Search Results (1,121)

Background: Brain microvascular endothelial cells (BMECs) form the blood–brain barrier (BBB), a highly selective interface that restricts paracellular diffusion and regulates the transport of nutrients and drugs into the central nervous system via specialized transporters and receptors. Tight junction-associated protein 1 (Tjap1), also termed protein incorporated later into tight junctions (Pilt), has been localized to tight junctions (TJs) in epithelial cells and to the trans-Golgi network in fibroblasts; however, its expression, subcellular localization, and functional significance in BMECs are still unknown. Methods: We characterized Tjap1 subcellular localization in mouse and human BMEC cell lines as well as primary mouse BMECs by immunofluorescence with and without pharmacological Golgi disruption by treatment with Brefeldin A, Golgicide A or Pitstop 2. CRISPR/Cas9-mediated Tjap1 knockout cells were generated and examined with regard to their Golgi morphology using immunostaining. Tjap1 mRNA localization was examined by RNAscope in situ hybridization. Quantitative real-time PCR and Western blot was performed to assess the expression of BBB-associated efflux transporters, solute carrier transporters, and cellular receptors in control and Tjap1 knockout cells. Results: Tjap1 predominantly localized to the cis-Golgi compartment, co-localizing with Gm130 rather than Tgn38, and was absent from TJs in BMECs. Tjap1 knockout induced pronounced Golgi fragmentation BMECs. Importantly, Tjap1 knockout significantly downregulated mRNA-expression of Abcb1a, Abcb1b, Abcc4, Slc2a1, Slc7a1, Slc7a5 and Tfrc, while Abcg2 was upregulated. At the protein level, a decrease in the protein levels of Abcb1, Abcc4, Slc2a1, Slc7a1, and Tfrc was observed in Tjap1 knockout cEND cells. Conclusions: In BMECs, Tjap1 is a cis-Golgi-associated protein required for the structural integrity of the Golgi apparatus. Its deletion is associated with Golgi fragmentation and significant alterations in the mRNA and protein expression of drug transporters and receptors at the BBB. These findings identify Tjap1 as a candidate regulator of both Golgi architecture and the BBB transporter profile in vitro, with potential implications for modulating drug transport across the BBB. Full article

Background: The blood–brain barrier (BBB) separates the circulation from the central nervous system (CNS) and serves to maintain brain homeostasis. The BBB comprises highly specialized brain endothelial cells (BECs) with unique properties that allow the BBB to maintain strict regulation of molecules entering and exiting the CNS. These characteristics include tight junctions, low endocytosis rates, and efflux and nutrient transporters. Breast cancer resistance protein (BCRP) is an efflux transporter found at the BBB that plays a key role in protecting the CNS. Together with other efflux transporters, BCRP contributes to multidrug-resistant cancers and difficulty delivering drugs and therapeutics to the brain and other organs. Methods: Using the hCMEC/D3 line, we utilized BCRP substrate rosuvastatin to effectively select for cells expressing high amounts of BCRP, thus generating hCMEC/D3-BCRP. To assess protein abundance, we utilized flow cytometry and confirmed expression via qPCR. To investigate BCRP efflux function in evolved hCMEC/D3-BCRP, we performed substrate accumulation assays with BCRP and P-gp substrates. Results: We found hCMEC/D3-BCRP had increased BCRP abundance and expression relative to parent hCMEC/D3. We also observed an increase in BCRP function via substrate accumulation of two BCRP substrates compared to parent hCMEC/D3. Conclusions: BCRP serves a protective role within the BBB and is a major hurdle in drug delivery. We generated a BCRP overexpression BEC cell line (hCMEC/D3-BCRP) under the influence of endogenous promoters. This cell line can be used to further investigate the role of BCRP in BECs and utilized in efflux transport studies. Full article

Hepatocellular carcinoma (HCC) is a highly heterogeneous malignancy characterized by poor prognosis and limited therapeutic response. Cancer stem cells (CSCs) contribute to tumor progression, therapeutic resistance, and tumor recurrence. Among transcriptional regulators potentially involved in these processes, Specificity Protein 1 (SP1) has emerged as a candidate integrator of oncogenic and epigenetic signaling networks. However, its contribution to CSC-associated phenotypes and drug resistance in HCC remains incompletely defined. In this study, we combined transcriptomic analyses of TCGA datasets with functional experiments in HCC cell lines (Huh7 and HepG2). SP1-associated transcriptional programs were targeted pharmacologically using mithramycin A (MIT-A) and genetically using siRNA-mediated knockdown. The effects were assessed by RNA sequencing, RT-qPCR, Western blotting, flow cytometry, and functional assays evaluating proliferation, migration, CSC-associated properties, and response to sorafenib. MIT-A treatment markedly reduced the expression of stemness-associated transcription factors (NANOG, OCT4, SOX2) and CSC markers (CD133, CD24), impaired CSC-related functions including ALDH activity and the Side Population phenotype, and inhibited cell proliferation and migration. MIT-A also sensitized both parental and sorafenib-resistant HCC cells to sorafenib, associated with modulation of apoptotic regulators and reduced transporter-mediated efflux activity. SP1 knockdown partially reproduced several of these effects, supporting a contribution of SP1-dependent transcriptional programs to these phenotypes. Overall, these findings identify SP1-associated transcriptional networks as potential regulators of CSC features and therapeutic resistance in HCC and support targeting SP1-associated transcriptional programs as a strategy to enhance sorafenib efficacy. Full article

Background/Objectives: With the ongoing efforts in supporting the discovery of novel targeted drug delivery systems for the upper region of the female reproductive tract (FRT), it is imperative to understand the local drug disposition pathways. We aim to obtain a comprehensive profile of the drug transporters and drug-metabolizing enzymes in the human ectocervix, uterus, and fallopian tubes, as these factors may substantially influence mucosal penetration, tissue exposure, drug disposition, and the risk of drug–drug interactions. Methods: Gene expression of 12 drug transporters and 21 drug-metabolizing enzymes was quantified using RT-qPCR. Protein expression of highly expressed transporters was assessed using immunohistochemistry (IHC). Results: Among the 12 transporters analyzed, the efflux transporters P-gp, BCRP, and MRP4 exhibited the highest expression across the ectocervix, endometrium, myometrium, and fallopian tubes, with P-gp consistently showing the greatest abundance in all evaluated FRT tissues. Expression of these transporters was significantly higher (6–17×) in myometrium compared with ectocervix. IHC demonstrated strong localization of P-gp, BCRP, and MRP4 to epithelial layers facing the lumen, as well as to stromal and vascular endothelial cells. For drug-metabolizing enzymes, all 21 phase I and II enzymes were detectable across the FRT, and 15 were expressed at comparatively higher levels across all tissue types. These included CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C19, CYP3A4, UGT1A1, UGT1A3, UGT1A4, UGT1A7, UGT1A8, UGT1A10, UGT2B4, UGT2B15, and UGT2B17. Conclusions: The gene expression and localization data obtained from this work may improve our understanding of drug disposition in the FRT, which will inform selection, design, and optimization of drugs intended for targeted delivery within the FRT. Full article

Intraoperative fluorescence-guided surgery is an important adjunct to brain tumor resection. However, fluorescent probe performance varies across molecularly and histopathologically distinct entities, including IDH-wildtype glioblastoma, metastatic brain tumors (MBTs), and primary central nervous system lymphoma (PCNSL), and the mechanisms underlying this variability remain poorly understood. We propose a mechanistic framework integrating biomechanical constraints, molecular barrier heterogeneity, and probe-specific pharmacokinetics to explain cross-tumor differences in fluorescence signal. Probe performance is conceptualized through three sequential bottlenecks: extravasation (blood–brain barrier/blood–tumor barrier permeability and transcytosis), interstitial penetration (extracellular matrix density and hydraulic resistance), and retention/clearance (efflux transporters and metabolic processing). An overlying optical layer, including tissue absorption, scattering, and autofluorescence, further modulates the detected signal. Tumor-specific molecular heterogeneity critically shapes these processes. In IDH-wildtype glioblastoma and legacy high-grade glioma cohorts, heterogeneous expression of ATP-binding cassette transporters has been associated with reduced intracellular accumulation of protoporphyrin IX after 5-aminolevulinic acid administration and may contribute to false-negative fluorescence in selected tumor regions. In MBTs, stage-dependent blood–tumor barrier integrity and vascular programs influence probe delivery, whereas in PCNSL, corticosteroid-sensitive restoration of endothelial barrier function may compromise the performance of leakage-dependent tracers. Together, this framework highlights how tumor biology, barrier function, and probe pharmacology jointly shape fluorescence contrast. Rational probe selection informed by tumor-specific transport and barrier constraints may improve intraoperative visualization of brain tumors and optimize surgical decision-making. Full article

The genus Bacillus is widely recognized for its metabolic versatility, enabling it to colonize extreme environments, including sites contaminated with metals. In this study, we report the genome of B. velezensis strain HM1, isolated from sulfur-rich mine tailings from silver mining activities in southwestern Mexico. Isolation was performed by heat treatment followed by selective cultivation in a medium enriched with mine tailings extract (metals and sulfates), resulting in a single dominant morphotype corresponding to strain HM1. Whole-genome sequencing was carried out using the Illumina NovaSeq platform (2 × 250 bp). The assembled genome of strain HM1 has a size of 4,044,128 bp, distributed across 20 contigs, with an N50 of 700,388 bp and an L50 of 3, and an average coverage of 66.8×. The GC content was 46.31%, with an estimated completeness of 99.81% and contamination of 0.01%. Genome analyses indicate that the assembly corresponds to a single chromosome, with no evidence of plasmid replicons. Genome annotation identified 3950 coding sequences (CDSs), 83 tRNAs, 11 rRNAs, 26 ncRNAs, and 4 sORFs. Phylogenomic analysis, together with genomic similarity metrics (ANI > 98.6%, AAI > 98.8%, dDDH > 87%), confirms its classification as Bacillus velezensis. Functionally, the genome encodes multiple genes involved in resistance to metals and metalloids (including ABC transporters, efflux pumps, and biotransformation enzymes), as well as a complete pathway for sulfate assimilation. Collectively, these genomic features reveal a broad repertoire of adaptive strategies employed by strain HM1 to thrive in metal-contaminated environments. Full article

MK-2048 is a potent second-generation HIV integrase inhibitor that has demonstrated acceptable safety and pharmacokinetics (PKs) in clinical trials of vaginal formulations. The substrate-type interactions between MK-2048 and the transporters/metabolizing enzymes that are highly expressed in the human female reproductive tract (FRT) were evaluated. The interactions between MK-2048 and P-gp/BCRP were investigated using a cellular bidirectional permeability assay, while those between MK-2048 and MRP4 were assessed using a vesicular uptake assay. Reaction phenotyping was performed to characterize the interactions between MK-2048 and CYP1A1 and CYP1B1. Using human cervicovaginal fluids (CVFs), MK-2048’s solubility was determined using a thermodynamic solubility method and its protein binding was determined using a rapid equilibrium dialysis method. Our study shows an efflux of MK-2048 in P-gp/BCRP-overexpressing MDCKII cells, which was reduced by a P-gp/BCRP inhibitor. Uptake of MK-2048 in MRP4/control vesicles was found to be ATP-independent. MK-2048 was metabolized by the CYP1A1 enzyme but not by CYP1B1. These data confirm that MK-2048 is a substrate of P-gp, BCRP, and CYP1A1, but is not a substrate of MRP4 or CYP1B1. MK-2048 displays low solubility and high protein binding in human CVF. This data suggests that MK-2048 may potentially interact with drugs that modulate the activity of P-gp, BCRP, and CYP1A1. Full article

The phenotypic plasticity of epithelial cells along the epithelial–mesenchymal (E-M) axis, or epithelial–mesenchymal transition (EMT), is a critical aspect of tumour progression and therapeutic resistance. During EMT, epithelial cells gradually acquire mesenchymal traits, facilitating vital functions in embryogenesis, wound healing, fibrosis, and tumour metastasis. This review article investigates the potential interplay between hyperglycaemia-induced metabolic stress and EMT in the context of therapeutic resistance. The study examines a complex, multifaceted network of molecular mechanisms regulating EMT, including specialised transcription factors and signalling pathways as well as growth factors, integrins, and matrix metalloproteinases in various epithelial carcinomas. Emerging findings have demonstrated the existence of EMT hybrid states along the continuum, possessing heightened metastatic potential and distinctive metabolic signatures that play critical roles in the development of therapeutic resistance in cancer cells. Hyperglycaemia has been particularly highlighted for its potential to promote EMT-driven therapeutic resistance through various interconnected mechanisms. Elevated glucose levels induce the increased production of reactive oxygen species (ROS), activation of EMT-promoting transcription factors, and a metabolic shift towards glycolysis. This hyperglycaemic stress involves upregulation of glucose transporters and glycolytic enzymes, creating feed-forward loops that support drug efflux mechanisms and help maintain the mesenchymal phenotype. Clinical data also indicate that hyperglycaemia in OSCC patients is associated with more advanced tumour stages, more extended hospital stays, less effective treatments, and higher rates of local recurrence and distant metastasis. Overall, these insights reveal a deleterious feed-forward loop in which hyperglycaemia promotes EMT-driven therapeutic resistance, with the strongest clinical evidence in oral squamous cell carcinoma (OSCC) and supportive data from pancreatic and breast cancers. Although glycaemic control represents a promising low-risk adjunctive approach, its clinical benefit remains to be validated in prospective interventional studies. Full article

Gastric cancer (GC) is one of the most aggressive malignancies with a dismal prognosis, late diagnosis, and limited therapy efficacy. Biologically, GC is associated with multiple barriers to therapeutic response including gastric mucosal layer, acidic tumor microenvironment (TME), high accumulation of extracellular matrix (ECM) components, and limited penetration depth of anticancer drugs into tumor tissue. Furthermore, inherent or acquired drug resistance associated with drug efflux transporters, deregulated autophagy, tumor heterogeneity, and cell survival pathways severely compromise treatment response. Nanotechnology has been widely used to develop next-generation nanotherapeutic delivery systems to overcome these biological barriers. Currently available nanoplatforms such as liposomes, polymeric nanoparticles, dendrimers, and inorganic nanocarriers have improved drug loading capacity, aqueous solubility, circulation time stability, tumor-targeted delivery, and sustained release of chemotherapeutics. Smart and stimuli-responsive nanocarriers can also take advantage of pathological hallmarks of tumors including low pH, redox potential, and overexpressed enzymes for enhanced selective delivery to the tumor site. Nanotherapeutics have also shown promise for co-delivery of multiple therapeutic agents to overcome drug resistance, manipulation of TME, and suppression of autophagy and apoptosis signaling pathways associated with drug resistance. This review discusses recent advances in nanotherapeutics for GC including approaches to overcome biological barriers and drug resistance and highlights translational gaps for clinical development. Full article

Background/Objectives: Bile acids, synthesized from cholesterol in the liver, are amphipathic molecules that play an integral role in lipid digestion and absorption, while also serving as systemic endocrine hormones. They continuously undergo enterohepatic circulation, where they interact with various transporter proteins. Dysregulated bile acid transport is associated with the pathogenesis of liver disease. This review summarizes the key findings relating to bile acid transport expression and activity in the pathogenesis of liver disease. Methods: A review of the literature was performed using PubMed and relevant terms including, but not limited to, “bile acid transporters”, “liver disease”, and “bile acid uptake and efflux”. Studies published in peer-reviewed journals relevant to this review were considered and reviewed. Results: Within the gut and liver, several key bile acid and xenobiotic transporters within the enterohepatic circulation are dysregulated. The directionality and extent of changes are cell- and disease-specific. Many of the regulatory processes are driven by changes in bile acid signaling, although further work is needed to expand on post-translational modification of bile acid transporters in liver disease. Conclusions: Bile acid transporters are dynamically regulated in liver diseases with distinct etiologies. Therefore, restoring BA transporter function represents an actionable therapeutic approach to liver disease. Full article

Metal homeostasis, which coordinates the influx and efflux of essential elements such as iron (Fe) and manganese (Mn) in chloroplasts, is essential for optimum photosynthesis, especially in metal-accumulating plants. Brassica juncea (Indian mustard) is a metal-tolerant species with a strong metal accumulation capacity, making it a suitable model for studying transition metal homeostasis. In this study, we identified two efflux transporters, BjYSL6.1 and BjYSL6.4, that localize in the endomembrane system of Schizosaccharomyces pombe and interact with the chloroplast Mn influx transporter BjNRAMP4.1 at the plasma membrane and within the chloroplasts. Bimolecular fluorescence complementation and split-ubiquitin yeast two-hybrid assays confirmed specific protein–protein interactions among these transporters, as well as with the membrane-bound thioredoxin BjHCF164, a known regulator of photosynthetic electron transport. Gene expression studies revealed that BjNRAMP4.1 and BjYSL6 isoforms are inversely regulated under Fe and Mn stress conditions, with BjNRAMP4.1 being strongly induced under deficiency, whereas BjYSL6.1 and BjYSL6.4 are downregulated. These findings suggest that a coordinated network involving BjNRAMP4.1, BjYSL6s, and BjHCF164 modulates metal influx and efflux at the chloroplast and plasma membrane interfaces, thereby maintaining metal homeostasis, which is critical for photosynthetic efficiency in B. juncea. Full article

Background/Objectives: Freshwater microalgae–bacteria consortia are increasingly utilized in wastewater treatment and biomass production. However, bacteria associated with the algal phycosphere may act as environmental reservoirs of multidrug-resistant (MDR) phenotypes and antibiotic resistance genes (ARGs), including resistance to last-resort antibiotics such as colistin. Methods: An axenic culture of the freshwater microalga Pectinodesmus pectinatus was established using a NaClO-based cleaning protocol. Three phycosphere-associated bacterial strains (Chryseobacterium sp., Pseudomonas monteilii, and Stenotrophomonas pavanii) were isolated and identified by 16S rRNA gene analysis. Antimicrobial susceptibility testing was performed using broth microdilution against 16 antibiotics. Whole-genome sequencing of the most resistant isolate, S. pavanii, was conducted using Oxford Nanopore technology, followed by genome annotation and in silico resistome analysis using CARD, AMRFinderPlus, and ResFinder. Results: Among the three isolates, S. pavanii exhibited the broadest resistance profile, including high minimum inhibitory concentrations (MICs) to multiple β-lactams and colistin (MIC ≥ 16 μg/mL). No plasmid-borne mcr genes were detected. Instead, the genome encoded multiple chromosomal determinants potentially associated with polymyxin non-susceptibility, including lipid A and lipopolysaccharide modification pathways (e.g., arn genes and eptA), outer-membrane maintenance and LPS transport systems, multidrug efflux pumps, and regulatory elements. Integration of genomic and phenotypic data suggested that the observed colistin non-susceptibility may be associated with intrinsic chromosomal determinants inferred from whole-genome analysis. Conclusions: This study demonstrates that the P. pectinatus phycosphere can harbor multidrug-resistant (MDR) bacteria, including strains exhibiting colistin non-susceptibility potentially associated with a repertoire of intrinsic chromosomal resistance mechanisms inferred from genomic analysis. Therefore, freshwater microalgae-based systems should be considered potential environmental reservoirs contributing to the dissemination of antimicrobial resistance. Full article

Background/Objectives: The escalating crisis of antibiotic resistance and the inherent limitations of conventional antibiotics necessitate the development of innovative therapeutic strategies. Targeted drug delivery (TDD) offers a powerful approach to enhance efficacy, minimize systemic toxicity, and circumvent bacterial resistance. This systematic review aims to evaluate the potential of unique bacterial transport systems (BTSs), surface specific receptors and intracellular enzymes as platforms for TDD via the “Trojan Horse” strategy (THS). Methods: A comprehensive literature review was conducted, focusing on studies that investigated the specificity and mechanisms of BTSs responsible for the uptake of metabolites that are essential for and unique to bacteria. This includes an analysis of transport systems for siderophores, bacteria-specific sugars, cell wall components, D-amino acids, and vitamins. We assessed preclinical and clinical examples of drug conjugates utilizing these pathways, as well as emerging platforms such as bacteriophage-derived proteins, antibody–antibiotic conjugates, and bacterial extracellular vesicles (EVs). Results: BTSs demonstrate high specificity for their cognate substrates, providing effective molecular gateways for TDD of drugs photosensitizers and diagnostic probes in form of conjugates. The siderophore–cephalosporin conjugate cefiderocol represents a clinically validated example, having received FDA approval. Preclinical studies further reveal that conjugates utilizing sugars (e.g., maltose, trehalose) and vitamins (e.g., B12) can significantly enhance antibiotic uptake and activity against both Gram-positive and Gram-negative pathogens, including drug-resistant strains. Emerging platforms like bacteriophage endolysins and engineered EVs show promise for overcoming biological barriers such as bacterial outer membranes and intracellular host niches. Conclusions: The THS leveraging BTSs represents a clinically viable and promising avenue for next-generation antibacterial therapies. Advantages of BTS include overcoming bacterial resistance, such as reduced membrane permeability and efflux pumps, enabling the “revival” of antibiotics that are poorly permeable or toxic, increasing their local concentration at the target site and reducing side effects on host cells. While significant progress has been made, a striking disconnect persists between the hundreds of conjugates demonstrating potent in vitro activity and the limited agent that has achieved clinical use. This in vitro–in vivo gap reflects, in large part, the early stage of this field rather than a fundamental failure. Further research is critically needed not only to identify novel BTSs and optimize drug-linker chemistry, but also to systematically address the translational barriers—including poor pharmacokinetics, immunogenicity, and unexpected toxicity—that have prevented most promising candidates from advancing beyond preclinical evaluation. Full article

Yeast harbour more than ten different multiple drug resistance (MDR) genes encoding transporters that extrude xenobiotics from the cytoplasm into the environment. These transporters, belonging to the ATP-binding cassette (ABC) or major facilitator superfamily (MFS), exhibit broad and significantly overlapping substrate specificities, though the precise boundaries of their individual substrate ranges remain undefined. During evolution, genes with overlapping functions tend either to specialize or to degenerate into pseudogenes. Here, we propose several explanations for how this apparent redundancy of MDR efflux pumps benefits cells, and we discuss the potential individual roles of the full MDR efflux pump repertoire in the model organism Saccharomyces cerevisiae. We posit that individual MDR transporters may vary in stability under challenging environmental conditions, in the energetic cost of their synthesis and maintenance, and in their degree of specialization toward particular classes of xenobiotics. Furthermore, given that ABC transporters and MFS transporters exploit distinct driving forces for xenobiotic efflux, each class may have its own vulnerabilities. We argue that deciphering the distinct roles of MDR proteins will reveal critical weaknesses in the MDR system and guide the development of strategies to overcome multidrug resistance in pathogenic fungi. Full article

Arsenic-contaminated groundwater is a major environmental concern, particularly in northern Argentina. Here, Microbacterium oxydans AE038-20, isolated from arsenic-rich groundwater, was investigated to elucidate its tolerance and transformation capacity. Growth assays showed that the strain tolerates inorganic arsenic [As(III), As(V)] and methylarsenite [MAs(III)] without significant inhibition. Speciation analyses revealed progressive oxidation of As(III) to As(V), reaching near-complete conversion after 10 days. Similarly, MAs(III) was fully oxidized to MAs(V). Genome sequencing identified ars-related determinants, including arsR, arsC, putative arsenite efflux systems, and arsP, supporting detoxification via arsenate reduction and arsenite efflux. Proteomic analyses confirmed the expression of proteins related to arsenic resistance, oxidative stress response, and metal transport. However, no canonical arsenite oxidases were detected at either the genomic or proteomic level. Despite this, M. oxydans AE038-20 exhibited clear arsenic oxidation activity. The detection of pigment-associated proteins and in vitro oxidation assays suggest an alternative mechanism potentially mediated by redox-active pigments. These findings highlight an alternative pathway for arsenic transformation in environmental bacteria. Full article