize that these transporters may confer not only resistance but also susceptibility, by dissipating the proton-motive force. This means to use an SMR transporter as a target; it merely needs to be present (as opposed to being the primary resistance mechanism). Here, we test this hypothesis with an SMR transporter found in Pseudomonas aeruginosa and find that it can perform both antiport (conferring resistance) and substrate-gated proton leak. Proton leak is detrimental to growth in Escherichia coli but not P. aeruginosa , suggesting that P. aeruginosa responds differently to or can altogether prevent ∆pH dissipation., Competing Interests: The authors declare no conflict of interest.

Lactococcus lactis is a crucial food-grade cell factory for secreting valuable peptides and proteins primarily via the Sec-dependent pathway. YidC, a membrane insertase, facilitates protein insertion into the lipid membrane for the translocation. However, the mechanistic details of how YidC affects protein secretion in L. lactis remain elusive. This study investigates the effects of deleting yidC1 / yidC2 on L. lactis phenotypes and protein secretion. Compared to the original strain, deleting yidC2 significantly decreased the relative biomass, electroporation efficiency, and F-ATP activity by 25%, 47%, and 33%, respectively, and weakened growth and stress resistance, whereas deleting yidC1 had a minimal impact. The absence of either yidC1 or yidC2 reduced target proteins secretion. Meanwhile, there is a considerable alteration in the transcription levels of genes involved in the secretion pathway, with secY transcription increasing over 135-fold. Our results provide a theoretical foundation for further improving target protein secretion and investigating the YidC function.

Antimicrobial resistance is rapidly increasing worldwide, highlighting the urgent need for pharmaceutical and nonpharmaceutical interventions to tackle different-to-treat bacterial infections. Tigecycline, a semi-synthesis glycylcycline for parenteral administration, is widely recognized as one of the few effective therapies available against pan-drug resistant Gram-negative pathogens. Regrettably, the efficacy of multiple drugs, including tigecycline, is currently being undermined due to the emergence of a recently discovered mobilized resistance-nodulation-division-type efflux pump gene cluster tmexCD1-toprJ1 . Herein, by employing untargeted metabolomic approaches, we reveal that the expression of tmexCD1-toprJ1 disrupts bacterial purine metabolism, with inosine being identified as a crucial biomarker. Notably, the supplementation of inosine effectively reverses tigecycline resistance in tmexCD1-toprJ1 -positive bacteria. Mechanistically, exogenous inosine enhanced bacterial proton motive force, which promotes the uptake of tigecycline. Furthermore, inosine enhances succinate biosynthesis by stimulating the tricarboxylic acid cycle. Succinate interacts with the two-component system EnvZ/OmpR and upregulates OmpK 36, thereby promoting the influx of tigecycline. These actions collectively lead to the increased intracellular accumulation of tigecycline. Overall, our study offers a distinct combinational strategy to manage infections caused by tmexCD-toprJ -positive bacteria., Importance: TMexCD1-TOprJ1, a mobilized resistance-nodulation-division-type efflux pump, confers phenotypic resistance to multiple classes of antibiotics. Nowadays, tmexCD-toprJ has disseminated among diverse species of clinical pathogens, exacerbating the need for novel anti-infective strategies. In this study, we report that tmexCD1-toprJ1 -negative and -positive bacteria exhibit significantly different metabolic flux and characteristics, especially in purine metabolism. Intriguingly, the addition of inosine, a purine metabolite, effectively restores the antibacterial activity of tigecycline by promoting antibiotic uptake. Our findings highlight the correlation between bacterial mechanism and antibiotic resistance, and offer a distinct approach to overcome tmexCD-toprJ -mediated multidrug resistance., Competing Interests: The authors declare no conflict of interest.

The ability to derive retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) has led to numerous advances in the field of retinal research, with great potential for the use of hPSC-derived RGCs for studies of human retinal development, in vitro disease modeling, drug discovery, as well as their potential use for cell replacement therapeutics. Of all these possibilities, the use of hPSC-derived RGCs as a human-relevant platform for in vitro disease modeling has received the greatest attention, due to the translational relevance as well as the immediacy with which results may be obtained compared to more complex applications like cell replacement. While several studies to date have focused upon the use of hPSC-derived RGCs with genetic variants associated with glaucoma or other optic neuropathies, many of these have largely described cellular phenotypes with only limited advancement into exploring dysfunctional cellular pathways as a consequence of the disease-associated gene variants. Thus, to further advance this field of research, in the current study we leveraged an isogenic hPSC model with a glaucoma-associated mutation in the Optineurin (OPTN) protein, which plays a prominent role in autophagy. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor AMPK, along with subsequent neurodegeneration in OPTN(E50K) RGCs differentiated from hPSCs, and have further validated some of these findings in a mouse model of ocular hypertension. Pharmacological inhibition of mTORC1 in hPSC-derived RGCs recapitulated disease-related neurodegenerative phenotypes in otherwise healthy RGCs, while the mTOR-independent induction of autophagy reduced protein accumulation and restored neurite outgrowth in diseased OPTN(E50K) RGCs. Taken together, these results highlighted that autophagy disruption resulted in increased autophagic demand which was associated with downregulated signaling through mTORC1, contributing to the degeneration of RGCs., (© 2024. The Author(s).)

Efflux pumps are well known to be an important mechanism for removing noxious substances such as antibiotics from bacteria. Given that many antibiotics function by accumulating inside bacteria, efflux pumps contribute to resistance. Efflux pump inactivation is a potential strategy to combat antimicrobial resistance, as bacteria would not be able to pump out antibiotics. We recently discovered that the impact of loss of efflux function is only apparent in actively growing cells. We demonstrated that the global transcriptome of Salmonella Typhimurium is drastically altered during slower growth leading to stationary-phase cells having a remodeled, less permeable envelope that prevents antibiotics entering the cell. Here, we investigated the effects of deleting the major efflux pump of Salmonella Typhimurium, AcrB, on global gene transcription across growth. We revealed that an acrB knockout entered stationary phase later than the wild-type strain SL1344 and displayed increased and prolonged expression of genes responsible for anaerobic energy metabolism. We devised a model linking efflux and membrane potential, whereby deactivation of AcrB prevents influx of protons across the inner membrane and thereby hyperpolarization. Knockout or deactivation of AcrB was demonstrated to increase membrane potential. We propose that the global transcription regulator ArcBA senses changes to the redox state of the quinol pool (linked to the membrane potential of the bacterium) and coordinates the shift from exponential to stationary phase via the key master regulators RpoS, Rsd, and Rmf. Inactivation of efflux pumps therefore influences the fundamental physiology of Salmonella , with likely impacts on multiple phenotypes.IMPORTANCEWe demonstrate for the first time that deactivation of efflux pumps brings about changes to gross bacterial physiology and metabolism. Rather than simply being a response to noxious substances, efflux pumps appear to play a key role in maintenance of membrane potential and thereby energy metabolism. This discovery suggests that efflux pump inhibition or inactivation might have unforeseen positive consequences on antibiotic activity. Given that stationary-phase bacteria are more resistant to antibiotic uptake, late entry into stationary phase would prolong antibiotic accumulation by bacteria. Furthermore, membrane hyperpolarization could result in increased generation of reactive species proposed to be important for the activity of some antibiotics. Finally, changes in gross physiology could also explain the decreased virulence of efflux mutants., Competing Interests: The authors declare no conflict of interest.

Infections caused by Mycobacterium spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane. Here, we focus on MmpL3 from Mycobacterium tuberculosis , an essential inner membrane transporter of the resistance-nodulation-division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro , inside macrophages, and in M. tuberculosis- infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections.IMPORTANCEMmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition., Competing Interests: The authors declare no conflict of interest.

Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are useful for scaling in vitro and animal data to humans for accurate prediction and interpretation of drug clearance and toxicity. Targeted DMET proteomics that relies on synthetic stable isotope-labeled surrogate peptides as calibrators is routinely used for the quantification of selected proteins; however, the technique is limited to the quantification of a small number of proteins. Although the global proteomics-based total protein approach (TPA) is emerging as a better alternative for large-scale protein quantification, the conventional TPA does not consider differential sequence coverage by identifying unique peptides across proteins. Here, we optimized the TPA approach by correcting protein abundance data by the sequence coverage, which was applied to quantify 54 DMETs for characterization of 1) differential tissue DMET abundance in the human liver, kidney, and intestine, and 2) interindividual variability of DMET proteins in individual intestinal samples ( n = 13). Uridine diphosphate-glucuronosyltransferase 2B7 (UGT2B7), microsomal glutathione S-transferases (MGST1, MGST2, and MGST3) carboxylesterase 2 (CES2), and multidrug resistance-associated protein 2 (MRP2) were expressed in all three tissues, whereas, as expected, four cytochrome P450s (CYP3A4, CYP3A5, CYP2C9, and CYP4F2), UGT1A1, UGT2B17, CES1, flavin-containing monooxygenase 5, MRP3, and P-glycoprotein were present in the liver and intestine. The top three DMET proteins in individual tissues were: CES1>CYP2E1>UGT2B7 (liver), CES2>UGT2B17>CYP3A4 (intestine), and MGST1>UGT1A6>MGST2 (kidney). CYP3A4, CYP3A5, UGT2B17, CES2, and MGST2 showed high interindividual variability in the intestine. These data are relevant for enhancing in vitro to in vivo extrapolation of drug absorption and disposition and can be used to enhance the accuracy of physiologically based pharmacokinetic prediction of systemic and tissue concentration of drugs. SIGNIFICANCE STATEMENT: This study quantified the abundance and compositions of drug-metabolizing enzymes and transporters in pooled human liver, intestine, and kidney microsomes as well as individual intestinal microsomes using an optimized global proteomics approach. The data revealed large intertissue differences in the abundance of these proteins and high intestinal interindividual variability in the levels of cytochrome P450s (e.g., CYP3A4 and CYP3A5), uridine diphosphate-glucuronosyltransferase 2B17, carboxylesterase 2, and microsomal glutathione S-transferase 2. These data are applicable for the prediction of first-pass metabolism and tissue-specific drug clearance., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

The oculocutaneous albinism II ( OCA2 ) gene encodes a melanosomal transmembrane protein involved in melanogenesis. Recent genome-wide association studies have identified several single nucleotide polymorphisms within OCA2 genes that are involved in skin pigmentation. Nevertheless, there have been no attempts to modulate this gene to improve skin discoloration. Accordingly, our aim was to identify compounds that can reduce OCA2 expression and to develop a formula that can improve skin brightness and reduce hyperpigmented spots. In this study, we investigated the effects of OCA2 expression reduction on melanin levels, melanosome pH, and autophagy induction through siRNA knockdown. Additionally, we identified several bioactives that effectively reduce OCA2 expression. Ultimately, in a clinical trial, we demonstrated that topical application of those compounds significantly improved skin tone and dark spots compared to vitamin C, a typical brightening agent. These findings demonstrate that OCA2 is a promising target for the development of efficacious cosmetics and therapeutics designed to treat hyperpigmentation.

Efficient interbacterial competitions and diverse defensive strategies employed by various bacteria play a crucial role in acquiring a hold within a dense microbial community. The gut symbiont Bacteroides fragilis secretes an antimicrobial ubiquitin homologue (BfUbb) that targets an essential periplasmic PPIase to drive intraspecies bacterial competition. However, the mechanisms by which BfUbb enters the periplasm and its potential for interspecies antagonism remain poorly understood. Here, we employ transposon mutagenesis and identify a highly conserved TonB-dependent transporter SusCD (designated as ButCD) in B. fragilis as the BfUbb transporter. As a putative protein-related nutrient utilization system, ButCD is widely distributed across diverse Bacteroides species with varying sequence similarity, resulting in distinct import efficiency of Bacteroides ubiquitin homologues (BUbb) and thereby determining the species-specific toxicity of BUbb. Cryo-EM structural and functional investigations of the BfUbb-ButCD complex uncover distinctive structural features of ButC that are crucial for its targeting by BfUbb. Animal studies further demonstrate the specific and efficient elimination of enterotoxigenic B. fragilis (ETBF) in the murine gut by BfUbb, suggesting its potential as a therapeutic against ETBF-associated inflammatory bowel disease and colorectal cancer. Our findings provide a comprehensive elucidation of the species-specific toxicity exhibited by BUbb and explore its potential applications., (© 2024. The Author(s).)

YidC is a membrane protein that plays an important role in inserting newly generated proteins into lipid membranes. The Sec-dependent complex is responsible for inserting proteins into the lipid bilayer in bacteria. YidC facilitates the insertion and folding of membrane proteins, both in conjunction with the Sec complex and independently. Additionally, YidC acts as a chaperone during the folding of proteins. Multiple investigations have conclusively shown that Gram-positive bacterial YidC has Sec-independent insertion mechanisms. Through the use of microsecond-level all-atom molecular dynamics (MD) simulations, we have carried out an in-depth investigation of the YidC protein originating from Gram-negative bacteria. This research sheds light on the significance of multiple domains of the YidC structure at a detailed molecular level by utilizing equilibrium MD simulations. Specifically, multiple models of YidC embedded in the lipid bilayer were constructed to characterize the critical role of the C2 loop and the periplasmic domain (PD) present in Gram-negative YidC, which is absent in its Gram-positive counterpart. Based on our results, the C2 loop plays a role in the overall stabilization of the protein, most notably in the transmembrane (TM) region, and it also has an allosteric influence on the PD region. We have found critical inter- and intradomain interactions that contribute to the stability of the protein and its function. Finally, our study provides a hypothetical Sec-independent insertion mechanism for Gram-negative bacterial YidC.

Acinetobacter baumannii is a notorious opportunistic pathogen responsible for healthcare-associated infections worldwide. Efflux pumps play crucial roles in mediating antimicrobial resistance, motility, and virulence. In this study, we present the identification and characterization of the new A. baumannii efflux pump SxtP belonging to the MFS superfamily ( m ajor f acilitator s uperfamily), along with its associated activator L ysR - type t ranscriptional r egulator (LTTR) SxtR, demonstrating their roles in sulfamethoxazole/trimethoprim (also known as co-trimoxazole or SXT) resistance, surface-associated motility and virulence., Competing Interests: The authors declare no conflict of interest.

The endoplasmic reticulum (ER) is shaped by abundant membrane curvature-generating proteins that include the REEP family member REEP5. The REEP1 subfamily, consisting of four proteins in mammals (REEP1-4), is less abundant and lack a N-terminal region. Mutations in REEP1 and REEP2 cause Hereditary Spastic Paraplegia, but the function of these four REEP proteins remains enigmatic. Here we show that REEP1-4 reside in a unique vesicular compartment and identify features that determine their localization. Mutations in REEP1-4 that compromise curvature generation, including those causing disease, relocalize the proteins to the bulk ER. These mutants interact with wild-type proteins to retain them in the ER, consistent with their autosomal-dominant disease inheritance. REEP1 vesicles contain the membrane fusogen atlastin-1, but not general ER proteins. We propose that REEP1-4 generate these vesicles themselves by budding from the ER, and that they cycle back to the ER by atlastin-mediated fusion. The vesicles may serve to regulate ER tubule dynamics., (© 2024. The Author(s).)

Ethnopharmacological Relevance: Fuzheng Touxie Jiedu Huayu Decoction (FTJHD) is a commonly used clinical formula that has been found effective in resisting multidrug resistance-Pseudomonas aeruginosa in previous in vivo and in vitro studies., Aim of the Study: To investigate the antimicrobial effects of FTJHD and its drug-containing serum alone or in combination with ceftazidime on difficult-to-treat multidrug resistance-P. aeruginosa (DTMDR-P. aeruginosa)., Materials and Methods: The antibacterial effects of FTJHD and its drug-containing alone or in combination with ceftazidime against DTMDR-P. aeruginosa were examined by the tube dilution method and bacterial growth curves. The changes in the bacterial ultrastructure were examined by transmission electron microscopy. The biofilm formation ability of bacteria was examined by crystal violet staining and scanning electron microscopy. The expression of the MexAB-OprM efflux pump and quorum sensing system genes were validated through quantitative polymerase chain reaction. Molecular docking was used to evaluate the interaction between active components and the MexAB-OprM efflux pump., Results: FTJHD-containing serums at 1-, 2-, 4-, and 8-fold concentrations reduced the minimal inhibitory concentration (MIC) of ceftazidime against DTMDR-P. aeruginosa from 128 μg/mL to 64 μg/mL. Sub-inhibitory concentrations of ceftazidime in combination with FTJHD and FTJHD-containing serum prolonged the lag period of bacterial growth and reduced bacterial numbers. Additionally, 1/2 MIC of ceftazidime combined with FTJHD-containing serum significantly inhibited the activity of the MexAB-OprM efflux pump and quorum sensing system, thus reducing biofilm formation while causing more severe damage to the bacteria. Molecular docking revealed a strong affinity of quercetin, baicalein, luteolin, kaempferol, and β-sitosterol for the efflux pump regulatory proteins OprM and MexR., Conclusion: FTJHD can exert synergistic anti-DTMDR-P. aeruginosa effects with ceftazidime by inhibiting biofilm formation mediated by the MexAB-OprM efflux pump and quorum sensing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background emergence of multidrug-resistant (MDR) bacterial strains is a public health concern that threatens global and regional security. Efflux pump-overexpressing MDR strains from clinical isolates are the best subjects for studying the mechanisms of MDR caused by bacterial efflux pumps. A Klebsiella pneumoniae strain overexpressing the OqxB-only efflux pump was screened from a clinical strain library to explore reverse OqxB-mediated bacterial resistance strategies. We identified non-repetitive clinical isolated K. pneumoniae strains using a matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry clinical TOF-II (Clin-TOF-II) and susceptibility test screening against levofloxacin and ciprofloxacin. And the polymorphism analysis was conducted using pulsed-field gel electrophoresis. Efflux pump function of resistant strains is obtained by combined drug sensitivity test of phenylalanine-arginine beta-naphthylamide (PaβN, an efflux pump inhibitor) and detection with ethidium bromide as an indicator. The quantitative reverse transcription PCR was performed to assess whether the oqxB gene was overexpressed in K. pneumoniae isolates. Additional analyses assessed whether the oqxB gene was overexpressed in K. pneumoniae isolates and gene knockout and complementation strains were constructed. The binding mode of PaβN with OqxB was determined using molecular docking modeling. Among the clinical quinolone-resistant K. pneumoniae strains, one mediates resistance almost exclusively through the overexpression of the resistance-nodulation-division efflux pump, OqxB. Crystal structure of OqxB has been reported recently by N. Bharatham, P. Bhowmik, M. Aoki, U. Okada et al. (Nat Commun 12:5400, 2021, https://doi.org/10.1038/s41467-021-25679-0). The discovery of this strain will contribute to a better understanding of the role of the OqxB transporter in K. pneumoniae and builds on the foundation for addressing the threat posed by quinolone resistance.IMPORTANCEThe emergence of antimicrobial resistance is a growing and significant health concern, particularly in the context of K. pneumoniae infections. The upregulation of efflux pump systems is a key factor that contributes to this resistance. Our results indicated that the K. pneumoniae strain GN 172867 exhibited a higher oqxB gene expression compared to the reference strain ATCC 43816. Deletion of oqxB led a decrease in the minimum inhibitory concentration of levofloxacin. Complementation with oqxB rescued antibiotic resistance in the oqxB mutant strain. We demonstrated that the overexpression of the OqxB efflux pump plays an important role in quinolone resistance. The discovery of strain GN 172867 will contribute to a better understanding of the role of the OqxB transporter in K. pneumoniae and promotes further study of antimicrobial resistance., Competing Interests: The authors declare no conflict of interest.

Thiamine (vitamin B 1 ) functions as an essential coenzyme in cells. Humans and other mammals cannot synthesise this vitamin de novo and thus have to take it up from their diet. Eventually, every cell needs to import thiamine across its plasma membrane, which is mainly mediated by the two specific thiamine transporters SLC19A2 and SLC19A3. Loss of function mutations in either of these transporters lead to detrimental, life-threatening metabolic disorders. SLC19A3 is furthermore a major site of drug interactions. Many medications, including antidepressants, antibiotics and chemotherapeutics are known to inhibit this transporter, with potentially fatal consequences for patients. Despite a thorough functional characterisation over the past two decades, the structural basis of its transport mechanism and drug interactions has remained elusive. Here, we report seven cryo-electron microscopy (cryo-EM) structures of the human thiamine transporter SLC19A3 in complex with various ligands. Conformation-specific nanobodies enable us to capture different states of SLC19A3's transport cycle, revealing the molecular details of thiamine recognition and transport. We identify seven previously unknown drug interactions of SLC19A3 and present structures of the transporter in complex with the inhibitors fedratinib, amprolium and hydroxychloroquine. These data allow us to develop an understanding of the transport mechanism and ligand recognition of SLC19A3., (© 2024. The Author(s).)

Trace metals are required as cofactors in metalloproteins that are essential in microbial metabolism and growth. The microbial requirements of diverse metals and the capabilities of prokaryotic taxa to acquire these metals remain poorly understood. We present here results from metagenomic observations over an entire productive season in the region off Kerguelen Island (Indian Sector of the Southern Ocean). We observed seasonal patterns in the abundance of prokaryotic transporters of seven trace elements (zinc [Zn], manganese [Mn], nickel [Ni], molybdenum [Mo], tungsten [W], copper [Cu] and cobalt [Co]) and the consecutive spring and summer phytoplankton blooms were strong drivers of these temporal trends. Taxonomic affiliation of the functional genes revealed that Rhodobacteraceae had a broad repertoire of trace metal transporters (Mn, Zn, Ni, W and Mo) and a more restricted set was observed for other prokaryotic groups, such as Flavobacteriaceae (Zn), Nitrincolaceae (Ni and W) and Thioglobaceae (Mo). The prevalence of trace metal transporters within a prokaryotic group, as determined on the family level, was overall confirmed in representative metagenome-assembled genomes. We discuss the potential involvement of prokaryotic groups in processes related to organic matter utilisation that require these metals and the consequences on carbon and trace metal cycling in surface waters of the Southern Ocean., (© 2024 The Author(s). Environmental Microbiology published by John Wiley & Sons Ltd.)

Immune surveillance by cytotoxic T cells eliminates tumor cells and cells infected by intracellular pathogens. This process relies on the presentation of antigenic peptides by Major Histocompatibility Complex class I (MHC-I) at the cell surface. The loading of these peptides onto MHC-I depends on the peptide loading complex (PLC) at the endoplasmic reticulum (ER). Here, we uncovered that MHC-I antigen presentation is regulated by ER-associated degradation (ERAD), a protein quality control process essential to clear misfolded and unassembled proteins. An unbiased proteomics screen identified the PLC component Tapasin, essential for peptide loading onto MHC-I, as a substrate of the RNF185/Membralin ERAD complex. Loss of RNF185/Membralin resulted in elevated Tapasin steady state levels and increased MHC-I at the surface of professional antigen presenting cells. We further show that RNF185/Membralin ERAD complex recognizes unassembled Tapasin and limits its incorporation into PLC. These findings establish a novel mechanism controlling antigen presentation and suggest RNF185/Membralin as a potential therapeutic target to modulate immune surveillance., (© 2024. The Author(s).)

Preeclampsia (PE) is a prevalent obstetric complication affecting approximately 3-5% of pregnancies worldwide and is a major cause of maternal and perinatal morbidity and mortality. Preeclampsia is considered a disease of the endothelial system that can progress to eclampsia, characterized by seizures. Early diagnosis and appropriate management are crucial to improving maternal and fetal outcomes, as preeclampsia can lead to severe complications such as placental abruption, fetal growth restriction, and stroke. The pathophysiology of PE is complex, involving a combination of genetic, acquired, and immunological factors. A central feature of the condition is inadequate placentation and impaired uteroplacental perfusion, leading to local hypoxia, endothelial dysfunction, vasoconstriction, and immunological dysregulation. Recent evidence suggests that dysregulation of ion transporters may play a significant role in the adaptation of uterine circulation during placentation. These transporters are essential for maintaining maternal-fetal homeostasis, influencing processes such as nutrient exchange, hormone synthesis, trophoblast cell migration, and the function of smooth muscle cells in blood vessels. In preeclampsia, adverse conditions like hypoxia and oxidative stress result in the downregulation of ion, solute, and water transporters, impairing their function. This review focuses on membrane transporters involved in PE, discussing functional alterations and their physiological implications. The goal of this investigation is to enhance understanding of how dysregulation of ion and small molecule transporters contributes to the development and progression of preeclampsia, underscoring the importance of exploring these signaling pathways for potential therapeutic interventions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mycobacterial membrane protein Large 3 (MmpL3) of Mycobacterium tuberculosis (Mtb) is crucial for the translocation of trehalose monomycolate (TMM) across the inner bacterial cell membrane, making it a promising target for anti-tuberculosis (TB) drug development. While several structural, microbiological, and in vitro studies have provided significant insights, the precise mechanisms underlying TMM transport by MmpL3 and its inhibition remain incompletely understood at the atomic level. In this study, molecular dynamic (MD) simulations for the apo form and seven inhibitor-bound forms of Mtb MmpL3 were carried out to obtain a thorough comprehension of the protein's dynamics and function. MD simulations revealed that the seven inhibitors in this work stably bind to the central channel of the transmembrane domain and primarily forming hydrogen bonds with ASP251, ASP640, or both residues. Through dynamical cross-correlation matrix and principal component analysis analyses, several types of coupled motions between different domains were observed in the apo state, and distinct conformational states were identified using Markov state model analysis. These coupled motions and varied conformational states likely contribute to the transport of TMM. However, simulations of inhibitor-bound MmpL3 showed an enlargement of the proton channel, potentially disrupting coupled motions. This indicates that inhibitors may impair MmpL3's transport function by directly blocking the proton channel, thereby hindering coordinated domain movements and indirectly affecting TMM translocation., (© 2024 The Protein Society.)

SAR11 bacteria are the most abundant microorganisms in the surface ocean 1 and have global biogeochemical importance 2-4 . To thrive in their competitive oligotrophic environment, these bacteria rely heavily on solute-binding proteins that facilitate uptake of specific substrates via membrane transporters 5,6 . The functions and properties of these transport proteins are key factors in the assimilation of dissolved organic matter and biogeochemical cycling of nutrients in the ocean, but they have remained largely inaccessible to experimental investigation. Here we performed genome-wide experimental characterization of all solute-binding proteins in a prototypical SAR11 bacterium, revealing specific functions and general trends in their properties that contribute to the success of SAR11 bacteria in oligotrophic environments. We found that the solute-binding proteins of SAR11 bacteria have extremely high binding affinity (dissociation constant >20 pM) and high binding specificity, revealing molecular mechanisms of oligotrophic adaptation. Our functional data have uncovered new carbon sources for SAR11 bacteria and enable accurate biogeographical analysis of SAR11 substrate uptake capabilities throughout the ocean. This study provides a comprehensive view of the substrate uptake capabilities of ubiquitous marine bacteria, providing a necessary foundation for understanding their contribution to assimilation of dissolved organic matter in marine ecosystems., (© 2024. The Author(s).)

Micro- and nanoplastics (MPs/NPs) constitute emerging and widely-distributed environmental contaminants to which humans are highly exposed. They possibly represent a threat for human health. In order to identify cellular/molecular targets for these plastic particles, we have analysed the effects of exposure to manufactured polystyrene (PS) MPs and NPs on in vitro activity and expression of human membrane drug transporters, known to interact with chemical pollutants. PS MPs and NPs, used at various concentrations (1, 10 or 100 µg/mL), failed to inhibit efflux activities of the ATP-binding cassette (ABC) transporters P-glycoprotein, MRPs and BCRP in ABC transporter-expressing cells. Furthermore, PS particles did not impair the transport of P-glycoprotein or BCRP substrates across intestinal Caco-2 cell monolayers. Uptake activities of solute carriers (SLCs) such as OCT1 and OCT2 (handling organic cations) or OATP1B1, OATP1B3, OATP2B1, OAT1 and OAT3 (handling organic anions) were additionally not altered by PS MPs/NPs in HEK-293 cells overexpressing these SLCs. mRNA expression of ABC transporters and of the SLCs OCT1 and OATP2B1 in Caco-2 cells and human hepatic HepaRG cells were finally not impaired by a 48-h exposure to MPs/NPs. Altogether, these data indicate that human drug transporters are unlikely to be direct and univocal targets for synthetic PS MPs/NPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Renal transporters (cotransporters, channels, and claudins) mediate homeostasis of fluids and electrolytes and are targets of hormonal and therapeutic regulators. Assessing renal transporter abundance with antibody probes by immunoblotting is an essential tool for mechanistic studies. Although journals require authors to demonstrate antibody specificity, there are no consensus guidelines for kidney sample preparation leading to lab-to-lab variability in immunoblot results. In this study, we determined the impact of sample preparation, specifically freeze-thawed (Frozen) versus freshly processed (Fresh) kidneys (female and male rats and mice) on immunoblot signal detection of 15 renal transporters and the impact of protease inhibitors during homogenization. In female Sprague-Dawley rat kidneys homogenized with aprotinin, Na 2 EDTA, PMSF, and phosphatase inhibitors, immunodetection signals were ∼50% lower in Frozen versus Fresh samples for most transporters. Inclusion of additional inhibitors (Roche cOmplete Protease Inhibitor, "+") only partially increased transporter immunoblot signals to near Fresh levels. In male Sprague-Dawley rats, immunoblot signal density was lower in Frozen+ versus Fresh+ despite additional inhibitors. In C57BL/6 male mice, immunoblot signals from proximal tubule transporters were lower in Frozen versus Fresh by ∼25-50% and greater in Frozen+. In contrast, immunodetection signal was equivalent in female Frozen+ versus female Fresh+ for claudin 2, villin, AQP1, NKCC2, NCC, ENaCβ, ENaCɣ, claudin 7, AQP2, NKAα1, and NKAβ1. Thus, kidney sample preparation variables, including freeze-thaw and protease inhibition, have substantial transporter-specific effects on quantification of renal transporter abundance by immunoblot. These findings underscore the critical importance of assessing and reporting the impact of sample preparation protocols on transporter recovery to ensure robust rigor and reproducibility. NEW & NOTEWORTHY Freeze-thawing kidneys before homogenization is widely accepted in renal research. This study demonstrates that if kidneys are freeze-thawed just once before homogenization, immunoblot signals are reduced in a transporter-specific manner in rats and mice dependent on sex and that immunoblot signals can be partially recovered by adding additional protease inhibitors. These findings underscore the critical importance of assessing the impact of sample preparation, including freeze-thaw versus fresh, to ensure robust rigor and reproducibility.

Nontuberculous mycobacteria (NTM), which include the Mycobacterium avium complex, are classified as difficult-to-treat pathogens due to their ability to quickly develop drug resistance against the most common antibiotics used to treat NTM infections. The overexpression of efflux pumps (EPs) was demonstrated to be a key mechanism of clarithromycin (CLA) resistance in NTM. Therefore, in this work, 24 compounds from an in-house library, characterized by chemical diversity, were tested as potential NTM EP inhibitors (EPIs) against Mycobacterium smegmatis mc 2  155 and M. avium clinical isolates. Based on the acquired results, 12 novel analogs of the best derivatives 1b and 7b were designed and synthesized to improve the NTM EP inhibition activity. Among the second set of compounds, 13b emerged as the most potent NTM EPI. At a concentration of 4 µg/mL, it reduced the CLA minimum inhibitory concentration by 16-fold against the clinical isolate M. avium 2373 overexpressing EPs as primary mechanism of CLA resistance., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

Background and Aims: The inconsistent data on thiamine status in obese subjects necessitates an examination of genes associated with intestinal absorption of thiamine. We aimed to reveal thiamine status in obese subjects and examine the expression of SLC19A2/3 genes encoding thiamine transporters and Sp1 transcription factor., Methods and Results: Thirty-five adult obese subjects and 11 healthy controls were included in this cross-sectional study. Small intestine epithelial cells were used for quantitative RT-PCR analysis of the gene expression. The daily thiamine and energy intake were assessed with a food frequency questionnaire. Thiamine phosphate esters were hydrolyzed to free thiamine, and liquid chromatography with a tandem mass spectrometry-based method was used to measure total thiamine in whole blood. Daily energy intake according to body weight and daily carbohydrate intake were not significantly different between groups after adjustment for sex. Although daily thiamine intake was significantly lower in the obesity group (p = 0.015), obese subjects had significantly higher whole blood thiamine levels than controls (44.96 ± 14.6 ng/mL and 33.05 ± 8.6 ng/mL, p = 0.002). There was a significant positive correlation between whole blood thiamine and BMI (r = 0.342, p = 0.020). SLC19A2 gene expression was lower in those with BMI ≥35 kg/m 2 (p = 0.036). A significant positive correlation was found between SLC19A2 expression and whole blood thiamine level (r = 0.310, p = 0.038)., Conclusion: A possible association between intestinal thiamine intake and total thiamine in whole blood was determined. The transcriptional changes of genes encoding the high-affinity membrane thiamine transporters, especially SLC19A2, probably play a role in this relationship., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2024 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Dysregulation of melanin homeostasis is implicated in causing skin pigmentation disorders, such as melasma due to hyperpigmentation and vitiligo due to hypopigmentation. Although the synthesis of melanin has been well studied, the removal of the formed skin pigment requires more research. We determined that β-mangostin, a plant-derived metabolite, induces the degradation of already-formed melanin in the mouse B16F10 cell line. The whitening effect of β-mangostin is mediated by macroautophagy/autophagy, as it was abolished by the knockdown of ATG5 or RB1CC1/FIP200, and by treatment with 3-methyladenine, a phosphatidylinositol 3-kinase complex inhibitor. However, the exact autophagy mechanism of melanosome degradation remains unknown. Selective autophagy for a specific cellular organelle requires specific E3-ligases and autophagic receptors for the target organelle. In this study, an E3-ligase, RCHY1, and an autophagy receptor, OPTN (optineurin), were identified as being essential for melanophagy in the β-mangostin-treated B16F10 cell line. As per our knowledge, this is the first report of a specific mechanism for the degradation of melanosomes, the target organelle of melanophagy. These findings are expected to broaden the scope of melanin homeostasis research and can be exploited for the development of therapeutics for skin pigmentation disorders.

Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H + -coupled antiporters EmrE and SugE, the lactose permease LacY, the Na + /H + antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as K M , I MAX , and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests: Andre Bazzone reports a relationship with Nanion Technologies GmbH that includes employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The majority of mitochondrial proteins are encoded in the nucleus, translated on cytosolic ribosomes, and subsequently targeted to the mitochondrial surface. Their further import into the organelle is facilitated by highly specialized protein translocases. Mitochondrial precursor proteins that are destined to the mitochondrial matrix and, to some extent, the inner membrane, utilize translocase of the inner membrane (TIM23). This indispensable import machinery has been extensively studied in yeast. The translocating unit of the TIM23 complex in yeast consists of two membrane proteins, Tim17 and Tim23. In contrast to previous findings, recent reports demonstrate the primary role of Tim17, rather than Tim23, in the translocation of newly synthesized proteins. Very little is known about human TIM23 translocase. Human cells have two orthologs of yeast Tim17, TIMM17A and TIMM17B. Here, using computational tools, we present the architecture of human core TIM23 variants with either TIMM17A or TIMM17B, forming two populations of highly similar complexes. The structures reveal high conservation of the core TIM23 complex between human and yeast. Interestingly, both TIMM17A and TIMM17B variants interact with TIMM23 and reactive oxygen species modulator 1 (ROMO1); a homolog of yeast Mgr2, a protein that can create a channel-like structure with Tim17. The high structural conservation of proteins that form the core TIM23 complex in yeast and humans raises an interesting question about mechanistic and functional differences that justify existence of the two variants of TIM23 in higher eukaryotes., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Staphylococcus aureus is a bacterium responsible for resistance to multiple drugs and the efflux system is widely studied among the resistance mechanisms developed by this species. The present study evaluates the inhibition of the MepA efflux pump by thiadiazine-derived compounds. For this purpose, thiadiazine-derived compounds (IJ-14 to IJ-20) were tested against S. aureus K2068 strains. Microdilution tests were initially conducted to assess the Minimum Inhibitory Concentration (MIC) of the compounds and their efflux pump inhibition activity. In addition, fluorimetry tests were performed using BrEt emission and tests were conducted to inhibit the expression of the mepA gene. This involved comparing the bacterial gene expression with the antibiotic alone to the gene expression after combining compounds (IJ-17 and IJ-20) with the antibiotic. Furthermore, membrane permeability assessment tests and in silico molecular docking tests were performed. It was observed that the IJ17 and IJ20 compounds exhibited direct activity against the tested strain. The IJ17 compound produced significant results in the gene inhibition tests, which was also evidenced through the membrane permeability alteration test. These findings suggest that thiadiazine-derived compounds have promising effects against one of the main resistance mechanisms, with the IJ17 compound presenting observable mechanisms of action., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The inner membrane of mitochondria contains hundreds of different integral membrane proteins. These proteins transport molecules into and out of the matrix, they carry out multifold catalytic reactions and they promote the biogenesis or degradation of mitochondrial constituents. Most inner membrane proteins are encoded by nuclear genes and synthesized in the cytosol from where they are imported into mitochondria by translocases in the outer and inner membrane. Three different import routes direct proteins into the inner membrane and allow them to acquire their appropriate membrane topology. First, mitochondrial import intermediates can be arrested at the level of the TIM23 inner membrane translocase by a stop-transfer sequence to reach the inner membrane by lateral insertion. Second, proteins can be fully translocated through the TIM23 complex into the matrix from where they insert into the inner membrane in an export-like reaction. Carriers and other polytopic membrane proteins embark on a third insertion pathway: these hydrophobic proteins employ the specialized TIM22 translocase to insert from the intermembrane space (IMS) into the inner membrane. This review article describes these three targeting routes and provides an overview of the machinery that promotes the topogenesis of mitochondrial inner membrane proteins., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Barley (Hordeum vulgare) is an important global cereal crop and a model in genetic studies. Despite advances in characterising barley genomic resources, few mutant studies have identified genes controlling root architecture and anatomy, which plays a critical role in capturing soil resources. Our phenotypic screening of a TILLING mutant collection identified line TM5992 exhibiting a short-root phenotype compared with wild-type (WT) Morex background. Outcrossing TM5992 with barley variety Proctor and subsequent SNP array-based bulk segregant analysis, fine mapped the mutation to a cM scale. Exome sequencing pinpointed a mutation in the candidate gene HvPIN1a, further confirming this by analysing independent mutant alleles. Detailed analysis of root growth and anatomy in Hvpin1a mutant alleles exhibited a slower growth rate, shorter apical meristem and striking vascular patterning defects compared to WT. Expression and mutant analyses of PIN1 members in the closely related cereal brachypodium (Brachypodium distachyon) revealed that BdPIN1a and BdPIN1b were redundantly expressed in root vascular tissues but only Bdpin1a mutant allele displayed root vascular defects similar to Hvpin1a. We conclude that barley PIN1 genes have sub-functionalised in cereals, compared to Arabidopsis (Arabidopsis thaliana), where PIN1a sequences control root vascular patterning., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Introduction: Determining whether a new drug is a substrate, inhibitor or inducer of efflux or uptake membrane transporters has become a routine process during drug discovery and development. In vitro assays are utilized to establish whether a new drug has the potential to be an object (substrate) or precipitant (inhibitor, inducer) in transporter-mediated clinical drug-drug interactions. The findings from these in vitro experiments are then used to determine whether further in vivo drug interaction studies are necessary for a new drug., Areas Covered: This article provides an update on in vitro transporter assays, focusing on new uses of transfected cells, time-dependent inhibition, transporter induction, and complex model systems., Expert Opinion: The newer in vitro assays add to the toolbox in defining new drugs as transporter substrates, inhibitors, or inducers. Complex models such as spheroids, organoids, and microphysiological systems require standardization and further research with model transporter substrates and inhibitors. In drug discovery, the more traditional transporter assays may be employed as substrate and inhibitor screening assays. In drug development, more complex cell models can be employed in later drug development to better understand how transporter(s) are involved in the absorption, distribution, and excretion of new drugs.

Antibiotic resistance has become a pressing global health crisis, with bacterial infections increasingly difficult to treat due to the emergence of multidrug resistance. This study aims to identify potential chalcone molecules that interact with two key multidrug efflux pumps, AcrB and EmrD, of Escherichia coli, using advanced computational tools. In silico ADMET (absorption, distribution, metabolism, excretion, and toxicity), drug-likeness prediction, molecular docking, and molecular dynamics simulation analyses were conducted on a ligand library comprising 100 chalcone compounds against AcrB (PDB: 4DX5) and EmrD (PDB: 2GFP). The results demonstrated that Elastichalcone A (PubChem CID 102103730) exhibited a remarkable binding affinity of -9.9 kcal/mol against AcrB, while 4'-methoxy-4-hydroxychalcone (PubChem CID 5927890) displayed a binding affinity of -9.8 kcal/mol against EmrD. Both ligands satisfied drug-likeness rules and possessed favorable pharmacokinetic profiles. Molecular dynamics simulation of the AcrB-Elastichalcone A complex remained stable over 100 ns, with minimal fluctuations in root-mean-square deviation and root-mean-square fluctuation. The screened ligand library demonstrated good drug-likeness and pharmacokinetic properties. Moreover, the MM/PB(GB)SA calculation indicated the tight binding and thermodynamic stability of the simulated protein-ligand complexes. Overall, this study highlights the potential of chalcones as promising candidates for targeting multidrug efflux pumps, offering a potential strategy to overcome antibiotic resistance. Further exploration and optimization of these compounds may lead to the development of effective therapeutics against multidrug-resistant bacterial infections.Communicated by Ramaswamy H. Sarma.

The liver plays a pivotal role in drug disposition owing to the expression of transporters accounting for the uptake at the sinusoidal membrane and the efflux across the basolateral and canalicular membranes of hepatocytes of many different compounds. Moreover, intracellular mechanisms of phases I and II biotransformation generate, in general, inactive compounds that are more polar and easier to eliminate into bile or refluxed back toward the blood for their elimination by the kidneys, which becomes crucial when the biliary route is hampered. The set of transporters expressed at a given time, i.e., the so-called transportome, is encoded by genes belonging to two gene superfamilies named Solute Carriers (SLC) and ATP-Binding Cassette (ABC), which account mainly, but not exclusively, for the uptake and efflux of endogenous substances and xenobiotics, which include many different drugs. Besides the existence of genetic variants, which determines a marked interindividual heterogeneity regarding liver drug disposition among patients, prevalent diseases, such as cirrhosis, non-alcoholic steatohepatitis, primary sclerosing cholangitis, primary biliary cirrhosis, viral hepatitis, hepatocellular carcinoma, cholangiocarcinoma, and several cholestatic liver diseases, can alter the transportome and hence affect the pharmacokinetics of drugs used to treat these patients. Moreover, hepatic drug transporters are involved in many drug-drug interactions (DDI) that challenge the safety of using a combination of agents handled by these proteins. Updated information on these questions has been organized in this article by superfamilies and families of members of the transportome involved in hepatic drug disposition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales. However, tigecycline resistance in Klebsiella pneumoniae has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical K. pneumoniae isolates. A total of 153 clinical K. pneumoniae isolates were collected, and identified 15 tigecycline-resistant and three tigecycline-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Total RNAs from K. pneumoniae ATCC13883 and the laboratory-induced tigecycline-resistant strain were extracted and sequenced on an Illumina platform. Differentially expressed genes and regulatory small RNAs (sRNAs) were analyzed and validated in clinical isolates of K. pneumoniae using quantitative real-time PCR. RNA sequencing results showed that mdtABC efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of mdtABC was observed in a clinical K. pneumoniae isolate, which increased tigecycline minimum inhibitory concentrations (MICs) and was involved in tigecycline heteroresistance. Sequencing analysis of sRNA demonstrated that candidate sRNA-120 directly interacted with the mdtABC operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of K. pneumoniae. The sRNA-120 deletion strain displayed increased mRNA levels of mdtA, mdtB, and mdtC and an increase in MICs of tigecycline. The complemented strain of sRNA-120 restored the mRNA levels of these genes and the susceptibility to tigecycline. RNA antisense purification and parallel reaction monitoring mass spectrometry were performed to verify the interactions between sRNA-120 and mdtABC. Collectively, our study highlights that the post-transcriptional repression of mdtABC through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical K. pneumoniae isolates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Plants synthesize hundreds of small secretory peptides, which are perceived by the receptor-like kinase (RLK) family at the cell surface. Various signaling peptide-RLK pairs ensure plant adaptation to distinct environmental conditions. Here, we report that SERINE RICH ENDOGENOUS PEPTIDE (SCOOP) immune peptides modulate root growth and development by regulating PIN-FORMED (PIN)-regulated polar auxin transport in Arabidopsis. The SCOOP4 and SCOOP12 treatments impaired root gravitropic growth, auxin redistribution in response to gravistimulation, and PIN abundance in the PM. Furthermore, genetic and cell biological analyses revealed that these physiological and cellular effects of SCOOP4 and SCOOP12 peptides are mediated by the receptor MALE DISCOVERER1-INTERACTING RECEPTOR LIKE KINASE2 (MIK2) and the downstream mitogen-activated kinase MPK6. Biochemical evidence indicates that MPK6 directly phosphorylates the cytosolic loop of PIN proteins. Our work established a link between the immune signaling peptide SCOOPs and root growth pathways, providing insights into the molecular mechanisms underlying plant root adaptive growth in the defense response., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Background: SQ109 is a promising candidate drug for the treatment of patients with drug-resistant tuberculosis (DR-TB). The purpose of this study was to investigate the activity of SQ109 against clinical isolates of Mycobacterium tuberculosis (MTB) from patients with multidrug-resistant TB (MDR-TB) and pre-extensively drug-resistant TB (pre-XDR-TB), and to explore new drug-resistant mechanisms of SQ109., Methods: We evaluated the in vitro activity of SQ109 against clinical isolates from patients with MDR-TB and pre-XDR-TB using minimal inhibitory concentration (MIC) assay. The drug-resistant gene, mmpL3 of SQ109-resistant strains was sequenced, and a quantitative real-time PCR assay was used to analyze 28 efflux pump genes in SQ109-resistant strains without mmpL3 mutations. The role of candidate efflux pumps mmpL5 and mmpL7 on the MIC of SQ109 was evaluated using recombinantly cloned MmpL5 and MmpL7 expressed in Mycobacterium smegmatis., Results: The MIC 90 , MIC 95 and MIC 99 values of SQ109 for 225 clinical isolates of MTB were 0.25 mg/L, 0.5 mg/L and 1.0 mg/L, respectively. Among the pre-XDR strains, six showed resistance to SQ109 despite the absence of gene mutations in mmpL3. In six resistant pre-XDR strains, the MIC of SQ109 decreased with the use of an efflux pump inhibitor, and there was significant upregulation of mmpL5 and mmpL7 in two strains after exposure to SQ109. The presence of MmpL7 in Mycobacterium smegmatis resulted in decreased susceptibility to SQ109, with the MIC increasing from 16 mg/L to 32 mg/L., Conclusions: Our data demonstrated that SQ109 exhibited excellent levels of in vitro activity against MTB. MmpL7 may be a potential gene for MTB resistance to SQ109, providing a useful target for detecting SQ109 resistance in MTB., (© 2024. The Author(s).)

Biofilms in nature often exist as communities. In this study, an experimental mixed-species community consisting of Pseudomonas aeruginosa, Pseudomonas protegens and Klebsiella pneumoniae was used to investigate how AI-2 transporters affect interspecies interactions and composition. The K. pneumoniae lsrB/lsrD deletion mutants had a 10-25-fold higher concentration of extracellular AI-2 compared to the wild-type. Although these deletion mutants produced monospecies biofilms of similar biomass, the substitution of these mutants for the parental strain significantly altered composition. Dual-species biofilm assays demonstrated that the changes in composition were due to the cumulative effect of pairwise interactions. It was further revealed that K. pneumoniae being present physically in the consortium was important in AI-2 mediating composition in the consortium, and that AI-2 transporters were crucial in achieving maximum biomass in the community. In conclusion, these findings demonstrate that AI-2 transporters mediate interspecies interactions and is important in maintaining the compositional equilibrium of the community., (© 2024. The Author(s).)

SUMMARYThe discovery of bacterial efflux pumps significantly advanced our understanding of how bacteria can resist cytotoxic compounds that they encounter. Within the structurally and functionally distinct families of efflux pumps, those of the Resistance-Nodulation-Division (RND) superfamily are noteworthy for their ability to reduce the intracellular concentration of structurally diverse antimicrobials. RND systems are possessed by many Gram-negative bacteria, including those causing serious human disease, and frequently contribute to resistance to multiple antibiotics. Herein, we review the current literature on the structure-function relationships of representative transporter proteins of tripartite RND efflux pumps of clinically important pathogens. We emphasize their contribution to bacterial resistance to clinically used antibiotics, host defense antimicrobials and other biocides, as well as highlighting structural similarities and differences among efflux transporters that help bacteria survive in the face of antimicrobials. Furthermore, we discuss technical advances that have facilitated and advanced efflux pump research and suggest future areas of investigation that will advance antimicrobial development efforts., Competing Interests: The authors declare no conflict of interest.

The MtrCDE efflux pump of Neisseria gonorrhoeae exports a wide range of antimicrobial compounds that the gonococcus encounters at mucosal surfaces during colonization and infection and is a known gonococcal virulence factor. Here, we evaluate the role of this efflux pump system in strain FA1090 during in vivo human male urethral infection with N. gonorrhoeae using a controlled human infection model. With the strategy of competitive infections initiated with mixtures of wild-type FA1090 and an isogenic mutant FA1090 strain that does not contain a functional MtrCDE pump, we found that the presence of the efflux pump is not required for an infection to be established in the human male urethra. This finding contrasts with previous studies of in vivo infection in the lower genital tract of female mice, which demonstrated that mutant gonococci of a different strain (FA19) lacking a functional MtrCDE pump had a significantly reduced fitness compared to their wild-type parental FA19 strain. To determine if these conflicting results are due to strain or human vs. mouse differences, we conducted a series of systematic competitive infections in female mice with the same FA1090 strains as in humans, and with FA19 strains, including mutants that do not assemble a functional MtrCDE efflux pump. Our results indicate the fitness advantage provided by the MtrCDE efflux pump during infection of mice is strain dependent. Owing to the equal fitness of the two FA1090 strains in men, our experiments also demonstrated the presence of a colonization bottleneck of N. gonorrhoeae in the human male urethra, which may open a new area of inquiry into N. gonorrhoeae infection dynamics and control. TRIAL REGISTRATION. Clinicaltrials.gov NCT03840811., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The complexity of branching and curvilinear morphology of a complete mitochondrial network within each cell is challenging to analyze and quantify. To address this challenge, we developed an image analysis technique using persistent homology with a multiparameter filtration framework, combining image processing techniques in mathematical morphology. We show that such filtrations contain both topological and geometric information about complex cellular organelle structures, which allows a software program to extract meaningful features. Using this information, we also develop a connectivity index that describes the morphology of the branching patterns. As proof of concept, we utilize this approach to study how mitochondrial networks are altered by genetic changes in the Optineurin gene. Mutations in the autophagy gene Optineurin (OPTN) are associated with primary open-angle glaucoma (POAG), amyotrophic lateral sclerosis (ALS), and Paget's disease of the bone, but the pathophysiological mechanism is unclear. We utilized the proposed mathematical morphology-based multiparameter filtration and persistent homology approach to analyze and quantitatively compare how changes in the OPTN gene alter mitochondrial structures from their normal interconnected, tubular morphology into scattered, fragmented pieces., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Chung et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Proinflammatory cytokines, elevated during inflammation caused by infection and/or autoimmune disorders, result in reduced clearance of drugs eliminated primarily by cytochrome P450 enzymes (CYPs). However, the effect of cytokines on hepatic drug transporter expression or activity has not been well-studied. Here, using plated human hepatocytes (PHHs; n = 3 lots), we investigated the effect of interleukin (IL)-6, IL-1 β , tumor necrosis factor- α (TNF- α ), and interferon-γ (IFN-γ), on the mRNA expression and activity of hepatic drug transporters. PHHs were incubated for 72 hours at their pathophysiologically relevant plasma concentrations, both individually (0.01, 0.1, 1, 10 ng/ml) or as a cocktail (i.e., when each was combined at 0.1 or 1 ng/ml). Following cytokine cocktail exposure (1 ng/ml), significant downregulation of mRNA expression of organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, sodium/taurocholate cotransporting polypeptide (NTCP), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), multidrug and toxin extrusion protein 1, multidrug resistance proteins (MRP) 2, 3, and 4 was observed. While the mRNA expression of organic anion transporter (OAT) 2 and organic cation transporter (OCT) 1 was downregulated in two lots, it was upregulated in one lot. In agreement (mostly), the 1 ng/ml cytokine cocktail reduced OATP1B1/3, OATP2B1, OAT2, OCT1, and NTCP activity by 75%, 44%, 82%, 47%, and 80%, respectively. Interestingly, upregulation of OAT2 and OCT1 mRNA in one donor did not translate into the same directional change in activity. Although significant interlot variability was observed, in general, the above effects, using individual cytokines, could be attributed to IL-1 β , TNF- α , and IFN-γ. SIGNIFICANCE STATEMENT: To date, this is the first comprehensive study to investigate the effect of four major proinflammatory cytokines, both individually and as a cocktail, on the mRNA expression and activity of human hepatic drug transporters. The data obtained can be used in the future to predict transporter-mediated drug clearance changes during inflammation through physiologically based pharmacokinetic modeling and simulation., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Key Message: Promoters of moso bamboo silicon transporter genes PeLsi1-1 and PeLsi1-2 contain elements in response to hormone, silicon, and abiotic stresses, and can drive the expression of PeLsi1-1 and PeLsi1-2 in transgene Arabidopsis. Low silicon 1 (Lsi1) transporters from different species have been shown to play an important role in influxing silicon from soil. In previous study, we cloned PeLsi1-1 and PeLsi1-2 from Phyllostachys edulis and verified that PeLsi1-1 and PeLsi1-2 have silicon uptake ability. Furthermore, in this study, the promoters of PeLsi1-1(1910 bp) and PeLsi1-2(1922 bp) were cloned. Deletion analysis identified the key regions of the PeLsi1-1 and PeLsi1-2 promoters in response to hormone, silicon, and abiotic stresses. RT-qPCR analysis indicated that PeLsi1-1 and PeLsi1-2 were regulated by hormones, salt stress and osmotic stress. In addition, we found that the driving activity of the PeLsi1-1 and PeLsi1-2 promoters was regulated by 2 mM K 2 SiO 3 and PeLsi1-1-P3 ~ P4 and PeLsi1-2-P4 ~ 5 were the regions regulated by silicon. Overexpression of PeLsi1-1 or PeLsi1-2 driven by 35S promoter in Arabidopsis resulted in a threefold increase of Si accumulation, whereas transgenic plants showed deleterious symptoms and dwarf seedlings and shorter roots under 2 mM Si treatment. When the 35S promoter was replaced by PeLsi1-1 or PeLsi1-2 promoter, a similar Si absorption was achieved and the transgene plants grew normally. This study, therefore, demonstrates that the promoters of PeLsi1-1 and PeLsi1-2 are indeed effective in driving the expression of moso bamboo Lsi1 genes and leading to silicon uptake., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The rapid growth of cyanobacteria, particularly Microcystis aeruginosa, poses a significant threat to global water security. The proliferation of toxic Microcystis aeruginosa raises concerns due to its potential harm to human health and socioeconomic impacts. Dense blooms contribute to spatiotemporal inorganic carbon depletion, promoting interest in the roles of carbon-concentrating mechanisms (CCMs) for competitive carbon uptake. Despite the importance of HCO 3 - transporters, genetic evaluations and functional predictions in M. aeruginosa remain insufficient. In this study, we explored the diversity of HCO 3 - transporters in the genomes of 46 strains of M. aeruginosa, assessing positive selection for each. Intriguingly, although the Microcystis BicA transporter became a partial gene in 23 out of 46 genomic strains, we observed significant positive sites. Structural analyses, including predicted 2D and 3D models, confirmed the structural conservation of the Microcystis BicA transporter. Our findings suggest that the Microcystis BicA transport likely plays a crucial role in competitive carbon uptake, emphasizing its ecological significance. The ecological function of the Microcystis BicA transport in competitive growth during cyanobacterial blooms raises important questions. Future studies require experimental confirmation to better understand the role of the Microcysits BicA transporter in cyanobacterial blooms dynamics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The original UT-1 transporter gene was initially identified in the spiny dogfish ( Squalus acanthias ), but localization of the UT-1 protein was not determined. Subsequent UT-1 expression was shown to localize to the collecting tubule (CT) of the shark nephron in other shark species, with expression in a closely related chimaera species also located additionally at a lower level in the intermediate-I segment (IS-I) of the nephron. In spiny dogfish, two UT-1 splice variants are known (UT-1 long and short), and there was also a second UT-1 gene described (here termed Brain UT). In this study, a second splice variant of the second Brain UT gene was discovered. Expression profiles (mRNA) of UT-1 long and short and Brain UT were determined in a number of spiny dogfish tissues. Quantitative PCR in kidney samples showed that the level of the short variant of UT-1 was around 100 times higher than the long variant, which was itself expressed around 10 times higher than Brain UT cDNA/mRNA (in kidney). For the long variant, there was a significantly higher level of mRNA abundance in fish acclimatized to 75% seawater. Ultimately, three UT-1 antibodies were made that could bind to both the UT-1 short and long variant proteins. The first two of these showed bands of appropriate sizes on Western blots of around 52.5 and 46 kDa. The second antibody had some additional lower molecular weight bands. The third antibody was mainly bound to the 46 kDa band with faint 52.5 kDa staining. Both the 52.5 and 46 kDa bands were absent when the antibodies were pre-blocked with the peptide antigens used to make them. Across the three antibodies, there were many similarities in localization but differences in subcellular localization. Predominantly, antibody staining was greatest in the intermediate segment 1 (IS-I) and proximal (PIb) segments of the first sinus zone loop of the nephron, with reasonably strong expression also found at the start and middle of the late distal tubule (LDT; second sinus zone loop). While some expression in the collecting tubule (CT) could not be ruled out, the level of staining seemed to be low or non-existent in convoluted bundle zone nephron segments such as the CT. Hence, this suggests that spiny dogfish have a fundamentally different mode of urea absorption in comparison to that found in other shark species, potentially focused more on the nephron sinus zone loops than the CT.

Visceral cestodiases, like cysticercoses and echinococcoses, are caused by cystic larvae from parasites of the Cestoda class and are endemic or hyperendemic in many areas of the world. Current therapeutic approaches for these diseases are complex and present limitations and risks. Therefore, new safer and more effective treatments are urgently needed. The Niemann-Pick C1 (NPC1) protein is a cholesterol transporter that, based on genomic data, would be the solely responsible for cholesterol uptake in cestodes. Considering that human NPC1L1 is a known target of ezetimibe, used in the treatment of hypercholesterolemia, it has the potential for repurposing for the treatment of visceral cestodiases. Here, phylogenetic, selective pressure and structural in silico analyses were carried out to assess NPC1 evolutive and structural conservation, especially between cestode and human orthologs. Two NPC1 orthologs were identified in cestode species (NPC1A and NPC1B), which likely underwent functional divergence, leading to the loss of cholesterol transport capacity in NPC1A. Comparative interaction analyses performed by molecular docking of ezetimibe with human NPC1L1 and cestode NPC1B pointed out to similarities that consolidate the idea of cestode NPC1B as a target for the repurposing of ezetimibe as a drug for the treatment of visceral cestodiases., (© 2024. The Author(s).)

Truncating genetic variants of SORL1 , encoding the endosome recycling receptor SORLA, have been accepted as causal of Alzheimer's disease (AD). However, most genetic variants observed in SORL1 are missense variants, for which it is complicated to determine the pathogenicity level because carriers come from pedigrees too small to be informative for penetrance estimations. Here, we describe three unrelated families in which the SORL1 coding missense variant rs772677709, that leads to a p.Y1816C substitution, segregates with Alzheimer's disease. Further, we investigate the effect of SORLA p.Y1816C on receptor maturation, cellular localization, and trafficking in cell-based assays. Under physiological circumstances, SORLA dimerizes within the endosome, allowing retromer-dependent trafficking from the endosome to the cell surface, where the luminal part is shed into the extracellular space (sSORLA). Our results showed that the p.Y1816C mutant impairs SORLA homodimerization in the endosome, leading to decreased trafficking to the cell surface and less sSORLA shedding. These trafficking defects of the mutant receptor can be rescued by the expression of the SORLA 3Fn-minireceptor. Finally, we find that iPSC-derived neurons with the engineered p.Y1816C mutation have enlarged endosomes, a defining cytopathology of AD. Our studies provide genetic as well as functional evidence that the SORL1 p.Y1816C variant is causal for AD. The partial penetrance of the mutation suggests this mutation should be considered in clinical genetic screening of multiplex early-onset AD families., Competing Interests: Competing interests statement:Dr. Petsko is a member of the Scientific Advisory Boards of Amicus Therapeutics, Retromer Therapeutics, MeiraGTx, Aevum Therapeutics and Proclara Biosciences. Dr. Andersen is a consultant for Retromer Therapeutics. Dr. Petsko has equity exceeding $5,000 in Retromer Therapeutics and MeiraGTx. Dr. Andersen has filed a patent on how the SORL1 minigene can enhance the endosome recycling pathway, including its positive effect on the SORL1 mutant protein (p.Y1816C).

In Candida albicans, Cdr1 pumps azole drugs out of the cells to reduce intracellular accumulation at detrimental concentrations, leading to azole-drug resistance. Milbemycin oxime, a veterinary anti-parasitic drug, strongly and specifically inhibits Cdr1. However, how Cdr1 recognizes and exports azole drugs, and how milbemycin oxime inhibits Cdr1 remain unclear. Here, we report three cryo-EM structures of Cdr1 in distinct states: the apo state (Cdr1 Apo ), fluconazole-bound state (Cdr1 Flu ), and milbemycin oxime-inhibited state (Cdr1 Mil ). Both the fluconazole substrate and the milbemycin oxime inhibitor are primarily recognized within the central cavity of Cdr1 through hydrophobic interactions. The fluconazole is suggested to be exported from the binding site into the environment through a lateral pathway driven by TM2, TM5, TM8 and TM11. Our findings uncover the inhibitory mechanism of milbemycin oxime, which inhibits Cdr1 through competition, hindering export, and obstructing substrate entry. These discoveries advance our understanding of Cdr1-mediated azole resistance in C. albicans and provide the foundation for the development of innovative antifungal drugs targeting Cdr1 to combat azole-drug resistance., (© 2024. The Author(s).)