Objective To investigate the impact of Toxoplasma gondii type I, II and III rhoptry protein 16 (ROP16) on programmed cell death ligand 1 (PD-L1) expression in lung adenocarcinoma cells, and to examine the effects of T. gondii type I ROP16 protein on the relative PD-L1 expression, the relative PD-L1 distribution on the cell membrane surface, and the binding of programmed cell death 1 (PD-1) to PD-L1 in lung adenocarcinoma cells. Methods Lentiviral vectors overexpressing T. gondii type I, II and III ROP16 proteins were generated, and transfected into the human lung adenocarcinoma A549 cell line. A549 cells were used as a blank control group, and A549 cells transfected with an empty lentiviral expression vector were used as a negative control group, while A549 cells transfected with lentiviral vectors overexpressing T. gondii type I, II and III ROP16 proteins served as experimental groups. Stably transfected cells were selected with puromycin and verified using Western blotting, quantitative real-time PCR (RT-qPCR), and immunofluorescence assays. The PD-L1 expression was quantified at translational and transcriptional levels using Western blotting and RT-qPCR assays in A549 cells in the five groups, and the relative PD-L1 distribution was detected on the A549 cell membrane surface using flow cytometry. In addition, the effect of T. gondii type I ROP16 protein on the PD-1/PD-L1 binding was measured in A549 cells using enzyme-linked immunosorbent assay (ELISA). Results The relative ROP16 protein expression was 0, 0, 1.546 ± 0.091, 1.822 ± 0.047 and 2.334 ± 0.089 in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 1 339.00,P < 0.001), and the relative ROP16 mRNA expression was 2.153 ± 0.949, 2.436 ± 1.614, 14.343 ± 0.020, 12.577 ± 0.285 and 15.090 ± 0.420 in the blank control group, negative control group and the T. gondii type I, II and III ROP16 protein overexpression groups, respectively (F = 483.50,P < 0.001). The ROP16 expression was higher in the T. gondii type I, II and III ROP16 protein overexpression groups than in the blank control group at both translational and transcriptional levels (allP values < 0.001). Immunofluorescence assay revealed that T. gondii type I, II and III ROP16 proteins were predominantly localized in A549 cell nuclei. Western blotting showed that the relative PD-L1 protein expression was 0.685 ± 0.109, 0.589 ± 0.114, 1.007 ± 0.117, 0.572 ± 0.151, and 0.426 ± 0.116 in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 9.46,P < 0.05), and RT-qPCR assay quantified that the relative PD-L1 mRNA expression was 1.012 ± 0.190, 1.281 ± 0.465, 1.950 ± 0.175, 0.889 ± 0.251, and 0.230 ± 0.192 in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 14.18,P < 0.05). The PD-L1 expression was higher in the T. gondii type IROP16 protein overexpression group than in the blank control group at both translational and transcriptional levels (both P values < 0.05). Flow cytometry detected that the relative distributions of PD-L1 protein were (10.83 ± 0.60)%, (11.23 ± 0.20)%, and (14.61 ± 0.50)% on the A549 cell membrane surface (F = 28.31, P < 0.05), and the relative distribution of PD-L1 protein was higher in the T. gondii type IROP16 protein overexpression group than in the blank control group and negative control group (both P values < 0.001). ELISA measured significant differences in the absorbance (A) value among the T. gondii type IROP16 protein overexpression group, the blank control group and the negative control group if the concentrations of the recombinant PD-1 protein were 0.04 (F = 10.45, P < 0.05), 0.08 μg/mL (F = 11.68, P < 0.05) and 0.12 μg/mL (F = 52.68, P < 0.05), and the A value was higher in the T. gondii type IROP16 protein overexpression group than in the blank control group and the negative control group (both P values < 0.05), indicating that T. gondii type IROP16 protein promoted the PD-L1/PD-1 binding in A549 cells in a concentration-dose manner. Conclusions T. gondii type IROP16 protein overexpression may up-regulate PD-L1 expression in A549 cells at both transcriptional and translational levels and the relative PD-L1 distribution on the A549 cell membrane surface, and affect the PD-1/PD-L1 binding in a concentration-dependent manner.

Objective To investigate the effects of Toxoplasma gondii type Iand IIrhoptry protein 16 (ROP16) on the polarization and inflammatory response of mouse alveolar macrophages, so as to provide the scientific evidence for unveiling the immunoregulatory mechanisms following T. gondii infection in host cells and the clinical diagnosis and treatment of pulmonary toxoplasmosis. MethodsMouse alveolar macrophages served as blank controls, and mouse alveolar macrophages transfected with the empty lentiviral expression vector served as negative controls, and mouse alveolar macrophages transfected with lentiviral vectors overexpressing T. gondii type I and II ROP16 served as the type I and II ROP16 overexpression groups. Following puromycin selection, stably transfected cells that overexpressed type Iand IIROP16 were generated, observed for green fluorescence expression under a fluorescence microscope and verified using PCR, Western blotting and real-time quantitative reverse transcription PCR (RT-qPCR) assays. The expression of ROP16, inducible nitric oxide synthase (iNOS), arginase (Arg)-1, mannose receptor (CD206), cluster of differentiation 86 (CD86), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), caspase-1, apoptosis-associated speck-like protein containing a CARD (ASC), and interleukin (IL)-1β proteins was determined in mouse alveolar macrophages using Western blotting assay, and the mRNA levels of ROP16, iNOS, IL-1β, IL-4, IL-12, IL-18, Arg-1, IL-10, IL-6, tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β were detected in mouse alveolar macrophages using RT-qPCR assay. Results Fluorescence microscopy showed 90% of mouse alveolar macrophages producing green fluorescent signals in the type Iand II ROP16 overexpression groups and the negative control group. The relative ROP16 protein expression was 1.000 ± 0.000, 1.003 ± 0.020, 1.349 ± 0.055, and 1.376 ± 0.080 in mouse alveolar macrophages in the blank control group, negative control group, and type Iand IIROP16 overexpression groups (F = 35.30, P < 0.01), and the relative ROP16 mRNA expression was 1.007 ± 0.172, 2.030 ± 0.356, 1 409.579 ± 75.960, and 1 413.581 ± 27.712 in the blank control group, negative control group, and type Iand II ROP16 overexpression groups (F = 811.00, P < 0.01). The ROP16 expression was significantly higher in the type Iand IIROP16 overexpression groups than in the blank control group at both protein and mRNA levels (all P value < 0.01). Western blotting assay detected significant differences among the four groups in terms of iNOS, Arg-1, CD86, CD206, NLRP3, caspase-1, ASC, and IL-1β protein expression (F = 124.70, 82.40, 79.82, 919.40, 84.74, 39.85, 2 354.00 and 65.96, all P values < 0.05), and the expression of Arg-1, CD206, NLRP3, caspase-1, ASC, and IL-1β proteins was significantly higher in the type I ROP16 overexpression group than in the blank control group (all P values < 0.001), while the expression of iNOS, CD86, NLRP3, caspase-1, ASC, and IL-1β proteins was significantly higher in the type II ROP16 overexpression group than in the blank control group (all P values < 0.01). RT-qPCR assay detected significant differences among the four groups in terms of iNOS, IL-1β, IL-4, IL-12, IL-18, Arg-1, IL-10, IL-6, TNF-α, and TGF-β mRNA expression (F = 407.00, 1 528.00, 833.10, 267.90, 989.80, 161.80, 461.10, 5 529.00, 849.60 and 8 836.00, all P values < 0.05), and the Arg-1, IL-4, IL-10, and TGF-β mRNA expression was significantly higher in the type I ROP16 overexpression group than in the blank control group (all P values < 0.001), while the iNOS, IL-1β, IL-12, IL-18, IL-6, and TNF-α mRNA expression was significantly higher in the type II ROP16 overexpression group than in the blank control group (all P values < 0.001). Conclusions T. gondii type IROP16 may induce M2-dominant phenotypes of mouse alveolar macrophages, and type II ROP16 may induce M1-dominant phenotypes of mouse alveolar macrophages. Both T. gondii type I and II ROP16 may activate NLRP3, and mediate the activation of ASC, caspase-1 and IL-1β to promote inflammatory responses.

Objective To explore the efficacy of progressive exercise training based on the modified Medical Research Council dyspnea scale (mMRC) grading in respiratory rehabilitation for patients with chronic obstructive pulmonary disease (COPD) at a primary healthcare setting. Methods A total of 106 patients with COPD admitted to Zhuanqiao Community Health Service Center in Shanghai from Aug.1, 2022 to Jul. 30, 2024 were selected as research subjects. They were randomly divided into a study group and a control group in a 1∶1 ratio, with 53 patients in each group. The control group received conventional treatment, while the study group received conventional treatment combined with progressive exercise training. After 4 weeks of continuous treatment, the changes in the 6-minute walk test (6MWT), COPD assessment test (CAT) score, mMRC grading, Global Initiative for Chronic Obstructive Lung Disease (GOLD) grading and pulmonary function were compared between the two groups. Results Patients in both groups showed improvements in 6MWT distance, CAT score, mMRC grading, GOLD grading, and pulmonary function compared to baseline (P＜0.05). Moreover, the study group had better improvements in 6MWT distance, CAT score, mMRC grading, GOLD grading, and pulmonary function than the control group (P＜0.05). Conclusions Conventional treatment combined with progressive exercise training based on mMRC grading can enhance the effect of respiratory rehabilitation in patients with COPD, particularly in improving pulmonary function and exercise tolerance.

OBJECTIVE: To investigate the role of α7-nicotinic acetylcholine receptor (α7-nAChR) in the regulation of neuronal activity and expression of synapse-related proteins in the thalamic reticular nucleus (TRN) of insomnia rats treated by acupuncture. METHODS: A total of 36 male Sprague-Dawley (SD) rats of clean grade were randomly divided into a control group, a model group, an acupuncture group, and an acupuncture+antagonist group, with 9 rats in each group. The model group, the acupuncture group, and the acupuncture+antagonist group were treated with intraperitoneal injection of p-chlorophenylalanine (PCPA) to establish insomnia model. After successful modeling, the acupuncture group and the acupuncture+antagonist group received acupuncture at bilateral Neiguan (PC6) and Zusanli (ST36) once daily for 5 consecutive days. Thirty min before each acupuncture session, the acupuncture+antagonist group was intraperitoneally injected with methyllycaconitine citrate (MLA), an α7-nAChR antagonist, at a dosage of 5 mg/kg while the acupuncture group received the same volume of 0.9% sodium chloride solution. The rats' daytime spontaneous activity was observed. Neuronal discharge in the TRN was detected using neuroelectrophysiological methods. Immunofluorescence staining was used to detect parvalbumin-positive (PV⁺) neurons and co-expression of PV⁺ and postsynaptic density protein-95 (PSD-95) in the TRN. RESULTS: Compared with the control group, the model group showed increased daytime spontaneous activity (P<0.01); decreased average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); decreased neuronal discharge frequency (P<0.01), prolonged inter-discharge intervals (P<0.01) in the TRN; reduced number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.01). Compared with the model group, the acupuncture group showed decreased daytime spontaneous activity (P<0.01); increased average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); increased neuronal discharge frequency (P<0.01), shortened inter-discharge intervals (P<0.01) in the TRN; increased number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.05). Compared with the acupuncture group, the acupuncture+antagonist group exhibited increased daytime spontaneous activity (P<0.01); reduced average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); decreased neuronal discharge frequency (P<0.05), prolonged inter-discharge intervals (P<0.05) in the TRN; reduced number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.01). CONCLUSION α7-nAChR are involved in mediating the regulatory effect of acupuncture on circadian rhythm disturbances in PCPA-induced insomnia rats. Blocking α7-nAChR attenuates the activating effect of acupuncture on TRN neurons, and reduces the expression of PSD-95 protein on GABAergic neurons.
Animals ; Male ; Acupuncture Therapy ; alpha7 Nicotinic Acetylcholine Receptor/genetics* ; Rats, Sprague-Dawley ; Rats ; Sleep Initiation and Maintenance Disorders/physiopathology* ; Neurons/metabolism* ; Humans ; Thalamic Nuclei/physiopathology* ; Acupuncture Points ; Disks Large Homolog 4 Protein

Pelvic organ prolapse (POP), whose etiology is influenced by genetic and clinical risk factors, considerably impacts women's quality of life. However, the genetic underpinnings in non-European populations and comprehensive risk models integrating genetic and clinical factors remain underexplored. This study constructed the first polygenic risk score (PRS) for POP in the Chinese population by utilizing 20 disease-associated variants from the largest existing genome-wide association study. We analyzed a discovery cohort of 576 cases and 623 controls and a validation cohort of 264 cases and 200 controls. Results showed that the case group exhibited a significantly higher PRS than the control group. Moreover, the odds ratio of the top 10% risk group was 2.6 times higher than that of the bottom 10%. A high PRS was significantly correlated with POP occurrence in women older than 50 years old and in those with one or no childbirths. As far as we know, the integrated prediction model, which combined PRS and clinical risk factors, demonstrated better predictive accuracy than other existing PRS models. This combined risk assessment model serves as a robust tool for POP risk prediction and stratification, thereby offering insights into individualized preventive measures and treatment strategies in future clinical practice.
Humans ; Female ; Pelvic Organ Prolapse/epidemiology* ; Middle Aged ; Risk Assessment/methods* ; China/epidemiology* ; Multifactorial Inheritance ; Aged ; Risk Factors ; Genome-Wide Association Study ; Genetic Predisposition to Disease ; Case-Control Studies ; Adult ; Polymorphism, Single Nucleotide ; Genetic Risk Score ; East Asian People

Bone defect has always been a major clinical challenge because of its great difficulty and long period of treatment. Drynariae Rhizoma is a commonly used medicine in osteology and traumatology of traditional Chinese medicine， and its active ingredients（mainly flavonoids） facilitate osteoblast differentiation of bone marrow mesenchymal stem cells， osteoclast proliferation， vascular-osteogenic coupling， and inhibit osteoclast activity to promote bone mineralization， and repair and reconstruction of bone defect. As a good substitute for bone regeneration drugs， the active constituents of Drynariae Rhizoma can be loaded on scaffold materials of tissue engineering， which greatly improves the bioavailability of the drug. Meanwhile， the sustained-release microspheres also solve some problems such as sudden drug release from the scaffolds， and the composite scaffolds with active ingredient of Drynariae Rhizoma prepared by them have good ossification activity and osteoinduction， with precise bone repair effects， which meet the diverse performance requirements of bone grafts and have a promising clinical application prospect.

Objective:To summarize and analyze the clinical outcome of minimally invasive McKeown esophagectomy for esophageal squamous cell carcinoma.Methods:We retrospectively analyzed the clinical data of patients with esophageal squamous cell carcinoma who received minimally invasive McKeown esophagectomy in Peking Union Medical College Hospital from January 2017 to December 2022. Improved anesthesia methods, monitoring of recurrent laryngeal nerve, minimally invasive gastrostomy, and jejunostomy techniques were introduced in surgical procedure. We evaluated perioperative data and long-term follow-up survival in these patients.Results:A total of 226 esophageal squamous cell carcinoma patients who met the inclusion and exclusion criteria were enrolled, of which 48.2% received neoadjuvant therapy. The mean operation time was( 327 ± 68) min, with a total of 40.5(33, 50) lymph nodes and 27(19, 33) thoracic lymph nodes harvested. The postoperative hospital stay was 9(7, 12) days, and the postoperative complication rate was 36.3%. In terms of learning curve, after 50 patients intraoperative blood loss, postoperative hospital stay, and recurrent laryngeal nerve injury rate were significantly decreased. The number of total lymph nodes, thoracic lymph nodes, and the 106tbl harvested was significantly increased. The median follow-up time was 23.5(14, 47) months, with a loss of follow-up rate of 3.5%. The overall 2-year and 5-year survival rates were 82.6% and 71.8%, respectively.Conclusion:Improved minimally invasive McKeown esophagectomy for esophageal squamous cell carcinoma are safe and acceptable. Learning curve can be shortened, with increased lymph node harvested and decreased postoperative complications, which improving the short-term and long-term outcomes of patients.

Objective:To analyze and summarize the oncological outcomes after laparoscopic radical trachelectomy (LRT) for early stage cervical cancer.Methods:The clinical data and follow-up results of 148 patients with early stage cervical cancer who underwent LRT in Renji Hospital, School of Medicine, Shanghai Jiao Tong University from July 2014 to June 2023 were collected, while tumor outcomes and postoperative pregnancy were analyzed retrospectively.Results:(1) General situation: the median age of 148 patients with LRT was 33 years (range: 19-42 years). Pathological type: 111 cases of squamous cell carcinoma, 36 cases of adenocarcinoma, 1 case of adenosquamous carcinoma. International Federation of Gynecology and Obstetrics (2018) stage: 17 cases of stage Ⅰa1 with lympho-vascular space invasion, 25 cases of stage Ⅰa2, 102 cases of stage Ⅰb1, and 4 cases of stage Ⅰb2. (2) Tumor outcomes: 148 patients were followed up regularly after LRT, and the median follow-up time was 59 months (range: 2-104 months). During the follow-up period, 5 cases of tumor recurred (including 1 death), and the median recurrence time was 10 months (range: 4-33 months). Among them, there were 3 cases of pelvic metastasis, 1 case of distant metastasis, and 1 case of both pelvic and distant metastasis. Both 3-year and 5-year disease-free survival rates of 148 patients were 94.5%, and the 5-year overall survival rate was 98.9%. (3) Postoperative pregnancy: among 148 patients with LRT, 67 patients had pregnancy requirements, followed up for 1 year, and 20 of them were pregnant, with a pregnancy rate of 29.9% (20/67). Among the 20 pregnant patients, 2 cases early abortion, 1 case mid-term abortion, and 17 cases gave birth (including 4 cases of premature birth and 13 cases of full-term birth).Conclusion:Under the condition of strict control of surgical indications, guaranteed surgical scope and tumor-free operation, LRT in patients with early cervical cancer has a good outcome.

The biological clock(also known as the circadian rhythm)is the fundamental reliance for all organisms on Earth to adapt and survive in the Earth's rotation environment.Circadian rhythm is the most basic regulatory mechanism of life activities,and plays a key role in maintaining normal physiological and biochemical homeostasis,disease occurrence and treatment.Recent studies have shown that the biologi-cal clock plays an important role in the development of oral tissues and in the occurrence and treatment of oral diseases.Since there is cur-rently no guiding literature on the research methods of biological clock in stomatology,researchers mainly conduct research based on pub-lished references,which has led to controversy about the research methods of biological clock in stomatology,and there are many confusions about how to rationally apply the research methods of circadia rhythms.In view of this,this expert consensus summarizes the characteristics of the biological clock and analyzes the shortcomings of the current biological clock research in stomatology,and organizes relevant experts to summarize and recommend 10 principles as a reference for the rational implementation of the biological clock in stomatology research.

Objective To investigate the effect of long non-coding RNA(lncRNA)macrophage migration inhibitory factor antisense RNA1(MIF-AS1)on the malignant biological behavior of prostate cancer(PC)cells by regulating the miR-423-5p/pyrroline-5-carboxylic acid reductase 1(PYCR1)axis.Methods PC3 cells were cultured in vitro to knock down the expression of MIF-AS1 or down-regulate the expression of miR-423-5p.The expression of MIF-AS1,miR-423-5p and PYCR1 mRNA in tumor tissues and adjacent tissues and cells of PC patients were detected.The cell proliferation,apoptosis,migration,and invasion were detected and the expression of PYCR1 protein was detected by Western blot.The relationships between miR-423-5p,IF-AS1 and PYCR1 were verified.Results The MIF-AS1 and PYCR1 mRNA were observed to be highly expressed in the tumor tissues,while miR-423-5p was lowly expressed.Silenced MIF-AS1 inhibited the proliferation,migration and invasion of PC3 cells and up-regulated miR-423-5p induced cell apoptosis(P＜0.05).Inhibition of miR-423-5p expression reversed the inhibitory effect of silencing MIF-AS1 on malignant behavior of PC3 cells(P＜0.05).miR-423-5p was correlated with MIF-AS1 and PYCR1 by targeted regulation.Conclusion Silencing MIF-AS1 may inhibit the expression of PYCR1 by up-regulating miR-423-5p,thereby inhibiting the malignant behavior of PC cells.