The circadian clock plays a critical role in regulating various physiological processes, including gene expression, metabolic regulation, immune response, and the sleep-wake cycle in living organisms. Post-translational modifications (PTMs) are crucial regulatory mechanisms to maintain the precise oscillation of the circadian clock. By modulating the stability, activity, cell localization and protein-protein interactions of core clock proteins, PTMs enable these proteins to respond dynamically to environmental and intracellular changes, thereby sustaining the periodic oscillations of the circadian clock. Different types of PTMs exert their effects through distincting molecular mechanisms, collectively ensuring the proper function of the circadian system. This review systematically summarized several major types of PTMs, including phosphorylation, acetylation, ubiquitination, SUMOylation and oxidative modification, and overviewed their roles in regulating the core clock proteins and the associated pathways, with the goals of providing a theoretical foundation for the deeper understanding of clock mechanisms and the treatment of diseases associated with circadian disruption.
Protein Processing, Post-Translational/physiology* ; Circadian Rhythm/physiology* ; Humans ; Animals ; CLOCK Proteins/physiology* ; Circadian Clocks/physiology* ; Phosphorylation ; Acetylation ; Ubiquitination ; Sumoylation

This study aimed to explore the effects and mechanisms of total extracts from Anthriscus sylvestris on pulmonary hypertension in rats. Sixty male SD rats were divided into normal(NC) group, model(M) group, positive drug sildenafil(Y) group, low-dose A. sylvestris(ES-L) group, medium-dose A. sylvestris(ES-M) group, and high-dose A. sylvestris(ES-H) group. On day 1, rats were intraperitoneally injected with monocrotaline(60 mg·kg~(-1)) to induce pulmonary hypertension, and the rat model was established on day 28. From days 15 to 28, intragastric administration of the respective treatments was performed. After modeling and treatment, small animal echocardiography was used to detect the right heart function of the rats. Arterial blood gas was measured using a blood gas analyzer. Hematoxylin and eosin(HE) staining and Masson staining were performed to observe cardiopulmonary pathological damage. Flow cytometry was used to detect apoptosis in the lung and myocardial tissues and reactive oxygen species(ROS) levels. Western blot was applied to detect the expression levels of transforming growth factor-β1(TGF-β1), phosphorylated mothers against decapentaplegic homolog 3(p-Smad3), Smad3, tissue plasminogen activator(t-PA), and plasminogen activator inhibitor-1(PAI-1) in lung tissue. A blood routine analyzer was used to measure inflammatory immune cell levels in the blood. Enzyme-linked immunosorbent assay(ELISA) was used to detect the expression levels of P-selectin and thromboxane A2(TXA2) in plasma. The results showed that, compared with the NC group, right heart hypertrophy index, right ventricular free wall thickness, right heart internal diameter, partial carbon dioxide pressure(PaCO_2), apoptosis in cardiopulmonary tissue, and ROS levels were significantly increased in the M group. In contrast, the ratio of pulmonary blood flow acceleration time(PAT)/ejection time(PET), right cardiac output, change rate of right ventricular systolic area, systolic displacement of the tricuspid ring, oxygen partial pressure(PaO_2), and blood oxygen saturation(SaO_2) were significantly decreased in the M group. After administration of the total extract of A. sylvestris, right heart function and blood gas levels were significantly improved, while apoptosis in cardiopulmonary tissue and ROS levels significantly decreased. Further testing revealed that the total extract of A. sylvestris significantly decreased the levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and PAI-1 proteins in lung tissue, while increasing the expression of t-PA. Additionally, the extract reduced the levels of inflammatory cells such as leukocytes, lymphocytes, granulocytes, and monocytes in the blood, as well as the levels of P-selectin and TXA2 in plasma. Metabolomics results showed that the total extract of A. sylvestris significantly affected metabolic pathways, including arginine biosynthesis, tyrosine metabolism, and taurine and hypotaurine metabolism. In conclusion, the total extract of A. sylvestris may exert an anti-pulmonary hypertension effect by inhibiting the TGF-β1/Smad3 signaling pathway, thereby alleviating immune-inflammatory responses and thrombosis.
Animals ; Male ; Smad3 Protein/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Rats, Sprague-Dawley ; Rats ; Signal Transduction/drug effects* ; Hypertension, Pulmonary/genetics* ; Thrombosis/immunology* ; Drugs, Chinese Herbal/administration & dosage* ; Humans ; Apoptosis/drug effects*

OBJECTIVE: To establish an in vitro cell model simulating acute graft-versus-host disease (aGVHD) bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells (MSCs). METHODS: The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model, respectively. Bone marrow transplantation (BMT) mouse model (n=20) was established by being injected with bone marrow cells (1×10⁷ per mouse) from donor mice within 4-6 hours after receiving a lethal dose (8.0 Gy, 72.76 cGy/min) of γ ray general irradiation. A mouse model of aGVHD (n=20) was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells (1×10⁷ per mouse) and spleen lymphocytes (2×10⁶ per mouse). The blood was removed from the eyeballs and the mouse serum was aspirated on the 7^th day after modeling. Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2%, 5% and 10% BMT mouse serum and aGVHD mouse serum in the medium, respectively. The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining. The expression levels of related proteins PPARγ and CEBPα were detected by Western blot. The expression differences of key adipogenic transcription factors including PPARγ, CEBPα, FABP4 and LPL were determined by real-time quantitative PCR (RT-qPCR). RESULTS: An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established. Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10% compared with BMT serum. Western blot experiments showed that adipogenesis-related proteins PPARγ and CEBPα expressed in MSCs were down-regulated. Further RT-qPCR assay showed that the production of PPARγ, CEBPα, FABP4 and LPL, the key transcription factors for adipogenic differentiation of MSC, were significantly reduced. CONCLUSION The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.
Animals ; Mesenchymal Stem Cells/cytology* ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Adipogenesis ; Female ; Cell Differentiation ; Graft vs Host Disease/blood* ; Bone Marrow Cells/cytology* ; PPAR gamma/metabolism* ; Disease Models, Animal ; CCAAT-Enhancer-Binding Protein-alpha/metabolism*

OBJECTIVE: To investigate the relationship between physical activity and genetic risk and their combined effects on the risk of developing chronic obstructive pulmonary disease. METHODS: This prospective cohort study included 318,085 biobank participants from the UK. Physical activity was assessed using the short form of the International Physical Activity Questionnaire. The participants were stratified into low-, intermediate-, and high-genetic-risk groups based on their polygenic risk scores. Multivariate Cox regression models and multiplicative interaction analyses were used. RESULTS: During a median follow-up period of 13 years, 9,209 participants were diagnosed with chronic obstructive pulmonary disease. For low genetic risk, compared to low physical activity, the hazard ratios ( HRs) for moderate and high physical activity were 0.853 (95% confidence interval [ CI]: 0.748-0.972) and 0.831 (95% CI: 0.727-0.950), respectively. For intermediate genetic risk, the HRs were 0.829 (95% CI: 0.758-0.905) and 0.835 (95% CI: 0.764-0.914), respectively. For participants with high genetic risk, the HRs were 0.809 (95% CI: 0.746-0.877) and 0.818 (95% CI: 0.754-0.888), respectively. A significant interaction was observed between genetic risk and physical activity. CONCLUSION Moderate or high levels of physical activity were associated with a lower risk of developing chronic obstructive pulmonary disease across all genetic risk groups, highlighting the need to tailor activity interventions for genetically susceptible individuals.
Humans ; Pulmonary Disease, Chronic Obstructive/epidemiology* ; Exercise ; Male ; Female ; Middle Aged ; Prospective Studies ; Aged ; Genetic Predisposition to Disease ; Risk Factors ; United Kingdom/epidemiology* ; Incidence ; Adult

Objective To analyze the acupoint selection rules of acupuncture for the treatment of tic disorders in children based on data mining techniques.Methods A computerized search was conducted for the clinical research literature on acupuncture treatment of tic disorders in children included in the CNKI,Wanfang,VIP,SinoMed,and PubMed databases from January 1992 to December 2022.A database was established by Excel 2019 to count the commonly used treatment methods and analyze the high-frequency application methods acupuncture(high-frequency acupoints,channel entry of acupoints,acupoint association rules,and acupoint clustering),auricular point seed-pressing(high-frequency auricular points,and acupoint association rules),and the high frequency division of cluster needling of scalp point.Results A total of 190 valid literature articles were included,involving 270 acupuncture prescriptions;among them,184 acupoints were counted in the acupuncture method,with a total application frequency of 1 906 times,and the high-frequency application of the acupoints in descending order were Baihui(DU20),Taichong(LR3),Fengchi(GB20),Hegu(LI4),Sanyinjiao(SP6),Neiguan(PC6),Shenmen(HT7),Zusanli(ST36),Yintang(EX-HN3),Sishencong(EX-HN1);and the high-frequency meridians were governor vessol,foot taiyang stomach meridian,foot taiyang stomach meridian,foot shaoyang gallbladder meridian,hand taiyang large intestine meridian,foot taiyang bladder meridian,foot jueyin gallbladder meridian;three sets of strong association rules and five clusters of acupoints were analyzed by SPSS modeler 18.0 and IBM SPSS Statistics 26.0 software.There were 29 acupoints of auricular point seed-pressing,application total frequency was 206 times,high-frequency application of auricular points in descending order of Shenmen(HT7),liver,heart,subcortex,kidney;four groups of acupoint strong association rules were obtained through the analysis of SPSS modeler 18.0 software.A total of 14 zones were involved in the application of cephalic acupoint plexus zoning,of which the high-frequency zones were parietal anterior temporal diagonal,parietal parietal 1,and chorea tremor control zone.Conclusion Acupuncture treatment of tic disorders in children,according to its pathogenesis(liver hyperactivity,kidney depletion,spleen deficiency,phlegm disturbance,etc.)and tic site,select acupoints compatibility,and mostly choose yang meridian acupoints,which is related to the nature and treatment characteristics of wind pathogen.Children's tic disorders are closely related to emotional disorders,therefore acupuncture and auricular acupoints all emphasize the method of soothing the liver and clearing the heart,and regulating the emotional state.Cluster needling of scalp point mostly used parietal temporal anterior oblique line,parietal 1 line,and dance tremor control area for the treatment of tic disorders.For children,auricular point seed-pressing and cluster needling of scalp point has the minimun of pain,the effect of treatment is long,and it is not easy to have dangerous situations such as bent needle,broken needle and so on.

Objective:To establish a mesenchymal stem cell(MSC)-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease(aGVHD).Methods:Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors,and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients.The recipient mouse received a lethal dose(8.0 Gy,72.76 cGy/min)of total body γ irradiation,and injected with donor mouse derived bone marrow cells(1× 107/mouse)in 6-8 hours post irradiation to establish a bone marrow transplantation(BMT)mouse model(n=20).In addition,the recipient mice received a lethal dose(8.0 Gy,72.76 cGy/min)of total body γ irradiation,and injected with donor mouse derived bone marrow cells(1 × 107/mouse)and spleen lymphocytes(2 × 106/mouse)in 6-8 hours post irradiation to establish a mouse aGVHD model(n=20).On the day 7 after modeling,the recipient mice were anesthetized and the blood was harvested post eyeball enucleation.The serum was collected by centrifugation.Mouse MSCs were isolated and cultured with the addition of 2％,5％,and 10％recipient serum from BMT group or aGVHD group respectively.The colony-forming unit-fibroblast(CFU-F)experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC.The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining.In addition,the expression of self-renewal-related genes including Oct-4,Sox-2,and Nanog in MSC was detected by real-time fluorescence quantitative PCR(RT-qPCR).Results:We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD.CFU-F assay showed that,on day 7 after the culture,compared with the BMT group,MSC colony formation ability of aGVHD serum concentrations groups of 2％and 5％was significantly reduced(P＜0.05);after the culture,at day 14,compared with the BMT group,MSC colony formation ability in different aGVHD serum concentration was significantly reduced(P＜0.05).The immunofluorescence staining showed that,compared with the BMT group,the proportion of MSC surface molecules CD29+and CD 105+cells was significantly dereased in the aGVHD serum concentration group(P＜0.05),the most significant difference was at a serum concentration of 10％(P＜0.001,P＜0.01).The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4,Sox-2,and Nanog was decreased,the most significant difference was observed at an aGVHD serum concentration of 10％(P＜0.01,P＜0.001,P＜0.001).Conclusion:By co-culturing different concentrations of mouse aGVHD serum and mouse MSC,we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability,which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

Objective:To investigate the early clinical characteristics of elderly patients with severe burns and the risk factors on prognosis.Methods:This study was a retrospective case series study. Clinical data of 124 elderly patients with severe burns who met the inclusion criteria and were admitted to the 12 hospitals from January 2015 to December 2020 were collected, including 4 patients from the Fourth People's Hospital of Dalian, 5 patients from Fujian Medical University Union Hospital, 22 patients from Guangzhou Red Cross Hospital of Jinan University, 5 patients from Heilongjiang Provincial Hospital, 27 patients from the First Affiliated Hospital of Naval Medical University, 9 patients from the First Affiliated Hospital of Nanchang University, 10 patients from Affiliated Hospital of Nantong University, 9 patients from Tongren Hospital of Wuhan University & Wuhan Third Hospital, 12 patients from the 924 th Hospital of PLA, 6 patients from Zhangjiagang First People's Hospital, 4 patients from Taizhou Hospital of Zhejiang Province, and 11 patients from Zhengzhou First People's Hospital. The patients' overall clinical characteristics, such as gender, age, body mass index, total burn area, full-thickness burn area, inhalation injury, causative factors, whether combined with underlying medical diseases, and admission time after injury were recorded. According to the survival outcome within 28 days after injury, the patients were divided into survival group (89 cases) and death group (35 cases). The following data of patients were compared between the two groups, including the basic data and injuries (the same as the overall clinical characteristics ahead); the coagulation indexes within the first 24 hours of injury such as prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time, D-dimer, fibrinogen degradation product (FDP), international normalized ratio (INR), and fibrinogen; the blood routine indexes within the first 24 hours of injury such as white blood cell count, platelet count, neutrophil-to-lymphocyte ratio, monocyte count, red blood cell count, hemoglobin, and hematocrit; the organ function indexes within the first 24 hours of injury such as direct bilirubin, total bilirubin, urea, serum creatinine, aspartate aminotransferase, alanine aminotransferase, total protein, albumin, globulin, blood glucose, triglyceride, total cholesterol, alkaline phosphatase, creatine kinase, electrolyte indexes (potassium, sodium, chlorine, calcium, magnesium, and phosphorus in blood), uric acid, myoglobin, and brain natriuretic peptide; the infection and blood gas indexes within the first 24 hours of injury such as procalcitonin, C-reactive protein, pH value, oxygenation index, base excess, and lactate; treatment such as whether conducted with mechanical ventilation, whether conducted with continuous renal replacement therapy, whether conducted with anticoagulation therapy, whether applied with vasoactive drugs, and fluid resuscitation. The analysis was conducted to screen the independent risk factors for the mortality within 28 days after injury in elderly patients with severe burns. Results:Among 124 patients, there were 82 males and 42 females, aged 60-97 years, with body mass index of 23.44 (21.09, 25.95) kg/m 2, total burn area of 54.00% (42.00%, 75.00%) total body surface area (TBSA), and full-thickness burn area of 25.00% (10.00%, 40.00%) TBSA. The patients were mainly combined with moderate to severe inhalation injury and caused by flame burns. There were 43 cases with underlying medical diseases. The majority of patients were admitted to the hospital within 8 hours after injury. There were statistically significant differences between patients in the 2 groups in terms of age, total burn area, full-thickness burn area, and inhalation injury, and PT, APTT, D-dimer, FDP, INR, white blood cell count, platelet count, urea, serum creatinine, blood glucose, blood sodium, uric acid, myoglobin, and urine volume within the first 24 hours of injury (with Z values of 2.37, 5.49, 5.26, 5.97, 2.18, 1.95, 2.68, 2.68, 2.51, 2.82, 2.14, 3.40, 5.31, 3.41, 2.35, 3.81, 2.16, and -3.82, respectively, P<0.05); there were statistically significant differences between two groups of patients in whether conducted with mechanical ventilation and whether applied with vasoactive drugs (with χ2 values of 9.44 and 28.50, respectively, P<0.05). Age, total burn area, full-thickness burn area, serum creatinine within the first 24 hours of injury, and APTT within the first 24 hours of injury were the independent risk factors for the mortality within 28 days after injury in elderly patients with severe burns (with odds ratios of 1.17, 1.10, 1.10, 1.09, and 1.27, 95% confidence intervals of 1.03-1.40, 1.04-1.21, 1.05-1.19, 1.05-1.17, and 1.07-1.69, respectively, P<0.05). Conclusions:The elderly patients with severe burns had the injuries mainly from flame burns, often accompanied by moderate to severe inhalation injury and enhanced inflammatory response, elevated blood glucose levels, activated fibrinolysis, and impaired organ function in the early stage, which are associated with their prognosis. Age, total burn area, full-thickness burn area, and serum creatinine and APTT within the first 24 hours of injury are the independent risk factors for death within 28 days after injury in this population.

Objective To observe the clinical efficacy and safety of olanzapine tablets combined with magnesium valproate sustained-release tablets in the treatment of adolescent depressed patients.Methods Adolescents with depression were divided into control group and treatment group by simple random method.The control group was treated with oral olanzapine tablets with 5 mg·d-1 as the starting dose.After 1 week of treatment,the drug dose was adjusted according to the symptoms and kept within 20 mg·d-1.The treatment group was given oral magnesium valproate sustained-release tablet combined treatment on the basis of the control group,with 0.5 g as the initial dose,and the maximum dose was adjusted according to clinical symptoms after 1 week of treatment,and the maximum dose was no more than 1 g·d-1.Both groups were treated for 12 weeks.The clinical efficacy,excitatory amino acid(EAA),connectin level,intestinal fatty acid binding protein(Ⅰ-FABP),Hamilton depression scale(HAMD),Bech-Rafaelsen Mania Rating Scale(BRMS)and safety of the two groups were compared.Results Sixty-three cases were included in the treatment group and control group,respectively.After treatment,the total effective rates of the treatment group and the control group were 92.06％(58 cases/63 cases)and 79.37％(50 cases/63 cases),respectively,and the difference was statistically significant(P＜0.05).After treatment,the levels of EAA in the treatment group and the control group were(29.98±3.44)and(27.97±3.88)μg·mL-1;the levels of zonulin were(189.45±19.56)and(182.33±19.89)ng·mL-1;the levels of Ⅰ-FABP were(99.27±9.13)and(103.84±9.36)pg·mL-1,respectively;the HAMD scores of the treatment group and the control group were 9.88±1.03 and 10.74±1.95;the BRMS scores were 5.08±0.32 and 5.32±0.51,respectively.Compared with the control group,the differences of above indexes in the treatment group were statistically significant(all P＜0.05).The main adverse drug reactions in the two groups were weight gain,dry mouth,and drowsiness.The total incidences of adverse drug reactions in the treatment group and the control group were 12.70％and 15.87％,respectively,and the difference was not statistically significant(P＞0.05).Conclusion Olanzapine tablets combined with magnesium valproate sustained-release tablets can effectively increase plasma Ⅰ-FABP,EAA,and zonulin levels in adolescent depressed patients,and improve HAMD and BRMS scores,with good safety.

Objective: To analyze the trends of the age of menarche among Chinese Han girls aged 9 to 18 years from 2010 to 2019. Methods: Data were extracted from the Chinese National Surveys on Students' Constitution and Health in 2010, 2014 and 2019. A total of 253 037 Han girls aged 9 to 18 years with complete data on menarche were selected in this study. They were asked one-on-one about their menstrual status, age and residence information. The median age of menarche was estimated by probability regression. U tests were used to compare the difference in median age at menarche in different years. Results: The median age at menarche (95%CI) among Chinese Han girls was 12.47 (12.09-12.83) years in 2010, 12.17 (11.95-12.38) years in 2014 and 12.05 (10.82-13.08) years in 2019, respectively. Compared with that in 2010, the median age at menarche in 2019 decreased by 0.42 years (U=-77.27, P<0.001). The annual average changes were -0.076 years from 2010 to 2014 (U=-57.19, P<0.001) and -0.023 years from 2014 to 2019 (U=-21.41, P<0.001), respectively. The average annual changes in urban areas in the periods of 2010 to 2014 and 2014 to 2019 were -0.071 years and 0.006 years, respectively, while those in rural areas were -0.082 years and -0.053 years, respectively. The average annual changes in the regions of north, northeast, east, south central, southwest and northwest were -0.064, -0.099, -0.091, -0.080, -0.096 and -0.041 years in the period of 2010 to 2014 and 0.001, -0.040, -0.002, -0.005, -0.043 and -0.081 years in the period of 2014 to 2019. Conclusion: The age of menarche among Chinese Han girls aged 9 to 18 years shows an advanced trend from 2010 to 2019, and the trends in urban and rural areas and different regions have different characteristics.
Female ; Humans ; Menarche ; Probability ; East Asian People ; Child ; Adolescent

OBJECTIVES: To study the clinical features of children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant infection. METHODS: A retrospective analysis was performed on the medical data of 201 children with coronavirus disease 2019 (COVID-19) who were hospitalized and diagnosed with SARS-CoV-2 Omicron variant infection in Quanzhou First Hospital from March 14 to April 7, 2022. Among the 201 children, there were 34 children with asymptomatic infection and 167 with symptomatic infection. The two groups were compared in terms of clinical features, results of experimental examinations, and outcome. RESULTS: Of all the 201 children, 161 (80.1%) had a history of exposure to COVID-19 patients and 132 (65.7%) had a history of COVID-19 vaccination. Among the 167 children with symptomatic infections, 151 had mild COVID-19 and 16 had common COVID-19, with no severe infection or death. Among the 101 children who underwent chest CT examination, 16 had ground glass changes and 20 had nodular or linear opacities. The mean time to nucleic acid clearance was (14±4) days for the 201 children with Omicron variant infection, and the symptomatic infection group had a significantly longer time than the asymptomatic infection group [(15±4) days vs (11±4) days, P<0.05]. The group vaccinated with one or two doses of COVID-19 vaccine had a significantly higher positive rate of IgG than the group without vaccination (P<0.05). The proportions of children with increased blood lymphocyte count in the symptomatic infection group was significantly lower than that in the asymptomatic infection group (P<0.05). Compared with the asymptomatic infection group, the symptomatic infection group had significantly higher proportions of children with increased interleukin-6, increased fibrinogen, and increased D-dimer (P<0.05). CONCLUSIONS Most of the children with Omicron variant infection have clinical symptoms, which are generally mild. The children with symptomatic infection are often accompanied by decreased or normal blood lymphocyte count and increased levels of interleukin-6, fibrinogen, and D-dimer, with a relatively long time to nucleic acid clearance. Some of them had ground glass changes on chest CT.
Child ; Humans ; Asymptomatic Infections ; COVID-19/virology* ; COVID-19 Vaccines ; Fibrinogen ; Interleukin-6 ; Nucleic Acids ; Retrospective Studies ; SARS-CoV-2