ObjectiveTo explore the clinical features and causative genes of short stature children with unknown etiology， providing evidence for precise clinical diagnosis and treatment. MethodsThe study recruited children with suspected but undiagnosed short stature from the pediatric endocrinology department in our hospital between January 2018 and August 2022. A retrospective analysis was performed on the clinical manifestations， laboratory test and whole exome sequencing （WES） results. Causative genes were classified and analyzed according to different pathogenic mechanisms. ResultsA total of 48 children （30 boys and 18 girls） were enrolled， aged 7.73 ± 3.97 years， with a height standard deviation score （ HtSDS） of -3.63 ± 1.67. Of the patients， 33 （68.8%） suffered from facial anomalies， 31 （64.6%） from skeletal abnormalities， 26 ［54.2%， 61.5% of whom born small for gestational age （SGA）］ from perinatal abnormalities， 24 ［50.0%， 87.5% of whom with growth hormone （GH） peak concentration below normal］ from endocrine disorders and 21（43.8%） had a family history of short stature. Laboratory tests showed that GH peak concentration following stimulation test was （9.72 ± 7.25） ng/mL， IGF-1 standard deviation score was -0.82 ± 1.42， the difference between bone age and chronological age was -0.93 ± 1.39 years. Of the 25 cases with mutant genes found by WES， 14 （56.0%） had pathogenic mutation， 6 （24.0%） likely pathogenic mutation， and 5 （20.0%） mutation of uncertain significance. Pathogenic and likely pathogenic variants were identified in 14 genes， including 10 affecting intracellular signaling pathways （PTPN11， RAF1， RIT1， ARID1B， ANKRD11， CSNK2A1， SRCAP， CUL7， SMAD4 and FAM111A） and 4 affecting extracellular matrix （ECM） components or functions （ACAN， FBN1， COL10A1 and COMP）. ConclusionsA rare monogenic disease should be considered as the possible etiology for children with severe short stature accompanied by facial anomalies， disproportionate body types， skeletal abnormalities， SGA， GH peak concentration below normal and a family history of short stature. WES played an important role in identifying the monogenic causes of short stature. This study indicated that affecting growth plate cartilage formation through intracellular signaling pathways and ECM components or functions was the main mechanism of causative genes leading to severe short stature in children. Further research may help discover and study new pathogenic variants and gene functions.

Objective:To investigate the regulatory role of RNA m 6A methyltransferase (METTL14) in ultraviolet B (UVB) radiation-induced skin injury, and to preliminarily explore the potential of targeted inhibition of METTL14 for treating UVB-induced skin injury. Methods:A UVB radiation-induced skin injury model was established by exposing C57BL/6J mice to 150 mJ/cm 2 UVB, and was assessed and scored with HE staining and Masson staining. UVB radiation-induced cell injury models were established by exposing human immortalized keratinocytes (HaCaT) and human skin fibroblasts (WS1) to 10 and 30 mJ/cm 2 UVB, respectively. The m 6A levels in the mouse skin and cell models after UVB exposure were quantified by colorimetric assay, and m 6A-related enzymes in cells were measured by Western blot. HaCaT and WS1 cell lines overexpressing METTL14 were constructed using recombinant adenoviral vectors, and the overexpression effects were tested by Western blot. The METTL14 overexpression cells were examined for their m 6A levels, proliferative abilities after UVB exposure (by clone formation assay), and changes in apoptosis (by flow cytometry). The model mice with UVB-induced skin injury in the treatment groups received subcutaneous injection of the METTL14 inhibitor S-adenosylhomocysteine (SAH) solution (1 mg/kg, 5 mg/kg) twice consecutively before and after irradiation; and the mice were assessed and scored for skin injury with HE staining and Masson staining. Results:On the 4th day after 150 mJ/cm 2 UVB irradiation, the mice showed remarkable skin injury, pathologically featuring inflammatory infiltration, tissue structure disorganization, and collagen fiber degradation, reaching the maximum score; and the m 6A level in the skin was significantly downregulated ( t = 3.07, P < 0.05). At 24 h after 10 and 30 mJ/cm 2 irradiation, HaCaT and WS1 cells showed significantly reduced survival rates ( t = 7.64, 7.15, P < 0.05), significantly downregulated m 6A levels ( t = 4.78, 4.36, P <0.05), and significantly time-dependent downregulation of METTL14 protein expression ( t = 6.39, 4.76, P < 0.05). In HaCaT and WS1 cells, METTL14 overexpression significantly up-regulated m 6A levels ( t = 7.66, 3.67, P < 0.05), significantly inhibited the clone-forming ability of cells after UVB irradiation ( t = 6.29, 3.84, P < 0.05), and significantly increased the rate of cell apoptosis ( t = 3.48, 9.54, P < 0.05). Compared with those in the normal saline group, the model mice with UVB-induced skin injury in the SAH treatment group (5 mg/kg) showed significantly decreased pathological scores of skin injury ( t = 3.21, 4.27, 5.81, P < 0.05), with milder inflammatory infiltration, more orderly tissue structure, and less collagen fiber degradation. Conclusions:METTL14 can increase the sensitivity of skin cells to UVB radiation, and targeted inhibition of METTL14 can effectively alleviate UVB radiation-induced skin injury, which may be a potential new target for the treatment of UVB radiation-induced skin injury.

Objective To analyze the clinical manifestations and gene mutations of rare causes of primary adrenal insufficiency (PAI) in childhood.Methods The clinical features,laboratory tests and gene mutation of 13 patients with PAI in our hospital from September 2010 to August 2017 were analyzed retrospectively.Patients with congenital adrenal hyperplasia,X-linked adrenoleukodystrophy with neurological onset or a clear family history,and autoimmune adrenal insufficiency were excluded.Results The median age of 13 cases (12 males,1 female) was 3 years and 10 months.Medical history or clinical manifestations on the first visit included hyperpigmentation,electrolyte imbalance/salt-wasting crisis,gastrointestinal symptoms,and fatigue,etc.All developments of external genitalia were normal.All cases presented with decreased serum cortisol and increased ACTH levels.Some of the cases showed decreased aldosterone level and plasma renin activity,while 17α-hydroxyprogesterone,testosterone,and androstenedione were in the normal range.Part of cases revealed delayed bone age and adrenal atrophy.Three gene mutations were detected in 13 patients,including NR0B 1 gene (9/13),ABCD 1 gene (3/13),and CYP 11A 1 gene (1/13).NR0B1,and ABCD1 gene mutations were pathogenic mutations,consistent with clinical characteristics.CYP11A1 gene mutation was heterozygote,which cannot fully explain the clinical features.Conclusion PAI in childhood presents common clinical manifestations of adrenal insufficiency,e.g.hyperpigmentation and electrolyte imbalance/sah-wasting crisis,but without specificity.Gene mutational analysis is necessary for precise diagnosis and prognosis estimation.NR0B1 and ABCD1 gene mutations were common in childhood with rare causes of PAI.

Objective To study the perioperative comprehensive rapid rehabilitation care for single hole thoracoscopic lobectomypatients.Methods Clinical data of 68 cases with single-hole thoracoscopic lung resection were retrospectively analyzed, and were randomly divided into observation group(n=34) and control group(n=34), given perioperative rapid rehabilitation comprehensive nursing and routine nursing, respectively.Results Observation group had lower incidence rate and higher nursing satisfaction, and there were no died patients and all 68 patients were recovered after actively symptomatic treatment and careful nursing.Conclusion Single hole thoracoscopic pulmonary lobectomy is featured by relieving pain, shorten treatment courses, rapid recovery and shorter hospitalization time.

Objective To study the perioperative comprehensive rapid rehabilitation care for single hole thoracoscopic lobectomypatients.Methods Clinical data of 68 cases with single-hole thoracoscopic lung resection were retrospectively analyzed, and were randomly divided into observation group(n=34) and control group(n=34), given perioperative rapid rehabilitation comprehensive nursing and routine nursing, respectively.Results Observation group had lower incidence rate and higher nursing satisfaction, and there were no died patients and all 68 patients were recovered after actively symptomatic treatment and careful nursing.Conclusion Single hole thoracoscopic pulmonary lobectomy is featured by relieving pain, shorten treatment courses, rapid recovery and shorter hospitalization time.

Objective To study the influence of residual methylene blue after plasma viral inactivation on the human immune cell function by using the peripheral blood mononuclear cell(PBMC) .Methods PBMC were isolated by adopting the Ficoll‐Hypaque density gradient centrifugation method and co‐cultured for 72 h in presence of specific T cell stimulating factors(Anti‐CD3/28 and Anti‐CD28) ,with or without different concentration of methylene blue .The culture supernatant was collected and detected the cyto‐kines secretion situation by ELISA .After 66 h culture ,CCK‐8 dye was added and continueously cultured for 4-6 h ,the prolifera‐tion was determined at A450 .Results The high‐concentration doses of methylene blue (1 .25 ,2 .5 ,5 μmol/L groups) had signifi‐cantly inhibiting effect on the proliferation of PBMC stimulated by Anti‐CD3/28(P< 0 .01) ,its OD value was decreased from 0 .897 ± 0 .385 to 0 .632 ± 0 .334 ,0 .524 ± 0 .254 and 0 .445 ± 0 .287 respectively ,showing certain dose dependent effect .The high concentrations of methylene blue (1 .25 ,2 .5 ,5 μmol/L groups) could down‐regulate interleukin(IL)‐17a ,IL‐10 and interferon (IFN)‐γ secreted by anti‐CD28 induced PBMC ,moreover showing a dose dependent effect .1 .25 ,2 .5 ,5 μmol/L methylene blue af‐fected the IL‐17a level secreted by PBMC from (406 ± 57)pg/mL descending to (276 ± 38) ,(192 ± 31) ,(134 ± 24)pg/mL respec‐tively ;affected PBMC to secrete IL‐10 ,its level was reduced from (184 ± 15) pg/mL to (132 ± 13) ,(110 ± 12) ,(42 ± 8)pg/mL ;af‐fected PBMC to secrete IFN‐γ,its level was deduced from (4 512 ± 187)pg/mL to (2 876 ± 143) ,(2 234 ± 153) ,(1 988 ± 112)pg/mL respectively .Conclusion High concentrations of methylene blue (≥1 .25 μmol/L ) has the significant inhibiting effect on the proliferation and cytokine secretion functions of PBMC .In other words ,the residual methylene blue concentration in viral inactiva‐tion plasma (≤0 .33 μmol/L) has no obvious effect on the immune function of PBMC ,but whether this concentration of methylene blue having the effect on human pure T cell immune function needs to be further evaluated and studied .

Objective To investigate the expression of neive growth factor (NGF) in the ectopic endometrium in adenomyosis patients,and explore the relationship between NGF expression and innervation or pain scales.Methods From Mar.2009 to Oct.2009,45 adenomyosis patients undergoing hysterectomy in Obstetrics and Gynecology Hospital of Fudan University were enrolled in this study,which were classified into 33 cases in pain group and 12 cases in non-pain group based on symptom.The degree of dysmenoreal,chronic pelvic pain and dyspareunia was evaluated by visual analogue scale,including no pain,mild to moderate pain and severe pain group.In the mean time,26 patients with leiomyoma or cervical intraepithelial neoplasia Ⅲ (CIN Ⅲ)undergoing hysterectomy were defined as control group.Ectopic endometrium from experimental group and eutopic endometrium from control group were collected in the surgery.The expression of NGF was examined by immunohistochemistry.The density of protein gene product (PGP)9.5 positive nerve fibers was detected by immuno-fluorescence.Results The NGF level and the density of PGP 9.5 positive nerve fibers in adenomyosis pain group (0.25 ± 0.08,16 ± 8) were higher than adenomyosis painless (0.19 ± 0.05,P =0.007 ; 11 ± 5,P =0.018) and control group (0.18 ± 0.05,P =0.000;9 ± 4,P =0.000).The NGF level and the density of PGP9.5 positive nerve fibers in severe dysmenorrheal group(0.29 ± 0.07,19 ± 10) were higher than mild to moderate dysmenorrheal (0.22 ± 0.07,P =0.018 ; 13 ± 4,P =0.035) and painless group (0.18 ± 0.05,P =0.000 ; 11 ± 5,P =0.006) of adenomyosis patients.There was no difference of NGF level and the density of PGP 9.5 positive nerve fibers in chronic pelvic pain group and no chronic pelvic pain group of adenomyosis patients,so was dyspareunia group and no dyspareunia group.Conclusion The increased NGF level of adenomyosis nodules and improving innervation might be involved in the mechanism of adenomyosis related pain.

Objective To investigate the relationship between insulin resistance and endogenous androgens at early and late phase of postmenopause.Methods A total of 105 women with early postmenopause (≤5 years since menopause) and 107 women with late postmenopause (≥ 10 years since menopause) were enrolled in this study.In the mean time,those women were classified into normal weight [body mass index (BMI),BMI ＜24 kg/m2] group and overweight (BMI≥24 kg/m2) group.Sex hormonebinding globulin (SHBG),testosterone (T),dehydroepiandrosterone-sulfate (DHEA-S),fasting blood glucose(FBG),fasting insulin (FINS)levels were measured and then calculated free androgen index(FAI) and homeostatic model assessment of insulin resistance (HOMA-IR).The relationship between sex hormones and insulin resistance was analyzed by partial correlation and multiple linear regression analyses.Results Compared to early postmenopausal women,late postmenopausal women had higher FINS [(7.9 ± 6.6) mU/L versus (6.6 ±4.0) mU/L] and HOMA-IR(2.1 ± 1.9 versus 1.7 ± 1.1),but they had lower DHEA-S [(0.9 ± 0.5) mg/L versus (1.1 ± 0.5) mg/L,all P ＜ 0.05)].Both in early postmenopausal and late postmenopausal groups,overweight women had higher HOMA-IR (early group,2.2 ± 1.0 versus 1.2 ±0.9 ; late group,2.8 ± 2.6 versus 1.6±1.1)and FINS early group[(6.9±2.9) mU/L versus (4.6±2.0) mU/L] ;late group [(10.2 ± 9.3) mU/L versus (6.4 ± 3.6) mU/L] than those at women with normal weight group(all P ＜ 0.05).In early postmenopausal group,overweight women had lower SHBG [(52 ±37) nmol/L versus (71 ±37) nmol/L] and higher FAI(2.5 ±2.1) versus (1.3 ± 1.1) than those at normal weight women group(all P ＜ 0.05).In late postmenopausal group,overweight women had higher DHEA-S (1.0 ± 0.5) mg/L versus (0.8 ± 0.4) mg/L (P ＜ 0.05).The analyses suggested that in early postmenopausal group,SHBG was correlated negatively with FINS and HOMA-IR (β =-0.386,P ＜ 0.05 ;β =-0.553,P ＜0.05),DHEA-S was correlated positively with FBG (β =0.348,P ＜ 0.05) in early postmenopausal group.FAI was correlated positively with FBG in late postmenopausal group (β =0.505,P ＜ 0.05).Conclusions The increased androgenic activities are associated with insulin resistance after of menopause.These correlations are different at different stages of postmenopause,which SHBG levels correlate with high risk of insulin resistance and DHEA-S levels correlates with high blood glucose levels at early postmenopause and FAI correlates with high blood glucose levels at late postmenopause.

Objective To explore the effects of daidzein (DA) on the expressions of estrogen receptors (ER) and peroxisome proliferator-activated recepor γ (PPARγ) in osteoblasts and the influence of estrogen on these effects.Methods A mouse osteoblastic cell line MC3T3-E1 cultured in α-MEM containing 2％ FBS was treated by 0.1 and 10 μmol/L DA.ER antagonist ICI182780 and PPARγ antagonist GW9662 in 0.1 μmol/L was added as required,and an equivalent amount of phosphate buffer solution (PBS) was used as control.For the study on estrogen effect,the cells were treated by DA in the serum-free medium with or without 10 nmol/L 17β-estradiol (E2).The expressions of ERa,ERβ and PPARγ were determined by real-time RT-PCR and Western blot analysis,respectively.Results DA inhibited ER,expression but stimulated PPARγ expression in the cells at the concentration of 0.1 and 10 μmol/L.The down-regulation of ERα by DA could be blocked by ICI182780,whereas the up-regulation of PPARγ could be repressed by GW9662 in transcription levels.Furthermore,the inhibitory effect of DA on ERβ expression was markedly enhanced,while its stimulatory effect on PPARγ expression was almost lost in serum-free medium with 10 nmol/L 17βestradiol as determined by real-time RT-PCR.Conclusions Besides its direct roles in ERs and PPARγ mediated gene transcriptions,DA could exert indirect effect on cellular pharmacological responses by altering ER and PPARγ expressions.The predominant influence on receptors expression probably involved in the time-related biphasic effects of DA on osteogenesis,which was supposedly influenced by estrogen level.

To investigate different doses of daidzein (DAI) in regulating bone formation of osteoblasts, and the regulating mechanisms of estrogen receptors (ERs) and peroxisome proliferator-activated receptor γ (PPARγ) in bone formation.