Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma

Purpose: Methylated cell-free fetal DNA (cffDNA) in maternal plasma can potentially be used as a biomarker for accurate noninvasive prenatal testing (NIPT) of fetal disorders. Recovery and purification of cffDNA are key steps for downstream applications. In this study, we aimed to developed and evaluated different aspects of an optimized method and compared its efficiency with common methods used for extraction of methylated cffDNA. Methods: Single factor experiments, Plackett-Burman (PB) design, and response surface methodology (RSM) were conducted for conventional Triton/Heat/Phenol (cTHP) method optimization. The total cell-free DNA (cfDNA) was extracted from pooled maternal plasma using the optimized method called the Triton/Heat/Phenol/Glycogen (THPG), cTHP method, a column-based kit, and a magnetic bead-based kit. In the next step, methylated cfDNA from the extracted total cfDNA was enriched using a methylated DNA immunoprecipitation (MeDIP) kit. Real-time quantitative polymerase chain reaction was performed on the RASSF1 gene and hyper region to determine the genomic equivalents per milliliter (GEq/ml) values of the methylated cfDNA and cffDNA, respectively. Results: The optimum values of the significant factors affecting cfDNA extraction from 200 μl of plasma were 3% SDS, 1% Triton X-100, 0.9 μg/μl glycogen, and 0.3 M sodium acetate. The GEq/ml values of methylated cffDNA extracted using the THPG method were significantly higher than for the tested extraction methods (p < 0.001). Conclusions: Our results indicate that the THPG method is more efficient than the other tested methods for extraction of low copy number methylated cffDNA from a small volume of maternal plasm

DNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropica

Background: The vector-borne disease leishmaniasis is transmitted to humans by infected female sand flies, which transmits Leishmania parasites together with saliva during blood feeding. In Iran, cutaneous leishmaniasis (CL) is caused by Leishmania (L.) major and L. tropica, and their main vectors are Phlebotomus (Ph.) papatasi and Ph. sergenti, respectively. Previous studies have demonstrated that mice immunized with the salivary gland homogenate (SGH) of Ph. papatasi or subjected to bites from uninfected sand flies are protected against L. major infection. Methods and results: In this work we tested the immune response in BALB/c mice to 14 different plasmids coding for the most abundant salivary proteins of Ph. sergenti. The plasmid coding for the salivary protein PsSP9 induced a DTH response in the presence of a significant increase of IFN-γ expression in draining lymph nodes (dLN) as compared to control plasmid and no detectable PsSP9 antibody response. Animals immunized with whole Ph. sergenti SGH developed only a saliva-specific antibody response and no DTH response. Mice immunized with whole Ph. sergenti saliva and challenged intradermally with L. tropica plus Ph. sergenti SGH in their ears, exhibited no protective effect. In contrast, PsSP9-immunized mice showed protection against L. tropica infection resulting in a reduction in nodule size, disease burden and parasite burden compared to controls. Two months post infection, protection was associated with a significant increase in the ratio of IFN-γ to IL-5 expression in the dLN compared to controls. Conclusion: This study demonstrates that while immunity to the whole Ph. sergenti saliva does not induce a protective response against cutaneous leishmaniasis in BALB/c mice, PsSP9, a member of the PpSP15 family of Ph. sergenti salivary proteins, provides protection against L. tropica infection. These results suggest that this family of proteins in Ph. sergenti, Ph. duboscqi and Ph. papatasi may have similar immunogenic and protective properties against different Leishmania species. Indeed, this anti-saliva immunity may act as an adjuvant to accelerate the cell-mediated immune response to co-administered Leishmania antigens, or even cause the activation of infected macrophages to remove parasites more efficiently. These findings highlight the idea of applying arthropod saliva components in vaccination approaches for diseases caused by vector-borne pathogens. © 2019, Public Library of Science. All rights reserved

The effects of personal, environmental, and genetic factors on epidemic of coronavirus disease-19: A review of the current literature

Coronavirus disease (COVID)-19 is a viral disease that broke out in late 2019 in Wuhan, China. The World Health Organization has been forced to declare a public health emergency due to the global outbreak of COVID-19. The concerns about the COVID-19 disease are the rapid increase in the number of patients as well as the number of deaths compared with severe acute respiratory syndrome disease. Given that there is a remarkable variability amongst people for COVID-19 infection, there really is the possibility that there will be genetic and environmental effects, it is a need for their role to be fully clarified as soon as possible. Numerous studies have been performed on the on the stability of COVID-19 virus in different environmental conditions including temperature and humidity. In this study, we aimed to discuss in detail the benefits and effects of these factors on COVID-19. Some studies have confirmed the relationship between environmental conditions and disease transmission and others have rejected. Furthermore, not all COVID-19 exposed people are infected and not all infected patients develop severe respiratory complications. It is quite likely that these disparities are genetically mediated, in part. People who may be occupationally exposed to this virus may be due to different reasons, including lack of health, lack of knowledge and attitude, and working conditions. Reducing human-to-human contact by increasing the level of public health in the community as well as maintaining social distance plays a key role in prevention of COVID-19 disease. However, many aspects of COVID-19 are still unknown and require further and extensive studies. © 2020 Salar Bakhtiyari, Amin Mirzaei, Mohsen Jalilian, Sajad Mazlomi,

Environmental Impact Assessment (EIA) and Optimal Site Selection of Municipal Solid Waste Landfill via SAW, GIS and Leopold Matrix (Case Study: Yazd)

Introduction: Waste production from any source would lead to the formation of various pollutants. The existence of these materials and in turn, every non-normative plan designed for waste disposal produces environmentally detrimental effects. Therefore, evaluating positive and negative effects of influential engineering- sanitary projects in environment seem to be necessary such as landfill construction for management of urban health. The present study aimed to evaluate positioning and of municipal solid waste landfills by geological information system (GIS). To this purpose, the required layers were prepared and profit sites were obtained by 3 steps: talent mapping, field survey to study characteristics of suitable areas and EIA by means of Leopold matrix for zonal ranking and selection of optimal location. Results: To depict talent map, the layers were combined by simple additive weighting method using various data including geology, land use, distance from the city boundaries, slope, distance from roads, vegetation, infiltration and hydrology. The resulted data suggested that four sites were posited in appropriate ranks. Conclusion: The study findings revealed, that site No.1 (X:256915 & Y:3540127) was determined as the optimum location for engineering - Sanitary landfill