Assessment of pathogenicity and functional characterization of APPL1 gene mutations in diabetic patients.

BACKGROUND: Adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) plays a crucial role in regulating insulin signaling and glucose metabolism. Mutations in the APPL1 gene have been associated with the development of maturity-onset diabetes of the young type 14 (MODY14). Currently, only two mutations [c.1655T>A (p.Leu552*) and c.281G>A p.(Asp94Asn)] have been identified in association with this disease. Given the limited understanding of MODY14, it is imperative to identify additional cases and carry out comprehensive research on MODY14 and APPL1 mutations. AIM: To assess the pathogenicity of APPL1 gene mutations in diabetic patients and to characterize the functional role of the APPL1 domain. METHODS: Patients exhibiting clinical signs and a medical history suggestive of MODY were screened for the study. Whole exome sequencing was performed on the patients as well as their family members. The pathogenicity of the identified APPL1 variants was predicted on the basis of bioinformatics analysis. In addition, the pathogenicity of the novel APPL1 variant was preliminarily evaluated through in vitro functional experiments. Finally, the impact of these variants on APPL1 protein expression and the insulin pathway were assessed, and the potential mechanism underlying the interaction between the APPL1 protein and the insulin receptor was further explored. RESULTS: A total of five novel mutations were identified, including four missense mutations (Asp632Tyr, Arg633His, Arg532Gln, and Ile642Met) and one intronic mutation (1153-16A>T). Pathogenicity prediction analysis revealed that the Arg532Gln was pathogenic across all predictions. The Asp632Tyr and Arg633His variants also had pathogenicity based on MutationTaster. In addition, multiple alignment of amino acid sequences showed that the Arg532Gln, Asp632Tyr, and Arg633His variants were conserved across different species. Moreover, in in vitro functional experiments, both the c.1894G>T (at Asp632Tyr) and c.1595G>A (at Arg532Gln) mutations were found to downregulate the expression of APPL1 on both protein and mRNA levels, indicating their pathogenic nature. Therefore, based on the patient's clinical and family history, combined with the results from bioinformatics analysis and functional experiment, the c.1894G>T (at Asp632Tyr) and c.1595G>A (at Arg532Gln) mutations were classified as pathogenic mutations. Importantly, all these mutations were located within the phosphotyrosine-binding domain of APPL1, which plays a critical role in the insulin sensitization effect. CONCLUSION: This study provided new insights into the pathogenicity of APPL1 gene mutations in diabetes and revealed a potential target for the diagnosis and treatment of the disease.

[Effects of TrkB-BDNF signal pathway on synthesis and secretion of vascular endothelial growth factor in human neuroblastoma cells].

OBJECTIVE: To study the effects of TrkB-BDNF signal pathway on the synthesis and secretion of vascular endothelial growth factor (VEGF) in human neuroblastoma cells (NB). METHODS: TrkB protein expression in SY5Y cells before and after all-trans-retinoicacid (ATRA) treatment was detected by Western blot. P-TrkB protein expression in SY5Y cells before and after the treatment of ATRA along with BDNF was also detected by Western blot. VEGF concentrations in the SY5Y cell culture supernatants were measured using ELISA after the treatment with ATRA, BDNF, tyrosine kinase inhibitor K252a and PI3k inhibitor LY294002. RESULTS: TrkB protein was undetectable in SY5Y cells before ATRA treatment. After the treatment of 1, 10 and 100 nM/L ATRA for five days, TrkB protein was expressed in SY5Y cells and the TrkB protein level increased with the increasing ATRA concentration. P-TrkB protein was not expressed in SY5Y cells treated only with 10 nM/L ATRA, but it was detectable after the treatment of ATRA along with BDNF. VEGF concentrations in the group treated with ATRA+BDNF were significantly higher than those in the untreated control and the ATRA alone treatment groups (P<0.01). VEGF concentrations in the K252a pretreated ATRA+BDNF group were significantly lower than those in the group treated with ATRA+BDNF (P<0.05). VEGF concentrations in the LY294002 treatment group (ATRA+LY294002+BDNF group) were also significantly lower than those in the group treated with ATRA+BDNF (P<0.01). CONCLUSIONS: Activation of TrkB-BDNF signal pathway may increase the synthesis and secretion of VEGF in human NB cells. The synthesis and secretion of VEGF can be inhibited by blocking TrkB-BDNF signal pathway with K252a or blocking the TrkB-BDNF downstream signal pathway PI3K/Akt with LY294002.

Research advances on the advantages and disadvantages of SMILE and FS-LASIK on correction of myopia / 国际眼科杂志(Guoji Yanke Zazhi)

In modern society, the most popular surgery for correction of myopia and myopic astigmatism are small incision lenticule extraction ( SMILE ) and femtosecond assisted laser in situ keratomileusis ( FS-LASIK ) . FS-LASIK is widely accepted by myopic patients and corneal refractive surgeons for its excellent safety, efficacy, stability and predictability. With the use of femtosecond laser, SMILE makes the corneal refractive surgery enter a full femtosecond era and become one of the most popular refractive surgery in the world, which is a novel minimally invasive corneal refractive surgery and characterized by flap - free, minimally invasive, small incision, femtosecond. But there is still some controversy about visual acuity, stability of diopter, corneal high order aberration, contrast sensitivity, dry eye, corneal biomechanical property and complication after SMILE and FS-LASIK in correction of myopia. This article reviews the advantages and disadvantages after SMILE and FS-LASIK in correction of myopia and myopic astigmatism among all above mentioned aspects.

Change of optical coherence tomography and multifocal electroretinography in myopia patients / 国际眼科杂志(Guoji Yanke Zazhi)

Myopia is the highest incidence of eye disease in society, it threated people`s eye health seriously.At present, many researchers measure the structural changes of retina which caused by the extension of eye axial due to the refractive error according to the optical coherence tomography(OCT).Besides, multifocal electroretinography(mf-ERG) was used to detect the function of retina in myopia.Global flash mf-ERG can evaluate the function of retina including both outer and inner retina.Some researchers focus on the correlation between structural and functional changes in myopia using OCT and mf-ERG.More researches are needed to clarify the structural and functional changes in myopia.

Expression of interleukin-9 in murine cornea with herpetic stromal keratitis / 中华实验眼科杂志

Background Herpetic stromal keratitis (HSK) is an immunopathologic eye disorder mediated by CD4+ T lymphocytes.Interleukin-9 (IL-9) is a cytokine linked to the process of many immune inflammatory diseases,but whether IL-9 is involved in the immunopathology of HSK remains unclear.Objective This study was to investigate the expression of IL-9 in murine cornea during the development of HSK and its relationship with the degree of HSK.Methods One hundred and eighty clean BALB/c mice were divided into the normal control group (20 mice) and experimental group (160 mice).HSK models were established by scratching on the surface of the cornea followed by inoculation of 1 × 106 plague forming unit(PFU) herpes simplex virus type 1 (HSV-1) KOS strain.Change of ocular surface was examined under the slit lamp biomicroscopy before and I day,3,5,7,8,10,14,21 days after inoculation of HSV-1.The corneas of mice were collected on the time points mentioned above.The relative expression level of IL-9 mRNA in the cornea of mice was detected by real-time PCR.Immunocytochemical localization of IL-9 protein in murine cornea was viewed by a confocal microscope after preparation of the corneal cryostat sections.All experimental manipulations were undertaken in accordance with the institutional guidelines for the care and use of laboratory animals.Results Punctiform,dendriform or geographic defect in corneal epithelium were seen 3 days and healing 6 days after inoculation of HSV-1.However,edema and opacity of the corneal stroma appeared 7 days and peaked 14 days following the inoculation.Real-time PCR assay showed that very little of IL-9 mRNA (A260/A280) was expressed in the cornea in the mice of the control group.But in 1 day,3,5,7,8,10 and 14 days,the relative level of IL-9 mRNA was 6.37±0.45,5.66±0.53,3.93±0.35,3.62±0.34,3.23±0.18,2.57±0.14,2.19±0.20,with a significant difference among the various time points (F=92.764,P=0.000),and IL-9 mRNA in 1 day,3,5,7,8,10 and 14 days after inoculation was significantly higher than before inoculation (P＜0.05);while no markedly difference was found in IL-9 mRNA expression between the 21-day group after inoculation and the normal control group (P =0.340).IL-9 protein was expressed in the epithelial layer,stromal layer and endothelial cell layer of the corneas,with a stronger green fluorescence in the HSK mice,but no expression of IL-9 protein was seen in the control mice.Conclusions HSK model can be successfully created by corneal scratching and inoculation of HSV-1 KOS strain in BALB/c mouse.IL-9 is involved in the pathogenesis and development of HSK mediated by immunoresponse.