Background Rhodopsin (RHO) gene is the most common disease gene for autosomal dominant retinitis pigmentosa (adRP),one of the main pathogenesis is that misfolded mutant RHO proteins accumulate in the endoplasmic reticulum and cause endoplasmic reticulum stress (ERS).Objective This study aimed to determine the genetic basis for a consanguineous Chinese Han adRP family.Methods This study procedure complied with Helsinki Declaration.All participants in the family were investigated under the informed consent.Regular ocular examination was performed on the patients in this family.Next-generation sequencing (NGS) was carried out to screen the mutations in 189 genes associated with hereditary retinal diseases (HRDs).After being analyzed and filtered,variations detected by NGS were validated by Sanger sequencing and evaluating of pathogenicity.The wild-type RHOWT and mutant RHOP53Rwere cloned into the vector pEGFP-N1.Then the two plasmids were transfected into adult retinal pigmentosa epithelium cell line(ARPE19) and human embryo kidney 293 line (HEK293) to observe the location of rhodopsin-GFP fusion protein in cells,and the expression of ERS related protein XBP1 in the cells was detected by quantitative-PCR and Western blot.Results This family included 5 generations with the typical adRP characteristics.Genetic analysis identified a heterozygous variation,p.P53R in RHO gene,which was fully cosegregated in the family.Wild-type RHOWT-GFP fusion proteins showed the green fluorescence on the endoplasmic reticulum and cytomembrane,but the misfolded mutant RHO-GFP fusion protein gathered only in endoplasmic reticulum.Compared to wild-type RHOWT,the XBP1 was activated and increased by (1.28 ±0.09) fold.The introns of 26 bases in XBP1 mRNA were removed in the HEK293 cells with mutant RHO-GFP fusion protein,and the expression of XBP1 was stronger in the HEK293 cells with mutant RHO-GFP than that in HEK293 cells with wild type RHO-GFP and cells with blank pEGFP-N1 plasmid.Conclusions Heterozygous variant RHO p.P53R is very likely the pathogenical mutation in the adRP family.The RHOP53R mutant rhodopsin protein can not be delivery effectively from the endoplasmic reticulum to the cell membrane,and these proteins accumulate in the endoplasmic reticulum,which causes ERS.

Learning guidance in pathology teaching can help to improve the students' learning ability. Learning guidance can be mainly done in two aspects:developing and fostering the students' learning ability on their own and guiding the students to review scientifically.

Objective To study the predictive value for adverse cardiac events (ACE) of left ventricular diastolic function measured by G-MPI. Methods Patients assessed by 2 days rest-stressed G-MPI during March 2012 to May 2013 in Beijing Hospital were collected and observed for the cumulative survival rate without ACE after G-MPI. ACE included cardiac death, non-fatal MI and late revascularization (60 d after the examination). Using quantitative gated SPECT (QGS) to get the LVEF and left ventricular peak filling rate (PFR). Using 17 segments, 5-point scoring system to get the summed stress score (SSS). Cox regression analysis was used to identify the independent predictors for ACE, and Kaplan-Meier method was used to investigate the cumulative survival rate without ACE.χ2 test was also used. Results A total of 11 patients who underwent early revascularization (within 60 d after G-MPI) were excluded and 139 patients (76 males, 63 females; average age 68.73 years) were assigned to different groups for outcome analysis, and 9 (6.5%, 9/139) patients had ACE in the whole group. Cox regression analysis showed that SSS, LVEF, PFR were the independent predictors for ACE (all P<0.05). Patients with PFR<2.1 EDV/s had a higher incidence of ACE than those with PFR≥2.1 EDV/s (14.3%(7/49) vs 2.2%(2/90), χ2=7.63, P<0.05). Kaplan-Meier survival analysis showed that the cumulative survival rate without ACE was lower in patients with PFR<2.1 EDV/s than that in patients with PFR≥2.1 EDV/s (67.7% vs 95.0%), and lower in patients with LVEF<50% than that in patients with LVEF≥50%(32.0% vs 90.8%), and also lower in patients with SSS≥8 than those with SSS<8(62.0% vs 94.7%; χ2 values: 11.92,11.27, 10.40, all P<0.01).Combining PFR with SSS and LVEF respectively, Kaplan-Meier survival analysis showed that the cumulative survival rate without ACE in patients with LVEF≥50% was higher than that in patients with LVEF<50% when PFR<2.1 EDV/s (76.7% vs 30.8%), and also higher in patients with SSS<8 than that in patients with SSS≥8 when PFR≥2.1 EDV/s (100% vs 72.2%; χ2 values: 7.14, 13.09, both P<0.01). Conclusion PFR of left ventricular diastolic function measured by G-MPI is one of the independent predictors for ACE, and it could effectively improve the predictive value for ACE if combined with LVEF and SSS.

Objective:To determine the effect of Notch1 signaling pathway on the differentiation and function of Th17 cells in murine psoriasis model.Methods: BALB/c mice were randomly divided into psoriasis model group and control group.Murine psoriasis model was established by topical 5% imiquimod application in combination with intraperitoneal injection of α-2b interferon.The CD4+ T lymphocytes were isolated by magnetic activated cell sorter (MACS).Flow cytometric analysis (FCM) was performed to detect the percentage of Th17 cells.Real-time RT-PCR was employed to measure the mRNA levels of RORγt,IL-17A,Notch1 and Hes-1.The CD4+ T lymphocytes were then divided into γ-secretase inhibitor DAPT groups and control group,and the expression differences of Notch1 signaling molecule and its target gene Hes-1 mRNA levels,Th17 cell percentage,RORγt and IL-17A mRNA levels,and IL-17A concentrations in cell-free supernatant were detected.Results: The expression levels of Th17 cell percentage and RORγt,IL-17A,Notch1 and Hes-1 mRNA in CD4+ T lymphocytes of murine psoriasis model were significantly higher than control mouse[(2.97±0.86)% vs.(0.65±0.11)%,t=15.083;(5.75±0.61) vs.(1.57±0.43),t=21.630;(7.83±0.97) vs.(1.63±0.31),t=25.348;(7.10±1.37) vs.(1.47±0.34),t=17.386;(7.30±1.15) vs.(1.67±0.48),t=18.840,respectively,all P<0.01].Compared with control group,Th17 cell percentage,mRNA expression levels of Notch1,Hes-1,RORγt and IL-17A,and IL-17A concentrations in cell-free supernatant from cultured CD4+ T lymphocytes of murine psoriasis model were dramatically decreased in DAPT treated groups in a dose-dependent way (F=74.368,89.719,126.572,94.558,124.323 and 123.231 respectively,all P<0.01).Conclusion: Notch1 signaling pathway can regulate the differentiation and function of Th17 cells in murine psoriasis model,and may have potential value for the target immunotherapy of psoriasis.

Objective To explore the change of S-100? and glial fibrillary acidic protein(GFAP)in serum after seizure and medication in children with epilepsy.Methods Serum protein level of S-100? and GFAP were determined by double antibody sandwish enzyme-linked immunosorbent assay(ELISA)in 41 cases with epilepsy and 30 healthy children.The specimen of venous blood were taken by 24 hours after seizure,4 weeks,12 weeks after medicine and their supernate preserved at-80 ℃ after centrifugat.Results Twenty-four hours after seizure,protein level of S-100?,GFAP in serum was significantly higher than that of control group(Pa0.05).Four weeks after medication,protein level of S-100?,GFAP in serum of epileptic group decreased,but still higher than that in control group,and the difference was significant(P

Adult ; Diagnosis, Differential ; Female ; Glycogen ; metabolism ; Glycogen Storage Disease Type II ; pathology ; Humans ; Liver ; metabolism ; pathology ; Muscle, Skeletal ; metabolism ; pathology ; Muscular Dystrophies, Limb-Girdle ; pathology ; Pyomyositis ; pathology ; Young Adult

OBJECTIVETo investigate osthole effect on femoral tissue resorption activity of rat in vitro.

METHODSSix SD rats weighted (80 ± 5) g were used to isolate and culture femoral tissue (diaphyses and metaphysis) in vitro. The cultured tissue were devided into control group, estradiol group and osthole group. The femoral tissue was treated with final concentration of 1 x 10(-5) mol/L osthole and 1 x 10(-8) mol/L estradiol culture in vitro at 48 hours after cultured. Tartrate-resistant acid phosphatase (StrACP) activity, glucose and Lactic acid content, StrACP, MCSF (Macrophage colony stimulating factor) and CTSK (Cathepsin K) mRNA was detected by Real-Time RT-PCR were detected.

RESULTSConcetration of Alkaline phosphatase activity were 2226 and 2498 in 1 x 10(-5) mol/L osthole and 1 x 10(-8) mol/L estradiol respectively. As compared with control group, the activity of StrACP of 1 x 10(-5) mol/L osthole and 1 x 10(-8) mol/L estradiol were inhibited at 6, 9, 12 days (P < 0.05); under treatment of in l x 10(-5) mol/L osthole, the content of Lactic acid were increased and the content of glucose were decreased at 3, 6, 9 days (P < 0.05); StrACP, MCSF and CTSK mRNA expression level were inhibited at 6, 9 days (P < 0.05).

CONCLUSIONOsthole can inhibit bone resorption and raise the level of nutrition metabolism of femurs tissue.

Acid Phosphatase ; metabolism ; Animals ; Bone Resorption ; prevention & control ; Coumarins ; pharmacology ; Estradiol ; pharmacology ; Femur ; drug effects ; Glucose ; analysis ; Lactic Acid ; analysis ; Male ; Rats ; Rats, Sprague-Dawley

Objective To construct eukaryotic enhanced green fluorescent protein (EGFP) expressing recombinant plasmid, pEGFP-C1-hnRNP A1, which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleo-protein A1), and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the 3′-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoRⅠ/BamHⅡdouble enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A 1 with poly (A)+mRNA (the marker of the stress granules), or DCP1a (the marker of processome) were detected by RNA fluores-cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested cor-rectly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly(A)+mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effec-tively. EGFP tagged hnRNP A1 takes part in forming stress granules.

BackgroundChoriodal neovascularization is an important ocular manifestation of angiogenesis in eyes,which derives from the choroid capillaries.Recent studies have found that complement activation is playing a key role in the laser-induced CNV.Because of the key position of CFB in the alternative pathway,bytargeting CFB and blocking the alternative pathway may provide an approach to observe the role of this alternative pathway in the generation of CNV.Objective This study was to investigate the inhibitory effect of reconstructed complement factor B (CFB)-small interfering ribonucleicacid(siRNA)on choroidal neovascularization (CNV)and its mechanism. Methods Experimental CNV was induced by laser photocoagulation in 96 eyes of 48 clean Brown Norway rats.The rats were randomly divided into 4 groups.25,50 and 75 μg B factor siRNA were injected via caudal vein on 1 day,3,5 days after photocoagulation in different dose groups,and normal saline solution was injected at the same way in experimental control group.Other 12 normal rats were used as blank control group.Fundus fluorescein angiography(FFA) was performed on 3,7,14,21,28 days after injection of CFB-siRNA and CNV was scored.The expressions of vascular endothelial growth factor(VEGF) and factor Ⅷ in choroid were detected by immunochemistry.The expressions of CFB-siRNA,VEGF,transforming growth factor β2( TGF-β2 )proteins in choroid were determined using immunochemistry in 7,14,21,28 days,and the expressions of mRNA of CFB-siRNA,VEGF,TGF-β2 were examined by reverse transcription polymerase chain reaction(RT-PCR). ResultsFFA revealed that the CNV rates in various doses of CFB-siRNA groups were significant lower than those of experimental control group in various time points(P＜0.05),and those in 75 μg B factor siRNA were decreased in comparison with 25 μg B factor siRNA (P＜0.05).Immunochemistry showed that the intensities of the VEGF and factor Ⅶ expression in various doses of CFB-siRNA groups were weaker than the blank control group ( P ＜ 0.05 ).Compared with the control group,the expression of CFB reduced in 7 days,and then approached to the level near the control group.Fourteen to twenty-one days after injection of CFB-siRNA,VEGF and TGF-β2 depressions in different doses of CFB-siRNA groups were lower than blank control group( P＜0.05 ).CFB expression in choroid showed the lower levels in CFB-siRNA injection group compared with blank control group in from 7 through 21 days (P＜0.05).RT-PCR displayed the gradual increase of CFB mRNA and curve-like changes of VEGF and TGF-β2 with time prolong. Conclusions Recombinated CFB-siRNA can effectively inhibit laser-induced CNV by down-regulating the expression of VEGF and factor Ⅷ.Alternative pathway of complement plays an important role in the production of CNV.

Objective To evaluate the diagnostic value of these alternant indexes by compare the results of routine blood test of early herpes zoster(HZ),acute cholecystitis and acute myocardial infarction patients.Methods According to some inclu-sion and exclusion criteria,78 HZ patients,59 acute cholecystitis and 51 acute myocardial infarction patients from October 2013 to March 2017 were included.The receiver operator characteristics(ROC)were used to evaluate the diagnostic value of the blood corpuscle indexes.Results White blood cell count(WBC),blood platelet count(PLT)and neutrophilic granulo-cyte percentage(NE%)of HZ patients were lower than the acute cholecystitis and acute myocardial infarction patients(P<0.001).Lymphocyte percentage(LYM%)and monocyte percentage(MO%)were higher than the others'.The NE% index had a certain diagnostic value(AUC=0.848,95%CI:0.792~0.905).There was a highest specificity in LYM% index(spe-cificity=0.963).Conclusion There were some clinical significance of routine blood cell count results on the early HZ diag-nosis,which can reduce the missed diagnosis and misdiagnosis rate of an early diagnosis herpes zoster.