Resistance Protein Research Articles

Rifampicin and its derivatives: stability, disposition, and affinity towards pregnane X receptor employing 2D and 3D primary human hepatocytes

Rifampicin is a model ligand of the pregnane X receptor (PXR), the nuclear receptor involved in the regulation of cytochrome P450 3A4 (CYP3A4). Rifampicin forms several degradation products and metabolites of which 25-desacetylrifampicin is the most abundant in vivo. Here, we aimed to study both the stability and metabolism of rifampicin in media and 2D and 3D primary human hepatocytes (PHHs). Additionally, we analyzed interactions of rifampicin derivatives with PXR. We described that rifampicin gradually degrades by more than 50 % in the medium partly into quinone over 72 h. We observed 25-desacetylrifampicin in 2D PHHs but not in 3D PHHs. Contrary, rifampicin was converted into quinone in a one-direction process in media of 3D PHHs. The potency of rifampicin and its derivatives to activate human PXR was arranged as follows: 3-formylrifamycin SV > rifampicin quinone > rifampicin > rifampicin N-oxide > 25-desacetylrifampicin, respectively, but none activates mouse and rat PXR. The binding differences between rifampicin and 25-desacetylrifampicin were modeled in silico. Finally, we showed that overexpressed uptake organic anion transporting polypeptide 1B1 (OATP1B1) potentiated activation of PXR by rifampicin and rifampicin quinone, but overexpressed efflux multidrug resistance protein 1 (MDR1) decreased PXR activation by all derivatives.

Bioenergetics disruption, oxidative stress, and inflammation as underlying mechanisms of tramadol-induced nephrotoxicity.

Tramadol (TR), a commonly prescribed pain reliever for moderate to severe pain, has been associated with kidney damage. This study investigates TR-induced nephrotoxicity mechanisms, focusing on its effects on renal proximal tubular cells (PTCs). The study findings demonstrate that TR disrupts PTC bioenergetic processes, leading to oxidative stress and inflammation. Significant toxicity to PTCs was observed with estimated effective concentration 50 values of 9.8 and 11.5 µM based on 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, respectively. TR also interferes with the function of PTC transporters, including organic cation uptake transporter 1, organic cation transporter 2, P-glycoprotein, and multidrug resistance protein 2. Furthermore, bioenergetics assays showed that TR reduced the activities of mitochondrial complexes I and III, adenosine triphosphate production, mitochondrial membrane potential, and oxygen consumption rate while increasing lactate release. TR also increased the production of reactive oxygen species, lipid peroxidation thiobarbituric acid reactive substances end products, and the expression of the NRf2 gene while decreasing reduced glutathione (GSH-R) stores and catalase and superoxide dismutase antioxidant activities. Additionally, TR increased the production of inflammatory cytokines (TNF-α and IL-6) and their coding genes expression. Interestingly, the study found that antioxidants like GSH-R, inhibitors of IL-6 and TNF-α, and mitochondrial activating Co-Q10 could protect cells against TR-induced cytotoxicity. The study suggests that TR causes nephrotoxicity by disrupting bioenergetic processes, causing oxidative stress and inflammation, but antioxidants and agents targeting mitochondria may have protective and curative potential.

Effect of Genetic Polymorphisms of ABCB1, ABCG2, and SLC22A1 on the Steady-State Plasma Concentrations of Lamotrigine in Treatment-Resistant Depressed Patients Treated With Lamotrigine Augmentation Therapy.

The authors have demonstrated that a plasma lamotrigine concentration of 12.7 μmol/L may be a threshold for a good therapeutic response to lamotrigine augmentation therapy in treatment-resistant depressed patients. Lamotrigine is a substrate of P-glycoprotein, breast cancer resistant protein and organic cation transporter 1, which are encoded by ABCB1 , ABCG2 , and SLC22A1 , respectively. There have been several polymorphisms that affect its function. The present study investigated the relationship between these polymorphisms and the steady-state plasma concentrations (Css) of lamotrigine in treatment-resistant depressed patients receiving lamotrigine as augmentation therapy. One hundred twenty-nine treatment-resistant depressed patients were included in this study. Treatment resistance is defined as lack of therapeutic response to at least 3 psychotropics despite adequate doses and duration. Their diagnoses were as follows: major depressive disorder (n = 58), bipolar II disorder (n = 52), and bipolar I disorder (n = 19). Lamotrigine augmentation therapy for 8 weeks was conducted. The final lamotrigine doses were 75 mg/d for 39 patients with valproate and 100 mg/d for 90 without it. Blood was sampled at 8:00 am after the 8th week of treatment. Plasma lamotrigine levels were quantified by using LC/MS/MS. The polymorphisms of ABCB1 1236C>T, 2677G>T/A, 3435C>T, ABCG2 421C>A, and SLC22A1 1222G>A were detected by polymerase chain reaction analyses. No significant relationships were observed between these polymorphisms and the Css of lamotrigine in the patients with or without valproate. The present study suggests that these genetic polymorphisms do not play a role in controlling the Css of lamotrigine in treatment-resistant depressed patients treated with lamotrigine augmentation therapy.

Nosocomial transmission of fluconazole-resistant Candida glabrata bloodstream isolates revealed by whole-genome sequencing.

The prevalence of fluconazole-resistant bloodstream infections caused by Candida glabrata is increasing globally, but the transmission of these resistant strains within hospitals has rarely been documented. Through whole-genome sequencing and epidemiological analyses, this study identified two potential clusters of C. glabrata bloodstream infections within the same hospital, revealing the transmission of clonal C. glabrata strains with different levels of fluconazole susceptibility or distinct transcription factor pleiotropic drug resistance protein 1 (Pdr1p) single-nucleotide polymorphism profiles among patients receiving antifungal therapy.

Synthesis and Antimicrobial Activity of Newly Synthesized Nicotinamides.

Antioxidants are promising compounds with antimicrobial activity against drug-resistant pathogens, especially when combined with conventional antimicrobials. Our study aimed to characterize the structure of nicotinamides synthesized from nicotinic acid and thiocarbohydrazones and to evaluate their antibacterial and antifungal activity. Seven nicotinic acid hydrazides (NC 1-7) were synthesized using mono-thiocarbohydrazones with hydroxyl group substituents, along with quinolone, phenolic, and pyridine rings known for their antimicrobial activity. The in vitro antimicrobial activity of NC 1-7, at concentrations ranging from 0.001 to 1 mM, was tested against Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (NCIMB 9111), and Candida albicans (ATCC 24433) using the broth microdilution method per EUCAST 2024 guidelines. Microorganism survival percentages were calculated based on optical density, and target fishing using the PharmMapper database identified potential molecular targets. The results showed that P. aeruginosa was most susceptible to the compounds, while C. albicans was the least susceptible. NC 3 significantly inhibited P. aeruginosa and K. pneumoniae growth at 0.016 mM, while higher concentrations were required for S. aureus, E. faecalis, and C. albicans. NC 5 was most effective against gram-positive bacteria at 0.03 mM. Only NC 4 completely inhibited C. albicans below 1 mM. NC 3, with the lowest concentration for 50% growth inhibition (0.016-0.064 mM), showed promising antibacterial potential against specific AMR-related proteins (bleomycin resistance protein, HTH-type transcriptional regulator QacR, and streptogramin A acetyltransferase), suggesting that this class of compounds could enhance or restore the activity of established antibiotics.

MiR-149-3p Enhances Drug Sensitivity of AML Cells by Inhibiting Warburg Effect Through PI3K/AKT Pathway.

Acute myeloid leukemia (AML) is a kind of heterogeneous hematologic malignancy with high incidence, which is usually treated by intensive and maintenance treatment with large dose of conventional chemotherapy drugs. However, cell resistance is still an unsolved problem. The abnormal expression of miRNAs is closely related to the pathogenesis and progression of AML, and affects the drug resistance of cancer cells. miR-149-3p plays an important role in the resistance of cancer cells to cisplatin, and plays an excellent anti-tumor activity. By studying the function of miR-149-3p, it is expected to find new therapeutic methods to reverse chemotherapy resistance. In order to explore the mechanism of action of miR-149-3p on AML chemotherapeutic drug sensitivity, we explored the relationship between the Warburg effect and AML chemotherapeutic drug resistance. Based on AML cells, transfection of miR-149-3p inhibitor/NC and Warburg effect inhibitor (2DG) and PI3K/AKT pathway inhibitor (LY294002) were used to investigate the mechanism of IFN-γ regulating chemotherapy resistance of AML cells through Warburg effect. Down-regulation of miR-149-3p significantly inhibited drug sensitivity of AML cells. Down-regulation of miR-149-3p significantly promoted proliferation and invasion of AML cells while inhibiting apoptosis by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax. Down-regulation of miR-149-3p significantly promoted the expression of Warburg effect-related proteins hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), and Glucose transporter 1 (GLUT1), glucose consumption, lactic acid, and intracellular ATP production. After inhibiting the Warburg effect with 2DG, the effect of miR-149-3p was inhibited, suggesting that upregulation of miR-149-3p reversed AML cell resistance by inhibiting the Warburg effect. In addition, miR-149-3p interacted with AKT1. Down-regulation of miR-149-3p increased the expression of inosine phosphate 3 kinase (PI3K), protein kinase B (AKT), and multi-drug resistance protein (MDR1). LY294002 inhibited the expression of these proteins, and down-regulation of miR-149-3p reversed the effect of LY294002 and improved the drug resistance of cells. Upregulation of miR-149-3p expression may potentially be a therapeutic target for AML resistance. It has been shown to inhibit PI3K/AKT pathway activation, thereby inhibiting the Warburg effect, and affecting cell proliferation, apoptosis, and drug resistance.

Human enteroid monolayers as a potential alternative for Ussing chamber and Caco-2 monolayers to study passive permeability and drug efflux.

After oral administration, the intestine is the first site of drug absorption, making it a key determinant of the bioavailability of a drug, and hence drug efficacy and safety. Existing non-clinical models of the intestinal barrier in vitro often fail to mimic the barrier and absorption of the human intestine. We explore if human enteroid monolayers are a suitable tool for intestinal absorption studies compared to primary tissue (Ussing chamber) and Caco-2 cells. Bidirectional drug transport was determined in enteroid monolayers, fresh tissue (Ussing chamber methodology) and Caco-2 cells. Apparent permeability (Papp) and efflux ratios for enalaprilat (paracellular), propranolol (transcellular), talinolol (P-glycoprotein (P-gp)) and rosuvastatin (Breast cancer resistance protein (BCRP)) were determined and compared between all three methodologies and across intestinal regions. Bulk RNA sequencing was performed to compare gene expression between enteroid monolayers and primary tissue. All three models showed functional efflux transport by P-gp and BCRP with higher basolateral to apical (B-to-A) transport compared to apical-to-basolateral (A-to-B). B-to-A Papp values were similar for talinolol and rosuvastatin in tissue and enteroids. Paracellular transport of enalaprilat was lower and transcellular transport of propranolol was higher in enteroids compared to tissue. Enteroids appeared show more region- specific gene expression compared to tissue. Fresh tissue and enteroid monolayers both show active efflux by P-gp and BCRP in jejunum and ileum. Hence, the use of enteroid monolayers represents a promising and versatile experimental platform to complement current in vitro models.

Wheat Leaf Rust Fungus Effector Protein Pt1641 Is Avirulent to TcLr1.

Wheat leaf rust fungus is an obligate parasitic fungus that can absorb nutrients from its host plant through haustoria and secrete effector proteins into host cells. The effector proteins are crucial factors for pathogenesis as well as targets for host disease resistance protein recognition. Exploring the role of effector proteins in the pathogenic process of Puccinia triticina Eriks. (Pt) is of great significance for unraveling its pathogenic mechanisms. We previously found that a cysteine-rich effector protein, Pt1641, is highly expressed during the interaction between wheat and Pt, but its specific role in pathogenesis remains unclear. Therefore, this study employed techniques such as heterologous expression, qRT-PCR analysis, and host-induced gene silencing (HIGS) to investigate the role of Pt1641 in the pathogenic process of Pt. The results indicate that Pt1641 is an effector protein with a secretory function and can inhibit BAX-induced programmed cell death in Nicotiana benthamiana. qRT-PCR analyses showed that expression levels of Pt1641 were different during the interaction between the high-virulence strain THTT and low-virulence strains FGD and Thatcher, respectively. The highest expression level in the low-virulence strain FGD was four times that of the high-virulence strain THTT. The overexpression of Pt1641 in wheat near-isogenic line TcLr1 induced callose deposition and H2O2 production on TcLr1. After silencing Pt1641 in the Pt low-virulence strain FGD on wheat near-isogenic line TcLr1, the pathogenic phenotype of Pt physiological race FGD on TcLr1 changed from ";" to "3", indicating that Pt1641 plays a non-toxic function in the pathogenicity of FGD to TcLr1. This study helps to reveal the pathogenic mechanism of wheat leaf rust and provides important guidance for the mining and application of Pt avirulent genes.

The Influence of Chitosan Derivatives in Combination with Bacillus subtilis Bacteria on the Development of Systemic Resistance in Potato Plants with Viral Infection and Drought.

Viral diseases of potatoes are among the main problems causing deterioration in the quality of tubers and loss of yield. The growth and development of potato plants largely depend on soil moisture. Prevention strategies require comprehensive protection against pathogens and abiotic stresses, including modeling the beneficial microbiome of agroecosystems combining microorganisms and immunostimulants. Chitosan and its derivatives have great potential for use in agricultural engineering due to their ability to induce plant immune responses. The effect of chitosan conjugate with caffeic acid (ChCA) in combination with Bacillus subtilis 47 on the transcriptional activity of PR protein genes and changes in the proteome of potato plants during potato virus Y (PVY) infection and drought was studied. The mechanisms of increasing the resistance of potato plants to PVY and lack of moisture are associated with the activation of transcription of genes encoding PR proteins: the main protective protein (PR-1), chitinase (PR-3), thaumatin-like protein (PR-5), protease inhibitor (PR-6), peroxidase (PR-9), and ribonuclease (PR-10), as well as qualitative and quantitative changes in the plant proteome. The revealed activation of the expression of marker genes of systemic acquired resistance and induced systemic resistance under the influence of combined treatment with B. subtilis and chitosan conjugate indicate that, in potato plants, the formation of resistance to viral infection in drought conditions proceeds synergistically. By two-dimensional electrophoresis of S. tuberosum leaf proteins followed by MALDI-TOF analysis, 10 proteins were identified, the content and composition of which differed depending on the experiment variant. In infected plants treated with ChCA, the synthesis of proteinaceous RNase P 1 and oxygen-evolving enhancer protein 2 was enhanced in conditions of normal humidity, and 20 kDa chaperonin and TMV resistance protein N-like was enhanced in conditions of lack of moisture. The virus coat proteins were detected, which intensively accumulated in the leaves of plants infected with potato Y-virus. ChCA treatment reduced the content of these proteins in the leaves, and in plants treated with ChCA in combination with Bacillus subtilis, viral proteins were not detected at all, both in conditions of normal humidity and lack of moisture, which suggests the promising use of chitosan derivatives in combination with B. subtilis bacteria in the regulation of plant resistance.

Effect of midostaurin on the pharmacokinetics of P-gp, BCRP, and CYP2D6 substrates: assessing potential drug-drug interactions in healthy participants : Brief title: Drug-drug interaction of midostaurin.

Midostaurin, approved for FLT3-mutated acute myeloid leukemia and advanced systemic mastocytosis, is mainly metabolized by cytochrome P450 (CYP) 3A4. Midostaurin exhibited potential inhibitory effects on P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion-transporting polyprotein 1B1, and CYP2D6 in in vitro studies. This study investigated the pharmacokinetic (PK) effects of midostaurin on P-gp (digoxin), BCRP (rosuvastatin) and CYP2D6 (dextromethorphan) substrates in healthy adults. This was an open-label, single-sequence, phase I clinical study evaluating the effect of single-dose midostaurin (100mg) on the PK of digoxin and rosuvastatin (Arm 1), and dextromethorphan (Arm 2). Participants were followed up for safety 30 days after last dose. In addition, the effect of midostaurin on the PK of dextromethorphan metabolite (dextrorphan) was assessed in participants with functional CYP2D6 genes in Arm 2. The effect of midostaurin on digoxin was minor and resulted in total exposure (AUC) and peak plasma concentration (Cmax) that were only 20% higher. The effect on rosuvastatin was mild and led to an increase in AUCs of approximately 37-48% and of 100% in Cmax. There was no increase in the primary PK parameters (AUCs and Cmax) of dextromethorphan in the presence of midostaurin. The study treatments were very well tolerated with no occurance of severe adverse events (AEs), AEs of grade ≥ 2, or deaths. Midostaurin showed only a minor inhibitory effect on P-gp, a mild inhibitory effect on BCRP, and no inhibitory effect on CYP2D6. Study treatments were well tolerated in healthy adults.

Agave-Laurate-Bioconjugated Fructans Decrease Hyperinsulinemia and Insulin Resistance, Whilst Increasing IL-10 in Rats with Metabolic Syndrome Induced by a High-Fat Diet.

Metabolic syndrome (MetS) comprises a cluster of metabolic risk factors, which include obesity, hypertriglyceridemia, high blood pressure, and insulin resistance. The purpose of this study was to evaluate the effects of laurate-bioconjugated fructans on pro- and anti-inflammatory cytokines in Wistar rats with MetS induced by a high-fat diet. Laurate-bioconjugated fructans were synthesized with agave fructans, immobilized lipase B, and vinyl laureate as the acylant. Groups were fed a standard diet (NORMAL), a high-fat diet (HFD), or a high-fat diet plus laurate-bioconjugated fructans (FL PREV) for 9 weeks. A fourth group received a high-fat diet for 6 weeks, followed by simultaneous exposure to a high-fat diet and laurate-bioconjugated fructans for 3 additional weeks (FL REV). The dose of laurate-bioconjugated fructans was 130 mg/kg. Laurate-bioconjugated fructans reduced food and energy intake, body weight, body mass index, abdominal circumference, adipose tissue, adipocyte area, serum triglycerides, insulin, insulin resistance, and C-reactive protein but they increased IL-10 protein serum levels and mRNA expression. The impact of laurate-bioconjugated fructans on zoometric and metabolic parameters supports their potential as therapeutic agents to improve obesity, obesity comorbidities, insulin resistance, type 2 diabetes mellitus, and MetS.

Dysregulation of Human Hepatic Drug Transporters by Proinflammatory Cytokines.

Proinflammatory cytokines, elevated during inflammation caused by infection and/or autoimmune disorders, result in reduced clearance of drugs eliminated primarily by cytochrome P450 enzymes (CYPs). However, the effect of cytokines on hepatic drug transporter expression or activity has not been well-studied. Here, using plated human hepatocytes (PHHs; n = 3 lots), we investigated the effect of interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), on the mRNA expression and activity of hepatic drug transporters. PHHs were incubated for 72 hours at their pathophysiologically relevant plasma concentrations, both individually (0.01, 0.1, 1, 10 ng/ml) or as a cocktail (i.e., when each was combined at 0.1 or 1 ng/ml). Following cytokine cocktail exposure (1 ng/ml), significant downregulation of mRNA expression of organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, sodium/taurocholate cotransporting polypeptide (NTCP), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), multidrug and toxin extrusion protein 1, multidrug resistance proteins (MRP) 2, 3, and 4 was observed. While the mRNA expression of organic anion transporter (OAT) 2 and organic cation transporter (OCT) 1 was downregulated in two lots, it was upregulated in one lot. In agreement (mostly), the 1 ng/ml cytokine cocktail reduced OATP1B1/3, OATP2B1, OAT2, OCT1, and NTCP activity by 75%, 44%, 82%, 47%, and 80%, respectively. Interestingly, upregulation of OAT2 and OCT1 mRNA in one donor did not translate into the same directional change in activity. Although significant interlot variability was observed, in general, the above effects, using individual cytokines, could be attributed to IL-1β, TNF-α, and IFN-γ. SIGNIFICANCE STATEMENT: To date, this is the first comprehensive study to investigate the effect of four major proinflammatory cytokines, both individually and as a cocktail, on the mRNA expression and activity of human hepatic drug transporters. The data obtained can be used in the future to predict transporter-mediated drug clearance changes during inflammation through physiologically based pharmacokinetic modeling and simulation.

Coproporphyrin-I as a Selective OATP1B Biomarker Can Be Used to Delineate the Mechanisms of Complex Drug-Drug Interactions: Cedirogant Case Study.

Cedirogant is an inverse agonist of retinoic acid-related orphan receptor gamma thymus developed for the treatment of chronic plaque psoriasis. Cedirogant induces cytochrome P450 (CYP) 3A4 while inhibiting P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1, and OATP1B3 in vitro. Static drug-drug interactions (DDIs) predictions suggested possible clinical induction of CYP3A4, and inhibition of P-gp, BCRP, and OATP1B1, leading to challenges in interpreting DDI studies between cedirogant and substrates of CYP3A, P-gp, BCRP, and OATP1B1/3. Here the effects of cedirogant on the pharmacokinetics of two statin drugs were investigated in healthy participants. Coproporphyrin-I (CP-I), a selective endogenous OATP1B biomarker, was used to assess the impact of cedirogant on OATP1B. Cedirogant (375 mg once daily) increased rosuvastatin maximum plasma concentration (Cmax) and area under the plasma concentration curve (AUCtau) by 141% and 55%, respectively when co-administered, whereas atorvastatin Cmax increased by 40% with no effect on its AUCtau compared with administration of rosuvastatin/atorvastatin alone. Cedirogant did not increase CP-I exposures, indicating no clinical OATP1B inhibition. The increased rosuvastatin exposure and minimal change in atorvastatin exposure with co-administration of cedirogant is attributed to BCRP inhibition and interplay between P-gp/BCRP inhibition and CYP3A induction, respectively. Correlation analysis with data from two investigational drugs (glecaprevir and flubentylosin) demonstrated that OATP1B1 R-value of > 1.5 and [Cmax,u]/[OATP1B1 IC50] of > 0.1 are associated with > 1.25-fold increase in CP-I Cmax ratio. This demonstrates the utility of CP-I in disentangling mechanisms underlying a complex DDI involving multiple transporters and enzymes and proposes refined criteria for static OATP1B inhibition predictions.

Plasma, brain and spinal cord concentrations of caffeine are reduced in the SOD1G93A mouse model of amyotrophic lateral sclerosis following oral administration

Modifications to the small intestine and liver are known to occur during the symptomatic disease period of amyotrophic lateral sclerosis (ALS), a member of the motor neuron disease (MND) family of neurodegenerative disorders. How these modifications impact on oral absorption and pharmacokinetics of drugs remains unknown. In this study, model drugs representing different mechanisms of intestinal transport (caffeine for passive diffusion, digoxin for P-glycoprotein efflux, and sulfasalazine for breast cancer resistance protein efflux) were administered via oral gavage to postnatal day 114–120 male and female SOD1G93A mice (model of familial ALS) and wild-type (WT) littermates. Samples of blood, brain and spinal cord were taken at either 15, 30, 60 or 180 min after administration. In addition, the in vivo gastric emptying of 70 kDa fluorescein isothiocyanate-dextran (FITC-dextran) and the ex vivo intestinal permeability of caffeine were assessed. The area under the plasma concentration–time curves (AUCplasma) of digoxin and sulfasalazine were not significantly different between SOD1G93A and WT mice for both sexes. However, the AUCplasma of caffeine was significantly lower (female: 0.79-fold, male: 0.76-fold) in SOD1G93A compared to WT mice, which was associated with lower AUCbrain (female: 0.76-fold, male: 0.80-fold) and AUCspinal cord (female: 0.81-fold, male: 0.82-fold). The AUCstomach of caffeine was significantly higher (female: 1.5-fold, male: 1.9-fold) in SOD1G93A compared to WT mice, suggesting reduced gastric emptying in SOD1G93A mice. In addition, there was a significant reduction in gastric emptying of FITC-dextran (0.66-fold) and ex vivo intestinal permeability of caffeine (0.52-fold) in male SOD1G93A compared to WT mice. Reduced systemic and brain/spinal cord exposure of caffeine in SOD1G93A mice may therefore result from alterations to gastric emptying and small intestinal permeability. Specific dosing requirements may therefore be required for certain medicines in ALS to ensure that they remain in a safe and effective concentration range.

In silico and biological analyses of missense variants of the human biliary efflux transporter ABCC2: effects of novel rare missense variants.

The ATP-dependent biliary efflux transporter ABCC2, also known as multidrug resistance protein 2 (MRP2), is essential for the cellular disposition and detoxification of various xenobiotics including drugs as well as endogenous metabolites. Common functionally relevant ABCC2 genetic variants significantly alter drug responses and contribute to side effects. The aim of this study was to determine functional consequences of rare variants identified in subjects with European ancestry using in silico tools and in vitro analyses. Targeted next-generation sequencing of the ABCC2 gene was used to identify novel variants in European subjects (n = 143). Twenty-six in silico tools were used to predict functional consequences. For biological validation, transport assays were carried out with membrane vesicles prepared from cell lines overexpressing the newly identified ABCC2 variants and estradiol β-glucuronide and carboxydichlorofluorescein as the substrates. Three novel rare ABCC2 missense variants were identified (W227R, K402T, V489F). Twenty-five in silico tools predicted W227R as damaging and one as potentially damaging. Prediction of functional consequences was not possible for K402T and V489F and for the common linked variants V1188E/C1515Y. Characterisation in vitro showed increased function of W227R, V489F and V1188E/C1515Y for both substrates, whereas K402T function was only increased for carboxydichlorofluorescein. In silico tools were unable to accurately predict the substrate-dependent increase in function of ABCC2 missense variants. In vitro biological studies are required to accurately determine functional activity to avoid misleading consequences for drug therapy.

The processed C-terminus of AvrRps4 effector suppresses plant immunity via targeting multiple WRKYs.

Pathogens generate and secrete effector proteins to the host plant cells during pathogenesis to promote virulence and colonization. If the plant carries resistance (R) proteins that recognize pathogen effectors, effector-triggered immunity (ETI) is activated, resulting in a robust immune response and hypersensitive response (HR). The bipartite effector AvrRps4 from Pseudomonas syringae pv. pisi has been well studied in terms of avirulence function. In planta, AvrRps4 is processed into two parts. The C-terminal fragment of AvrRps4 (AvrRps4C) induces HR in turnip and is recognized by the paired resistance proteins AtRRS1/AtRPS4 in Arabidopsis. Here, we show that AvrRps4C targets a group of Arabidopsis WRKY, including WRKY46, WRKY53, WRKY54, and WRKY70, to induce its virulence function. Indeed, AvrRps4C suppresses the general binding and transcriptional activities of immune-positive regulator WRKY54 and WRKY54-mediated resistance. AvrRps4C interferes with WRKY54's binding activity to target gene SARD1 in vitro, suggesting WRKY54 is sequestered from the SARD1 promoter by AvrRps4C. Through the interaction of AvrRps4C with four WRKYs, AvrRps4 enhances the formation of homo-/heterotypic complexes of four WRKYs and sequesters them in the cytoplasm, thus inhibiting their function in plant immunity. Together, our results provide a detailed virulence mechanism of AvrRps4 through its C-terminus.

Overcoming ABCB1 mediated multidrug resistance in castration resistant prostate cancer

Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. PCa that relapses after hormonal therapies, referred to as castration resistant PCa (CRPC), often presents with metastases (mCRPC) that are the major cause of mortality. The few available therapies for mCRPC patients include taxanes docetaxel (DTX) and cabazitaxel (CBZ). However, development of resistance limits their clinical use. Mechanistically, resistance arises through upregulation of multidrug resistance (MDR) proteins such as MDR1/ABCB1, making ABCB1 an attractive therapeutic target. Yet, ABCB1 inhibitors failed to be clinically useful due to low specificity and toxicity issues. To study taxanes resistance, we produced CBZ resistant C4-2B cells (RC4-2B) and documented resistance to both CBZ and DTX in cell culture and in 3D prostaspheres settings. RNAseq identified increased expression of ABCB1 in RC4-2B, that was confirmed by immunoblotting and immunofluorescent analysis. ABCB1-specific inhibitor elacridar reversed CBZ and DTX resistance in RC4-2B cells, confirming ABCB1-mediated resistance mechanism. In a cell-based screen using a curated library of cytotoxic drugs, we found that DNA damaging compounds Camptothecin (CPT) and Cytarabine (Ara-C) overcame resistance as seen by similar cytotoxicity in parental C4-2B and resistant RC4-2B. Further, these compounds were cytotoxic to multiple PC cells resistant to taxanes with high ABCB1 expression and, therefore, can be used to conquer the acquired resistance to taxanes in PCa. Finally, inhibition of cyclin-dependent kinases 4/6 (CDK4/6) with small molecule inhibitors (CDK4/6i) potentiated cytotoxic effect of CPT or Ara-C in both parental and resistant cells. Overall, our findings indicate that DNA damaging agents CPT and Ara-C alone or in combination with CDK4/6i can be suggested as a new treatment regimen in CRPC patients, including those that are resistant to taxanes.

Colibactin leads to a bacteria-specific mutation pattern and self-inflicted DNA damage.

Colibactin produced primarily by Escherichia coli strains of the B2 phylogroup cross-links DNA and can promote colon cancer in human hosts. Here, we investigate the toxin's impact on colibactin producers and on bacteria cocultured with producing cells. Using genome-wide genetic screens and mutation accumulation experiments, we uncover the cellular pathways that mitigate colibactin damage and reveal the specific mutations it induces. We discover that although colibactin targets A/T-rich motifs, as observed in human colon cells, it induces a bacteria-unique mutation pattern. Based on this pattern, we predict that long-term colibactin exposure will culminate in a genomic bias in trinucleotide composition. We test this prediction by analyzing thousands of E. coli genomes and find that colibactin-producing strains indeed show the predicted skewness in trinucleotide composition. Our work reveals a bacteria-specific mutation pattern and suggests that the resistance protein encoded on the colibactin pathogenicity island is insufficient in preventing self-inflicted DNA damage.

Convergent evolution in toxin detection and resistance provides evidence for conserved bacterial–fungal interactions

Microbes rarely exist in isolation and instead form complex polymicrobial communities. As a result, microbes have developed intricate offensive and defensive strategies that enhance their fitness in these complex communities. Thus, identifying and understanding the molecular mechanisms controlling polymicrobial interactions is critical for understanding the function of microbial communities. In this study, we show that the gram-negative opportunistic human pathogen Pseudomonas aeruginosa, which frequently causes infection alongside a plethora of other microbes including fungi, encodes a genetic network which can detect and defend against gliotoxin, a potent, disulfide-containing antimicrobial produced by the ubiquitous filamentous fungus Aspergillus fumigatus. We show that gliotoxin exposure disrupts P. aeruginosa zinc homeostasis, leading to transcriptional activation of a gene encoding a previously uncharacterized dithiol oxidase (herein named as DnoP), which detoxifies gliotoxin and structurally related toxins. Despite sharing little homology to the A. fumigatus gliotoxin resistance protein (GliT), the enzymatic mechanism of DnoP from P. aeruginosa appears to be identical that used by A. fumigatus. Thus, DnoP and its transcriptional induction by low zinc represent a rare example of both convergent evolution of toxin defense and environmental cue sensing across kingdoms. Collectively, these data provide compelling evidence that P. aeruginosa has evolved to survive exposure to an A. fumigatus disulfide-containing toxin in the natural environment.

Pharmacogenetic determinants of tenofovir diphosphate and lamivudine triphosphate concentrations in people with HIV/HBV coinfection.

The nucleos(t)ide analogs require phosphorylation to the pharmacologically active anabolites in cells. We investigated the hypothesis that single-nucleotide polymorphisms (SNPs) in genes that encode transporters and phosphodiesterase (PDE) enzymes involved in tenofovir (TFV), disoproxil fumarate (TDF), and lamivudine (3TC) disposition will be associated with concentrations of their phosphate anabolites and virologic response. Individuals with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) coinfection receiving TDF/3TC-containing antiretroviral therapy were enrolled. Steady-state TFV diphosphate (TFV-DP) and 3TC triphosphate (3TC-TP) concentrations in peripheral blood mononuclear cells (PBMCs) and dried blood spot samples were quantified. The relationship between genetic variants and TFV-DP and 3TC-TP concentrations as well as with virologic response were examined using multivariable linear regression. Of the 136 participants (median age 43 years; 63% females), 6.6% had HBV non-suppression, and 7.4% had HIV non-suppression. The multidrug resistance protein 2 (encoded by ABCC2 rs2273697) SNP was associated with 3TC-TP concentrations in PBMCs. The human organic anion transporter-1 (encoded by SLC28A2) rs11854484 SNP was associated with HIV non-suppression, and when evaluated together with SNPs with marginal associations (ABCC2 rs717620 and PDE1C rs30561), participants with two or three variants compared to those with none of these variants had an adjusted odds ratio of 48.3 (confidence interval, 4.3-547.8) for HIV non-suppression. None of the SNPs were associated with HBV non-suppression. Our study identified ABCC2 SNP to be associated with 3TC-TP concentrations in PBMCs. Also, a combination of genetic variants of drug transporters and PDE was associated with HIV non-suppression.