Molecular epidemiological study of Scrub Typhus in residence, farm and forest habitats from Yunnan Province, China.

The number of people suffering from scrub typhus, which is not of concern, is increasing year by year, especially in Yunnan Province, China. From June 1, 2021 to August 15, 2022, a total of 505 mammalian samples were collected from farm, forest, and residential habitats with high incidence of scrub typhus in Yunnan, China, for nPCR (nested PCR) and qPCR (quantitative real-time PCR) detection of Orientia tsutsugamushi. A total of 4 orders of murine-like animals, Rodentia (87.52%, n = 442), Insectivora (10.29%, n = 52), Lagomorpha (1.79%, n = 9) and Scandentia (0.40%, n = 2) were trapped. Comparing the qPCR infection rates in the three habitats, it was no significant difference that the infection rate of residential habitat (44.44%) and that of the farm habitat (45.05%, P>0.05), which is much larger than that of the forest habitat (3.08%) (P<0.001). Three genotypes (Karp-like, Kato-like and TA763-like) of O. tsutsugamushi were found from Yunnan, China in this study.

Metagenomic analysis of individual mosquito viromes reveals the geographical patterns and drivers of viral diversity.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Here, using a meta-transcriptomic approach, we determined the viromes of 2,438 individual mosquitoes (81 species), spanning ~4,000 km along latitudes and longitudes in China. From these data we identified 393 viral species associated with mosquitoes, including 7 (putative) species of arthropod-borne viruses (that is, arboviruses). We identified potential mosquito species and geographic hotspots of viral diversity and arbovirus occurrence, and demonstrated that the composition of individual mosquito viromes was strongly associated with host phylogeny. Our data revealed a large number of viruses shared among mosquito species or genera, enhancing our understanding of the host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, perhaps reflecting long-distance mosquito dispersal. Together, these results greatly expand the known mosquito virome, linked viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the biogeography and diversity of viruses in insect vectors.

Family resilience, patient-reported symptoms in young stroke dyads: The effect of caregiver readiness and social support.

AIMS AND OBJECTIVES: To investigate empirically the direct effect and potential mechanism of family resilience on patient-reported outcomes among young stroke dyads in China. BACKGROUND: Young patients with stroke have been becoming an important public health issue. According to relevant theories and previous studies, we found that family resilience might play an important role in patient's symptoms. However, it is less clear about the specific relationship and potential mechanisms of these two variables. DESIGN: We used a prospective cross-sectional design. METHODS: A multi-item questionnaire was used to assess the constructs of interest. Researchers progressively constructed and validated conditional process models. The PROCESS macro was used to verify the research hypotheses. RESULTS: A total of 560 questionnaires were collected in this study. We found that family resilience of stroke patients and their spouses had a direct effect on the physical, psychological and social aspects of patient-reported symptoms. We further revealed that caregiver preparedness partially mediated the relationship between family resilience and patient's symptoms in stroke patient-spouse dyads, while perceived social support moderated the relationship between caregiver preparedness and patient's symptoms. Finally, we observed that the impact of caregiver readiness and social support on patients' symptoms predominantly manifested in physical and physiological outcomes. CONCLUSIONS: Our research provides evidence about the positive impact of family resilience on patient-reported symptoms in young stroke dyads. Meanwhile, it further revealed how caregiver preparedness and perceived social support may play out in the relationship. PRACTICE IMPLICATIONS: Our research introduces a novel perspective and pathway to enhance short-term recovery outcomes for patients. It also furnishes clinicians and nurses with evidence to guide the implementation of interventions aimed at improving patient health outcomes and facilitating smoother transitions from the hospital to home. IMPACT: What problem did the study address? Families play a crucial role in a patient's recovery process from illness, with family resilience serving as an important force for families to overcome adversity. However, the impact on patient symptoms and the underlying mechanisms of this relationship are uncertain. Empirical research is required to validate these aspects. What were the main findings? Family resilience has a positive impact on the physical, psychological and social aspects of patient-reported symptoms in young stroke dyads. Both the actor effect and partner effect are supported. The impact of caregiver readiness and social support on patient-reported symptoms is primarily observed in physical and physiological outcomes. Where and on whom will the research have an impact? This study offers a novel approach to enhance the short-term recovery of stroke patients. The researchers believe that the findings of this study will play an even more significant role during patients' transition from the hospital to home. REPORTING METHOD: This study followed the STROBE statement of cross-sectional studies. PATIENT OR PUBLIC CONTRIBUTION: The study was conducted by patients, their spouses, healthcare professionals and the research team.

Bio-based anode material production for lithium-ion batteries through catalytic graphitization of biochar: the deployment of hybrid catalysts.

Producing sustainable anode materials for lithium-ion batteries (LIBs) through catalytic graphitization of renewable biomass has gained significant attention. However, the technology is in its early stages due to the bio-graphite's comparatively low electrochemical performance in LIBs. This study aims to develop a process for producing LIB anode materials using a hybrid catalyst to enhance battery performance, along with readily available market biochar as the raw material. Results indicate that a trimetallic hybrid catalyst (Ni, Fe, and Mn in a 1:1:1 ratio) is superior to single or bimetallic catalysts in converting biochar to bio-graphite. The bio-graphite produced under this catalyst exhibits an 89.28% degree of graphitization and a 73.95% conversion rate. High-resolution transmission electron microscopy (HRTEM) reveals the dissolution-precipitation mechanism involved in catalytic graphitization. Electrochemical performance evaluation showed that the trimetallic hybrid catalyst yielded bio-graphite with better electrochemical performances than those obtained through single or bimetallic hybrid catalysts, including a good reversible capacity of about 293 mAh g-1 at a current density of 20 mA/g and a stable cycle performance with a capacity retention of over 98% after 100 cycles. This study proves the synergistic efficacy of different metals in catalytic graphitization, impacting both graphite crystalline structure and electrochemical performance.

Sympathetic skin response for early detection of type 2 diabetic peripheral neuropathy and nephropathy.

BACKGROUND: Diabetic peripheral neuropathy (DPN) and diabetic nephropathy (DN) are common complications of type 2 diabetes mellitus (T2DM). Although nerve conduction studies (NCS) and sympathetic skin response (SSR) can detect DPN, the more sensitive method for early diagnosis remains unclear. Furthermore, whether DPN can be used as a predictor for diabetic nephropathy needs clarification. METHODS: We evaluated nerve conduction studies, sympathetic skin response, and the diabetic nephropathy indicator microalbuminuria (MAU) in 192 patients with type 2 diabetes mellitus and 50 healthy controls. RESULTS: Patients with type 2 diabetes mellitus showed a lower sensory nerve conduction velocity (SCV), sensory active nerve potential (SNAP), motor nerve conduction velocity (MCV), and compound motor action potential (CMAP) than the controls on NCS. Abnormal rates for nerve conduction studies and sympathetic skin response were 75.0% and 83.3%, respectively, in patients with type 2 diabetes mellitus. Interestingly, 54.2% of patients with normal nerve conduction studies had an abnormal sympathetic skin response. Moreover, we found a positive correlation between sympathetic skin response and microalbuminuria for the first time. The abnormal rate of microalbuminuria was 53.8%, lower than that of abnormal nerve conduction studies or sympathetic skin response patients. CONCLUSION: Sympathetic skin response is a more sensitive method than nerve conduction studies for the early diagnosis of diabetic peripheral neuropathy. Abnormal sympathetic skin response might serve as an indicator for early diabetic nephropathy. Additionally, diabetic peripheral neuropathy may occur earlier than diabetic nephropathy in the development of type 2 diabetes mellitus.

A highly sensitive and anti-freezing conductive strain sensor based on polypyrrole/cellulose nanofiber crosslinked polyvinyl alcohol hydrogel for human motion detection.

Electro-conductive hydrogels emerge as a stretchable conductive materials with diverse applications in the synthesis of flexible strain sensors. However, the high-water content and low cross-links density cause them to be mechanically destroyed and freeze at subzero temperatures, limiting their practical applications. Herein, we report a one-pot strategy by co-incorporating cellulose nanofiber (CNF), Poly pyrrole (PPy) and glycerol with polyvinyl alcohol (PVA) to prepare hydrogel. The addition of PPy endowed the hydrogel with good conductivity (∼0.034 S/m) compared to the no PPy@CNF group (∼0.0095 S/m), the conductivity was increased by 257.9 %. The hydrogel exhibits comparable ionic conductivity at -18 °C as it does at room temperature. It's attributed to the glycerol as a cryoprotectant and the formation of hydrated [Zn(H2O)n]2+ ions via strong interaction between Zn2+ and water molecules. Moreover, the cellulose nanofiber intrinsically assembled into unique hierarchical structures allow for strong hydrogen bonds between adjacent cellulose and PPy polymer chains, greatly improve the mechanical strength (stress∼0.65 MPa, strain∼301 %) and excellent viscoelasticity (G'max âˆ¼ 82.7 KPa). This novel PPy@CNF-PVA hydrogel exhibits extremely high Gauge factor (GF) of 2.84 and shows excellent sensitivity, repeatability and stability. Therefore, the hydrogel can serve as reliable and stable strain sensor which shows excellent responsiveness in human activities monitoration.

Van Krevelen diagrams based on machine learning visualize feedstock-product relationships in thermal conversion processes.

Feedstock properties play a crucial role in thermal conversion processes, where understanding the influence of these properties on treatment performance is essential for optimizing both feedstock selection and the overall process. In this study, a series of van Krevelen diagrams were generated to illustrate the impact of H/C and O/C ratios of feedstock on the products obtained from six commonly used thermal conversion techniques: torrefaction, hydrothermal carbonization, hydrothermal liquefaction, hydrothermal gasification, pyrolysis, and gasification. Machine learning methods were employed, utilizing data, methods, and results from corresponding studies in this field. Furthermore, the reliability of the constructed van Krevelen diagrams was analyzed to assess their dependability. The van Krevelen diagrams developed in this work systematically provide visual representations of the relationships between feedstock and products in thermal conversion processes, thereby aiding in optimizing the selection of feedstock and the choice of thermal conversion technique.

Application of Minimally Invasive Surgery-Multidisciplinary Team in Advanced and Recurrent Gynecological Cancers: 10-Year Exploration and Practice.

Objectives: The treatment of advanced and recurrent gynecological cancers (ARGCs) remains more difficult evens. This assay aims to introduce the application of minimally invasive surgery-multidisciplinary team (MIS-MDT) as well as a comprehensive evaluation and treatment program of ARGC. Materials and Methods: The diagnosis and treatment model of MDT collaboration has become a new model of clinical cancer treatment. In my country, it is in the start-up and trial stage. Our team began to explore surgical treatment of recurrent gynecological cancers in 2011 and has been committed to MDT treatment of ARGC for more than 3 years. Results: So far, 61 patients have completed MDT treatment (28 of them were advanced gynecological cancer patients, 33 of them were recurrent gynecological cancer patients). Among them, MDT involved 43 times in gastrointestinal surgery, 21 times in urology, 5 times in the department of intractable abdominal diseases, and 5 times in other departments. After surgery, 58 patients (95%) restarted adjuvant therapy such as radiotherapy and chemotherapy. In addition, 32 patients (52.5%) underwent genetic and molecular testing, of which 14 patients (23%) accepted targeted and immunotherapy based on the testing results. After MIS-MDT treatment, the median progression-free survival of these patients was >30 months, respectively. Conclusion: These patients have achieved good results after surgery of MDT. With continuous accumulation and summarization, we have systematically reviewed the diagnosis and treatment model of ARGC and guided clinical work as the model of Tongji Tenth Hospital (comprehensive evaluation and treatment).

Automatic Kidney Stone Composition Analysis Method Based on Dual-energy CT.

BACKGROUND: The composition of kidney stones is related to the hardness of the stones. Knowing the composition of the stones before surgery can help plan the laser power and operation time of percutaneous nephroscopic surgery. Moreover, patients can be treated with medications if the kidney stone is compounded by uric acid before treatment, which can relieve the patients of the pain of surgery. However, although the literature generally reports the kidney stone composition analysis method base on dual-energy CT images, the accuracy of these methods is not enough; they need manual delineation of the kidney stone location, and these methods cannot analyze mixed composition kidney stones. OBJECTIVE: This study aimed to overcome the problem related to identifying kidney stone composition; we need an accurate method to analyze the composition of kidney stones. METHODS: In this paper, we proposed the automatic kidney stone composition analysis algorithm based on a dual-energy CT image. The algorithm first segmented the kidney stone mask by deep learning model, then analyzed the composition of each stone by machine learning model. RESULTS: The experimental results indicate that the proposed algorithm can segment kidney stones accurately (AUC=0.96) and predict kidney stone composition accurately (mean Acc=0.86, mean Se=0.75, mean Sp=0.9, mean F1=0.75, mean AUC=0.83, MR (Exact match ratio)=0.6). CONCLUSION: The proposed method can predict the composition and location of kidney stones, which can guide its treatment. Experimental results show that the weighting strategy can improve kidney stone segmentation performance. In addition, the multi-label classification model can predict kidney stone composition precisely, including the mixed composition kidney stones.

Metagenomic analysis of individual mosquitos reveals the ecology of insect viruses.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Using a meta-transcriptomic approach, we analysed the virome of 2,438 individual mosquitos (79 species), spanning ~4000 km along latitudes and longitudes in China. From these data we identified 393 core viral species associated with mosquitos, including seven (putative) arbovirus species. We identified potential species and geographic hotspots of viral richness and arbovirus occurrence, and demonstrated that host phylogeny had a strong impact on the composition of individual mosquito viromes. Our data revealed a large number of viruses shared among mosquito species or genera, expanding our knowledge of host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, possibly facilitated by long-distance mosquito migrations. Together, our results greatly expand the known mosquito virome, linked the viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the ecology of viruses of insect vectors.

Individual bat virome analysis reveals co-infection and spillover among bats and virus zoonotic potential.

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within individual bats, and hence the frequency of virus co-infection and spillover among them. We characterize the mammal-associated viruses in 149 individual bats sampled from Yunnan province, China, using an unbiased meta-transcriptomics approach. This reveals a high frequency of virus co-infection (simultaneous infection of bat individuals by multiple viral species) and spillover among the animals studied, which may in turn facilitate virus recombination and reassortment. Of note, we identify five viral species that are likely to be pathogenic to humans or livestock, based on phylogenetic relatedness to known pathogens or in vitro receptor binding assays. This includes a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV and SARS-CoV-2. In vitro assays indicate that this recombinant virus can utilize the human ACE2 receptor such that it is likely to be of increased emergence risk. Our study highlights the common occurrence of co-infection and spillover of bat viruses and their implications for virus emergence.

Prevalence and Characteristics of Novel Pathogenic Leptospira Species in Bats in Yunnan Province, China.

Leptospirosis has been identified as a zoonotic disease caused by pathogenic spirochetes of the bacterial genus Leptospira. Rodents are considered the primary hosts of these bacteria, whereas many recent studies suggest that bats may serve as potential natural reservoirs. However, studies on pathogenic spirochetes hosted by bat populations still need to be completed in China. In this study, a total of 276 bats belonging to five genera collected in Yunnan Province (Southwest China) from 2017 to 2021 were included in the screening. Pathogenic spirochetes were detected by PCR amplification and sequencing targeting four genes (rrs, secY, flaB, and LipL32), resulting in 17 positive samples. Phylogenetic analysis based on multi-loci concatenated sequences, inferred by MLST approach, identified the strains as two novel Leptospira species within the pathogenic group. Of note, only Rousettus leschenaultii was found to harbor these spirochetes, suggesting it may be one of the potential natural reservoirs in circulating leptospires in this region. Nevertheless, the pathogenesis and transmission dynamics still need to be fully understood, requiring in-depth studies on other animals and the surrounding population.

Nelson Bay Reovirus Isolated from Bats and Blood-Sucking Arthropods Collected in Yunnan Province, China.

Nelson Bay reovirus (NBV) is an emerging zoonotic virus that can cause acute respiratory disease in humans. These viruses are mainly discovered in Oceania, Africa, and Asia, and bats have been identified as their main animal reservoir. However, despite recent expansion of diversity for NBVs, the transmission dynamics and evolutionary history of NBVs are still unclear. This study successfully isolated two NBV strains (MLBC1302 and MLBC1313) from blood-sucking bat fly specimens (Eucampsipoda sundaica) and one (WDBP1716) from the spleen specimen of a fruit bat (Rousettus leschenaultii), which were collected at the China-Myanmar border area of Yunnan Province. Syncytia cytopathic effects (CPE) were observed in BHK-21 and Vero E6 cells infected with the three strains at 48 h postinfection. Electron micrographs of ultrathin sections showed numerous spherical virions with a diameter of approximately 70 nm in the cytoplasm of infected cells. The complete genome nucleotide sequence of the viruses was determined by metatranscriptomic sequencing of infected cells. Phylogenetic analysis demonstrated that the novel strains were closely related to Cangyuan orthoreovirus, Melaka orthoreovirus, and human-infecting Pteropine orthoreovirus HK23629/07. Simplot analysis revealed the strains originated from complex genomic reassortment among different NBVs, suggesting the viruses experienced a high reassortment rate. In addition, strains successfully isolated from bat flies also implied that blood-sucking arthropods might serve as potential transmission vectors. IMPORTANCE Bats are the reservoir of many viral pathogens with strong pathogenicity, including NBVs. Nevertheless, it is unclear whether arthropod vectors are involved in transmitting NBVs. In this study, we successfully isolated two NBV strains from bat flies collected from the body surface of bats, which implies that they may be vectors for virus transmission between bats. While the potential threat to humans remains to be determined, evolutionary analyses involving different segments revealed that the novel strains had complex reassortment histories, with S1, S2, and M1 segments highly similar to human pathogens. Further experiments are required to determine whether more NBVs are vectored by bat flies, their potential threat to humans, and transmission dynamics.

Sclerotherapy for congenital vascular malformations with mixing foam of polidocanol and liquid of bleomycin.

OBJECTIVE: To evaluate the efficacy of sclerotherapy for congenital vascular malformation (CVM) using a combination of polidocanol foam and bleomycin liquid. METHODS: A retrospective review of a prospectively collected data on patients who had sclerotherapy for CVM from May 2015 to July 2022 was performed. RESULTS: A total of 210 patients with a mean age of 24.8 ± 2.0 years were included. Venous malformation (VM) was the most common type of CVM, accounting for 81.9% (172/210) of all patients. At 6 months follow-up, the overall clinical effective rate was 93.3% (196/210), and 50% (105/210) of patients were clinically cured. The clinical effective rates in VM, lymphatic, and arteriovenous malformation group were 94.2%, 100%, and 100%. CONCLUSION: Sclerotherapy using a combination of polidocanol foam and bleomycin liquid is an effective and safe treatment for venous and lymphatic malformations. It is a promising treatment option with satisfactory clinical outcome in arteriovenous malformations.

Maximizing the Recycling of Iron Ore Pellets Fines Using Innovative Organic Binders.

This research work focuses on the practicality of using organic binders for the briquetting of pellet fines. The developed briquettes were evaluated in terms of mechanical strength and reduction behavior with hydrogen. A hydraulic compression testing machine and thermogravimetric analysis were incorporated into this work to investigate the mechanical strength and reduction behavior of the produced briquettes. Six organic binders, namely Kempel, lignin, starch, lignosulfonate, Alcotac CB6, and Alcotac FE14, in addition to sodium silicate, were tested for the briquetting of pellet fines. The highest mechanical strength was achieved using sodium silicate, Kempel, CB6, and lignosulfonate. The best combination of binder to attain the required mechanical strength even after 100% reduction was found to be a combination of 1.5 wt.% of organic binder (either CB6 or Kempel) with 0.5 wt.% of inorganic binder (sodium silicate). Upscaling using an extruder gave propitious results in the reduction behavior, as the produced briquettes were highly porous and attained pre-requisite mechanical strength.

[Mammalian DMRTs: Structure, function and relationship with cancer].

DMRT, a gene family related to sexual determination, encodes a large group of transcription factors (DMRTs) with the double-sex and mab-3 (DM) domain (except for DMRT8), which is able to bind to and regulate DNAs. Current studies have shown that the DMRT gene family plays a critical role in the development of sexual organs (such as gender differentiation, gonadal development, germ cell development, etc.) as well as extrasexual organs (such as musculocartilage development, nervous system development, etc.). Additionally, it has been suggested that DMRTs may be involved in the cancer development and progression (such as prostate cancer, breast cancer, lung cancer, etc.). This review summarizes the research progress about the mammalian DMRTs' structure, function and its critical role in cancer development, progression and therapy (mainly in human and mice), which suggests that DMRT gene could be a candidate gene in the study of tumor formation and therapeutic strategy.

Repressing IRS1/2 by NT157 inhibits the malignant behaviors of ovarian cancer through inactivating PI3K/AKT/mTOR pathway and inducing autophagy.

Insulin receptor substrate 1 and 2 (IRS1/2) have been found involved in many cancers development and their inhibitors exert significant tumor-suppressive effects. Here, we tried to explore the function of NT157, an IGF1R-IRS1/2 inhibitor, in ovarian cancer. We treated ovarian cancer cells with varying doses of NT157. The MTT assay was employed to evaluate cell proliferation and colony formation assay was used for detecting colony-forming ability. TUNEL assay was adopted to test cell apoptosis. Cell invasion was checked by the Transwell assay. The expression of apoptosis-related proteins, autophagy markers, IRS1/2, and PI3K/AKT/mTOR pathway was compared by Western blot, immunofluorescence, or qRT-PCR. As indicated by the data, NT157 abated the viability, proliferation, and induced autophagy of ovarian cancer cells. Overexpressing IRS1/2 attenuated the tumor-suppressive effect of NT157 and heightened the PI3K/AKT/mTOR pathway activation. Inhibition of the PI3K/AKT/mTOR pathway enhanced the tumor-suppressive effect of NT157 and facilitated NT157-mediated autophagy. However, the autophagy inhibitor 3-MA partly reversed NT-157-mediated antitumor effects. In conclusion, this study disclosed that NT157 suppressed the malignant phenotypes of ovarian cancer cells by inducing autophagy and hampering the expression of IRS1/2 and PI3K/AKT/mTOR pathway.

Recognizing pathology of renal tumor from macroscopic cross-section image by deep learning.

OBJECTIVES: This study aims to develop and evaluate the deep learning-based classification model for recognizing the pathology of renal tumor from macroscopic cross-section image. METHODS: A total of 467 pathology-confirmed patients who received radical nephrectomy or partial nephrectomy were retrospectively enrolled. The experiment of distinguishing malignant and benign renal tumor are conducted followed by performing the multi-subtypes classification models for recognizing four subtypes of benign tumor and four subtypes of malignant tumors, respectively. The classification models used the same backbone networks which are based on the convolutional neural network (CNN), including EfficientNet-B4, ResNet-18, and VGG-16. The performance of the classification models was evaluated by area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and accuracy. Besides, we performed the quantitative comparison among these CNN models. RESULTS: For the model to differentiate the malignant tumor from the benign tumor, three CNN models all obtained relatively satisfactory performance and the highest AUC was achieved by the ResNet-18 model (AUC = 0.9226). There is not statistically significance between EfficientNet-B4 and ResNet-18 architectures and both of them are significantly statistically better than the VGG-16 model. The micro-averaged AUC, macro-averaged sensitivity, macro-averaged specificity, and micro-averaged accuracy for the VGG-16 model to distinguish the malignant tumor subtypes achieved 0.9398, 0.5774, 0.8660, and 0.7917, respectively. The performance of the EfficientNet-B4 is not better than that of VGG-16 in terms of micro-averaged AUC except for other metrics. For the models to recognize the benign tumor subtypes, the EfficientNet-B4 ranked the best performance, but had no significantly statistical difference with other two models with respect to micro-averaged AUC. CONCLUSIONS: The classification results were relatively satisfactory, which showed the potential for clinical application when analyzing the renal tumor macroscopic cross-section images. Automatically distinguishing the malignant tumor from benign tumor and identifying the subtypes pathology of renal tumor could make the patient-management process more efficient.

CYP3A4 and CYP3A5 Expression is Regulated by CYP3A4*1G in CRISPR/Cas9-Edited HepG2 Cells.

Functional CYP3A4*1G (G>A, rs2242480) in cytochrome P450 3A4 (CYP3A4) regulates the drug-metabolizing enzyme CYP3A4 expression. The objective of this study was to investigate whether CYP3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5 in gene-edited human HepG2 cells. CYP3A4*1G GG and AA genotype HepG2 cells were established using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) single nucleotide polymorphism technology and homology-directed repair in the CYP3A4*1G GA HepG2 cell line. In CYP3A4*1G GG, GA, and AA HepG2 cells, CYP3A4*1G regulated expression of CYP3A4 and CYP3A5 mRNA and protein in an allele-dependent manner. Of note, significantly decreased expression level of CYP3A4 and CYP3A5 was observed in CYP3A4*1G AA HepG2 cells. Moreover, the results after RIF treatment showed that CYP3A4*1G decreased the induction level of CYP3A4 and CYP3A5 mRNA expression in CYP3A4*1G AA HepG2 cells. At the same time, CYP3A4*1G decreased CYP3A4 enzyme activity and tacrolimus metabolism, especially in CYP3A4*1G GA HepG2 cells. In summary, we successfully constructed CYP3A4*1G GG and AA homozygous HepG2 cell models and found that CYP3A4*1G regulates both basal and RIF-induced expression and enzyme activity of CYP3A4 and CYP3A5 in CRISPR/Cas9 CYP3A4*1G HepG2 cells. SIGNIFICANCE STATEMENT: Cytochrome P450 (CYP) 3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5. This study successfully established CYP3A4*1G (G>A, rs2242480), GG, and AA HepG2 cell models using CRISPR/Cas9, thus providing a powerful tool for studying the mechanism by which CYP3A4*1G regulates the basal and RIF-induced expression of CYP3A4 and CYP3A5.