Molecular Biology: Open Access

Proliferative diabetic retinopathy (PDR) is the advanced stage of diabetic retinopathy in which neovascularization occurs at the retina. Angiogenesis, the formation of new vessels from the existing one involves sequential step starting from the degradation of basement membrane by matrix metalloproteinases (MMPs). The elevated expression of MMP2 is considered as the one of the important parameter in the progress of PDR. Therefore, it is important to inhibit the activity of MMP2 in the retina of the PDR patients for the management of the diseases. In this concern we examined the anti angiogenic potential of antisense MMP2 oligonucleotide using chicken late CAM (chorio allantoic membrane) using various techniques. CAM has been used as a model for retinal research by ophthalmologist for many years due to its similarities with retina in the tissue thickness and blood vessels formation. MMP2 ASO inhibited blood vessels growth at the treated area and their length and size and reduced the thickness of the CAM compared to control. MMP2 ASO enhanced the accumulation of fibroblast and flattened endothelial cells and reduced the expression of MMP2 at gene and protein level. The chorionic epithelial layer of the CAM got flattened after the treatment with MMP2 ASO as an indication of lack of blood vessels formation and growth at the treated area. Altogether, our results demonstrates that MMP2 ASO is able to inhibit neovascularization effectively and therefore can be a suitable therapeutic molecule in the management of pathological neovascularization.

Grapevine degeneration in grapevines caused by Grapevine Fanleaf Virus (GFLV) has been documented in many viticulture regions worldwide. It is one of the major economically important virus diseases affecting the longevity of grapevines and reducing the fruit yield and fruit quality. To diminish the time required for some diagnostic assays, including reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP) and also double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) into a minimum level, an innovative immunocapture RT-LAMP (IC-RT-LAMP) and immunocapture RT-PCR (IC-RT-PCR) protocol on the basis of GFLV genome were used and the reaction conditions were optimized for rapid detection of GFLV. In this regard, DAS-ELISA was employed first to validate the existence of the virus. All six RT-LAMP primers (i.e., F3, B3, FIP, BIP, LF and LB) together with RT-PCR primers (F and B) were designed on the basis of coat protein (CP) gene of GFLV. LAMP method, on the whole, had the following advantages over the other mentioned procedures: (I) fascinatingly, no need of RNA extraction; (II) no requirement of expensive and sophisticated tools for amplification and detection; (III) no post-amplification treatment of the amplicons; and (IV) a flexible and easy detection approach, which is visually detected by naked eyes using diverse visual dyes.

Osteogenesis imperfecta (OI) is a heterogeneous rare connective tissue disorder commonly caused by mutations in the collagen type 1 gene. It is a worldwide extensive disorder regardless of age, gender or ethnic group for a children and adults. Typical clinical features are brittle bone, high frequency of fractures and bone deformities. The other observed signs are blue sclera, dentinogenesis imperfect and otosclerosis. In this review, author make a systematic overview, including the mechanisms, classification, diagnostic methods, related to human concerned disease and treatment. The review also focuses on the OI related so many health concern diseases. In OI patients maintaining of health is very important otherwise, the bone deformities and collagen defects common to OI it can affect various internal organs, leading to major or minor secondary problems. Individuals and optimization of OI treatment in children and adults remain a challenge, because available treatments do not target then underlying collagen defect. Treatment includes physiotherapy, surgical procedures and pharmacology therapy. In this brief review, author mainly discusses current knowledge of pharmacology therapies and possible future therapies for treatment of OI.

Nebracetam has been recently proposed to have a neuroprotective action and cognitive enhancing effect being characteristic of a nootropic drug, while the mechanisms remained ambiguity. In this study, we investigated the protective effects of nebracetam on hippocampus neurons injury-induced by β-amyloid protein(Aβ25-35)and its mechanisms. Hippocampus neurons were treated with nebracetam (0.05 mM, 0.2 mM or 0.8 mM) or Aβ25-35 (20 uM/L). We found that Nebracetam significantly reduced apoptotic induced by Aβ25-35 and increase in the total number of dendritic spines and dendritic spine density in a dose-dependent manner. RT-PCR assay and Western blotting analysis revealed that nebracetam increased the expressions of myeloid cell leukemia-1 (Mcl-1), B-cell lymphoma-2 (Bcl-2) and activity regulated cytoskeleton associated protein (Arc) in Aβ25-35-treated hippocampus neurons. Cotreatment nebracetam with MRTF-A (Myocardin-related transcription factor-A) siRNA reversed Mcl-1, Bcl-2 and Arc mRNA and protein levels. What’s more, the luciferase assays indicated that the transcriptional activities of Mcl-1, Bcl-2 and Arc genes were significantly abolished by MRTF-A siRNA while it showed no changes on activities of mut Mcl- 1-promoter-luc, mut Bcl-2-promoter-luc and mut Arc-promoter-luc. Additionally, the up-regulation of Mcl-1, Bcl-2 and Arc protein expressions in nebracetam-treated group was inhibited by extracellular signal regulated protein kinase 1/2 (ERK1/2) inhibitor PH98059. These results demonstrated that nebracetam inhibited Aβ25-35-induced hippocampus neurons injury by enhancing the transactivity of Mcl-1, Bcl-2 and Arc, which may actively based in MRTF-A-CArGdependent manner by thwarting the ERK1/2 pathway.

Serum α-fetoprotein (AFP) has been used as an early diagnostic biomarker of hepatocellular carcinoma (HCC), however the sensitivity and specificity of detection are poor. Therefore, new markers remain to be explored. MicroRNAs (miRNAs) are a class of small RNAs which could contribute to tumorigenesis by interaction with targeted mRNAs. MiRNAs are abundant in the circulating blood and are potentially useful for early diagnosis of hepatocellular carcinoma. In this study, we screened a group of candidate miRNAs (miR-182-5p, miR-363-3p, and miR-378a-3p) in serum, which were found to be up-regulated in HCC patients compared with in the healthy controls. Analysis of the receiver operating characteristic (ROC) curve suggested that these three serum miRNAs had merits in the diagnosis of HCC (AUC=0.802, 0.845, 0.880, respectively). Furthermore, these candidate miRNAs can also differentiate HCC patients with negative AFP level from the healthy controls (AUC=0.865, 0.930, 0.970, respectively). In conclusion, our results suggested that serum miR-182-5p, miR-363-3p, and miR-378a-3p might be applied as potential non-invasive biomarkers for HCC diagnosis, which is especially important for AFP-negative patients.

Introduction: Progressive Multifocal Leukoencephalopathy (PML) is a severe and often fatal CNS disease caused by JC human polyomavirus infection. Diagnosis of PML is based upon suggestive clinical symptoms and brain images, supported by the presence of JC virus genome in CSF samples.

Objective: The main objective of our study was the search for an alternative method for JC virus DNA detection in CSF samples, with sensitivity and specificity characteristics close to that of standard techniques, but feasible at any clinical laboratory.

Methods: In order to evaluate the effect of topoisomerase I treatment in the detection of JC virus genome, and its feasibility in laboratory diagnosis of PML, 129 CSF samples were examined for the presence of JC virus DNA by a nested-PCR protocol, with and without previous treatment with topoisomerase I. All CSF samples were also evaluated through a real-time PCR protocol.

Results: Eleven CSF samples presented detectable JC virus DNA with all used protocols. On 9 CSF samples, JC virus DNA was only detectable with topoisomerase I modified nested-PCR and real-time PCR protocols. Real-time PCR was the only protocol able to detect JC virus genome in 4 CSF samples. One CSF sample revealed the presence of the expected amplified fragment only when tested with topoisomerase I modified nested-PCR protocol.

Conclusion: The results of the present study point towards the benefit of using topoisomerase I DNA treatment before amplification reactions in JC virus DNA detection on CSF samples, and confirm that topoisomerase I modified nested-PCR protocol represents a good alternative method to detect JC virus DNA in CSF samples from patients with clinical signs and brain images suggestive of PML.