Recursive dynamics of GspE through machine learning enabled identification of inhibitors.

Type II secretion System has been increasingly recognized as a key driver of virulence in many pathogenic bacteria including Achromobacter xylosoxidans. ATPase GspE is the powerhouse of the T2SS. It powers the entire secretion process by binding with ATP and hydrolyzing it. Therefore, targeting it was thought to have a profound effect on the normal functioning of the whole T2SS. A. xylosoxidans is a Gram-negative bacterium that poses a rising concern to immunocompromised people. It is responsible for many opportunistic infections mostly in people with cystic fibrosis. Due to its intrinsic and acquired resistance mechanisms, it is challenging to treat. In this current study, an extensive machine learning-enabled computational investigation was carried out. Drug libraries were screened using machine learning random forest algorithm trained on non-redundant dataset of 8722 antibacterial compounds with reported IC50 values. Active compounds were then further subjected to molecular docking. To unravel the dynamics and better understand the stability of complexes, the top complexes were subjected to MD Simulations followed by various post-simulation analyses including Trajectory analysis, Atom Contacts, SASA, Hydrogen Bond, RDF, binding free energy calculations, PCA, and AFD analysis. Findings from the study unanimously unveiled Asinex-BAS00263070-28551 as the best inhibitor as it instigated the recursive dynamics of the target by making key hydrogen bond interactions with Walker A motif, suggesting it could serve as the promising drug candidate against GspE. Further experimental in-vivo and in-vitro validation is still required to authenticate the therapeutic effects of these drugs.

Identifying risk factors for recurrent multidrug resistant tuberculosis based on patient's record data from 2016 to 2021: retrospective study.

Globally, the prevalence of multidrug-resistant tuberculosis (MDR-TB) has been increasing recently. This is a major public health concern, as MDR-TB is more difficult to treat and has poorer outcomes compared to drug-sensitive tuberculosis. The main objective of the study was to identify risk factors for recurrent multidrug-resistant tuberculosis, at Alert Specialized Hospital, Addis Ababa, by using different parametric shared frailty models. From January 2016 to December 2021, a retrospective study was conducted on MDR-TB patients at Alert Specialized Hospital in Addis Ababa. The data for the study were collected from the medical records of MDR-TB patients at the hospital during this time period. Gamma and inverse-Gaussian shared frailty models were used to analyze the dataset, with the exponential, Weibull, and lognormal distributions included as baseline hazard functions. The data were analyzed using R statistical software. The median recurrence time of the patients was 12 months, and 149 (34.3%) had recurrences. The clustering effect was statistically significant for multiple drug-resistant tuberculosis patients' recurrence. According to the Weibull-Inverse-Gaussian model, factors that reduced time to MDR-TB recurrence included lower weight (ɸ = 0.944), smoking (ɸ = 0.045), alcohol use (ɸ = 0.631), hemoptysis (ɸ = 0.041), pneumonia (ɸ = 0.564), previous anti-TB treatment (ɸ = 0.106), rural residence (ɸ = 0.163), and chronic diseases like diabetes (ɸ = 0.442) were associated with faster recurrence. While, higher education (ɸ = 3.525) and age (ɸ = 1.021) extended time to recurrence. For weight increment, smokers and alcohol users, clinical complications of hemoptysis and pneumonia, patients with pulmonary disease who had a history of previous anti-TB treatment, and being rural residents are prognostic factors. There was a significant clustering effect at the Alert Specialized Hospital in Addis Ababa, Ethiopia. The Weibull-Inverse Gaussian Shared Frailty Model was chosen as the best model for predicting the time to recurrence of MDR-TB.

Synergistic pH-responsive MUC-1 aptamer-conjugated Ag/MSN Janus nanoparticles for targeted chemotherapy, photothermal therapy, and gene therapy in breast cancer.

Drug resistance in cancer treatment, primarily attributed to the overexpression of the multidrug resistance (MDR) gene, significantly hampers the effectiveness of chemotherapy. This mechanism, driven by the increased production of P-glycoprotein (P-gp) efflux pumps, highlights the urgent need for innovative strategies to combat drug resistance in cancer patients. This study explores the application of antisense technology to suppress MDR gene expression, while addressing the challenges of instability and limited cellular uptake associated with antisense oligonucleotides. We synthesized Janus silver-mesoporous silica nanoparticles (Ag/MSN JNPs) using a sol-gel method, characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS), revealing uniformly sized, dumbbell-shaped nanoparticles with an average size of 285 ± 5.12 nm. Doxorubicin (DOX) was loaded into the porous structure of the mesoporous silica, and JNPs were functionalized with chitosan (CS) to incorporate P-gp antisense and a MUC-1 aptamer, serving as a pH-responsive gatekeeper. Our findings indicate that the Ap-As-DOX-JNPs achieved a remarkable 89 ± 0.59 % cell death in drug-resistant MCF-7/ADR cells after 48 h, alongside an 80 % reduction in P-gp expression. The combination of DOX, antisense technology, and photothermal therapy utilizing these JNPs demonstrates a promising strategy to effectively overcome drug resistance. Notably, normal MCF-7 cells exhibited reduced viability from 39.11 ± 1.12 % to 30.05 ± 1.07 % when treated with DOX-JNPs under near-infrared (NIR) irradiation. These results underscore the potential of utilizing MUC-1 aptamer-conjugated Janus nanoparticles in conjunction with chitosan as a gatekeeper to enhance the efficacy of chemotherapy, photothermal therapy, and gene therapy in overcoming multidrug resistance in cancer treatment.

The VEGFA-Induced MAPK-AKT/PTEN/TGFß Signal Pathway Enhances Progression and MDR in Gastric Cancer.

BACKGROUND/OBJECTIVES: Gastric cancer (GC) is a globally frequent cancer, in particular leading in mortality caused by digestive tract cancers in China. Vascular endothelial growth factor A (VEGFA) is excessively expressed in cancers including GC; its involvement in GC development, particularly in multidrug resistance (MDR), and the signal route it affects in GC remain unknown. To explore the roles VEGFA plays during progression and MDR formation in GC, we studied its function in a VEGFA-deleted GC cell platform. METHODS: We initially assessed the importance of VEGFA in GC and MDR using database analysis. Then, using CCK8, wound healing, transwell, scanning electron microscopy, immunofluorescence, flow cytometry, and other techniques, the alterations in tumor malignancy-connected cell behaviors and microstructures were photographed and evaluated in a VEGFA-gene-deleted GC cell line (VEGFA-/-SGC7901). Finally, the mechanism of VEGFA in GC progression and MDR was examined by Western blot. RESULTS: Database analysis revealed a strong correlation between high VEGFA expression and a poor prognosis for GC. The results showed that VEGFA deletion reduced GC cell proliferation and motility and altered microstructures important for motility, such as the depolymerized cytoskeleton. VEGFA deletion inhibited the growth of pseudopodia/filopodia and suppressed the epithelial-mesenchymal transition (EMT). The occurrence of MDR is induced by overactivation of the MAPK-AKT and TGFß signaling pathways, while PTEN inhibits these pathways. CONCLUSIONS: All findings suggested that VEGFA acts as a cancer enhancer and MDR inducer in GC via the MAPK-AKT/PTEN/TGFß signal pathway.

Antiproliferative Activity of Cephalotaxus Esters: Overcoming Chemoresistance.

INTRODUCTION: Omacetaxine, a semisynthetic form of Homoharringtonine (HHT), was approved for the treatment of Chronic Myeloid Leukemia (CML). Previously, we have published the synthesis of this natural alkaloid and three of its derivatives: Deoxyharringtonine (DHT), Deoxyhomoharringtonine (DHHT), and Bis(demethyl)-deoxyharringtonine (BDHT), and reported its refractory activity against the HL-60/RV+ cells over-expressing P-glycoprotein 1 (MDR1). METHODS: In this study, we have explored the extent of this resistance by first expanding the panel of established cell lines and using a panel of 21 leukemia patient-derived primary cells. RESULTS: Herein, we have reported consistent resistance to HTT of K562-derived cells and to mitoxantrone of MES-SA/MX2-derived cells; all of them have been found to overexpress MDR1, while we have found U87MG-ABCG2 and H69AR cells to be very sensitive to HTT. In contrast, DHT, DHHT, and BDHT seemingly overcame this resistance due to the changes made to the acyl chain of HTT, rendering the derivatives less susceptible to efflux. Surprisingly, the leukemia primary cells were very sensitive to HHT and its derivatives with low nanomolar potencies, followed by a new class of CDC7 kinase inhibitors, the anthracycline class of topoisomerase inhibitors, the DNA intercalator actinomycin-D, and the vinca alkaloid class of microtubule inhibitors. The mechanism of cell death induced by HTT and DHHT was found to be mediated via caspase 3 cleavage, leading to apoptosis. CONCLUSION: Taken together, our results confirm that HHT is a substrate for MDR1. It opens the door to a new opportunity to clinically evaluate HHT and its derivatives for the treatment of AML and other cancers.

Diagnostic accuracy of active pulmonary tuberculosis screening during detention admission: a systematic review.

Individuals entering incarceration are at high risk for infectious diseases, other ill conditions, and risky behavior. Typically, the status of active pulmonary tuberculosis (PTB) is not known at the time of admission. Early detection and treatment are essential for effective TB control. So far, no study has compared the diagnostic accuracy of various TB screening tools in detention using a network meta-analysis (NMA). We aimed to investigate the diagnostic accuracy of active PTB screening tests upon detention admission. We searched PubMed, Global Index Medicus, the Cochrane Library electronic databases, and grey literature for publications reporting detention TB entry screening in March 2022 and January 2024. Inclusion was non-restrictive regarding time, language, location, reference standards, or screening tests. Eligible study designs comprised comparative, observational, and diagnostic studies. Publications had to report TB screening of individuals entering confinement and provide data for diagnostic accuracy calculations. The QUADAS-2 tool was designed to assess the quality of primary diagnostic accuracy studies. This systematic review was registered with PROSPERO (CRD42022307863) and conducted without external funding. We screened a total of 2,455 records. Despite extensive searching, no studies met our inclusion criteria. However, we identified evidence revealing key differences in screening algorithm application. In conclusion, more diagnostic accuracy data on TB screening algorithms for detention admission worldwide needs to be collected. We recommend that global TB initiatives set up multi-site studies to investigate the diagnostic accuracy of TB screening on admission in low- and high-prevalence criminal justice systems. Further network meta-analyses of these studies could inform policymakers and public health experts to establish or fine-tune TB control in detention settings.

Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer.

PURPOSE: All patients with metastatic breast cancer (MBC) expressing estrogen receptor-α (ESR1) will eventually develop resistance to endocrine therapies. In up to 40% of patients, this resistance is caused by activating mutations in the ligand-binding domain (LBD) of ESR1. Accumulating clinical evidence indicate adverse outcomes for these patients, beyond that expected by resistance to endocrine therapy. Here we aimed to study the role of ESR1 mutations in conferring chemoresistance in BC cells. METHODS: MCF-7 cells harboring Y537S and D538G ESR1 mutations (mut-ER) were employed to study the response to chemotherapy drugs, paclitaxel and doxorubicin, using viability and apoptotic assay in vitro, and tumor growth in vivo. JNK/c-Jun/MDR1 pathway was studied using qRT-PCR, western-blot, gene-reporter and ChIP assays. MDR1 expression was analyzed in clinical samples using IHC. RESULTS:  Cell harboring ESR1 mutations displayed relative chemoresistance compared to WT-ER, evidenced by higher viability and reduced apoptosis as well as resistance to paclitaxel in vivo. To elucidate the underlying mechanism, MDR1 expression was examined and elevated levels were observed in mut-ER cells, and in clinical BC samples. MDR1 is regulated by the c-Jun pathway, and we showed high correlation between these two genes in BC using TCGA databases. Accordingly, we detected higher JNK/c-Jun expression and activity in ESR1-mutated cells, as well as increased occupancy of c-Jun in MDR1 promoter. Importantly, JNK inhibition decreased MDR1 expression and restored sensitivity to chemotherapy. CONCLUSIONS: Taken together, these data indicate that ESR1 mutations confer chemoresistance through activation of the JNK/MDR1 axis. These finding suggest a novel treatment option for BC tumors expressing ESR1 mutations.

Delivery of doxorubicin by Fe3O4 nanoparticles, reduces multidrug resistance gene expression in ovarian cancer cells.

BACKGROUND: Ovarian cancer is one of the most common malignancy in women with significant mortality rate due to the resistance to chemotherapy drugs. Doxorubicin (DOX) is a chemotropic agent in ovarian cancer treatment. Overexpression of multidrug resistance (MDR) genes, such as ABCB1, in cancer cells after chemotherapy is one of main problems in clinical applications. Here we have compared the efficiency of doxorubicin-loaded (NIPAAM-DMAEMA) Fe3O4 nanocomposite (DOX-NANO) against DOX on ABCB1(MDR1) gene expression in the ovarian cancer cell line. MATERIALS AND METHODS: The cell viability of SKOV-3 cells were evaluated by MTT assay. Real Time PCR was used to measure the expression level of MDR1. MTT data were normalized in 10 different attribute weighting models, also to reveal the interaction between DOX, ABCB1, and ovarian cancer genes, Pathway Studio Database (Elsevier) was used. RESULTS: Cell viability of SKOV-3cells was significantly decreased after 24, 48 and 72 hours (P < 0.0001) of either DOX with IC50 22.38, 0.61 and 0.072 µg/ml or DOX-NANO treatment with IC50 11.54, 1.01, 0.0126 µg/ ml respectively. TREATMENT: Notable decrease in the expression of MDR gene, ABCB1, was observed 48 hours after treatment with DOX-NANO (P < 0.0001) with 26 % in the assessed with control group. Meta-analysis showed the concentration of 10 µg/ml variables was the second most significant feature, whereas the concentration of 0.01 µg/ml recognized the lowest weights. Also, LGALS3 is an extra cellular receptor with upregulation in ovarian cancer that interacts with ABCB1. CONCLUSION: Our findings highlight the beneficial effects of DOX delivery in ovarian cancer cells by nanocomposite as efficient drug delivery method. DOX-NANO is a promising therapeutic reagent to overcome chemotherapy resistance in ovarian cancer.

Multi-drug resistant Pseudomonas aeruginosa isolation is an independent risk factor for recurrent hemoptysis after bronchial artery embolization in patients with idiopathic bronchiectasis: a retrospective cohort study.

BACKGROUND: Currently, there is a lack of research on multi-drug resistant Pseudomonas aeruginosa (MDR-PA) isolation in bronchiectasis-related hemoptysis. The aim of this study to analyze the risk factors for recurrent hemoptysis following bronchial artery embolization (BAE) and compare the recurrent hemoptysis-free rates between MDR-PA, non-MDR-PA, and non-PA isolation. METHODS: A retrospective study was performed of patients diagnosed with idiopathic bronchiectasis-related recurrent hemoptysis who underwent BAE at an university-affiliated hospital. Patients were categorized based on PA susceptibility tests into non-PA, non-MDR-PA, and MDR-PA groups. Univariate and multivariate Cox regression were conducted to identify independent risk factors for recurrent hemoptysis. The Kaplan-Meier curves was conducted to compare recurrent hemoptysis-free rates after BAE for non-PA, non-MDR-PA, and MDR-PA. RESULTS: A total of 432 patients were included. 181 (41.90%) patients experienced recurrent hemoptysis during a median follow-up period of 25 months. MDR-PA isolation (adjusted hazard ratio (aHR) 2.120; 95% confidence interval (CI) [1.249, 3.597], p = 0.005) was identified as an independent risk factor for recurrent hemoptysis. Antibiotic treatment (aHR 0.666; 95% CI [0.476, 0.932], p = 0.018) reduced the risk of recurrent hemoptysis. The cumulative recurrent hemoptysis-free rates for non-PA, non-MDR-PA, and MDR-PA were as follows: at 3 months, 88.96%, 88.24%, and 75.86%, respectively; at 1 year, 73.13%, 69.10%, and 51.72%; and at 3 years, 61.91%, 51.69%, and 41.10% (p = 0.034). CONCLUSION: MDR-PA isolation was an independent risk factor of recurrent hemoptysis post-BAE. Reducing the occurrence of MDR-PA may effectively decrease the recurrence rates of hemoptysis.

Serum metabonomics reveal the effectiveness of human placental mesenchymal stem cell therapy for primary sclerosing cholangitis.

BACKGROUND: The metabolic patterns of human placental-derived mesenchymal stem cell (hP-MSC) treatment for primary sclerosing cholangitis (PSC) remain unclear, and therapeutic effects significantly vary due to individual differences. Therefore, it is crucial to investigate the serological response to hP-MSC transplantation through small molecular metabolites and identify easily detectable markers for efficacy evaluation. METHODS: Using Mdr2-/- mice as a PSC model and Mdr2+/+ mice as controls, the efficacy of hP-MSC treatment was assessed based on liver pathology, liver enzymes, and inflammatory factors. Serum samples were collected for 12C-/13C-dansylation and DmPA labeling LC-MS analysis to investigate changes in metabolic pathways after hP-MSC treatment. Key metabolites and regulatory enzymes were validated by qRT-PCR and Western blotting. Potential biomarkers of hP-MSC efficacy were identified through correlation analysis and machine learning. RESULTS: Collectively, the results of the liver histology, serum liver enzyme levels, and inflammatory factors supported the therapeutic efficacy of hP-MSC treatment. Based on significant differences, 41 differentially expressed metabolites were initially identified; these were enriched in bile acid, lipid, and hydroxyproline metabolism. After treatment, bile acid transport was accelerated, whereas bile acid production was reduced; unsaturated fatty acid synthesis was upregulated overall, with increased FADS2 and elongase expression and enhanced fatty acid ß-oxidation; hepatic proline 4-hydroxylase expression was decreased, leading to reduced hydroxyproline production. Correlation analysis of liver enzymes and metabolites, combined with time trends, identified eight potential biomarkers: 2-aminomuconate semialdehyde, L-1-pyrroline-3-hydroxy-5-carboxylic acid, L-isoglutamine, and maleamic acid were more abundant in model mice but decreased after hP-MSC treatment. Conversely, 15-methylpalmitic, eicosenoic, nonadecanoic, and octadecanoic acids were less abundant in model mice but increased after hP-MSC treatment. CONCLUSIONS: This study revealed metabolic regulatory changes in PSC model mice after hP-MSC treatment and identified eight promising biomarkers, providing preclinical evidence to support therapeutic applications of hP-MSC.

In Vitro Antibacterial Activity of Adiantum capillus Veneris Extraction with Methanol, Chloroform, and Ether Solvents against Methicillin-resistant Staphylococcus aureus.

BACKGROUND: The increasing problem of multi-drug resistant (MDR) pathogens is a worldwide concern, especially in the pharmaceutical industry. At the same time, medicinal plants have renewed interest because of their wide variety of bioactive phytochemicals, which could be used to develop new antimicrobial drugs. This renewed interest is partly due to the growing resistance to traditional drugs and their associated side effects. METHODS: The objective of this study is to assess the antimicrobial properties of the total extract and various fractions of Adiantum capillus veneris against Methicillin-resistant Staphylococcus aureus (MRSA). The aerial parts of Adiantum capillus veneris were subjected to extraction using methanol, chloroform, and ether, and the resulting extracts were tested for their antimicrobial activity against MRSA. Additionally, essential oil was obtained from the aerial parts using a Clevenger apparatus and boiling water. Furthermore, Gas Chromatography-Mass Spectrometry (GC/MS) was utilized to analyze the phytochemicals isolated from the extracts of Adiantum capillus veneris. RESULTS: The essential oil was obtained through distillation and then analyzed using GC/MS. The antimicrobial activity was evaluated using the agar diffusion method. CONCLUSION: GC/MS analysis revealed that the composition was primarily phytol (59.9%), constituting 99.3% of phyto-constituents. However, both the total extract and the individual fractions exhibited no inhibitory effects against MRSA strains.

Synthesis and evaluation of novel tetrahydroisoquinoline-benzo[h]chromen-4-one conjugates as dual ABCB1/CYP1B1 inhibitors for overcoming MDR in cancer.

The emergence of multidrug resistance (MDR) in malignant tumors is one of the major threats encountered currently by many chemotherapeutic agents. Among the various mechanisms involved in drug resistance, P-glycoprotein (P-gp, ABCB1), a member of the ABC transporter family that significantly increases the efflux of various anticancer drugs from tumor cells, and the metabolic enzyme CYP1B1 are widely considered to be two critical targets for overcoming MDR. Unfortunately, no MDR modulator has been approved by the FDA to date. In this study, based on pharmacophore hybridization, bioisosteric and fragment-growing strategies, we designed and synthesized 11 novel tetrahydroisoquinoline-benzo[h]chromen-4-one conjugates as dual ABCB1/CYP1B1 inhibitors. Among them, the preferred compound A10 exhibited the best MDR reversal activity (IC50 = 0.25 µM, RF = 44.4) in SW620/AD300 cells, being comparable to one of the most potent third-generation P-gp inhibitors WK-X-34. In parallel, this dual ABCB1/CYP1B1 inhibitory effect drives compound A10 exhibiting prominent drug resistance reversal activity to doxorubicin (IC50 = 4.7 µM, RF = 13.7) in ABCB1/CYP1B1-overexpressing DOX-SW620/AD300-1B1 resistant cells, which is more potent than that of the CYP1B1 inhibitor ANF. Furthermore, although compound A2 possessed moderate ABCB1/CYP1B1 inhibitory activity, it showed considerable antiproliferative activity towards drug-resistant SW620/AD300 and MKN45-DDP-R cells, which may be partly related to the increase of PUMA expression to promote the apoptosis of the drug-resistant MKN45-DDP-R cells as confirmed by proteomics and western blot assay. These results indicated that the tetrahydroisoquinoline-benzo[h]chromen-4-one conjugates may provide a fundamental scaffold reference for further discovery of MDR reversal agents.

Systemic Codelivery of Thymoquinone and Doxorubicin by Targeted Mesoporous Silica Nanoparticle Sensitizes Doxorubicin-Resistant Breast Cancer by Interfering between the MDR1/P-gp and miR 298 Crosstalk.

Multi drug resistance (MDR) in breast carcinoma still poses a significant impairment to successful chemotherapy. As the arsenal of anticancer agents increases with improved preclinical methods, the growth of therapeutic drug combinations is now unprecedented. The malignancies addressed by mono drugs often fail to limit cancer progression, resulting in resistant cancer, thereby offering combinatorial therapies a terrific edge over monodrug regimes. However, the selection of drug combinations required enough preliminary evidence for their synergistic effect. The fundamental mechanisms of MDR to chemotherapeutics are associated with the overexpression of membrane efflux pumps, alternations in drug targets, and increased drug metabolism. Unfortunately, it is very difficult for drugs to overcome resistance produced on their own or by another different drug action. In this context, herein, we report a simple delivery system for coencapsulation and intracellular codelivery of dual-drug thymoquinone (TQ) and doxorubicin (DOX) to resensitize DOX-resistant MDA MB231 cell line (231 R). The 231 R cell line developed in our lab showed an enhanced expression of the ATP-binding cassette (ABC) transporters P-gp1/MDR-1 and a declined miR-298 expression. The present delivery system is based on amine-functionalized mesoporous silica nanoparticles (MSNs), in which the side chain amine functional group was used to react with the carbonyl group of TQ, which acts as a pro-drug system (TQ-MSN) to release TQ and DOX simultaneously. DOX was encapsulated later into the above TQ-MSN by a simple diffusion method. The drugs containing MSNs were further coated with a hyaluronic acid-conjugated PEG-PLGA polymer (HA@TQ-DOX-MSN). This simple nanostrategy interferes with the MDR-1/miR-298 cross-talk, thereby allowing a significant reduction in drug efflux from the cell and highlighting a promising nanotechnology-based combinatorial delivery approach in managing breast cancer chemoresistance.

Myosin Vb Traffics P-Glycoprotein to the Apical Membrane of Intestinal Epithelial Cells.

BACKGROUND & AIMS: The xenobiotic efflux pump P-glycoprotein is highly expressed on the apical membrane of the gastrointestinal tract, where it regulates the levels of intracellular substrates. P-glycoprotein is altered in disease, but the mechanisms that regulate the levels of P-glycoprotein are still being explored. The molecular motor myosin Vb (Myo5b) traffics diverse cargo to the apical membrane of intestinal epithelial cells. We hypothesized that Myo5b was responsible for the delivery of P-glycoprotein to the apical membrane of enterocytes. METHODS: We used multiple murine models that lack functional Myo5b or the myosin binding partner Rab11a to analyze P-glycoprotein localization. Pig and human tissue were analyzed to determine P-glycoprotein localization in the setting of MYO5B mutations. Intestinal organoids were used to examine P-glycoprotein trafficking and to assay P-glycoprotein function when MYO5 is inhibited. RESULTS: In mice lacking Myo5b or the binding partner Rab11a, P-glycoprotein was improperly trafficked and had decreased presence in the brush border of enterocytes. Immunostaining of a pig model lacking functional Myo5b and human biopsies from a patient with an inactivating mutation in Myo5b also showed altered localization of intestinal P-glycoprotein. Human intestinal organoids expressing the motorless MYO5B tail domain had colocalization with P-glycoprotein, confirming that P-glycoprotein was trafficked by MYO5B in human enterocytes. Inhibition of MYO5 in human intestinal cell lines and organoids resulted in decreased P-glycoprotein capacity. Additionally, inhibition of MYO5 in human colon cancer cells diminished P-glycoprotein activity and increased cell death in response to a chemotherapeutic drug. CONCLUSIONS: Collectively, these data demonstrate that Myo5b is necessary for the apical delivery of P-glycoprotein.

Evaluation of Novel Diaza Cage Compounds as MRP Modulators in Cancer Cells.

AIM: Novel MRP modulators are needed to combat MRP-mediated multidrug resistance (MDR) in cancer cells. BACKGROUND: Anticancer drug resistance is the main problem in cancer therapy. Causative multidrug efflux pumps are attractive target structures for the development of inhibitors of their activity. OBJECTIVE: We synthesized novel cage dimeric 1,4-dihydropyridines to evaluate them as MRP modulators in cancer cells targeting MRP1, MRP2, and MRP4. METHOD: Cage compounds were synthesized by solution dimerization of monomeric 1,4-dihydropyridines and a final functionalization reaction. The MRP modulation was determined in cellular efflux assays by the use of the flow cytometry technique as well as cellular fluorescent measurements with each fluorescent substrate of the efflux pumps. RESULTS: Difluoro phenyl and methoxy or dimethoxy benzyl substitutions were most favourable for the MRP1 and MRP2 inhibition, whereas monofluor phenyl and dimethoxy benzyl substitutions were most favourable for the MRP4 inhibition. CONCLUSION: Effective inhibitors were identified that were demonstrated to restore the respective cancer cell line sensitivity for the anticancer drug as a proof-of-concept that encourages further preclinical studies.

Differential Effect of Simulated Microgravity on the Cellular Uptake of Small Molecules.

The space environment can affect the function of all physiological systems, including the properties of cell membranes. Our goal in this study was to explore the effect of simulated microgravity (SMG) on the cellular uptake of small molecules based on reported microgravity-induced changes in membrane properties. SMG was applied to cultured cells using a random-positioning machine for up to three hours. We assessed the cellular accumulation of compounds representing substrates of uptake and efflux transporters, and of compounds not shown to be transported by membrane carriers. Exposure to SMG led to an increase of up to 60% (p < 0.01) in the cellular uptake of efflux transporter substrates, whereas a glucose transporter substrate showed a decrease of 20% (p < 0.05). The uptake of the cathepsin activity-based probe GB123 (MW, 1198 g/mol) was also enhanced (1.3-fold, p < 0.05). Cellular emission of molecules larger than ~3000 g/mol was reduced by up to 50% in SMG (p < 0.05). Our findings suggest that short-term exposure to SMG could differentially affect drug distribution across membranes. Longer exposure to microgravity, e.g., during spaceflight, may have distinct effects on the cellular uptake of small molecules.

Raman Spectroscopy for Instant Bladder Tumor Diagnosis: System Development and In Vivo Proof-Of-Principle Study in Accordance with the European Medical Device Regulation (MDR2017/745).

This work reports on an in vivo Raman-based endoscopy system, invaScope, enabling Raman measurements of healthy and tumor bladder tissue during an endoscopic procedure in the operating theatre. The presented study outlines the progression from the initial concept (validated through previously performed ex vivo studies) to the approval and implementation of a clinical investigational device according to the requirement within the framework of the European Medical Device Regulation (MDR2017/745). The study's primary objective was to employ the invaScope Raman system within the bladder, capturing in vivo spectroscopic Raman data followed by standard histo- and cytopathological examinations of urological tissue (considered the gold standard). The collected data were analyzed and correlated with histopathological findings post-procedure. Additionally, the study aimed to assess the feasibility of using diagnostic equipment, probes, and software for application in a clinical setting, evaluating usability aspects that are important during surgical procedures. This research represents a pivotal step toward advancing Raman spectroscopy for routine clinical use in characterizing bladder lesions.

Reversible chemoresistance of pancreatic cancer grown as spheroids.

Better in vitro models are needed to identify active drugs to treat pancreatic adenocarcinoma (PAC) patients. We used 3D hanging drop cultures to produce spheroids from five PAC cell lines and tested nine FDA-approved drugs in clinical use. All PAC cell lines in 2D culture were sensitive to three drugs (gemcitabine, docetaxel and nab-paclitaxel), however most PAC (4/5) 3D spheroids acquired profound chemoresistance even at 10 µM. In contrast, spheroids retained sensitivity to the investigational drug triptolide, which induced apoptosis. The acquired chemoresistance was also transiently retained when cells were placed back into 2D culture and six genes potentially associated with chemoresistance were identified by microarray and confirmed using quantitative RT-PCR. We demonstrate the additive effect of gemcitabine and erlotinib, from the 12 different combinations of nine drugs tested. This comprehensive study shows spheroids as a useful multicellular model of PAC for drug screening and elucidating the mechanism of chemoresistance.

The role of efflux transporters in cytotoxicity and intracellular concentration of chlorpyrifos and chlorpyrifos oxon in human cell lines.

In this study, we investigated the role of two efflux transporters, p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), in the cytotoxicity and intracellular accumulation of the organophosphate pesticide chlorpyrifos (CPF) and its active metabolite, CPF-oxon (CPFO), in a human-derived liver cell line (HepG2) and kidney epithelial cell line (HK-2). The cytotoxicity to CPF and CPFO differed between cell lines where HK-2 had lower IC50 values which could be attributed to lower basal expression and inducibility of metabolizing enzymes, transporters, and nuclear receptors in HK-2 cells. In HepG2 cells, co-exposure of CPF with a specific inhibitor of either P-gp or BCRP enhanced the cytotoxicity of CPF while co-exposure of CPFO with VRP enhanced the cytotoxicity of CPFO, suggesting the role of these transporters in the elimination CPF and CPFO. Inhibition of efflux transporters did not affect the cytotoxicity of CPF and CPFO in HK-2 cells. Co-incubation of CPF with P-gp and BCRP inhibitors increased the intracellular concentration of CPF in HepG2 cells suggesting that both transporters play a role in limiting the cellular accumulation of CPF in HepG2 cells. Our results provide evidence that inhibition of efflux transporters can enhance CPF-induced toxicity through enhanced cellular accumulation and raises additional questions regarding how pesticide-transporter interactions may influence toxicity of mixtures containing pesticides and other environmental chemicals.

Synergy of histone acetyltransferase inhibitor (HATi) with quercetin inhibits biofilm formation in Candida tropicalis.

Histone acetyltransferase inhibitors (HATi) are mechanism-based inhibitors that show promise in the treatment of several illnesses, including diabetes, hyperlipidemia, cancer, and Alzheimer's disease. The work emphasizes the significance of HATi as a possible treatment strategy against Candida species biofilms. Here, in this study, we found that combining a HATi, anacardic acid (AA), and quercetin, a known flavonoid, significantly prevented biofilm formation by C. tropicalis. We further show that C. tropicalis exhibited a considerable downregulation of drug-resistance gene expression (CDR1 and MDR1) when co-administrated. Additionally, in silico studies revealed that the AA interacts strongly with a histone acetyltransferase, Rtt109, which may account for the observed biofilm inhibitory effect. In conclusion, the study illustrates how HATi may be used to potentiate the inhibitory action of phytoactives or antifungals against drug-resistant yeast infections.