ADAMTS13 gene; a novel splicing site mutation in a case with thrombotic thrombocytopenic purpura

A plasma protease, ADAMTS13, cleaves the von Willebrand factor (VWF) and its deficiency is associated with the pathogenesis of thrombotic thrombocytopenic purpura (TTP). According to the Human Gene Mutation Database (HGMD), about 150 mutations have been identified in the ADAMTS13 gene. A 23-year-old man, with hematuria and gingival bleeding was admitted to our University Hospital. Four years ago he was diagnosed with a TTP history. During these years, he was under intermittent plasma exchange. A blood sample was taken for genetic study. He effectively responded to one session of fresh frozen plasma replacement and plasma exchange. Genetic study indicated that this case carries two heterozygous mutations in ADAMTS13 gene; a novel splicing variant (c.2610+5G>A) and a nonsense p.Arg910X mutation that previously is reported to relate to TTP. The novel variant predicted to result in an aberrant ADAMTS13 transcript processing

Dysregulated levels of glycogen synthase kinase-3β (GSK-3β) and miR-135 in peripheral blood samples of cases with nephrotic syndrome

Background. Glycogen synthase kinase-3 (GSK-3β) is a serine/threonine kinase with multifunctions in various physiological procedures. Aberrant level of GSK-3β in kidney cells has a harmful role in podocyte injury. Methods. In this article, the expression levels of GSK-3β and one of its upstream regulators, miR-135a-5p, were measured in peripheral blood mononuclear cells (PBMCs) of cases with the most common types of nephrotic syndrome (NS); focal segmental glomerulosclerosis (FSGS) and membranous glomerulonephritis (MGN). In so doing, fifty-two cases along with twenty-four healthy controls were included based on the strict criteria. Results. Levels of GSK-3β mRNA and miR-135 were measured with quantitative realtime PCR. There were statistically significant increases in GSK-3β expression level in NS (P = 0.001), MGN (P = 0.002), and FSGS (P = 0.015) groups compared to the control group. Dysregulated levels of miR-135a-5p in PBMCs was not significant between the studied groups. Moreover, a significant decrease was observed in the expression level of miR-135a-5p in the plasma of patients with NS (P = 0.020), MGN (P = 0.040), and FSGS (P = 0.046) compared to the control group. ROC curve analysis approved a diagnostic power of GSK-3β in discriminating patients from healthy controls (AUC: 0.72, P = 0.002) with high sensitivity and specificity. Conclusions. Dysregulated levels of GSK-3β and its regulator miR-135a may participate in the pathogenesis of NS with different etiology. Therefore, more research is needed for understanding the relationship between them

The Use of Nanomaterials in Tissue Engineering for Cartilage Regeneration; Current Approaches and Future Perspectives

The repair and regeneration of articular cartilage represent important challenges for orthopedic investigators and surgeons worldwide due to its avascular, aneural structure, cellular arrangement, and dense extracellular structure. Although abundant efforts have been paid to provide tissue-engineered grafts, the use of therapeutically cell-based options for repairing cartilage remains unsolved in the clinic. Merging a clinical perspective with recent progress in nanotechnology can be helpful for developing efficient cartilage replacements. Nanomaterials, < 100 nm structural elements, can control different properties of materials by collecting them at nanometric sizes. The integration of nanomaterials holds promise in developing scaffolds that better simulate the extracellular matrix (ECM) environment of cartilage to enhance the interaction of scaffold with the cells and improve the functionality of the engineered-tissue construct. This technology not only can be used for the healing of focal defects but can also be used for extensive osteoarthritic degenerative alterations in the joint. In this review paper, we will emphasize the recent investigations of articular cartilage repair/regeneration via biomaterials. Also, the application of novel technologies and materials is discussed

Fenugreek (Trigonella Foenum-Graecum) Induced Interstitial Nephritis

Herbal medicine has grabbed the public interest and there is a general belief of their safety and life compatibility. There are growing proportion of reported side effects of herbal medicine in past years. We reported a 62-year-old woman who developed acute interstitial nephritis (AIN) accompanied by autoimmune hemolytic anemia (AIHA), three weeks after daily ingestion of heat extract of fenugreek. The patient\u27s renal, hematologic and liver panels were improved after steroid pulse therapy and fenugreek discontinuation. Fenugreek contains high amount of flavonoid which might be a main culprit for AIN and AIHA

An update to DNA ladder assay for apoptosis detection

Introduction: A growing interest in apoptosis, programmed cell death, in the last years is observed and leads to better understanding of molecular mechanisms during cell–cell signaling, cell-environment interaction and screening of drugs. This in turn results in emerging of new assays and development of more accurate kits for fast and early detection of apoptosis. However, their sensitivity and reliability have often been scrutinized. Here we introduce a rapid and improved method of DNA ladder apoptosis assay for evaluating apoptosis in mammalian cells. Methods: NIH-3T3 cell line was used in this study. After treatment of cells with apoptotic agent, 500 µM H2O2 at 48 hours, DNA was extracted. Then an update protocol of DNA ladder assay was applied for detection of apoptosis. Flow cytometry and DAPI staining were performed to verify apoptosis. Results: Primary and late apoptosis in the H2O2-treated cells was determined by flow cytometry analysis. DAPI Staining used to show DNA damage and DNA ladder assay using 1.5% gel electrophoresis showed fragmentation in the DNA of treated cells. Conclusion: In this research we aimed to improve DNA ladder assay to the high quality detection of apoptosis in mammalian cells. In our strategy, employing a practical DNA extraction protocol, DNA ladder assay could be applied as an easy/fast method for apoptosis detection. This improved method is able to detect apoptosis in a cost effective/timely manner without need for commercial kits and special equipment

COVID-19 interactions with angiotensin-converting enzyme 2 (ACE2) and the kinin system; looking at a potential treatment

The novel coronavirus disease 2019 (COVID-19) is a rapidly expanding infection around the world. The world Health Organization (WHO) in March 2020 announced the Coronavirus pandemic. This infection causes many deaths on daily basis. Therapeutic options are currently limited. It is revealed that COVID-19 binds to human angiotensin-converting enzyme 2 (ACE2) to enter the host cells. One of the activities of ACE2 is hydrolyzing the active bradykinin metabolite [des-Arg973] BK (DABK). A decreased activity or reducing expression of ACE2 by the virus impairs the inactivation of DABK. This enhances its signaling through the bradykinin B1 receptor (BKB1R) and could lead to fluid extravasation and leukocyte recruitment to the lung. Targeting the bradykinin system by either blocking the bradykinin production or blocking bradykinin receptors may open a new potential therapeutic window for the treatment of COVID-19 induced acute respiratory distress syndrome (ARDS) particularly before patients enter the irreversible stages

A microRNA isolation method from clinical samples

Introduction: microRNAs (miRNAs) are considered to be novel molecular biomakers that could be exploited in the diagnosis and treatment of different diseases. The present study aimed to develop an efficient miRNA isolation method from different clinical specimens. Methods: Total RNAs were isolated by Trizol reagent followed by precipitation of the large RNAs with potassium acetate (KCH3COOH), polyethylene glycol (PEG) 4000 and 6000, and lithium chloride (LiCl). Then, small RNAs were enriched and recovered from the supernatants by applying a combination of LiCl and ethanol. The efficiency of the method was evaluated through the quality, quantity, and integrity of the recovered RNAs using the A260/280 absorbance ratio, reverse transcription PCR (RT-PCR), and quantitative real-time PCR (q-PCR). Results: Comparison of different RNA isolation methods based on the precipitation of DNA and large RNAs, high miRNA recovery and PCR efficiency revealed that applying potassium acetate with final precipitation of small RNAs using 2.5 M LiCl plus ethanol can provide high yield and quality small RNAs that can be exploited for clinical purposes. Conclusion: The current isolation method can be applied for most clinical samples including cells, formalin-fixed and paraffin-embedded (FFPE) tissues and even body fluids with a wide applicability in molecular biology investigations

Highly Effective DNA Extraction Method from Fresh, Frozen, Dried and Clotted Blood Samples

Introduction: Today, with the tremendous potential of genomics and other recent advances in science, the role of science to improve reliable DNA extraction methods is more relevant than ever before. The ideal process for genomic DNA extraction demands high quantities of pure, integral and intact genomic DNA (gDNA) from the sample with minimal co-extraction of inhibitors of downstream processes. Here, we report the development of a very rapid, less-hazardous, and high throughput protocol for extracting of high quality DNA from blood samples. Methods: Dried, clotted and ethylene diamine tetra-acetic acid (EDTA) treated fresh and frozen blood samples were extracted using this method in which the quality and integrity of the extracted DNA were corroborated by agarose gel electrophoresis, PCR reaction and DNA digestion using restricted enzyme. The UV spectrophotometric and gel electrophoresis analysis resulted in high A260/A280 ratio (>1.8) with high intactness of DNA. Results: PCR and DNA digestion experiments indicated that the final solutions of extracted DNA contained no inhibitory substances, which confirms that the isolated DNA is of good quality. Conclusion: The high quality and quantity of current method, no enzymatic processing and accordingly its low cost, make it appropriate for DNA extraction not only from human but also from animal blood samples in any molecular biology labs