MicroRNA-5195-3p alleviates high glucose‑induced injury in human ARPE-19 cells by targeting GMFB

Hyperglycemia is generally considered to be an important cause of diabetic retinopathy (DR). The aim of the present study was to investigate the role of miR-5195-3p in high glucose (HG)-induced human retinal pigment epithelial ARPE-19 cell injury. Here, we first found that the expression level of miR-5195-3p was significantly downregulated in HG-stimulated ARPE-19 cells using reverse transcription quantitative PCR. Overexpression of miR-5195-3p attenuated the impaired cell viability, increased apoptosis and pro-inflammatory cytokines secretion in ARPE-19 cells under HG condition using CCK-8 assay, flow cytometry and ELISA assay, respectively. Luciferase reporter assay showed that miR-5195-3p could specifically bind to the 3’UTR of glia maturation factor-β (GMFB). GMFB overexpression reversed, while knockdown enhanced the protective effects of miR-5195-3p overexpression against HG-induced ARPE-19 cell injury. In summary, miR-5195-3p targeting GMFB might be a potential therapeutic target for DR.

Glia Maturation Factor Beta as a Novel Biomarker and Therapeutic Target for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common types of cancer. The novel sensitive biomarkers and therapeutic targets are urgently needed for the early diagnosis of HCC and improvement of clinical outcomes. Glia maturation factor-β (GMFB) is a growth and differentiation factor for both glia and neurons and has been found to be tightly involved in inflammation and neurodegeneration conditions. In our study, the expression level of GMFB was significantly up-regulated in patients with HCC and positively co-expression with tumor node metastases (TNM) stage and histopathological grade of HCC. The high expression level of GMFB was remarkably associated with poor overall survival, which mainly occurred in males rather than females. Multivariate analysis revealed GMFB to be an independent prognostic factor for overall survival in patients with HCC. Results of Gene Ontology (GO) and KEGG pathways analysis showed that down-regulation of pathways related to protein translation and mitochondria function were enriched. Protein-protein interaction analysis revealed the central role of mitochondria protein in HCC. The downregulation of genes involved in glycolysis and gluconeogenesis was observed among the co-expression genes of GMFB. Knockdown of GMFB in Hep3B significantly inhibited proliferation, migration, and invasion of Hep3B cells, and also downregulated the expression levels of some of metal matrix proteinase (MMP), increased mtDNA copy number and loss of mitochondrial transmembrane potential. GMFB influences the malignancy rate of HCC possibly through regulation of the expression of MMPs, mtDNA function and glycolysis. We proposed that GMFB was a promising HCC diagnostic and prognostic biomarker and therapeutic target in HCC.

Assessment of Zinc-alfa2 Glycoprotein (ZAG) and Lipase Maturation Factor 1 (LMF1) concentration in children with chronic kidney disease

ZAG (zinc-α2-glycoprotein) - adipokine, may participate in the mechanism of malnutrition in chronic kidney disease (CKD) as cachexia factor. The transmembrane protein of the endoplasmic reticulum - lipase maturation factor 1 (LMF1) is necessary for the secretion and enzymatic activity of lipases and lowering triglycerides level. The aim of the study was to evaluate these markers - ZAG and LMF1, their potential importance in CKD in children. The study included 59 children and adolescents aged 10.7±5.0 years with CKD. Compared with healthy children, serum and urine ZAG levels were higher in children with CKD. A similar relationship was obtained in the comparison of girls and boys between the above groups. We showed a reduced serum and urine concentration of LMF1 in children with CKD. Additionally, ZAG and LMF1 levels in children below 10 years of age and above 10 were no different. There was also no correlation between these markers and serum creatinine (except negative correlation of urinary ZAG), albumin, cholesterol, triglycerides. LMF1 concentration correlated positively with vitamin D level in dialyzed patients. To conclude, elevated serum ZAG levels in children with CKD document that selective kidney damage results in the rise of ZAG concentration, however the specific role of this marker in malnutrition was not documented. Reduced serum LMF1 concentration in children with CKD, did not correlate with standard parameters used to assess lipid metabolism and severity of CKD. The usefulness of LMF1 as the marker of the lipid metabolism disturbances in children with CKD was not proven.

Glia Maturation Factor-γ (GMFG) Promotes Ovarian Tumorigenesis and Chemoresistance via Activation of the FAK Signaling-Associated Focal Adhesion Dynamics

Background: Glia maturation factor-γ (GMFG) is reported to regulate actin cytoskeleton remodeling through the facilitation of actin debranching and nucleation suppression, which may be associated with cellular malignancy, but the role of GMFG in tumorigenesis remains largely unknown. Methods: By overexpression or silencing of GMFG in ovarian cancer cell lines, we show that GMFG enhances in vitro ovarian cancer cell proliferation, migration, invasion, and paclitaxel resistance and accelerates in vivo tumor growth and intraperitoneal metastasis in xenograft animal models. Results: The mechanistic study demonstrates that GMFG activates the FAK/Talin/Paxillin/Src signaling molecules via binding to p-FAK (Tyr397) and p-Talin (Ser425), whereas cell proliferation, migration and paclitaxel resistance induced by GMFG can be inversely suppressed by the chemical inhibition of p-FAK (Tyr397). Additionally, patients with high expression of GMFG exhibited a poor progression-free survival (PFS) (HR = 1.2, 95%CI: 1.05−1.37, P = 0.0069), and were significantly correlated with lymph node metastasis (P = 0.002) and venous invasion (P = 0.028). Conclusion: Our study suggests that GMFG may activate FAK signaling via binding to p-FAK (tyr397) and p-Talin (ser425) to promote ovarian tumorigenesis and chemoresistance. These findings indicate a functional interaction between GMFG and FAK pathway in ovarian tumorigenesis and chemoresistance. Thus, targeting the oncogenic GMFG-FAK axis may be a promising therapeutic strategy for ovarian cancer.

The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

Signaling by the B cell antigen receptor (BCR) initiates actin remodeling. The assembly of branched actin networks that are nucleated by the Arp2/3 complex exert outward force on the plasma membrane, allowing B cells to form membrane protrusions that can scan the surface of antigen-presenting cells (APCs). The resulting Arp2/3 complex-dependent actin retrograde flow promotes the centripetal movement and progressive coalescence of BCR microclusters, which amplifies BCR signaling. Glia maturation factor γ (GMFγ) is an actin disassembly-protein that releases Arp2/3 complex-nucleated actin filaments from actin networks. By doing so, GMFγ could either oppose the actions of the Arp2/3 complex or support Arp2/3 complex-nucleated actin polymerization by contributing to the recycling of actin monomers and Arp2/3 complexes. We now show that reducing the levels of GMFγ in human B cell lines via transfection with a specific siRNA impairs the ability of B cells to spread on antigen-coated surfaces, decreases the velocity of actin retrograde flow, diminishes the coalescence of BCR microclusters into a central cluster at the B cell-APC contact site, and decreases APC-induced BCR signaling. These effects of depleting GMFγ are similar to what occurs when the Arp2/3 complex is inhibited. This suggests that GMFγ cooperates with the Arp2/3 complex to support BCR-induced actin remodeling and amplify BCR signaling at the immune synapse.

Structural basis for inhibition of the AAA-ATPase Drg1 by diazaborine

The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2′-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.

Abstract P-31: Assembly of the Complex of the 30S Ribosomal Subunit and the Ribosome Maturation Factor P from Staphylococcus aureus for Structural Studies by Cryo-Electron Microscopy

Background: Staphylococcus aureus (S. aureus) is one of the main human pathogens causing numerous nosocomial soft tissue infections and is among the best-known causes of bacterial infections. The bacterial 70S ribosome consists of two subunits, designated the 30S (small) and 50S (large) subunits. The small subunit (30S) consists of 16S ribosomal RNA (rRNA), from which the assembly of 30S begins, and 21 ribosomal proteins (r-proteins). The ribosome maturation factor P (RimP protein) binds to the free 30S subunit. Strains lacking RimP accumulate immature 16S rRNA, and fewer polysomes and an increased amount of unassociated 30S and 50S subunits compared to wild-type strains are observed in the ribosomal profile. Structural studies of the 30S subunit complex and the ribosome maturation factor RimP will make it possible in the future to develop an antibiotic that slows down or completely stops the translation of Staphylococcus aureus, which will complicate the synthesis and isolation of its pathogenic factors. Here we present the protocol of the in vitro reconstruction of S. aureus 30S ribosome subunit in a complex with RimP for further structural studies by cryo-electron microscopy. Methods: Recombinant RimP protein from S. aureus was expressed in E. coli and purified by Ni-NTA chromatography and size exclusion chromatography. Reconstitution of the 30S–RimP complex was performed by mixing RimP protein with 30S ribosome. Unbound RimP protein was removed by Amicon Ultra Concentration (Merk KGaA, Darmstadt, Germany) with a cut-off limit of 100 kDa. The presence of RimP protein in the resulting 30S-RimP complex was confirmed by SDS-PAGE, and the quality of the final sample was analyzed by the negative staining EM. Results: Finally, by in vitro reconstruction, the 30S-RimP complex from S. aureus was obtained for further structural studies by cryo-electron microscopy.

Sperm cryopreservation impacts the early development of equine embryos by downregulating specific transcription factors

Equine embryos were obtained by insemination with either fresh or frozen-thawed spermatozoa at 8, 10 and 12 h post spontaneous ovulation, maintaining the pairs mare-stallion for the type of semen used. Next generation sequencing (NGS) was performed in all embryos and bioinformatic and enrichment analysis performed on the 21,058 identified transcripts. A total of 165 transcripts were downregulated in embryos obtained with cryopreserved spermatozoa respect embryos resulting from an insemination with fresh spermatozoa (p=0.021, q=0.1). The enrichment analysis using human orthologs using g:profiler on the downregulated transcripts marked an enrichment in transcription factors (TFs) in mRNAs downregulated in embryos obtained after insemination with cryopreserved spermatozoa. The 12 mRNAs (discriminant variables) most significantly downregulated in these embryos included among others, the chromatin-remodeling ATPase INO80, Lipase maturation factor 1 LMF1, the mitochondrial mRNA pseudouridine synthase RPUSD3, LIM and cysteine-rich domains protein 1, LMCD1. Sperm cryopreservation also caused a significant impact on the embryos at 8 to 10 days of development, but especially in the transition from 10 to 12 days. Overall, our findings provide strong evidence that insemination with cryopreserved spermatozoa poses a major impact in embryo development that may compromise its growth and viability, probably due to modifications in sperm proteins induced by cryopreservation. Identification of specific factors in the spermatozoa causing these changes may improve cryopreservation.