Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling

Background The microbiota of the lower female genital tract plays an important role in women’s health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT. Methods DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition. Results DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.

Methodologies to Isolate and Purify Clinical Grade Extracellular Vesicles for Medical Applications

The use of extracellular vesicles (EV) in nano drug delivery has been demonstrated in many previous studies. In this study, we discuss the sources of extracellular vesicles, including plant, salivary and urinary sources which are easily available but less sought after compared with blood and tissue. Extensive research in the past decade has established that the breadth of EV applications is wide. However, the efforts on standardizing the isolation and purification methods have not brought us to a point that can match the potential of extracellular vesicles for clinical use. The standardization can open doors for many researchers and clinicians alike to experiment with the proposed clinical uses with lesser concerns regarding untraceable side effects. It can make it easier to identify the mechanism of therapeutic benefits and to track the mechanism of any unforeseen effects observed.

Assessment of Physico-Chemical Parameters and Heavy Metal of Drinking Water Samples from Some Iranian Universities and Surveyed the Efficacy of Several Water Purification Methods on Removal of Heavy Metal

Water quality is the most important factor for consumers because it has a significant impact on their health. So, monitoring and controlling water quality is of particular significance in different human communities such as universities. In this study, the physicochemical parameters such as turbidity, electrical conductivity, hardness, alkalinity, calcium and magnesium ions, and heavy metals including lead, cadmium, nickel, arsenic, and chromium in drinking water were determined in nine Iranian universities. Then, the efficacy of several treatment methods was evaluated in the removal of heavy metal, including the boiling process in two stages: 5 minutes boiling and 5 hours boiling and filtration process. The results revealed that the physicochemical quality of drinking water in all universities was appropriate. Hardness levels in drinking water samples of all universities were higher than the WHO standards. However, this parameter was lower than the national standard of Iran (500 mg/l). Nevertheless, cadmium content was not detected in any samples, nickel content was below the permissible limit, and chromium content was below the permissible limit in all samples. However, its value was higher than the allowable limit in the university with a code of 6. Lead content was higher than the limit in samples with codes 3, 5-1, and 5-4, and arsenic content was higher than the limit in samples with codes 6, 1-1, 1-2, and 2. Water purification methods, including boiling and water purifier, were satisfactory and significantly reduced pollutants. According to the present study, it is suggested to check drinking water quality in universities randomly, and the 5-minute boiling method can be recommended as a cost-effective solution for water purification.

A Comprehensive Insight into Fungal Enzymes: Structure, Classification, and Their Role in Mankind’s Challenges

Enzymes have played a crucial role in mankind’s challenges to use different types of biological systems for a diversity of applications. They are proteins that break down and convert complicated compounds to produce simple products. Fungal enzymes are compatible, efficient, and proper products for many uses in medicinal requests, industrial processing, bioremediation purposes, and agricultural applications. Fungal enzymes have appropriate stability to give manufactured products suitable shelf life, affordable cost, and approved demands. Fungal enzymes have been used from ancient times to today in many industries, including baking, brewing, cheese making, antibiotics production, and commodities manufacturing, such as linen and leather. Furthermore, they also are used in other fields such as paper production, detergent, the textile industry, and in drinks and food technology in products manufacturing ranging from tea and coffee to fruit juice and wine. Recently, fungi have been used for the production of more than 50% of the needed enzymes. Fungi can produce different types of enzymes extracellularly, which gives a great chance for producing in large amounts with low cost and easy viability in purified forms using simple purification methods. In the present review, a comprehensive trial has been advanced to elaborate on the different types and structures of fungal enzymes as well as the current status of the uses of fungal enzymes in various applications.

The Extraction, Functionalities and Applications of Plant Polysaccharides in Fermented Foods: A Review

Plant polysaccharides, as prebiotics, fat substitutes, stabilizers, thickeners, gelling agents, thickeners and emulsifiers, have been immensely studied for improving the texture, taste and stability of fermented foods. However, their biological activities in fermented foods are not yet properly addressed in the literature. This review summarizes the classification, chemical structure, extraction and purification methods of plant polysaccharides, investigates their functionalities in fermented foods, especially the biological activities and health benefits. This review may provide references for the development of innovative fermented foods containing plant polysaccharides that are beneficial to health.

THE ROLE OF DIFFERENT MEDIA FOR THE URDHVA PATANA OF PARADA

In our ancient literature of Rasashastra, Parada or Rasa is considered the supreme Dravya out of all Rasoparasa’s, Dhatu’s etc1and described to be of divine origin. The importance of Parada is highlighted mainly for Rasachikitsa. Mercury always comes with natural impurities i.e., “Naisargika dosha”, when mercury is put to trade and commercial use it is adulterated with cheaper metals like Naga and Vanga for more commercial gains. As a result, mercury in the market is often available with this kind of adulterants, which are considered as “Yougi- ka Dosha”. Consumption of impure mercury is considered highly toxic and hazardous. It can be made into medi- cine by adopting proper Shodhana procedures. In Rasashastra process of Shodhana is having greater im- portance. Hence a wide range of purification methods is described for each metal and mineral including Parada. The literary meaning of Shodhana is purification, but in Rasashastra Shodana is a Samskara which essentially improves the potency along with detoxifying the metal or mineral. In our classics, various methods have been told for Shodhana of Parada, in which Patana plays a very important role2. All the different classical references of Urdhva Patana Samskaras are compiled and critically analysed to know their role in Shodhana of Parada. Various media told are Tamra churna, Sarja Kshara, Yava Kshara, Hingu, Panchalavana and Kaashtoaushadis such as Kumari, Nisha churna etc. An attempt is made to analyse the role of various media for the Urdhva Patana of Parada. Keywords: Parada, Shodhana, Urdhva Patana, Mercury

Mesenchymal stem cells-derived exosomes for drug delivery

Exosomes are extracellular vesicles secreted by various cells, mainly composed of lipid bilayers without organelles. In recent years, an increasing number of researchers have focused on the use of exosomes for drug delivery. Targeted drug delivery in the body is a promising method for treating many refractory diseases such as tumors and Alzheimer's disease (AD). Finding a suitable drug delivery carrier in the body has become a popular research today. In various drug delivery studies, the exosomes secreted by mesenchymal stem cells (MSC-EXOs) have been broadly researched due to their immune properties, tumor-homing properties, and elastic properties. While MSC-EXOs have apparent advantages, some unresolved problems also exist. This article reviews the studies on MSC-EXOs for drug delivery, summarizes the characteristics of MSC-EXOs, and introduces the primary production and purification methods and drug loading methods to provide solutions for existing problems and suggestions for future studies.