Biotin

Adduction of a nitric oxide moiety (NO·) to cysteine(s) termed S-nitrosylation (SNO) is a novel mechanism for NO to regulate protein function directly. However, endothelial SNO-protein network affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture (SILAC) for comparing vascular endothelial growth factor (VEGFA)- and NO donor- responsive endothelialnitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-12C6 14N4-Arg and L-12C6 14N2-Lys) or "heavy" (L-13C6 15N4-Arg and L-13C6 15N2-Lys) amino acids. The "light" cells were treated with a NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the "heavy" cells received vehicle as control. Equal amounts of cellular proteins from the "light" (GSNO- or VEGFA-treated) and "heavy" cells were mixed for labeling SNO-proteins by biotin-switch technique and then trypsin-digested.

Responses to stocking density (SD), dietary biotin concentration and litter condition were evaluated on 2016 Ross 308 male broilers in the fattening period (day 22-day 42). The birds were placed in 48 pens with either dry or wet litter to simulate the final stocking density of 30 kg (12 broilers/m2 ; normal stocking density, NSD) and 40 kg (16 broilers/m2 ; high stocking density, HSD) of body weight (BW)/m2 floor space. A corn-soybean meal-based diet was supplemented with biotin to provide a normal (NB; 155 μg/kg) or high (HB, 1521 μg/kg) level of dietary biotin. There were six repetitions per treatment. The inappropriate moisture content of litter associated with HSD was avoided (p < 0.05) by good management (SD difference: dry litter, 6.65% vs. wet litter, 13.23%; 42 days), which made it advantageous (p < 0.01) for footpad (SD difference: dry litter, 0.118 vs. wet litter, 0.312; weekly average value) and hock health (SD difference: dry litter, 0.

Cytosolic Dynamin-related protein 1 (Drp1) is required for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used gene-editing technology to create a suite of cell lines lacking adaptors MiD49, MiD51, MFF and Fis1. Increased mitochondrial connectivity was observed following loss of individual adaptors while this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49/MiD51 showing a more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we find that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission.

Electron beam (e-beam) lithography was employed to prepare one protein immobilized hydrogel encapsulated inside another by first fabricating protein-reactive hydrogels of orthogonal reactivity and subsequently conjugating the biomolecules. Exposure of thin films of eight arm star poly(ethylene glycol) (PEG) functionalized with biotin (Biotin-PEG), alkyne (Alkyne-PEG) or aminooxy (AO-PEG) end-groups to e-beam radiation resulted in cross-linked hydrogels with the respective functionality. It was determined via confocal microscopy that a nominal size exclusion effect exists for streptavidin immobilized on Biotin-PEG hydrogels of feature sizes ranging from 5 to 40 μm. AO-PEG was subsequently patterned as an encapsulated core inside a contiguous outer shell of Biotin-PEG. Similarly, Alkyne-PEG was patterned as a core inside an AO-PEG shell. The hydrogel reactive end-groups were conjugated to dyes or proteins of complementary reactivity, and the three-dimensional (3-D) spatial orientation was determined for both configurations using confocal microscopy.