Current Molecular Medicine - Volume 19, Issue 7, 2019

The demands for genotyping techniques with acceptable precision, accuracy, cost-effectiveness in high throughput formats made driving forces for continuous development of novel technologies. A wide range of mutation detection techniques based on polymerase chain reaction (PCR) have been introduced. The best alternatives were the isothermal amplification technologies that those did not require a thermal cycler. In this review, we aimed to describe the most known isothermal amplification techniques for SNP genotyping.

Background: In the present study, the interaction between new drug Rubraca and CNT(4,4-8) nanotube by Density Functional Theory (DFT) calculations in an aqueous medium for first time have been investigated. Method and Results: According to calculations, the intermolecular hydrogen bonds take place between active positions of the molecule Rubraca and hydrogen atoms of the nanotube that plays an important role in the stability of the complex CNT(4,4- 8)/Rubraca. The non-bonded interaction effects of the molecule Rubraca with CNT(4,4- 8) nanotube on the electronic properties, chemical shift tensors and natural charge have been also detected. The natural bond orbital (NBO) analysis suggested that the molecule Rubraca as an electron donor and the CNT(4,4-8) nanotube plays the role an electron acceptor at the complex CNT(4,4-8)/Rubraca. The electronic spectra of the Rubraca drug and the complex CNT(4,4-8)/Rubraca were also calculated by Time Dependent Density Functional Theory (TD-DFT) for the investigation of adsorption effect of the Rubraca drug over nanotube. Conclusion: The use of CNT(4,4-8) nanotube for Rubraca delivery to the diseased cells have been established.

Background: Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a congenital rare metabolic disease with broad clinical phenotypes and variable evolution. This inborn error of metabolism is caused by mutations in the ETFA, ETFB or ETFDH genes, which encode for the mitochondrial ETF and ETF:QO proteins. A considerable group of patients has been described to respond positively to riboflavin oral supplementation, which constitutes the prototypic treatment for the pathology. Objectives: To report mutations in ETFA, ETFB and ETFDH genes identified in Portuguese patients, correlating, whenever possible, biochemical and clinical outcomes with the effects of mutations on the structure and stability of the affected proteins, to better understand MADD pathogenesis at the molecular level. Methods: MADD patients were identified based on the characteristic urinary profile of organic acids and/or acylcarnitine profiles in blood spots during newborn screening. Genotypic, clinical and biochemical data were collected for all patients. In silico structural analysis was employed using bioinformatic tools carried out in an ETF:QO molecular model for the identified missense mutations. Results: A survey describing clinical and biochemical features of eight Portuguese MADD patients was made. Genotype analysis identified five ETFDH mutations, including one extension (p.X618QextX*14), two splice mutations (c.34+5G>C and c.405+3A>T) and two missense mutations (ETF:QO-p.Arg155Gly and ETF:QO-p.Pro534Leu), and one ETFB mutation (ETFβ- p.Arg191Cys). Homozygous patients containing the ETFDH mutations p.X618QextX*14, c.34+5G>C and ETF:QO-p.Arg155Gly, all presented severe (lethal) MADD phenotypes. However, when any of these mutations are in heterozygosity with the known ETF:QO-p.Pro534Leu mild variant, the severe clinical effects are partly and temporarily attenuated. Indeed, the latter destabilizes an ETF-interacting loop, with no major functional consequences. However, the position 155 in ETF:QO is localized at the ubiquinone binding and membrane interacting domain, and is thus expected to perturb protein structure and membrane insertion, with severe functional effects. Structural analysis of molecular models is therefore demonstrated to be a valuable tool to rationalize the effects of mutations in the context of the clinical phenotype severity. Conclusion: Advanced molecular diagnosis, structural analysis and clinical correlations reveal that MADD patients harboring a severe prognosis mutation in one allele can actually revert to a milder phenotype by complementation with a milder mutation in the other allele. However, such patients are nevertheless in a precarious metabolic balance which can revert to severe fatal outcomes during catabolic stress or secondary pathology, thus requiring strict clinical follow-up.

Background: An increasing number of studies have described the pathological changes of placenta tissues in gestational diabetes mellitus (GDM), although the underlying mechanisms involved in this process remain uncertain. The aim of the present study was to verify the possible role of microRNA-137 (miR)-137 and FNDC5 in regulating the biological function of trophoblasts in high glucose (HG) conditions during the GDM period. Methods: Expression levels of miR-137 and FNDC5 were measured in placenta specimens, the HG-treated trophoblast cell line HTR-8/SVneo and miR-137- overexpressing HTR-8/SVneo cells using reverse transcription quantitative-PCR or western blotting. The viability of HTR-8/SVneo cells was tested using a Cell Counting kit- 8 (CCK8) assay, with cell migration assessed using scratch and transwell assays. Results: It was observed that the expression levels of miR-137 were increased and the expression levels of FNDC5 were decreased in the placenta tissues of women with severe GDM and in HG-exposed HTR-8/SVneo cells. In addition, upregulating miR-137 in HTR-8/SVneo cells downregulated the expression levels of FNDC5. The viability and migration of HTR-8/SVneo cells were suppressed by increased miR-137 expression levels, and upregulating FNDC5 in miR-137-overexpressing HTR-8/SVneo cells resulted in the reversal of all these effects. Conclusions: The data from the present study suggest that miR-137 suppresses the viability and migration of trophoblasts via downregulating FNDC5 in GDM, which may contribute to the pathology of placenta tissues and occurrence of adverse pregnancy outcomes.

Background: The incidence of fungal infections has increased significantly. Specifically the cases of candida albicans infection are increasing day by day and their resistance to clinically approved drugs is a major concern for humans. Various classes of antifungal drugs are available in the market for the treatment of these infections but unfortunately, none of them is able to treat the infection. Objectives: Thus, in the present investigation, we have repurposed the well-known drug (Fluvastatin) in the treatment of Candida albicans infections by using in silico, in vitro and ex vivo techniques. Material and Methods: Computational and in vitro techniques. Results: Firstly, we developed and validated a simple model of CYP45014α-lanosterol demethylase of Candida albicans by using crystal structure of Mycobacterium tuberculosis (1EA1). Further, fluvastatin was docked with a validated model of CYP45014α-lanosterol demethylase and revealed good binding affinity as that of fluconazole. In vitro results (Percentage growth retardation, Fungal growth kinetics, Biofilm test and Post antifungal test) have shown good antifungal activity of fluvastatin. Finally, the results of MTT assay have shown non-cytotoxic effect of fluvastatin in murine splenocytes and thymocytes. Conclusion: However, further in vivo studies are required to confirm the complete role of fluvastatin as an antifungal agent.

Objective: We aimed to investigate the potential risk factors for residual lesions after uterine artery chemotherapy and embolization (UACE) in combination with dilatation and curettage (D) in patients with cesarean scar pregnancy (CSP). Settings: Retrospective case-control study. Method: Univariate analysis and logistic analysis were applied to analyze these data to assess the risk factor of residue after UACE in combination with D Results: Gestational age, human chorionic gonadotropin (HCG) level, and the gestation sac (GS) evagination to the bladder were the risk factors for the postoperative lesion. The most relevant was GS evagination to the bladder, followed by the preoperative HCG level and the gestational age. We defined the cut-off value of gestational age as 28 days with a sensitivity and specificity of 100 and 0, respectively. Conclusion: In cases with GS evagination to bladder and HCG of >28,113.65 mIU/ml as well as a gestational age of >28 days, the possibility of residual lesions was high.

Background: Portal vein thrombosis (PVT) is a common complication of endstage hepatocellular carcinoma (HCC). The aim of our study was to evaluate the role of Homocysteine (Hcy) in HCC patient with PVT. Hcy is a sulphur amino-acid involved in two pathways, trans-sulphuration and remethylation, that involve vitamins B6, B12 and folates. Methods: We recruited 54 patients with HCC and PVT, 60 patients with HCC and without PVT and 60 control subjects. We measured serum levels of Hcy, folate, vitamins B6 and B12. Results: The comparison between HCC patients with PVT versus HCC without PVT was shown that mean values of Hcy were 6.4 nmol/L (p<0.0073) higher, LDL cholesterol were 4.8 mg/dl (p<0.0079) lower, vitamin B6 were 4.6 nmol/L(p=0.0544) lower, vitamins B 12 were 22.1 pg/ml (p=0.0001) lower. Conclusion: High serum levels of Hcy are an established thrombotic risk factor in the general population. We found significantly higher levels of Hcy in HCC patients with PVT versus both HCC patients without PVT and controls.