Objective To isolate and cultivate Long-Evans rat bone mesenchymal stem cells (BMSCs) and identify their biological characteristics. Methods BMSCs were obtained from femurs of Long-Evan rats and dealt with 0.85 NH4Cl. The cell growth curve and cell cycle were measured and surface antigens were detected by flow cytometry. Osteoblast differentiation was studied by Alizarin red staining. Results BMSCs had active proliferative ability. The growth velocity of passage 5 cells was slower than that of passage 3 cells. The cell cycle analysis showed that 93.79 of BMSCs was at G0/G1 phase. BMSCs were positive for CD29 and CD44, but negative for CD45. Alizarin red staining of BMSCs after osteoblast induction was positive. Conclusion Highly purified BMSCs obtained from Long-Evan rats by 0.85 NH4Cl show stable biological properties.

OBJECTIVE To evaluate the genotoxicity of naproxen (NPX) impurities acetylnerolin (Ace). METHODS The genotoxicity of Ace was predicted by ADMET, Derek and Sarah with the quanti?tative structure-activity relationship (QSAR). The chromosomal aberration and bacterial reverse-muta?tion (Ames) tests were performed to verify the above results. In chromosomal aberration tests, CHL cells were incubated with Ace 10, 20 and 40 mg · L-1 for 4 h in the presence or absence of metabolic activation system solution (S9 mix). Methyl methane sulfonate (MMS) 20 mL · L-1 without S9 mix and cyclophosphamide (CP) 12 mg · L-1 with S9 mix served as positive control. The number of chromo?somes in each aberrant metaphase (including fissure, exchange, ring, break and polyploid) was counted and recorded, when the distortion rate less than 5％was considered negative and more than 10％was considered positive. In Ames test, the potential mutagenicity was evaluated using five strains of S. typhimurium ( TA97,TA98,TA100,TA102 and TA1535). They were treated with Ace 5, 25, 125 and 625μg per plate with or without S9 mix and incubated for 48-72 h. When without S9 mix, Dexon 50μg per plate served as positive control for TA97 and TA98, MMS 2.0μL per plate served as positive control for TA100 and TA102, and sodium azide 1.5μg per plate served as positive control for TA1535. When with S9 mix, 2-AF 100 μg per plate served as positive control for TA97, TA98 and TA100, 1, 8-dihydroxyanthraquinone (100μg per plate) served as positive control for TA102 and CP 50μg per plate served as positive control for TA1525. When the number of colonies was at least two-fold that of the negative control, the compound was considered mutagenic. RESULTS Although the Derek and Sarah software predicted that the NPX impurities were not genotoxic, ADMET data showed that Ace could induce chromosomal aberrations. The distortion rate of Ace 40 mg · L-1 was greater than 5％, but less than 10％. The distortion rate of Ace was less than 5％when<20 mg·L-1. Consistent with the results of ADMET, Ace might induce chromosomal aberrations. Ames test results showed that Ace did not signifi?cantly increase the number of bacteria (5-625μg per plate) compared with the negative control. Contrary to the ADMET results, Ace had no mutagenicity. CONCLUSION Ace has potential chromosomal muta?genicity. For life-long usage of NPX, the content of Ace should be reduced from 0.15％of conventional impurities to 0.015％.

Objective To explore the distribution of dendritic cells (DCs) and the expression of adhesion molecules in rat kidney with unilateral ureteral obstruction (UUO),as well as the regulatory effect of anti-P-selectin lectin-EGF domain monoelonal antibody (PsL-EGFmAb) on adhesion,maturation and function of human DCs cultured in vitro.Methods UUO rat models were established,which were divided into sham group (n=6),untreated group (n=18)and treated group with PsL-EGFmAb(n=18).DCs were analyzed with Axioplan 2 microscopy,while P-selectin being observed by immunohistochemistry.CD34~+ stem cells were isolated from cord blood and cultured in 20%IMDM medium with SCF,GM-CSF,TGF-?1,Flt-3L and TNF-?in vitro.During development, PsL-EGFmAb was added and IL-10 served as control.FACS was performed to detect the expression of HLA-DR,CD1a,CD11c,CD54,CD83,CD80,CD86,CD209 (DC-SIGN) and CD62-P,-E,-L (P-,E-,L-selectin) on DCs.RT-PCR was performed to detect the expression of NF-_KB P50, P65 mRNA.MLR was performed to detect the stimulatory effect of DCs on T cell proliferation and ELISA to determine IL-12p70 amount.Results Comparing with Sham group,the expression of P- selectin was up-regulated among tubulointerstitium mainly on renal tubular epithelial cell after unilateral ureteral obstruction on day 1,while CD1a~+CD80~+DCs being also found in renal interstitium. The expression of P-seleetin and CD1a~+CD80~+DCs was increased evidently on day 7,and correlated with the degree of renal tubulointerstitial fibrosis closely.However,these changes became less conspicuous in rat treated with PsL-EGFmAb.In vitro experiment showed on day 5 after cultured with the induction of TNF-?,immature DCs highly expressed C-type lectin DC-SIGN of pattern recognition receptors;the expression of co-stimulatory molecules such as CD11c,CD83,CD80 and CD86 on mature DCs was up-regulated in paralleling with the mRNA level of NF-_KB;the secretion of IL-12 was enhanced,as well as displaying the features of antigen-presenting cells with a higher ability to induce proliferation of T lymphocytes in vitro.In addition,L-selectin expressed highly on immature DCs,but lowly on mature DCs,neither of two DCs expressed P- and E-selectin.Compared with the IL-10 treated group,PsL-EGFmAb had an inhibitory effect on DC-SIGN of DCs with down- regulating the mRNA level of NF-_KB.PsL-EGFmAb could also inhibited CD11c,CD83,CD80, CD86 expression,reduced secretion of IL-12,and inhibited T cell proliferation stimulated by DCs in vitro.Conclusion DCs may play a critical role on initiating the inflammatory injury of renal tubulointerstitium,and the inhibitory effect of PsL-EGFmAb on DC maturation and function correlated with the inhibition of DC-SIGN,which is mainly mediated through NF-_KB signaling pathway.

Objective: To report a family diagnosed with Allan-Herndon-Dudley syndrome (AHDS) due to SLC16A2 gene mutation and to summarize the phenotypes, genotypes, diagnosis, treatment, and prognosis. Methods: The clinical features of a family of AHDS diagnosed in Xiangya Hospital of Central South University in November 2017 were analyzed. Related literature was searched at Online Mendelian Inheritance in Man (OMIM), PubMed, CNKI and Wanfang database (from the establishment of databases to June 2018) by using "Allan-Herndon-Dudley syndrome" , and "AHDS" as keywords and the case reports from April 2013 to June 2018 were reviewed. Results: The proband was a boy aged 8 months who presented with global developmental retardation, inability to hold up the head, disability to sit independently or grab, no language development, elongated face, big ears, esotropia, scoliosis, hypotonia in the trunk, hypertonia in extremities, and hyperreflexia. Brain magnetic resonance imaging (MRI) showed widening of the extracerebral space and delayed myelination. Thyroid function tests revealed increased FT3, decreased FT4 and normal TSH. Whole exome sequencing (WES) revealed the SLC16A2 gene c.431-1 (IVS1) G>C hemizygous mutation. The infant's mother and grandmother are carriers, but whose father had no related mutation. One uncle from maternal side had severe psychomotor retardation as well as dystonia and died at one year of age with unknown etiology. A total of 97 articles were retrieved in which 19 case reports were reviewed. Forty-two cases (22 from 8 families and 20 sporadic) were reported. Among these 42 cases (all males), all of them presented with moderate to severe cognitive dysfunction, 15 with seizures; 36 were bedridden, only 4 could walk; 31 had no language development, 2 could speak sentences, 4 could speak few words, 1 had babbling sounds. Furthermore,16 had microcephaly, 18 had facial dysmorphism, 6 had esotropia, 2 had hearing loss,14 had scoliosis, 11 had joint contracture, 30 had low body weight/muscle wasting, 37 had hypotonia in trunk or extremities, 32 had progressive spastic paraplegia or hypertonia. In terms of thyroid function, 33 had abnormal results, within whom 30 had increased T3, 25 had decreased T4 and 3 had increased TSH. Brain MRI showed delayed myelination in 22 cases, within which one normalized with development. Genetic tests showed that 31 had missense mutation (14 sporadic), 5 had deletion mutation (3 sporadic, and 1 due to frameshift mutation), 5 had insertion mutation (2 sporadic), and 1 had repeated mutation. The prognosis was poor as patients often died of recurrent respiratory tract infection. Conclusions The main clinical manifestations of AHDS are severe global developmental retardation, hypotonia, spastic paraplegia, abnormal serum levels of thyroid hormone and delayed brain myelination. SLC16A2 c. 431-1 (IVS1) G > C mutation is accountable for this disease.

Objective To investigate the expression of peroxisome proliferator-activated receptor γ (PPARγ) and uncoupling protein-1 (UCP-1) after sleeve gastrectomy in Zucker rats and to discuss the weight loss mechanisms.Metbods 30 male Zucker rats aged 10 weeks were randomly divided into 3 groups:the operation group (10 rats),the sham operation group(10 rats) and the diet-pairing group (10 rats).The rats were decapitated to retrieve the retroperitoneal adipose.mRNA and protein expressions of PPARγ and UCP-1 were detected by RT-PCR and Western blot.Results As for the operation group,the weight decreased significantly after the operation compared to the other two groups((250±5.8) g,(370±10.0) g,(310±9.6) g,respectively,P＜0.05).The expressions of PPARγ and UCP-1 gene of mRNA and protein were all significantly higher in the operation group (P＜0.05).Conclusions SG can up-regulate the expressions of thermogenic gene PPARγand UCP-1 in adipose in Zucker rats,browning the white adipose tissue,which was one of the important mechanisms of weight loss.

Objective To investigate mRNA expression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) after gastric bypass surgery (GBS) in rats with type 2 diabetes mellitus (T2DM).Methods 36 male Goto-Kakizaki rats,aged 12 weeks,were randomly divided into GBS,sham operation with diet restriction (SO),and sham operation alone(control) groups(n=12 per group).The blood lipid levels and fasting plasma glucose (FPG)levels in rats before and 8 weeks after surgery were measured and compared.The insulin sensitivity index (ISI)was calculated.Real-time polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of PEPCK mRNA and protein in hepatocytes at 8 weeks after operation.Results 8 weeks after operation,the blood lipid levels [TC(1.25±0.08) mmol/L,TG (0.93±0.10) mmol/L,FFA(0.88±0.12) mmoUL] in GBS group were significantly lower than those before operation [TC (2.31 ±0.52) mmol/L,TG(1.44±0.27) mmol/L,FFA (1.08±0.06) mmol/L] (P＜0.05).The fasting blood glucose levels in GBS decreased from (11.73±0.37) mmol/L to (5.13±0.22) mmol/L (P＜0.05),and ISI in GBS group increased from (-5.78±0.10) to (-4.64±0.15) (P＜0.05).PEPCKmR-NA (3.97±0.30) and protein (1.60±0.31) expression significantly reduced (P＜0.05).Conclusion GBS can reduce blood glucose in T2DM rats while improving glucose tolerance and hyperglycemia,and the mechanism appears to be associated with a decrease of hepatic PEPCK mRNA and protein expression.

Objective To investigate the expression of hormone sensitive lipase (HSL) and carnitine acyl transferase-1 (CAT-1) in Zucker rats after sleeve gastrectomy and to discuss the weight loss mechanism.Methods 30 male Zucker rats aging 10 weeks were randomly divided into 3 groups:the operation group (10 rats),the sham operation group (10 rats) and the diet-pairing group (10 rats).The rats were decapitated to retrieve the retroperitoneal adipose,mRNA and protein expression of HSL and CAT-1 gene of were detected by RTPCR and Western blot.Results As for the operation group,the weight decreased significantly after the operation comparing to the other two groups ((250±5.8) g,(370±10.0) g,(310±9.6) g,P＜0.05).mRNA and protein expressions of HSL and CAT-1 gene were all significantly higher in the operation group (P＜0.05).Conclusion SG can up-regulate the expressions of lipolysis gene HSL and CAT-1 in adipose in Zucker rats,promoting fat hydrolysis and increasing the energy expenditure,which is one of the important mechanisms of weight loss.

OBJECTIVE: To study the clinical features of the diseases associated with aminoacyl-tRNA synthetases (ARS) deficiency. METHODS: A retrospective analysis was performed of the clinical and gene mutation data of 10 children who were diagnosed with ARS gene mutations, based on next-generation sequencing from January 2016 to October 2019. RESULTS: The age of onset ranged from 0 to 9 years among the 10 children. Convulsion was the most common initial symptom (7 children). Clinical manifestations included ataxia and normal or mildly retarded intellectual development (with or without epilepsy; n=4) and onset of epilepsy in childhood with developmental regression later (n=2). Some children experienced disease onset in the neonatal period and had severe epileptic encephalopathy, with myoclonus, generalized tonic-clonic seizure, and convulsive seizure (n=4); 3 had severe delayed development, 2 had feeding difficulty, and 1 had hearing impairment. Mutations were found in five genes: 3 had novel mutations in the AARS2 gene (c.331G>C, c.2682+5G>A, c.2164C>T, and c.761G>A), 2 had known mutations in the DARS2 gene (c.228-16C>A and c.536G>A), 1 had novel mutations in the CARS2 gene (c.1036C>T and c.323T>G), 1 had novel mutations in the RARS2 gene (c.1210A>G and c.622C>T), and 3 had novel mutations in the AARS gene (c.1901T>A, c.229C>T, c.244C>T, c.961G>C, c.2248C>T, and Chr16:70298860-70316687del). CONCLUSIONS A high heterogeneity is observed in the clinical phenotypes of the diseases associated with the ARS deficiency. A total of 14 novel mutations in 5 genes are reported in this study, which enriches the clinical phenotypes and genotypes of the diseases associated with ARS deficiency.
Amino Acyl-tRNA Synthetases ; genetics ; Child ; Epilepsy ; Humans ; Mutation ; Phenotype ; Retrospective Studies

To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken (Gallus gallus) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/β and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/β level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/β. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.
Apoptosis* ; Aspirin ; Cell Survival ; Chickens* ; Heat Stress Disorders ; Heat-Shock Proteins* ; Hot Temperature* ; HSP90 Heat-Shock Proteins ; In Vitro Techniques* ; Proto-Oncogene Proteins c-akt ; Reactive Oxygen Species ; Transducers