Journal: bioRxiv

Article Title: Genome-wide screen for enhanced noncanonical amino acid incorporation in yeast

doi: 10.1101/2022.04.28.489958

Figure Lengend Snippet: Overview of screening campaign. (A) Diagram of the general architecture of the four unique plasmid systems transformed into the ~6,000-member yeast knockout (YKO) collection containing the reporter and orthogonal translation system (OTS). The reporters consisted of a BFP linked to GFP by a peptide linker containing an amber (TAG) stop codon. Two reporters, BXG and BXG-altTAG, that differ by the location of the stop codon within the peptide linker were used. The OTS contained an aaRS and suppressor tRNA CUA . Two aaRSs, TyrOmeRS and LeuOmeRS, were used. (B) Structure of ncAA, O -methyl-L-tyrosine (OmeY), used for screens. (C) General schematic of the screening process. A pooled YKO collection transformed with the reporter/OTS was induced in the presence of 0, 0.1, or 1 mM OmeY. FACS was used to isolate cells with high GFP expression, which indicated successful OmeY incorporation in the reporter (or, in some cases, canonical amino acid incorporation). Following multiple rounds of screening, the barcodes of isolated colonies from enriched populations were Sanger sequenced to identify their respective gene deletions. (D) Representative flow cytometry overlay dot plots comparing readthrough of naïve (presort) and enriched (post sort) populations of three sorting paths. Populations were induced with the same concentration of OmeY used for screening.

Article Snippet: NcAAs were purchased from these companies: p -acetyl-L-phenylalanine (SynChem); p -azido-L-phenylalanine (Chem-Impex International); O -methyl-L-tyrosine (Chem-Impex International); p -propargyloxy-L-phenylalanine (Iris Biotech); 4-azidomethyl-L-phenylalanine (SynChem); 4-iodo-L-phenylalanine (AstaTech); N ε -Boc-L-lysine (Chem-Impex International).

Techniques: Plasmid Preparation, Transformation Assay, Knock-Out, Expressing, Isolation, Flow Cytometry, Concentration Assay

Journal: Regenerative Therapy

Article Title: Orexin-A increases the differentiation of human olfactory sensory neurons through orexin receptor type 1

doi: 10.1016/j.reth.2024.10.014

Figure Lengend Snippet: Immunofluorescence of orexin receptors and dose-response analyses of orexin-A. (a) Immunofluorescence images of OSNs stained with OX1R and OX2R (green). (b) Schematic showing the proposed mechanism of orexin-mediated differentiation of OSNs and dose-response analyses of suvorexant. Western blot densitometric analysis of (c) ASCL1, (d) βIII Tubulin, and (e) OMP, respectively. (p > 0.05, compared to controls, n = 5).

Article Snippet: The orexin receptor type 1 (OX1R) antagonist, SB-674042 (HY-10898, MedChem Express, NJ, USA), and orexin receptor type 2 (OX2R) antagonist, TCS-OX2-29 (HY-100452, MedChem Express) were diluted in the dimethyl sulfoxide, DMSO with 1 μg/mL and 10 μg/mL, respectively.

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Regenerative Therapy

Article Title: Orexin-A increases the differentiation of human olfactory sensory neurons through orexin receptor type 1

doi: 10.1016/j.reth.2024.10.014

Figure Lengend Snippet: The effect of OX1R antagonists (SB674042) and OX2R antagonists (TCS-OX2-29) on the differentiation of OSN. The expression of various markers was assessed through western blots (a and g) and subsequently analyzed densitometrically for ADCY3, Golf, ASCL1, βIII tubulin, and OMP (b-f and h-l). (Asterisks indicate p < 0.05, compared to controls, n = 5–6).

Article Snippet: The orexin receptor type 1 (OX1R) antagonist, SB-674042 (HY-10898, MedChem Express, NJ, USA), and orexin receptor type 2 (OX2R) antagonist, TCS-OX2-29 (HY-100452, MedChem Express) were diluted in the dimethyl sulfoxide, DMSO with 1 μg/mL and 10 μg/mL, respectively.

Techniques: Expressing, Western Blot