Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells

Subcellular fractionation approaches remain an indispensable tool among a large number of biochemical methods to facilitate the study of specific intracellular events and characterization of protein functions. During apoptosis, the best-known form of programmed cell death, numerous proteins are translocated into and from the nucleus. Therefore, suitable biochemical techniques for the subcellular fractionation of apoptotic cells are required. However, apoptotic bodies and cell fragments might contaminate the fractions upon using the standard protocols. Here, we compared different nucleus/cytoplasm fractionation methods and selected the best-suited approach for the separation of nuclear and cytoplasmic contents. The described methodology is based on stepwise lysis of cells and washing of the resulting nuclei using non-ionic detergents, such as NP-40. Next, we validated this approach for fractionation of cells treated with various apoptotic stimuli. Finally, we demonstrated that nuclear fraction could be further subdivided into nucleosolic and insoluble subfractions, which is crucial for the isolation and functional studies of various proteins. Altogether, we developed a method for simple and efficient nucleus/cytoplasm fractionation of both normal and apoptotic cells.

Preanalytical Stability of CSF Total and Oligomeric Alpha-Synuclein

Background: The role of cerebrospinal fluid (CSF) alpha-synuclein as a potential biomarker has been challenged mainly due to variable preanalytical measures between laboratories. To evaluate the impact of the preanalytical factors contributing to such variability, the different subforms of alpha-synuclein need to be studied individually.Method: We investigated the effect of exposing CSF samples to several preanalytical sources of variability: (1) different polypropylene (PP) storage tubes; (2) use of non-ionic detergents; (3) multiple tube transfers; (4) multiple freeze-thaw cycles; and (5) delayed storage. CSF oligomeric- and total-alpha-synuclein levels were estimated using our in-house sandwich-based enzyme-linked immunosorbent assays.Results: Siliconized tubes provided the optimal preservation of CSF alpha-synuclein proteins among other tested polypropylene tubes. The use of tween-20 detergent significantly improved the recovery of oligomeric-alpha-synuclein, while multiple freeze-thaw cycles significantly lowered oligomeric-alpha-synuclein in CSF. Interestingly, oligomeric-alpha-synuclein levels remained relatively stable over multiple tube transfers and upon delayed storage.Conclusion: Our study showed for the first-time distinct impact of preanalytical factors on the different forms of CSF alpha-synuclein. These findings highlight the need for special considerations for the different forms of alpha-synuclein during CSF samples’ collection and processing.

Mixed fruit wastes as a potential source for Bacteriocin Producing Lactic acid bacterium – Lactobacillus saniviri NKSS1

Mixed fruit juices contain microflora on the surface of fruits during the harvest and post-harvest practices. The presence of useful organisms like lactic acid bacteria from the mixed fruit wastes was explored in this study since these microbes use these wastes as a nutrient source for their growth. The lactic acid bacteria isolated using MRS medium was identified as Lactobacillus saniviri NKSS1 by 16s rRNA analysis. The bacteriocin produced by Lactobacillus saniviri NKSS1 showed inhibitory effect against the food pathogen (Listeria monocytogenes) and clinical pathogen (Acinetobacter baumannii). Optimization of bacteriocin production from Lactobacillus saniviri NKSS1 was achieved at 24 h of incubation, temperature at 35 °C with the initial medium pH of 6.5. The carbon & nitrogen sources like dextrose (3% w/v) and yeast extract (0.75% w/v) enhanced the production of bacteriocin in MRS medium. Antimicrobial activity was reduced in the partially purified bacteriocin when incubated at 95 °C for 2 h but it retained its activity in the pH range of 5.5 to 8.5. Whereas, metals like CuSO4 and MgSO4 at (0.5 % w/v) interfered with the antagonistic activity of partially purified bacteriocin. Ionic detergents like SDS and CTAB partially decreased the antimicrobial activity, while other non-ionic detergents inhibited the antimicrobial activity completely. The molecular weight of partially purified bacteriocin from Lactobacillus saniviri NKSS1 was found to be 10.9 kDa. It can be concluded that bacteriocin of L. saniviri NKSS1 holds a promising potential for extension of shelf-life and improvement of microbiological safety in food industries.