Expression of the cobalamin transporters cubam and MRP1 in the canine ileum-Upregulation in chronic inflammatory enteropathy.

Chronic inflammatory enteropathy (CIE) in dogs, a spontaneous model of human inflammatory bowel disease (IBD), is associated with a high rate of cobalamin deficiency. The etiology of hypocobalaminemia in human IBD and canine CIE remains unknown, and compromised intestinal uptake of cobalamin resulting from ileal cobalamin receptor deficiency has been proposed as a possible cause. Here, we evaluated the intestinal expression of the cobalamin receptor subunits, amnionless (AMN) and cubilin (CUBN), and the basolateral efflux transporter multi-drug resistance protein 1 (MRP1) in 22 dogs with CIE in comparison to healthy dogs. Epithelial CUBN and AMN levels were quantified by confocal laser scanning microscopy using immunohistochemistry in endoscopic ileal biopsies from dogs with (i) CIE and normocobalaminemia, (ii) CIE and suboptimal serum cobalamin status, (iii) CIE and severe hypocobalaminemia, and (iv) healthy controls. CUBN and MRP1 expression was quantified by RT-qPCR. Receptor expression was evaluated for correlation with clinical patient data. Ileal mucosal protein levels of AMN and CUBN as well as mRNA levels of CUBN and MRP1 were significantly increased in dogs with CIE compared to healthy controls. Ileal cobalamin receptor expression was positively correlated with age, clinical disease activity index (CCECAI) score, and lacteal dilation in the ileum, inversely correlated with serum folate concentrations, but was not associated with serum cobalamin concentrations. Cobalamin receptor downregulation does not appear to be the primary cause of hypocobalaminemia in canine CIE. In dogs of older age with severe clinical signs and/or microscopic intestinal lesions, intestinal cobalamin receptor upregulation is proposed as a mechanism to compensate for CIE-associated hypocobalaminemia. These results support oral supplementation strategies in hypocobalaminemic CIE patients.

Clinical characteristics and follow-up of a newborn with Dubin-Johnson Syndrome: A clinical case report.

BACKGROUND: Dubin-Johnson syndrome (DJS) is a rare autosomal recessive liver disorder, characterized by conjugated hyperbilirubinemia. This case report investigates the clinical characteristics and longitudinal outcomes of a neonate diagnosed with DJS. METHODS: A newborn presented with elevated bilirubin levels and abnormal liver enzyme readings. Comprehensive genetic evaluation was conducted, which included peripheral blood sample collection from the infant and both parents after obtaining informed consent and high-throughput trio exome sequencing was performed. The genetic analysis revealed 2 significant mutations in the ABCC2 gene on chromosome 10: the insertion mutation c.4237(exon30)_c.4238(exon30)ins CT, inherited from the father, and the missense mutation c.517(exon5)G > A, inherited from the mother. Both mutations were classified as pathogenic according to the ACMG 2015 guidelines, indicating a compound heterozygous inheritance pattern. The patient's treatment regimen included phototherapy, which was initiated to address her jaundice upon admission. To support liver function and regulate gut activity, oral ursodeoxycholic acid (20 mg/kg/dose, twice a day) and probiotics were administered. Additionally, a postdischarge medication plan involving a low-dose regimen of phenobarbital (3.5 mg/kg/dose, twice a day) was implemented for 2 weeks. RESULTS: During a 2-year follow-up after discharge, the infant's bilirubin levels significantly decreased, and liver enzymes, including GGT, progressively normalized. CONCLUSION: This case report enhances the understanding of DJS in neonates by emphasizing the clinical ramifications of compound heterozygous mutations within the ABCC2 gene and documenting the evolution of the disease. The gradual normalization of liver function tests suggests potential compensatory mechanisms in response to the genetic abnormalities in neonates with DJS. The correlation between the patient's genetic profile of compound heterozygosity and her milder clinical phenotype warrants attention, suggesting that this specific genetic configuration may be associated with less severe manifestations of the disease. The necessity for long-term follow-up is highlighted, recognizing that intercurrent stress conditions could influence the hepatic profile and potentially exacerbate symptoms. Such sustained observation is crucial to further delineate the genomic and clinical landscape of DJS, offering opportunities to refine prognostic and therapeutic approaches.

The induced and intrinsic resistance of Escherichia coli to sanguinarine is mediated by AcrB efflux pump.

IMPORTANCE: The use of plant extracts is increasing as an alternative to synthetic compounds, especially antibiotics. However, there is no sufficient knowledge on the mechanisms and potential risks of antibiotic resistance induced by these phytochemicals. In the present study, we found that stable drug resistant mutants of E. coli emerged after repetitive exposure to sanguinarine and demonstrated that the AcrB efflux pump contributed to the emerging of induced and intrinsic resistance of E. coli to this phytochemical. Our results offered some insights into comprehending and preventing the onset of drug-resistant strains when utilizing products containing sanguinarine.

Acacia senegal Budmunchiamines as a Potential Adjuvant for Rejuvenating Phenicol Activities towards Escherichia coli-Resistant Strains.

The continuous emergence of bacterial resistance alters the activities of different antibiotic families and requires appropriate strategies to solve therapeutic impasses. Medicinal plants are an attractive source for researching alternative and original therapeutic molecules. In this study, the fractionation of natural extracts from A. senegal and the determination of antibacterial activities are associated with molecular networking and tandem mass spectrometry (MS/MS) data used to characterize active molecule(s). The activities of the combinations, which included various fractions plus an antibiotic, were investigated using the "chessboard" test. Bio-guided fractionation allowed the authors to obtain individually active or synergistic fractions with chloramphenicol activity. An LC-MS/MS analysis of the fraction of interest and molecular array reorganization showed that most identified compounds are Budmunchiamines (macrocyclic alkaloids). This study describes an interesting source of bioactive secondary metabolites structurally related to Budmunchiamines that are able to rejuvenate a significant chloramphenicol activity in strains that produce an AcrB efflux pump. They will pave the way for researching new active molecules for restoring the activity of antibiotics that are substrates of efflux pumps in enterobacterial-resistant strains.

Evaluation of efflux pump inhibitory activity of some plant extracts and using them as adjuvants to potentiate the inhibitory activity of some antibiotics against Staphylococcus aureus.

Background: Antibiotic-resistant pathogens became a real global threat to human and animal health. This needs to concentrate the efforts to minimize and control these organisms. Efflux pumps are considered one of the important strategies used by bacteria to exclude harmful materials from the cell. Inhibition of these pumps can be an active strategy against multidrug resistance pathogens. There are two sources of efflux pump inhibitors that can be used, chemical and natural inhibitors. The chemical origin efflux pump inhibitors have many toxic side effects while the natural origin is characterized by a wide margin of safety for the host cell. Aim: In this study, the ability of some plant extracts like (propolis show rosemary, clove, capsaicin, and cumin) to potentiate the inhibitory activity of some antibiotics such as (ciprofloxacin, erythromycin, gentamycin, tetracycline, and ampicillin) against Staphylococcus aureus pathogen were tested. Methods: Efflux pump inhibitory activity of the selected plant extracts was tested using an ethidium bromide (EtBr) accumulation assay. Results: The results have shown that Propolis has a significant synergistic effect in combination with ciprofloxacin, erythromycin, and gentamycin. While it has no effect with tetracycline or ampicillin. Also, no synergic effect was noticed in a combination of the minimum inhibitory concentration for the selected plant extracts (rosemary, clove, capsaicin, and cumin) with any of the tested antibiotics. Interestingly, according to the results of the EtBr accumulation assay, Propolis has potent inhibitory activity against the S. aureus (MRS usa300) pump system. Conclusion: This study suggests that Propolis might act as a resistance breaker that is able to restore the activity of ciprofloxacin, erythromycin, and gentamycin against S. aureus strains, in case of the efflux-mediated antimicrobial resistance mechanisms.

Computational assessment of phytochemicals of medicinal plants from Mexico as potential inhibitors of Salmonella enterica efflux pump AcrB protein.

The AcrAB-TolC efflux pump (EP) confers multidrug resistance to Salmonella enterica, a major etiological agent of foodborne infections. Phytochemicals that inhibit the functions of AcrAB-TolC EP present ideal candidates for reversal of antibiotic resistance. Progressive technological advancements, have facilitated the development of computational methods that offer a rapid low-cost approach to screen and identify phytochemicals with inhibitory potential against EP. In this study, 71 phytochemicals derived from plants used for medicinal purposes in Mexico were screened for their potential as inhibitors of Salmonella AcrB protein using in silico approaches including molecular docking and molecular dynamics (MD) simulation. Consequently, naringenin, 5-methoxypsoralen, and licarin A were identified as candidate inhibitors of AcrB protein. The three phytochemicals bound distal/deep pocket (DP) and hydrophobic trap (HPT) residues of AcrB protein critical for interactions with inhibitors, with estimated binding free energies of -95.5 kJ/mol, -97.4 kJ/mol, and -143.8 kJ/mol for naringenin, 5-methoxypsoralen, and licarin A, respectively. Data from the 50 ns MD simulation study revealed stability of the protein-ligand complex and alterations in the AcrB protein DP conformation upon binding of phytochemicals to the DP and HPT regions. Based on the estimated binding free energy and interactions with three out of five residues lining the hydrophobic trap, licarin A demonstrated the highest inhibitory potential, supporting its further application as a candidate for overcoming drug resistance in pathogens. Communicated by Ramaswamy H. Sarma.

Combinatory therapy of MRP1-targeted photoimmunotherapy and liposomal doxorubicin promotes the antitumor effect for chemoresistant small cell lung cancer.

Small cell lung cancer (SCLC), considered a mortal recalcitrant cancer, is a severe healthcare issue because of its poor prognosis, early metastasis, drug resistance and limited clinical treatment options. In our previous study, we established a MRP1-targeted antibody-IR700 system (Mab-IR700) for near infrared photoimmunotherapy (NIR-PIT) which exhibited a promising therapeutic effect on drug resistant H69AR cells both in vitro and in vivo, though the tumor growth suppression effect did not last long with a single round of PIT treatment. To achieve a better anticancer effect, we have combined Mab-IR700-mediated NIR-PIT with liposomal doxorubicin (Doxil®) and investigated the in vitro and in vivo cytotoxicity by using a H69AR/3T3 cell co-culture model in which 3T3 cells were used to mimic stromal cells. Cytotoxicity experiments demonstrated the specificity of Mab-IR700 to H69AR cells, while cytotoxicity and flow cytometry experiments confirmed that H69AR cells were doxorubicin-resistant. Compared with Mab-IR700-mediated PIT or Doxil-mediated chemotherapy, the combination therapy exhibited the best cell killing effect in vitro and superior tumor growth inhibition and survival prolongation effect in vivo. Super enhanced permeability and retention (SUPR) effect was observed in both co-culture spheroids and tumor-bearing mice. Owing to an approximately 9-fold greater accumulation of Doxil within the tumors, NIR-PIT combined with Doxil resulted in enhanced antitumor effects compared to NIR-PIT alone. This photoimmunochemotherapy is a practical strategy for the treatment of chemoresistant SCLC and should be further investigated for clinical translation.

Identification and characterization of a heme exporter from the MRP family in Drosophila melanogaster.

BACKGROUND: The heme group constitutes a major functional form of iron, which plays vital roles in various biological processes including oxygen transport and mitochondrial respiration. Heme is an essential nutrient, but its pro-oxidant nature may have toxic cellular effects if present at high levels, and its synthesis is therefore tightly regulated. Deficiency and excess of heme both lead to pathological processes; however, our current understanding of metazoan heme transport is largely limited to work in mammals and the worm Caenorhabditis elegans, while functional analyses of heme transport in the genetically amenable Drosophila melanogaster and other arthropods have not been explored. RESULTS: We implemented a functional screening in Schneider 2 (S2) cells to identify putative heme transporters of D. melanogaster. A few multidrug resistance-associated protein (MRP) members were found to be induced by hemin and/or involved in heme export. Between the two plasma membrane-resident heme exporters CG4562 and CG7627, the former is responsible for heme transit across the intestinal epithelium. CG4562 knockdown resulted in heme accumulation in the intestine and lethality that could be alleviated by heme synthesis inhibition, human MRP5 (hMRP5) expression, heme oxygenase (HO) expression, or zinc supplement. CG4562 is mainly expressed in the gastric caeca and the anterior part of the midgut, suggesting this is the major site of heme absorption. It thus appears that CG4562 is the functional counterpart of mammalian MRP5. Mutation analyses in the transmembrane and nucleotide binding domains of CG4562 characterized some potential binding sites and conservative ATP binding pockets for the heme transport process. Furthermore, some homologs in Aedes aegypti, including that of CG4562, have also been characterized as heme exporters. CONCLUSIONS: Together, our findings suggest a conserved heme homeostasis mechanism within insects, and between insects and mammals. We propose the fly model may be a good complement to the existing platforms of heme studies.

Influence of 6-shogaol potentiated on 5-fluorouracil treatment of liver cancer by promoting apoptosis and cell cycle arrest by regulating AKT/mTOR/MRP1 signalling.

Currently, chemoresistance seriously attenuates the curative outcome of liver cancer. The purpose of our work was to investigate the influence of 6-shogaol on the inhibition of 5-fluorouracil (5-FU) in liver cancer. The cell viability of cancer cells was determined by MTT assay. Liver cancer cell apoptosis and the cell cycle were examined utilizing flow cytometry. Moreover, qRT-PCR and western blotting was used to analyse the mRNA and protein expression levels, respectively. Immunohistochemistry assays were used to examine multidrug resistance protein 1 (MRP1) expression in tumour tissues. In liver cancer cells, we found that 6-shogaol-5-FU combination treatment inhibited cell viability, facilitated G0/G1 cell cycle arrest, and accelerated apoptosis compared with 6-shogaol or 5-FU treatment alone. In cancer cells cotreated with 6-shogaol and 5-FU, AKT/mTOR pathway- and cell cycle-related protein expression levels were inhibited, and MRP1 expression was downregulated. AKT activation or MRP1 increase reversed the influence of combination treatment on liver cancer cell viability, apoptosis and cell cycle arrest. The inhibition of AKT activation to the anticancer effect of 6-shogaol-5-FU could be reversed by MRP1 silencing. Moreover, our results showed that 6-shogaol-5-FU combination treatment notably inhibited tumour growth in vivo. In summary, our data demonstrated that 6-shogaol contributed to the curative outcome of 5-FU in liver cancer by inhibiting the AKT/mTOR/MRP1 signalling pathway.

Multidrug resistance-associated protein 2 (MRP2) is an efflux transporter of EGCG and its metabolites in the human small intestine.

Green tea polyphenols have various beneficial effects on human health, such as antiobesity and anti-carcinogenesis. (-)-Epigallocatechin-gallate (EGCG) is one of the major potent green tea catechins; however, detailed mechanisms of EGCG transport and metabolism in the human small intestine remain unknown due to lack of a suitable model. We investigated metabolite profiles of EGCG in the fresh human duodenal biopsy, cryopreserved human duodenal mucosal enterocytes and Caco-2 cells, and found that EGCG was readily metabolized into methylated and sulphate conjugates, which are major metabolites in these models. Next, we examined possible efflux transporters of EGCG and its metabolites using specific inhibitors of MRP2, P-gp and BCRP in Caco-2 cell monolayers. MRP2 was thereby identified as an efflux transporter, and further analysis using MRP2-knockout Caco-2 cells and vesicular transport assays confirmed that MRP2 is a selective efflux transporter of EGCG and its metabolites. Assuming that functional inhibition of MRP2 would result in efficient uptake of EGCG, we screened for MRP2 functional blockade and identified quercetin, which led to increased intracellular accumulation and basal transport of EGCG in Caco-2 cells. This result suggested that co-administration of quercetin and EGCG would enable efficient transport of EGCG in the human intestine. Therefore, we performed co-oral administration of quercetin and EGCG in human subjects to examine whether this occurred in humans. These studies demonstrated that MRP2 is a selective transporter of EGCG and conjugates and Caco-2 is a model to examine transport mechanisms and metabolites of polyphenols in the human small intestine.

MRP5 and MRP9 play a concerted role in male reproduction and mitochondrial function.

Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter because mrp-5 mutants are unviable due to their inability to export heme from the intestine to extraintestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent in C. elegans, raising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO (DKO) mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes, dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes, and RNA sequencing shows significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.

Minocycline Counteracts Ectopic Calcification in a Murine Model of Pseudoxanthoma Elasticum: A Proof-of-Concept Study.

Pseudoxanthoma elasticum (PXE) is an intractable Mendelian disease characterized by ectopic calcification in skin, eyes and blood vessels. Recently, increased activation of the DNA damage response (DDR) was shown to be involved in PXE pathogenesis, while the DDR/PARP1 inhibitor minocycline was found to attenuate aberrant mineralization in PXE cells and zebrafish. In this proof-of-concept study, we evaluated the anticalcifying properties of minocycline in Abcc6-/- mice, an established mammalian PXE model. Abcc6-/- mice received oral minocycline supplementation (40 mg/kg/day) from 12 to 36 weeks of age and were compared to untreated Abcc6-/- and Abcc6+/+ siblings. Ectopic calcification was evaluated using X-ray microtomography with three-dimensional reconstruction of calcium deposits in muzzle skin and Yasue's calcium staining. Immunohistochemistry for the key DDR marker H2AX was also performed. Following minocycline treatment, ectopic calcification in Abcc6-/- mice was significantly reduced (-43.4%, p < 0.0001) compared to untreated Abcc6-/- littermates. H2AX immunostaining revealed activation of the DDR at sites of aberrant mineralization in untreated Abcc6-/- animals. In conclusion, we validated the anticalcifying effect of minocycline in Abcc6-/- mice for the first time. Considering its favorable safety profile in humans and low cost as a generic drug, minocycline may be a promising therapeutic compound for PXE patients.

Upregulation of USP22 and ABCC1 during Sorafenib Treatment of Hepatocellular Carcinoma Contribute to Development of Resistance.

Sorafenib is a small molecule that blocks tumor proliferation by targeting the activity of multi-kinases for the treatment of advanced hepatocellular carcinoma (HCC). Increasing sorafenib resistance following long-term treatment is frequently encountered. Mechanisms underlying sorafenib resistance remain not completely clear. To further understand the mechanism of sorafenib resistance in HCC, we established sorafenib-resistant cell lines by slowly increasing sorafenib concentration in cell culture medium. Upregulation of USP22 and ABCC1 were found in Sorafenib-resistant cells. Sorafenib-resistant cells treated with USP22 siRNA showed significant reduction in endogenous mRNA and protein levels of ABCC1. During sorafenib treatment, upregulation of USP22 increases ABCC1 expression and subsequently contributes to sorafenib resistance in HCC cells. Immunohistochemical analysis revealed a positive correlation between USP22 and ABCC1 expression in tissue samples from sorafenib-resistant patients (Pearson's correlation = 0.59, p = 0.03). Our findings indicate that upregulation of USP22 and ABCC1 expression during treatment contribute to sorafenib resistance in HCC cells and that USP22 has strong potential as a therapeutic target for overcoming sorafenib resistance in HCC patients.

Vitamin D and Calcium Supplementation Accelerate Vascular Calcification in a Model of Pseudoxanthoma Elasticum.

Arterial calcification is a common feature of pseudoxanthoma elasticum (PXE), a disease characterized by ABCC6 mutations, inducing a deficiency in pyrophosphate, a key inhibitor of calcium phosphate crystallization in arteries. METHODS: we analyzed whether long-term exposure of Abcc6-/- mice (a murine model of PXE) to a mild vitamin D supplementation, with or without calcium, would impact the development of vascular calcification. Eight groups of mice (including Abcc6-/- and wild-type) received vitamin D supplementation every 2 weeks, a calcium-enriched diet alone (calcium in drinking water), both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Aorta and kidney artery calcification was assessed by 3D-micro-computed tomography, Optical PhotoThermal IR (OPTIR) spectroscopy, scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS) and Yasue staining. RESULTS: at 6 months, although vitamin D and/or calcium did not significantly increase serum calcium levels, vitamin D and calcium supplementation significantly worsened aorta and renal artery calcification in Abcc6-/- mice. CONCLUSIONS: vitamin D and/or calcium supplementation accelerate vascular calcification in a murine model of PXE. These results sound a warning regarding the use of these supplementations in PXE patients and, to a larger extent, patients with low systemic pyrophosphate levels.

Virtual high-throughput screening and simulation studies of compounds from selected traditionally important medicinal plants for the identification of potential inhibitors of AcrB.

One of the significant human health concerns today is the emergence of drug-resistant bacteria and their propagation worldwide, rendering all available treatment options useless. One of the molecular mechanisms behind the evolution and dissemination of multidrug-resistant species is the overexpression of efflux pumps. AcrB is a major component of the AcrAB-TolC efflux pump belonging to the RND division and responsible for the extrusion of antibiotics from the bacterial cell leading to resistance. In this study, we applied the reverse chemogenomics technique to find inhibitors of AcrB to combat the issue of drug resistance. A total of 102 compounds from five different plants having medicinal properties were passed through Lipinski filter and ADMET screening was done to check their drug likeliness before docking against the AcrB protein and based on the -cDocker energy scores and toxicity analysis report, the compounds with best values were analyzed. A comparison was made between them and known inhibitors as well as antibiotics. Heat maps, frequency histograms, 2 D diagrams were generated, and the molecules were simulated for 60 ns using GROMACS. From the study, we have found Dihydrocapsaicin and Garcinexanthone-A to be a potential efflux pump inhibitors having all the characters of a promising drug candidate.Communicated by Ramaswamy H. Sarma.

Repurposing Approved Drugs as Fluoroquinolone Potentiators to Overcome Efflux Pump Resistance in Staphylococcus aureus.

Staphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

Biliary excretion of arsenic by human HepaRG cells is stimulated by selenide and mediated by the multidrug resistance protein 2 (MRP2/ABCC2).

Millions of people worldwide are exposed to unacceptable levels of arsenic, a proven human carcinogen, in drinking water. In animal models, arsenic and selenium are mutually protective through formation and biliary excretion of seleno-bis (S-glutathionyl) arsinium ion [(GS)2AsSe]-. Selenium-deficient humans living in arsenic-endemic regions are at increased risk of arsenic-induced diseases, and may benefit from selenium supplementation. The influence of selenium on human arsenic hepatobiliary transport has not been studied using optimal human models. HepaRG cells, a surrogate for primary human hepatocytes, were used to investigate selenium (selenite, selenide, selenomethionine, and methylselenocysteine) effects on arsenic hepatobiliary transport. Arsenite + selenite and arsenite + selenide at different molar ratios revealed mutual toxicity antagonism, with the latter being higher. Significant levels of arsenic biliary excretion were detected with a biliary excretion index (BEI) of 14 ± 8%, which was stimulated to 32 ± 7% by selenide. Consistent with the formation and biliary efflux of [(GS)2AsSe]-, arsenite increased the BEI of selenide from 0% to 24 ± 5%. Arsenic biliary excretion was lost in the presence of selenite, selenomethionine, and methylselenocysteine. Sinusoidal export of arsenic was stimulated ∼1.6-fold by methylselenocysteine, but unchanged by other selenium forms. Arsenic canalicular and sinusoidal transport (±selenide) was temperature- and GSH-dependent and inhibited by MK571. Knockdown experiments revealed that multidrug resistance protein 2 (MRP2/ABCC2) accounted for all detectable biliary efflux of arsenic (±selenide). Overall, the chemical form of selenium and human MRP2 strongly influenced arsenic hepatobiliary transport, information critical for human selenium supplementation in arsenic-endemic regions.

Study on mechanism of low bioavailability of black tea theaflavins by using Caco-2 cell monolayer.

This study aimed to clarify the bioavailability mechanism of theaflavins by using the Caco-2 monolayer in vitro model. Prior to the transport of theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3'-gallate (TF3'G), and theaflavin-3, 3'-digallate (TFDG), we found the cytotoxicity of theaflavins was in the order of TF3'G > TFDG > TF3G > TF, suggesting the galloyl moiety enhances the cytotoxicity of theaflavins. Meantime, the galloyl moiety made theaflavins unstable, with the stability in the order of TF > TFDG > TF3G/TF3'G. Four theaflavins showed poor bioavailability with the Papp values ranging from 0.44 × 10-7 to 3.64 × 10-7 cm/s in the absorptive transport. All the theaflavins showed an efflux ratio of over 1.24. And it is further confirmed that P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs) and breast cancer resistance protein (BCRP) were all shown to contribute to the efflux transport of four theaflavins, with P-gp playing the most important role, followed by MRPs and BCRP. Moreover, theaflavins increased the expression of P-gp, MRP1, MPR3, and BCRP while decreased the expression of MRP2 at the transcription and translation levels. Additionally, the gallated theaflavins were degraded into simple theaflavins and gallic acids when transported through Caco-2 monolayers. Overall, the structural instability, efflux transporters, and cell metabolism were all responsible for the low bioavailability of four theaflavins in Caco-2 monolayers.

Antioxidant effect of ascorbic acid against cisplatin-induced nephrotoxicity and P-glycoprotein expression in rats.

Cisplatin (CDDP) is a highly potent anticancer drug that is widely used in the treatment of several cancers. CDDP-induced nephrotoxicity (CIN) is one of the most significant adverse effects, and oxidative stress is thought to be one of the mechanisms underlying CIN. Although there are some studies available on the variability in transporter expression in the kidney after a single CDDP dose, none have reported the change in renal transporter expression after multiple CDDP dose administrations. P-glycoprotein (P-gp), a transporter, is reported to be induced by oxidative stress. Ascorbic acid is a vitamin with antioxidant potential and therefore, may regulate the expression of P-gp transporter and affect CIN. In the present study, our aim was to assess the variability in expression of several renal transporters after multiple CDDP dose administrations and the antioxidant effect of ascorbic acid against transporter expression and CIN. Multiple doses of CDDP affected markers of kidney injury and antioxidants in the kidneys. Also, the expression of P-gp, breast cancer resistance protein, and multidrug resistance-associated protein 4 was upregulated by CDDP. Using a normal kidney cell line, we demonstrated that ascorbic acid attenuated CDDP-induced cytotoxicity due to its high superoxide scavenging ability. CDDP and ascorbic acid were injected into rats once a week for three weeks, and it was observed that co-administration of ascorbic acid attenuated CIN and regulated antioxidant marker. In addition, ascorbic acid reduced P-gp expression, which was upregulated by CDDP. In conclusion, ascorbic acid may attenuate CIN and reverse P-gp-mediated changes in drug pharmacokinetics.

Yinzhihuang Oral Liquid Ameliorates Hyperbilirubinemia Induced by δ-Aminolevulinic Acid and Novobiocin in Neonatal Rats.

Yinzhihuang oral liquid (YZH) is a traditional Chinese medicine that has been widely used in Asia to prevent and treat neonatal hyperbilirubinemia, but the published preclinical studies on its anti-hyperbilirubinemia effect are conducted in adult animals, partly due to the lack of preclinical neonatal hyperbilirubinemia animal models. In the present study, we tested six reagents to induce hyperbilirubinemia in neonatal rats, and established two appropriate neonatal hyperbilirubinemia rat models by subcutaneous injection of δ-Aminolevulinic acid (ALA, 200 mg/kg) or novobiocin (NOVO, 200 mg/kg). Oral treatment of YZH (80, 160 and 320 mg/kg) significantly decreased serum conjugated bilirubin levels in ALA-treated neonatal rats and serum unconjugated bilirubin levels in NOVO-treated neonatal rats, respectively. Additionally, pre-treatment of YZH also prevented the increase of serum bilirubin levels in both ALA- and NOVO-treated rats. Mechanistically, YZH significantly up-regulated the mRNA expression of genes involved in hepatic bilirubin disposition (organic anion-transporting polypeptide 1b2, Oatp1b2; multidrug resistance-associated protein 2, Mrp2) and bilirubin conjugation (UDP-glucuronosyltransferase 1a1, Ugt1a1). Additionally, YZH up-regulated the mRNA expression of cytochrome P450 1A1 (Cyp1a1), the target gene of aryl hydrocarbon receptor (AhR), and increased the nuclear protein levels of AhR in livers of neonatal rats. YZH and its two active ingredients, namely baicalin (BCL) and 4'-hydroxyacetophenone (4-HT), up-regulated the mRNA expression of AhR target genes (CYP1A1 and UGT1A1) and increased nuclear protein levels of AhR in HepG2 cells. In conclusion, the present study provides two neonatal hyperbilirubinemia animal models and evaluates the anti-hyperbilirubinemia effect and mechanisms of YZH in neonatal animals.