Project description:Ionizing radiation (IR) therapy for malignant tumors can damage adjacent tissues, leading to severe wound complications. Plasma-derived exosome treatment has recently emerged as a safe and impactful cell-free therapy. Herein, we aimed to determine whether plasma-derived exosomes could improve the healing of post-radiation wound. Rat plasma-derived exosomes (RP-Exos) were locally injected on cutaneous wounds created on the backs of irradiated rats and boosted the healing process as well as the deposition and remodeling of the extracellular matrix with collagen formation. Subsequently, the effects of RP-Exos were further evaluated on irradiated fibroblasts in vitro. The results suggested that exosomes promoted fibroblast proliferation, migration, cell cycle progression, and cell survival. Moreover, transcriptome sequencing, analysis, and quantitative polymerase chain reaction validation were performed to identify the underlying molecular mechanisms. RP-Exos enhanced the expression of cell proliferation and radioresistance-related genes, and yet downregulated ferroptosis pathway in irradiated fibroblasts. Inhibition of ferroptosis by RP-Exos was further confirmed through colorimetric assay, fluorescence probe and flow cytometry in ferroptosis-induced fibroblasts. Our results suggest that RP-Exos regulate cell proliferation and ferroptosis in radiated fibroblasts, thereby boosting the healing of radiated wounds. These findings support plasma-derived exosomes as a potential therapeutic method for post-radiation wound complications.
Project description:<p>Clinical metabolic phenotyping employs metabolomics and lipidomics to detect and measure thousands of metabolites and lipids within human samples. This approach aims to identify metabolite and lipid changes between phenotypes (e.g. disease status) that aid understanding of biochemical mechanisms driving the phenotype. Sample preparation is a critical step in clinical metabolic phenotyping: it must be reproducible and give a high extraction yield of metabolites and lipids. Here, we assessed the extraction of polar metabolites from human urine and polar metabolites and lipids from human plasma for analysis by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics and lipidomics. We evaluated several monophasic (urine and plasma) and biphasic (plasma) extractions, and we also tested alterations to (a) solvent-biofluid incubation time and temperature during monophasic extraction, and (b) phase partitioning time during biphasic extraction. Extracts were analysed by three UHPLC-MS assays: (i) HILIC for urine and plasma, (ii) C18 aqueous reversed phase for urine, and (iii) C18 reversed phase for plasma lipids, and the yield and reproducibility of each method was assessed. For HILIC UHPLC-MS plasma and urine analysis, monophasic 50:50 methanol:acetonitrile had the most detected putatively-identified polar metabolites. If lipid removal from the plasma polar HILIC extract is required, then the biphasic methanol/chloroform/water method is recommended. For C18 (aqueous) UHPLC-MS urine analysis, 50:50 methanol:water had high reproducibility and yield. For C18 UHPLC-MS plasma lipidomics, monophasic 100% isopropanol had the highest detection response of all annotated lipid classes. Increasing monophasic incubation time and temperature had little benefit on metabolite and lipid yield and reproducibility.</p>
Project description:Bacteria was grown at 30C in 3 different conditions, i.e. SYN (syngas and minimal medium- ATCC no 1789), AC (0.3% acetate in minimal medium- ATCC no 1789) and TSB (tryptic soy broth). After harvesting by centrifugation, O. carboxidovorans pellets (1g) were lysed in 100 mM Tris-Cl pH 8.0, 2% Triton X-100, 2.6 mg/ml sodium azide, 8 mM PMSF by sonication on ice (4 pulses of 15 s duration each). For each condition of growth 4 samples (1g pellets) were separately treated (i.e. lysed and processed further). The supernatants were treated with 50% cold TCA, and the precipitated protein washed with acetone. The pellets were resuspended in solubilization buffer (7M urea, 20 mM tris-Cl, pH 8.0, 5 mM EDTA, 5 mM MgCl2, 4% CHAPS), and protein concentration was determined using the Plus One 2-D Quant Kit (Amersham) following the manufacturers instructions. Protein samples from each treatment were stored at -80 C. One hundred micrograms of each protein sample was resuspended in 0.1 M ammonium bicarbonate, 5% HPLC grade ACN, reduced in 5 mM DTT (65 C, 5 min), alkylated in 10 mM iodoacetamide (30 C, 30 min), and then trypsin digested until there was no visible pellet (1:50 w/w 37 C, 16 h). Peptides were desalted using a peptide microtrap (Michrom BioResources, Auburn, CA) and eluted using a 0.1% TFA, 95% ACN solution. Desalted peptides were dried in a vacuum centrifuge and resuspended in 20 ?l of 0.1% formic acid. Peptides were separated by strong cation exchange (SCX) liquid chromatography (LC) followed by reverse phase (RP) LC coupled directly in line with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). 2DLC ESI MS/MS was done exactly as described (1). All searches were done using TurboSEQUEST (Bioworks Browser 3.2; Thermo Electron). Mass spectra and tandem mass spectra were searched against all annotated proteins from the strain OM5 including all the annotated plasmid-encoded proteins. Cysteine carbamidomethylation and methionine oxidation (single and double) were included in the search strategy. We used the reverse database functionality in Bioworks 3.2 and searched MS2 data against a reversed OM5 database using identical search criteria.
Project description:Lameness due to femoral head separation (FHS) is a production and welfare issue in commercial poultry. FHS is an idiopathic disorder, which is attributed to a myriad of factors but in order to improve bone health, broiler breeders must be prognosed for disease susceptibility and selected against FHS using biomarkers. Proteins from plasma of blood, which can be obtained using minimally invasive methods represent an ideal, rich source of biomarkers which might be different in susceptible or affected birds. The peptide and proteins in plasma of healthy (HLTH) and affected birds (FHS) were compared using Matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS) and Liquid chromatography and tandem mass spectrometry (LC-MS/MS). The peptide profile of HLTH and FHS were compared using ClinPro tools and the differentially expressed peptides were isolated by Reverse phase liquid chromatography fractionation (C18-RP-HPLC) and identified using peptide mass fingerprinting. Peptides derived from fibrinogen precursor and fetuin were reduced in FHS birds. Based on the proteomic analysis, proteins such as Gallinacin 10, Apolipoprotein A-1 and Hemoglobin chains are elevated in FHS while Alpha 1-acid glycoprotein is reduced in FHS birds. Our study shows that bodyweight, lipid profile and the above mentioned proteins could be useful as a biomarker for improvement of bone health. These proteins indicate that blood lysis, antimicrobial defense and lipid disorder but lack of an inflammatory response might be consequential to FHS.