Mobile electrophoresis kit for high school students: Scientific practices with innovation.

Gel electrophoresis (GE) is the most preferred and adapted technique for the separation and identification of biological molecules like proteins/peptides and nucleic acids from diverse types of organisms. All over the world, researchers, educators, and students aspiring to work in biochemistry and molecular biology disciplines use the polyacrylamide gel electrophoresis (PAGE) technique for resolving proteins/nucleic acids for understanding the structure and function of any cell. A simple PAGE technique requires a wide range of chemicals/reagents along with a well-equipped and well-spaced laboratory. We have developed a compact and impeccable mobile electrophoresis kit suitable for any vertically oriented PAGE technique. This comprehensive and portable laboratory set-up provides the complete advantages of safety, cost-efficiency, space management, and utility to the researchers for high-throughput research. All new equipment of the mobile electrophoresis kit is fabricated using inexpensive and off-the-rack components. Overall performance of the mobile kit was verified through a practical exercises executed by high school students with positive outcomes.

Sustainable biodiesel production from microalgae Graesiella emersonii through valorization of garden wastes-based vermicompost.

Biodiesel production from microalgae has gained significant interest recently due to the growing energy demand and non-renewable nature of petroleum. However, high cost of production and environmental health related issues like excess use of inorganic fertilizers, eutrophication are the major constraints in commercial-scale biodiesel production. Besides this, solid wastes (garden-based) management is also a global concern. In the present study, to overcome these limitations vermicompost extract was tested as nutrient source to enhance growth performance and lipid production from a freshwater microalga (Graesiella emersonii MN877773). Garden wastes were first converted into vermicompost manure and its extract (aerobic and anaerobically digested) was prepared. The efficacy of the extract was then tested in combination with BG11 medium. The mixotrophic cultivation of microalgae in anaerobically digested vermicompost extract at 50:50 combination with BG11 medium enhanced the cell biomass (0.64 g d. wt. L-1) and lipid productivity (3.18 mg L-1 day-1) of microalgae by two times. Moreover, the combination also improved the saturated (methyl palmitate) and monounsaturated fatty acids (oleic acid) content in the test algae. The quality of biodiesel also complies with all the properties of biodiesel standard provided by India, the USA, and Europe except the cold filter plugging property. The combination was also found to improve the cell biomass (0.041 g L-1) as compared to BG11 medium in mass-scale cultivation. Hence, the study proved that G. emersonii grown in media supplemented with garden waste-based vermicompost extract had significant potential for mass-scale biodiesel and bioproduct production.

Immunomodulatory and antimicrobial non-mulberry Antheraea mylitta silk fibroin accelerates in vitro fibroblast repair and regeneration by protecting oxidative stress.

The antimicrobial nature of Antharaea mylitta silk-fibroin (SF) is reported but antioxidant potential and the immunomodulatory role towards the fibroblast cell repair process is not explored. Polyurethane is reported to have inflammatory potential by mononuclear cells directed cytokine release, which can guide fibroblast repair. Present study demonstrates the conjunctive effect of inflammatory PU/SF to regulate the favorable shift from pro-inflammatory to anti-inflammatory cytokine stimulation for accelerated fibroblast repair. Minimal inhibitory concentration of SF was determined against pathogenic strains and the effect of SF was investigated for fibroblast NIH3T3 cell adhesion. SF doses (8, 8.5, 9 mg mL-1) were found to be greater than both the IC50 of DPPH scavenging and the ED50 for NIH3T3 proliferation. Anti-lipid peroxidase (ALP) activity of SF doses and citric acid-treated NIH3T3 cells were compared under hydrogen peroxide (H2O2) induced oxidative stress. 9 mg mL-1 SF showed greater ALP activity than the citric acid standard. SF-driven protection to oxidative damage was measured by viable cell fraction in trypan blue dye exclusion assay where 9 mg mL-1 SF showed the highest viability (p ≤ 0.05). 9 mg mL-1 SF was blended with PU for scaffold (w/v = 2 : 5, 2 : 7, 2 : 9) fabrication. The protective effect of PU/SF (2 : 5, 2 : 7, 2 : 9) against oxidative stress was verified by damaged cell survival in MTT assay and DNA quantification. The highest number of cells survived on PU/SF (2 : 9) at all intervals (p ≤ 0.01) upon oxidative damage; PU/SF (2 : 9) was also fabricated by employing the immobilization technique. Immobilized PU/SF (2 : 9) exhibited a greater zone of microbial inhibition, a higher extent of inhibition to microbial adherence, and caused more LDH release from bacterial cell membrane due to membrane rupture, resulting in bacterial cell death (E. coli, K. pneumoniae, P. aeruginosa, S. aureus) compared to the experimental results shown by blended PU/SF (2 : 9). The protective nature of PU/SF (2 : 9) against oxidative stress was ensured through the LDH activity of damaged NIH3T3 cells. Initial raised IL-6, TNF-alpha (pro-inflammatory cytokines) and lowered IL-8, IL-10 (anti-inflammatory cytokine) profiles coupled with fallen IL-6, TNF-alpha, and elevated IL-8, IL-10 at later hours synergistically progress the inflammatory phase of in vitro scratch wound repair in mononuclear culture treated by PU/SF (2 : 9).

Anti-inflammatory effect of epidermal growth factor conjugated silk fibroin immobilized polyurethane ameliorates diabetic burn wound healing.

Few studies are reported on immunomodulatory potential of non-mulberry (Antheraea mylitta) silk fibroin (SF) combined with polyurethane (PU) in diabetic wound healing. In this study, PU/SF (Antheraea mylitta) scaffolds were fabricated by blending and immobilization techniques. Effective SF dosage was determined and incorporated according to minimum inhibitory concentrations against wound associated bacterial strains while fabricating scaffold. Dermal fibroblast NIH3T3 cells were seeded on epidermal growth factor (EGF) treated and untreated PU/SF scaffolds, fabricated by blending and immobilization techniques. Fibroblast seeded PU/SF scaffolds were investigated for anti-inflammatory response in wound recovering potential comparing with Acticoat™ in third degree burn of streptozotocin induced diabetic rats. At 16th, 24th days, promising healing was achieved with faster granulation, enhanced collagenization, patterned re-epithelialization by EGF treated cellular immobilized PU/SF in normal, hyperglycemic burn. Biomarkers of different healing stages, CD31 (haemostasis), Ki67 (proliferative), alpha-sma, COL III (maturation) were examined. Since hyperglycemic burn is characterized by inflated pro-inflammatory cytokines, serum, tissue IL-6,8,10 were recorded, which revealed timely restoration of inflated IL-6,8 and protection against IL-10 elevation by cellular immobilized PU/SF compared to Acticoat™ (p ≤ 0.05), control (p ≤ 0.01). E-cadherin (gap junction protein), MMP 9 response suggested anti-inflammatory role of PU/SF on accelerated healing of thermal injury as potent dermal substitute.

User-friendly tool kits for protein gel electrophoresis techniques: A training program for high school students.

Recent advancements in biochemical sciences have helped the researchers to explore the molecular logic of life inclusive of its multifarious expressions and explain many facts about the structure and functions of cellular macromolecules. Due to its simple and cost-effective nature, polyacrylamide gel electrophoresis (PAGE) has become the most favored technique for qualitative and quantitative examination of macromolecules. Major drawbacks of such modifications are the cost and operational complexities faced by naïve students. Many interlinking laboratory equipment are needed in the school laboratories for the conduct of even simple scientific experiment. Some of these costly modern equipment are inaccessible for students of small laboratories, and their alternatives are not easily available. Many of these laboratory equipment required for routine gel electrophoresis technique can be fabricated in their simplest form using off-the-shelf components. A short term biochemistry training program was executed for high school students to provide them "hands-on" training using newly modified equipment, which was proved to be an exciting way of learning biochemical gel separation techniques. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):566-577, 2018.

A tetrad apparatus for protein gel casting, electrophoresis, staining, and scanning techniques with dual sensors for automatic detection of gel polymerization and protein migration.

Recent advancements in biochemical sciences have facilitated researchers to explore the structure and function of macro molecules in a cell. PAGE is one of the most favored and adapted laboratory techniques. Due to its simple and economical procedures, several variants or new modifications are routinely observed in the basic electrophoresis technique that comprises gel casting, electrophoresis, staining, and imaging process which consequently necessitates additional apparatuses/components in the laboratory. Operation of these additional apparatuses/components lengthens the pre- and postelectrophoresis procedures involving many intermittent tedious and time-consuming steps. A universal apparatus that can facilitate all such associated techniques is lacking and is of utmost importance for fast and effective results. An apparatus that can perform synchronized action of slab gel casting (16 × 16 cm), electrophoresis (SDS-PAGE), dye staining (Coomassie), and imaging (scanning) techniques with real-time monitoring through sensor technology is described in this article. The estimated cost (∼$150) of fabrication of the apparatus is very economical and simple assembly procedure of the main apparatus can be completed within ∼30 min after fabrication.

Simple and rapid system for two-dimensional gel electrophoresis technique: A laboratory exercise for high school students.

Polyacrylamide gel electrophoresis (PAGE) is the most classical technique favored worldwide for resolution of macromolecules in many biochemistry laboratories due to its incessant advanced developments and wide modifications. These ever-growing advancements in the basic laboratory equipments lead to emergence of many expensive, complex, and tricky laboratory equipments. Practical courses of biochemistry at high school or undergraduate levels are often affected by these complications. Two dimensional gel electrophoresis technique (2D-PAGE) used for resolving thousands of proteins in a gel is a combination of isoelectric focusing (first dimension gel electrophoresis technique) and sodium-dodecylsulphate PAGE (second dimension gel electrophoresis technique or SDS-PAGE). Two different laboratory equipments are needed to carry out effective 2D-PAGE technique, which also invites extra burden to the school laboratory. Here, we describe a low cost, time saving and simple gel cassette for protein 2D-PAGE technique that uses easily fabricated components and routine off-the-shelf materials. The performance of the apparatus was verified in a practical exercise by a group of high school students with positive outcomes. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):237-244, 2018.

Multi-gel casting apparatus for vertical polyacrylamide gels with in-built solution flow system and liquid level detectors.

PAGE is the most widely used technique for the separation and biochemical analysis of biomolecules. The ever growing field of proteomics and genomics necessitates the analysis of many proteins and nucleic acid samples to understand further about the structure and function of cells. Simultaneous analysis of multiple protein samples often requires casting of many PAGE gels. Several variants of multi-gel casting/electrophoresis apparatuses are frequently used in research laboratories. Requirement of supplementary gels to match the growing demand for analyzing additional protein samples sometimes become a cause of concern. Available apparatuses are not amenable to and therefore, not recommended for any modification to accommodate additional gel casting units other than what is prescribed by the manufacturer. A novel apparatus is described here for casting multiple PAGE gels comprising four detachable components that provide enhanced practicability and performance of the apparatus. This newly modified apparatus promises to be a reliable source for making multiple gels in less time without hassle. Synchronized functioning of unique components broaden the possibilities of developing inexpensive, safe, and time-saving multi-gel casting apparatus. This apparatus can be easily fabricated and modified to accommodate desired number of gel casting units. The estimated cost (∼$300) for fabrication of the main apparatus is very competitive and effortless assembly procedure can be completed within ∼30 min.

Simple and cost effective apparatus for silver staining of polyacrylamide gels with sequential reagents addition and real time monitoring.

Highly reproducible results in molecular biology depend a lot on effective staining and destaining methods. Silver staining of polyacrylamide DNA and protein gel has been adopted widely in the molecular biology laboratories for detecting a very low nanogram range of sample. An efficient staining of a polyacrylamide gel requires a number of well controlled and highly sensitive steps that often becomes tiresome when done manually or when there are a number of gels to be stained simultaneously. Since, silver staining is a multistep procedure that requires proper fixation and exchange of substance, a reliable protocol is necessary and a simple apparatus may be an added advantage to carry out the steps with ease and safety. Here, we describe a simple and cost effective device made from off-the-shelf components for some established silver staining protocols. Staining is done on a tray while six graduated bottles with a liquid delivery stopcock each, is connected to the tray through silicon tubing. The used up solution is drained off completely from the staining tray through a liquid outlet stopcock using vacuum pressure. The system is fixed with a camera connected to a computer for effective control of the staining process in each step. The apparatus provides the researchers with efficient staining and real time monitoring of gels without the need for handling toxic chemicals.