INPP5E基因影响小鼠胚胎神经管闭合的分子机制研究

目的探讨肌醇多聚磷酸5-磷酸酶(INPP5E)基因影响小鼠胚胎神经管闭合的分子机制。方法采用前期复制的神经管缺陷(NTD)小鼠模型,在体视显微镜下观察胚胎表型、苏木精-伊红染色情况,测量胚胎顶臀长、体重等指标;重亚硫酸盐测序法检测NTD组及对照组胚胎发育第11.5天小鼠胚胎神经组织中INPP5E基因启动子区DNA甲基化情况;实时荧光定量聚合酶链反应和Western blotting检测胚胎神经组织中INPP5E蛋白和mRNA相对表达量;超高效液相色谱串联质谱法检测胚胎神经组织中叶酸(FA)、5-甲基四氢叶酸(5-MeTHF)、5-甲酰四氢叶酸(5-FoTHF)及同型半胱氨酸(Hcy)水平。结果NTD组小鼠胚胎神经组织INPP5E基因启动子区甲基化水平、INPP5E蛋白和mRNA相对表达量在胚胎发育第11.5天时较对照组低(P<0.05)。NTD组胚胎神经组织的FA和Hcy水平较对照组升高(P<0.05),而5-MeTHF和5-FoTHF水平较对照组低(P<0.05)。结论INPP5E基因在调节小鼠胚胎神经管闭合中起重要作用。FA代谢紊乱引起的INPP5E基因启动子区甲基化水平降低,可能通过影响其表达水平参与NTD的发生。

Complement factors C1q, C3 and C5b-9 in the posterior sclera of guinea pigs with negative lens-defocused myopia

· AIM: To investigate the expression of complement factors in the posterior scleral fibroblasts of guinea pigs with negative lens-defocused myopia.· METHODS: Eighteen guinea pigs were assigned randomly to two groups: the negative lens-defocused group(NLD group, n =9) and the normal control without treatment group(NC group, n =9). The effect of myopic induction was compared in three subgroups: eyes treated with a-10.00 D negative lens in the NLD group(NL group), eyes treated with a plano(0 D) lens in the NLD group(PL group), and untreated right eyes in the NC group(NC group). The following analyses were conducted at four weeks: examination of the refractive error via retinoscopy, assessment of complement C5b-9expression in the posterior scleral fibroblasts using immunohistochemistry, and measurements of complement C1 q and C3 protein levels in the posterior sclera by Western blot.·RESULTS: After an induction period of four weeks, a significant myopic shift was detected in the eyes of the NL group, relative to that of the PL and NC groups(P 【0.05). Data analysis showed a significant increase in the percentage of C5b-9 immunopositive fibroblasts in the posterior sclera of the NL group eyes, compared to the PL group(q =11.50, P 【0.001). Significantly higher levels of C1q(q =4.94, P =0.01) and C3(q =4.07, P =0.03)protein were detected in the posterior sclera of NL group eyes, compared to the PL group. There were no significant difference between the PL and NC groups for C5b-9(q =2.44, P =0.10), C1q(q =1.55, P =0.53) and C3(q =0.98, P =0.77) in the posterior sclera.·CONCLUSION: The data from present study provide evidence of the up-regulation of C5b-9, C1 q and C3 in the posterior scleral fibroblasts in a NLD myopic animal model. The results suggest that the complement system may be involved in the development of myopia.

Clinical features and in vivo confocal microscopy assessment in 12 patients with ocular cicatricial pemphigoid

AIM： To describe the clinical features and microstructural characteristics assessed by in vivo confocal microscopy（IVCM） in patients with ocular cicatricial pemphigoid（OCP）.· METHODS： A descriptive, uncontrolled case series study. Patients diagnosed with OCP were examined by clinical history, slit-lamp biomicroscopy features and IVCM images. The results of direct immunofluorescence（DIF） biopsies and indirect immunofluorescence（IIF） were also recorded. Local and systemic immunosuppressive therapy were administered and adjusted according to response.·RESULTS： A total of 12 consecutive OCP patients（7male, 5 female; mean age 60.42 ±10.39y） were recruited.All patients exhibited bilateral progressive conjunctival scarring and recurrent chronic conjunctivitis was the most frequent clinical pattern. The mean duration of symptoms prior to diagnosis of OCP was 2.95 ±2.85y（range： 5mo to 10y）. The Foster classification varied from stage I to IV and 20 eyes（83%） were within or greater than Foster stage Ⅲ on presentation. Two of the 12patients（17%） demonstrated positive DIF; 3 of the 12（25%） patients reported positive IIF. The mean duration of the follow-up period was 20.17 ±11.88mo（range： 6 to48mo）. IVCM showed variable degrees of abnormality in the conjuctiva-cornea and conjuctival scarring was detected in all the involved eyes. Corneal stromal cell activation and dendritic cell infiltration presented asocular surface inflammation, ocular surface keratinization along with the destroyed Vogt palisades was noted in eyes with potential limbal stem cell deficiency. After treatment, remission of ocular surface inflammation was achieved in all the patients, 18 eyes（75%） remained stable, 6 eyes（25%） had recurrent conjunctivitis and cicatrization in 2 eyes（8%） was progressing.· CONCLUSION： As an autoimmune disease, OCP manifests as variable degrees of clinical and laboratory abnormalities with both local and systemic immunosuppressive treatment playing important roles in disease therapy. IVCM can be as a valuable non-invasive technique to assess ocular surface changes in a cellular level with a potential value for providing diagnostic evidence and monitoring therapeutic effects during follow-up.

Bubble Formation in Apatite Structures by He-Ion Irradiation at High Temperature

Apatite ceramics Ca10(PO4)6 X2(X=F,OH)were prepared by the standard solid state sintering method and irradiated with He ions under a fluence of 5×10^16 ions/cm^2 at 450℃.Irradiation induced formation and growth of the He bubbles were observed by a transmission electron microscope.Hydroxyapatite Ca10(PO4)6(OH)2 and fluoroapatite Ca10(PO4)6 F2 with different He bubble morphologies indicate the influence of OH^-/F^-substitution on the He-ion annealing efficiency,as well as the structure itself,which affects the process of He bubble evolution and formation.The grain boundaries also act as sinks to accumulate He bubbles.No obvious irradiation damage but slight intensity reduction and left shift of diffraction peaks were observed according to the grazing incidence x-ray diffraction and Raman spectra characterizations,indicating that defects of interstitials and vacancies were generated.

Correlation of OPG/RANKL expression in gingival crevicular fluid with inflammatory factors, free radical generation and bone metabolism in patients with diabetes mellitus complicated by periodontitis

Objective:To study the correlation of OPG/RANKL expression in gingival crevicular fluid with inflammatory factors, free radical generation and bone metabolism in patients with diabetes mellitus complicated by periodontitis.Methods:Patients with diabetes mellitus complicated by periodontitis who were treated in Xi'an No. 4 Hospital between March 2015 and May 2017 were selected as the periodontitis group for the research, and healthy volunteers who underwent periodontal examination during the same period were selected as the control group. The gingival crevicular fluid was collected to determine the expression of OPG/RANKL, the contents of inflammatory factors and bone metabolism molecules as well as the generation of free radicals.Results: OPG protein expression, OPG/RANKL expression ratio as well as ICTP and Runx2 contents in gingival crevicular fluid of periodontitis group were significantly lower than those of control group whereas RANKL protein expression, TNF-α, IL-1β, IL-17, HMGB1, DKK1 and MMP1 contents as well as ROS, MDA and 8-OHdG generation were significantly higher than those of control group;OPG/RANKL expression ratio in gingival crevicular fluid of periodontitis group was negatively correlated with TNF- , IL-1β, IL-17, HMGB1, DKK1 and MMP1 contents as well as ROS, MDA and 8-OHdG generation, and positively correlated with ICTP and Runx2 contents.Conclusion: The excessive activation of inflammation and oxidative stress in periodontal tissue of patients with diabetes mellitus complicated by periodontitis can cause OPG/RANKL expression disorder and then lead to bone metabolism disorder.

RNF187 governs the maintenance of mouse GC-2 cell development by facilitating histone H3 ubiquitination at K57/80

RING finger 187(RNF187),a ubiquitin-ligating(E3)enzyme,plays a crucial role in the proliferation of cancer cells.However,it remains unclear whether RNF187 exhibits comparable functionality in the development of germline cells.To investigate thepotential involvement of RNF187 in germ cell development,we conducted interference and overexpression assays using GC-2 cells,a mouse spermatocyte-derived cell line.Our findings reveal that the interaction between RNF187 and histone H3 increases theviability,proliferation,and migratory capacity of GC-2 cells.Moreover,we provide evidence demonstrating that RNF187 interactswith H3 and mediates the ubiquitination of H3 at lysine 57(K57)or lysine 80(K80),directly or indirectly resulting in increasedcellular transcription.This is a study to report the role of RNF187 in maintaining the development of GC-2 cells by mediatinghistone H3 ubiquitination,thus highlighting the involvement of the K57 and K80 residues of H3 in the epistatic regulation of genetranscription.These discoveries provide a new theoretical foundation for further comprehensive investigations into the functionof RNF187 in the reproductive system.

A thermosensitive chitosan-based hydrogel for controlled release of insulin

Present study aims at synthesizing a thermosensitive hydrogel for controlled release of insulin. According to a modified method, hydroxybutyl chitosan （HBC） hydrogel possessed thermal sensitivity is prepared which can form hydrogel at over 25℃. The HBC hydrogel is non-cytotoxic to mice fibroblasts cells （L929）. Insulin is 100% entrapped in the hydrogel, 38% of which is released in vitro from the concentration of 5% hydrogel after 48 h, whereas by enzymolysis with lysozyme, 80% of the total insulin is released after 48h. This study suggests that HBC hydrogel could be utilized for controlled release of insulin in a non-invasive manner.

Strawberry Notch 1 (SBN01) promotes proliferation of spermatogonial stem cells via the noncanonical Wnt pathway in mice

While it is known that spermatogonial stem cells (SSCs) initiate the production of male germ cells, the mechanisms of SSC self-renewal, proliferation, and differentiation remain poorly understood. We have previously identified Strawberry Notch 1 (SBN01), a vertebrate strawberry notch family protein, in the proteome profile for mouse SSC maturation and differentiation, revealing SBN01 is associated with neonatal testicular development. To explore further the location and function of SBN01 in the testes, we performed Sbnol gene knockdown in mice to study the effects of SBN01 on neonatal testicular and SSC development. Our results revealed that SBN01 is required for neonatal testicular and SSC development in mice. Particularly, in vitro Sbnol gene knockdown with morpholino oligonucleotides caused a reduction of SSCs and inactivation of the noncanonical Wnt pathway, through Jun N-terminal kinases. Our study suggests SBN01 maintains SSCs by promoting the noncanonical Wnt pathway.

PTEN Lipid Phosphatase Activity Enhances Dengue Virus Production through Akt/FoxO1/Maf1 Signaling

Dengue virus(DENV) is an arthropod-borne viral pathogen and a global health burden. Knowledge of the DENV-host interactions that mediate virus pathogenicity remains limited. Host lipid metabolism is hijacked by DENV for virus replication in which lipid droplets(LDs) play a key role during the virus lifecycle. In this study, we reveal a novel role for phosphatase and tensin homolog deleted on chromosome 10(PTEN) in LDs-mediated DENV infection. We demonstrate that PTEN expression is downregulated upon DENV infection through post-transcriptional regulation and, in turn, PTEN overexpression enhances DENV replication. PTEN lipid phosphatase activity was found to decrease cellular LDs area and number through Akt/FoxO1/Maf1 signaling, which, together with autophagy, enhanced DENV replication and virus production. We therefore provide mechanistic insight into the interaction between lipid metabolism and the DENV replication cycle.