Activity Of Transport Proteins Research Articles

Trace element zinc metabolism and its relation to tumors

Zinc is an essential trace element in the human body, playing a crucial role in cellular metabolism.Dysregulation of zinc homeostasis can lead to abnormal cellular metabolism, contributing to diseases and closely related to tumor development. Adequate zinc intake can maintain zinc homeostasis in the body and support normal cellular metabolism. This review discusses the metabolic processes of zinc in the human body and its close relationship with tumorigenesis. It briefly describes zinc absorption, transport, storage, and release, as well as its important role in gene expression, signal transduction, oxidative stress, immune response, and apoptosis. It focuses on the abnormal cellular metabolism caused by excessive or insufficient zinc, the relationship between zinc homeostasis disruption and metabolic syndrome, and the mechanisms involved in tumor development. It analyzes how changes in the expression and activity of zinc transporters may lead to disrupted zinc homeostasis in tumor tissues. It points out that zinc deficiency is associated with various cancers, including prostate cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer, ovarian cancer, esophageal squamous cell carcinoma, and breast cancer. The summary emphasizes that zinc metalloproteins could serve as potential targets for cancer therapy, and regulating the expression and activity of zinc transport proteins may offer new methods and strategies for clinical cancer treatment.

Natural Bioactive Compounds: Emerging Therapies for Hyperuricemia.

Hyperuricemia is a crucial feature of metabolic syndrome, characterized by elevated uric acid that causes urate crystal deposits in joints, kidneys, and subcutaneous tissues, resulting in gout and hyperuricemic nephropathy. The primary causes of uric acid metabolism disorder include overproduction and reduced excretion. The majority of uric acid in human body is derived from the breakdown of purine nucleotides. Overproduction of uric acid can result from increased concentration or activity of xanthine oxidase, the key enzyme responsible for uric acid synthesis. Alterations in the activity of proteins responsible for uric acid reabsorption and excretion can also affect serum uric acid. Many bioactive compounds derived from natural plants have been shown to inhibit xanthine oxidase activity to reduce uric acid production, modulate the activity of transport proteins to promote uric acid excretion, or alleviate oxidative stress and inflammation through various signaling pathways. These properties have garnered significant attention from researchers. In this paper, we first introduce the pathophysiological mechanisms of hyperuricemia, then summarize bioactive compounds with urate-lowering effects, and discuss their potential applications in treating hyperuricemia and its complications.

Understanding fungal and plant active urea transport systems: Keys from Aspergillus nidulans and beyond

Urea is present in all ecosystems, as a result of the metabolism of different organisms and also of human activity, being the world's most common form of nitrogen fertilizer. Fungi and plants can use urea as a nitrogen source, taking it up from the environment through specialized active transport proteins. These proteins belong to a subfamily of urea/H+ symporters included in the Solute:Sodium Symporter (SSS) family of transporters. In this review we summarize the current knowledge on this group of transporters, based on our previous studies on Aspergillus nidulans UreA. We delve into its transcriptional and post-translational regulation, structure-function relationships, transport mechanism, and certain aspects of its biogenesis. Recent findings suggest that this urea transporter subfamily is more expanded than originally thought, with representatives found in organisms as diverse as Archaea and mollusks, which raises questions on evolutionary aspects. A. nidulans ureA knockout strains provide a valuable platform for expressing urea transporters from diverse sources, facilitating their characterization and functional analysis. In this context, given the close relationship between plant and fungal active urea transporters, this knowledge could serve to develop strategies to improve the efficiency of applied urea as fertilizer.

Molecular mechanisms and diagnostic model of glioma-related epilepsy

Epilepsy is one of the most common symptoms in patients with gliomas; however, the mechanisms underlying its interaction are not yet clear. Moreover, epidemiological studies have not accurately identified patients with glioma-related epilepsy (GRE), and there is an urgent need to identify the molecular mechanisms and markers of its occurrence. We analyzed the demographics, transcriptome, whole-genome, and methylation sequences of 997 patients with glioma, to determine the genetic differences between glioma and GRE patients and to determine the upregulated molecular function, cellular composition, biological processes involved, signaling pathways, and immune cell infiltration. Twelve machine learning algorithms were refined into 113 combinatorial algorithms for building diagnostic recognition models. A total of 342 patients with GRE were identified with WHO grade 2 (174), grade 3 (107), and grade 4 (61). The mean age of the patients with GREs, with IDH mutations (n = 217 [63%]) and 1p19q non-codeletion (n = 169 [49%]), was 38 years old. GRE molecular functions were mainly passive transmembrane transporter protein activity, ion channel activity, and gated channel activity. Cellular components were enriched in the cation-channel and transmembrane transporter complexes. Cerebral cortical development regulates the membrane potential and synaptic organization as major biological processes. The signaling pathways mainly focused on cholinergic, GABAergic, and glutamatergic synapses. LASSO, combined with Random Forest, was the best diagnostic model and identified nine diagnostic genes. This study provides new insights and future perspectives for resolving the molecular mechanisms of GRE.

Translation arrest cancellation of VemP, a secretion monitor in Vibrio, is regulated by multiple cis and trans factors, including SecY

VemP is a secretory protein in the Vibrio species that monitors cellular protein-transport activity through its translation arrest, allowing expression of the downstream secD2-secF2 genes in the same operon, which encode components of the protein translocation machinery. When cellular protein-transport function is fully active, secD2/F2 expression remains repressed as VemP translation arrest is canceled immediately. The VemP arrest-cancellation occurs on the SecY/E/G translocon in a late stage in the translocation process and requires both trans-factors, SecD/F and PpiD/YfgM, and a cis-element, Arg-85 in VemP; however, the detailed molecular mechanism remains elusive. This study aimed to elucidate how VemP passing through SecY specifically monitors SecD/F function. Genetic and biochemical studies showed that SecY is involved in the VemP arrest-cancellation and that the arrested VemP is stably associated with a specific site in the protein-conducting pore of SecY. VemP-Bla reporter analyses revealed that a short hydrophobic segment adjacent to Arg-85 plays a critical role in the regulated arrest-cancellation with its hydrophobicity correlating with the stability of the VemP arrest. We identified Gln-65 and Pro-67 in VemP as novel elements important for the regulation. We propose a model for the regulation of the VemP arrest cancellation by multiple cis-elements and trans-factors with different roles.

P016 METAPROTEOMICS ANALYSIS IN HEALTHY INDIVIDUALS REVEALS A HIGH CARDIOVASCULAR RISK PROFILE ACROSS BLOOD PRESSURE, DIET, HUMAN AND MICROBIAL PROTEINS

Background/Objective: The gut microbiota is a crucial link between diet and cardiovascular health by producing beneficial metabolites, such as short-chain fatty acids (SCFAs), through fibre fermentation. However, most studies have focused on microbial DNA to infer microbiota function. Here, we employed metaproteomics, a novel method to capture gut human and microbiota protein expression profiles concurrently. We aimed to investigate the crosstalk between gut microbiota, diet, host gut health, and blood pressure (BP). Methods: Mass spectrometry-based metaproteomic analysis was performed on 61 faecal samples with paired ambulatory BP monitoring, food intake (food frequency questionnaire), and demographic data. Results: We identified 21,594 microbial and 1,072 human proteins. Traditional risk factors (age, BMI, sex, BP) explained only ∼10% of the proteome variance. However, unsupervised clustering analysis of both human and microbial proteome revealed two distinct clusters (low-risk and high-risk) with significantly different 24-hour systolic BP (mean±SD, low-risk 116±13mmHg vs high-risk: 126±15mmHg, P=0.016), women prevalence (72.4% vs 40.6%, P=0.025), and diet quality (assessed as Athlete Diet Index, 61.6±6.7 vs 57.8±7.6, P=0.044). In the human proteome, the low-risk group had lower expression of the VEGFA-VEGFR2 signalling pathway and tight junction proteins and higher SCFA production (particularly propionate), suggesting an anti-inflammatory gut environment. In the microbial proteome, the low-risk group had higher activity of inorganic ion transport and metabolism-related proteins, with increased expression of acetate kinase that produces both the SCFAs acetate and propionate, particularly in primary fibre-fermenting bacteria, including Phocaeicola dorei and Blautia wexlerae. Finally, via co-expression networks, we identified profilin-1 (PFN1), which contributes to hypertension and atherosclerosis, as a crucial human protein associated with several bacteria present in the high-risk group. Conclusions: Employing an unsupervised clustering based on human and microbial proteomics, we identified two groups of participants with varying traditional and emerging cardiovascular risk factors. This was explained by complex interactions among diet, gut microbiota, and BP. This underscores the need for sophisticated models to understand multifactorial risk factors for cardiovascular disease.

Unveiling gut microbiota's role: Bidirectional regulation of drug transport for improved safety.

Drug safety is a paramount concern in the field of drug development, with researchers increasingly focusing on the bidirectional regulation of gut microbiota in this context. The gut microbiota plays a crucial role in maintaining drug safety. It can influence drug transport processes in the body through various mechanisms, thereby modulating their efficacy and toxicity. The main mechanisms include: (1) The gut microbiota directly interacts with drugs, altering their chemical structure to reduce toxicity and enhance efficacy, thereby impacting drug transport mechanisms, drugs can also change the structure and abundance of gut bacteria; (2) bidirectional regulation of intestinal barrier permeability by gut microbiota, promoting the absorption of nontoxic drugs and inhibiting the absorption of toxic components; (3) bidirectional regulation of the expression and activity of transport proteins by gut microbiota, selectively promoting the absorption of effective components or inhibiting the absorption of toxic components. This bidirectional regulatory role enables the gut microbiota to play a key role in maintaining drug balance in the body and reducing adverse reactions. Understanding these regulatory mechanisms sheds light on novel approaches to minimize toxic side effects, enhance drug efficacy, and ultimately improve drug safety. This review systematically examines the bidirectional regulation of gut microbiota in drug transportation from the aforementioned aspects, emphasizing their significance in ensuring drug safety. Furthermore, it offers a prospective outlook from the standpoint of enhancing therapeutic efficacy and reducing drug toxicity, underscoring the importance of further exploration in this research domain. It aims to provide more effective strategies for drug development and treatment.

Lipopolysaccharide Triggers Luminal Acidification to Promote Defense Against Bacterial Infection in Vaginal Epithelium

The vaginal epithelium plays pivotal roles in host defense against pathogen invasion, contributing to the maintenance of an acidic microenvironment within the vaginal lumen through the activity of acid-base transport proteins. However, the precise defense mechanisms employed by the vaginal epithelium following bacterial infection remain incompletely understood. This study demonstrated that the bacterial lipopolysaccharide (LPS) potentiated net proton efflux by up-regulating the expression of Na+-H+ exchanger 1 (NHE1), without affecting other acid-base transport proteins in vaginal epithelial cells. Pharmacological inhibition or genetic knockdown of Toll-like receptor-4 (TLR4) and the extracellular-signal-regulated protein kinase (ERK) signaling pathway effectively counteracted the up-regulation of NHE1 and the enhanced proton efflux triggered by LPS in vaginal epithelial cells. In vivo studies revealed that LPS administration led to luminal acidification through the up-regulation of NHE1 expression in the rat vagina. Moreover, inhibition of NHE exhibited impaired defense against acute bacterial infection in the rat vagina. These findings collectively indicated the active involvement of vaginal epithelial cells in facilitating luminal acidification during acute bacterial infection, offering potential insights into the treatment of bacterial vaginosis.

The deubiquitinating enzymes-related signature predicts the prognosis and immunotherapy response in breast cancer.

Breast cancer is a prevalent disease that has a dismal prognosis for patients and a bad outlook for treatments. Ubiquitination is a reversible biological process that regulates protein production and degradation, as well as plays a vital role in protein transport, localization, and biological activity. We obtained the breast cancer patient sample data and used a machine learning technique to create a novel index called Deubiquitinating enzyme related index (DUBRI) by gathering genes associated to deubiquitinating enzymes. Based on DUBRI, we systematically analyze patients' prognosis, clinical characteristics, tumor immune microenvironment, chemotherapy response and immunotherapy response. Finally, the function of OTUB2 was explored in breast cancer cells. DUBRI, which consists of five deubiquitinating enzyme genes (OTUB2, USP41, MINDY2, YOD1, and PSMD7), is a reliable predictor of survival in breast cancer patients. We found that the high DUBRI group presented higher levels of immune cell infiltration. We performed molecular docking prediction of core target proteins in deubiquitinating enzymes. In vitro experiments verified that knockdown of OTUB2 could inhibit the proliferation and migration of breast cancer. The DUBRI discovered in this research may effectively evaluate the outlook of breast cancer patients and identify groups of patients who would gain advantages from immunotherapy, offering vital knowledge for the future targeted treatment of breast cancer patients.

Proteomic analysis of brain tissue from ducks with meningitis caused by Riemerella anatipestifer infection

Riemerella anatipestifer is a Gram-negative, rod-shaped bacterium that is flagellated, non-budded, and encapsulated, measuring approximately 0.4 μm × 0.7 μm. After infecting ducklings with R. anatipestifer, the hosts exhibited pathological changes, such as bacterial meningitis, fibrinous pericarditis, and fibrinous peripheral hepatitis. The pathogenesis of meningitis caused by R. anatipestifer has not yet been elucidated. To investigate the key molecules or proteins involved in R. anatipestifer's penetration of the blood-brain barrier (BBB) and the subsequent development of duck meningitis, a duck meningitis model was established and characterized. Duckling brain tissues were collected and analyzed using 4D label-free proteomic technology. Differentially expressed proteins were analyzed using a series of bioinformatics methods and verified using RT-qPCR and Western-Blot. The results showed that the differentially expressed proteins were primarily related to intracellular transport, transport protein activity, and transmembrane transport protein activity, and were mainly enriched in pathways associated with reducing intercellular connections and adhesion and increasing cell migration and apoptosis. Thus, it is suggested that R. anatipestifer may penetrate the BBB via transcellular and paracellular pathways, causing neurological diseases such as meningitis. This study is the first to analyze R. anatipestifer-infected duckling brain tissue using proteomics, thus providing a direction for further research into the mechanisms of R. anatipestifer's penetration of the BBB.

Vitamin B12-Related Biomarkers.

Adult vitamin B12 (B12) deficiency may present itself with nonspecific mainly neurological symptoms, and thus plasma biomarkers are often judged to be of major importance in the further diagnostic process. Four biomarkers are of special relevance: total B12, holotranscobalamin (the part of B12 bound to the active transport protein, transcobalamin, also named holoTC or active B12) and the 2 so-called metabolic markers that accumulate if B12 is lacking, methylmalonic acid (MMA) and homocysteine. This article briefly reviews the inherent limitation of biomarkers, discusses its use in establishing the diagnosis and cause of B12 deficiency, and when following or discontinuing treatment with B12. The review is based on published papers, but also on knowledge gained from working within the area. It is concluded that a combination of a B12 and a metabolic marker, for example, total B12 and MMA, may prove most useful in daily practice. An unexpectedly high level of total B12 is most often of no clinical importance, though sometimes related to the presence of underlying cancer. Measurement of total B12 is of limited value in patients on treatment with pharmacological doses of B12 but may be helpful if B12 treatment is discontinued.

Comprehensive Transcriptome and Proteome Analyses Reveal the Drought Responsive Gene Network in Potato Roots.

The root system plays a decisive role in the growth and development of plants. The water requirement of a root system depends strongly on the plant species. Potatoes are an important food and vegetable crop grown worldwide, especially under irrigation in arid and semi-arid regions. However, the expected impact of global warming on potato yields calls for an investigation of genes related to root development and drought resistance signaling pathways in potatoes. In this study, we investigated the molecular mechanisms of different drought-tolerant potato root systems in response to drought stress under controlled water conditions, using potato as a model. We analyzed the transcriptome and proteome of the drought-sensitive potato cultivar Atlantic (Atl) and the drought-tolerant cultivar Qingshu 9 (Q9) under normal irrigation (CK) and weekly drought stress (D). The results showed that a total of 14,113 differentially expressed genes (DEGs) and 5596 differentially expressed proteins (DEPs) were identified in the cultivars. A heat map analysis of DEGs and DEPs showed that the same genes and proteins in Atl and Q9 exhibited different expression patterns under drought stress. Weighted gene correlation network analysis (WGCNA) showed that in Atl, Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)-enriched pathways were related to pyruvate metabolism and glycolysis, as well as cellular signaling and ion transmembrane transporter protein activity. However, GO terms and KEGG-enriched pathways related to phytohormone signaling and the tricarboxylic acid cycle were predominantly enriched in Q9. The present study provides a unique genetic resource to effectively explore the functional genes and uncover the molecular regulatory mechanism of the potato root system in response to drought stress.

Advance in Iron Metabolism, Oxidative Stress and Cellular Dysfunction in Experimental and Human Kidney Diseases.

Kidney diseases pose a significant global health issue, frequently resulting in the gradual decline of renal function and eventually leading to end-stage renal failure. Abnormal iron metabolism and oxidative stress-mediated cellular dysfunction facilitates the advancement of kidney diseases. Iron homeostasis is strictly regulated in the body, and disturbance in this regulatory system results in abnormal iron accumulation or deficiency, both of which are associated with the pathogenesis of kidney diseases. Iron overload promotes the production of reactive oxygen species (ROS) through the Fenton reaction, resulting in oxidative damage to cellular molecules and impaired cellular function. Increased oxidative stress can also influence iron metabolism through upregulation of iron regulatory proteins and altering the expression and activity of key iron transport and storage proteins. This creates a harmful cycle in which abnormal iron metabolism and oxidative stress perpetuate each other, ultimately contributing to the advancement of kidney diseases. The crosstalk of iron metabolism and oxidative stress involves multiple signaling pathways, such as hypoxia-inducible factor (HIF) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways. This review delves into the functions and mechanisms of iron metabolism and oxidative stress, along with the intricate relationship between these two factors in the context of kidney diseases. Understanding the underlying mechanisms should help to identify potential therapeutic targets and develop novel and effective therapeutic strategies to combat the burden of kidney diseases.

#1838 SGLT2 computational structural studies investigating polypharmacy

Abstract Background and Aims Sodium Glucose Cotransporter 2 (SGLT2) is the main active transport protein involved in sodium and glucose reabsorption in the kidney. SGLT2 inhibitors (SGLT2i) are widely recommended for patients with diabetes, heart failure and chronic kidney disease (CKD). Many multimorbid patients will be prescribed these compounds resulting in polypharmacy. We have performed a computational drug repurposing screen to identify other licensed drugs capable of binding at or near the SGLT2i active site aiming to identify compounds that could either compete with SGLT2i or inhibit sodium and glucose transport. Method The library of BNF listed compounds was obtained from NCBI PubChem. D-I-TASSER was used to generate monomeric structural models and MODELLER was used to incorporate MAP-17 and empaglifozin from a reference structure (PDB 7VSI). CHARM-GUI was used to insert the protein into a membrane. The centroid of a 5 nanosecond GROMACS molecular dynamics (MD) equilibration was used for docking studies using PLANTS. A bespoke tool was used to identify compounds interacting with key protein residues. CHARM-GUI and bespoke software were used to prepare membrane-bound systems to run in GROMACS using GPU's in Google Colab. 10 nanosecond simulations were undertaken (300 Kelvin and 1 bar) to discriminate between binding and non-binding events. Results The SGLT2-MAP17 structure in the inward-open conformation obtained showed good agreement with published structures. Existing SGLT2i (empaglifozin, dapaglifozin, canaglifozin, ertuglifozin) all feature in the top 1% of docked compounds in the repurposing screen. 17 compounds were investigated by MD, with all remaining bound to the protein in simulation. Ceftriaxone, tobramycin, clindamycin, fluvastatin, atorvastatin and ticagrelor were among the compounds with potentially significant interactions. It is not clear whether the stable ligand interactions identified here would result in inhibition of sodium and glucose transport, or if the interactions might provide competitive inhibition for SGLT2i compounds currently used. The compounds identified have no hitherto recognised interactions with SGLT2i; nor do they result in adverse effects suggesting inhibition of the protein. This study is limited by considering only the protein-ligand interaction and not wider pharmacokinetic or pharmacodynamic factors. Conclusion The indication of interactions with several compounds likely to be prescribed alongside SGLT2 inhibitors, such as antibiotics, statins and antiplatelet agents, warrants further investigation of the potential for polypharmacological complications.

Identification of disease-specific genes related to immune infiltration in nonalcoholic steatohepatitis using machine learning algorithms.

To identify disease signature genes associated with immune infiltration in nonalcoholic steatohepatitis (NASH), we downloaded 2 publicly available gene expression profiles, GSE164760 and GSE37031, from the gene expression omnibus database. These profiles represent human NASH and control samples and were used for differential genes (DEGs) expression screening. Two machine learning methods, the Least Absolute Shrinkage and Selection Operator regression model and Support Vector Machine Recursive Feature Elimination, were used to identify candidate disease signature genes. The CIBERSORT deconvolution algorithm was employed to analyze the infiltration of 22 immune cell types in NASH. Additionally, we constructed a NASH cell model using HepG2 cells treated with oleic acid and free fatty acids. The construction of the cell model was verified using oil red O staining, and Western blotting was used to detect the protein expression of the disease signature genes in both control and model groups. As a result, a total of 262 DEGs were identified. These DEGs were primarily associated with metal ion transmembrane transporter activity, sodium ion transmembrane transporter protein activity, calcium ion, and neuroactive ligand-receptor interactions. FOS, IGFBP2, dual-specificity phosphatase 1 (DUSP1), and IKZF3 were identified as disease signature genes of NASH by the least absolute shrinkage and selection operator and Support Vector Machine Recursive Feature Elimination algorithms for DEGs analysis. The receiver operating characteristic curves showed that FOS, IGFBP2, DUSP1, and IKZF3 had good diagnostic value (area under receiver operating characteristic curve > 0.8). These findings were validated in the GSE89632 dataset and through cellular assays. Immunocyte infiltration analysis revealed that NASH was associated with CD8 T cells, CD4 T cells, follicular helper T cells, resting NK cells, eosinophils, regulatory T cells, and γδ T cells. The FOS, IGFBP2, DUSP1, and IKZF3 genes were specifically associated with follicular helper T cells. Lipid droplet aggregation significantly increased in HepG2 cells treated with oleic acid and free fatty acids, indicating successful construction of the cell model. In this model, the expression of FOS, IGFBP2, and DUSP1 was significantly decreased, while that of IKZF3 was significantly elevated (P < .01, P < .001) compared with the control group. Therefore, FOS, IGFBP2, DUSP1, and IKZF3 can be considered as disease signature genes associated with immune infiltration in NASH.

Better maintenance of enzymatic capacity and higher levels of substrate transporter proteins in skeletal muscle of aging female mice.

This study investigated sex-specific differences in high-energy phosphate, glycolytic, and mitochondrial enzyme activities and also metabolite transporter protein levels in the skeletal muscles of adult (5 months old), middle-aged (12 months old), and advanced-aged (24 months old) mice. While gastrocnemius glycogen content increased with age regardless of sex, gastrocnemius triglyceride levels increased only in advanced-aged female mice. Aging decreased creatine kinase and adenylate kinase activities in the plantaris muscle of both sexes and in the soleus muscle of male mice but not in female mice. Irrespective of sex, phosphofructokinase and lactate dehydrogenase (LDH) activities decreased in the plantaris and soleus muscles. Additionally, hexokinase activity in the plantaris muscle and LDH activity in the soleus muscle decreased to a greater extent in aged male mice compared with those in aged female mice. Mitochondrial enzyme activities increased in the plantaris muscle of aged female mice but did not change in male mice. The protein content of the glucose transporter 4 in the aged plantaris muscle and fatty acid translocase/cluster of differentiation 36 increased in the aged plantaris and soleus muscles of both sexes, with a significantly higher content in female mice. These findings suggest that females possess a better ability to maintain metabolic enzyme activity and higher levels of metabolite transport proteins in skeletal muscle during aging, despite alterations in lipid metabolism. Our data provide a basis for studying muscle metabolism in the context of age-dependent metabolic perturbations and diseases that affect females and males differently.

Extracellular Vesicles as Surrogates for the Regulation of the Drug Transporters ABCC2 (MRP2) and ABCG2 (BCRP).

Drug efflux transporters of the ATP-binding-cassette superfamily play a major role in the availability and concentration of drugs at their site of action. ABCC2 (MRP2) and ABCG2 (BCRP) are among the most important drug transporters that determine the pharmacokinetics of many drugs and whose overexpression is associated with cancer chemoresistance. ABCC2 and ABCG2 expression is frequently altered during treatment, thus influencing efficacy and toxicity. Currently, there are no routine approaches available to closely monitor transporter expression. Here, we developed and validated a UPLC-MS/MS method to quantify ABCC2 and ABCG2 in extracellular vesicles (EVs) from cell culture and plasma. In this way, an association between ABCC2 protein levels and transporter activity in HepG2 cells treated with rifampicin and hypericin and their derived EVs was observed. Although ABCG2 was detected in MCF7 cell-derived EVs, the transporter levels in the vesicles did not reflect the expression in the cells. An analysis of plasma EVs from healthy volunteers confirmed, for the first time at the protein level, the presence of both transporters in more than half of the samples. Our findings support the potential of analyzing ABC transporters, and especially ABCC2, in EVs to estimate the transporter expression in HepG2 cells.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

Colorectal cancer with low SLC35A3 is associated with immune infiltrates and poor prognosis

The expression level of SLC35A3 is associated with the prognosis of many cancers, but its role in colorectal cancer (CRC) is unclear. The purpose of our study was to elucidate the role of SLC35A3 in CRC. The expression levels of SLC35A3 in CRC were evaluated through tumor immune resource assessment (TIMER), The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), International Cancer Genome Consortium (ICGC), Human Protein Atlas (HPA), qRT-PCR, and immunohistochemical evaluation. TCGA, GEO, and ICGC databases were used to analyze the diagnostic and prognostic value of SLC35A3 in CRC. A overall survival (OS) model was constructed and validated based on the expression level of SLC35A3 and multivariable analysis results. The cBioPortal tool was used to analyze SLC35A3 mutation in CRC. The UALCAN tool was used to analyze the promoter methylation level of SLC35A3 in colorectal cancer. In addition, the role of SLC35A3 in CRC was determined through GO analysis, KEGG analysis, gene set enrichment analysis (GSEA), immune infiltration analysis, and immune checkpoint correlation analysis. In vitro experiments validated the function of SLC35A3 in colorectal cancer cells. Compared with adjacent normal tissues and colonic epithelial cells, the expression of SLC35A3 was decreased in CRC tissues and CRC cell lines. Low expression of SLC35A3 was associated with N stage, pathological stage, and lymphatic infiltration, and it was unfavorable for OS, disease-specific survival (DSS), recurrence-free survival (RFS), and post-progression survival (PPS). According to the Receiver Operating Characteristic (ROC) analysis, SLC35A3 is a potential important diagnostic biomarker for CRC patients. The nomograph based on the expression level of SLC35A3 showed a better predictive model for OS than single prognostic factors and TNM staging. SLC35A3 has multiple types of mutations in CRC, and its promoter methylation level is significantly decreased. GO and KEGG analysis indicated that SLC35A3 may be involved in transmembrane transport protein activity, cell communication, and interaction with neurotransmitter receptors. GSEA revealed that SLC35A3 may be involved in energy metabolism, DNA repair, and cancer pathways. In addition, SLC35A3 was closely related to immune cell infiltration and immune checkpoint expression. Immunohistochemistry confirmed the positive correlation between SLC35A3 and helper T cell infiltration. In vitro experiments showed that overexpression of SLC35A3 inhibited the proliferation and invasion capability of colorectal cancer cells and promoted apoptosis. The results of this study indicate that decreased expression of SLC35A3 is closely associated with poor prognosis and immune cell infiltration in colorectal cancer, and it can serve as a promising independent prognostic biomarker and potential therapeutic target.

Study on the mechanism of salt relief and growth promotion of Enterobacter cloacae on cotton

AimsIn-depth studies on plant ion uptake and plant growth-promoting rhizobacteria (PGPR) at the molecular level will help to further reveal the effects of PGPR on plants and their interaction mechanisms under salt stress.MethodsCotton was inoculated with a PGPR-Enterobacter cloacae Rs-35, and the ion uptake capacity, membrane transporter protein activity, and expression of key genes were determined under salt stress. Changes in the endogenous hormone content of cotton were also determined. Further, the genome-wide metabolic pathway annotation of E. cloacae Rs-35 and its differential enrichment pathway analysis of multi-omics under salinity environments were performed.ResultsIn a pot experiment of saline-alkali soil, E. cloacae Rs-35-treated cotton significantly increased its uptake of K+ and Ca2+ and decreased uptake of Na+, elevated the activity of the H+-ATPase, and increased the sensitivity of the Na+/H+ reverse transporter protein on the vesicle membrane. Meanwhile, inoculation with E. cloacae Rs-35 could promote cotton to maintain the indole-3-acetic acid (IAA) content under salt stress. Genome-wide annotation showed that E. cloacae Rs-35 was respectively annotated to 31, 38, and 130 related genes in osmotic stress, phytohormone and organic acid metabolism, and ion uptake metabolic pathway. Multi-omics differences analysis showed that E. cloacae Rs-35 were enriched to tryptophan metabolism, multiple amino acid biosynthesis, carbon and glucose synthesis, and oxidative phosphorylation metabolic pathways at the transcriptome, proteome, and metabolome.ConclusionE. cloacae Rs-35 can promote cotton balance cell ion concentration, stabilize intracellular IAA changes, stimulate induction of systemic tolerance, and promote the growth of cotton plants under salt stress.