Journal: Journal of Extracellular Biology

Article Title: TDO2‐augmented fibroblasts secrete EVs enriched in immunomodulatory Y‐derived small RNA

doi: 10.1002/jex2.73

Figure Lengend Snippet: NT4 is a short non‐coding RNA fragment with immunomodulatory function derived from human Y RNA‐4 (hY4). (a) Schematic of the hY4 sequence and functional regions. (b) Sequence of NT4 and its position within the hY4 fragment EV‐YF1. (c) qPCR showing NT4 enrichment in CCM of fibroblasts transduced with TDO2‐activating lentivirus (TDO2‐CCM) compared to those from fibroblasts transduced with a lentivirus containing a control vector (VC‐CCM). (d) QPCR of NT4 from SEC‐isolated EVs compared to the EV‐depleted fraction. (e) Absolute quantification (NT4 copies/10 3 particles) of TDO2‐CCM, VC‐CCM, SEC‐isolated TDO2‐EVs and the EV‐depleted fraction. (f) RNAse protection assay demonstrating NT4 is encapsulated inside TDO2‐EVs. (g) Broad expression of the monocyte marker, CD68, in bone marrow‐derived macrophages. (h) Equivalent uptake of TDO2‐ and HDF‐EVs (EV ceramide dye; red, phalloidin: green and nuclei: blue) by bone marrow derived macrophages at 30, 60 and 180 min post exposure as measured by internalisation of red signals inside macrophages and, (i) area under the curve measurement of cumulative signal at all three time points. (j) Representative image of TDO2‐ and HDF‐EVs (EV ceramide dye; red, phalloidin: green and nuclei: blue) by bone marrow derived macrophages at 1‐h post‐exposure. (k–m) TDO2‐EV and NT4 pre‐treatment lowered expression of inflammatory genes il1b and il6 , as well as pro‐senescence p21 / cdkn1a . Suppression of NT4 in TDO2‐CCM abrogated the suppressive effect of TDO2‐CCM on il1b , il6 and p21/cdkn1a in LPS‐stimulated macrophages. Scale bars: 100 μm. Statistical analysis between two groups was done using Student's T test and comparisons between three or more groups was done using one‐way ANOVA with Sidak's multiple comparison test. Error bars represent standard deviation,* p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: RNA was isolated using the urine EV RNA isolation kit (Norgen Biotek, cat# 47200). qPCR was performed to assess NT4 expression levels.

Techniques: Derivative Assay, Sequencing, Functional Assay, Transduction, Control, Plasmid Preparation, Isolation, Quantitative Proteomics, Rnase Protection Assay, Expressing, Marker, Comparison, Standard Deviation

Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

Article Title: Comprehensive, robust, and sensitive UPLC-MS/MS analysis of free biogenic monoamines and their metabolites in urine

doi: 10.1016/j.jchromb.2018.09.012

Figure Lengend Snippet: Long-term stability investigations in human urine samples ( n = 30) and Lyphochek Quantitative Urine Controls (Level 1 Normal and Level 2 Abnormal, n = 24 for both).

Article Snippet: To assess long-term stability, both Lyphochek Quantitative Urine Controls (Bio-Rad Laboratories, Inc. CA, n = 24) and experimental urine samples (n = 30) were reanalyzed after storage at –80 °C for > 200 days subsequent to the first measurement.

Techniques: