OliTag-seq enhances in cellulo detection of CRISPR-Cas9 off-targets.

The potential for off-target mutations is a critical concern for the therapeutic application of CRISPR-Cas9 gene editing. Current detection methodologies, such as GUIDE-seq, exhibit limitations in oligonucleotide integration efficiency and sensitivity, which could hinder their utility in clinical settings. To address these issues, we introduce OliTag-seq, an in-cellulo assay specifically engineered to enhance the detection of off-target events. OliTag-seq employs a stable oligonucleotide for precise break tagging and an innovative triple-priming amplification strategy, significantly improving the scope and accuracy of off-target site identification. This method surpasses traditional assays by providing comprehensive coverage across various sgRNAs and genomic targets. Our research particularly highlights the superior sensitivity of induced pluripotent stem cells (iPSCs) in detecting off-target mutations, advocating for using patient-derived iPSCs for refined off-target analysis in therapeutic gene editing. Furthermore, we provide evidence that prolonged Cas9 expression and transient HDAC inhibitor treatments enhance the assay's ability to uncover off-target events. OliTag-seq merges the high sensitivity typical of in vitro assays with the practical application of cellular contexts. This approach significantly improves the safety and efficacy profiles of CRISPR-Cas9 interventions in research and clinical environments, positioning it as an essential tool for the precise assessment and refinement of genome editing applications.

Characterization of BCP/PreC/C region quasispecies in treatment-naive patients with different phases of HBV infection using next-generation sequencing.

BACKGROUND: To analysis of quasispecies (QS) changes and high-frequency mutations in the BCP/PreC/C region of patients at different phases of hepatitis B virus (HBV) infection and provides novel biomarkers for the diagnosis of chronic hepatitis B (CHB) patients. METHODS: With the application of next-generation sequencing technology, we were able to sequence the HBV BCP/PreC/C regions in 40 patients, each at different phases of the HBV infection. The heterogeneity of QS and the frequency of mutations were calculated using MEGA 7 software. RESULTS: Our results show that the complexity and diversity of the BCP/PreC/C QS in HBeAg-positive CHB patients are significantly higher than those in HBeAg-positive chronic infection patients, while HBeAg-negative chronic infection patients had significantly higher QS complexity and diversity than HBeAg-negative CHB patients. In addition, HBeAg-negative patients showed reduced complexity but increased diversity compared with HBeAg-positive patients. Receiver operating characteristic curves showed that G1764A, C2102T, dN and complexity of QS could be used as potential biomarkers for diagnosing HBeAg-positive CHB, while the A2189C, dS and complexity of QS could be used as potential biomarkers for diagnosing HBeAg-negative chronic hepatitis. Finally, our study also found that G1896A and A2159G may be hotspot mutations affecting HBeAg seroconversion. CONCLUSION: Our research elucidates the evolution of HBV by analyzing QS heterogeneity and mutation patterns, offering novel serum biomarkers for enhancing clinical diagnosis and disease prognosis. This comprehensive approach sheds light on the intricate dynamics of HBV progression and paves the way for more precise medical interventions.

A Gas Therapy Strategy for Intestinal Flora Regulation and Colitis Treatment by Nanogel-Based Multistage NO Delivery Microcapsules.

Current approaches to treating inflammatory bowel disease focus on the suppression of overactive immune responses, the removal of reactive intestinal oxygen species, and regulation of the intestinal flora. However, owing to the complex structure of the gastrointestinal tract and the influence of mucus, current small-molecule and biologic-based drugs for treating colitis cannot effectively act at the site of colon inflammation, and as a result, they tend to exhibit low efficacies and toxic side effects. In this study, nanogel-based multistage NO delivery microcapsules are developed to achieve NO release at the inflammation site by targeting the inflammatory tissues using the nanogel. Surprisingly, oral administration of the microcapsules suppresses the growth of pathogenic bacteria and increases the abundance of probiotic bacteria. Metabolomics further show that an increased abundance of intestinal probiotics promotes the production of metabolites, including short-chain fatty acids and indole derivatives, which modulate the intestinal immunity and restore the intestinal barrier via the interleukin-17 and PI3K-Akt signaling pathways. This work reveals that the developed gas therapy strategy based on multistage NO delivery microcapsules modulates the intestinal microbial balance, thereby reducing inflammation and promoting intestinal barrier repair, ultimately providing a new therapeutic approach for the clinical management of colitis.

Corticosteroids impair epithelial regeneration in immune-mediated intestinal damage.

Corticosteroid treatment (CST) failure is associated with poor outcomes for patients with gastrointestinal (GI) graft-versus-host disease (GVHD). CST is intended to target the immune system, but the glucocorticoid receptor (GR) is widely expressed, including within the intestines, where its effects are poorly understood. Here, we report that corticosteroids (CS) directly targeted intestinal epithelium, potentially worsening immune-mediated GI damage. CS administered to mice in vivo and intestinal organoid cultures ex vivo reduced epithelial proliferation. Following irradiation, immediate CST mitigated GI damage but delayed treatment attenuated regeneration and exacerbated damage. In a murine steroid-refractory (SR) GVHD model, CST impaired epithelial regeneration, worsened crypt loss, and reduced intestinal stem cell (ISC) frequencies. CST also exacerbated immune-mediated damage in organoid cultures with SR, GR-deficient T cells or IFN-γ. These findings correlated with CS-dependent changes in apoptosis-related gene expression and STAT3-related epithelial proliferation. Conversely, IL-22 administration enhanced STAT3 activity and overcame CS-mediated attenuation of regeneration, reducing crypt loss and promoting ISC expansion in steroid-treated mice with GVHD. Therefore, CST has the potential to exacerbate GI damage if it fails to control the damage-inducing immune response, but this risk may be countered by strategies augmenting epithelial regeneration, thus providing a rationale for clinical approaches combining such tissue-targeted therapies with immunosuppression.

The roles of autophagy in the treatment of diabetic nephropathy with rapamycin.

BACKGROUND: Rapamycin is known to induce autophagy, promote cell survival and inhibit the progression of diabetic nephropathy (DN). OBJECTIVES: The aim of this study was to examine the role of autophagy in the treatment of DN with rapamycin to provide the basis for the DN treatment with rapamycin. MATERIAL AND METHODS: Human mesangial cells (HMC) were cultured in a constant temperature incubator with 5% CO2, at 37°C and saturated humidity. Cells were divided into 5 groups and the 5-ethynyl-2-deoxyuridine (EdU) cell proliferation assay was used to determine cell proliferation. Flow cytometry was used to determine cell apoptosis, while GFP-RFP-LC3 showed autophagy flow. Western blot was employed to detect the expression of autophagy-related proteins LC3-II/LC3-I and P62. Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of type IV collagen fiber (Col4), hyaluronic acid (HA) and laminin (LA) in the extracellular matrix (ECM). RESULTS: Cell proliferation was the lowest in the hyperglycemic group. Additionally, the hyperglycemic group displayed the lowest number of autolysosomes compared to other groups. In contrast, the rapamycin group exhibited the highest number of autolysosomes. The LC3-II/LC3-I ratio was also the lowest in the hyperglycemic group, measuring 0.53 (0.50-0.58), while the expression level of P62 was significantly higher in that group at 0.98 (0.95-1.01) compared to other groups. Upon the introduction of rapamycin, the LC3-II/LC3-I ratio was significantly increased at 2.21 (1.95-2.21), and P62 was significantly decreased 0.38 (0.38-0.39) compared to the hyperglycemic group. Both changes were statistically significant, with p-values of 0.034 and 0.010, respectively. Enzyme-linked immunosorbent assay was employed to detect Col4, HA and LA content. The study findings demonstrated significantly higher levels of glucose in the hyperglycemic group in comparison to other groups. In contrast, the rapamycin group exhibited significantly lower levels of glucose than the hyperglycemic group, yet the difference was not statistically significant. CONCLUSIONS: Hyperglycemic can inhibit the autophagic activity of HMC, promote cell apoptosis, enhance ECM accumulation, and facilitate the DN progression. In contrast, rapamycin can elicit autophagy, decrease mesangial matrix proliferation, and therefore impede DN progression.

Approaches for Performance Verification Toward Standardization of Peripheral Blood Regulatory T-Cell Detection by Flow Cytometry.

CONTEXT.­: Regulatory T-cell (Treg) detection in peripheral blood, based on flow cytometry, is invaluable for diagnosis and treatment of immune-mediated diseases. However, there is a lack of reliable methods to verify the performance, which is pivotal towards standardization of the Tregs assay. OBJECTIVE.­: To conduct standardization studies and verify the performance of 3 commercially available reagent sets for the Tregs assay based on flow cytometry and agreement analysis for Treg detection across the different reagent sets. DESIGN.­: The analytical performance of Tregs assay using reagent sets supplied by 3 manufacturers was evaluated after establishing the gating strategy and determining the optimal antibody concentration. Postcollection sample stability was evaluated, as well as the repeatability, reproducibility, reportable range, linearity, and assay carryover. Agreement between the different assays was assessed via Bland-Altman plots and linear regression analysis. The relationship between the frequency of CD4+CD25+CD127low/- Tregs and CD4+CD25+Foxp3+ Tregs was evaluated. RESULTS.­: The postcollection sample stability was set at 72 hours after collection at room temperature. The accuracy, repeatability, reproducibility, and accuracy all met the requirements for clinical analysis. Excellent linearity, with R2 ≥0.9 and no assay carryover, was observed. For reportable range, a minimum of 1000 events in the CD3+CD4+ gate was required for Tregs assay. Moreover, the results for Tregs labeled by antibodies from the 3 manufacturers were in good agreement. The percentage of CD4+CD25+CD127low/- Tregs was closely correlated with CD4+CD25+Foxp3+ Tregs. CONCLUSIONS.­: This is the first study to evaluate systematically the measurement performance of Tregs in peripheral blood by flow cytometry, which provides a practical solution to verifying the performance of flow cytometry-based immune monitoring projects in clinical practice.

Multi-omics reveals deoxycholic acid modulates bile acid metabolism via the gut microbiota to antagonize carbon tetrachloride-induced chronic liver injury.

Deoxycholic acid (DCA) serves essential functions in both physiological and pathological liver processes; nevertheless, the relationship among DCA, gut microbiota, and metabolism in chronic liver injury remain insufficiently understood. The primary objective of this study is to elucidate the potential of DCA in ameliorating chronic liver injury and evaluate its regulatory effect on gut microbiota and metabolism via a comprehensive multi-omics approach. Our study found that DCA supplementation caused significant changes in the composition of gut microbiota, which were essential for its antagonistic effect against CCl4-induced chronic liver injury. When gut microbiota was depleted with antibiotics, the observed protective efficacy of DCA against chronic liver injury became noticeably attenuated. Mechanistically, we discovered that DCA regulates the metabolism of bile acids (BAs), including 3-epi DCA, Apo-CA, and its isomers 12-KLCA and 7-KLCA, IHDCA, and DCA, by promoting the growth of A.muciniphila in gut microbiota. This might lead to the inhibition of the IL-17 and TNF inflammatory signaling pathway, thereby effectively countering CCl4-induced chronic liver injury. This study illustrates that the enrichment of A. muciniphila in the gut microbiota, mediated by DCA, enhances the production of secondary bile acids, thereby mitigating chronic liver injury induced by CCl4. The underlying mechanism may involve the inhibition of hepatic IL-17 and TNF signaling pathways. These findings propose a promising approach to alleviate chronic liver injury by modulating both the gut microbiota and bile acids metabolism.

Clinical internship environment and caring behaviours among nursing students: A moderated mediation model.

BACKGROUND: Caring behaviour is critical for nursing quality, and the clinical internship environment is a crucial setting for preparing nursing students for caring behaviours. Evidence about how to develop nursing students' caring behaviour in the clinical environment is still emerging. However, the mechanism between the clinical internship environment and caring behaviour remains unclear, especially the mediating role of moral sensitivity and the moderating effect of self-efficacy. RESEARCH OBJECTIVE: This study aimed to examine the mediating effect of moral sensitivity and the moderating function of self-efficacy on the association between the clinical internship environment and caring behaviours. RESEARCH DESIGN: A cross-sectional design used acceptable validity scales. The hypothesised moderated mediation model was tested in the SPSS PROCESS macro. PARTICIPANTS AND RESEARCH CONTEXT: This survey collected data from 504 nursing students in an internship at a teaching hospital in Changsha, China. ETHICAL CONSIDERATIONS: This study was pre-approved by the ethics committee of the medical school (No. E2022210). Informed consent was obtained from all students. RESULTS: The clinical internship environment (B = 0.450, 95% CI = [0.371, 0.530]) and moral sensitivity (B = 1.352, 95% CI = [1.090, 1.615]) had positive direct effects on nursing students' caring behaviours. Clinical internship environment also indirectly influenced students' caring behaviours via moral sensitivity (B = 0.161, 95% CI = [0.115, 0.206]). In addition, self-efficacy played a moderating role between the clinical internship environment and caring behaviours (B = 0.019, 95% CI = [0.007, 0.031]), as well as the relationship between the clinical internship environment and moral sensitivity (B = 0.006, 95% CI = [0.003, 0.010]). CONCLUSION: Moral sensitivity mediates the effect of the clinical internship environment on caring behaviour, and self-efficacy strengthens both direct and indirect effects. This study emphasises the importance of self-efficacy in developing moral sensitivity and caring behaviours in nursing students.

Oncostatin M Induces IFITM1 Expression to Inhibit Hepatitis B Virus Replication Via JAK-STAT Signaling.

BACKGROUND & AIMS: Functional cure is achieved by a limited number of patients with chronic hepatitis B (CHB) after nucleotide analogue(s) and interferon treatment. It is urgent to develop therapies that can help a larger proportion of patients achieve functional cure. The present study was designed to explore the anti-hepatitis B virus (HBV) potency of interleukin-6 family cytokines and to characterize the underlying mechanisms of the cytokine displaying the highest anti-HBV potency. METHODS: HBV-infected cells were used to screened the anti-HBV potency of interleukin-6 family cytokines. The concentration of oncostatin M (OSM) in patients with chronic HBV infection was examined by enzyme-linked immunosorbent assay. The underlying mechanism of OSM anti-HBV was explored through RNA-seq. C57BL/6 mice injected with rAAV8-1.3HBV were used to explore the suppression effect of OSM on HBV in vivo. RESULTS: OSM is the most effective of the interleukin-6 family cytokines for suppression of HBV replication (percentage of average inhibition: hepatitis B surface antigen, 34.44%; hepatitis B e antigen, 32.52%; HBV DNA, 61.57%). Hepatitis B e antigen-positive CHB patients with high OSM levels had lower hepatitis B surface antigen and hepatitis B e antigen than those with low levels. OSM activated JAK-STAT signaling pathway promoting the formation of STAT1-IRF9 transcription factor complex. Following this, OSM increased the expression of various genes with known functions in innate and adaptive immunity, which was higher expression in patients with CHB in immune clearance phase than in immune tolerance phase (data from GEO: GSE65359). Interferon-induced transmembrane protein 1, one of the most differentially expressed genes, was identified as an HBV restriction factor involved in OSM-mediated anti-HBV effect. In vivo, we also found OSM significantly inhibited HBV replication and induced expression of antiviral effector interferon-induced transmembrane protein 1. CONCLUSIONS: Our study shows that OSM remodels the immune response against HBV and exerts potent anti-HBV activity, supporting its further development as a potential therapy for treating CHB.

Targeted quantitative lipidomic uncovers lipid biomarkers for predicting the presence of compensated cirrhosis and discriminating decompensated cirrhosis from compensated cirrhosis.

OBJECTIVES: This study aimed to characterize serum lipid metabolism and identify potential biomarkers for compensated cirrhosis (CC) predicting and decompensated cirrhosis (DC) discrimination using targeted quantitative lipidomics and machine learning approaches. METHODS: Serum samples from a cohort of 120 participants was analyzed, including 90 cirrhosis patients (45 CC patients and 45 DC patients) and 30 healthy individuals. Lipid metabolic profiling was performed using targeted LC-MS/MS. Two machine learning methods, least absolute shrinkage and selection operator (LASSO), and random forest (RF) were applied to screen for candidate metabolite biomarkers. RESULTS: The metabolic profiling analysis showed a significant disruption in patients with CC and DC. Compared to the CC group, the DC group exhibited a significant upregulation in the abundance of glycochenodeoxycholic acid (GCDCA), glyco-ursodeoxycholic acid (GUDCA), glycocholic acid (GCA), phosphatidylethanolamine (PE), N-acyl-lyso-phosphatidylethanolamine (LNAPE), and triglycerides (TG), and a significant downregulation in the abundance of ceramides (Cer) and lysophosphatidylcholines (LPC). Machine learning identified 11 lipid metabolites (abbreviated as BMP11) as potential CC biomarkers with excellent prediction performance, with an AUC of 0.944, accuracy of 94.7 %, precision of 95.6 %, and recall of 95.6 %. For DC discrimination, eight lipids (abbreviated as BMP8) were identified, demonstrating strong efficacy, with an AUC of 0.968, accuracy of 92.2 %, precision of 88.0 %, and recall of 97.8 %. CONCLUSIONS: This study unveiled distinct lipidomic profiles in CC and DC patients and established robust lipid-based models for CC predicting and DC discrimination.

Detection of serum large and middle hepatitis B virus surface proteins: A novel potential diagnostic and prognostic biomarker for chronic hepatitis B.

BACKGROUND: The significance of large (LHB) and middle (MHB) HBV surface proteins in chronic hepatitis B (CHB) remains uncertain. This study investigates the role of LHB and MHB in different infection phases and liver diseases. METHODS: Serum samples from 217 patients with HBV chronic infection, CHB, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) were subjected to quantification of LHB and MHB using ELISA. RESULTS: Positive correlations were observed among LHB, MHB, and LHB/HBsAg, with HBV serum markers including HBsAg, HBeAg, and HBV DNA. (P < 0.0001). In HBeAg-positive chronic infection, LHB and MHB were higher than in HBeAg-positive CHB (P < 0.01). In HBeAg-negative chronic infection, LHB and MHB were lower than in HBeAg-negative CHB (P < 0.01). ROC analysis identified LHB and MHB as potential discriminators of CHB and chronic infection. LC and HCC exhibited lower LHB, MHB, and MHB/HBsAg than CHB (P < 0.05). Multivariate analysis found that age and the MHB/HBsAg serve as independent factors for the progression of CHB to end stage of liver disease. CONCLUSIONS: LHB and MHB emerge as novel biomarkers distinguishing chronic infection and CHB. MHB/HBsAg shows promise as a predictor for CHB progression.

A new prognostic model for glioblastoma multiforme based on coagulation-related genes.

Background: Glioblastoma multiforme (GBM) is the most aggressive, common, and lethal type of primary brain tumor. Multiple cancers have been associated with abnormalities in the coagulation system that facilitate tumor invasion and metastasis. In GBM, the prognostic value and underlying mechanism of coagulation-related genes (CRGs) have not been explored. Methods: RNA sequencing (RNA-seq) and clinical information on GBM were obtained from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA), respectively. Following the identification of differentially expressed CRGs (DECRGs) between GBM and control samples, the survival-related DECRGs were selected via univariate and multivariate Cox regression analyses to establish a prognostic signature. The prognostic performance and clinical utility of the prognostic signature were assessed by the Kaplan-Meier (KM) analysis and receiver operating characteristic (ROC) curve analysis, and a nomogram was constructed. The signature genes-related underlying mechanisms were analyzed according to gene set enrichment analysis (GSEA), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and single-cell analysis. Finally, the difference in immune cell infiltration, stromal score, immune score, and Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) score were compared between different risk groups. Results: A 5-gene prognostic signature (PLAUR, GP6, C5AR1, SERPINA5, F2RL2) was established for overall survival (OS) prediction of GBM patients. The predicted efficiency of the prognostic signature was confirmed in TGGA-GBM dataset and validated in the CGGA-GBM dataset, revealing that it could differentiate GBM patients from controls well, and high risk score was accompanied with poor prognosis. Moreover, biological process (BP) and signaling pathway analyses showed that signature genes were mainly enriched in the functions of blood coagulation and tumor invasion and metastasis. Moreover, high-risk patients exhibited higher levels of immune cell infiltration, stromal score, immune score, and ESTIMATE score than that of low-risk patients. Conclusions: An analysis of coagulation-related prognostic signatures was conducted in this study, as well as how signature genes may affect GBM progress, providing information that might provide new ideas for the development of GBM-related molecular targeted therapies.

Curative effect of wrist-ankle acupuncture on postpartum abdominal pain and its influence on serum ß-EP level in puerpera. / è •è¸é’ˆæ²»ç–—äº§åŽè ¹ç—›çš„ä¸´åºŠç–—æ•ˆåŠå¯¹äº§å¦‡è¡€æ¸ ß-EP水平的影响.

OBJECTIVES: To observe the clinical effect of wrist-ankle acupuncture on postpartum abdominal pain and its influence on serum beta-endorphin (ß-EP) level in puerpera. METHODS: Seventy patients with postpartum abdominal pain were randomly divided into an acupuncture + herbal medication group (35 cases, 1 case dropped out) and a herbal medication group (35 cases, 2 cases dropped out). In the herbal medication group, 1 day after delivery, modified shenghua decoction was taken orally, one dose a day. In the acupuncture + herbal medication group, on the basis of herbal medication, wrist-ankle acupuncture was given at the Lower 1 and Lower 2 of the ankles, once daily. The duration of treatment was 3 days in the two groups. Before and after treatment, the score of visual analogue scale (VAS) for pain, serum ß-EP level, uterine fundus height, postpartum conditions of lochia and the uterine recovery at 42 days postpartum were compared in the patients of the two groups. RESULTS: At each time point after treatment (24 h, 48 h and 72 h after delivery), VAS scores and the uterine fundus height were reduced as compared with those before treatment (2 h after delivery) in the two groups (P<0.05); these indexes in the acupuncture + herbal medication group were lower than those in the herbal medication group (P<0.05). After treatment (72 h after delivery), ß-EP levels in the serum were increased when compared with those before treatment in the two groups (P<0.05), and the ß-EP level in the acupuncture + herbal medication group was higher than that in the herbal medication group (P<0.05). The volume of postpartum lochia discharge in the acupuncture + herbal medication group was higher than that in the herbal medication group (P<0.05), while the duration of postpartum lochia discharge and the total time of lochia discharge were shorter (P<0.05). Regarding the recovery of the uterus at 42 days postpartum, there was no statistical significance between the two groups (P>0.05). CONCLUSIONS: Wrist-ankle acupuncture obviously reduces the degree of postpartum abdominal pain and promotes the lochia discharge and the uterine recovery. The effect mechanism may be related to the up-regulation of serum ß-EP level and the increase of pain threshold so that analgesia is obtained.

Advance Progress in Assembly Mechanisms of Carrier-Free Nanodrugs for Cancer Treatment.

Nanocarriers have been widely studied and applied in the field of cancer treatment. However, conventional nanocarriers still suffer from complicated preparation processes, low drug loading, and potential toxicity of carriers themselves. To tackle the hindrance, carrier-free nanodrugs with biological activity have received increasing attention in cancer therapy. Extensive efforts have been made to exploit new self-assembly methods and mechanisms to expand the scope of carrier-free nanodrugs with enhanced therapeutic performance. In this review, we summarize the advanced progress and applications of carrier-free nanodrugs based on different types of assembly mechanisms and strategies, which involved noncovalent interactions, a combination of covalent bonds and noncovalent interactions, and metal ions-coordinated self-assembly. These carrier-free nanodrugs are introduced in detail according to their assembly and antitumor applications. Finally, the prospects and existing challenges of carrier-free nanodrugs in future development and clinical application are discussed. We hope that this comprehensive review will provide new insights into the rational design of more effective carrier-free nanodrug systems and advancing clinical cancer and other diseases (e.g., bacterial infections) infection treatment.

A tissue-intrinsic IL-33/EGF circuit promotes epithelial regeneration after intestinal injury.

Intestinal stem cells (ISCs) maintain the epithelial lining of the intestines, but mechanisms regulating ISCs and their niche after damage remain poorly understood. Utilizing radiation injury to model intestinal pathology, we report here that the Interleukin-33 (IL-33)/ST2 axis, an immunomodulatory pathway monitored clinically as an intestinal injury biomarker, regulates intrinsic epithelial regeneration by inducing production of epidermal growth factor (EGF). Three-dimensional imaging and lineage-specific RiboTag induction within the stem cell compartment indicated that ISCs expressed IL-33 in response to radiation injury. Neighboring Paneth cells responded to IL-33 by augmenting production of EGF, which promoted ISC recovery and epithelial regeneration. These findings reveal an unknown pathway of niche regulation and crypt regeneration whereby the niche responds dynamically upon injury and the stem cells orchestrate regeneration by regulating their niche. This regenerative circuit also highlights the breadth of IL-33 activity beyond immunomodulation and the therapeutic potential of EGF administration for treatment of intestinal injury.

Tracing the geographical origin of tobacco at two spatial scales by stable isotope and element analyses with chemometrics.

Tobacco is a widely cultivated cash crop, but it is often smuggled and sold illegally. Unfortunately, there is currently no way to verify the origin of tobacco in China. In an effort to address this issue, we conducted a study using stable isotopes and elements from 176 tobacco samples at both provincial and municipal scales. Our findings revealed significant differences in δ13C, K, Cs, and 208/206Pb at the provincial-level, and Sr, Se, and Pb at the municipal level. We created a heat map at the municipal level, which showed a similar cluster classification to geographic grouping and provided an initial assessment of tobacco origins. Using OPLS-DA modeling, we achieved a 98.3% accuracy rate for the provincial scale and 97.6% for the municipal scale. It is worth noting that the importance of rankings of variables varied depending on the spatial scale of the evaluation. This study offers the first traceability fingerprint dataset of tobacco and has the potential to combat mislabeling and fraudulent conduct by identifying the geographical origin of tobacco.

Physiological Function of RDL1 and RDL2 Subunits of the Ionotropic GABA Receptor in the Spodoptera litura with the CRISPR/Cas9 System In Vivo.

In insect ionotropic γ-aminobutyric acid receptor (iGABAR) subunits, only resistance to dieldrin (RDL) can be individually and functionally expressed in vitro. In lepidopteran, two to three RDL subtypes are identified; however, their physiological roles have not been distinguished in vivo. In this study, SlRdl1 and SlRdl2 of S. litura were individually knocked out using CRISPR/Cas9, respectively. The mortality and larval and pupal duration of KOSlRdl1 and KOSlRdl2 were increased. The flight time and distance were increased by 43.30%-80.66% and 58.96%-198.22%, respectively, in KOSlRdl1. The GABA-induced current was significantly decreased by 53.57%-74.28% and 46.91%-63.34% in the ventral nerve cord, and the GABA titer was significantly reduced by 17.65%-28.05% and 19.85%-42.46% in KOSlRdl1 and KOSlRdl2, respectively. In conclusion, SlRdl1 and SlRdl2 are necessary for the transmission of GABA-induced neural signals; however, only SlRdl1 could regulate the flight capability of S. litura. Our results provided a new avenue to study lepidopteran iGABARs.

Comparison of intracorporeal and extracorporeal anastomosis in left hemicolectomy: updated meta-analysis of retrospective control trials.

BACKGROUND: The feasibility and effectiveness of selecting an intracorporeal or extracorporeal technique in left hemicolectomy remain poorly understood. This meta-analysis aimed to evaluate the difference between the two approaches regarding intraoperative and postoperative outcomes. METHODS: A thorough exploration of online databases (PubMed, Embase, Cochrane, and Web of Science) was executed to identify randomized controlled trials, cohort studies, and case control studies. The outcomes contained four aspects: intraoperative outcomes, postoperative complications, postoperative patient conditions, and postoperative outcomes. All of these data were analyzed using RevMan 5.4. Seven retrospective control trials (intracorporeal, 396 patients; extracorporeal, 426 patients) were evaluated. RESULTS: Compared to the extracorporeal group, the intracorporeal group demonstrated superiority in incision length (P = 0.005), overall complications (P = 0.01), time to first flatus (P < 0.001), time to first stool (P = 0.005), time to first diet (P < 0.001) and hospital stay duration (P = 0.001). CONCLUSIONS: The intracorporeal technique is associated with superiority over the extracorporeal technique in reducing postoperative complications, promoting postoperative recovery of gastrointestinal function, and reducing hospital stay duration.

[Three-dimensional gingival morphology in the aesthetic zone of maxillary anterior teeth: a cone-beam CT analysis].

PURPOSE: To measure and investigate the relationship of three-dimensional gingival morphology on the labial side of the maxillary anterior teeth by using cone-beam CT(CBCT) in conjunction with a novel radiocontrast agent. METHODS: Thirty periodontal healthy subjects were enrolled. The composition of light-cured gingival barrier resin and iohexol injection was applied to the measurement area, then a positioning wire was set up, and CBCT was used to assess supracrestal gingiva tissue (SGT), gingiva thickness (GT) and width of keratinized gingiva (KGW). The differences in each parameter between different gingival biotypes were compared. SPSS 25.0 software package was used for data analysis. RESULTS: The mean distance of SGT was greater for central incisors than canines (P＜0.05). The central incisors had the thickest GT in the maxillary anterior region, while the canines had the thinnest(P＜0.01). The GT of male central and lateral incisors was significantly thicker than that of females(P＜0.05), and the KGW was significantly wider than that of canines (P＜0.05). GT-SGT, KGW-SGT and GT-KGW all had a positive correlation (r=0.315, 0.287,0.406, P＜0.01). The thick gingival type was greater than the thin gingival type in the KGW of lateral incisors and canines and the SGT height of canines (P＜0.05). CONCLUSIONS: There were significant differences in the measuring results of GT, KGW, and SGT in the maxillary anterior region under different gingival biotypes, and individualized treatment strategies can be formulated based on gingival biotypes.

Morphology of the metapleural gland and its associated novel atrial cone gland in Strumigenys ants.

The metapleural gland is a unique feature of the ant phenotype, but diversity in its anatomy and function across the ants is not well documented or understood. We studied the morphology of the metapleural gland in 20 mainly Oriental Strumigenys species using histology, scanning and transmission electron microscopy, and micro-CT. The gland is formed by a cluster of class-3 cells, their secretion is transported through a bundle of ducts into the sclerotized atrium and is guided along a series of parallel cuticular ridges towards the atrial opening. Among the examined species, queens have more gland cells than conspecific workers, while the examined males do not have the gland. The social parasite S. mutica has the most developed metapleural gland. In addition, we describe a novel class-1 atrial cone gland which is associated with the metapleural gland. The epithelium of this cone gland forms an invagination into the ventral atrium of the metapleural gland. The cuticular cone may be lacking in some Afrotropical and Neotropical species, although these may still contain the epithelial gland. The functional and evolutionary drivers of morphological variation in the exocrine system across species form interesting questions for future work.