Comparative study of susceptibility to methylmercury cytotoxicity in cell types composing rat peripheral nerves: a higher susceptibility of dorsal root ganglion neurons.

Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.

Contribution of the transcription factor SfGATAe to Bt Cry toxin resistance in Spodoptera frugiperda through reduction of ABCC2 expression.

Insect resistance evolution poses a significant threat to the advantages of biopesticides and transgenic crops utilizing insecticidal Cry-toxins from Bacillus thuringiensis (Bt). However, there is limited research on the relationship between transcriptional regulation of specific toxin receptors in lepidopteran insects and their resistance to Bt toxins. Here, we report the positive regulatory role of the SfGATAe transcription factor on the expression of the ABCC2 gene in Spodoptera frugiperda. DNA regions in the SfABCC2 promoter that are vital for regulation by SfGATAe, utilizing DAP-seq technology and promoter deletion mapping. Through yeast one-hybrid assays, DNA pull-down experiments, and site-directed mutagenesis, we confirmed that the transcription factor SfGATAe regulates the core control site PBS2 in the ABCC2 target gene. Tissue-specific expression analysis has revealed that SfGATAe is involved in the regulation and expression of midgut cells in the fall armyworm. Silencing SfGATAe in fall armyworm larvae resulted in reduced expression of SfABCC2 and decreased sensitivity to Cry1Ac toxin. Overall, this study elucidated the regulatory mechanism of the transcription factor SfGATAe on the expression of the toxin receptor gene SfABCC2 and this transcriptional control mechanism impacts the resistance of the fall armyworm to Bt toxins.

Utilization of Diverse Molecules as Receptors by Cry Toxin and the Promiscuous Nature of Receptor-Binding Sites Which Accounts for the Diversity.

By 2013, it had been shown that the genes cadherin-like receptor (Cad) and ATP-binding cassette transporter subfamily C2 (ABCC2) were responsible for insect resistance to several Cry1A toxins, acting as susceptibility-determining receptors, and many review articles have been published. Therefore, this review focuses on information about receptors and receptor-binding sites that have been revealed since 2014. Since 2014, studies have revealed that the receptors involved in determining susceptibility vary depending on the Cry toxin subfamily, and that binding affinity between Cry toxins and receptors plays a crucial role. Consequently, models have demonstrated that ABCC2, ABCC3, and Cad interact with Cry1Aa; ABCC2 and Cad with Cry1Ab and Cry1Ac; ABCC2 and ABCC3 with Cry1Fa; ABCB1 with Cry1Ba, Cry1Ia, Cry9Da, and Cry3Aa; and ABCA2 with Cry2Aa and Cry2Ba, primarily in the silkworm, Bombyx mori. Furthermore, since 2017, it has been suggested that the binding sites of BmCad and BmABCC2 on Cry1Aa toxin overlap in the loop region of domain II, indicating that Cry toxins use various molecules as receptors due to their ability to bind promiscuously in this region. Additionally, since 2017, several ABC transporters have been identified as low-efficiency receptors that poorly induce cell swelling in heterologously expressing cultured cells. In 2024, research suggested that multiple molecules from the ABC transporter subfamily, including ABCC1, ABCC2, ABCC3, ABCC4, ABCC10, and ABCC11, act as low-efficiency receptors for a single Cry toxin in the midgut of silkworm larvae. This observation led to the hypothesis that the presence of such low-efficiency receptors contributes to the evolution of Cry toxins towards the generation of highly functional receptors that determine the susceptibility of individual insects. Moreover, this evolutionary process is considered to offer valuable insights for the engineering of Cry toxins to overcome resistance and develop countermeasures against resistance.

Factors Influencing Mortality in Children with Central Nervous System Tumors: A Cohort Study on Clinical Characteristics and Genetic Markers.

Multidrug resistance (MDR) commonly leads to cancer treatment failure because cancer cells often expel chemotherapeutic drugs using ATP-binding cassette (ABC) transporters, which reduce drug levels within the cells. This study investigated the clinical characteristics and single nucleotide variant (SNV) in ABCB1, ABCC1, ABCC2, ABCC4, and ABCG2, and their association with mortality in pediatric patients with central nervous system tumors (CNST). Using TaqMan probes, a real-time polymerase chain reaction genotyped 15 SNPs in 111 samples. Patients were followed up until death or the last follow-up day using the Cox proportional hazards model. An association was found between the rs1045642 (ABCB1) in the recessive model (HR = 2.433, 95% CI 1.098-5.392, p = 0.029), and the ICE scheme in the codominant model (HR = 9.810, 95% CI 2.74-35.06, p ≤ 0.001), dominant model (HR = 6.807, 95% CI 2.87-16.103, p ≤ 0.001), and recessive model (HR = 6.903, 95% CI 2.915-16.544, p = 0.038) significantly increased mortality in this cohort of patients. An association was also observed between the variant rs3114020 (ABCG2) and mortality in the codominant model (HR = 5.35, 95% CI 1.83-15.39, p = 0.002) and the dominant model (HR = 4.421, 95% CI 1.747-11.185, p = 0.002). A significant association between the ICE treatment schedule and increased mortality risk in the codominant model (HR = 6.351, 95% CI 1.831-22.02, p = 0.004, HR = 9.571, 95% CI 2.856-32.07, p ≤ 0.001), dominant model (HR = 6.592, 95% CI 2.669-16.280, p ≤ 0.001), and recessive model (HR = 5.798, 95% CI 2.411-13.940, p ≤ 0.001). The genetic variants rs3114020 in the ABCG2 gene and rs1045642 in the ABCB1 gene and the ICE chemotherapy schedule were associated with an increased mortality risk in this cohort of pediatric patients with CNST.

SMARCA4 (BRG1) activates ABCC3 transcription to promote hepatocellular carcinogenesis.

AIMS: Hepatocellular carcinoma (HCC) is a lead cause of cancer-related deaths. In the present study we investigated the role of Brahma-related gene 1 (BRG1), a chromatin remodeling protein, in HCC the pathogenesis focusing on identifying novel transcription targets. METHODS AND MATERIALS: Hepatocellular carcinogenesis was modeled in mice by diethylnitrosamine (DEN). Cellular transcriptome was evaluated by RNA-seq. RESULTS: Hepatocellular carcinoma was appreciably retarded in BRG1 knockout mice compared to wild type littermates. Transcriptomic analysis identified ATP Binding Cassette Subfamily C Member 3 (ABCC3) as a novel target of BRG1. BRG1 over-expression in BRG1low HCC cells (HEP1) up-regulated whereas BRG1 depletion in BRG1high HCC cells (SNU387) down-regulated ABCC3 expression. Importantly, BRG1 was detected to directly bind to the ABCC3 promoter to activate ABCC3 transcription. BRG1 over-expression in HEP1 cells promoted proliferation and migration, both of which were abrogated by ABCC3 silencing. On the contrary, BRG1 depletion in SNU387 cells decelerated proliferation and migration, both of which were rescued by ABCC3 over-expression. Importantly, high BRG1/ABCC3 expression predicted poor prognosis in HCC patients. Mechanistically, ABCC3 regulated hepatocellular carcinogenesis possibly by influencing lysosomal homeostasis. SIGNIFICANCE: In conclusion, our data suggest that targeting BRG1 and its downstream target ABCC3 can be considered as a reasonable approach for the intervention of hepatocellular carcinoma.

New Paralogs of the Heliothis virescens ABCC2 Transporter as Potential Receptors for Bt Cry1A Proteins.

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.

Histological Findings in the Eyes of Abcc6 Knockout Rat Model of Pseudoxanthoma Elasticum.

Purpose: To describe the ocular findings of murine pseudoxanthoma elasticum (PXE) models with ATP-binding cassette subfamily C member 6 (Abcc6) gene knockout. Methods: This experiment was conducted in four Abcc6-/- rats and compared with six wild-type Abcc6+/+ control rats. The animals underwent necropsy at 6 months of age. Histological examination of the eyes was performed. Results: Histological examination of eight eyes from four Abcc6-/- rats revealed multiple nodular foci of calcification in the uvea, sclera, and conjunctiva, focally in perivascular distribution, as well as linear and nodular calcification of Bruch's membrane. Calcific foci were not associated with inflammation in the knockout rats. There was no evidence of calcification in control eyes. Discussion: The Abcc6-/- rat model shows that PXE can affect multiple ocular tissues beyond the calcification in Bruch's membrane noted in human eyes. Nodular calcific foci probably correspond to comet lesions seen in patients with PXE. The presence of ectopic calcium without inflammation distinguishes it from inflammatory calcium deposition in atherosclerosis. Further studies are needed to determine why PXE does not cause inflammatory infiltration. Translational Relevance: The Abcc6-/- murine model may be suitable for studying ocular PXE pathophysiology and ectopic calcification and developing effective therapies.

Mechanisms of lipopolysaccharide protection in tumor drug-induced macrophage damage.

Malignant tumors contribute significantly to human mortality. Chemotherapy is a commonly used treatment for tumors. However, due to the low selectivity of chemotherapeutic drugs, immune cells can be damaged during antitumor treatment, resulting in toxicity. Lipopolysaccharide (LPS) can stimulate immune cells to respond to foreign substances. Here, we found that 10 ng/mL LPS could induce tolerance to antitumor drugs in macrophages without altering the effect of the drugs on tumor cells. Differentially expressed genes (DEGs) were identified between cells before and after LPS administration using transcriptome sequencing and found to be mainly associated with ATP-binding cassette (ABC)-resistant transporters and glutathione S-transferase (GST). LPS was shown by qRT-PCR and western blotting to promote the expression of ABCC1, GSTT1, and GSTP1 by 38.3 %, 194.8 %, and 27.0 %. Furthermore, three inhibitors (inhibitors of GST, glutathione synthesis, and ABCC1) were used for further investigation, showing that these inhibitors reduced macrophage survival rates by 44.0 %, 52.3 %, and 43.3 %, while the intracellular adriamycin content increased by 28.9 %, 42.9 %, and 51.3 %, respectively. These findings suggest that the protective mechanism of LPS on macrophages is associated with increased GST activity, the consumption of glutathione, and increased expression of ABCC1 protein. Therefore, LPS has a potential role in enhancing immunity.

Characterization of Plasmodium Falciparum Resistance Genes to Common Antimalarial Drugs in Semi-urban Areas of Burkina Faso.

INTRODUCTION: Malaria still remains the most frequent parasitic disease on the world with, in 2022, 249 million cases and 608,000 deaths worldwide. Malaria control is compromised by the spread of the parasite's resistance to available antimalarials. The objective of our study is to characterize the Plasmodium falciparum resistance genes to common antimalarial drugs in semi-urban areas of Burkina Faso. MATERIALS AND METHODS: This is a prospective cross-sectional study whose collection took place from June to October 2021 and from June to October 2022 in five health facilities in Burkina Faso. The molecular analysis based on PCR-RFLP took place from January to June 2023 at Centre National de Recherche et de Formation (CNRFP) to determine resistance genes such as Pfcrt, Pfmdr1, Pfdhps, and Pfdhfr. RESULTS: A total of 150 samples were analyzed giving a prevalence of 46.67, 1.33, 0.67, 20, 82, and 4.67%, for Pfcrt 76 T, Pfmdr1 86Y, Pfdhps 437G, Pfdhfr 51I, Pfdhfr 59R, and Pfdhfr 108N mutations, respectively. There are no mutations observed Pfdhps 540E and Pfdhfr 164L positions. However, mutation on Pfdhfr 59R position was the most common. In addition, triple mutation (Pfdhps 437G + Pfdhfr 59R + Pfdhfr 108N) was found with a low frequency which is 0.67%. CONCLUSION: Surveillance of Plasmodium falciparum resistance markers to antimalarial drugs, remains one of the priorities in the context of the control or malaria elimination.

Deciphering the molecular and functional basis of TMexCD1: the plasmid-encoded efflux pump of resistance-nodulation-division superfamily.

Horizontal gene transfer has been demonstrated to be an important driver for the emergency of multidrug-resistant pathogens. Recently, a transferable gene cluster tmexCD1-toprJ1 of the resistance-nodulation-division (RND) superfamily was identified in the plasmids of animal-derived Klebsiella pneumoniae strains, with a higher efflux capacity for various drugs than the Escherichia coli AcrAB-TolC homolog system. In this study, we focused on the differences in the inner membrane pump of these two systems and identified some key residues that contribute to the robust efflux activity of the TMexCD1 system. With the aid of homologous modeling and molecular docking, eight residues from the proximal binding pocket (PBP) and nine from the distal binding pocket (DBP) were selected and subjected to site-directed mutagenesis. Several of them, such as S134, I139, D181, and A290, were shown to be important for substrate binding in the DBP region, and all residues in PBP and DBP showed certain substrate preferences. Apart from the conservative switch loop (L613-623TMexD1) previously identified in the E. coli AcrB (EcAcrB), a relatively unconservative loop (L665-675TMexD1) at the bottom of PBP was proposed as a critical element for the robust activity of TMexD1, due to variations at sites E669, G670, N673, and S674 compared to EcAcrAB, and the significantly altered efflux activity due to their mutations. The conservation and flexibility of these key factors can contribute to the evolution of the RND efflux pumps and thus serve as potential targets for developing inhibitors to block the widespread of the TMexCD1 system.

Trends of Plasmodium falciparum molecular markers associated with resistance to artemisinins and reduced susceptibility to lumefantrine in Mainland Tanzania from 2016 to 2021.

BACKGROUND: Therapeutic efficacy studies (TESs) and detection of molecular markers of drug resistance are recommended by the World Health Organization (WHO) to monitor the efficacy of artemisinin-based combination therapy (ACT). This study assessed the trends of molecular markers of artemisinin resistance and/or reduced susceptibility to lumefantrine using samples collected in TES conducted in Mainland Tanzania from 2016 to 2021. METHODS: A total of 2,015 samples were collected during TES of artemether-lumefantrine at eight sentinel sites (in Kigoma, Mbeya, Morogoro, Mtwara, Mwanza, Pwani, Tabora, and Tanga regions) between 2016 and 2021. Photo-induced electron transfer polymerase chain reaction (PET-PCR) was used to confirm presence of malaria parasites before capillary sequencing, which targeted two genes: Plasmodium falciparum kelch 13 propeller domain (k13) and P. falciparum multidrug resistance 1 (pfmdr1). RESULTS: Sequencing success was ≥ 87.8%, and 1,724/1,769 (97.5%) k13 wild-type samples were detected. Thirty-seven (2.1%) samples had synonymous mutations and only eight (0.4%) had non-synonymous mutations in the k13 gene; seven of these were not validated by the WHO as molecular markers of resistance. One sample from Morogoro in 2020 had a k13 R622I mutation, which is a validated marker of artemisinin partial resistance. For pfmdr1, all except two samples carried N86 (wild-type), while mutations at Y184F increased from 33.9% in 2016 to about 60.5% in 2021, and only four samples (0.2%) had D1246Y mutations. pfmdr1 haplotypes were reported in 1,711 samples, with 985 (57.6%) NYD, 720 (42.1%) NFD, and six (0.4%) carrying minor haplotypes (three with NYY, 0.2%; YFD in two, 0.1%; and NFY in one sample, 0.1%). Between 2016 and 2021, NYD decreased from 66.1% to 45.2%, while NFD increased from 38.5% to 54.7%. CONCLUSION: This is the first report of the R622I (k13 validated mutation) in Tanzania. N86 and D1246 were nearly fixed, while increases in Y184F mutations and NFD haplotype were observed between 2016 and 2021. Despite the reports of artemisinin partial resistance in Rwanda and Uganda, this study did not report any other validated mutations in these study sites in Tanzania apart from R622I suggesting that intensified surveillance is urgently needed to monitor trends of drug resistance markers and their impact on the performance of ACT.

Distribution patterns of molecular markers of antimalarial drug resistance in Plasmodium falciparum isolates on the Thai-Myanmar border during the periods of 1993-1998 and 2002-2008.

BACKGROUND: Polymorphisms of Plasmodium falciparum chloroquine resistance transporter (pfcrt), Plasmodium falciparum multi-drug resistance 1 (pfmdr1) and Plasmodium falciparum kelch 13-propeller (pfk13) genes are accepted as valid molecular markers of quinoline antimalarials and artemisinins. This study investigated the distribution patterns of these genes in P. falciparum isolates from the areas along the Thai-Myanmar border during the two different periods of antimalarial usage in Thailand. RESULTS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect pfcrt mutations at codons 76, 220, 271, 326, 356, and 371 as well as pfmdr1 mutation at codon 86. The prevalence of pfcrt mutations was markedly high (96.4-99.7%) in samples collected during both periods. The proportions of mutant genotypes (number of mutant/total isolate) at codons 76, 220, 271, 326, 356 and 371 in the isolates collected during 1993-1998 (period 1) compared with 2002-2008 (period 2) were 97.9% (137/140) vs. 97.1% (401/413), 97.9% (140/143) vs. 98.8% (171/173), 97.2% (139/143) vs. 97.1% (333/343), 98.6% (140/142) vs. 99.7% (385/386), 96.4% (134/139) vs. 98.2% (378/385) and 97.8% (136/139) vs. 98.9% (375/379), respectively. Most isolates carried pfmdr1 wild-type at codon 86, with a significant difference in proportions genotypes (number of wild type/total sample) in samples collected during period 1 [92.9% (130/140)] compared with period 2 [96.9% (379/391)]. Investigation of pfmdr1 copy number was performed by real-time PCR. The proportions of isolates carried 1, 2, 3 and 4 or more than 4 copies of pfmdr1 (number of isolates carried correspondent copy number/total isolate) were significantly different between the two sample collecting periods (65.7% (90/137) vs. 87.8% (390/444), 18.2% (25/137) vs. 6.3%(28/444), 5.1% (7/137) vs. 1.4% (6/444) and 11.0% (15/137) vs. 4.5% (20/444), for period 1 vs. period 2, respectively). No pfk13 mutation was detected by nested PCR and nucleotide sequencing in all samples with successful analysis (n = 68). CONCLUSIONS: The persistence of pfcrt mutations and pfmdr1 wild-types at codon 86, along with gene amplification in P. falciparum, contributes to the continued resistance of chloroquine and mefloquine in P. falciparum isolates in the study area. Regular surveillance of antimalarial drug resistance in P. falciparum, incorporating relevant molecular markers and treatment efficacy assessments, should be conducted.

ABCC4 suppresses glioblastoma progression and recurrence by restraining cGMP-PKG signalling.

BACKGROUND: Cyclic nucleotides are critical mediators of cellular signalling in glioblastoma. However, the clinical relevance and mechanisms of regulating cyclic nucleotides in glioblastoma progression and recurrence have yet to be thoroughly explored. METHODS: In silico, mRNA, and protein level analyses identified the primary regulator of cyclic nucleotides in recurrent human glioblastoma. Lentiviral and pharmacological manipulations examined the functional impact of cyclic nucleotide signalling in human glioma cell lines and primary glioblastoma cells. An orthotopic xenograft mice model coupled with aspirin hydrogels verified the in vivo outcome of targeting cyclic nucleotide signalling. RESULTS: Elevated intracellular levels of cGMP, instead of cAMP, due to a lower substrate efflux from ATP-binding cassette sub-family C member 4 (ABCC4) is engaged in the recurrence of glioblastoma. ABCC4 gene expression is negatively associated with recurrence and overall survival outcomes in glioblastoma specimens. ABCC4 loss-of-function activates cGMP-PKG signalling, promoting malignancy in glioblastoma cells and xenografts. Hydrogels loaded with aspirin, inhibiting glioblastoma progression partly by upregulating ABCC4 expressions, augment the efficacy of standard-of-care therapies in orthotopic glioblastoma xenografts. CONCLUSION: ABCC4, repressing the cGMP-PKG signalling pathway, is a tumour suppressor in glioblastoma progression and recurrence. Aspirin hydrogels impede glioblastoma progression through ABCC4 restoration and constitute a viable translational approach.

The role of ABCC10/MRP7 in anti-cancer drug resistance and beyond.

Multidrug resistance protein 7 (MRP7), also known as ATP-binding cassette (ABC) transporter subfamily C10 (ABCC10), is an ABC transporter that was first identified in 2001. ABCC10/MRP7 is a 171 kDa protein located on the basolateral membrane of cells. ABCC10/MRP7 consists of three transmembrane domains and two nucleotide binding domains. It mediates multidrug resistance of tumor cells to a variety of anticancer drugs by increasing drug efflux and results in reducing intracellular drug accumulation. The transport substrates of ABCC10/MRP7 include antineoplastic drugs such as taxanes, vinca alkaloids, and epothilone B, as well as endobiotics such as leukotriene C4 (LTC4) and estradiol 17 ß-D-glucuronide. A variety of ABCC10/MRP7 inhibitors, including cepharanthine, imatinib, erlotinib, tariquidar, and sildenafil, can reverse ABCC10/MRP7-mediated MDR. Additionally, the presence or absence of ABCC10/MRP7 is also closely related to renal tubular dysfunction, obesity, and other diseases. In this review, we discuss: 1) Structure and functions of ABCC10/MRP7; 2) Known substrates and inhibitors of ABCC10/MRP7 and their potential therapeutic applications in cancer; and 3) Role of ABCC10/MRP7 in non-cancerous diseases.

Novel Screening System for Biliary Excretion of Drugs Using Human Cholangiocyte Organoid Monolayers with Directional Drug Transport.

It has recently been reported that cholangiocyte organoids can be established from primary human hepatocytes. The purpose of this study was to culture the organoids in monolayers on inserts to investigate the biliary excretory capacity of drugs. Cholangiocyte organoids prepared from hepatocytes had significantly higher mRNA expression of CK19, a bile duct epithelial marker, compared to hepatocytes. The organoids also expressed mRNA for efflux transporters involved in biliary excretion of drugs, P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP). The subcellular localization of each protein was observed. These results suggest that the membrane-cultured cholangiocyte organoids are oriented with the upper side being the apical membrane side (A side, bile duct lumen side) and the lower side being the basolateral membrane side (B side, hepatocyte side), and that each efflux transporter is localized to the apical membrane side. Transport studies showed that the permeation rate from the B side to the A side was faster than from the A side to the B side for the substrates of each efflux transporter, but this directionality disappeared in the presence of inhibitor of each transporter. In conclusion, the cholangiocyte organoid monolayer system has the potential to quantitatively evaluate the biliary excretion of drugs. The results of the present study represent an unprecedented system using human cholangiocyte organoids, which may be useful as a screening model to directly quantify the contribution of biliary excretion to the clearance of drugs.

Natural Product Baohuoside I Impairs the Stability and Membrane Location of MRP2 Reciprocally Regulated by SUMOylation and Ubiquitination in Hepatocytes.

Epimedii Folium (EF) is a botanical dietary supplement to benefit immunity. Baohuoside I (BI), a prenylated flavonoid derived from EF, has exhibited the cholestatic risk before. Here, the mechanism of BI on the stability and membrane localization of liver MRP2, a bile acid exporter in the canalicular membrane of hepatocytes, was investigated. The fluorescent substrate of MRP2, CMFDA was accumulated in sandwich-cultured primary mouse hepatocytes (SCH) under BI stimulation, followed by reduced membrane MRP2 expression. BI triggered MRP2 endocytosis associated with oxidative stress via inhibition of the NRF2 signaling pathway. Meanwhile, BI promoted the degradation of MRP2 by reducing its SUMOylation and enhancing its ubiquitination level. Co-IP and fluorescence colocalization experiments all proved that MRP2 was a substrate protein for SUMOylation for SUMO proteins. CHX assays showed that SUMO1 prolonged the half-life of MRP2 and further increased its membrane expression, which could be reversed by UBC9 knockdown. Correspondingly, MRP2 accumulated in the cytoplasm by GP78 knockdown or under MG132 treatment. Additionally, the SUMOylation sites of MRP2 were predicted by the algorithm, and a conversion of lysines to arginines at positions 940 and 953 of human MRP2 caused its decreased stability and membrane location. K940 was further identified as the essential ubiquitination site for MRP2 by an in vitro ubiquitination assay. Moreover, the decreased ubiquitination of MRP2 enhanced the SUMOylation MRP2 and vice versa, and the crosstalk of these two modifiers could be disrupted by BI. Collectively, our findings indicated the process of MRP2 turnover from the membrane to cytoplasm at the post-translational level and further elucidated the novel toxicological mechanism of BI.

The 75-99 C-Terminal Peptide of URG7 Protein Promotes α-Synuclein Disaggregation.

Up Regulation Gene seven (URG7) is the pseudogene 2 of the transporter ABCC6. The translated URG7 protein is localized with its single transmembrane α-helix in the endoplasmic reticulum (ER) membrane, orienting the N- and C-terminal regions in the lumen and cytoplasm, respectively, and it plays a crucial role in the folding of ER proteins. Previously, the C-terminal region of URG7 (PU, residues 75-99) has been shown to modify the aggregation state of α-synuclein in the lysate of HepG2 cells. PU analogs were synthesized, and their anti-aggregation potential was tested in vitro on α-synuclein obtained using recombinant DNA technology. Circular dichroism (CD), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and microscopic techniques were used to assess the sample's behavior. The results show that the peptides studied by themselves are prone to clathrate-like structure formation of variable stability. Aggregation of α-synuclein is accompanied by desolvation of its peptide chain and an increase in intermolecular ß-sheets. The PU analogs all interact with α-synuclein aggregates and those possessing the most stable clathrate-like structures have the highest disaggregating effect. These findings suggest that the C-terminal region of URG7 may have a role in interacting and modulating α-synuclein structures and could be used to generate interesting therapeutic candidates as disaggregators of α-synuclein.

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.

Significant Impact of AcrB Amino Acid Polymorphism at Residue 716 on Susceptibility to Tigecycline and Other Antibiotics in Klebsiella pneumoniae.

AcrAB-TolC is a multidrug RND-type efflux pump that is widespread in Gram-negative bacteria. As the substrate-binding subunit, AcrB was shown to modulate antimicrobial resistance in Escherichia coli, but the influence of AcrB mutation on Klebsiella pneumoniae, a major clinical pathogen, has not been well-studied. The finding of an R716L mutation in AcrB in a clinical tigecycline-nonsusceptible K. pneumoniae S1 strain inspired us to probe the role of AcrB residue 716 in antimicrobial resistance. This residue was subsequently subjected to saturation mutagenesis, followed by antibiotic susceptibility tests, survival assays, and antibiotic accumulation assays, showing strong influences of AcrB mutation on antimicrobial resistance. In particular, resistance levels to azithromycin, tetracycline, tigecycline, and cefoxitin were significantly changed by AcrB mutation at residue 716. Mutations to charged residues, polar residues, and residues that disrupt secondary structures have particularly reduced the antimicrobial susceptibility of bacteria, except for azithromycin, and the impact is not due to the abolishment of the efflux function of the pump. Therefore, it is concluded that residue 716 is an important residue that significantly influences antimicrobial resistance in K. pneumoniae, adding to our understanding of antimicrobial resistance mechanisms in this key clinical pathogen.

Restoring microRNA-34a overcomes acquired drug resistance and disease progression in human breast cancer cell lines via suppressing the ABCC1 gene.

PURPOSE: Breast cancer is one of the leading types of cancer diagnosed in women. Despite the improvements in chemotherapeutic cure strategies, drug resistance is still an obstacle leading to disease aggressiveness. The small non-coding RNA molecules, miRNAs, have been implicated recently to be involved as regulators of gene expression through the silencing of mRNA targets that contributed to several cellular processes related to cancer metastasis. Hence, the present study aimed to investigate the beneficial role and mechanism of miRNA-34a-based gene therapy as a novel approach for conquering drug resistance mediated by ATP-binding cassette (ABC) transporters in breast cancer cells, besides exploring the associated invasive behaviors. MATERIAL AND METHODS: Bioinformatics tools were used to predict miRNA ABC transporter targets by tracking the ABC transporter pathway. After the establishment of drug-resistant breast cancer MCF-7 and MDA-MB-231 sublines, cells were transfected with the mimic or inhibitor of miRNA-34a-5p. The quantitative expression of genes involved in drug resistance was performed by QRT-PCR, and the exact ABC transporter target specification interaction was confirmed by dual-luciferase reporter assay. Furthermore, flow cytometric analysis was utilized to determine the ability of miRNA-34a-treated cells against doxorubicin uptake and accumulation in cell cycle phases. The spreading capability was examined by colony formation, migration, and wound healing assays. The apoptotic activity was estimated as well. RESULTS: Our findings firstly discovered the mechanism of miRNA-34a-5p restoration as an anti-drug-resistant molecule that highly significantly attenuates the expression of ABCC1 via the direct targeting of its 3'- untranslated regions in resistant breast cancer cell lines, with a significant increase of doxorubicin influx by MDA-MB-231/Dox-resistant cells. Additionally, the current data validated a significant reduction of metastatic potentials upon miRNA-34a-5p upregulation in both types of breast cancer-resistant cells. CONCLUSION: The ectopic expression of miRNA-34a ameliorates the acquired drug resistance and the migration properties that may eventually lead to improved clinical strategies and outcomes for breast cancer patients. Additionally, miRNA-34a could be monitored as a diagnostic/prognostic biomarker for resistant conditions.