A novel 4-aminoquinoline chemotype with multistage antimalarial activity and lack of cross-resistance with PfCRT and PfMDR1 mutants.

Artemisinin-based combination therapy (ACT) is the mainstay of effective treatment of Plasmodium falciparum malaria. However, the long-term utility of ACTs is imperiled by widespread partial artemisinin resistance in Southeast Asia and its recent emergence in parts of East Africa. This underscores the need to identify chemotypes with new modes of action (MoAs) to circumvent resistance to ACTs. In this study, we characterized the asexual blood stage antiplasmodial activity and resistance mechanisms of LDT-623, a 4-aminoquinoline (4-AQ). We also detected LDT-623 activity against multiple stages (liver schizonts, stage IV-V gametocytes, and ookinetes) of Plasmodium's life cycle, a feature unlike other 4-AQs such as chloroquine (CQ) and piperaquine (PPQ). Using heme fractionation profiling and drug uptake studies in PfCRT-containing proteoliposomes, we observed inhibition of hemozoin formation and PfCRT-mediated transport, which constitute characteristic features of 4-AQs' MoA. We also found minimal cross-resistance to LDT-623 in a panel of mutant pfcrt or pfmdr1 lines, but not the PfCRT F145I mutant that is highly resistant to PPQ resistance yet is very unfit. No P. falciparum parasites were recovered in an in vitro resistance selection study, suggesting a high barrier for resistance to emerge. Finally, a competitive growth assay comprising >50 barcoded parasite lines with mutated resistance mediators or major drug targets found no evidence of cross-resistance. Our findings support further exploration of this promising 4-AQ.

Circular RNA circABCC4 as the ceRNA facilitates renal carcinoma progression.

PURPOSE: This study explores the role of circular RNA derived from the Multidrug Resistance Protein 4 (MRP4/ABCC4) gene, which is markedly elevated in renal cell carcinoma (RCC). Our objective is to clarify how this circular RNA contributes to the progression and development of RCC. METHODS: We quantified the presence of circular ABCC4 RNA in tissue samples, plasma and urine from patients diagnosed with RCC. In addition, the impact of this circular RNA on RCC tumour growth was assessed through studies in RCC cell lines and in animal models mimicking the disease. RESULTS: Our findings reveal that circular ABCC4 RNA, specifically the variant containing exons 25-29 (circABCC4e), is upregulated in RCC cell lines and tissues. This upregulation correlates with advanced tumor stages in RCC patients, suggesting circABCC4e's potential as a biomarker for RCC progression. Furthermore, the reduction in circABCC4e levels following tumor resection indicates its potential utility in monitoring treatment response. The mechanism by which circABCC4e promotes RCC tumor growth through the antagonism of tumor-suppressive microRNAs highlights its significance in RCC pathogenesis. These insights may inform the development of diagnostic and therapeutic strategies for RCC. CONCLUSION: This study demonstrates that circABCC4e accelerates RCC progression by inhibiting tumor-suppressive microRNAs. Its role as a diagnostic and prognostic biomarker for RCC underscores its potential value in improving RCC management.

Association Between ABCC2 -24C>T and Nab-Paclitaxel-induced Peripheral Neuropathy in Japanese Patients With Pancreatic Cancer.

BACKGROUND/AIM: Nab-paclitaxel is used to treat patients with pancreatic cancer. However, it frequently induces peripheral neuropathy. Notably, pharmacokinetic factors may be associated with neuropathic symptoms as the onset depends on the cumulative dose. Therefore, we prospectively examined the association between the cumulative dose of nab-paclitaxel at the onset of peripheral neuropathy and polymorphisms of hepatic transporter genes. PATIENTS AND METHODS: Patients with pancreatic cancer receiving nab-paclitaxel (125 mg/m2) and gemcitabine (1,000 mg/m2) were enrolled. Peripheral neuropathy was assessed using the Common Terminology Criteria for Adverse Events (CTCAE), Patient-Reported Outcomes CTCAE (PRO-CTCAE), and Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity (FACT/GOG-Ntx), every 2-12 weeks, and every 4 weeks thereafter. solute carrier organic anion transporter family member 1B1 (SLCO1B1) 521T>C, 388A>G; SLCO1B3 rs11045585; ATP-binding cassette transporters, subfamily B, member 1 (ABCB1) 1236C>T, 2677G>T/A, 3435C>T; ABCC1 rs2644983; ABCC2 24C>T; and ABCG2 421C>A were analyzed by direct sequencing. Correlations between transporter genotypes and cumulative dose at symptom onset were assessed using Kaplan-Meier and log-rank tests. RESULTS: In total, 25 patients were enrolled. The lowest median cumulative dose for nab-paclitaxel at peripheral neuropathy onset using PRO-CTCAE was 593 mg. By CTCAE it was 800 mg, and by FACT/GOG-Ntx it was 1,090 mg (p<0.0001). At symptom onset, patients with ABCC2 -24C/T genotype had received a significantly lower median cumulative dose by PRO-CTCAE (540 mg) than those with C/C (720 mg) (p=0.0188). However, the other polymorphisms studied were not associated with symptoms. CONCLUSION: Herein, we found for the first time that ABCC2 -24C/T genotype was significantly associated with the onset of nab-paclitaxel-induced peripheral neuropathy detected with PRO-CTCAE.

Optimization of loop mediated isothermal amplification assay (LAMP) for detection of chloroquine resistance in P. vivax malaria.

Chloroquine is still used as a first-line treatment for uncomplicated Plasmodium vivax malaria in India and resistance to this therapy can act as a major hurdle for malaria elimination. It is difficult to monitor drug-efficacy and drug resistance through in vivo and in vitro studies in case of Plasmodium vivax so analysis of molecular markers serves as an important tool to track resistance. Molecular methods that are currently in use for detecting single nucleotide polymorphisms in resistant genes including Polymerase chain reaction (PCR), Realtime-Polymerase chain reaction require highly sophisticated labs and are time consuming. So, with this background the study has been designed to optimize Loop Mediated Isothermal Amplification Assay to detect single nucleotide polymorphisms in chloroquine resistance gene of Plasmodium vivax in field settings. Eighty-eight Plasmodium vivax positive samples were collected. Pvmdr1 gene was amplified for all the samples and sequenced. Obtained sequences were analyzed for the presence of single nucleotide polymorphisms in the target gene. Further Loop Mediated Isothermal Amplification Assay primer sets were designed for the target mutants and the assay was optimized. Clinical as well as analytical sensitivity and specificity for the assay was calculated. Double mutants with variations at T958M and F1076L were detected in 100% of the Plasmodium vivax clinical isolates with haplotype M958 Y976 Y1028 L1076. Designed primers for Loop Mediated Isothermal Amplification Assay successfully detected both the mutants (T958M and F1076L) in 100% of the isolates and do not show cross-reactivity with other strains. So, the assay was 100% sensitive and specific for detecting single nucleotide polymorphisms in the target Pvmdr1 gene. Limit of detection was found to be 0.9 copies/µl and lowest DNA template concentration detected by designed assay was 1.5 ng/µL. Observed prevalence of single nucleotide polymorphisms in Pvmdr 1 gene is indicating a beginning of trend towards chloroquine resistance in Plasmodium vivax. The present study optimized LAMP for detecting single nucleotide polymorphisms in Plasmodium vivax cases in field settings, thus would help in finding significant hubs of emerging chloroquine drug resistance and ultimately helping in the management of suitable antimalarial drug policy.

CircABCC1 reduces endometrial receptivity via METTL3/FAM155B axis.

OBJECTIVE: Impaired endometrial receptivity is the main cause of embryo implantation failure. Little information is available on the role of circRNAs in endometrial receptivity. Here, the effect of circABCC1 on endometrial receptivity and its mechanism were investigated. METHODS: GEO database was screened for key biomarkers for recurrent implantation failure (RIF). Endometrial epithelial cells (EECs) were cultured and transfected with circABCC1- and/or FAM155B-related vectors, followed by CCK-8 detection of cell proliferation, western blotting detection of receptivity-related factors LIF and DKK1, and ELISA detection of LIF secretion. An in vitro adhesion model was established to detect trophoblast adhesion to EECs. RIP was used to detect the binding of METTL3 to circABCC1 and FAM155B mRNA, and MeRIP-qPCR was used to detect m6A modification of FAM155B mRNA. RESULTS: CircABCC1 and FAM155B were highly expressed in patients with RIF. CircABCC1 or FAM155B overexpression reduced EEC proliferation, LIF and DKK1 expression, LIF secretion, and trophoblast adhesion; circABCC1 or FAM155B knockdown led to the opposite results. CircABCC1 and METTL3 positively regulated FAM155B expression. METTL3 bound circABCC1 and FAM155B mRNA. METTL3 overexpression increased m6A modification of FAM155B mRNA. FAM155B overexpression partially eliminated circABCC1 knockdown-induced promotion of EEC proliferation, LIF and DKK1 expression, LIF secretion, and trophoblast adhesion. CONCLUSION: CircABCC1 binds to METTL3 to regulate FAM155B mRNA modification and promote FAM155B expression, thereby inhibiting EEC proliferation and reducing endometrial receptivity.

Targeting METTL3 enhances the chemosensitivity of non-small cell lung cancer cells by decreasing ABCC2 expression in an m6A-YTHDF1-dependent manner.

Patients with non-small cell lung cancer (NSCLC) are easily resistant to first-line chemotherapy with paclitaxel (PTX) or carboplatin (CBP). N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) has crucial functions in m6A modification and tumorigenesis. However, its role in chemoresistance of NSCLC is still elusive. Here, we demonstrated that METTL3 inhibitor STM2457 significantly reduced the IC50 values of PTX or CBP in NSCLC cells, and they showed a synergistic effect. Comparing with monotherapy, a combination of STM2457 and PTX or CBP exhibited more potent in vitro and in vivo anti-tumor efficacy. In addition, we found that ATP binding cassette subfamily C member 2 (ABCC2) was responsively elevated in cytomembrane after PTX or CBP treatment, and targeting METTL3 could reverse this effect. Mechanistically, targeting METTL3 decreased the m6A modification of ABCC2 mRNA and accelerated its mRNA degradation. Further studies revealed that YTHDF1 could bind and stabilize the m6A-modified mRNA of ABCC2, while YTHDF1 knockdown promoted it mRNA degradation. These results, taken together, demonstrate that targeting METTL3 enhances the sensitivity of NSCLC cells to PTX or CBP by decreasing the cytomembrane-localized ABCC2 in an m6A-YTHDF1-dependent manner, and suggest that METTL3 may be a potential therapeutic target for acquired resistance to PTX or CBP in NSCLC.

Pharmacogenetic insights into ABCB1, ABCC2, CYP1A2, and CYP2B6 variants with epilepsy susceptibility among Egyptian Children: A retrospective case-control study.

BACKGROUND: Pediatric epilepsy is a complicated neuropsychiatric disorder that is characterized by recurrent seizures and unusual synchronized electrical activities within brain tissues. It has a substantial effect on the quality of life of children, thus understanding of the hereditary considerations influencing epilepsy susceptibility and the response to antiepileptic medications is crucial. This study focuses on assessing the correlation of the ABCB1, ABCC2, CYP1A2, and CYP2B6 genetic polymorphisms with the susceptibility to epileptic seizures and their contributions to antiepileptic medication throughout the course of the disease. METHODS: This study included 134 Egyptian epileptic children, comprising 67 drug-responsive and 67 drug-resistant patients, along with 124 healthy controls matching for age, gender, and geographical district. Genotyping of the rs2032582, rs717620, rs2273697, rs762551, and rs3745274 variants was performed using the PCR technique. Statistical analyses, including haplotype, multivariate, logistic regression, and bioinformatics approaches, were conducted to evaluate the associations within the disease. RESULTS: The ABCC2*rs717620 (T allele) revealed an increased risk of epilepsy compared to healthy controls (OR = 2.12, p-value < 0.001), with the rs717620 (C/T + T/T genotypes) showing significant differences between drug-responsive and drug-resistant patients (p-value < 0.05). Moreover, the ABCC2*rs2273697 (A allele) indicated a decreased risk of epileptic seizures compared to healthy controls (OR = 0.51, p-value = 0.033), with the rs2273697 (G/A + A/A genotypes) indicating a significant association with drug-resistant patients (OR = 0.21, p-value = 0.002). The rs717620*T/rs2273697*G haplotype was significantly correlated with an elevated risk of epileptic seizures within drug-responsive patients (OR = 2.26, p-value = 0.019). Additionally, the CYP1A2*rs762551 (A allele) represented a protective effect against epilepsy susceptibility (OR = 0.50, p-value < 0.001), with the rs762551 (G/A + A/A genotypes) disclosing a substantial association with a decreased risk of epileptic seizures among drug-resistant patients compared to drug-responsive patients (OR = 0.07, p-value < 0.001). Conversely, the ABCB1*rs2032582 (G allele) and the CYP2B6*rs3745274 (T allele) did not attain a significant difference with the epilepsy risk compared to healthy controls (p-value > 0.05). CONCLUSIONS: The findings of our study emphasize the importance of pharmacogenetic screening in epilepsy research, particularly regarding to drug-resistant patients. The ABCC2*rs717620 variant conferred a significant correlation with elevated risk of epileptic seizures, while the ABCC2*rs2273697 and CYP1A2*rs762551 variants confirmed their contributions as protective markers against epilepsy development. Conversely, the ABCB1*rs2032582 and CYP2B6*rs3745274 alleles were not considered as independent risk factors with the course of epilepsy disease.

Efflux pumps mediate changes to fundamental bacterial physiology via membrane potential.

Efflux pumps are well known to be an important mechanism for removing noxious substances such as antibiotics from bacteria. Given that many antibiotics function by accumulating inside bacteria, efflux pumps contribute to resistance. Efflux pump inactivation is a potential strategy to combat antimicrobial resistance, as bacteria would not be able to pump out antibiotics. We recently discovered that the impact of loss of efflux function is only apparent in actively growing cells. We demonstrated that the global transcriptome of Salmonella Typhimurium is drastically altered during slower growth leading to stationary-phase cells having a remodeled, less permeable envelope that prevents antibiotics entering the cell. Here, we investigated the effects of deleting the major efflux pump of Salmonella Typhimurium, AcrB, on global gene transcription across growth. We revealed that an acrB knockout entered stationary phase later than the wild-type strain SL1344 and displayed increased and prolonged expression of genes responsible for anaerobic energy metabolism. We devised a model linking efflux and membrane potential, whereby deactivation of AcrB prevents influx of protons across the inner membrane and thereby hyperpolarization. Knockout or deactivation of AcrB was demonstrated to increase membrane potential. We propose that the global transcription regulator ArcBA senses changes to the redox state of the quinol pool (linked to the membrane potential of the bacterium) and coordinates the shift from exponential to stationary phase via the key master regulators RpoS, Rsd, and Rmf. Inactivation of efflux pumps therefore influences the fundamental physiology of Salmonella, with likely impacts on multiple phenotypes.IMPORTANCEWe demonstrate for the first time that deactivation of efflux pumps brings about changes to gross bacterial physiology and metabolism. Rather than simply being a response to noxious substances, efflux pumps appear to play a key role in maintenance of membrane potential and thereby energy metabolism. This discovery suggests that efflux pump inhibition or inactivation might have unforeseen positive consequences on antibiotic activity. Given that stationary-phase bacteria are more resistant to antibiotic uptake, late entry into stationary phase would prolong antibiotic accumulation by bacteria. Furthermore, membrane hyperpolarization could result in increased generation of reactive species proposed to be important for the activity of some antibiotics. Finally, changes in gross physiology could also explain the decreased virulence of efflux mutants.

Resistance to 5-fluorouracil: The molecular mechanisms of development in colon cancer cells.

Colon cancer is a significant health problem worldwide as it is one of the most common and deadliest cancers. The standard approach for the treatment of colon cancer is 5-fluorouracil (5-FU) based chemotherapy, which is limited by the development of resistance to this drug. Therefore, our study aimed to establish 5-FU resistance in SW-480 and HT-29 colon cancer cells and to precisely determine the molecular mechanisms and biomarkers that contribute to its development, both after short-term exposure and in cells with already developed resistance (SW-480-5FUR and HT-29-5FUR). The expression of various molecules involved in the different mechanisms of resistance development was monitored at the gene (qPCR) and protein (immunocytochemistry) levels. Based on the obtained results, alterations in the 5-FU anabolic pathway, biotransformation, drug efflux, mismatch repair, and apoptosis process together contributed to the development of 5-FU resistance in SW-480 and HT-29 colon cancer cells. In addition, UMPS, ABCC1, ABCC5, and MLH1, as well as the disturbed ratio of pro-apoptotic BAX and anti-apoptotic BCL2, should be taken into consideration as potential targets for the discovery of 5-FU resistance-related biomarkers in colon cancer cells. We suggest that future investigations focus on further validation of these findings by additional in vitro and in vivo testing, which is a limitation of our study.

IRE1α-XBP1s axis regulates SREBP1-dependent MRP1 expression to promote chemoresistance in non-small cell lung cancer cells.

BACKGROUND: Inositol-requiring enzyme 1 (IRE1) is an endoplasmic reticulum (ER)-resident transmembrane protein that senses ER stress and mediates an essential arm of the unfolded protein response (UPR). IRE1 reduces ER stress by upregulating the expression of multiple ER chaperones through activation of X-box-binding protein 1 (XBP1). Emerging lines of evidence have revealed that IRE1-XBP1 axis serves as a multipurpose signal transducer during oncogenic transformation and cancer development. In this study, we explore how IRE1-XBP1 signaling promotes chemoresistance in lung cancer. METHODS: The expression patterns of UPR components and MRP1 were examined by Western blot. qRT-PCR was employed to determine RNA expression. The promoter activity was determined by luciferase reporter assay. Chemoresistant cancer cells were analyzed by viability, apoptosis. CUT & Tag (Cleavage under targets and tagmentation)-qPCR analysis was used for analysis of DNA-protein interaction. RESULTS: Here we show that activation of IRE1α-XBP1 pathway leads to an increase in MDR-related protein 1 (MRP1) expression, which facilitates drug extrusion and confers resistance to cytotoxic chemotherapy. At the molecular level, XBP1-induced c-Myc is necessary for SREBP1 expression, and SREBP1 binds to the MRP1 promoter to directly regulate its transcription. CONCLUSIONS: We conclude that IRE1α-XBP1 had important role in chemoresistance and appears to be a novel prognostic marker for lung cancer.

Potentiation of growth suppression and modulation of multidrug resistance by gamma and beta interferons in MDA-MB-231 breast cancer cell line.

Multi-drug resistance (MDR) might be acquired by the cancer cells during chemotherapy, and ATP-binding cassette (ABC) transporters play a significant role in MDR. Interferon-γ (IFN-γ) and IFN-ß can inhibit cancer cell proliferation; however, the effects and mechanism of these cytokines on the growth and MDR are still unclear. To investigate the effects of IFN-γ and IFN-ß, alone or in combination, on viability, resistance, and the expression of ABC transporters of the MDA-MB-231 breast cancer cell line. Using the MDA-MB-231 cell line, we assessed the effects of 20, 100, and 500 IU/ml of IFN-γ and IFN-ß, alone or in combination, on cell viability by methyl thiazolyl tetrazolium (MTT) assay; and then we performed the Uptake and Efflux experiment to evaluate the effect of these IFNs on the cell resistance. Then, using quantitative real-time PCR, we evaluated changes in the expression of ABCB1, ABCC1, and ABCG2 mRNA levels. We discovered that IFN-γ and IFN-ß might both reduce viability, either alone or in combination. The combination of IFNs also displayed synergistic responses, particularly when utilizing equivalent dosages of 500 or 100 IU/ml. The combination of IFN-γ and IFN-ß resulted in a significant increase in Doxorubicin accumulation and down-regulation of the ABCC1 gene at the mRNA level. Our study suggested that equal doses of IFN-γ and IFN-ß in combination might result in potentiated responses against cancer, especially, along with chemotherapy agents.

The mrp-3 gene is involved in haem efflux and detoxification in a blood-feeding nematode.

BACKGROUND: Haem is essential but toxic for metazoan organisms. Auxotrophic nematodes can acquire sufficient haem from the environment or their hosts in the meanwhile eliminate or detoxify excessive haem through tightly controlled machinery. In previous work, we reported a role of the unique transporter protein HRG-1 in the haem acquisition and homeostasis of parasitic nematodes. However, little is known about the haem efflux and detoxification via ABC transporters, particularly the multiple drug resistance proteins (MRPs). RESULTS: Here, we further elucidate that a member of the mrp family (mrp-3) is involved in haem efflux and detoxification in a blood-feeding model gastrointestinal parasite, Haemonchus contortus. This gene is haem-responsive and dominantly expressed in the intestine and inner membrane of the hypodermis of this parasite. RNA interference of mrp-3 resulted in a disturbance of genes (e.g. hrg-1, hrg-2 and gst-1) that are known to be involved in haem homeostasis and an increased formation of haemozoin in the treated larvae and lethality in vitro, particularly when exposed to exogenous haem. Notably, the nuclear hormone receptor NHR-14 appears to be associated the regulation of mrp-3 expression for haem homeostasis and detoxification. Gene knockdown of nhr-14 and/or mrp-3 increases the sensitivity of treated larvae to exogenous haem and consequently a high death rate (> 80%). CONCLUSIONS: These findings demonstrate that MRP-3 and the associated molecules are essential for haematophagous nematodes, suggesting novel intervention targets for these pathogens in humans and animals.

Molecular survey of pfmdr-1, pfcrt, and pfk13 gene mutations among patients returning from Plasmodium falciparum endemic areas to Turkey.

BACKGROUND: In recent years, there has been an increasing trend in the number of imported Plasmodium falciparum cases in Turkey. To improve treatment success and to better understand malaria epidemiology among imported cases, it is necessary to determine anti-malarial drug resistance. This study aimed to survey polymorphisms of resistance genes in imported P. falciparum patients using archived thin smear preparations and EDTA blood samples. METHODS: A total of 100 imported P. falciparum patients admitted to Bakirköy Dr. Sadi Konuk Research and Training Hospital between 2017 and 2022 were included in this study. DNA extraction was performed using an archived slide and EDTA blood samples that were microscopically diagnosed. After confirming the samples by real-time PCR, the pfmdr1, pfcrt, and pfk13 genes were amplified and sequenced. Single nucleotide polymorphisms (SNPs) were screened using Geneious R9 software, with the reference P. falciparum clone 3D7 isolate. RESULTS: All studied samples were confirmed to be P. falciparum using real-time PCR. Nested PCR was conducted and the pfcrt (92 samples), pfmdr1 (91 samples), and pfk13 (93 samples) genes were successfully amplified. Sequence analysis revealed the highest mutation rate in the pfmdr1 (74.5%) gene, with the identification of five haplotypes: NYSND (wild-type, 23%), NFSND (56%), NYSDD (2.2%), NFSDD (15.4%), and YFSND (3.4%)]. The pfcrt mutation was identified in 11 samples (12.2%), whereas the pfk13 mutation was found in only two samples. CONCLUSION: This study is the first molecular survey of anti-malarial drug resistance genes in Turkey. With the increasing number of imported Plasmodium malaria cases and recent reports of sporadic indigenous P. falciparum cases, malaria is becoming a growing concern in Turkey. Although molecular screening for resistance markers in P. falciparum malaria is not routinely conducted, the data from this study will enhance treatment success rates and contribute to global malaria elimination.

Generalized Arterial Calcification of Infancy (GACI).

Generalized arterial calcification of infancy (GACI) is an ultra-rare autosomal recessive disorder associated with pathogenic variants in ENPP1, the major gene involved in this condition, and in ABCC6, which is involved in a small fraction of affected individuals. Loss-of-function pathogenic variants of ENPP1 and ABCC6 lead to perturbations in the PPi/Pi ratio, thereby promoting hydroxyapatite mineralization in peripheral tissues. GACI is initially characterized by an abnormal ectopic mineralization process in arteries and soft tissue. Nearly half of the patients die within the first 6 months of life from cardiovascular complications, hence the poor prognosis associated with an early diagnosis. In recent years, progress has been made in our understanding of the long-term natural history of GACI, the intricate symptoms due to vascular calcifications, the overmineralization of soft tissues, of hypophosphatemia designated as ARHR2, and of the consequences such as undermineralization of the skeleton, but also of the features possibly seen in pseudoxanthoma elasticum (PXE). Indeed, GACI, PXE, and ARHR2 share common pathophysiological pathways and clinical features beyond the vascular calcifications. Treatment options for severe forms of GACI are mostly based on symptomatic management, including the option of starting bisphosphonates early after birth, such as etidronate and pamidronate, analogues of PPi. Follow-up within an expert and coordinated multidisciplinary team includes treatment of arterial hypertension, calcitriol and phosphorus adjustments, hearing aids, and early detection of possible angioid streaks. It is hoped that ongoing basic and clinical research will lead to the development of effective therapies that specifically target the abnormal PPi regulation and the other mechanisms involved in this disorder.

Downregulation of APN1 and ABCC2 mutation in Bt Cry1Ac-resistant Trichoplusia ni are genetically independent.

The resistance to the insecticidal protein Cry1Ac from the bacterium Bacillus thuringiensis (Bt) in the cabbage looper, Trichoplusia ni, has previously been identified to be associated with a frameshift mutation in the ABC transporter ABCC2 gene and with altered expression of the aminopeptidase N (APN) genes APN1 and APN6, shown as missing of the 110-kDa APN1 (phenotype APN1¯) in larval midgut brush border membrane vesicles (BBMV). In this study, genetic linkage analysis identified that the APN1¯ phenotype and the ABCC2 mutation in Cry1Ac-resistant T. ni segregated independently, although they were always associated under Cry1Ac selection. The ABCC2 mutation and APN1¯ phenotype were separated into two T. ni strains respectively. Bioassays of the T. ni strains with Cry1Ac determined that the T. ni with the APN1¯ phenotype showed a low level resistance to Cry1Ac (3.5-fold), and the associated resistance is incompletely dominant in the background of the ABCC2 mutation. Whereas the ABCC2 mutation-associated resistance to Cry1Ac is at a moderate level, and the resistance is incompletely recessive in the genetic background of downregulated APN1. Analysis of Cry1Ac binding to larval midgut BBMV indicated that the midgut in larvae with the APN1¯ phenotype had reduced binding affinity for Cry1Ac, but the number of binding sites remained unchanged, and the midgut in larvae with the ABCC2 mutation had both reduced binding affinity and reduced number of binding sites for Cry1Ac. The reduced Cry1Ac binding to BBMV from larvae with the ABCC2 mutation or APN1¯ phenotype correlated with the lower levels of resistance.IMPORTANCEThe soil bacterium Bacillus thuringiensis (Bt) is an important insect pathogen used as a bioinsecticide for pest control. Bt genes coding for insecticidal proteins are the primary transgenes engineered into transgenic crops (Bt crops) to confer insect resistance. However, the evolution of resistance to Bt proteins in insect populations in response to exposure to Bt threatens the sustainable application of Bt biotechnology. Cry1Ac is a major insecticidal toxin utilized for insect control. Genetic mechanisms of insect resistance to Cry1Ac are complex and require to be better understood. The resistance to Cry1Ac in Trichoplusia ni is associated with a mutation in the ABCC2 gene and also associated with the APN expression phenotype APN1¯. This study identified the genetic independence of the APN1¯ phenotype from the ABCC2 mutation and isolated and analyzed the ABCC2 mutation-associated and APN1¯ phenotype-associated resistance traits in T. ni to provide new insights into the genetic mechanisms of Cry1Ac resistance in insects.

microRNA maintains nutrient homeostasis in the symbiont-host interaction.

Endosymbionts provide essential nutrients for hosts, promoting growth, development, and reproduction. However, the molecular regulation of nutrient transport from endosymbiont to host is not well understood. Here, we used bioinformatic analysis, RNA-Sequencing, luciferase assays, RNA immunoprecipitation, and in situ hybridization to show that a bacteriocyte-distributed MRP4 gene (multidrug resistance-associated protein 4) is negatively regulated by a host (aphid)-specific microRNA (miR-3024). Targeted metabolomics, microbiome analysis, vitamin B6 (VB6) supplements, 3D modeling/molecular docking, in vitro binding assays (voltage clamp recording and microscale thermophoresis), and functional complementation of Escherichia coli were jointly used to show that the miR-3024/MRP4 axis controls endosymbiont (Serratia)-produced VB6 transport to the host. The supplementation of miR-3024 increased the mortality of aphids, but partial rescue was achieved by providing an external source of VB6. The use of miR-3024 as part of a sustainable aphid pest-control strategy was evaluated by safety assessments in nontarget organisms (pollinators, predators, and entomopathogenic fungi) using virus-induced gene silencing assays and the expression of miR-3024 in transgenic tobacco. The supplementation of miR-3024 suppresses MRP4 expression, restricting the number of membrane channels, inhibiting VB6 transport, and ultimately killing the host. Under aphids facing stress conditions, the endosymbiont titer is decreased, and the VB6 production is also down-regulated, while the aphid's autonomous inhibition of miR-3024 enhances the expression of MRP4 and then increases the VB6 transport which finally ensures the VB6 homeostasis. The results confirm that miR-3024 regulates nutrient transport in the endosymbiont-host system and is a suitable target for sustainable pest control.

Oral pyrophosphate protects Abcc6-/- mice against vascular calcification induced by chronic kidney disease.

One of the hallmarks of chronic kidney disease (CKD) is the development of vascular calcification. Inorganic pyrophosphate is a potent inhibitor of calcification, and previous studies have reported low plasma pyrophosphate levels in hemodialysis patients. A long-term mouse model of CKD-accelerated vascular calcification was developed to study pyrophosphate metabolism and to test whether oral pyrophosphate supplementation attenuates the propensity for arterial calcification. CKD was induced by repeated injections of aristolochic acid in wild-type and Abcc6-/- mice, which tend to develop vascular calcifications. CKD accelerated the development of vascular calcifications in Abcc6-/- mice, in the aorta and small renal arteries, and decreased circulating pyrophosphate levels. Oral pyrophosphate supplementation for 6 months attenuated CKD-induced vascular calcification in this model. These results show that oral pyrophosphate may be of interest in preventing vascular calcification in patients with CKD. KEY MESSAGES: Chronic kidney disease accelerates the development of vascular calcification in pyrophosphate-deficient mice. Oral pyrophosphate supplementation for 6 months attenuates chronic kidney disease-induced vascular calcification in a mouse model. Oral pyrophosphate may be of interest in preventing vascular calcification in patients with chronic kidney disease.

Precision Medicine in Childhood Cancer: The Influence of Genetic Polymorphisms on Vincristine-Induced Peripheral Neuropathy.

Cancer is the leading cause of disease-related death among children. Vincristine (VCR), a key component of childhood cancer treatment protocols, is associated with the risk of peripheral neuropathy (PN), a condition that may be reversible upon drug discontinuation but can also leave lasting sequelae. Single nucleotide polymorphism (SNP) in genes involved in VCR pharmacokinetics and pharmacodynamics have been investigated in relation to an increased risk of PN. However, the results of these studies have been inconsistent. A retrospective cohort study was conducted to investigate the potential association of drug transporter genes from the ATP-binding cassette (ABC) family and the centrosomal protein 72 (CEP72) gene with the development of PN in 88 Caucasian children diagnosed with cancer and treated with VCR. Genotyping was performed using real-time PCR techniques for the following SNPs: ABCB1 rs1128503, ABCC1 rs246240, ABCC2 rs717620, and CEP72 rs924607. The results indicated that age at diagnosis (OR = 1.33; 95% CI = 1.07-1.75) and the ABCC1 rs246240 G allele (OR = 12.48; 95% CI = 2.26-100.42) were associated with vincristine-induced peripheral neuropathy (VIPN). No association was found between this toxicity and CEP72 rs924607. Our study provides insights that may contribute to optimizing childhood cancer therapy in the future by predicting the risk of VIPN.

ABCC2 induces metabolic vulnerability and cellular ferroptosis via enhanced glutathione efflux in gastric cancer.

BACKGROUND: Although it is traditionally believed that ATP binding cassette subfamily C member 2 (ABCC2) is a multidrug resistance-associated protein correlated with a worse prognosis, our previous and several other studies demonstrated the contrary to be true in gastric cancer (GC). We aim to explore the underlying mechanism of this discovery. METHODS: Our study utilized whole-exome sequencing (WES), RNA sequencing, and droplet digital PCR (ddPCR) analysis of 80 gastric cancer samples, along with comprehensive immunohistochemical (IHC) analysis of 1044 human GC tissue samples.By utilizing CRISPRCas9 to genetically modify cell lines with the ABCC2-24C > T (rs717620) point mutation and conducting dual-luciferase reporter assays, we identified that transcription factors SOX9 and ETS1 serve as negative regulators of ABCC2 expression. Seahorse assay and mass spectrometry were used to discover altered metabolic patterns. Gain and loss-of-function experiments in GC cell lines and preclinical models were carried out to validate ABCC2 biological function. RESULTS: ABCC2 high expression correlated with better prognosis, and rs717620 can influence ABCC2 expression by disrupting the binding of ETS1 and SOX9. Gain and loss-of-function experiments in GC cell lines demonstrated amino acid deprivation reduces proliferation, migration, and drug resistance in ABCC2-high GC cells. ABCC2 leads to reduced intracellular amino acid pools and disruption of cellular energy metabolism. This phenomenon depended on ABCC2-mediated GSH extrusion, resulting in alterations in redox status, thereby increasing the cell's susceptibility to ferroptosis. Furthermore, patient-derived organoids and patient-derived tumor-like cell clusters were used to observe impact of ABCC2 on therapeutic effect. In the xenograft model with high ABCC2 expression, we observed that constricting amino acid intake in conjunction with GPX4 inactivation resulted in notable tumor regression. CONCLUSIONS: Our findings demonstrate a significant role of ABCC2 in amino acid metabolism and ferroptosis by mediating GSH efflux in GC. This discovery underlines the potential of combining multiple ferroptosis targets as a promising therapeutic strategy for GC with high ABCC2 expression. HIGHLIGHTS: ABCC2 plays a crucial role in inducing metabolic vulnerability and ferroptosis in gastric cancer through enhanced glutathione efflux. The ABCC2 24C > T polymorphism is a key factor influencing its expression. These results highlight the potential of ABCC2 as a predictive biomarker and therapeutic target in gastric cancer.

ABCC1 Is a ΔNp63 Target Gene Overexpressed in Squamous Cell Carcinoma.

The transcription factor ΔNp63 plays a pivotal role in maintaining the integrity of stratified epithelial tissues by regulating the expression of distinct target genes involved in lineage specification, cell stemness, cell proliferation and differentiation. Here, we identified the ABC transporter subfamily member ABCC1 as a novel ΔNp63 target gene. We found that in immortalized human keratinocytes and in squamous cell carcinoma (SCC) cells, ∆Np63 induces the expression of ABCC1 by physically occupying a p63-binding site (p63 BS) located in the first intron of the ABCC1 gene locus. In cutaneous SCC and during the activation of the keratinocyte differentiation program, ∆Np63 and ABCC1 levels are positively correlated raising the possibility that ABCC1 might be involved in the regulation of the proliferative/differentiative capabilities of squamous tissue. However, we did not find any gross alteration in the structure and morphology of the epidermis in humanized hABCC1 knock-out mice. Conversely, we found that the genetic ablation of ABCC1 led to a marked reduction in inflammation-mediated proliferation of keratinocytes, suggesting that ABCC1 might be involved in the regulation of keratinocyte proliferation upon inflammatory/proliferative signals. In line with these observations, we found a significant increase in ABCC1 expression in squamous cell carcinomas (SCCs), a tumor type characterized by keratinocyte hyper-proliferation and a pro-inflammatory tumor microenvironment. Collectively, these data uncover ABCC1 as an additional ∆Np63 target gene potentially involved in those skin diseases characterized by dysregulation of proliferation/differentiation balance.