Advances of microbial xylanases in the application of flour industries: A comprehensive review.

Microbial xylanase has a wide range of applications, and many researchers favoring its utilization as an alternative to improve flour products. Wheat flour is the main raw material of flour products, although the content of arabinoxylan is not high in flour products, but it has a great influence on the quality of flour products, microbial xylanase can act on wheat arabinoxylan, so as to play the role of flour product improvement. This review carries out a description of the research progress on the application of xylanases in flour products in terms of xylanase properties, different families of xylanases and improvement mechanisms of xylanases in flour products. According to the properties of various microbial sources of xylanases, the suitable xylanase can be added to flour products, and the effect of xylanase toward wheat arabinoxylan in flour can be used to improve the quality of flour products. The molecular modification based on the properties of xylanase and the crystal structure of different families of xylanase and their substrate specificity toward wheat arabinoxylan are discussed. The article reviews the information about microbial xylanases in order to achieve better results in flour products and to provide a theoretical basis for their industrial application.

Polyvinyl Alcohol (PVA)-Based Hydrogels: Recent Progress in Fabrication, Properties, and Multifunctional Applications.

Polyvinyl alcohol (PVA)-based hydrogels have attracted significant attention due to their excellent biocompatibility, tunable mechanical properties, and ability to form stable three-dimensional networks. This comprehensive review explores the recent advancements in PVA-based hydrogels, focusing on their unique properties, fabrication strategies, and multifunctional applications. Firstly, it discusses various facile synthesis techniques, including freeze/thaw cycles, chemical cross-linking, and enhancement strategies, which have led to enhanced mechanical strength, elasticity, and responsiveness to external stimuli. These improvements have expanded the applicability of PVA-based hydrogels in critical areas such as biomedical, environmental treatment, flexible electronics, civil engineering, as well as other emerging applications. Additionally, the integration of smart functionalities, such as self-healing capabilities and multi-responsiveness, is also examined. Despite progress, challenges remain, including optimizing mechanical stability under varying conditions and addressing potential toxicity of chemical cross-linkers. The review concludes by outlining future perspectives, emphasizing the potential of PVA-based hydrogels in emerging fields like regenerative medicine, environmental sustainability, and advanced manufacturing. It underscores the importance of interdisciplinary collaboration in realizing the full potential of these versatile materials to address pressing societal challenges.

Hydrologic changes induced by groundwater abstraction lead to arsenic mobilization in shallow aquifers.

Intensive groundwater abstraction leads to hydrologic changes of groundwater. Nevertheless, the effects of hydrologic change on groundwater arsenic (As) mobilization remain controversial. Here, we investigated fluctuations in water levels and their effects on As mobilization in the shallow aquifer of the Hetao Basin. Results showed that large groundwater level fluctuations and high horizontal hydraulic gradients occurred in irrigation seasons. In the groundwater near the wetland with higher surface water levels than groundwater levels, biological index values of dissolved organic matter (DOM) ranged from 0.54 to 0.72, and a positive correlation between δ18O values and dissolved organic carbon (DOC) was observed, indicating that groundwater DOM was mainly sourced from surface water. The degradation of allochthone labile DOM drove the reductive dissolution of As-bearing Fe(III) oxides to Fe(II). Both DOC and humification indices of DOM exhibited positive correlations with horizontal hydraulic gradients downstream of the study area, implying that the humified organic matter flushed from aquifer sediments contributed to groundwater DOM. The humified DOM controlled by hydraulic conditions participated in the redox reactions mainly by shuttling electrons to As-bearing Fe(III) oxides. These findings highlight distinct roles of hydrologic changes induced by groundwater abstraction in As mobilization.

Reducing CRISPR-Cas9 off-target effects by optically controlled chemical modifications of guide RNA.

A photocatalytic click chemistry approach, offering a significant advancement over conventional methods in RNA function modulation is described. This innovative method, utilizing light-activated small molecules, provides a high level of precision and control in RNA regulation, particularly effective in intricate cellular processes. By applying this strategy to CRISPR-Cas9 gene editing, we demonstrate its effectiveness in enhancing gene editing specificity and markedly reducing off-target effects. Our approach employs a vinyl ether modification in RNA, which activated under visible light with a phenanthrenequinone derivative, creating a CRISPR-OFF switch that precisely regulates CRISPR system activity. This method not only represents an advancement in genomic interventions but also offers broad applications in gene regulation, paving the way for safer and more reliable gene editing in therapeutic genomics.

Highly efficient synthetic bacterial consortium for biodegradation of aromatic volatile organic compounds: Behavior and mechanism.

Aromatic volatile organic compounds (VOCs) are prevalent pollutants in chemically contaminated sites, posing threats to ecological safety and human health. To address the challenge of achieving low-carbon, low-cost, green, and sustainable in-situ remediation at these sites, a highly efficient synthetic bacterial consortium was constructed for biodegradation of selected pollutants (i.e., benzene, toluene, ethyl benzene, m-xylene, chlorobenzene, p-chlorotoluene, and p-chlorotrifluorotoluene). Under optimized conditions, the consortium achieved a total degradation efficiency of 77%. Biodegradation of benzene, toluene, ethyl benzene, and m-xylene followed first-order kinetics, while p-chlorotoluene and p-chlorotrifluorotoluene followed zero-order kinetics. The mechanisms were analyzed using microbiome technology at genetic, protein, and metabolic levels, identifying key enzymes and differences in protein expression and related metabolites. Carbon dioxide measurements and fluorescence spectrum analysis elucidated the transformation pathways. These findings underscore the consortium's significant potential for achieving effective, eco-friendly, and sustainable bioremediation of aromatic VOCs in chemically contaminated environments.

Plant-derived biomass-based hydrogels for biomedical applications.

Hydrogels made of plant-derived biomass have gained popularity in biomedical applications because they are frequently affordable, readily available, and biocompatible. Finding the perfect plant-derived biomass-based hydrogels for biomedicine that can replicate essential characteristics of human tissues in regard to structure, function, and performance has proved to be difficult. In this review, we summarize some of the major contributions made to this topic, covering basic ideas and different biomass-based hydrogels made of cellulose, hemicellulose, and lignin. Also included is an in-depth discussion regarding the biosafety and toxicity assessments of biomass-based hydrogels. Finally, this review also highlights important scientific debates and major obstacles regarding biomass-based hydrogels for biomedical applications.

A sensitive electrochemical biosensor based on Pd@PdPtCo mesoporous nanopolyhedras as signal amplifiers for assay of cardiac troponin I.

Cardiac troponin I (cTnI) has been widely used in clinical diagnosis of acute myocardial infarction (AMI). Herein, a sensitive electrochemical biosensor for cTnI analysis was designed, in which the simple synthesized Pd@PdPtCo mesoporous nanopolyhedras (MNPs) were utilized as signal amplifiers. The mesoporous polyhedral structure of Pd@PdPtCo MNPs endows them with more specific surface area and more active sites, as well as the synergistic effect between multiple metal elements, all of which increase the electrocatalytic performance of Pd@PdPtCo MNPs in efficiently oxidizing hydroquinone (HQ) to benzoquinone (BQ). Experimental results showed that Pd@PdPtCo MNPs had better performance in oxidation of HQ to BQ compared with their corresponding monometallic and bimetallic nanomaterials. With the aid of the interaction between antigens and antibodies, the peak current of HQ to BQ showed an upward trend with increasing concentration of cTnI, thus the quantitative detection of cTnI could be achieved. Under optimal conditions, the biosensor prepared in this work has a wider linear range (1.0 × 10-4-200 ng mL-1) and a lower detection limit (0.031 pg mL-1) than other sensors reported in literatures, coupled by good stability and high sensitivity. More importantly, it also performed well in complex serum environment, proving that the electrochemical sensor has a practical application potential in this field.

Oil-Resistant Underoil Superhydrophilic Metallic Foams for Lampblack Prefiltration.

Superwetting/repelling coatings have been utilized to address the issue of oil contamination on lampblack prefiltration metallic foam by both academia and industry. Nevertheless, the widely adopted superamphiphobic coatings are currently costly and suffer from poor wear resistance. In this study, we propose an oil-resistant underoil superhydrophilic (LSH) coating by a dip-coating method. The subsequent heating process at 200 °C for 5 min strengthens the designed coating based on lithium polysilicate cross-linking reinforcement. The LSH coating with a minimal water contact angle up to 3.4° under soybean oil can spontaneously achieve oil desorption within 7 s under water. Moreover, the coating retains its superhydrophilicity after enduring 900 friction cycles under a 500 g load or being immersed in 50 °C soapy water for 48 h. Hence, the LSH coating with great durability on metallic foam for lampblack prefiltration resulted in a 9.3% decrease in the oil absorption weight ratio after a 17-day cooking test. This work underscores the potential application of the LSH coating in lampblack prefiltration components, presenting promising technological advancements in self-cleaning for the catering industry.

Precise Regulation of Interlayer Stacking Modes in Trinuclear Copper Organic Frameworks for Efficient Photocatalytic Reduction of Uranium(VI).

The interlayer stacking modes of 2D covalent-organic frameworks (COFs) directly influence their structural features, ultimately determining their functional output. However, controllably modulating the interlayer stacking structure in traditional 2D metal-free COFs, based on the same building blocks, remains challenging. Here, two trinuclear copper organic frameworks are synthesized successfully with different interlayer stacking structures: eclipsed AA stacking in Cu3-PA-COF-AA and staggered ABC stacking in Cu3-PA-COF-ABC, using the same monomers. Remarkably, various functionalities, including porosity and electronic and optical properties, can be effectively regulated by interlayer stacking. As a result, Cu3-PA-COF-AA and Cu3-PA-COF-ABC exhibit significantly different activities toward the photoreduction of U(VI), presenting a promising strategy for removing radioactive uranium pollution. Due to its broader visible-light absorption range and superior photogenerated carrier migration and separation efficiency, Cu3-PA-COF-AA achieves a U(VI) removal ratio of 93.6% without additional sacrificial agents in an air atmosphere-≈2.2 times higher than that of Cu3-PA-COF-ABC (42.0%). To the best of the knowledge, this is the first study to elucidate the effect of interlayer stacking in COFs on the photocatalytic activity of U(VI) reduction. This finding may inspire further exploration of the structure-function relationship in COFs as photocatalysts and their potential for photoinduced removal of radionuclides.

Analysis of the Relationship Between Primary Tumor Site and Clinicopathological Characteristics and Survival Prognosis of Breast Cancer Patients Based on SEER Database.

PURPOSE: This study aimed to analyze the association between the primary tumor site and clinicopathological characteristics and survival prognosis of breast cancer (BC) patients using a large population database. METHODS: BC patients screened in the Surveillance, Epidemiology, and End Results (SEER) database were categorized into 6 groups based on primary sites. Descriptive statistics, Kaplan-Meier curves, Cox regression models, forest plots were used to assess the effect of primary sites on overall survival (OS) and breast cancer-specific survival (BCSS). Multivariate Cox proportional analyses were conducted to calculate hazard ratios (HRs) and adjusted subgroups' hazard ratios (AHRs). Nomograms were utilized to predict OS and BCSS. RESULTS: Among 193,043 BC patients, the highest incidence was found in the upper outer quadrant (52.60%). Central portion patients are associated with more clinical features indicating a poor prognosis, and had worse OS and BCSS than other sites. Univariate and multifactorial Cox analyses showed associations between OS/BCSS and various factors. Subgroup analyses revealed differences in OS and BCSS between central portion and upper outer quadrant varied among age, T and N stage. The nomogram was established to predict the survival of central portion BC patients. CONCLUSIONS: Primary tumor site is associated with clinicopathological features and prognosis of BC, may be influenced by age at diagnosis and T and N stage. Central portion BC patients have worse prognosis due to older age at diagnosis, higher T stage and higher likelihood of lymph node metastasis. Early diagnosis and treatment may help to improve survival of central portion BC.

Unveiling a Potent Small Molecule Disruptor for RNA G-Quadruplexes Tougher Than DNA G-Quadruplex Disruption.

This research presents a unique small molecule characterized by its ability to effectively disrupt RNA G-quadruplexes (G4s), which are notably more stable than their DNA counterparts. We conducted a comprehensive series of in vitro experiments to thoroughly assess the disruptive capabilities of this molecule on RNA G4s. These experiments included comparisons with established G4 stabilizers and DNA G4 disruptors, providing a multifaceted evaluation of the molecule's efficacy. Our extensive in vitro analyses demonstrated that this molecule effectively alters G4 structures and interactions with the BG4 protein, a well-recognized G4-specific antibody. These findings underscore the molecule's potential to modulate G4-protein interactions, indicating promising applications for manipulating cellular functions associated with G4 dynamics in future research.

The role of cholesterol metabolism in lung cancer.

Elevated serum cholesterol metabolism is associated with a reduced risk of lung cancer. Disrupted cholesterol metabolism is evident in both lung cancer patients and tumor cells. Inhibiting tumor cell cholesterol uptake or biosynthesis pathways, through the modulation of receptors and enzymes such as liver X receptor and sterol-regulatory element binding protein 2, effectively restrains lung tumor growth. Similarly, promoting cholesterol excretion yields comparable effects. Cholesterol metabolites, including oxysterols and isoprenoids, play a crucial role in regulating cholesterol metabolism within tumor cells, consequently impacting cancer progression. In lung cancer patients, both the cholesterol levels in the tumor microenvironment and within tumor cells significantly influence cell growth, proliferation, and metastasis. The effects of cholesterol metabolism are further mediated by the reprogramming of immune cells such as T cells, B cells, macrophages, myeloid-derived suppressor cells, among others. Ongoing research is investigating drugs targeting cholesterol metabolism for clinical treatments. Statins, targeting the cholesterol biosynthesis pathway, are widely employed in lung cancer treatment, either as standalone agents or in combination with other drugs. Additionally, drugs focusing on cholesterol transportation have shown promise as effective therapies for lung cancer. In this review, we summarized current research regarding the rule of cholesterol metabolism and therapeutic advances in lung cancer.

Organic Photoredox-Catalyzed Site-Selective Alkylation of Glycine Derivatives and Peptides via Infrequent 1,2-Hydrogen Atom Transfer of Amidyl Radicals.

Herein, we disclose a visible-light-driven photoredox-catalyzed protocol for site-selective alkylation of glycine derivatives via 1,2-hydrogen atom transfer, which is distinguished by metal free and mild conditions, high chemoselectivity, and good functional group compatibility. This protocol provides a unique approach for synthesizing valuable α,ß-diamino acid derivatives. Furthermore, the potential synthetic merit of this transformation is proven by a scale-up reaction and late-stage functionalization of peptides.

GDSL in Lilium pumilum (LpGDSL) Confers Saline-Alkali Resistance to the Plant by Enhancing the Lignin Content and Balancing the ROS.

In order to explore the response mechanism of Lilium pumilum (L. pumilum) to saline-alkali stress, we successfully cloned LpGDSL (GDSL lipase, Gly-Asp-Ser-Leu) from L. pumilum. The qRT-PCR results indicated that the LpGDSL expression was higher in the leaves of L. pumilum, and the expression of the LpGDSL reached the highest level at 12 h in leaves under 11 mM H2O2, 200 mM NaCl, 25 mM Na2CO3, and 20 mM NaHCO3. The bacteriophage overexpressing LpGDSL was more tolerant than the control under different NaHCO3 contents. Overexpressed and wild-type plants were analyzed for phenotype, chlorophyll content, O2- content, H2O2 content, lignin content, and so on. Overexpressed plants had significantly higher resistance than the wild type and were less susceptible to saline-alkali stress. The yeast two-hybrid and BiFC assays demonstrated the existence of an interaction between LpGDSL and LpBCP. The yeast one-hybrid assay and transcriptional activation assay confirmed that B3 transcription factors could act on LpGDSL promoters. Under saline-alkali stress, L. pumilum will promote the expression of LpGDSL, which will then promotes the accumulation of lignin and the scavenging of reactive oxygen species (ROS) to reduce its damage, thus improving the saline-alkali resistance of the plant.

Characterization of lncRNA and mRNA profiles in the process of repairing peripheral nerve defects with cell-matrixed nerve grafts.

BACKGROUND: Decellularized extracellular matrix (dECM) is an intriguing natural biomaterial that has garnered significant attention due to its remarkable biological properties. In our study, we employed a cell-matrixed nerve graft for the repair of sciatic nerve defects in rats. The efficacy of this approach was assessed, and concurrently, the underlying molecular regulatory mechanisms were explored to elucidate how such grafts facilitate nerve regeneration. Long noncoding RNAs (lncRNAs) regulate mRNA expression via multiple mechanisms, including post-transcriptional regulation, transcription factor effects, and competitive binding with miRNAs. These interactions between lncRNAs and mRNAs facilitate precise control of gene expression, allowing organisms to adapt to varying biological environments and physiological states. By investigating the expression profiles and interaction dynamics of mRNAs and lncRNAs, we can enhance our understanding of the molecular mechanisms through which cell-matrixed nerve grafts influence neural repair. Such studies are pivotal in uncovering the intricate networks of gene regulation that underpin this process. RESULTS: Weighted gene co-expression network analysis (WGCNA) utilizes clustering algorithms, such as hierarchical clustering, to aggregate genes with similar expression profiles into modules. These modules, which potentially correspond to distinct biological functions or processes, can subsequently be analyzed for their association with external sample traits. By correlating gene modules with specific conditions, such as disease states or responses to treatments, WGCNA enables a deeper understanding of the genetic architecture underlying various phenotypic traits and their functional implications. We identified seven mRNA modules and five lncRNA modules that exhibited associations with treatment or time-related events by WGCNA. We found the blue (mRNAs) module which displayed a remarkable enrichment in "axonal guidance" and "metabolic pathways", exhibited strong co-expression with multiple lncRNA modules, including blue (related to "GnRH secretion" and "pyrimidine metabolism"), green (related to "arginine and proline metabolism"), black (related to "nitrogen metabolism"), grey60 (related to "PPAR signaling pathway"), and greenyellow (related to "steroid hormone biosynthesis"). All of the top 50 mRNAs and lncRNAs exhibiting the strongest correlation were derived from the blue module. Validation of key molecules were performed using immunohistochemistry and qRT-PCR. CONCLUSION: Revealing the principles and molecular regulatory mechanisms of the interaction between materials and biological entities, such as cells and tissues, is a direction for the development of biomimetic tissue engineering technologies and clinically effective products.

Sintilimab combined with bevacizumab in relapsed or persistent ovarian clear cell carcinoma (INOVA): a multicentre, single-arm, phase 2 trial.

BACKGROUND: Ovarian clear cell carcinoma rarely responds to second-line chemotherapy, the recommended treatment for relapsed epithelial ovarian cancer. Here, we report the activity and safety of sintilimab in combination with bevacizumab in patients with relapsed or persistent ovarian clear cell carcinoma. METHODS: In the prospective, multicentre, single-arm, phase 2 INOVA trial, patients aged 18-75 years with histologically confirmed relapsed or persistent ovarian clear cell carcinoma were enrolled from eight tertiary hospitals in China. Eligible patients had an Eastern Cooperative Oncology Group performance status score of 0-2 and previous exposure to at least one cycle of platinum-containing chemotherapy. Enrolled patients received sintilimab (200 mg) and bevacizumab (15 mg/kg) intravenously every 3 weeks until disease progression. The primary endpoint was objective response rate assessed by independent central review based on Response Evaluation Criteria in Solid Tumours version 1.1. Eligible enrolled patients who received at least one cycle of treatment and had at least one tumour response assessment following the baseline assessment per protocol were included in the activity analysis. Patients who received at least one dose of study drug were included in the safety analysis. The study is registered with ClinicalTrials.gov (NCT04735861) and is ongoing. FINDINGS: Between April 8, 2021, and July 3, 2023, 51 patients were screened and 41 patients received at least one dose of sintilimab in combination with bevacizumab. Response evaluation was completed in 37 patients. Objective responses were observed in 15 patients (objective response rate 40·5%; 95% CI 24·8-57·9), of which five (14%) were complete responses and ten (27%) were partial responses. At data cutoff (Jan 29, 2024), the median follow-up was 16·9 months (IQR 7·5-23·4). Three (7%) patients developed grade 3 treatment-related adverse events including one patient with proteinuria, one patient with myocarditis, and one patient with rash. No treatment-related adverse events of worse than grade 3 severity were recorded. Treatment-related serious adverse events occurred in two (5%) patients including one patient with immune-related myocarditis and another with hypertension and renal dysfunction. No treatment-related deaths occurred. INTERPRETATION: Sintilimab in combination with bevacizumab showed promising anti-tumour activity and manageable safety in patients with relapsed or persistent ovarian clear cell carcinoma. Larger, randomised trials are warranted to compare this low-toxicity, chemotherapy-free combinatorial regimen with standard chemotherapy. FUNDING: National Key Technology Research and Development Program of China, National Natural Science Foundation of China, Beijing Xisike Clinical Oncology Research Foundation, and Innovent Biologics. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Enhancing colorimetric efficiency: nanozyme-activated peroxymonosulfate for in situ 3-aminophenol detection.

A novel colorimetric approach specifically designed to effectively identify the presence of 3-aminophenol (3-AP) in environmental water is introduced. Briefly, a nitrogen-doped carbon-supported cobalt nanozyme (Co@CN-1) was synthesized and utilized to improve the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of peroxymonosulfate (PMS). Comparative catalytic reactions confirmed that the performance of PMS as an activator exceeds that of hydrogen peroxide catalytically by a factor of 3.5. The catalytic reaction parameters underwent optimization, further resulting in the derivation of a linear detection equation for 3-AP, expressed as inhibition rate (IR%) = 3.35[3-AP]-4.36 (0-20 µM, R2 = 0.994) and IR% = 1.43[3-AP] + 31.87 (20-36 µM, R2 = 0.992), with the limit of detection (LOD) of 2.84 µM. The linear relationship between 3-AP concentration and the conversion of color to grayscale value (GSV) was established by smartphones, expressed as GSV = 1.28[3-AP] + 147.10 (R2 = 0.972). Density functional theory calculations revealed that Co acts as the preferred active site for donating electrons in PMS activation. This work provides a rapid and accurate approach for monitoring 3-AP concentration, enabling real-time analysis and potentially contributing to environmental and ecological studies.

Hematite tailings to high-purity silica: Mechanistic studies and life cycle assessment analysis.

This study aimed to recover high-purity silica from hematite tailings (HTs) using superconducting high-gradient magnetic separation (S-HGMS) technology. This process involved converting silica into a silicone-rich concentrate and subsequently employing a fluorine-free mixed acid to leach the silicon-rich concentrate to remove impurities and achieve refinement and purification. The optimization of the S-HGMS process was conducted using the "Box-Behnken Design" method, resulting in the following optimal conditions: a pulp concentration of 50 g/L, a magnetic velocity ratio of 0.076 T s/m, and a pulp velocity of 500 mL/min. These conditions yielded a silica grade range of 61.905% in the HTs to 91.818% in the silicon-rich concentrate, with corresponding recovery rates of 53.031%. Under the optimized leaching process, this resulted in an increase in the silica content from 91.818% in the silicon-rich concentrate to 99.938% in high-purity silica. Additionally, by analyzing the production process of 1 kg of high-purity silica from HTs using the process LCA method, environmental hotspots were identified, and corresponding solutions were proposed. This approach is vital for efficient utilization of HTs as a resource. This process has low energy consumption and is environmentally friendly, enabling the reduction of hematite tailings. It has a wide range of applications and offers substantial economic benefits, rendering it a promising candidate for industrial applications.

Changes in insulin utilization in China from 2020 to 2022.

AIM: To assess the variation in patterns of use of insulin and other antidiabetic medicines across China, both geographically and over time. MATERIALS AND METHODS: Nationally, we calculated the relative change in antidiabetic medicine purchases between the first and last quarters of 2020 through 2022 based on the number of defined daily doses procured per quarter. We used annual data to analyse differences in antidiabetic medicine use and patterns across seven regions of China. Considering large regional variations, we used multifactor linear regression to preliminarily explore the possible factors influencing this variation. RESULTS: Nationally, the procurement of antidiabetic medicines and insulin increased from 2020 to 2022, while the proportion of insulin among antidiabetic medicines remained stable at approximately 22%. Among all insulins, premixed insulin (human) was ranked first. Of the three subgroups of insulin, analogues were the most preferred and had the largest procurement, but different categories showed different trends in terms of purchases and proporation. Regionally, the growth rate of antidiabetic medicines, the proportion of insulin procurement and the preferred types of insulin across the seven regions were different. Regarding preliminary influencing factors, the level of education and owning a domestically funded producer had a positive effect on insulin procurement. CONCLUSIONS: From 2020 to 2022, the procurement of insulin increased, which may be due to the increased attention for diabetes from the country and residents.However, the proportion of insulin among all antidiabetic medicines was essentially unchanged, while the use of some non-insulin hypoglycemic drugs increased significantly, especially the SGLT2i and GLP-1 RA. Given the economic and cultural diversity, Insulin procurement and utilization patterns varied greatly across the regions. Owning domestic enterprises potentially influences the procurement of insulin. Enhancing education to further improve the self-management of patients with diabetes is essential.

[Construction of HEK293T cell line stably expressing TRPM2 channel based on PiggyBac transposition system and its application in drug screening for cerebral ischemia and other diseases]. / 基于PiggyBac转座子优化稳定表达细胞系的新型瞬时受体电位M2通道抑制剂筛选.

OBJECTIVES: To establish a cell line stably expressing the transient receptor potential melastatin 2 (TRPM2) channel for screening TRPM2 inhibitors based on PiggyBac transposition system. METHODS: A plasmid PiggyBac-human TRPM2 (pPB-hTRPM2) eukaryotic expression vector was constructed using PiggyBac transposition system. The plasmid and a helper plasmid were co-transfected into HEK293T cells to express TRPM2, which was identified by fluorescence and patch-clamp assays. The high throughput screening performance was assessed with the Z ´ factor. Calcium imaging and patch clamp techniques were employed to assess the initial activity of eleven compound molecules, confirming the inhibitory effects of the primary molecules on TRPM2. The protective effect of the screened compounds on damaged cells was validated using the oxygen-glucose deprivation/reperfusion (OGD/R) injury model and CCK-8 kit. The level of cellular reactive oxygen species (ROS) was detected by flow cytometry. The neuroprotective effects of the compounds were evaluated using a transient middle cerebral artery occlusion (tMCAO) mouse model. RESULTS: The HEK293T cells transfected with pPB-hTRPM2-EGFP showed high TRPM2 expression. Puromycin-resistant cells, selected through screening, exhibited robust fluorescence. Whole-cell patch results revealed that induced cells displayed classical TRPM2 current characteristics comparable to the control group, showing no significant differences (P>0.05). With a Z ´ factor of 0.5416 in calcium imaging, the model demonstrated suitability for high-throughput screening of TRPM2 inhibitors. Calcium imaging and electrophysiological experiments indicated that compound 6 significantly inhibited the TRPM2 channel. Further experiments showed that 1.0 µmol/L of compound 6 enhanced cell viability (P<0.05) and reduced the level of ROS (P<0.05) of SH-SY5Y under OGD/R injury. 0.3 and 1.0 mg/kg of compound 6 reduced the cerebral infarction volume in tMCAO mice (both P<0.05). CONCLUSIONS: A stable TRPM2 gene expressing cell line has been successfully established using PiggyBac gene editing in this study. TRPM2 channel inhibitors were screened through calcium imaging and patch clamp techniques, and an inhibitor compound 6 was identified. This compound can alleviate cell damage after OGD/R by reducing cellular ROS levels and has a protective effect against cerebral ischemia-reperfusion injury in mice.