OBJECTIVE To analyze and identify the metabolites of pirtobrutinib （PTN） in rats， and clarify the possible metabolic pathways of PTN in rats. METHODS Six rats were intragastrically administered with 10 mg/kg PTN suspension. Blood samples were collected from the rats 30 minutes before administration and at 0.25， 0.5， 1， 2， 4， 6， 8， 12， 24 hours after administration. Urine and feces samples were collected 12 hours before administration and 24 hours after administration. UHPLC- Orbitrap Exploris 240 system combined with Compound Discoverer 3.0 and Xcalibur 2.0 software were adopted for structural identification and metabolic pathway analysis of PTN metabolites in rat plasma， urine， and feces. RESULTS A total of 29 PTN metabolites were identified， including 17， 19 and 22 metabolites in plasma， urine and feces， respectively. The metabolic pathways of PTN mainly included oxidation， sulfation， glucuronidation， etc.， and its metabolites were mostly combination products of two or more different metabolic forms. In detail， a total of 26 metabolites were associated with phase Ⅰ metabolic reactions （14 oxidation metabolites， 9 reduction/dehydrogenation metabolites， 8 demethylation metabolites， and 5 hydrolysis metabolites）. Meanwhile， a total of 20 products were involved in phase Ⅱ metabolites （14 sulfation metabolites and 8 glucuronic acid binding metabolites）. CONCLUSIONS PTN exhibits a diverse range of metabolites in rat fecal samples， with the primary metabolic pathways being oxidation， sulfation， glucuronidation， and others.

Objective To investigate the relationship between tooth loss and the occurrence of esophageal cancer in a natural village in Wenfeng District, Anyang City, Henan Province. Methods A prospective cohort study was conducted to observe the occurrence of tooth loss and esophageal cancer among the asymptomatic residents of the natural village for 16 years from January 2008 to July 2024. Data were analyzed by chi-square test, binary logistic regression, and restricted cubic spline. Results Among the total population of 711 cases, 136 cases were lost to follow-up and 575 cases were included in the final statistics, including 45 cases with esophageal cancer. Significant statistical difference was found between esophageal cancer patients with and without tooth loss (P<0.05). Logistic regression analysis showed that tooth loss was associated with the occurrence of esophageal cancer (OR=3.977, 95%CI: 1.543-10.255). After the adjustment for confounders, tooth loss remained significantly associated with the occurrence of esophageal cancer (OR=3.038, 95%CI: 1.035-8.914). A nonlinear dose-response relationship was observed between the number of teeth lost and the incidence of esophageal cancer. When the number of teeth lost was less than 12, the risk of esophageal cancer increased with the number of teeth lost. When more than 12 teeth were lost, the risk of esophageal cancer decreased as the number of teeth lost increased. Conclusion Tooth loss is a risk factor for the occurrence of esophageal cancer in this natural village. When the number of teeth lost is less than 12, the risk of esophageal cancer increases with the number of teeth lost.

OBJECTIVE: To observe the effects of moxibustion at "Feishu" (BL13) and "Xinshu" (BL15) on the circular RNA of exon 2-5 of the Pan3 gene (circPAN3), forkhead box O3 (FOXO3), and Bcl-2/adenovirus E1B19kDa-interacting protein 3 (BNIP3) in rats with chronic heart failure (CHF), and explore the potential mechanisms of moxibustion in alleviating myocardial fibrosis. METHODS: Ten rats of 60 male SPF-grade SD rats were randomly assigned into a normal group. The remaining rats underwent left anterior descending coronary artery (LAD) ligation to establish the CHF model. Forty successfully modeled rats were randomly divided into a model group, a moxibustion group, a rapamycin (RAPA) group, and a moxibustion+RAPA group, with 10 rats in each group. The moxibustion group received mild moxibustion at bilateral "Feishu" (BL13) and "Xinshu" (BL15), 30 min per session. The RAPA group received intraperitoneal injection of the autophagy activator RAPA (1 mg/kg). The moxibustion+RAPA group first received RAPA injection, followed by mild moxibustion at bilateral "Feishu" (BL13) and "Xinshu" (BL15). All interventions were administered once daily for 4 consecutive weeks. After the intervention, cardiac ultrasound was used to measure ejection fraction (EF) and left ventricular fractional shortening (FS). Serum placental growth factor (PLGF) level was determined by ELISA. Myocardial tissue morphology and collagen volume were assessed using hematoxylin-eosin (HE) staining and Masson's trichrome staining. The expression levels of circPAN3, FOXO3, and BNIP3 mRNA in myocardial tissue were detected by real-time PCR, while FOXO3 and BNIP3 protein expression levels were analyzed by Western blot. RESULTS: Compared with the normal group, the model group exhibited myocardial cell disorder, severe fibrosis, and increased collagen volume (P<0.01), along with significantly decreased EF, FS, and circPAN3 mRNA expression in myocardial tissue (P<0.01), and the serum PLGF level, as well as FOXO3 and BNIP3 mRNA and protein expression in myocardial tissue were increased (P<0.01). Compared with the model group, the moxibustion group showed reduced myocardial fibrosis, decreased collagen volume (P<0.01), increased EF, FS, and circPAN3 mRNA expression in myocardial tissue (P<0.01), and decreased serum PLGF level as well as FOXO3 and BNIP3 mRNA and protein expression in myocardial tissue (P<0.01). Compared with the model group, the RAPA group showed further deterioration in these parameters (P<0.01). Compared with the RAPA group, the moxibustion+RAPA group exhibited alleviation of myocardial fibrosis, reduced collagen volume (P<0.01), increased EF, FS, and circPAN3 mRNA expression in myocardial tissue (P<0.01), and decreased serum PLGF level as well as FOXO3 and BNIP3 mRNA and protein expression in myocardial tissue (P<0.01). CONCLUSION Moxibustion could alleviate myocardial fibrosis in CHF rats, possibly through upregulation of myocardial circPAN3 expression, downregulation of FOXO3 and BNIP3 expression, and inhibition of excessive myocardial autophagy.
Animals ; Moxibustion ; Heart Failure/metabolism* ; Male ; Rats ; Rats, Sprague-Dawley ; Myocardium/pathology* ; RNA, Circular/metabolism* ; Membrane Proteins/metabolism* ; Forkhead Box Protein O3/metabolism* ; Acupuncture Points ; Humans ; Fibrosis/genetics* ; Chronic Disease/therapy* ; Mitochondrial Proteins

Objective To investigatethe relationship between gastroesophageal reflux disease (GERD) symptoms and clinicopathological characteristics, p53 expression, and survival of Chinese patients with esophageal adenocarcinoma. Methods A total of 3882 patients with esophageal adenocarcinoma, 970 matched patients with esophageal squamous cell carcinoma, and 970 matched patients with gastric cardia adenocarcinoma were included to compare the incidence of GERD symptoms. Patients with esophageal adenocarcinoma were divided into two groups according to the presence and absence of GERD symptoms. The differences in clinicopathological characteristics and p53 expression between the two groups were compared by chi-square test, and the differences in survival between the two groups were compared by landmark analysis. Results The incidence of GERD symptoms increased successively in esophageal squamous cell carcinoma, esophageal adenocarcinoma, and gastric cardia adenocarcinoma. In patients with esophageal adenocarcinoma, the proportions of male, less than 65 years old, lower thoracic tumors, tumors without squamous cell carcinoma, poorly differentiation, early tumors (stage 0-I), and p53-positive tumors in the group with GERD symptoms were higher than those in the group without GERD symptoms; moreover, the long-term survival (≥15 years) was better. Conclusion GERD symptom is an important clinical sign of esophageal adenocarcinoma. It is closely related to specific clinicopathological characteristics, p53 overexpression, and good long-term prognosis.

Objective The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains is exacerbating the global burden of tuberculosis (TB), highlighting the urgent need for new treatment strategies for TB. Methods The recombinant adenovirus vaccine expressing cyclic di-adenosine monophosphate (c-di-AMP) phosphodiesterase B (CnpB) (rAd-CnpB), was administered to normal mice via mucosal immunization, either alone or in combination with drug therapy, to treat Mtb respiratory infections in mice.Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of antibodies in serum and bronchoalveolar lavage fluid (BALF). Real-time quantitative PCR was performed to assess the transcription levels of cytokines interferon γ(IFN-γ) and interleukin 10(IL-10) in mouse lungs. Flow cytometry was used to determine the proportions of CD4⁺ and CD8⁺ T cell subsets in the lungs and spleens. ELISA was employed to measure the levels of cytokines IFN-γ, IL-2, IL-10, inflammatory factors IL-6, and tumor necrosis factor α (TNF-α) secreted by spleen cells following antigen stimulation. The bacteria loads in the lungs and spleens of Mtb-infected mice were enumerated by plate counting methods. Resluts Intranasal immunization with rAd-CnpB induced high titers of IgG in mouse serum and the production of IgG and IgA in BALF, along with alterations in T lymphocyte subsets in the lungs and spleens. Administration of rAd-CnpB, either alone or in combination with drugs, to Mtb-infected mice significantly increased serum IgG levels as well as IgA and IgG levels in BALF. rAd-CnpB immunization promoted the secretion of CnpB-specific cytokines and inflammatory factors by splenocytes in Mtb-infected mice. However, rAd-CnpB immunotherapy, either alone or combined with drugs, did not significantly affect the bacterial loads in the lungs and spleens of mice with Mtb respiratory infections. Conclusion Mucosal immunization with rAd-CnpB induced significant mucosal, humoral and cellular immune responses in mice, and significantly enhanced CnpB-specific cellular immune responses in Mtb-infected mice.
Animals ; Adenoviridae/immunology* ; Mycobacterium tuberculosis/genetics* ; Mice ; Female ; Phosphoric Diester Hydrolases/genetics* ; Tuberculosis Vaccines/administration & dosage* ; Tuberculosis/prevention & control* ; Mice, Inbred BALB C ; Cytokines ; Lung/microbiology* ; Immunization ; Bronchoalveolar Lavage Fluid/immunology* ; Immunity, Mucosal

The 5-hydroxytryptamine 2A receptor (5-HT_2AR) is one of the key targets in the development of novel antidepressants. To develop new antidepressants targeting the 5-HT_2A receptor, this study established a high-throughput screening method for drugs targeting the 5-HT_2A receptor based on the principle of detecting calcium flux signals. The immunofluorescence assay and western blotting were employed to evaluate receptor expression levels in the 5-HT_2AR-CHO cell line. The reaction system parameters, including cell seeding density, DMSO concentration, and dye incubation time, were optimized with Z'-factor and signal window values as evaluation indicators. The specificity, precision, stability, and applicability of the method were assessed. Results indicated that the 5-HT_2AR-CHO cell line stably expressed high levels of the 5-HT_2A receptor. The optimized screening method involved a reaction system with 10 000 cells/well, 0.2% DMSO, and 2 h incubation with Calcium 6 dye. The method demonstrated excellent specificity, with inter-batch precision below 10% for the detection of 5-hydroxytryptamine (5-HT) at low, medium, and high concentrations. Testing four compounds that target the 5-HT_2A receptor- agonists 2,5-dimethoxy-4-iodoamphetamine (DOI), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), and lysergic acid diethylamide (LSD), along with the antagonist MDL100907-yielded Z'-factors (at EC₈₀) greater than 0.85 and signal window values over 0.91. The EC₅₀ values of these compounds were in the nanomolar range, and their potency rank order aligned with previously reported data, confirming the reliability of the established method. When being applied to the detection of 38 known active compounds, the method efficiently identified 5-HT_2A receptor agonists and antagonists while showing no response to non-target compounds. In conclusion, this study successfully constructs a high-throughput screening approach for 5-HT_2A receptor-targeting drugs based on calcium flux signals. The method possesses strong specificity, high sensitivity, and robust stability, being suitable for screening antidepressants targeting the 5-HT_2A receptor.
High-Throughput Screening Assays/methods* ; Receptor, Serotonin, 5-HT2A/metabolism* ; Animals ; CHO Cells ; Cricetulus ; Calcium Signaling/drug effects* ; Antidepressive Agents/pharmacology* ; Humans ; Serotonin 5-HT2 Receptor Antagonists/pharmacology* ; Calcium/metabolism*

Screening and evaluating the diseases responding specifically to traditional Chinese medicine （TCM） will help to highlight the advantages of TCM treatment， and the evaluation method should be standardized with consideration to the unique characteristics of the diseases. The incidence of Sjögren's Syndrome （SS） is increasing year by year， while the pathogenesis of this disease remains unclear. Modern therapies for this disease include biological agents and immunosuppressants， which generally have unsatisfactory efficacy. The TCM treatment of SS focuses on the harmony of the physical and mental health. The Rheumatology Branch of the China Association of Chinese Medicine organizes experts in TCM， Western medicine， and evidence-based medicine to form working groups. Delphi method and bibliometric method were used for analysis， and SS was selected as a disease responding specifically to TCM. Furthermore， the evaluation system was established for this disease， and the consensus regarding this disease was reached after seminar discussion. This paper summarized the whole process of the evaluation of the advantages of TCM treatment of SS. First， because TCM atomization is widely used in clinical practice and enriches TCM administration methods， this therapy is included after other non-drug therapies were taken as characteristic therapies. Second， the evaluation indicators of therapeutic effect should be determined with consideration to international acceptance and the current research status. Third， the expression method should be accurate， standardized， and objective， highlight the natural advantages of TCM， and avoid arbitrary extension. This paper provides a reference for clinicians to explore other diseases responding specifically to TCM.

The foveal avascular zone(FAZ)is the most sensitive region of the retina, which is interconnected by the macular capillary plexus. Its morphology can indirectly reflect the alterations of macular microcirculation. With strong repeatability and reliability, optical coherence tomography angiography(OCTA)can non-invasively visualize and quantify the FAZ. The great value of OCTA makes it an important supplemental examination tool in ophthalmology and other professions. The area and perimeter of FAZ have been demonstrated to be an effective clinical diagnostic indicator in high myopia, diabetic retinopathy, glaucoma and other ocular diseases. In recent years, the geometry of FAZ has also proven to have clinical value. The parameters describing the geometry of FAZ, such as circularity index, acircularity index and axial ratio, provide a new perspective for ocular disease research. The comprehensive investigation of the morphological characteristics of the FAZ is helpful to explore the pathological mechanism of the occurrence and development of ocular diseases, predict preclinical changes, make pathological stages of the disease precise, and provide a theoretical basis for monitoring the disease's progression and assessing patients' visual prognosis.

Objective:To explore the genetic basis of two children with unexplained psychomotor developmental delay and facial dysmorphisms suggestive of Coffin-Siris syndrome (CSS).Methods:A boy and a girl suspected for CSS at the 980th Hospital of the People′s Liberation Army Joint Service Support Force respectively in July 2019 and January 2021, and seven members from their families, were selected as the study subjects. Clinical data and family history of the children were collected, and detailed physical examination was carried out, in addition with laboratory and related auxiliary examinations. Potential variants and copy number variations (CNVs) were detected by whole exome sequencing (WES) and copy number variation sequencing (CNV-seq).Results:Child 1, an 8-month-old female, had featured microcephaly, atrial septal defect, curving of fifth finger/toe, and low limb muscle tone. Child 2 was a 2.5-year-old male with language delay, social impairment, dense hair but no curving of the fifth fingers. Genetic testing revealed that child 1 had loss of heterozygosity for exons 8 to 21 of the ARID1B gene, which was unreported previously. Family verification showed that both of her parents were of the wild type. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG) and American Society of Molecular Pathology (AMP), the variant was rated as pathogenic (PVS1+ PS2+ PM2-supporting). Child 2 was found to harbor a heterozygous c. 4263-6 (IVS17) T>G variant of the ARID1B gene. Transcriptome sequencing confirmed that the variant can affect the normal splicing, resulting in retention of a 5 bp sequence in intron 17. Family verification showed that both of his parents were of the wild type. Based on the guidelines from the ACMG, the variant was rated as pathogenic (PS2+ PM2-supporting+ PP3+ PS3). Conclusion:WES and RNA-seq have confirmed the diagnosis of CSS in both children. Discovery of the novel variants has expanded the spectrum of pathogenic mutations underlying CSS, and provided a basis for the genetic counseling.

Objective:To analyze the current adoption of palliative care by patients with unresectable metastatic colorectal cancer (mCRC) in China.Methods:From 1 March 2023 to 30 June 2023, a questionnaire survey was conducted by random sampling. An exclusive research platform for the Blue Book on Clinical Diagnosis and Treatment of Metastatic Colorectal Cancer. An online questionnaire was sent to medical oncologists (including chief physicians, associate chief physicians, attending physicians and residents) in general hospitals and oncology hospitals in four major regions of East, Central, South and Northeast China. The questionnaire contained 28 questions requesting basic information about doctors, the number of patients with mCRC, the status of treatment from first to fourth line and beyond, points concerning treatment of pain in patients with mCRC, and expectations for the future. A medical team was responsible for the quality control of data collected, whereas statisticians performed the data cleaning and sorting and statistical analysis.Results:A total of 300 clinical questionnaires were collected, including 217 (72%) from doctors in general hospitals and 83 (28%) from doctors in oncology hospitals. Senior physicians (including associate chief physicians and chief physicians) accounted for 65% of the respondents, attending physicians 30%, and residents 5%. Within 3 months (average for each month), 46.4±26.6% patients were diagnosed with recurrent or unresectable mCRC by each physician, 51.6±26.8% of the patients being in cancer hospitals and 44.4±26.3% in general hospitals. One hundred percent of patients receiving first-line treatment received palliative care, as did 80.3% of those receiving second-line treatment, 58.2% of those receiving third-line treatment, and 35.1% of those receiving ≥fourth-line treatment. The primary factor governing selection of first-line treatment was guideline recommendations, whereas comorbidities and the patients' physical status dictated second line to fourth line treatment. Standard first-line treatment was administered to 93.8% of eligible patients, standard second-line treatment to 94.3%; and standard third-line treatment to 73.5%. First-line therapy included targeted therapy in 63.6% of patients and immunotherapy in 2.8%; second-line therapy included targeted therapy in 63.0% of patients and immunotherapy in 2.0%; third-line therapy included targeted therapy in 59.2% of patients and immunotherapy in 2.2%; and fourth-line therapy included targeted therapy in 48.7% of patients and immunotherapy in 3.1%. First-line treatment lasted an average of 9.6 months, second-line treatment 6.7 months, third-line treatment 4.9 months, and fourth-line treatment 3.7 months. More than 70% of the patients maintained a good quality of life after receiving first and second-line treatment and more than 60% of them had ECOG performance scores of 0–1. After receiving third- and fourth-line treatment, 50%–60% of patients maintained a good quality of life and 40%–50% of them maintained ECOG performance scores of 0–1. The survey also revealed that the main deficiencies in treatment were limited effectiveness of third-line treatment, insufficient availability and opportunity for clinical research, popularity of new drugs or new drug combination strategies, and limited channels for participation in multidisciplinary diagnosis and treatment. Clinicians reported looking forward to participating in more clinical research on new drugs, hearing about the experience of experts in the field, and discovery of new targets and new drugs that increased the options for posterior line treatment of colorectal cancer.Conclusions:This report objectively summarizes the current situation, treatment difficulties, and expectations of frontline physicians concerning management of mCRC, thus providing a basis for decision-making and future direction for the diagnosis and research on treatment of mCRC.