Objective:To explore the effect of miRNA-511-3p (miR-511-3p) on the proliferation and apoptosis of lung cancer cells, and the possible role of ATP2A2 and endoplasmic reticulum stress therein.Methods:Lung cancer tissues and paracancerous normal tissues (>2 cm from the tumor) were retrospectively collected from 69 lung cancer patients who were admitted to Huizhou Central People's Hospital from January 2020 to March 2022. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the transcript-level relative expression of miR-511-3p in cancer and paracancerous tissues, as well as immortalized lung epithelial cell line BEAS-2B, human lung cell line CCD-19Lu, and human lung carcinoma cell lines A549, H1975 and H1299. The lung cancer cell line A549 with the lowest relative expression level of miR-511-3p was selected for subsequent experiments. The cells were divided into miR control group (transfected with miR-511-3p irrelevant sequence), miR-511-3p overexpression group (transfected with miR-511-3p mimic), and miR-511-3p knockdown group (transfected with miR-511-3p repressor); and the additional A549 cells were taken and divided into ATP2A2 overexpression control group (transfected with its empty plasmid), ATP2A2 overexpression group (transfected with ATP2A2 overexpression plasmid), and ATP2A2 knockdown control group (transfected with irrelevant small interfering RNA plasmid) and ATP2A2 knockdown group (transfected with ATP2A2 small interfering RNA plasmid). The transcript-level relative expression of miR-511-3p and ATP2A2 in A549 cells in each group was detected by qRT-PCR; the relative expressions of ATP2A2 protein and endoplasmic reticulum stress marker proteins in each group were detected by Western blotting; the targeting relationship between miR-511-3p and ATP2A2 mRNA was verified by dual-luciferase reporter gene assay; CCK-8 assay was used to detect the proliferation ability of A549 cells in each group (expressed as absorbance value); flow cytometry was used to detect the percentage of apoptotic cells in A549 cells in each group; inorganic phosphorus colorimetry was used to detect the activity of Ca 2+-ATPase in A549 cells in each group; fluorescent probe assay was used to detect the Ca 2+ concentration in A549 cells in each group. Results:The transcript-level relative expression of miR-511-3p in lung cancer tissues and paracancerous tissues of 69 patients were 0.08±0.03 and 0.17±0.12, respectively, and the difference was statistically significant ( t= 6.04, P<0.05). The percentage of apoptotic cells in A549 cells in the miR-511-3p overexpression group, miR-511-3p knockdown group and miR control group was (58.1±6.1)%, (11.0±1.3)% and (22.0±2.1)%, respectively. The percentage of apoptotic cells in the miR-511-3p overexpression group was higher than that in the miR control group, and the percentage of apoptotic cells in the miR-511-3p knockdown group was lower than that in the control group, and the differences were statistically significant ( t values were 9.70 and 7.64, respectively, both P<0.05). After 24, 48 and 72 h of culture, the proliferation ability of A549 cells in the miR-511-3p overexpression group was lower than that in the miR control group, the proliferation ability of A549 cells in the miR-511-3p knockdown group was higher than that in the miR control group, and the differences were statistically significant (all P < 0.05). The percentage of apoptotic cells in A549 cells of ATP2A2 knockdown group and ATP2A2 knockdown control group were (58.2±1.5)% and (23.8±1.0)%, respectively, and the difference was statistically significant ( t= 33.94, P < 0.05); the percentage of apoptotic cells in A549 cells of ATP2A2 overexpression group and ATP2A2 overexpression control group were (13.8±2.0)% and (23.8±1.0)%, respectively, and the difference was statistically significant ( t= 7.96, P < 0.05). The transcript-level relative expression of ATP2A2 in A549 cells of miR-511-3p overexpression group was lower than that of miR control group, the transcript-level relative expression of ATP2A2 in A549 cells of miR-511-3p knockdown group was higher than that of control group, and the differences were statistically significant ( t values were 5.76 and 6.40, respectively, both P < 0.05); the relative expression of ATP2A2 protein in A549 cells of miR-511-3p overexpression group was lower than that of its control group, and the relative expression of ATP2A2 protein in A549 cells of miR-511-3p knockdown group was higher than that of its control group, and the differences were statistically significant (both P < 0.05). Dual-luciferase reporter gene assay verified the targeting relationship between miR-511-3p and ATP2A2 mRNA. The percentage of apoptotic cells in A549 cells in the control group, miR-511-3p overexpression group, ATP2A2 overexpression group, and miR - 511 - 3p overexpression+ ATP2A2 overexpression group were (21.5±3.0)%, (58.1±5.0)%, (13.3±1.2)%, and (20.5±4.0)%, respectively, and the difference was statistically significant (F= 73.28, P < 0.001); the percentage of apoptotic cells in A549 cells in the control group, miR-511-3p knockdown group, ATP2A2 knockdown group, and miR-511-3p knockdown+ ATP2A2 knockdown group were (23.5±3.0)%, (11.3±1.2)%, (60.1±7.0)%, and (25.6±5.0)%, respectively, and the difference was statistically significant ( F= 78.45, P < 0.001). The Ca 2+-ATPase activity in A549 cells of ATP2A2 overexpression group was higher than that of its control group, the Ca 2+-ATPase activity in A549 cells of ATP2A2 knockdown group was lower than that of its control group, and the differences were statistically significant ( t values were 4.61 and 6.07, respectively, both P < 0.05); the intracellular Ca 2+ concentration in A549 cells of ATP2A2 overexpression group was lower than that of its control group, the intracellular Ca 2+ concentration in A549 cells of ATP2A2 knockdown group was higher than that of its control group, and the differences were statistically significant ( t values were 3.30 and 3.95, respectively, both P < 0.05). The Ca 2+-ATPase activity in the miR-511-3p overexpression group was lower than that in the miR control group, the Ca 2+-ATPase activity in the miR- 511-3p knockdown group was higher than that in the miR control group, and the differences were statistically significant ( t values were 6.54 and 4.16, respectively, both P < 0.05); the intracellular Ca 2+ concentration in A549 cells of miR-511-3p overexpression group was higher than that of miR control group, the intracellular Ca 2+ concentration in A549 cells of miR - 511 - 3p knockdown group was lower than that of miR control group, and the differences were statistically significant ( t values were 3.60 and 6.23, respectively, both P < 0.05). The relative expressions of endoplasmic reticulum stress markers GRP78, PERK, p - eIF2a, ATF4, and CHOP proteins in A549 cells with knockdown of ATP2A2 or overexpression of miR-511-3p were higher than those in the corresponding control groups, and the differences were statistically significant (all P < 0.05); the relative expressions of all proteins in A549 cells with overexpression of ATP2A2 or knockdown of miR-511-3p were lower than those in the corresponding control groups (all P < 0.05). Conclusions:Changes in miR-511-3p level may affect the proliferation and apoptosis of lung cancer cells, and the mechanism may be that it affects the apoptosis of lung cancer cells by targeting ATP2A2 to regulate the endoplasmic reticulum stress.

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is the main pathogen that causes COVID-19,which is fast-mutating and highly transmissible.The infection has led to a global epidemic.As the main preventive and control measure,vaccination plays a critical role in fighting a-gainst COVID-19.Although a large number of epitope-based and mucosal vaccines have been stud-ied,few peptide epitope vaccines targeting the mucosa and their functional evaluation have been re-ported.In this study,we used SARS-CoV-2 structural protein M peptide epitope predicted by the IEDB database as an antigenic target to design the MS-3S gene containing 3 050 and 1 229 signal peptides and DCpep optimized for insertion into MS2 phage coat proteins.The expression plasmid pSIP:MS-3S was constructed by cloning the PCR fragments seamlessly and was transformed into Lactiplantibacillus plantarum 18 to obtain the recombinant bacterium LP18:MS-3S.Expression conditions such as induction time,inducer concentration,rotational speed and initial pH were opti-mized.The intranasal immunization experiments were performed to examine the vaccine efficacy.The results showed that the 916 bp-long target gene MS-3S modified and optimized was amplified and used to successfully construct the recombinant bacterial strain LP18:MS-3S.The optimal con-ditions for recombinant protein expression were obtained and verified by Western blot,flow cy-tometry,immunofluorescence and other detection methods.The optimal expression conditions were determined as follows:induction time was 4 h with 100 pg/L of SppIP as the optimal induction concentration.Antibody-specific for the epitope was verified by ELISA experiments in serum,alve-olar lavage fluid and fecal dilutions of mice.In summary,a recombinant bacterial strain expressing the epitope antigen of the SARS-CoV-2 M protein peptide was constructed.The obtained protein can induce the body to produce humoral and mucosal immunity,which lays the foundation for the development of a vaccine candidate for the mucosal immunity of COVID-19.

ObjectiveTo investigate the preventive and therapeutic effects of Tiaogan Huaxian pills combined with entecavir on hepatic fibrosis in chronic hepatitis B with liver Qi stagnation， spleen deficiency， and blood stasis syndrome and its effect on diffusion-weighted imaging （DWI）. MethodClinical data of 117 patients with liver disease who visited the Department of Hepatology at the First Affiliated Hospital of Guangxi University of Chinese Medicine from January 2021 to April 2022 were retrospectively analyzed. According to different treatment plans， they were divided into a control group （59 cases） and a treatment group （58 cases）. Both groups of patients received entecavir-based etiology treatment， and the treatment group added Tiaogan Huaxian pills on the basis of basic treatment. Both groups were treated for 24 weeks. Before and after treatment， the two groups were compared in terms of alanine aminotransferase （ALT）， advanced surgical technologies （AST）， total bilirubin （TBil）， hepatitis B virus （HBV）-DNA conversion rate， liver stiffness measurement （LSM）， four items of liver fibrosis （hyaluronidase， type Ⅲ pro-collagen， type Ⅳ collagen， and laminin）， the fibrosis index based on four factors （FIB-4）， the aspartate aminotransferase to platelet ratio index （APRI）， the apparent diffusion coefficient （ADC） value in magnetic resonance imaging （MRI）， and traditional Chinese medicine symptom scores， so as to analyze the efficacy of the two groups. ResultBefore treatment， there was no significant difference in ALT， AST， TBil， LSM， four items of liver fibrosis， FIB-4， APRI， HBV-DNA conversion rate， ADC value， and traditional Chinese medicine symptom scores between the two groups. After treatment， both groups of patients showed significant reductions in ALT， AST， TBil， LSM， hyaluronidase， type Ⅲ pro-collagen， type Ⅳ collagen， laminin， FIB-4， and APRI （P<0.05） and a significant increase in ADC value （P<0.05） and HBV-DNA conversion rate （P<0.01）. The traditional Chinese medicine symptom score of the treatment group decreased significantly （P<0.05）. Compared with the control group after treatment， the effective rate of clinical traditional Chinese medicine in the treatment group was 91.38% （53/58）， which was significantly higher than that of the control group （54.23%， 32/59） （Z=-4.325， P<0.01）. In the treatment group， ALT， AST， TBil， LSM， hyaluronidase， type Ⅲ pro-collagen， type Ⅳ collagen， laminin， FIB-4， APRI， and traditional Chinese medicine symptom scores all decreased significantly （P<0.05）， and the increase in ADC values was more significant （P<0.05）， while the difference in HBV-DNA conversion rate was not statistically significant. There were no serious adverse reactions or events in either group. ConclusionTiaogan Huaxian pills combined with entecavir have significant clinical efficacy in the treatment of hepatic fibrosis in chronic hepatitis B， which can reduce liver inflammation activity， delay hepatic fibrosis progression， and reduce traditional Chinese medicine symptom scores. It is worthy of clinical promotion and application.

The methodological framework for interventional clinical research of Chinese medicine （CM） under the research mode of syndrome dominating disease provides a set of technical principles and methods to design， evaluate， and implement of this kind. It consists of three main parts including general principles， research points and key design elements， with a total of 25 items. This methodological framework proposes implementing requirements and recommendations in a variety of aspects， including basic norms to be followed in relevant studies， perspectives for selecting research topics， as well as the technological details on study population （P）， intervention （I） and comparison（C）， outcome measurement （O）， time frame （T） of treatment and follow-up， sample orientation （prospective versus retrospective）， study design （S） format and type. To provide practical guidance for future studies， this article clearly explains each items of the methodological framework through some supportive cases.

Objective:To investigate the sleep status of preschool children with a coordination development disorder (DCD).Methods:Implementing stratified cluster sampling, 38, 900 questionnaires were distributed in 1000 classes of 200 kindergartens randomly selected in 11 prefecture-level cities of China′s Zhejiang Province. A total of 35, 464 valid responses were returned. On the basis of the responses, 2251 of the children were of (6.35%) were deemed DCD-positive, while the rest formed a DCD-negative group. The Children′s Family Social Environment and Sleep Health Questionnaire (1-6 years old) and the children′s sleep habits questionnaire were employed to investigate their sleep status.Results:①Regular sleeping time and duration, and falling asleep within 20 minutes were significantly less common in the DCD-positive group. In the negative group, shifting sleep place at night, finding it hard to sleep in strange environments, waking up ≥2 times a night, being awoken by others in the morning, difficulty in waking up and getting up in the morning, and bad mood after waking were significantly less frequent. ②In addition to delayed bedtime, the incidence of refusal to go to bed, fear of sleeping in the dark or alone, bedwetting, restless sleep, sleepwalking, talking in sleep, being awakened by nightmares, teeth grinding, loud snoring, apnea, dyspnea, crying and sweating after waking up at night, and insufficient sleep were all significantly higher in the DCD-positive group. ③The DCD-positive group also reported more sleeping in the daytime on weekends, more time on playing electronic products along with shorter sleep duration at night. There was, however, no significant difference between the two groups in sleep duration on school days and all day on weekends.Conclusion:Preschool children with a DCD have different sleep habits from those developing normally. They are more likely to suffer from sleep problems which affect their sleep duration.

OBJECTIVE To evaluate the efficacy and safety of PD-1/PD-L1 inhibitors for neoadjuvant treatment of bladder cancer， and to provide evidence-based reference for clinical treatment. METHODS Retrieved from PubMed， Cochrane Library， Embase， American Society of Clinical Oncology Meeting Library， CNKI， VIP and Wanfang database， etc.， the randomized controlled trials （RCTs）， non-RCT， case-control studies， cohort studies， etc. about PD-1/PD-L1 inhibitors for neoadjuvant treatment of bladder cancer were collected from the inception to Jan 31st， 2023. After literature screening， data extraction and quality evaluation， RevMan 5.3 software was used to perform meta-analysis of single-group rates； sensitivity analysis and publication bias analysis were conducted using Stata12 software. RESULTS A total of 25 studies were included in this discussion， involving 940 patients. The results of meta-analysis showed that the pathologic complete response （pCR） rate was 32% [OR＝0.32， 95%CI （0.22， 0.45）， P＝0.006]， downstaging rate was 52% [OR＝0.52， 95%CI （0.45， 0.60）， P＝0.55]， and the incidence of ≥grade 3 immune-related adverse events （irAEs） was 16% [OR＝0.16， 95%CI （0.11， 0.22）， P＜0.000 01]. Subgroup analysis showed that the patients receiving PD-1/PD-L1 inhibitors alone had a pCR rate of 25% and a incidence of Grade≥3 irAEs of 9%； the patients receiving combined immunotherapy had a pCR rate of 29% and a incidence of Grade≥3 irAEs of 28%； the patients receiving PD-1/PD-L1 inhibitors combined with chemotherapy had a pCR rate of 43% and a incidence of Grade≥3 irAEs of 12%； PD-L1 positive patients had a pCR rate of 44%， and PD-L1 negative patients had a pCR rate of 25%. The results of the sensitivity analysis showed that the study was robust. The results of the publication bias analysis showed that there was no significant publication bias. CONCLUSIONS PD-1/PD-L1 inhibitors are effective and safe for adjuvant treatment of bladder cancer.

Hyperuricemic nephropathy （HN）， a secondary renal damage common in clinical practice， is characterized by early concealing and continuous progression. The understanding of HN in traditional Chinese medicine （TCM） is from a macroscopic perspective. According to the TCM theory， HN is caused by the combination of external pathogens and internal injuries， with the main pathogenesis being root deficiency combined with superficial excess and deficiency-excess in complexity. In western medicine， the understanding of HN is from the microscopic perspective， which holds that the occurrence of HN is the result of inflammation， oxidative stress， renin-angiotensin-aldosterone system （RAAS） activation， and metabolic abnormalities. The TCM syndromes of HN include internal dampness and heat， obstruction in dampness and turbidity， deficiency of spleen and kidney， and deficiency of kidney yin. Accordingly， the prescriptions should clear heat and dampness， remove dampness and turbidity， tonify spleen and kidney， and nourish kidney yin， respectively. In addition to TCM prescriptions， single herbal medicines and their extracts， Chinese patent medicines， and external applications of Chinese medicines have played a significant role in the treatment of HN， promoting the application of TCM in the treatment of HN. Moreover， the integrated traditional Chinese and western medicine has also played a role in the treatment of HN， enriching the treatment schemes of HN. Different from common kidney diseases such as acute and chronic glomerulonephritis and nephrotic syndrome， HN with particularity should be carefully differentiated in clinical practice. This article systematically summarizes the research progress in the treatment of traditional Chinese medicine and integrated traditional Chinese and western medicine on hyperuricemic nephropathy with TCM and integrated traditional Chinese and western medicine， aiming to enrich the system and theory of HN treatment and further guide the clinical practice.

Objective To investigate the efficacy of continuous hepatic arterial infusion chemotherapy (HAIC) with the FOLFOX regimen and its multimodality therapeutic regimen in the treatment of patients with advanced hepatocellular carcinoma, as well as the influencing factors for prognosis. Methods A retrospective analysis was performed for the clinical data of 66 patients with advanced hepatocellular carcinoma who received continuous HAIC with FOLFOX regimen in Nanfang Hospital, Southern Medical University, from September 2018 to November 2021. The patients were observed in terms of objective response rate (ORR), disease control rate (DCR), median progression-free survival (mPFS), and median overall survival (mOS) after treatment, and treatment-related adverse reactions were recorded. For the patients with portal vein tumor thrombus, the effect of the treatment on portal vein tumor thrombus was assessed. The Kaplan-Meier method was used for survival analysis, and the Cox regression analysis was used to investigate the influencing factors for prognosis. Results According to the RECIST1.1 criteria, FOLFOX-HAIC and its multimodality therapeutic regimen achieved an ORR of 33.3% (22/66) and a DCR of 86.4% (57/66) in the treatment of 66 patients with advanced hepatocellular carcinoma, with an mPFS time of 8.2 months and an mOS time of 22.1 months. Among the 39 patients with portal vein tumor thrombus, 2 achieved complete remission, 8 achieved partial remission, 24 achieved stable disease, and 5 had disease progression, with an ORR of 25.6% (10/39) and a DCR of 87.2% (34/39). The main adverse reactions included gastrointestinal reactions (16.7%, 11/66), pyrexia (12.1%, 8/66), liver area pain (10.6%, 7/66), bone marrow suppression (3.0%, 2/66), and contrast agent allergy (3.0%, 2/66), and there were no grade > Ⅳ toxic or side effects or deaths caused by such complications. The Cox regression analysis showed that extrahepatic metastasis (hazard ratio [ HR ]=2.668, 95% confidence interval [ CI ]: 1.357-5.245, P < 0.05) and prothrombin time (PT) ( HR =1.282, 95% CI : 1.080-1.630, P < 0.05) were independent risk factors for PFS, and aspartate aminotransferase level ( HR =1.008, 95% CI : 1.002-1.013, P < 0.05) and PT ( HR =1.303, 95% CI : 1.046-1.630, P < 0.05) were independent risk factors for OS. Conclusion FOLFOX-HAIC and its multimodality therapeutic regimen has a certain clinical effect with controllable adverse reactions in the treatment of advanced hepatocellular carcinoma.

Objective:To analyze the clinical and pathological characteristics of chronic hepatitis B (CHB) patients with normal alanine aminotransferase (ALT) and not receiving antiviral therapy.Methods:This study retrospectively included CHB patients diagnosed by liver biopsy at the Third Hospital of Hebei Medical University from January 2008 to December 2022. According to the HBV DNA and HBeAg status of "immune tolerance period and immune control period", these patients were divided into three groups: chronic HBV carrier group, inactive HBsAg carrier group and indeterminate group including the patients that did not meet the inclusion criteria of the above two groups. Kruskal-Wallis H test was used for comparison of continuous data between multiple groups. Mann-Whitney U test was used for comparison of continuous data and ordered categorical data between two groups. Chi-square test or Fisher′s exact test was used for comparison of categorical data between two groups. Results:A total of 284 CHB patients with normal ALT were enrolled. There were 64, 88 and 132 cases in the chronic HBV carrier group, inactive HBsAg carrier group and indeterminate group, respectively. Histopathological analysis revealed that there were 182 (64.08%) cases with pathological inflammation grade (G) and/or fibrosis stage (S)≥2, 155 (54.58%) with S≥2 and 120 (42.25%) with G≥2. The proportion of patients with G and/or S≥2 in the indeterminate group [70.45% (93/132)] was higher than that in the chronic HBV carrier group [48.44% (31/64)] and inactive HBsAg carrier group [65.91% (58/88)] (both P<0.05). Patient′s age and the ratio of patients with S≥2 in the chronic HBV carrier group [33 years old, 39.06% (25/64)] were smaller than those in the inactive HBsAg carrier group [39 years old, 56.82% (50/88)] and the indeterminate group [39 years old, 60.61% (80/132)] (all P<0.05). Patients in the inactive HBsAg carrier group (19 U/L) had lower ALT levels than those in the chronic HBV carrier group (26 U/L) and the indeterminate group (23 U/L) (both P<0.05). The proportion of patients with cytoplasmic/cytoplasmic nuclear-type HBcAg was higher in patients with G and/or S≥2 than in patients with G and S<2 [73.08% (57/78) vs 32.08% (17/53), P<0.05], and the proportion of patients with cytoplasmic/cytoplasmic nuclear-type HBcAg increased gradually with age. The proportion of patients with cytoplasmic/cytoplasmic nuclear-type HBcAg was higher in patients with G and/or S≥2 than in patients with G and S<2 in the chronic HBV carrier status and indeterminate groups [93.33% (28/30) vs 43.33%(13/30), P<0.05; 59.46% (22/37) vs 12.50% (2/16); both P<0.05]. There was a statistically significant difference in the incidence of significant liver injury between patients≤ 30 years old and >30 years old [52.7% (39/74) vs 68.1% (143/210), P<0.05]. Conclusions:Significant liver injury occurred in 64.08% (182/284) of CHB patients with normal ALT not receiving antiviral therapy, which required the attention of clinicians. Among CHB patients with normal ALT, the expression site of HBcAg in hepatocytes was related to the occurrence of significant liver injury and could be expected to serve as an important indicator for predicting the patient′s status and the necessity of antiviral treatment. CHB patients with positive HBV DNA who were older than 30 years required antiviral treatment, and CHB patients≤30 years with normal ALT and significant hepatic tissue damage also required antiviral treatment.

In order to investigate developmental coordination disorder (DCD) of kindergarten children in Zhejiang province, 200 ordinary kindergartens were randomly selected by stratified random sampling in 11 prefecture-level cities of Zhejiang Province, and 38 900 children from 1 000 classes in each grade were then randomly selected into the study from June 2019 to December 2019. The Little DCD Questionnaire and a self-designed questionnaire were used to screen the DCD of those children. There were 36 807 valid questionnaires, and 6.50% (2 391/36 807) of them were positive results. The results showed that boy, age ≤5 years, overweight or obesity, left handedness, comorbidity with motor or developmental disorders and premature infants were risk factors of DCD in children. As for parents and families, maternal gestational age<20 years, maternal overweight or obesity before pregnancy, low-middle level education of parents, direct family and low income of family were also associated with DCD in children. Therefore, it is necessary to conduct early prevention and intervention strategies targeting on identified risk factors among relevant population.