Benchwarmer (BNCH) gene encodes an orphan transmembrane transporter belonging to the Major Facilitator Superfamily (MFS), facilitating the transport of ions, amino acids, simple sugars and recently lysolipids. The loss of BNCH function caused lethality in several animal models with neurodegeneration and senescence. At the cellular level, dysregulation of BNCH leads to adverse phenotypes of lysosome and also autophagy (i.e. dyshomeostasis, accumulation of carbohydrates and sphingolipids, and enlarged lysosome). However, the molecular function and ligand of BNCH protein remain to be unrevealed. This study aims to create a radical substitution change in human BNCH coding gene to knock out the protein functions. More specifically, lysine (K) was used to replace the glutamic acid residue 164 (E164K) which is conserved in many animals (fly, zebrafish, mouse and human) and this E164K mutation recapitulated BNCH mutant phenotype. In conclusion, BNCH harboring E164K (BNCH*) was successfully produced by site-directed mutagenesis and cloned into pcDNA.3.1 vector. The construct was transformed into E. coli OmniMAX and that provides a valuable cell assay to search for the molecular ligand of BNCH.
Staphyloxanthin (STX) is a carotenoid pigment produced by Staphylococcus aureus to protect the bacteria from oxidation stress. Eliminating Staphyloxanthin from S. aureus cell membrane by inducing the pigment photolysis using 460nm light, then killing weakened bacteria with an H2O2 solution could be a new approach to develop anti - Staphylococcus aureus procedure. A model to prove the feasibility of this combination to kill Methicillin-Resistant Staphylococcus Aureus (MRSA) bacteria on a flat surface was tested. Material and method: Staphyloxanthin pigment extracted from MRSA bacteria was treated with 460nm light at different light intensities to evaluate the photolysis potential of 460nm light. The change in MRSA shape after 460nm light treatment was also investigated by Scanning electron microscopy. Using glass cover-slips as an emulated model for contact surface in public, the combination of different 460nm light intensities and H2O2 0.75% was tested on the surface loaded with MRSA living cells, and the number of MRSA cells survived after treatment was enumerated. Result: Higher intensity and longer light treatment yielded a higher photolysis effect, as 100 and 200 mW/cm2 light intensity could degrade 77.50% to 83.45% of STX pigment after 20 minutes of irradiation. Also, MRSA cells had significant changes as more wrinkles and bumps appeared under high-intensity 460nm light. When tested on the flat surface of the coverslip, the strongest MRSA eradication effect can be observed in the combination of 200 mW/cm2 light treatment with 0.75% H2O2 solution, as 100% MRSA cells were completely killed after 20 minutes of treatment.
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