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Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by N-myristoyltransferases (NMT). Myristoylation is emerging as a promising cancer therapeutic target; however, the molecular determinants of sensitivity to NMT inhibition or the mechanism by which it induces cancer cell death are not completely understood. We report that NMTs are a novel therapeutic target in lung carcinoma cells with LKB1 and/or KEAP1 mutations in a KRAS-mutant background. Inhibition of myristoylation decreases cell viability in vitro and tumor growth in vivo. Inhibition of myristoylation causes mitochondrial ferrous iron overload, oxidative stress, elevated protein poly (ADP)-ribosylation, and death by parthanatos. Furthermore, NMT inhibitors sensitized lung carcinoma cells to platinum-based chemotherapy. Unexpectedly, the mitochondrial transporter translocase of inner mitochondrial membrane 17 homolog A (TIM17A) is a critical target of myristoylation inhibitors in these cells. TIM17A silencing recapitulated the effects of NMT inhibition at inducing mitochondrial ferrous iron overload and parthanatos. Furthermore, sensitivity of lung carcinoma cells to myristoylation inhibition correlated with their dependency on TIM17A. This study reveals the unexpected connection between protein myristoylation, the mitochondrial import machinery, and iron homeostasis. It also uncovers myristoylation inhibitors as novel inducers of parthanatos in cancer, and the novel axis NMT-TIM17A as a potential therapeutic target in highly aggressive lung carcinomas.SignificanceKRAS-mutant lung carcinomas with LKB1 and/or KEAP1 co-mutations have intrinsic therapeutic resistance. We show that these tumors are sensitive to NMT inhibitors, which slow tumor growth in vivo and sensitize cells to platinum-based chemotherapy in vitro. Inhibition of myristoylation causes death by parthanatos and thus has the potential to kill apoptosis and ferroptosis-resistant cancer cells. Our findings warrant investigation of NMT as a therapeutic target in highly aggressive lung carcinomas.

Multidrug resistance protein 4 (MRP4) is a broadly expressed ATP-binding cassette transporter that is unique among the MRP subfamily for transporting prostanoids, a group of signaling molecules derived from unsaturated fatty acids. To better understand the basis of the substrate selectivity of MRP4, we used cryogenic-electron microscopy to determine six structures of nanodisc-reconstituted MRP4 at various stages throughout its transport cycle. Substrate-bound structures of MRP4 in complex with PGE1, PGE2 and the sulfonated-sterol DHEA-S reveal a common binding site that accommodates a diverse set of organic anions and suggest an allosteric mechanism for substrate-induced enhancement of MRP4 ATPase activity. Our structure of a catalytically compromised MRP4 mutant bound to ATP-Mg2+ is outward-occluded, a conformation previously unobserved in the MRP subfamily and consistent with an alternating-access transport mechanism. Our study provides insights into the endogenous function of this versatile efflux transporter and establishes a basis for MRP4-targeted drug design.

Orphan solute carrier (SLC) represents a group of membrane transporters whose exact functions and substrate specificities are not known. Elucidating the function and regulation of orphan SLC transporters is not only crucial for advancing our knowledge of cellular and molecular biology but can potentially lead to the development of new therapeutic strategies. Here, we provide evidence for the biological function of a ubiquitous orphan lysosomal SLC, the Major Facilitator Superfamily Domain-containing Protein 1 (MFSD1), which has remained phylogenetically unassigned. Targeted metabolomics revealed that dipeptides containing either lysine or arginine residues accumulate in lysosomes of cells lacking MFSD1. Whole-cell patch-clamp electrophysiological recordings of HEK293-cells expressing MFSD1 on the cell surface displayed transport affinities for positively charged dipeptides in the lower mM range, while dipeptides that carry a negative net charge were not transported. This was also true for single amino acids and tripeptides, which MFSD1 failed to transport. Our results identify MFSD1 as a highly selective lysosomal lysine/arginine/histidine-containing dipeptide exporter, which functions as a uniporter.

Successful acclimation to copper (Cu) deficiency involves a fine balance between Cu import and export. In the green alga Chlamydomonas reinhardtii, Cu import is dependent on a transcription factor, Copper Response Regulator 1 (CRR1), responsible for activating genes in Cu-deficient cells. Among CRR1 target genes are two Cu transporters belonging to the CTR/COPT gene family (CTR1 and CTR2) and a related soluble protein (CTR3). The ancestor of these green algal proteins was likely acquired from an ancient chytrid and contained conserved cysteine-rich domains (named the CTR-associated domains, CTRA) that are predicted to be involved in Cu acquisition. We show by reverse genetics that Chlamydomonas CTR1 and CTR2 are canonical Cu importers albeit with distinct affinities, while loss of CTR3 did not result in an observable phenotype under the conditions tested. Mutation of CTR1, but not CTR2, recapitulates the poor growth of crr1 in Cu-deficient medium, consistent with a dominant role for CTR1 in high-affinity Cu(I) uptake. On the other hand, the overaccumulation of Cu(I) (20 times the quota) in zinc (Zn) deficiency depends on CRR1 and both CTR1 and CTR2. CRR1-dependent activation of CTR gene expression needed for Cu over-accumulation can be bypassed by the provision of excess Cu in the growth medium. Over-accumulated Cu is sequestered into the acidocalcisome but can become remobilized by restoring Zn nutrition. This mobilization is also CRR1-dependent, and requires activation of CTR2 expression, again distinguishing CTR2 from CTR1 and consistent with the lower substrate affinity of CTR2.One sentence summaryRegulation of Cu uptake and sequestration by members of the CTR family of proteins in Chlamydomonas.

Our previous study identified 52 antiplasmodial peptaibols isolated from fungi. To understand their antiplasmodial mechanism of action, we conducted phenotypic assays, assessed the in vitro evolution of resistance, and performed a transcriptome analysis of the most potent peptaibol, HZ NPDG-I. HZ NPDG-I and 2 additional peptaibols were compared for their killing action and stage dependency, each showing a loss of digestive vacuole (DV) content via ultrastructural analysis. HZ NPDG-I demonstrated a stepwise increase in DV pH, impaired DV membrane permeability, and the ability to form ion channels upon reconstitution in planar membranes. This compound showed no signs of cross resistance to targets of current clinical candidates, and 3 independent lines evolved to resist HZ NPDG-I acquired nonsynonymous changes in the P. falciparum multidrug resistance transporter, pfmdr1. Conditional knockdown of PfMDR1 showed varying effects to other peptaibol analogs, suggesting differing sensitivity.

Plants sense abscisic acid (ABA) using chemical-induced dimerization (CID) modules, including the receptor PYR1 and HAB1, a phosphatase inhibited by ligand-activated PYR1. This system is unique because of the relative ease with which ligand recognition can be reprogrammed. To expand the PYR1 system, we designed an orthogonal * module, which harbors a dimer interface salt bridge; X-ray crystallographic, biochemical and in vivo analyses confirm its orthogonality. We used this module to create PYR1*MANDI/HAB1* and PYR1*AZIN/HAB1*, which possess nanomolar sensitivities to their activating ligands mandipropamid and azinphos-ethyl. Experiments in Arabidopsis thaliana and Saccharomyces cerevisiae demonstrate the sensitive detection of banned organophosphate contaminants using living biosensors and the construction of multi-input/output genetic circuits. Our new modules enable ligand-programmable multi-channel CID systems for plant and eukaryotic synthetic biology that can empower new plant-based and microbe-based sensing modalities.

BackgroundTrichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized.ResultsTo address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose.ConclusionThis study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Cancer is a leading cause of death worldwide, accounting for about one among six deaths, so the quest for new and improved therapies is of crucial importance. The discovery of cisplatin as an anticancer agent has paved the way for the development of other metal-based therapeutic agents and Re(I)-based candidates have been recently found to show promising results. It is known as well that chirality plays a central role in the interactions of metal-based drugs with intrinsically chiral biomolecules such as membrane transport proteins or DNA. To further exploit this property, we have developed a series of diastereomeric dinuclear Re(I) complexes with chiral ligands containing pinene-bipyridine units. These complexes offer unique insights into the relation between stereochemistry and biological activity. Single-crystal X-ray diffraction studies, spectroscopic analysis, including UV-Vis and circular dichroism (CD), confirmed the chiral structures of these complexes. Biological activity assessments were carried out against various cancer cell lines, with a particular focus on breast and colon cancer. The diastereomers exhibited distinct anticancer activities, with some displaying promising results. Notably, one diastereomer showed exceptional cytotoxicity against HCT116 and MCF-7 cancer cells. This research underscores the significance of chirality in the design of novel anticancer agents, providing insights into the potential of dinuclear Re(I) complexes as effective candidates for cancer treatment. [ABSTRACT FROM AUTHOR]

The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B12 transport protein BtuB by specifically binding coenzyme B12 (AdoCbl) as its natural ligand. The B12 sensing riboswitch class is known to accept various B12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl‐binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in‐line probing. Chemical modifications at sidechain b of vitamin B12 show larger effects indicating crucial B12‐RNA interactions. When introducing the same modification to AdoCbl the influence of any side‐chain modification tested is reduced. This renders the impact of the adenosyl‐ligand for B12‐btuB riboswitch recognition clearly beyond the known role in affinity. [ABSTRACT FROM AUTHOR]

Hot springs have tremendous significance due to their divulging physiochemical features. In the recent past, metagenomics has emerged as a unique methodology to explore microbiota as well as new biocatalysts possessing advantageous biochemical properties from hot springs. In the present study, metagenomics has been employed for microbial diversity exploration and identification of genes involved in various metabolic pathways among two hot springs, Manikaran and Tatapani, located in Himachal Pradesh, India. Taxonomic analysis of both metagenomes revealed the dominance of the Proteobacteria phylum. Genomic signatures of other bacterial phyla such as Chloroflexi, Actinobacteria, Bacteroidetes, Cyanobacteria, Planctomycetes, and Firmicutes were also found in significant abundance in both the metagenomes. The abundance of microorganisms belonging to genera, especially Nitrospira, Thauera, Meiothermus, Thiobacillus, Massilia, and Anaerolinea, was reported to be prevalent in the hot springs. A significant amount of metagenomic data remained taxonomically unclassified, which indeed emphasizes the scientific importance of these thermoaquatic niches. The functional potential analysis of both metagenomes revealed pathways related to carbohydrate metabolism, followed by amino acid metabolism, energy metabolism, genetic information processing, metabolism of cofactors and vitamins, membrane transporter, and signal transduction. Exploration of biomass-modifying biocatalysts enumerated the presence of glycoside hydrolases, glycosyl transferases, polysaccharide lyases, and carbohydrate esterases in the metagenomic data. Together, these findings offer an in-depth understanding of the microbial inhabitants in North-Western Himalayan hot springs and their underlying potential for various biotechnological and industrial applications. [ABSTRACT FROM AUTHOR]

Simple Summary: This review focuses on the current advancements in the development of inhibitors and the application of kinesin family member 20A (KIF20A) as a cancer immunotherapy target. KIF20A, a mitotic kinesin overexpressed in various cancers, plays a pivotal role in cell division and is increasingly recognized as a crucial factor in tumor progression and a potential target for novel cancer treatments. The analysis begins with a detailed examination of the gene expression and protein structure of KIF20A, highlighting its interaction with critical cellular components that influence key processes such as Golgi membrane transport and mitotic spindle assembly. The primary focus is on the development of specific KIF20A inhibitors, detailing their roles and the challenges encountered in enhancing their efficacy, such as achieving specificity, overcoming tumor resistance, and optimizing delivery systems. Additionally, it delves into the prognostic value of KIF20A across multiple cancer types, emphasizing its role as a novel tumor-associated antigen, which lays the groundwork for the development of targeted peptide vaccines. The therapeutic efficacy of these vaccines as demonstrated in recent clinical trials is discussed. Future directions are proposed, including the integration of precision medicine strategies to personalize treatments and the use of combination therapies to improve outcomes. By concentrating on the significant potential of KIF20A as both a direct target for inhibitors and an antigen in cancer vaccines, this review sets a foundation for future research aimed at harnessing KIF20A for effective cancer treatment. [ABSTRACT FROM AUTHOR]

Zinc (Zn) is an essential element for plants. Numerous proteins in different cellular compartments require Zn for their structure and function. Zn can be toxic when it accumulates in high levels in the cytoplasm. Therefore, Zn homeostasis at tissue, cell, and organelle levels is vital for plant growth. A part of the metal tolerance protein (MTP) / Cation Diffusion Facilitator (CDF) transporters functions as Zn transporters, exporting Zn from the cytosol to various membrane compartments. In Arabidopsis thaliana, MTP1, MTP2, MTP3, MTP4, MTP5, and MTP12 are classified as Zn transporters (Zn-CDF). In this study, we systematically analyzed the localization of GFP-fused Zn-CDFs in the leaf epidermal cells of Nicotiana benthamiana. As previously reported, MTP1 and MTP3 were localized to tonoplast, MTP2 to endoplasmic reticulum, and MTP5 to Golgi. In addition, we identified the localization of MTP4 to trans-Golgi Network (TGN). Since MTP4 is specifically expressed in pollen, we analyzed the localization of MTP4-GFP in the Arabidopsis pollen tubes and confirmed that it is in the TGN. We also showed the Zn transport capability of MTP4 in yeast cells. We then analyzed the phenotype of an mtp4 T-DNA insertion mutant under both limited and excess Zn conditions. We found that their growth and fertility were not largely different from the wild-type. Our study has paved the way for investigating the possible roles of MTP4 in metallating proteins in the secretory pathway or in exporting excess Zn through exocytosis. In addition, our system of GFP-fused MTPs will help study the mechanisms for targeting transporters to specific membrane compartments. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion injury (IRI) is an intrinsic risk associated with liver transplantation. Ex vivo hepatic machine perfusion (MP) is an emerging organ preservation technique that can mitigate IRI, especially in livers subjected to prolonged warm ischemia time (WIT). However, a method to quantify the biological response to WIT during MP has not been established. Previous studies used physiologically based pharmacokinetic (PBPK) modeling to demonstrate that a decrease in hepatic transport and biliary excretion of the tracer molecule sodium fluorescein (SF) could correlate with increasing WIT in situ. Furthermore, these studies proposed intracellular sequestration of the hepatocyte canalicular membrane transporter multidrug resistance-associated protein 2 (MRP2) leading to decreased MRP2 activity (maximal transport velocity; Vmax) as the potential mechanism for decreased biliary SF excretion. We adapted an extant PBPK model to account for ex vivo hepatic MP and fit a six-parameter version of this model to control time-course measurements of SF in MP perfusate and bile. We then identified parameters whose values were likely insensitive to changes in WIT and fixed them to generate a reduced model with only three unknown parameters. Finally, we fit the reduced model to each individual biological replicate SF time course with differing WIT, found the mean estimated value for each parameter, and compared them using a one-way ANOVA. We demonstrated that there was a significant decrease in the estimated value of Vmax for MRP2 at the 30-min WIT. These studies provide the foundation for future studies investigating real-time assessment of liver viability during ex vivo MP. NEW & NOTEWORTHY: We developed a computational model of sodium fluorescein (SF) biliary excretion in ex vivo machine perfusion and used this model to assess changes in model parameters associated with the activity of MRP2, a hepatocyte membrane transporter, in response to increasing warm ischemia time. We found a significant decrease in the parameter value describing MRP2 activity, consistent with a role of decreased MRP2 function in ischemia-reperfusion injury leading to decreased secretion of SF into bile. [ABSTRACT FROM AUTHOR]

Intraventricular haemorrhage is a common complication of premature birth. Survivors are often left with cerebral palsy, intellectual disability and/or hydrocephalus. Animal models suggest that brain tissue shrinkage, with subsequent vascular stretch and tear, is an important step in the pathophysiology, but the cause of this shrinkage is unknown. Clinical risk factors for intraventricular haemorrhage are biomarkers of hypoxic–ischaemic stress, which causes mature neurons to swell. However, immature neuronal volume might shift in the opposite direction in these conditions. This is because immature neurons express the chloride, salt and water transporter NKCC1, which subserves regulatory volume increases in non-neural cells, whereas mature neurons express KCC2, which subserves regulatory volume decreases. When hypoxic–ischaemic conditions reduce active ion transport and increase the cytoplasmic membrane permeability, the effects of these transporters are diminished. Consequentially, mature neurons swell (cytotoxic oedema), whereas immature neurons might shrink. After hypoxic–ischaemic stress, in vivo and in vitro multi-photon imaging of perinatal transgenic mice demonstrated shrinkage of viable immature neurons, bulk tissue shrinkage and blood vessel displacement. Neuronal shrinkage was correlated with age-dependent membrane salt and water transporter expression using immunohistochemistry. Shrinkage of immature neurons was prevented by prior genetic or pharmacological inhibition of NKCC1 transport. These findings open new avenues of investigation for the detection of acute brain injury by neuroimaging, in addition to prevention of neuronal shrinkage and the ensuing intraventricular haemorrhage, in premature infants. [ABSTRACT FROM AUTHOR]

Aim: Understanding how COVID‐19 impacts the expression of clinically relevant drug metabolizing enzymes and membrane transporters (DMETs) is vital for addressing potential safety and efficacy concerns related to systemic and peripheral drug concentrations. This study investigates the impact of COVID‐19 severity on DMETs expression and the underlying mechanisms to inform the design of precise clinical dosing regimens for affected patients. Methods: Transcriptomics analysis of 102 DMETs, 10 inflammatory markers, and 12 xenosensing regulatory genes was conducted on nasopharyngeal swabs from 50 SARS‐CoV‐2 positive (17 outpatients, 16 non‐ICU, and 17 ICU) and 13 SARS‐CoV‐2 negative individuals, clinically tested through qPCR, in the Greater Toronto area from October 2020 to October 2021. Results: We observed a significant differential gene expression for 42 DMETs, 6 inflammatory markers, and 9 xenosensing regulatory genes. COVID‐19 severity was associated with the upregulation of AKR1C1, MGST1, and SULT1E1, and downregulation of ABCC10, CYP3A43, and SLC29A4 expressions. Altogether, SARS‐CoV‐2‐positive patients showed an upregulation in CYP2C9, CYP2C19, AKR1C1, SULT1B1, SULT2B1, and SLCO4A1 and downregulation in FMO5, MGST3, ABCC5, and SLCO4C1 compared with SARS‐CoV‐2 negative individuals. These dysregulations were associated with significant changes in the expression of inflammatory and xenosensing regulatory genes driven by the disease. GSTM3, PPARA, and AKR1C1 are potential biomarkers of the observed DMETs dysregulation pattern in nasopharyngeal swabs of outpatients, non‐ICU, and ICU patients, respectively. Conclusion: The severity of COVID‐19 is associated with the dysregulation of DMETs involved in processing commonly prescribed drugs, suggesting potential disease–drug interactions, especially for narrow therapeutic index drugs. [ABSTRACT FROM AUTHOR]

In warm and humid regions, the productivity of sorghum is significantly limited by the fungal hemibiotrophic pathogen Colletotrichum sublineola, the causal agent of anthracnose, a problematic disease of sorghum (Sorghum bicolor (L.) Moench) that can result in grain and biomass yield losses of up to 50%. Despite available genomic resources of both the host and fungal pathogen, the molecular basis of sorghum-C. sublineola interactions are poorly understood. By employing a dual-RNA sequencing approach, the molecular crosstalk between sorghum and C. sublineola can be elucidated. In this study, we examined the transcriptomes of four resistant sorghum accessions from the sorghum association panel (SAP) at varying time points post-infection with C. sublineola. Approximately 0.3% and 93% of the reads mapped to the genomes of C. sublineola and Sorghum bicolor, respectively. Expression profiling of in vitro versus in planta C. sublineola at 1-, 3-, and 5-days post-infection (dpi) indicated that genes encoding secreted candidate effectors, carbohydrate-active enzymes (CAZymes), and membrane transporters increased in expression during the transition from the biotrophic to the necrotrophic phase (3 dpi). The hallmark of the pathogen-associated molecular pattern (PAMP)-triggered immunity in sorghum includes the production of reactive oxygen species (ROS) and phytoalexins. The majority of effector candidates secreted by C. sublineola were predicted to be localized in the host apoplast, where they could interfere with the PAMP-triggered immunity response, specifically in the host ROS signaling pathway. The genes encoding critical molecular factors influencing pathogenicity identified in this study are a useful resource for subsequent genetic experiments aimed at validating their contributions to pathogen virulence. This comprehensive study not only provides a better understanding of the biology of C. sublineola but also supports the long-term goal of developing resistant sorghum cultivars. [ABSTRACT FROM AUTHOR]

Epidemiological studies suggest that poor nutrition during pregnancy predisposes offspring to the development of lifestyle-related noncommunicable diseases and psychiatric disorders later in life. However, the molecular mechanisms underlying this predisposition are not well understood. In our previous study, using rats as model animals, we showed that behavioral impairments are induced by prenatal undernutrition. In this study, we identified solute carrier 22 family member 23 (Slc22a23) as a gene that is irreversibly upregulated in the rat brain by undernutrition during fetal development. Because the substrate of the SLC22A23 transporter has not yet been identified and the biological role of the Slc22a23 gene in vivo is not fully understood, we generated pan-Slc22a23 knockout rats and examined their phenotype in detail. The Slc22a23 knockout rats showed a lean phenotype, an increase in spontaneous locomotion, and improved endurance, indicating that they are not overweight and are even healthier in an ad libitum feeding environment. However, the knockout rats had reduced hippocampal volume, and the behavioral analysis suggested that they may have impaired cognitive function regarding novel objects. [ABSTRACT FROM AUTHOR]

This review discusses how research around the oxysterol-binding protein family has evolved. We briefly summarize how this protein family, designated OSBP-related (ORP) or OSBP-like (OSBPL) proteins, was discovered, how protein domains highly conserved among family members between taxa paved the way for understanding their mechanisms of action, and how insights into protein structural and functional features help to understand their versatility as lipid transporters. We also discuss questions and future avenues of research opened by these findings. The investigations on oxysterol-binding protein family serve as a real-life example of the notion that science often advances as a collective effort of multiple lines of enquiry, including serendipitous routes. While original articles invariably explain the motivation of the research undertaken in rational terms, the actual paths to findings may be less intentional. Fortunately, this does not reduce the impact of the discoveries made. Besides hopefully providing a useful account of ORP family proteins, we aim to convey this message. [ABSTRACT FROM AUTHOR]

SLC4A11 is the most abundant membrane transport protein in corneal endothelial cells. Its functional presence is necessary to support the endothelial fluid pump that draws fluid from the corneal stroma, preventing corneal edema. Several molecular actions have been proposed for SLC4A11 including H2O transport and cell adhesion. One of the most reproduced actions that SLC4A11 mediates is a H+ (or OH-) conductance that is enhanced in the presence of NH4Cl. The mechanism by which this occurs is controversial with some providing evidence in favor of NH3-H+ cotransport and others providing evidence for uncoupled H+ transport that is indirectly stimulated by the effects of NH4Cl upon intracellular pH and membrane potential. In the present study we provide new evidence and revisit previous studies, to support a model in which NH4Cl causes direct allosteric activation of SLC4A11 by means of an acidic shift in the intracellular pK (pKi) that governs the relationship between intracellular pH (pHi) and SLC4A11 H+-conductance. These findings have important implications for the assignment of a physiological role for SLC4A11. [ABSTRACT FROM AUTHOR]

Laminarin is a cytosolic storage polysaccharide of phytoplankton and macroalgae and accounts for over 10% of the world's annually fixed carbon dioxide. Algal disruption, for example, by viral lysis releases laminarin. The soluble sugar is rapidly utilized by free-living planktonic bacteria, in which sugar transporters and the degrading enzymes are frequently encoded in polysaccharide utilization loci. The annotation of flavobacterial genomes failed to identify canonical laminarin utilization loci in several particle-associated bacteria, in particular in strains of Maribacter. In this study, we report in vivo utilization of laminarin by Maribacter forsetii accompanied by additional cell growth and proliferation. Laminarin utilization coincided with the induction of an extracellular endo-laminarinase, SusC/D outer membrane oligosaccharide transporters, and a periplasmic glycosyl hydrolase family 3 protein. An ABC transport system and sugar kinases were expressed. Endo-laminarinase activity was also observed in Maribacter sp. MAR_2009_72, Maribacter sp. Hel_I_7, and Maribacter dokdonensis MAR_2009_60. Maribacter dokdonensis MAR_2009_71 lacked the large endolaminarinase gene in the genome and had no endo-laminarinase activity. In all genomes, genes of induced proteins were scattered across the genome rather than clustered in a laminarin utilization locus. These observations revealed that the Maribacter strains investigated in this study participate in laminarin utilization, but in contrast to many free-living bacteria, there is no co-localization of genes encoding the enzymatic machinery for laminarin utilization. [ABSTRACT FROM AUTHOR]

OCTN1 and OCTN2 are membrane transport proteins encoded by the SLC22A4 and SLC22A5 genes, respectively. Even though several transcripts have been predicted by bioinformatics for both genes, only one functional protein isoform has been described for each of them. Both proteins are ubiquitous, and depending on the physiopathological state of the cell, their expression is regulated by well-known transcription factors, although some aspects have been neglected. A plethora of missense variants with uncertain clinical significance are reported both in the dbSNP and the Catalogue of Somatic Mutations in Cancer (COSMIC) databases for both genes. Due to their involvement in human pathologies, such as inflammatory-based diseases (OCTN1/2), systemic primary carnitine deficiency (OCTN2), and drug disposition, it would be interesting to predict the impact of variants on human health from the perspective of precision medicine. Although the lack of a 3D structure for these two transport proteins hampers any speculation on the consequences of the polymorphisms, the already available 3D structures for other members of the SLC22 family may provide powerful tools to perform structure/function studies on WT and mutant proteins. [ABSTRACT FROM AUTHOR]

Rabies virus causes an estimated 59,000 annual fatalities worldwide and promising therapeutic treatments are necessary to develop. In this study, affinity tag-purification mass spectrometry was employed to delineate RABV glycoprotein and host protein interactions, and PDIA3/ERP57 was identified as a potential inhibitor of RABV infection. PDIA3 restricted RABV infection with follow mechanisms: PDIA3 mediated the degradation of RABV G protein by targeting lysine 332 via the selective macroautophagy/autophagy pathway; The PDIA3 interactor, AP3B1 (adaptor related protein complex 3 subunit beta 1) was indispensable in PDIA3-triggered selective degradation of the G protein; Furthermore, PDIA3 competitively bound with NCAM1/NCAM (neural cell adhesion molecule 1) to block RABV G, hindering viral entry into host cells. PDIA3 190-199 aa residues bound to the RABV G protein were necessary and sufficient to defend against RABV. These results demonstrated the therapeutic potential of biologics that target PDIA3 or utilize PDIA3 190-199 aa peptide to treat clinical rabies.Abbreviation: aa: amino acids; ANXA2: annexin A2; AP-MS: affinity tag purification-mass spectrometry; AP3B1: adaptor related protein complex 3 subunit beta 1; ATP6V1A: ATPase H+ transporting V1 subunit A; ATP6V1H: ATPase H+ transporting V1 subunit H; BafA1: bafilomycin A1; CHX: cycloheximide; co-IP: co-immunoprecipitation; DDX17: DEAD-box helicase 17; DmERp60: drosophila melanogaster endoplasmic reticulum p60; EBOV: Zaire ebolavirus virus; EV: empty vector; GANAB: glucosidase II alpha subunit; G protein: glycoprotein; GRM2/mGluR2: glutamate metabotropic receptor 2; HsPDIA3: homo sapiens protein disulfide isomerase family A member 3; IAV: influenza virus; ILF2: interleukin enhancer binding factor 2; KO: knockout; MAGT1: magnesium transporter 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MmPDIA3: mus musculus protein disulfide isomerase associated 3; NCAM1/NCAM: neural cell adhesion molecule 1; NGFR/p75NTR: nerve growth factor receptor; NGLY1: N-glycanase 1; OTUD4: OTU deubiquitinase 4; PDI: protein disulfide isomerase; PPIs: protein-protein interactions; RABV: rabies virus; RUVBL2: RuvB like AAA ATPase 2; SCAMP3: secretory carrier membrane protein 3; ScPdi1: Saccharomyces cerevisiae s288c protein disulfide isomerase 1; SLC25A6: solute carrier family 25 member 6; SQSTM1/p62: sequestosome 1; VSV: vesicular stomatitis virus. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to investigate the effect of selenium on gut microbiota in mice with breast cancer under a high-fat diet. Methods: A total of 12 female BALB/c mice were randomly divided into two groups: 4 T1 + selenium+ high-fat diet group and 4 T1 + high-fat diet group. Mice were injected with 4 T1 cells on the right 4th mammary fat pad and kept on a high-fat diet. Fecal samples were collected, and DNA was extracted for metagenomic sequencing and bioinformatics analysis. Relevant target genes and pathways were annotated and metabolically analyzed to explore the intervention effect of selenium on breast cancer in the high-fat diet state. Results: Selenium supplementation in the high-fat diet altered the composition and diversity of gut microbiota in mice with breast cancer. The gut microbial composition was significantly different in the selenium intervention group, with an increased abundance of Proteobacteria, Actinobacteria, and Verrucomicrobia phyla and species such as Helicobacter ganmani, Helicobacter japonicus, and Akkermansia muciniphila, while phyla, such as Bacteroidetes, Firmicutes, Deferribacteres, and Spirochaetes, and species, such as Prevotella sp. MGM2, Muribaculum intestinale, Lactobacillus murinus, and Prevotella sp. MGM1, were decreased. Functional analysis revealed differential expression of genes related to carbohydrate-active enzymes, pathogen-host interactions, cell communication, cell auto-induction, membrane transporters, and virulence factors. Furthermore, 37 COGs and 48 metabolites with rising metabolic potential in the selenium intervention group were predicted. Conclusion: Selenium alters the homeostasis of gut microbiota in mice with breast cancer on a high-fat diet, affecting their composition, abundance, and associated metabolism. These findings suggest that the mechanism involves interfering with gut microbiota homeostasis, leading to altered synthesis of tumor-associated proteins and fatty acids and inducing tumor cell apoptosis and pyroptosis. [ABSTRACT FROM AUTHOR]

This article discusses the challenges and importance of accurately annotating transporters in metabolic modeling. It emphasizes the need for investment in accurate functional characterization to improve predictive tools and benefit fields such as personalized medicine, metabolic engineering, and microbial ecology. The text also discusses the types of errors that can occur in transporter annotations and the databases and tools available for transporter annotation. The document is a list of references to various scientific articles and databases related to protein subcellular localization, phenotypic testing, gene function, transporter systems, metabolic modeling, and microbial communities. These resources provide valuable information for researchers studying these areas of biology and can be used as references for further exploration. [Extracted from the article]

RNA thermometers are temperature-sensing non-coding RNAs that regulate the expression of downstream genes. A well-characterized RNA thermometer motif discovered in bacteria is the ROSE-like element (repression of heat shock gene expression). ATP-binding cassette (ABC) transporters are a superfamily of transmembrane proteins that harness ATP hydrolysis to facilitate the export and import of substrates across cellular membranes. Through structure-guided bioinformatics, we discovered that ROSE-like RNA thermometers are widespread upstream of ABC transporter genes in bacteria. X-ray crystallography, biochemistry, and cellular assays indicate that these RNA thermometers are functional regulatory elements. This study expands the known biological role of RNA thermometers to these key membrane transporters. [ABSTRACT FROM AUTHOR]

As a selective and potent inhibitor targeting the isocitrate dehydrogenase‐2 (IDH2) mutant protein, enasidenib obtained approval from the US Food and Drug Administration (FDA) in 2017 for adult patients with acute myeloid leukemia (AML) with an IDH2 mutation. In vitro investigations demonstrated that enasidenib affects various drug metabolic enzymes and transporters. This current investigation aimed to assess enasidenib on the pharmacokinetics (PKs) of CYP substrates, including dextromethorphan (CYP2D6 probe drug), flurbiprofen (CYP2C9 probe drug), midazolam (CYP3A4 probe drug), omeprazole (CYP2C19 probe drug), and pioglitazone (CYP2C8 probe drug), in patients with AML or myelodysplastic syndrome. Results showed that following the co‐administration of enasidenib (100 mg, once daily) for 28 days, the PK parameters AUC(0‐∞) and Cmax of dextromethorphan increased by 1.37 (90% confidence interval (CI): 0.96, 1.96) and 1.24 (90% CI: 0.94, 1.65)‐fold, respectively, compared to dextromethorphan alone. For flurbiprofen, these parameters increased by 1.14 (90%CI: 1.01, 1.29) and 0.97 (90% CI 0.86, 1.08)‐fold, respectively, when compared to flurbiprofen alone. Conversely, midazolam exhibited decreases to 0.57 (90% CI 0.34, 0.97) and 0.77 (90% CI 0.39, 1.53)‐fold, respectively, in comparison to midazolam alone. The parameters for omeprazole increased by 1.86 (90% CI: 1.33, 2.60) and 1.47 (0.93, 2.31)‐fold, respectively, compared to omeprazole alone, while those for pioglitazone decreased to 0.80 (90% CI: 0.62, 1.03) and 0.87 (90% CI: 0.65, 1.16)‐fold, respectively, in comparison to pioglitazone alone. These findings provide valuable insights into dose recommendations concerning drugs acting as substrates of CYP2D6, CYP2C9, CYP3A4, CYP2C19, and CYP2C8 when administered concurrently with enasidenib. [ABSTRACT FROM AUTHOR]

Introduction: Thyroid hormone transporters are essential for thyroid hormones to enter target cells. Monocarboxylate transporter (MCT) 8 is a key transporter and is expressed at the blood–brain barrier (BBB), in neural cells and many other tissues. Patients with MCT8 deficiency have severe neurodevelopmental delays because of cerebral hypothyroidism and chronic sequelae of peripheral thyrotoxicosis. The T3 analog 3,3′,5-triiodothyroacetic acid (TRIAC) rescued neurodevelopmental features in animal models mimicking MCT8 deficiency and improved key metabolic features in patients with MCT8 deficiency. However, the identity of the transporter(s) that facilitate TRIAC transport are unknown. Here, we screened candidate transporters that are expressed at the human BBB and/or brain–cerebrospinal fluid barrier and known thyroid hormone transporters for TRIAC transport. Materials and Methods: Plasma membrane expression was determined by cell surface biotinylation assays. Intracellular accumulation of 1 nM TRIAC was assessed in COS-1 cells expressing candidate transporters in Dulbecco's phosphate-buffered saline (DPBS)/0.1% glucose or Dulbecco's modified Eagle's medium (DMEM) with or without 0.1% bovine serum albumin (BSA). Expression of Slc22a8 was determined by fluorescent in situ hybridization in brain sections from wild-type and Mct8/Oatp1c1 knockout mice at postnatal days 12, 21, and 120. Results: In total, 59 plasma membrane transporters were selected for screening of TRIAC accumulation (n = 40 based on expression at the human BBB and/or brain–cerebrospinal fluid barrier and having small organic molecules as substrates; n = 19 known thyroid hormone transporters). Screening of the selected transporter panel showed that 18 transporters facilitated significant intracellular accumulation of TRIAC in DPBS/0.1% glucose or DMEM in the absence of BSA. In the presence of BSA, substantial transport was noted for SLCO1B1 and SLC22A8 (in DPBS/0.1% glucose and DMEM) and SLC10A1, SLC22A6, and SLC22A24 (in DMEM). The zebrafish and mouse orthologs of these transporters similarly facilitated intracellular accumulation of TRIAC. Highest Slc22a8 mRNA expression was detected in mouse brain capillary endothelial cells and choroid plexus epithelial cells at early postnatal time points, but was reduced at P120. Conclusions: Human SLC10A1, SLCO1B1, SLC22A6, SLC22A8, and SLC22A24 as well as their mouse and zebrafish orthologs are efficient TRIAC transporters. These findings contribute to the understanding of TRIAC treatment in patients with MCT8 deficiency and animal models thereof. [ABSTRACT FROM AUTHOR]

Of the 450 cell membrane transporters responsible for shuttling substrates, nutrients, hormones, neurotransmitters, antioxidants, and signaling molecules, approximately nine are associated with clinically relevant drug–drug interactions (DDIs) due to their role in drug and metabolite transport. Therefore, a clinical study evaluating potential transporter DDIs is recommended if an investigational product is intestinally absorbed, undergoes renal or hepatic elimination, or is suspected to either be a transporter substrate or perpetrator. However, many of the transporter substrates and inhibitors administered during a DDI study also affect cytochrome P450 (CYP) activity, which can complicate data interpretation. To overcome these challenges, the assessment of endogenous biomarkers can help elucidate the mechanism of complex DDIs when multiple transporters or CYPs may be involved. This perspective article will highlight how creative study designs are currently being utilized to address complex transporter DDIs and the role of physiology-based -pharmacokinetic (PBPK) models can play. [ABSTRACT FROM AUTHOR]

Na+/Cl− cotransporter 2 (Ncc2 or Slc12a10) is a membrane transport protein that belongs to the electroneutral cation-chloride cotransporter family. The Slc12a10 gene (slc12a10) is widely present in bony vertebrates but is deleted or pseudogenized in birds, some bony fishes, and most mammals. Slc12a10 is highly homologous to Ncc (Slc12a3 or Ncc1); however, there are only a few reports measuring the activity of Slc12a10. In this study, we focused on zebrafish Slc12a10.1 (zSlc12a10.1) and analyzed its activity using Xenopus oocyte electrophysiology. Analysis using Na+-selective microelectrodes showed that intracellular sodium activity (aNai) in zSlc12a10.1 oocytes was significantly decreased in Na+- or Cl−-free medium and recovered when Na+ or Cl− was readded to the medium. Similar analysis using a Cl−-selective microelectrode showed that intracellular chloride activity (aCli) in zSlc12a10.1 oocytes significantly decreased in Na+- or Cl−-free medium and recovered when Na+ or Cl− was readded to the medium. When a similar experiment was performed with a voltage clamp, the membrane current did not change when aNai of zSlc12a10.1 oocytes was decreased in Na+-free medium. Molecular phylogenetic and synteny analyses suggest that gene duplication between slc12a10.2 and slc12a10.3 in zebrafish is a relatively recent event, whereas gene duplication between slc12a10.1 and the ancestral gene of slc12a10.2/slc12a10.3 occurred at least about 2 million years ago. slc12a10 deficiency was observed in species belonging to Ictaluridae, Salmoniformes, Osmeriformes, Batrachoididae, Syngnathiformes, Gobiesociformes, Labriformes, and Tetraodontiformes. These results indicate that zebrafish Slc12a10.1 is an electroneutral Na+/Cl−cotransporter and establish its evolutionary position among various teleost slc12a10 paralogs. NEW & NOTEWORTHY: Na+/Cl− cotransporter 2 (Slc12a10; Ncc2) is a protein highly homologous to Ncc (Slc12a3; Ncc1); however, there are only a few reports measuring the activity of Slc12a10. Electrophysiological analysis of Xenopus oocytes expressing zebrafish Slc12a10.1 showed that Slc12a10.1 acts as an electroneutral Na+/Cl−cotransporter. This is the third report on the activity of Slc12a10, following previous reports on Slc12a10 in eels. [ABSTRACT FROM AUTHOR]

Purpose: Incomplete partition type II (IP-II) is characterized by specific histological features and radiological appearance. It may occur in isolation or in association with an enlarged vestibular aqueduct (EVA). Among those with IP-II and EVA, a subset has a diagnosis of Pendred syndrome. This study aimed to explore the prevalence of isolated IP-II, IP-II with EVA, and cases with a genetic or syndromic basis in our cohort. Methods: From a large, multicentre database of dysplastic cochleae (446 patients, 892 temporal bones), those with imaging features of IP-II were examined in detail, including whether there was a genetic or syndromic association. Results: A total of 78 patients with IP-II were identified. Among these, 55 patients had bilateral IP-II and EVA (only 12 with typical Mondini triad), 8 with bilateral IP-II and normal VA, 2 with bilateral IP-II and unilateral EVA, and 13 with unilateral IP-II (9 with unilateral EVA). Among the group with bilateral IP-II and bilateral EVA in whom genetic analysis was available, 14 out of 29 (48%) had SLC26A4 mutations and a diagnosis of Pendred syndrome, 1 had a FOXI1 mutation, and a few other genetic abnormalities; none had KCNJ10 pathogenic variants. Conclusion: Bilateral IP-II-bilateral EVA may be seen in the context of Pendred syndrome (SLC26A4 or FOXI1 mutations) but, in the majority of our cohort, no genetic abnormalities were found, suggesting the possibility of unknown genetic associations. IP-II in isolation (without EVA) is favored to be genetic when bilateral, although the cause is often unknown. [ABSTRACT FROM AUTHOR]

Context: Calcium-dependent signaling in plants is responsible for several major cellular events, including the activation of the salinity-responsive pathways. Calcium binds to calcineurin B-like protein (CBL), and the resulting CBL-Ca2+ complex binds to CBL-interacting protein kinase (CIPK). The CBL-CIPK complex enhances the CIPK interaction with an upstream kinase. The upstream kinase phosphorylates CIPK that, in turn, phosphorylates membrane transporters. Phosphorylation influences transporter activity to kick-start many downstream functions, such as balancing the cytosolic Na+-to-K+ ratio. The CBL-CIPK interaction is pivotal for Ca2+-dependent salinity stress signaling. Methods: Computational methods are used to model the entire Arabidopsis thaliana CIPK24 protein structure in its autoinhibited and open-activated states. Arabidopsis thaliana CIPK24-CBL4 complex is predicted based on the protein–protein docking methods. The available structural and functional data support the CIPK24 and the CIPK24-CBL4 complex models. Models are energy-minimized and subjected to molecular dynamics (MD) simulations. MD simulations for 500 ns and 300 ns enabled us to predict the importance of conserved residues of the proteins. Finally, the work is extended to predict the CIPK24-CBL4 complex with the upstream kinase GRIK2. MD simulation for 300 ns on the ternary complex structure enabled us to identify the critical CIPK24-GRIK2 interactions. Together, these data could be used to engineer the CBL-CIPK interaction network for developing salt tolerance in crops. [ABSTRACT FROM AUTHOR]

Background: The fruit of Phyllanthus emblica L., a traditional medicine in China and India, is used to treat diabetes mellitus. Its water extract (WEPE) has demonstrated hypoglycemic effects in diabetic rats, but its mechanisms on glucose utilization and insulin resistance in skeletal muscle remain unclear. Therefore, this study aims to investigate the effects and underlying mechanisms of WEPE on glucose utilization and insulin resistance using C2C12 myotubes. Methods: Effects of WEPE on glucose uptake, GLUT4 translocation, and AMPK and AKT phosphorylation were investigated in C2C12 myotubes and palmitate-treated myotubes. An AMPK inhibitor and siRNA were used to explore the mechanisms of WEPE. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay, and protein expression and GLUT4 translocation were assessed via western blotting. Results: In normal myotubes, WEPE significantly stimulated glucose uptake and GLUT4 translocation to the plasma membrane at concentrations of 125 and 250 µg/mL. This was accompanied by an increase in the phosphorylation of AMPK and its downstream targets. However, both compound C and AMPK siRNA blocked the WEPE-induced GLUT4 translocation and glucose uptake. Moreover, pretreatment with STO-609, a calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) inhibitor, inhibited WEPE-induced AMPK phosphorylation and attenuated the WEPE-stimulated glucose uptake and GLUT4 translocation. In myotubes treated with palmitate, WEPE prevented palmitate-induced insulin resistance by enhancing insulin-mediated glucose uptake and AKT phosphorylation. It also restored the insulin-mediated translocation of GLUT4 from cytoplasm to membrane. However, these effects of WEPE on glucose uptake and GLUT4 translocation were blocked by pretreatment with compound C. Conclusions: WEPE significantly stimulated basal glucose uptake though CaMKKβ/AMPK pathway and markedly ameliorated palmitate-induced insulin resistance by activating the AMPK pathway in C2C12 myotubes. [ABSTRACT FROM AUTHOR]

The importance of fungi in ecological systems and pathogenicity hinges on their ability to search for nutrients, substrates, and hosts. Despite this, the question of whether fungal hyphae exhibit chemotropism toward them remains largely unresolved and requires close examination at the cellular level. Here, we designed a microfluidic device to assess hyphal chemotropism of Aspergillus nidulans in response to carbon and nitrogen sources, as well as pH. Within this device, hyphae could determine their growth direction in a two-layer flow with distinct compositions that were adjacent but non-mixing. Under conditions with and without a carbon source, hyphae changed growth direction to remain in the presence of a carbon source, but it was still difficult to distinguish between differences in growth and chemotropism. Although nitrogen sources such as ammonia and nitrate are important for growth, the hyphae indicated negative chemotropism to avoid them depending on the specific transporters. This fungus grows equally well at the colony level in the pH range of 4 to 9, but the hyphae exhibited chemotropism to acidic pH. The proton pump PmaA is vital for the chemotropism to acid pH, while the master regulatory for pH adaptation PacC is not involved, suggesting that chemotropism and adaptive growth via gene expression regulation are distinct regulatory mechanisms. Despite various plasma membrane transporters are distributed across membranes except at the hyphal tip, the control of growth direction occurs at the tip. Finally, we explored the mechanisms linking these two phenomena, tip growth and chemotropism. Fungi depend on their nutrient-seeking behavior for survival, yet how they find nutrients is not well understood. Using a microfluidic device, this study shows that hyphae of the fungus Aspergillus nidulans exhibit chemotropism towards nutrients, adjusting growth direction towards carbon sources and away from certain nitrogen sources and acidic pH. [ABSTRACT FROM AUTHOR]

Heteroleptic metal complexes containing CuII, CoII, and ZnII, incorporating curcumin and a Schiff base ligand (L), were synthesized and characterized, and their antifungal activity was evaluated. Their antifungal activities were investigated individually and in combination with fluconazole. Utilizing various analytical techniques such as UV-Vis, FT-IR, NMR, ESI-MS, TGA-DTG, elemental analyses, conductance, and magnetic susceptibility measurements, complex C1 ([Cu(Cur)LCl(H2O)]) was assigned a distorted octahedral geometry, while complexes C2 ([Co(Cur)LCl(H2O)]) and C3 ([Zn(Cur)LCl(H2O)]) were assigned octahedral geometries. Among these complexes, C2 exhibited the highest inhibitory activity against both FLC-susceptible and resistant strains of Candida albicans. Furthermore, C2 demonstrated candidicidal activity and synergistic interactions with fluconazole, effectively inhibiting the growth and survival of both FLC-resistant and FLC-sensitive C. albicans strains. The complex displayed a dose-dependent inhibition of drug efflux pumps in FLC-resistant C. albicans strains, indicating its potential to disrupt the cell membrane of these strains. The significant role of membrane efflux transporters in the development of antifungal drug resistance within Candida species has been extensively documented and our findings indicate that complex C2 specifically targets this crucial factor, thereby playing a pivotal role in mitigating drug resistance in C. albicans. [ABSTRACT FROM AUTHOR]

The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD. In this work, the authors present structural and functional evidence of lipid exchange within the gram-negative Mla pathway, broadening our understanding of lipid transport in bacteria and providing insight into the mechanism of a unique ABC transporter. [ABSTRACT FROM AUTHOR]

The key role of a thyroid hormone receptor in determining the maturation and diversity of cone photoreceptors reflects a profound influence of endocrine signaling on the cells that mediate color vision. However, the route by which hormone reaches cones remains enigmatic as cones reside in the retinal photoreceptor layer, shielded by the blood--retina barrier. Using genetic approaches, we report that cone differentiation is regulated by a membrane transporter for thyroid hormone, MCT8 (SLC16A2), in the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier. Mct8-deficient mice display hypothyroid-like cone gene expression and compromised electroretinogram responses. Mammalian color vision is typically facilitated by cone types that detect medium-long (M) and short (S) wavelengths of light but Mct8-deficient mice have a partial shift of M to S cone identity, resembling the phenotype of thyroid hormone receptor deficiency. RPE-specific ablation of Mct8 results in similar shifts in cone identity and hypothyroid-like gene expression whereas reexpression of MCT8 in the RPE in Mct8-deficient mice partly restores M cone identity, consistent with paracrine-like control of thyroid hormone signaling by the RPE. Our findings suggest that in addition to transport of essential solutes and homeostatic support for photoreceptors, the RPE regulates the thyroid hormone signal that pro-motes cone-mediated vision. [ABSTRACT FROM AUTHOR]

Background. ATP binding cassette transporter A1 (ABCA1) is a candidate gene within a POAG susceptibility locus by GWAS analysis, and it is involved in IOP modulation via the Cav1/eNOS/NO signaling pathway. We aim to examine the phenotype of ABCA1 deletion in the ABCA1 gene knockout (Abca1−/−) mice. Methods. The anterior segments of Abca1−/− eyes were imaged by slit‐lamp microscopy and anterior segment OCT. IOPs were measured by rebound tonometry. By perfusing enucleated eyes at various pressures, the aqueous humor outflow facility was determined. The mRNA expressions of ABCA1, Cav1, and eNOS were measured by RT‐qPCR. The protein expressions were analyzed by western blot and immunofluorescence staining. Results. There was no significant difference in the anterior segment morphology of Abca1−/− mice. IOP and aqueous humor outflow facility did not change in Abca1−/− mice compared with wild‐type mice. mRNA and protein expressions of ABCA1 were significantly lower in the outflow tissue of Abca1−/− eyes. The expressions of Cav1 and eNOS were both significantly upregulated in the outflow tissue of Abca1−/− eyes. Conclusion. ABCA1 deletion does not affect IOP and aqueous humor outflow function but the Cav1/eNOS/NO pathway is changed in Abca1−/− mice. The function of ABCA1 in aqueous humor outflow still requires further research. [ABSTRACT FROM AUTHOR]

We have recently shown that cancer cells of various origins take up extracellular citrate through the plasma membrane citrate carrier (pmCiC), a specific plasma membrane citrate transporter. Extracellular citrate is required to support cancer cell metabolism, in particular fatty acid synthesis, mitochondrial activity, protein synthesis and histone acetylation. In addition, cancer cells tend to acquire a metastatic phenotype in the presence of extracellular citrate. Our recent study also showed that cancer-associated stromal cells synthesise and release citrate and that this process is controlled by cancer cells. In the present study, we evaluated the expression of pmCiC, fibroblast activation protein-a (FAP) and the angiogenesis marker cluster of differentiation 31 (CD31) in human cancer tissues of different origins. In the cohort studied, we found no correlation between disease stage and the expression of FAP or CD31. However, we have identified a clear correlation between pmCiC expression in cancer cells and cancer-associated stroma with tumour stage. It can be concluded that pmCiC is increased in cancer cells and in cancersupporting cells in the tumour microenvironment at the later stages of cancer development, particularly at the metastatic sites. Therefore, pmCiC expression has the potential to serve as a prognostic marker, although further studies are needed. [ABSTRACT FROM AUTHOR]

Perhalogenated closo‐borates represent a new class of membrane carriers. They owe this activity to their chaotropicity, which enables the transport of hydrophilic molecules across model membranes and into living cells. The transport efficiency of this new class of cluster carriers depends on a careful balance between their affinity to membranes and cargo, which varies with chaotropicity. However, the structure–activity parameters that define chaotropic transport remain to be elucidated. Here, we have studied the modulation of chaotropic transport by decoupling the halogen composition from the boron core size. The binding affinity between perhalogenated decaborate and dodecaborate clusters carriers was quantified with different hydrophilic model cargos, namely a neutral and a cationic peptide, phalloidin and (KLAKLAK)2. The transport efficiency, membrane‐lytic properties, and cellular toxicity, as obtained from different vesicle and cell assays, increased with the size and polarizability of the clusters. These results validate the chaotropic effect as the driving force behind the membrane transport propensity of boron clusters. This work advances our understanding of the structural features of boron cluster carriers and establishes the first set of rational design principles for chaotropic membrane transporters. [ABSTRACT FROM AUTHOR]

Cohen Syndrome (CS) is a rare autosomal recessive disorder caused by biallelic mutations in the VPS13B gene. It is characterized by multiple clinical features, including acquired microcephaly, developmental delay, intellectual disability, neutropenia, and retinal degeneration. VPS13B is part of the bridge-like lipid transport (BLTP) protein family, which in mammals also includes VPS13A, -C, and -D. The proteins of this family are peripheral membrane proteins with different sub-cellular localization, but all share similar structural features and have been proposed to act as lipid transport proteins at organellar membrane contact sites. VPS13B is localized at the Golgi apparatus and is essential for the maintenance of organelle architecture. Here we present a review of the experimental data on the function of the protein at the cellular level, discussing the potential link with disease phenotype and review the studies on animal models recapitulating features of the human disease. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukaemia (CML) is a haematological malignancy characterized by the constitutive tyrosine kinase activity of the BCR‐ABL1 fusion protein. Flumatinib, a second‐generation tyrosine kinase inhibitor, has exhibited superior clinical efficacy compared to its precursor, imatinib. However, with increased clinical use, resistance to flumatinib has emerged as a significant challenge. To investigate the mechanisms of flumatinib resistance in CML, we induced the human CML cell line K562 using a flumatinib concentration gradient method in vitro, successfully establishing a flumatinib‐resistant K562/FLM cell line. This cell line exhibited cross‐resistance to imatinib and doxorubicin, but remained sensitive to the antiparasitic agent ivermectin, which possesses antitumoural effects. Through cellular experimentation, we explored the resistance mechanisms, which indicated that K562/FLM cells evade flumatinib cytotoxicity by enhancing autophagy, increasing the expression of membrane transport proteins, particularly P‐glycoprotein, ABCC1 and ABCC4, as well as enhancing phosphorylation of p‐EGFR, p‐ERK and p‐STAT3 proteins. Moreover, it was found that ivermectin effectively suppressed the expression of autophagy and transport proteins in K562/FLM cells, reduced the activity of the aforementioned phosphoproteins, and promoted apoptotic cell death. Collectively, the increased autophagy, higher expression of drug‐efflux proteins and hyperactivation of the EGFR/ERK/STAT3 signalling pathway were identified as pivotal elements promoting resistance to flumatinib. The significant effects of ivermectin might offer a novel therapeutic strategy to overcome flumatinib resistance and optimize the treatment outcomes of CML. [ABSTRACT FROM AUTHOR]

Heterotrophic microbial communities play a significant role in driving carbon fluxes in marine ecosystems. Despite their importance, these communities remain understudied in remote polar oceans, which are known for their substantial contribution to the biological drawdown of atmospheric carbon dioxide. Our research focused on understanding the environmental factors and genetic makeup of key bacterial players involved in carbon remineralization in the Weddell Sea, including its coastal polynyas. Our experiments demonstrated that the combination of labile organic matter supply and temperature increase synergistically boosted bacterial growth. This suggests that, besides low seawater temperature, carbon limitation also hinders heterotrophic bacterial activity. Through the analysis of metagenome‐assembled genomes, we discovered distinct genomic adaptation strategies in Bacteroidia and Gammaproteobacteria, both of which respond to organic matter. Both natural phytoplankton blooms and experimental addition of organic matter favoured Bacteroidia, which possess a large number of gene copies and a wide range of functional membrane transporters, glycoside hydrolases, and aminopeptidases. In contrast, the genomes of organic‐matter‐responsive Gammaproteobacteria were characterized by high densities of transcriptional regulators and transporters. Our findings suggest that bacterioplankton in the Weddell Sea, which respond to organic matter, employ metabolic strategies similar to those of their counterparts in temperate oceans. These strategies enable efficient growth at extremely low seawater temperatures, provided that organic carbon limitation is alleviated. [ABSTRACT FROM AUTHOR]

As the first‐in‐class, selective, and potent inhibitor of the isocitrate dehydrogenase‐2 (IDH2) mutant protein, enasidenib was approved by the US Food and Drug Administration (FDA) in 2017 for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with an IDH2 mutation. Known for its interactions with various cytochrome P450 (CYP) enzymes and transporters in vitro, a clinical pharmacokinetics (PK) trial was initiated to assess the impact of multiple doses of enasidenib on the single‐dose PK of sensitive probe substrates of several cytochrome P450 enzymes and transporters. In this study, a population pharmacokinetic analysis approach was employed to address challenges posed by high, nonzero baseline caffeine concentrations. Moreover, we integrated full Bayesian inference into this approach innovatively for a more detailed understanding of parameter uncertainty and greater modeling flexibility, alongside Student's t‐distribution for robust error modeling in handling the abnormal outlier caffeine concentration data observed in this trial. Our analyses demonstrated that multiple doses of enasidenib altered caffeine clearance to a clinically meaningful extent, as evidenced by an approximate 8‐fold decrease. This finding led to a specific recommendation in the package insert to avoid the concurrent use of certain CYP1A2 substrates with enasidenib, unless directed otherwise in the prescribing information. Furthermore, this research underlines the technical benefits of integrating full Bayesian inference and incorporating Student's t‐distribution for residual error modeling in the PK field. [ABSTRACT FROM AUTHOR]

• Metabolome analysis revealed that plasma concentration of 3-indoxyl sulfate was increased in Slc17a3−/− mice compared to that in wild-type mice. • Urinary excretion of 3-indoxyl sulfate was reduced in Slc17a3−/− mice compared to that in wild-type mice. • An uptake study using NPT4-expressing oocytes revealed that 3-indoxyl sulfate is a physiological substrate of NPT4. • Our findings suggest that NPT4 regulates systemic exposure to 3-indoxyl sulfate by regulating its urinary excretion. Sodium-phosphate transporter NPT4 (SLC17A3) is a membrane transporter for organic anionic compounds localized on the apical membranes of kidney proximal tubular epithelial cells and plays a role in the urinary excretion of organic anionic compounds. However, its physiological role has not been sufficiently elucidated because its substrate specificity is yet to be determined. The present study aimed to comprehensively explore the physiological substrates of NPT4 in newly developed Slc17a3−/− mice using a metabolomic approach. Metabolomic analysis showed that the plasma concentrations of 11 biological substances, including 3-indoxyl sulfate, were more than two-fold higher in Slc17a3−/− mice than in wild-type mice. Moreover, urinary excretion of 3-indoxyl sulfate was reduced in Slc17a3−/− mice compared to that in wild-type mice. The uptake of 3-indoxyl sulfate by NPT4-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. The calculated K m and V max values for NPT4-mediated 3-indoxyl sulfate uptake were 4.52 ± 1.18 mM and 1.45 ± 0.14 nmol/oocyte/90 min, respectively. In conclusion, the present study revealed that 3-indoxyl sulfate is a novel substrate of NPT4 based on the metabolomic analysis of Slc17a3−/− mice, suggesting that NPT4 regulates systemic exposure to 3-indoxyl sulfate by regulating its urinary excretion. [ABSTRACT FROM AUTHOR]

The clinical management of malignant tumours is challenging, often leading to severe adverse effects and death. Drug resistance (DR) antagonises the effectiveness of treatments, and increasing drug dosage can worsen the therapeutic index (TI). Current efforts to overcome DR predominantly involve the use of drug combinations, including applying multiple anti-cancerous drugs, employing drug sensitisers, which are chemical agents that enhance pharmacokinetics (PK), including the targeting of cellular pathways and regulating pertinent membrane transporters. While combining multiple compounds may lead to drug–drug interactions (DDI) or polypharmacy effect, the use of drug sensitisers permits rapid attainment of effective treatment dosages at the disease site to prevent early DR and minimise side effects and will reduce the chance of DDI as lower drug doses are required. This review highlights the essential use of TI in evaluating drug dosage for cancer treatment and discusses the lack of a unified standard for TI within the field. Commonly used benefit–risk assessment criteria are summarised, and the critical exploration of the current use of TI in the pharmaceutical industrial sector is included. Specifically, this review leads to the discussion of drug sensitisers to facilitate improved ratios of effective dose to toxic dose directly in humans. The combination of drug and sensitiser molecules might see additional benefits to rekindle those drugs that failed late-stage clinical trials by the removal of detrimental off-target activities through the use of lower drug doses. Drug combinations and employing drug sensitisers are potential means to combat DR. The evolution of drug combinations and polypharmacy on TI are reviewed. Notably, the novel binary weapon approach is introduced as a new opportunity to improve TI. This review emphasises the urgent need for a criterion to systematically evaluate drug safety and efficiency for practical implementation in the field. [ABSTRACT FROM AUTHOR]

This study aims to evaluate the impact of liver fibrosis stages of chronic infection with hepatitis C virus (HCV) on the in vivo activity of organic cation transporters (hepatic OCT1 and renal OCT2) using metformin (MET) as a probe drug. Participants allocated in Group 1 (n = 15, mild to moderate liver fibrosis) or 2 (n = 13, advanced liver fibrosis and cirrhosis) received a single MET 50 mg oral dose before direct-acting antiviral (DAA) drug treatment (Phase 1) and 30 days after achieving sustained virologic response (Phase 2). OCT1/2 activity (MET AUC0–24) was found to be reduced by 25% when comparing the two groups in Phase 2 (ratio 0.75 (0.61–0.93), p < 0.05) but not in Phase 1 (ratio 0.81 (0.66–0.98), p > 0.05). When Phases 1 and 2 were compared, no changes were detected in both Groups 1 (ratio 1.10 (0.97–1.24), p > 0.05) and 2 (ratio 1.03 (0.94–1.12), p > 0.05). So, this study shows a reduction of approximately 25% in the in vivo activity of OCT1/2 in participants with advanced liver fibrosis and cirrhosis after achieving sustained virologic response and highlights that OCT1/2 in vivo activity depends on the liver fibrosis stage of chronic HCV infection. [ABSTRACT FROM AUTHOR]

Genome co-editing technology is effective in breeding filamentous fungi for applications in the fermentation industry, achieving site-directed mutagenesis, the status of non-genetically modified organisms (non-GMOs), and wild-type-like growth phenotype. Prior to this study, thiI gene was found as a selectable marker for such genome co-editing in the filamentous fungus Aspergillus oryzae , while it cannot be reused via marker recycling. Therefore, we aimed to identify another marker gene to knock out another target gene via genome co-editing in A. oryzae. In this study, we focused on the membrane transporter gene nrtA (AO090012000623), which promotes uptake of nitrate (NO 3 -). It is known that, in nrtA knockout strain, chlorate (ClO 3 -), an analog of nitrate with antifungal activity, cannot be imported into the cytosol, which enables the mutant to grow in the presence of chlorate. Based on this information, knockout of the target gene wA was attempted using both nrtA - and wA -specific single-guide RNAs via genome co-editing with KClO 3 supplementation in A. oryzae laboratory strain RIB40 and industrial strain KBN616. Resultantly, wA knockout mutant was generated, and nrtA was identified as a selectable marker. Moreover, this genome co-editing system using nrtA was compatible with that using thiI , and thus, a double knockout mutant of two target genes wA and yA was constructed in RIB40 while maintaining non-GMO status and wild-type-like growth. As nrtA homologs have been found in several industrial Aspergillus species, genome co-editing using homolog genes as selectable markers is plausible, which would contribute to the widespread breeding of industrial strains of Aspergilli. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) induce oxidative stress in cells and are associated with various diseases, including autoimmune diseases. Ergothioneine (EGT) is a natural amino acid derivative derived from the ergot fungus and has been reported to exhibit an effective antioxidant function in many models of oxidative stress-related diseases. Recently, mutations in OCTN1, a membrane transporter of EGT, have been reported to be associated with rheumatoid arthritis. Therefore, we investigated the chondrocyte-protective function of EGT using a model of oxidative stress-induced injury of chondrocytes by hydrogen peroxide (H2O2). Human chondrocytes were subjected to oxidative stress induced by H2O2 treatment, and cell viability, the activity of lactate dehydrogenase (LDH) released into the medium, dead cell ratio, intracellular ROS production, and mitochondrial morphology were assessed. EGT improved chondrocyte viability and LDH activity in the medium and strongly suppressed the dead cell ratio. EGT also exerted protective effects on intracellular ROS production and mitochondrial morphology. These results provide evidence to support the protective effects of EGT on chondrocytes induced by oxidative stress. [ABSTRACT FROM AUTHOR]

Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9, and the resulting pin1 mutant (pin1-27) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed two independent pid null mutants, whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous pin1 mutants were also capable of rescuing pid nulls. Moreover, we inserted green fluorescent protein (GFP) into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the PIN1-GFPHDR line are similar to those in the previously reported PIN1-GFP transgenic lines. Interestingly, the PIN1-GFPHDR line also rescued various pid null mutant alleles in a semidominant fashion. We conclude that decreasing the number of functional PIN1 copies is sufficient to suppress the pid mutant phenotype, suggesting that PIN1 is likely part of a larger protein complex required for organogenesis.

Intestinal absorption is an important contributor to systemic cholesterol homeostasis. Niemann-Pick C1 Like 1 (NPC1L1) assists in the initial step of dietary cholesterol uptake, but how cholesterol moves downstream of NPC1L1 is unknown. We show that Aster-B and Aster-C are critical for nonvesicular cholesterol movement in enterocytes. Loss of NPC1L1 diminishes accessible plasma membrane (PM) cholesterol and abolishes Aster recruitment to the intestinal brush border. Enterocytes lacking Asters accumulate PM cholesterol and show endoplasmic reticulum cholesterol depletion. Aster-deficient mice have impaired cholesterol absorption and are protected against diet-induced hypercholesterolemia. Finally, the Aster pathway can be targeted with a small-molecule inhibitor to manipulate cholesterol uptake. These findings identify the Aster pathway as a physiologically important and pharmacologically tractable node in dietary lipid absorption.