Efflux Process Research Articles

The outer-membrane componentof the multi-drug efflux system –a structural analysis based onhydrophobicity distribution

The structure of a multi-drug efflux system (specifically the outer membrane part) is the focus of our analysis. The role of electrostatic interactions in the efflux process is well understood. The distribution of hydrophobicity in the periplasmic and membrane domains plays a significant role in both stabilisation within the membrane and in tunnel formation, which facilitates the transport of antibiotics. The analysis presented in this paper reveals the specificity of hydrophobicity distribution in relation to biological activity, as well as a possible mechanism for the folding process of proteins involved in multi-drug efflux. Our analysis is made possible by the application of the fuzzy oil drop model in its modified form (FOD-M).

Insights into Allosteric Inhibition of the AcrB Efflux Pump: Role of Distinct Binding Pockets, Protomer Preferences, and Crosstalk Disruption.

AcrB, a key component in bacterial efflux processes, exhibits distinct binding pockets that influence inhibitor interactions. In addition to the well-known distal binding pocket within the periplasmic domain, a noteworthy pocket amidst the transmembrane (TM) helices serves as an alternate binding site for inhibitors. The bacterial efflux mechanism involves a pivotal functional rotation of the TM protein, inducing conformational changes in each protomer and propelling drugs toward the outer membrane domain. Surprisingly, inhibitors binding to the TM domain display a preference for L protomers over T protomers. Metadynamics simulations elucidate that Lys940 in the TM domain of AcrB can adopt two conformations in L protomers, whereas the energy barrier for such transitions is higher in T protomers. This phenomenon results in stable inhibitor binding in l protomers. Upon a detailed analysis of unbinding pathways using random accelerated molecular dynamics and umbrella sampling, we have identified three distinct routes for ligand exit from the allosteric site, specifically involving regions within the TM domains─TM4, TM5, and TM10. To explore allosteric crosstalk, we focused on the following key residues: Val452 from the TM domain and Ala831 from the porter domain. Surprisingly, our findings reveal that inhibitor binding disrupts this communication. The shortest path connecting Val452 and Ala831 increases upon inhibitor binding, suggesting sabotage of the natural interdomain communication dynamics. This result highlights the intricate interplay between inhibitor binding and allosteric signaling within our studied system.

Mechanistic insights into P-glycoprotein ligand transport and inhibition revealed by enhanced molecular dynamics simulations

P-glycoprotein (P-gp) plays a crucial role in cellular detoxification and drug efflux processes, transitioning between inward-facing (IF) open, occluded, and outward-facing (OF) states to facilitate substrate transport. Its role is critical in cancer therapy, where P-gp contributes to the multidrug resistance phenotype. In our study, classical and enhanced molecular dynamics (MD) simulations were conducted to dissect the structural and functional features of the P-gp conformational states. Our advanced MD simulations, including kinetically excited targeted MD (ketMD) and adiabatic biasing MD (ABMD), provided deeper insights into state transition and translocation mechanisms. Our findings suggest that the unkinking of TM4 and TM10 helices is a prerequisite for correctly achieving the outward conformation. Simulations of the IF-occluded conformations, characterized by kinked TM4 and TM10 helices, consistently demonstrated altered communication between the transmembrane domains (TMDs) and nucleotide binding domain 2 (NBD2), suggesting the implication of this interface in inhibiting P-gp's efflux function. A particular emphasis was placed on the unstructured linker segment connecting the NBD1 to TMD2 and its role in the transporter's dynamics. With the linker present, we specifically noticed a potential entrance of cholesterol (CHOL) through the TM4-TM6 portal, shedding light on crucial residues involved in accommodating CHOL. We therefore suggest that this entry mechanism could be employed for some P-gp substrates or inhibitors. Our results provide critical data for understanding P-gp functioning and developing new P-gp inhibitors for establishing more effective strategies against multidrug resistance.

Abstract 2050: Slc43a3 And Intracellular Fatty Acid Trafficking

Background: Fatty acids (FAs) have important roles in many cellular functions. Dysregulation of FA flux is both a characteristic and effector of many prevalent clinical conditions, including diabetes mellitus, obesity as well as insulin resistance and/or metabolic syndrome. Adipocytes take up long chain fatty acids through diffusion and protein mediated transport, whereas fatty acid efflux is considered to occur by diffusion. Our previous study suggests the possibility of a protein facilitated/regulated process of FA efflux by demonstrating that the organic anion transport inhibitor DIDS strongly inhibits FA efflux in lipolytically-stimulated adipocytes, leading to an accumulation of intracellular FAs, while not affecting glycerol efflux. We hypothesize that there is potential regulator(s) for FA efflux. Methods and Results: In a screen of subcutaneous adipose samples from obese patients for expression levels of transporters without known function, Slc43a3 (a member of the solute carrier transporter family) shown increased expression after bariatric surgery. In addition, Slc43a3 regulates fatty acid flux in adipocytes. To study the mechanism of Slc43a3 function, clonal lines of preadipocyte OP9 cells were generated, in which green fluorescent protein has been knocked-in at the C-terminus of endogenous Slc43a3 using CRISPR/Cas9. In parallel, Slc43a3 knockout (KO) line has also been generated. Examination of the cells showed that expression of Slc43a3 induced in the cytoplasm of the adipocytes during differentiation. Slc43a3KO adipocytes accumulate bigger lipid droplets and less small lipid droplets. To study the functional significance of Slc43a3 in vivo, adipose tissue specific KO of Slc43a3 were generated with floxed Slc43a3 and tamoxifen induced adiponectin-Cre recombinase. The Slc43a3 KO mice have less body weight and less % body fat on a normal chow diet; however, no differences are noted when the mice are switched to high fat diet, indicating the lack of lipid signaling molecules in the Slc43a3 KO mice with normal chow diet. Functional analysis shows a blunted stimulated lipolysis in the Slc43a3 KO mice. Conclusions: Both in vitro and in vivo data confirmed an unique role of Slc43a3 in regulating fatty acid flux in adipose tissue.

Selenite resistance and biotransformation to SeNPs in Sinorhizobium meliloti 1021 and the synthetic promotion on alfalfa growth

Toxic selenite, commonly found in soil and water, can be transformed by microorganisms into selenium nanoparticles (SeNPs) as part of a detoxification process. In this study, a comprehensive investigation was conducted on the resistance and biotransformation of selenite in Sinorhizobium meliloti 1021 and the synergistic impact of SeNPs and the strain on alfalfa growth promotion was explored. Strain 1021 reduced 46% of 5mM selenite into SeNPs within 72hours. The SeNPs, composed of proteins, lipids and polysaccharides, were primarily located outside rhizobial cells and had a tendency to aggregate. Under selenite stress, many genes participated in multidrug efflux, sulfur metabolism and redox processes were significantly upregulated. Of them, four genes, namely gmc, yedE, dsh3 and mfs, were firstly identified in strain 1021 that played crucial roles in selenite biotransformation and resistance. Biotoxic evaluations showed that selenite had toxic effects on roots and seedlings of alfalfa, while SeNPs exhibited antioxidant properties, promoted growth, and enhanced plant’s tolerance to salt stress. Overall, our research provides novel insights into selenite biotransformation and resistance mechanisms in rhizobium and highlights the potential of SeNPs-rhizobium complex as biofertilizer to promote legume growth and salt tolerance.

Uptake of Fluorescein via a pH-Dependent Monocarboxylate Transporter by Human Kidney 2 (HK-2) Cells.

Herein, we investigated whether a fluorescent probe for an organic anion transporter (OAT), fluorescein (FLS), could be accumulated by human kidney 2 (HK-2) cells derived from human kidney proximal tubular epithelia. HK-2 cells took up FLS in a pH-dependent and concentration-dependent manner. FLS accumulation by HK-2 cells was inhibited by monocarboxylic acids, ibuprofen, rosuvastatin, and indoleacetic acid but not by typical substrates for OATs. A typical protonophore, carbonyl cyanide p-trichloromethoxyphenylhydrazone completely abolished FLS accumulation by HK-2 cells. The FLS efflux process from the preloaded HK-2 cells exhibited substantial trans-stimulation by the excess amount of extracellular FLS transport inhibitable monocarboxylate compounds such as 2,4-dichloro phenoxyacetic acid, fluvastatin, ibuprofen, indoleacetic acid, salicylic acid and rosuvastatin, indicating that the FLS transporter can recognize and accumulate them into the cells in a pH-dependent manner. The involvement of the FLS transporter in the reabsorption of monocarboxylic compounds was indicated by demonstrating that the pH-dependent FLS uptake is inhibited by various monocarboxylates in rabbit renal brush border membrane vesicles. pH-dependent FLS uptake was trans-stimulated by the inhibitable monocarboxylates. Collectively, the present data indicate that the pH-dependent transporters expressed in HK-2 cells are involved in the reabsorption of monocarboxylates from the urinary fluid into the tubular epithelia.

Translocation Processes of Pt(II)-Based Drugs through Human Breast Cancer Cell Membrane: In Silico Experiments.

Breast cancer is one of the most frequent modalities of cancer worldwide, with notable mortality. The medication based on platinum drugs (cisplatin (cddp), carboplatin (cpx), and oxaliplatin (oxa)) is a conventional chemotherapy despite severe side effects and the development of drug resistance. In order to provide a deeper molecular description of the influx and efflux processes of platinum drugs through breast cancer tissues, this study focuses on molecular dynamics (MD) simulations of the passive translocation process through a realistic plasma membrane prototype of human breast cancer cell (c_memb). The results showed that the permeation events were mainly mediated by neutral lipids (DOPC, DOPE, and cholesterol), producing a low and temporary membrane deformation. The drug insertion in the region of polar heads was the most favorable stage of the translocation mechanism, especially for cddp and oxa with potential wells of -8.6 and -9.8 kcal mol-1, respectively. However, the potentials of mean force (PMF) revealed unfavorable kinetics for the permeation of these drugs through lipid tails, with energy barriers of 28.3 (cddp), 32.2 (cpx), and 30.4 kcal mol-1 (oxa). The low permeability coefficients (P) of cpx and oxa, which were 3 and 1 orders of magnitude inferior than for cddp, resulted from the high energy barriers for their traslocation processes through the membrane. The obtained results provide a more accurate picture of the permeation of Pt(II)-based drugs through breast cancer cells, which may be relevant for the design and evaluation of new platinum complexes.

Tumor-associated macrophages confer colorectal cancer 5-fluorouracil resistance by promoting MRP1 membrane translocation via an intercellular CXCL17/CXCL22–CCR4–ATF6–GRP78 axis

Chemotherapy represents a major type of clinical treatment against colorectal cancer (CRC). Aberrant drug efflux mediated by transporters acts as a key approach for tumor cells to acquire chemotherapy resistance. Increasing evidence implies that tumor-associated macrophages (TAMs) play a pivotal role in both tumorigenesis and drug resistance. Nevertheless, the specific mechanism through which TAMs regulate drug efflux remains elusive. Here, we discovered that TAMs endow CRC cells with resistance to 5-fluorouracil (5-FU) treatment via a cell-cell interaction-mediated MRP1-dependent drug efflux process. Mechanistically, TAM-secreted C-C motif chemokine ligand 17 (CCL17) and CCL22, via membrane receptor CCR4, activated the PI3K/AKT pathway in CRC tumor cells. Specifically, phosphorylation of AKT inactivated IP3R and induced calcium aggregation in the ER, resulting in the activation of ATF6 and upregulation of GRP78. Accordingly, excessive GRP78 can interact with MRP1 and promote its translocation to the cell membrane, causing TAM-induced 5-FU efflux. Taken together, our results demonstrated that TAMs promote CRC chemotherapy resistance via elevating the expression of GRP78 to promote the membrane translocation of MRP1 and drug efflux, providing direct proof for TAM-induced drug resistance.

Antiatherogenic effect of blueberry fruit polyphenols extracts (Vaccinium angustifolium Ait) reduces oxidative stress and enhances the cholesterol efflux process

Antiatherogenic effect of blueberry fruit polyphenols extracts (Vaccinium angustifolium Ait) reduces oxidative stress and enhances the cholesterol efflux process

Comparative study on ABCB1-dependent efflux of anthracyclines and their metabolites: consequences for cancer resistance

1. ABCB1 (P-glycoprotein, MDR1) is one of the most important transporter involved in cancer multi-drug resistance. It also plays a significant role in cancer resistance against anthracyclines, an anticancer group of drugs, including doxorubicin and daunorubicin. Several intracellular enzymes metabolise anthracyclines to carbonyl-reduced, hydroxy metabolites, which have impaired cytotoxic properties. However, metabolite efflux by ABCB1 transporter is not well characterised, while it may be the mechanism responsible for the metabolites’ lack of activity. 2. In this study recombinant ABCB1 ATPase transporter assay; anthracyclines accumulation assay in resistant cells overexpressing ABCB1; and molecular modelling were used to investigate anthracyclines: doxorubicin and daunorubicin and their carbonyl-reduced metabolites (doxorubicinol, daunorubicinol) susceptibility for ABCB1-dependent efflux. 3. Based on the kinetics parameters of ATPase activity of ABCB1, it was found that daunorubicinol exerted an exceptionally high potential for being effluxed by the ABCB1 transporter. ABCB1 significantly affected the accumulation pattern of studied chemicals in resistant cancer cells. Doxorubicin and daunorubicinol accumulation were influenced by the activity of ABCB1 modulator – valspodar. 4. Results indicate that ABCB1 activity affects not only anthracyclines but also their metabolites. Therefore crosstalk between the process of anthracyclines metabolism and metabolite efflux may be the mechanism of impairing anticancer properties of anthracyclines metabolites.

An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202

BackgroundPolymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront.MethodsPandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling.ResultsThe MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter. Additionally, verapamil and CCCP exhibited significant inhibition via competition for binding sites.ConclusionsThese findings demonstrated that GI-M202a along with the MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistance.

Adaptive Laboratory Evolution Reveals the Selenium Efflux Process To Improve Selenium Tolerance Mediated by the Membrane Sulfite Pump in Saccharomyces cerevisiae.

Selenium (Se) is a micronutrient in most eukaryotes, and Se-enriched yeast is the most common selenium supplement. However, selenium metabolism and transport in yeast have remained unclear, greatly hindering the application of this element. To explore the latent selenium transport and metabolism mechanisms, we performed adaptive laboratory evolution under the selective pressure of sodium selenite and successfully obtained selenium-tolerant yeast strains. Mutations in the sulfite transporter gene ssu1 and its transcription factor gene fzf1 were found to be responsible for the tolerance generated in the evolved strains, and the selenium efflux process mediated by ssu1 was identified in this study. Moreover, we found that selenite is a competitive substrate for sulfite during the efflux process mediated by ssu1, and the expression of ssu1 is induced by selenite rather than sulfite. Based on the deletion of ssu1, we increased the intracellular selenomethionine content in Se-enriched yeast. This work confirms the existence of the selenium efflux process, and our findings may benefit the optimization of Se-enriched yeast production in the future. IMPORTANCE Selenium is an essential micronutrient for mammals, and its deficiency severely threatens human health. Yeast is the model organism for studying the biological role of selenium, and Se-enriched yeast is the most popular selenium supplement to solve Se deficiency. The cognition of selenium accumulation in yeast always focuses on the reduction process. Little is known about selenium transport, especially selenium efflux, which may play a crucial part in selenium metabolism. The significance of our research is in determining the selenium efflux process in Saccharomyces cerevisiae, which will greatly enhance our knowledge of selenium tolerance and transport, facilitating the production of Se-enriched yeast. Moreover, our research further advances the understanding of the relationship between selenium and sulfur in transport.

Smart pH- and Temperature-Sensitive Micelles Based on Chitosan Grafted with Fatty Acids to Increase the Efficiency and Selectivity of Doxorubicin and Its Adjuvant Regarding the Tumor Cells.

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

In vitro Raman imaging of human macrophages: Impact of eicosapentaenoic acid on the hydrolysis of cholesterol esters in lipid droplets

Atherosclerosis – a cardiovascular disease and the primary cause of morbidity and mortality in industrialized countries – is linked to the existence of atherosclerotic plaques characterized by cholesterol-laden macrophages called foam cells. In these cells, cholesterol esters associated with triglycerides form lipid droplets (LD). The only way to remove this excess cholesterol is to promote free cholesterol efflux from macrophages to specific acceptors. It has been shown recently that eicosapentaenoic acid (EPA) reduces efflux on cholesterol-loaded THP-1 macrophages in vitro due to decreased cholesterol esters hydrolysis. These in vitro observations could reflect EPA's difficulty in facilitating in vivo the antiatherogenic process of cholesterol efflux within advanced atherosclerotic plaques.This work aims to study in vitro the impact of EPA on cholesterol esters hydrolysis in the LD of human THP-1 macrophages using vibrational Raman microspectroscopy. For this, we used deuterated EPA and recorded spectral images at the cell scale after different hydrolysis times. Resultsshowed that EPA is involved in forming triglycerides and phospholipids of LD. Hydrolysis kinetics slowed down after 24 h, triglycerides increased, and the intensity of the characteristic bands linked to deuteration decreased. The size of LD without hydrolysis (H0) is higher than that after 24 h (H1) or 48 h (H2) of hydrolysis. The size decrease is sharper when going from H0 to H1 than from H1 to H2. Principal component analysis illustrated data’ projection according to the cellular compartment, the hydrolysis time, and the supplementation of the medium.

Small multidrug resistance protein EmrE phenotypically associates with OmpW, DcrB and YggM for osmotic stress protection by betaine in Escherichia coli.

The small multidrug resistance (SMR) protein EmrE resides in the inner membrane and provides resistance against a wide range of antiseptic quaternary cationic compounds (QCCs) for the Gram-negative bacterium Escherichia coli. We have reported previously that overexpression of the emrE gene results in the reduction of pH and osmotic tolerance, likely through EmrE-mediated biological QCC-based osmoprotectant efflux, indicating a potential physiological role for EmrE beyond providing drug resistance. EmrE is the most studied member of SMR transporter family; however, it is not known how the substrates translocated by EmrE move across the periplasm and through the outer membrane (OM). We have shown that the OM protein OmpW participates in the EmrE-mediated substrate efflux process and provided a hypothesis for the present study that additional OM and periplasmic proteins participate in the translocation process. To test the hypothesis, we conducted alkaline pH-based growth phenotype screens under emrE overexpression conditions. This screen identified 10 additional genes that appear to contribute to the EmrE-coupled osmoprotectant efflux: gspD, hofQ, yccZ, acrA, emrA, emrB, proX, osmF, dcrB and yggM. Further screening of these genes using a hyperosmotic growth phenotype assay in the presence and the absence of the osmoprotectant glycine betaine identified ompW and two periplasmic protein genes, dcrB and yggM, are mechanistically linked to EmrE.

A NiCoT family metal transporter of Mycobacterium tuberculosis (Rv2856/NicT) behaves as a drug efflux pump that facilitates cross-resistance to antibiotics.

Metals often act as a facilitator in the proliferation and persistence of antibiotic resistance. Efflux pumps play key roles in the co-selection of metal and antibiotic resistance. Here, we report the ability of a putative nickel/cobalt transporter (NiCoT family), Rv2856 or NicT of Mycobacterium tuberculosis (Mtb), to transport metal and antibiotics and identified some key amino acid residues that are important for its function. Ectopic expression of NicT in Escherichia coli CS109 resulted in the increase of intracellular nickel uptake. Additionally, enhanced tolerance towards several antibiotics (norfloxacin, sparfloxacin, ofloxacin, gentamicin, nalidixic acid and isoniazid) was observed with NicT overexpression in E. coli and Mycobacterium smegmatis. A comparatively lower intracellular accumulation of norfloxacin upon NicT overexpression than that of the cells without NicT indicated the involvement of NicT in an active efflux process. Although expression of NicT did not alter the sensitivity towards kanamycin, doxycycline, tetracycline, apramycin, neomycin and ethambutol, the presence of a sub-inhibitory dose of Ni2+ resulted in the manifestation of low-level tolerance towards these drugs. Further, substitution of four residues (H77I, D82I, H83L and D227I) in the conserved regions of NicT by isoleucine and leucine resulted in reduced to nearly complete loss of the transport function for both metals and antimicrobials. Therefore, the study suggests that nickel transporter Rv2856/NicT may actively export different drugs and the presence of nickel might drive the cross-resistance to some of the antibiotics.

Cellular transport of uranium and its cytotoxicity effects on CHO-k1 cells

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

Identification of diagnostic biomarkers and therapeutic targets in peripheral immune landscape from coronary artery disease

BackgroundPeripheral biomarkers are increasingly vital non-invasive methods for monitoring coronary artery disease (CAD) progression. Their superiority in early detection, prognosis evaluation and classified diagnosis is becoming irreplaceable. Nevertheless, they are still less explored. This study aimed to determine and validate the diagnostic and therapeutic values of differentially expressed immune-related genes (DE-IRGs) in CAD.MethodsWe downloaded clinical information and RNA sequence data from the GEO database. We used R software, GO, KEGG and Cytoscape to analyze and visualize the data. A LASSO method was conducted to identify key genes for diagnostic model construction. The ssGSEA analysis was used to investigate the differential immune cell infiltration. Besides, we constructed CAD mouse model (low-density lipoprotein receptor deficient mice with high fat diet) to discover the correlation between the screened genes and severe CAD progress. We further uncovered the role of IL13RA1 might play in atherosclerosis.ResultsA total of 762 differential genes were identified between the peripheral blood of 218 controls and 199 CAD patients, which were significantly associated with infection, immune response and neural activity. 58 DE-IRGs were obtained by overlapping the differentially expressed genes(DEGs) and immune-related genes downloaded from ImmpDb database. Through LASSO regression, CCR9, CER1, CSF2, IL13RA1, INSL5, MBL2, MMP9, MSR1, NTS, TNFRSF19, CXCL2, HTR3C, IL1A, and NR4A2 were distinguished as peripheral biomarkers of CAD with eligible diagnostic capabilities in the training set (AUC = 0.968) and test set (AUC = 0.859). The ssGSEA analysis showed that the peripheral immune cells had characteristic distribution in CAD and also close relationship with specific DE-IRGs. RT-qPCR test showed that CCR9, CSF2, IL13RA1, and NTS had a significant correlation with LDLR−/− mice. IL13RA1 knocked down in RAW264.7 cell lines decreased SCARB1 and ox-LDL-stimulated CD36 mRNA expression, TGF-β, VEGF-C and α-SMA protein levels and increased the production of IL-6, with downregulation of JAK1/STAT3 signal pathway.ConclusionsWe constructed a diagnostic model of advanced-stage CAD based on the screened 14 DE-IRGs. We verified 4 genes of them to have a strong correlation with CAD, and IL13RA1 might participate in the inflammation, fibrosis, and cholesterol efflux process of atherosclerosis by regulating JAK1/STAT3 pathway.

Two transporters mobilize magnesium from vacuolar stores to enable plant acclimation to magnesium deficiency.

Magnesium (Mg) is an essential metal for chlorophyll biosynthesis and other metabolic processes in plant cells. Mg is largely stored in the vacuole of various cell types and remobilized to meet cytoplasmic demand. However, the transport proteins responsible for mobilizing vacuolar Mg2+ remain unknown. Here, we identified two Arabidopsis (Arabidopsis thaliana) Mg2+ transporters (MAGNESIUM TRANSPORTER 1 and 2; MGT1 and MGT2) that facilitate Mg2+ mobilization from the vacuole, especially when external Mg supply is limited. In addition to a high degree of sequence similarity, MGT1 and MGT2 exhibited overlapping expression patterns in Arabidopsis tissues, implying functional redundancy. Indeed, the mgt1 mgt2 double mutant, but not mgt1 and mgt2 single mutants, showed exaggerated growth defects as compared to the wild type under low-Mg conditions, in accord with higher expression levels of Mg-starvation gene markers in the double mutant. However, overall Mg level was also higher in mgt1 mgt2, suggesting a defect in Mg2+ remobilization in response to Mg deficiency. Consistently, MGT1 and MGT2 localized to the tonoplast and rescued the yeast (Saccharomyces cerevisiae) mnr2Δ (manganese resistance 2) mutant strain lacking the vacuolar Mg2+ efflux transporter. In addition, disruption of MGT1 and MGT2 suppressed high-Mg sensitivity of calcineurin B-like 2 and 3 (cbl2 cbl3), a mutant defective in vacuolar Mg2+ sequestration, suggesting that vacuolar Mg2+ influx and efflux processes are antagonistic in a physiological context. We further crossed mgt1 mgt2 with mgt6, which lacks a plasma membrane MGT member involved in Mg2+ uptake, and found that the triple mutant was more sensitive to low-Mg conditions than either mgt1 mgt2 or mgt6. Hence, Mg2+ uptake (via MGT6) and vacuolar remobilization (through MGT1 and MGT2) work synergistically to achieve Mg2+ homeostasis in plants, especially under low-Mg supply in the environment.

Transcriptional profiling reveals ataxia telangiectasia mutated pathways regulate joint copper and arsenic toxicity for hepatic metalloplasia and anti-cancer therapies

AimsExposures to toxic metals, including arsenic (As), pose health risks but joint effects of physiologically needed metals, e.g., copper (Cu), are ill-defined for regulated metal-dependent cell proliferation (or metalloplasia). This study elucidated hepatic toxicities of As and Cu. Main methodsHuman HuH-7 cells were exposed to As and Cu and mRNA profiling obtained for molecular networks, regulators and signaling pathways. This followed biological testing of ATM signaling-related DNA damage response, mitochondrial dysfunction and lysosome activity using HuH-7 cells and primary hepatocytes. Free Cu ions were bound to 3-indole propionic acid for finding their contribution in toxicity. Key findingsThe As or As plus Cu toxicities in HuH-7 cells produced dimorphic down- or up-regulation patterns in mRNA profiles. Significant differences extended for ontologies in protein synthesis, intermediary metabolism, mitochondrial function, autophagy, or cell survival and growth. Bioassays revealed ATM signaling regulated As and Cu toxicity for oxidative phosphorylation, mitochondrial membrane potential, lysosomal activity, DNA damage response, and cell growth-arrest. Removal of reactive Cu ions decreased As and Cu toxicity. Primary hepatocytes withstood Cu and As toxicity better. SignificanceThis joint As and Cu toxicity offers further mechanisms for metalloplasia, carcinogenesis and tissue damage in other settings, e.g., during excess Cu accumulation in Wilson disease. Moreover, joint As and Cu toxicities are relevant for anti-cancer therapies, potentially including manipulations to increase intracellular Cu through altered uptake or efflux processes and incorporating ATM-related checkpoint inhibitors. Superior tolerance of healthy hepatocytes to Cu and As toxicity should improve safety margins for anti-cancer therapies.