Identification and Functional Assessment of Eight CYP3A4 Allelic Variants *39-*46 Detected in the Chinese Han Population.

Cytochrome P450 3A4 (CYP3A4), a key enzyme, is pivotal in metabolizing approximately half of the drugs used clinically. The genetic polymorphism of the CYP3A4 gene significantly influences individual variations in drug metabolism, potentially leading to severe adverse drug reactions (ADRs). In this study, we conducted a genetic analysis on CYP3A4 gene in 1163 Chinese Han individuals to identify the genetic variations that might affect their drug metabolism capabilities. For this purpose, a multiplex polymerase chain reaction (PCR) amplicon sequencing technique was developed, enabling us to perform the genotyping of CYP3A4 gene efficiently and economically on a large scale. As a result, a total of 14 CYP3A4 allelic variants were identified, comprising six previously reported alleles and eight new nonsynonymous variants that were nominated as new allelic variants *39-*46 by the PharmVar Association. Further, functional assessments of these novel CYP3A4 variants were undertaken by coexpressing them with cytochromes P450 oxidoreductase (CYPOR) in Saccharomyces cerevisiae microsomes. Immunoblot analysis indicated that with the exception of CYP3A4.40 and CYP3A4.45, the protein expression levels of most new variants were similar to that of the wild-type CYP3A4.1 in yeast cells. To evaluate their catalytic activities, midazolam was used as a probe drug. The results showed that variant CYP3A4.45 had almost no catalytic activity, whereas the other variants exhibited significantly reduced drug metabolism abilities. This suggests that the majority of the CYP3A4 variants identified in the Chinese population possess markedly altered capacities for drug metabolism. SIGNIFICANCE STATEMENT: In this study, we established a multiplex polymerase chain reaction (PCR) amplicon sequencing method and detected the maximum number of new CYP3A4 variants in a single ethnic population. Additionally, we performed the functional characterizations of these eight novel CYP3A4 allele variants in vitro. This study not only contributes to the understanding of CYP3A4 genetic polymorphism in the Chinese Han population but also holds substantial reference value for their potential clinical applications in personalized medicine.

Electrochemical filtration for drinking water purification: A review on membrane materials, mechanisms and roles.

Electrochemical filtration can not only enrich low concentrations of pollutants but also produce reactive oxygen species to interact with toxic pollutants with the assistance of a power supply, making it an effective strategy for drinking water purification. In addition, the application of electrochemical filtration facilitates the reduction of pretreatment procedures and the use of chemicals, which has outstanding potential for maximizing process simplicity and reducing operating costs, enabling the production of safe drinking water in smaller installations. In recent years, the research on electrochemical filtration has gradually increased, but there has been a lack of attention on its application in the removal of low concentrations of pollutants from low conductivity water. In this review, membrane substrates and electrocatalysts used to improve the performance of electrochemical membranes are briefly summarized. Meanwhile, the application prospects of emerging single-atom catalysts in electrochemical filtration are also presented. Thereafter, several electrochemical advanced oxidation processes coupled with membrane filtration are described, and the related working mechanisms and their advantages and shortcomings used in drinking water purification are illustrated. Finally, the roles of electrochemical filtration in drinking water purification are presented, and the main problems and future perspectives of electrochemical filtration in the removal of low concentration pollutants are discussed.

Esophageal cancer cell-derived small extracellular vesicles decrease circulating Tfh/Tfr via sEV-PDL1 to promote immunosuppression.

Esophageal cancer (EC) is a deadly malignancy. Small extracellular vesicles (sEVs) with programmed death ligand 1 (sEV-PDL1) induce immune escape to promote tumor progression. Furthermore, the imbalance between circulating follicular helper T (Tfh) and circulating follicular regulatory T (Tfr) cells is related to the progression of many malignant tumors. However, the role of the EC-derived sEV-PDL1 in circulating Tfh/Tfr is unknown. Circulating Tfh and Tfr cells were detected by flow cytometry. sEVs were isolated through differential centrifugation and cultured for cell expansion assays. Naïve CD4+ T cells were isolated, stimulated, and cultured with sEVs to evaluate the frequencies, phenotypes, and functions of Tfh and Tfr cells. The proportion of circulating Tfh in patients with EC was lower than that in healthy donors (HDs), whereas that of circulating Tfr was higher. The EC group showed significantly lower circulating Tfh/Tfr and a higher level of sEV-PDL1 than HDs. Notably, sEV-PDL1 was negatively correlated with circulating Tfh/Tfr in the EC group. In vitro assays, sEV-PDL1 inhibited Tfh expansion, enhanced the cytotoxic T lymphocyte-associated antigen 4+ (CTLA4+) Tfh cell percentage, decreased the levels of interleukin (IL)-21 and interferon-γ, and increased IL-10. sEV-PDL1 promoted the expansion and immunosuppressive functions of circulating Tfr; the increased percentages of CTLA4+ Tfr and inducible T cell co-stimulator+ Tfr were accompanied with high IL-10. However, applying an anti-PDL1 antibody significantly reversed this. Our results suggest a novel mechanism of sEV-PDL1-mediated immunosuppression in EC. Inhibiting sEV-PDL1 to restore circulating Tfh/Tfr balance provides a novel therapeutic approach for EC.

Distinguishing optimal candidates for primary tumor resection in patients with metastatic lung adenocarcinoma: A predictive model based on propensity score matching.

Background: Primary tumor resection is associated with survival benefits in patients with metastatic lung adenocarcinoma (mLUAD). However, there are no established methods to determine which individuals would benefit from surgery. Therefore, we developed a model to predict the patients who are likely to benefit from surgery in terms of survival. Methods: Data on patients with mLUAD were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. Depending on whether surgery was performed on the primary tumor, patients were categorized into two groups: cancer-directed surgery (CDS) and no-cancer-directed surgery (No-CDS). Propensity Score Matching (PSM) was utilized to address bias between the CDS and No-CDS groups. The prognostic impact of CDS was assessed using Kaplan-Meier analysis and Cox proportional hazard models. Subsequently, we constructed a nomogram to predict the potential for surgical benefits based on multivariable logistic regression analysis using preoperative factors. Results: A total of 89,039 eligible patients were identified, including 6.4% (5705) who underwent surgery. Following PSM, the CDS group demonstrated a significantly longer median overall survival (mOS) compared with the No-CDS group (23 [21-25] vs. 7 [7-8] months; P < 0.001). The nomogram showed robust performance in both the training and validation sets (area under the curve [AUC]: 0.698 and 0.717, respectively), and the calibration curves exhibited high consistency. The nomogram proved clinically valuable according to decision curve analysis (DCA). According to this nomogram, surgical patients were categorized into two groups: no-benefit candidates and benefit candidates groups. Compared with the no-benefit candidate group, the benefit candidate group was associated with longer survival (mOS: 25 vs. 6 months, P < 0.001). Furthermore, no difference in survival was observed between the no-benefit candidates and the no-surgery groups (mOS: 6 vs. 7 months, P = 0.9). Conclusions: A practical nomogram was developed to identify optimal CDS candidates among patients with mLUAD.

The CsmiR397a-CsLAC17 module regulates lignin biosynthesis to balance the tenderness and gray blight resistance in young tea shoots.

Lignin accumulation can enhance the disease resistance of young tea shoots (Camellia sinensis). It also greatly reduces their tenderness, which indirectly affects the quality and yield of tea. Therefore, the regulation of lignin biosynthesis appears to be an effective way to balance tenderness and disease resistance in young tea shoots. In this study, we identified a laccase gene, CsLAC17, that is induced during tenderness reduction and gray blight infection in young tea shoots. Overexpression of CsLAC17 significantly increased the lignin content in transgenic Arabidopsis, enhancing their resistance to gray blight and decreasing stem tenderness. In addition, we found that CsLAC17 was negatively regulated by the upstream CsmiR397a by 5'-RLM-RACE, dual-luciferase assay, and transient expression in young tea shoots. Interestingly, the expression of CsmiR397a was inhibited during tenderness reduction and gray blight infection of young tea shoots. Overexpression of CsmiR397a reduced lignin accumulation, resulting in decreased resistance to gray blight and increased stem tenderness in transgenic Arabidopsis. Furthermore, the transient overexpression of CsmiR397a and CsLAC17 in tea leaves directly confirms the function of the CsmiR397a-CsLAC17 module in lignin biosynthesis and its effect on disease resistance. These results suggest that the CsmiR397a-CsLAC17 module is involved in balancing tenderness and gray blight resistance in young tea shoots by regulating lignin biosynthesis.

Identification and in vitro functional assessment of 10 CYP2C9 variants found in Chinese Han subjects.

Cytochrome P450 2C9 (CYP2C9) participates in about 15% of clinical drug metabolism, and its polymorphism is associated with individual drug metabolism differences, which may lead to the adverse drug reactions (ADRs). In this study, 1163 Chinese Han individuals were recruited to investigate their distribution pattern of CYP2C9 gene and find out the variants that may affect their drug metabolic activities. We successfully developed a multiplex PCR amplicon sequencing method and used it for the genetic screening of CYP2C9 in a large scale. Besides the wild type CYP2C9*1, totally 26 allelic variants of CYP2C9 were detected, which included 16 previously reported alleles and 10 new non-synonymous variants that had not been listed on the PharmVar website. The characteristics of these newly detected CYP2C9 variants were then evaluated after co-expressing them with CYPOR in S. cerevisiae microsomes. Immunoblot analysis revealed that except for Pro163Ser, Glu326Lys, Gly431Arg and Ile488Phe, most of newly detected variants showed comparable protein expression levels to wild type in yeast cells. Two typical CYP2C9 probe drugs, losartan and glimepiride, were then used for the evaluation of metabolic activities of variants. As a result, 3 variants Thr301Met, Glu326Lys, and Gly431Arg almost lost their catalytic activities and most of other variants exhibited significantly elevated activities for drug metabolism. Our data not only enriches the knowledge of naturally occurring CYP2C9 variants in the Chinese Han population, but also provides the fundamental evidence for its potential clinical usage for personalized medicine in the clinic.

Characterization of 15 CYP2J2 variants identified in the Chinese Han population on the metabolism of ebastine and terfenadine in vitro.

Genetic polymorphism of the cytochrome P450 (CYP) gene can significantly influence the metabolism of endogenous and xenobiotic compounds. However, few studies have focused on the polymorphism of CYP2J2 and its impact on drug catalytic activity, especially in the Chinese Han population. In this study, we sequenced the promoter and exon regions of CYP2J2 in 1,163 unrelated healthy Chinese Han individuals using the multiplex PCR amplicon sequencing method. Then, the catalytic activities of the detected CYP2J2 variants were evaluated after recombinant expression in S. cerevisiae microsomes. As a result, CYP2J2*7, CYP2J2*8, 13 variations in the promoter region and 15 CYP2J2 nonsynonymous variants were detected, of which V15A, G24R, V68A, L166F and A391T were novel missense variations. Immunoblotting results showed that 11 of 15 CYP2J2 variants exhibited lower protein expression than wild-type CYP2J2.1. In vitro functional analysis results revealed that the amino acid changes of 14 variants could significantly influence the drug metabolic activity of CYP2J2 toward ebastine or terfenadine. Specifically, 4 variants with relatively higher allele frequencies, CYP2J2.8, 173_173del, K267fs and R446W, exhibited extremely low protein expression and defective catalytic activities for both substrates. Our results indicated that a high genetic polymorphism of CYP2J2 could be detected in the Chinese Han population, and most genetic variations in CYP2J2 could influence the expression and catalytic activity of CYP2J2. Our data significantly enrich the knowledge of genetic polymorphisms in CYP2J2 and provide new theoretical information for corresponding individualized medication in Chinese and other Asian populations.

Revisiting chemoresistance in ovarian cancer: Mechanism, biomarkers, and precision medicine.

Among the gynecological cancers, ovarian cancer is the most lethal. Its therapeutic options include a combination of chemotherapy with platinum-based compounds and cytoreductive surgery. Most ovarian cancer patients exhibit an initial response to platinum-based therapy, however, platinum resistance has led to up to 80% of this responsive cohort becoming refractory. Ovarian cancer recurrence and drug resistance to current chemotherapeutic options is a global challenge. Chemo-resistance is a complex phenomenon that involves multiple genes and signal transduction pathways. Therefore, it is important to elucidate on the underlying molecular mechanisms involved in chemo-resistance. This inform decisions regarding therapeutic management and help in the identification of novel and effective drug targets. Studies have documented the individual biomarkers of platinum-resistance in ovarian cancer that are potential therapeutic targets. This review summarizes the molecular mechanisms of platinum resistance in ovarian cancer, novel drug targets, and clinical outcomes.

Polymorphic mutations in the polb gene promoter and their impact on transcriptional activity.

BACKGROUND: DNA polymerase ß is one of the key enzymes involved in DNA damage repair and its proper expression is strictly controlled within different cells. We previously reported that three genetic mutations in the promoter region of the polb gene are prevalent in the Chinese Han population and two types of mutation are associated with thymic hyperplasia. The purpose of this study was to explore whether other mutated sites exist within the promoter region of the polb gene. METHODS: Genomic DNAs of 421 healthy Chinese Han individuals were extracted from whole blood samples and used for gene amplification of the promoter region of the polb gene. After gel purification, PCR amplicons were sequenced by the Sanger sequencing method and used for sequence alignment with the Lasergene program. PCR products with novel mutations were then subcloned into luciferase reporter plasmid pGL4.10 and transfected into 293T cells for dual-luciferase activity analysis. RESULTS: In total, 11 mutated sites were detected in the Chinese Han population and eight of these were reported for the first time. Using a dual luciferase reporter system, it was found that one novel mutation -142 C > G could decrease the transcription activity of the polb gene, whereas two novel mutations, -152_-151insC and -218 C > G, could significantly increase the transcription activity of the polb gene. CONCLUSIONS: High polymorphic sites could be found in the promoter region of polb gene and approximately half of them could influence its transcription activity.

Oncogenic LncRNA CASC9 in Cancer Progression.

BACKGROUND: Long non-coding RNAs (LncRNAs), with the length of over 200 nucleotides, that originate from intergenic, antisense, or promoter-proximal regions, are a large family of RNAs that lack coding capacity. Emerging evidences illustrated that LncRNAs played significant roles in a variety of cellular functions and biological processes in profuse human diseases, especially in cancers. Cancer susceptibility candidate 9 (CASC9), as a member of the LncRNAs group, firstly found its oncogenic function in esophageal cancer. In the following recent studies, a growing amount of human malignancies are verified to be correlated with CASC9, most of which are derived from the squamous epithelium tissue. This present review attempts to highlight the latest insights into the expression, functional roles, and molecular mechanisms of CASC9 in different human malignancies. METHODS: In this review, the latest findings related to the pathophysiological processes of CASC9 in human cancers were summarized and analyzed, and the associated studies collected in systematic retrieval of PubMed used lncRNA and CASA9 as keywords. RESULTS: CASC9 expression is identified to be aberrantly elevated in a variety of malignancies. The over-expression of CASC9 has been suggested to accelerate cell proliferation, migration, cell growth and drug resistance of cancer cells, while depressing cell apoptosis, revealing its role as an oncogene. Moreover, the current review demonstrated CASC9 as closely related to the neoplastic transformation of squamous epithelial cells and squamous metaplasia in non-squamous epithelial tissues. Finally, we discuss the limitations and tremendous diagnostic/ therapeutic potential of CASC9 in various human cancers. CONCLUSION: Long non-coding RNA CASC9 likely serve as useful disease biomarkers or therapeutic targets which be effectively applied in the treatment of different kinds of cancers.

CDKN2B-AS1: An Indispensable Long Non-coding RNA in Multiple Diseases.

BACKGROUND: In view of the roles of long non-coding RNA CDKN2B antisense RNA 1 (CDKN2BAS1) in various human diseases, we investigated the function of CDKN2B-AS1 and explored its therapeutic and prognostic target value in multiple biological processes. The aim of this review was to explore the molecular mechanism and clinical significance of CDKN2B-AS1 in various types of diseases. MATERIALS AND METHODS: In this review, the biological functions and mechanisms of lncRNA CDKN2B-AS1 in a variety of pathophysiological processes were summarized and analyzed. The correlated studies were collected via a systematic search of PubMed, Wiley Online Library, and ScienceDirect. RESULTS: CDKN2B-AS1 is a potential long non-coding RNA that has been shown to be aberrantly expressed in various malignancies, containing hepatocellular carcinoma, intrahepatic cholangiocarcinoma, esophageal squamous cell carcinoma, gastric cancer, colonic adenocarcinoma, cervical cancer, ovarian cancer, breast cancer, glioma, lung cancer, laryngeal squamous cell carcinoma and osteosarcoma, involving in the processes of tumor cells proliferation, migration, invasion and inhibition of tumor cells apoptosis. Besides, CDKN2B-AS1 has been proved implicated in numerous non-malignant diseases, such as idiopathic pulmonary fibrosis, endometriosis, inflammatory bowel disease, intracranial aneurysm, diabetes mellitus and its complications, primary open angle glaucoma, ischemic stroke, atherosclerosis, coronary artery diseases, hypertension and heart failure, participating in the procession of lipid, carbohydrate metabolism and inflammation regulation. CONCLUSION: Long non-coding RNA CDKN2B-AS1 likely serves as a promising therapeutic target or prognosis biomarker in multiple human diseases.

Pathophysiological Functions of the lncRNA TUG1.

BACKGROUND: Long non-coding RNAs (lncRNAs) with little or no coding capacity are associated with a plethora of cellular functions, participating in various biological processes. Cumulative study of lncRNA provides explanations to the physiological and pathological processes and new perspectives to the diagnosis, prevention, and treatment of some clinical diseases. Long non-coding RNA taurine-upregulated gene 1(TUG1) is one of the first identified lncRNAs associated with human disease, which actively involved in various physiological processes, including regulating genes at epigenetics, transcription, post-transcription, translation, and posttranslation. The aim of this review was to explore the molecular mechanism of TUG1 in various types of human diseases. METHODS: In this review, we summarized and analyzed the latest findings related to the physiologic and pathophysiological processes of TUG1 in human diseases. The related studies were retrieved and selected the last six years of research articles in PubMed with lncRNA and TUG1 as keywords. RESULTS: TUG1 is a valuable lncRNA that its dysregulated expression and regulating the biological processes were found in a variety of human diseases. TUG1 is found to exhibit aberrant expression in a variety of malignancies. Dysregulation of TUG1 has been shown to contribute to proliferation, migration, cell cycle changes, inhibited apoptosis, and drug resistance of cancer cells, which revealed an oncogenic role for this lncRNA, but some reports have shown downregulation of TUG1 in lung cancer samples compared with noncancerous samples. In addition, the molecular and biological functions of TUG1 in physiology and disease (relevant to endocrinology, metabolism, immunology, neurobiology) have also been highlighted. Finally, we discuss the limitations and tremendous diagnostic/therapeutic potential of TUG1 in cancer and other diseases. CONCLUSION: Long non-coding RNA-TUG1 likely served as useful disease biomarkers or therapy targets and effectively applied in different kinds of diseases, such as human cancer and cardiovascular diseases.

Retraction note to: Lentivirus-Mediated Short-Hairpin RNA Targeting Protein Phosphatase 4 Regulatory Subunit 1 Inhibits Growth in Breast Cancer.

[This retracts the article on p. 218 in vol. 18, PMID: 26472971.].

The over-expression of a chrysanthemum gene encoding an RNA polymerase II CTD phosphatase-like 1 enzyme enhances tolerance to heat stress.

The enzyme RNAPII CTD phosphatase-like 1 is known as a transcriptional regulator of the plant response to various abiotic stresses. Here, the isolation of CmCPL1, a chrysanthemum (Chrysanthemum morifolium) gene encoding this enzyme is described. Its predicted 955 residue gene product includes the FCPH catalytic domain, two double-stranded RNA binding motifs, and a nuclear localization signal. A sub-cellular localization assay confirmed that CmCPL1 was expressed in the nucleus. CmCPL1 transcription was shown to be significantly inducible by heat stress. The over-expression and knockdown of CmCPL1, respectively, increased and diminished the tolerance of chrysanthemum to heat stress, which maybe dependent on the regulation of CmCPL1 and on the expression of downstream heat stress-responsive genes.

Erratum: Lentivirus-Mediated Short-Hairpin RNA Targeting Protein Phosphatase 4 Regulatory Subunit 1 Inhibits Growth in Breast Cancer.

[This corrects the article on p. 218 in vol. 18, PMID: 26472971.].

Lentivirus-Mediated Short-Hairpin RNA Targeting Protein Phosphatase 4 Regulatory Subunit 1 Inhibits Growth in Breast Cancer.

PURPOSE: Protein phosphatase 4 regulatory subunit 1 (PP4R1), as an interaction partner of the catalytic serine/threonine-protein phosphatase 4 catalytic subunit has been shown to involve in cellular processes and nuclear factor κB signaling. However, the functions of PP4R1 in human breast cancers remain unclear. This study is designed to explore the effect of PP4R1 knockdown on the biological characteristics of breast cancer cells. METHODS: A lentivirus-mediated short hairpin RNA (shRNA) was designed to knockdown the expression of PP4R1 in ZR-75-30 breast cancer cells. The efficiency of lentivirus-mediated shRNA infection was determined using fluorescence microscopy to observe lentivirus-mediated green fluorescent protein expression and confirmed to be over 80%. PP4R1 expression in infected ZR-75-30 cells was detected by quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation and colony formation ability were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and colony formation assay, respectively. Flow cytometry was used to measure cell cycle progression and cell apoptosis. In addition, apoptosis makers, including poly-ADP-ribose polymerase (PARP) and caspase-3, were investigated in PP4R1-silenced ZR-75-30 cells by western blot assay. RESULTS: We successfully constructed lentivirus-mediated shRNA to target PP4R1 in ZR-75-30 cells. MTT assay and colony formation assay showed the loss of PP4R1 suppressed the proliferation of ZR-75-30 cells. Flow cytometry analysis indicated cell cycle arrest and increased cell apoptosis in PP4R1 knockdown cells. Further, the apoptosis response in cells depleted of PP4R1 was illustrated by downregulation of PARP and upregulation of caspase-3. CONCLUSION: Our results suggest that PP4R1 could promote breast cancer cell proliferation and might play a vital role in breast cancer occurrence.