GMFB/AKT/TGF-ß3 in Müller cells mediated early retinal degeneration in a streptozotocin-induced rat diabetes model.

Retinal degeneration, characterized by Müller cell gliosis and photoreceptor apoptosis, is considered an early event in diabetic retinopathy (DR). Our previous study proposed that GMFB may mediate diabetic retinal degeneration. This study identified GMFB as a sensitive and functional gliosis marker for DR. Compared to the wild type (WT) group, Gmfb knockout (KO) significantly improved visual function, attenuated gliosis, reduced the apoptosis of neurons, and decreased the mRNA levels of tumor necrosis factor α (Tnf-α) and interleukin-1ß (Il-1ß) in diabetic retinas. Tgf-ß3 was enriched by hub genes using RNA sequencing in primary WT and KO Müller cells. Gmfb KO significantly upregulated the transforming growth factor (TGF)-ß3 protein level via the AKT pathway. The protective effect of TGF-ß3 in the vitreous resulted in significantly improved visual function and decreased the number of apoptotic cells in the diabetic retina. The protection of Gmfb KO in primary Müller cells against high glucose (HG)-induced photoreceptor apoptosis was partially counteracted by TGF-ß3 antibody and administration of TGFBR1/2 inhibitors. Nuclear receptor subfamily 3 group C member 1 (NR3C1) binds to the promoter region of Gmfb and regulates Gmfb mRNA at the transcriptional level. NR3C1 was increased in the retinas of early diabetic rats but decreased in the retinas of late diabetic rats. N'-[(1E)-(3-Methoxyphenyl)Methylene]-3-Methyl-1H-Pyrazole-5-Carbohydrazide (DS-5) was identified as an inhibitor of GMFB, having a protective role in DR. We demonstrated that GMFB/AKT/TGF-ß3 mediated early diabetic retinal degeneration in diabetic rats. This study provides a novel therapeutic strategy for treating retinal degeneration in patients with DR.

The identification of a novel shared therapeutic target and drug across all insulin-sensitive tissues under insulin resistance.

Background: To identify key and shared insulin resistance (IR) molecular signatures across all insulin-sensitive tissues (ISTs), and their potential targeted drugs. Methods: Three datasets from Gene Expression Omnibus (GEO) were acquired, in which the ISTs (fat, muscle, and liver) were from the same individual with obese mice. Integrated bioinformatics analysis was performed to obtain the differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) was carried out to determine the "most significant trait-related genes" (MSTRGs). Enrichment analysis and PPI network were performed to find common features and novel hub genes in ISTs. The shared genes of DEGs and genes between DEGs and MSTRGs across four ISTs were identified as key IR therapeutic target. The Attie Lab diabetes database and obese rats were used to verify candidate genes. A medical drug-gene interaction network was conducted by using the Comparative Toxicogenomics Database (CTD) to find potential targeted drugs. The candidate drug was validated in Hepa1-6 cells. Results: Lipid metabolic process, mitochondrion, and oxidoreductase activity as common features were enriched from ISTs under an obese context. Thirteen shared genes (Ubd, Lbp, Hp, Arntl, Cfd, Npas2, Thrsp., Tpx2, Pkp1, Sftpd, Mthfd2, Tnfaip2, and Vnn3) of DEGs across ISTs were obtained and confirmed. Among them, Ubd was the only shared gene between DEGs and MSTRGs across four ISTs. The expression of Ubd was significantly upregulated across four ISTs in obese rats, especially in the liver. The IR Hepa1-6 cell models treated with dexamethasone (Dex), palmitic acid (PA), and 2-deoxy-D-ribose (dRib) had elevated expression of Ubd. Knockdown of Ubd increased the level of p-Akt. A lowing Ubd expression drug, promethazine (PMZ) from CTD analysis rescued the decreased p-Akt level in IR Hepa1-6 cells. Conclusion: This study revealed Ubd, a novel and shared IR molecular signature across four ISTs, as an effective biomarker and provided new insight into the mechanisms of IR. PMZ was a candidate drug for IR which increased p-Akt level and thus improved IR by targeting Ubd and downregulation of Ubd expression. Both Ubd and PMZ merit further clinical translational investigation to improve IR.

Tibial cortex transverse transport potentiates diabetic wound healing via activation of SDF-1/CXCR4 signaling.

Background: The current treatments for diabetic foot ulcers have disadvantages of slow action and numerous complications. Tibial cortex transverse transport (TTT) surgery is an extension of the Ilizarov technique used to treat diabetic foot ulcers, and can shorten the repair time of diabetic foot ulcers. This study assessed the TTT technique for its effectiveness in healing diabetic foot ulcer skin lesions and its related molecular mechanisms. Methods: Diabetic rat models were established by injecting healthy Sprague-Dawley rats with streptozotocin (STZ). The effects of TTT surgery on the model rats were assessed by recording changes in body weight, analyzing skin wound pictures, and performing H&E staining to assess the recovery of wounded skin. The numbers of endothelial progenitor cells (EPCs) in peripheral blood were analyzed by flow cytometry, and levels of CXCR4 and SDF-1 expression were qualitatively analyzed by immunofluorescence, immunohistochemistry, qRT-PCR, and western blotting. Results: Both the histological results and foot wound pictures indicated that TTT promoted diabetic wound healing. Flow cytometry results showed that TTT increased the numbers of EPCs in peripheral blood as determined by CD34 and CD133 expression. In addition, activation of the SDF-1/CXCR4 signaling pathway and an accumulation of EPCs were observed in skin ulcers sites after TTT surgery. Finally, the levels of SDF-1 and CXCR4 mRNA and protein expression in the TTT group were higher than those in a blank or fixator group. Conclusion: TTT promoted skin wound healing in diabetic foot ulcers possibly by activating the SDF-1/CXCR4 signaling pathway.

Rapidly determining the 3D structure of proteins by surface-enhanced Raman spectroscopy.

Despite great advances in protein structure analysis, label-free and ultrasensitive methods to obtain the natural and dynamic three-dimensional (3D) structures are still urgently needed. Surface-enhanced Raman spectroscopy (SERS) can be a good candidate, whereas the complexity originated from the interactions between the protein and the gradient surface electric field makes it extremely challenging to determine the protein structure. Here, we propose a deciphering strategy for accurate determination of 3D protein structure from experimental SERS spectra in seconds by simply summing SERS spectra of isolated amino acids in electric fields of different strength with their orientations in protein. The 3D protein structure can be reconstructed by comparing the experimental spectra obtained in a well-defined gap-mode SERS configuration with the simulated spectra. The gradient electric field endows SERS with a unique advantage to section biomolecules with atomic precision, which makes SERS a competent tool for monitoring biomolecular events under physiological conditions.

Effects of Asiaticoside Treatment on the Survival of Random Skin Flaps in Rats.

Background: Asiaticoside (AS) is extracted from the traditional herbal medicine Centella asiatica, and has angiogenic, antioxidant, anti-inflammatory, and wound-healing effects. We investigated the effects of AS on skin flap survival. Methods: Dorsal McFarlane flaps were harvested from 36 rats and divided into two groups: an experimental group treated with 40 mg/kg AS administered orally once daily, and a control group administered normal saline in an identical manner. On day 2, superoxide dismutase (SOD) and malondialdehyde (MDA) levels, and production of the cytokines tumor necrosis factor-α and interleukin (IL)-6 were evaluated. On day 7, tissue slices were stained with hematoxylin and eosin. The expression of vascular endothelial growth factor (VEGF), IL-6, and IL-1ß were immunohistochemically evaluated. Microcirculatory flow was measured using laser Doppler flowmetry. Flap angiography, using the lead oxide-gelatin injection technique, was performed with the aid of a soft X-ray machine. Results: The AS group exhibited greater mean flap survival area, improved microcirculatory flow, and higher expression levels of SOD and VEGF compared with the control group. However, MDA levels and the inflammatory response were significantly reduced. Conclusions: AS exhibits promise as a therapeutic option due to its effects on the viability and function of random skin flaps in rats.

Preparation of a novel monoclonal antibody against Avian leukosis virus subgroup J Gp85 protein and identification of its epitope.

Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that has caused huge economic losses in the poultry industry due to its great pathogenicity and transmission ability. However, the continuous emergence of new strains would bring challenges to diagnosis and control of ALV-J. .This study focuses on preparing the monoclonal antibody (MAb) against ALV-J Gp85 and identifying its epitope. The truncated ALV-J gp85 gene fragment was amplified and then cloned into expression vectors. Purified GST-Gp85 was used to immune mice and His-Gp85 was used to screen MAb. Finally, a hybridoma cell line named J16 that produced specific MAb against the ALV-J. Immunofluorescence assay showed that MAb J16 specifically recognized ALV-J rather than ALV-A or ALV-K infected DF-1 cells. To identify the epitope recognized by MAb J16, fourteen partially overlapping ALV-J Gp85 fragments were prepared and tested by Western blot. The results indicated that peptide 150-LIRPYVNQ-157 was the minimal epitope of ALV-J Gp85 recognized by MAb J16. Alignment analysis of Gp85 from different ALV subgroups showed that the epitope keep high conservation among 36 ALV-J strains, but significant different from that of ALV subgroup A, B, C, D, E and K. Overall, we prepared a MAb specific against ALV-J and identified peptide 150-LIRPYVNQ-157 as a novel specific epitope of ALV-J Gp85, which may assist in laying the foundation for specific ALV-J detection methods.

[Treatment of diabetic foot with vaccum sealing drainage combined with transverse tibial bone transport].

OBJECTIVE: To summarize the effectiveness and experience of Wanger grade 3-5 diabetic foot treated with vacuum sealing drainage (VSD) combined with transverse tibial bone transport. METHODS: Between March 2015 and January 2018, 21 patients with refractory diabetic foot who failed conservative treatment were treated with VSD combined with transverse tibial bone transport. There were 15 males and 6 females, aged 55-88 years (mean, 65 years). The diabetes history was 8-15 years (mean, 12.2 years). The duration of diabetic foot ranged from 7 to 84 days (mean, 35.3 days). The size of diabetic foot ulcer before operation ranged from 2 cm×2 cm to 8 cm×5 cm. According to Wanger classification, 8 cases were rated as grade 3, 11 cases as grade 4, and 2 cases as grade 5. Among the 21 cases, angiography of lower extremity before operation was performed in 5 cases, CT angiography of lower extremity in 16 cases, all of which indicated that the arteries below the knee were narrowed to varying degrees and not completely blocked. Preoperative foot skin temperature was (29.28±0.77)℃, C-reactive protein was (38.03±31.23) mg/L, leukocyte count was (9.44±2.21)×10 9/L, and the visual analogue scale (VAS) score was 6.8±1.5, and ability of daily living (Barthel index) was 54.3±10.3. RESULTS: After operation, 2 patients with Wanger grade 4 and smoking history failed treatment and had an major amputation (amputation above ankle joint) at 30 days and 45 days after operation, respectively. One patient with Wanger grade 5 and chronic heart failure died of cardiac arrest at 60 days after operation. The remaining 18 patients were followed up 6-24 months (mean, 9.2 months). The external fixator was removed at 40-62 days after operation, with an average of 46 days. All the wounds healed, with a healing time of 50-120 days (mean, 62.5 days). The pain of 18 patients' feet was relieved obviously, and there was no recurrence of ulcer in situ or other parts. There was no complication such as tibial fracture and ischemic necrosis of lower leg skin after operation. After ulcer healing, the foot skin temperature was (30.86±0.80)℃, C-reactive protein was (22.90±18.42) mg/L, VAS score was 2.4±1.2, and Barthel index was 77.3±4.6, all showing significant differences when compared with preoperative ones ( P<0.05); the leukocyte count was (8.91±1.72)×10 9/L, showing no significant difference ( t=1.090, P=0.291). CONCLUSION: VSD combined with transverse tibial bone transport can effectively promote the healing of Wanger grade 3-5 diabetic foot wounds, but smokers, unstable blood glucose control, and chronic heart failure patients have the risk of failure.

Theoretical Evaluation of the Influence of Molecular Packing Mode on the Intramolecular Reorganization Energy of Oligothiophene Molecules.

Accurate determination of the relationships among packing mode, molecular structure and charge transfer mobility for oligothiophene analogues has been significantly impeded, due to the lack of crystal structure information. In the current study, molecular dynamics (MD) were used to investigate the packing mode of non-, methyl- and ethyl-substituted poly(3-alkylthiophenes) (P3ATs). Obvious conformational changes were observed when comparing the packed and isolated oligothiophene molecules, indicating the important influence of packing mode on the geometric structures of these materials. Considering the crucial role played by reorganization energy (RE) in the charge transfer process, both quantum mechanics (QM) and quantum mechanics/molecular mechanics (QM/MM) were performed to examine the impact of different conformations on energy. Our simulations revealed that the geometric structures have distinct effects on the RE. Our data suggest that MD could give a reliable packing mode of oligothiophene analogues, and that QM/MM is indispensable for precisely estimating RE.