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1.
Small-protein Enrichment Assay Enables the Rapid, Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma.
Harney, DJ, Hutchison, AT, Su, Z, Hatchwell, L, Heilbronn, LK, Hocking, S, James, DE, Larance, M
Molecular & cellular proteomics : MCP. 2019;(9):1899-1915
Abstract
Unbiased and sensitive quantification of low abundance small proteins in human plasma (e.g. hormones, immune factors, metabolic regulators) remains an unmet need. These small protein factors are typically analyzed individually and using antibodies that can lack specificity. Mass spectrometry (MS)-based proteomics has the potential to address these problems, however the analysis of plasma by MS is plagued by the extremely large dynamic range of this body fluid, with protein abundances spanning at least 13 orders of magnitude. Here we describe an enrichment assay (SPEA), that greatly simplifies the plasma dynamic range problem by enriching small-proteins of 2-10 kDa, enabling the rapid, specific and sensitive quantification of >100 small-protein factors in a single untargeted LC-MS/MS acquisition. Applying this method to perform deep-proteome profiling of human plasma we identify C5ORF46 as a previously uncharacterized human plasma protein. We further demonstrate the reproducibility of our workflow for low abundance protein analysis using a stable-isotope labeled protein standard of insulin spiked into human plasma. SPEA provides the ability to study numerous important hormones in a single rapid assay, which we applied to study the intermittent fasting response and observed several unexpected changes including decreased plasma abundance of the iron homeostasis regulator hepcidin.
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Vegetarian-Based Dietary Patterns and their Relation with Inflammatory and Immune Biomarkers: A Systematic Review and Meta-Analysis.
Craddock, JC, Neale, EP, Peoples, GE, Probst, YC
Advances in nutrition (Bethesda, Md.). 2019;(3):433-451
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Abstract
Dietary patterns with substantial proportions of energy from plant sources have been associated with favorable biomarkers of low-grade inflammation. Less is known of the relation between vegetarian-based dietary patterns and markers of inflammation and immune status. This systematic review and meta-analysis aimed to determine the relation between vegetarian-based dietary patterns and inflammatory and immune markers (C-reactive protein, tumour necrosis factor α, fibrinogen, natural killer cells, leukocytes, lymphocytes, thrombocytes, interleukins, and immunoglobulins). PubMed, Medline, and Cochrane scientific databases were searched to identify relevant studies. Random effects meta-analyses were conducted to assess the weighted mean differences (WMDs) for each outcome variable between vegetarian and non-vegetarian groups. Thirty observational and 10 intervention studies were included in the review. Pooled effects of vegetarian-based dietary patterns were associated with significantly lower concentrations of CRP (WMD: -0.61 mg/L; 95% CI: -0.91, -0.32 mg/L; P = 0.0001), fibrinogen (WMD: -0.22 g/L; 95% CI: -0.41, -0.04 mg/L; P = 0.02), and total leukocyte (WMD: -0.62 × 10(3)/μL; 95% CI -1.13 × 10(3), -0.10 × 10(3)/μL; P = 0.02) compared with those following non-vegetarian dietary patterns in observational studies. Insufficient data were identified for a meta-analysis of intervention studies. This study provides evidence that vegetarian-based dietary patterns are associated with lowered serum C-reactive protein, fibrinogen, and total leukocyte concentrations. Future research should focus on large-scale intervention trials, contrasting differences in inflammation and immune status and function between vegetarian and non-vegetarian-based populations.
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A moderate increase in dietary zinc reduces DNA strand breaks in leukocytes and alters plasma proteins without changing plasma zinc concentrations.
Zyba, SJ, Shenvi, SV, Killilea, DW, Holland, TC, Kim, E, Moy, A, Sutherland, B, Gildengorin, V, Shigenaga, MK, King, JC
The American journal of clinical nutrition. 2017;(2):343-351
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Abstract
BACKGROUND Food fortification has been recommended to improve a population's micronutrient status. Biofortification techniques modestly elevate the zinc content of cereals, but few studies have reported a positive impact on functional indicators of zinc status. OBJECTIVE We determined the impact of a modest increase in dietary zinc that was similar to that provided by biofortification programs on whole-body and cellular indicators of zinc status. DESIGN Eighteen men participated in a 6-wk controlled consumption study of a low-zinc, rice-based diet. The diet contained 6 mg Zn/d for 2 wk and was followed by 10 mg Zn/d for 4 wk. To reduce zinc absorption, phytate was added to the diet during the initial period. Indicators of zinc homeostasis, including total absorbed zinc (TAZ), the exchangeable zinc pool (EZP), plasma and cellular zinc concentrations, zinc transporter gene expression, and other metabolic indicators (i.e., DNA damage, inflammation, and oxidative stress), were measured before and after each dietary-zinc period. RESULTS TAZ increased with increased dietary zinc, but plasma zinc concentrations and EZP size were unchanged. Erythrocyte and leukocyte zinc concentrations and zinc transporter expressions were not altered. However, leukocyte DNA strand breaks decreased with increased dietary zinc, and the level of proteins involved in DNA repair and antioxidant and immune functions were restored after the dietary-zinc increase. CONCLUSIONS A moderate 4-mg/d increase in dietary zinc, similar to that which would be expected from zinc-biofortified crops, improves zinc absorption but does not alter plasma zinc. The repair of DNA strand breaks improves, as do serum protein concentrations that are associated with the DNA repair process. This trial was registered at clinicaltrials.gov as NCT02861352.
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"Omics" of Selenium Biology: A Prospective Study of Plasma Proteome Network Before and After Selenized-Yeast Supplementation in Healthy Men.
Sinha, I, Karagoz, K, Fogle, RL, Hollenbeak, CS, Zea, AH, Arga, KY, Stanley, AE, Hawkes, WC, Sinha, R
Omics : a journal of integrative biology. 2016;(4):202-13
Abstract
Low selenium levels have been linked to a higher incidence of cancer and other diseases, including Keshan, Chagas, and Kashin-Beck, and insulin resistance. Additionally, muscle and cardiovascular disorders, immune dysfunction, cancer, neurological disorders, and endocrine function have been associated with mutations in genes encoding for selenoproteins. Selenium biology is complex, and a systems biology approach to study global metabolomics, genomics, and/or proteomics may provide important clues to examining selenium-responsive markers in circulation. In the current investigation, we applied a global proteomics approach on plasma samples collected from a previously conducted, double-blinded placebo controlled clinical study, where men were supplemented with selenized-yeast (Se-Yeast; 300 μg/day, 3.8 μmol/day) or placebo-yeast for 48 weeks. Proteomic analysis was performed by iTRAQ on 8 plasma samples from each arm at baseline and 48 weeks. A total of 161 plasma proteins were identified in both arms. Twenty-two proteins were significantly altered following Se-Yeast supplementation and thirteen proteins were significantly changed after placebo-yeast supplementation in healthy men. The differentially expressed proteins were involved in complement and coagulation pathways, immune functions, lipid metabolism, and insulin resistance. Reconstruction and analysis of protein-protein interaction network around selected proteins revealed several hub proteins. One of the interactions suggested by our analysis, PHLD-APOA4, which is involved in insulin resistance, was subsequently validated by Western blot analysis. Our systems approach illustrates a viable platform for investigating responsive proteomic profile in 'before and after' condition following Se-Yeast supplementation. The nature of proteins identified suggests that selenium may play an important role in complement and coagulation pathways, and insulin resistance.
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Earthing the human body influences physiologic processes.
Sokal, K, Sokal, P
Journal of alternative and complementary medicine (New York, N.Y.). 2011;(4):301-8
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Abstract
OBJECTIVES This study was designed to answer the question: Does the contact of the human organism with the Earth via a copper conductor affect physiologic processes? Subjects and experiments: Five (5) experiments are presented: experiment 1-effect of earthing on calcium-phosphate homeostasis and serum concentrations of iron (N = 84 participants); experiment 2-effect of earthing on serum concentrations of electrolytes (N = 28); experiment 3-effect of earthing on thyroid function (N = 12); experiment 4-effect of earthing on glucose concentration (N = 12); experiment 5-effect of earthing on immune response to vaccine (N = 32). Subjects were divided into two groups. One (1) group of people was earthed, while the second group remained without contact with the Earth. Blood and urine samples were examined. RESULTS Earthing of an electrically insulated human organism during night rest causes lowering of serum concentrations of iron, ionized calcium, inorganic phosphorus, and reduction of renal excretion of calcium and phosphorus. Earthing during night rest decreases free tri-iodothyronine and increases free thyroxine and thyroid-stimulating hormone. The continuous earthing of the human body decreases blood glucose in patients with diabetes. Earthing decreases sodium, potassium, magnesium, iron, total protein, and albumin concentrations while the levels of transferrin, ferritin, and globulins α1, α2, β, and γ increase. These results are statistically significant. CONCLUSIONS Earthing the human body influences human physiologic processes. This influence is observed during night relaxation and during physical activity. Effect of the earthing on calcium-phosphate homeostasis is the opposite of that which occurs in states of weightlessness. It also increases the activity of catabolic processes. It may be the primary factor regulating endocrine and nervous systems.
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Proteomic profiling of growth hormone-responsive proteins in human peripheral blood leukocytes.
Chung, L, Nelson, AE, Ho, KK, Baxter, RC
The Journal of clinical endocrinology and metabolism. 2009;(8):3038-43
Abstract
CONTEXT GH is a known modulator of the immune system, but the effect of exogenous GH administration on white blood cell proteins has not been investigated. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a powerful platform for the study of GH effects on immune system proteins. OBJECTIVE Our objective was to explore a novel approach for the detection of GH-responsive proteins in human leukocytes by proteomic analysis using SELDI-TOF MS. DESIGN We conducted a randomized double-blind, placebo-controlled GH administration study of 8 wk treatment followed by 6 wk washout. Pre- and posttreatment samples from 30 subjects were used for biomarker discovery. SETTING The study was performed at a clinical research facility. PARTICIPANTS We studied 30 recreationally trained healthy athletes. INTERVENTION Subjects received either recombinant human GH (2 mg/d sc; n = 22) or placebo (n = 8) for 8 wk. MAIN OUTCOME MEASURES Proteomic profiles were determined using CM10 weak cation-exchange protein chips, and some GH-regulated proteins were purified and identified by mass spectrometry and/or immunoblotting. RESULTS SELDI-TOF analysis revealed a number of GH-regulated peptides/proteins in the 3- to 22-kDa range that are either up- or down-regulated by GH. Several of these may be useful as biomarkers of GH action. The calcium-binding, proinflammatory calgranulins S100A8, S100A9, and S100A12 were all significantly down-regulated in response to GH treatment. CONCLUSION This study illustrates the novel use of human leukocyte proteomic profiling by SELDI-TOF MS and reveals the negative regulation of proinflammatory S100 proteins by GH in human white blood cells.
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An attempt to understand kidney's protein handling function by comparing plasma and urine proteomes.
Jia, L, Zhang, L, Shao, C, Song, E, Sun, W, Li, M, Gao, Y
PloS one. 2009;(4):e5146
Abstract
BACKGROUND With the help of proteomics technology, the human plasma and urine proteomes, which closely represent the protein compositions of the input and output of the kidney, respectively, have been profiled in much greater detail by different research teams. Many datasets have been accumulated to form "reference profiles" of the plasma and urine proteomes. Comparing these two proteomes may help us understand the protein handling aspect of kidney function in a way, however, which has been unavailable until the recent advances in proteomics technology. METHODOLOGY/PRINCIPAL FINDINGS After removing secreted proteins downstream of the kidney, 2611 proteins in plasma and 1522 in urine were identified with high confidence and compared based on available proteomic data to generate three subproteomes, the plasma-only subproteome, the plasma-and-urine subproteome, and the urine-only subproteome, and they correspond to three groups of proteins that are handled in three different ways by the kidney. The available experimental molecular weights of the proteins in the three subproteomes were collected and analyzed. Since the functions of the overrepresented proteins in the plasma-and-urine subproteome are probably the major functions that can be routinely regulated by excretion from the kidney in physiological conditions, Gene Ontology term enrichment in the plasma-and-urine subproteome versus the whole plasma proteome was analyzed. Protease activity, calcium and growth factor binding proteins, and coagulation and immune response-related proteins were found to be enriched. CONCLUSION/SIGNIFICANCE The comparison method described in this paper provides an illustration of a new approach for studying organ functions with a proteomics methodology. Because of its distinctive input (plasma) and output (urine), it is reasonable to predict that the kidney will be the first organ whose functions are further elucidated by proteomic methods in the near future. It can also be anticipated that there will be more applications for proteomics in organ function research.
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Evaluation of matrix-assisted laser desorption/ionization-time of flight mass spectrometry proteomic profiling: identification of alpha 2-HS glycoprotein B-chain as a biomarker of diet.
Mitchell, BL, Yasui, Y, Lampe, JW, Gafken, PR, Lampe, PD
Proteomics. 2005;(8):2238-46
Abstract
Biomarkers have the potential to impact a wide range of public health concerns, including early detection of diseases, drug discovery, and improved accuracy of monitoring effects of interventions. Given new technological developments, broad-based screening approaches will likely advance biomarker discovery at an accelerated pace. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) allows for the elucidation of individual protein masses from a complex mixture with high throughput. We have developed a method for identifying serum biomarkers using MALDI-TOF and statistical analysis. However, before applying this approach to screening of complex diseases, we evaluated the approach in a controlled dietary intervention study. In this study, MALDI-TOF spectra were generated using samples from a randomized controlled trial. During separate feeding periods, 38 participants ate a basal diet devoid of fruits and vegetables and a basal diet supplemented with cruciferous (broccoli) family vegetables. Serum samples were obtained at the end of each 7-day feeding period and treated to remove large, abundant proteins. MALDI-TOF spectra were analyzed using peak picking algorithms and logistic regression models. Our bioinformatics methods identified two significant peaks at m/z values of 2740 and 1847 that could classify participants based on diet (basal vs. cruciferous) with 76% accuracy. The 2740 m/z peak was identified as the B-chain of alpha 2-HS glycoprotein, a serum protein previously found to vary with diet and be involved in insulin resistance and immune function.
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Direct effect of plasma permeability factors from patients with idiopatic FSGS on nephrin and podocin expression in human podocytes.
Doublier, S, Musante, L, Lupia, E, Candiano, G, Spatola, T, Caridi, G, Zennaro, C, Carraro, M, Ghiggeri, GM, Camussi, G
International journal of molecular medicine. 2005;(1):49-58
Abstract
The presence of circulating plasma factors (PF) altering renal permeability to proteins has been previously described in patients with focal segmental glomerulosclerosis (FSGS). Since these patients show reduced nephrin and podocin expression at renal biopsy, we evaluated the effect of serum and PF from patients with FSGS on nephrin and podocin expression in human podocytes. We studied 7 sera from patients with steroid-resistant FSGS, 3 from patients with nephrotic syndrome caused by non-immune disease, and 6 from healthy subjects. PF was prepared from plasmapheresis eluates of 2 patients with post-transplant recurrence of FSGS. Purification procedure was based on protein A Sepharose chromatography and differential precipitation in ammonium sulphate. Nephrin and podocin expression was semi-quantitatively evaluated by immunofluorescence. We found that serum and PF from FSGS patients rapidly induced redistribution and loss of nephrin in podocytes. This effect was associated with cytoskeleton redistribution and inhibited by cytochalasin B and sodium azide. On the contrary, podocin expression was unchanged after incubation with serum and PF from FSGS patients for short periods, but markedly reduced at 24 h. Our results demonstrate that serum and PF from FSGS patients may directly affect nephrin and podocin in human podocytes, thus providing new insights into the mechanisms causing proteinuria in FSGS.
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In vitro resistance of Staphylococcus aureus to thrombin-induced platelet microbicidal protein is associated with alterations in cytoplasmic membrane fluidity.
Bayer, AS, Prasad, R, Chandra, J, Koul, A, Smriti, M, Varma, A, Skurray, RA, Firth, N, Brown, MH, Koo, SP, et al
Infection and immunity. 2000;(6):3548-53
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Abstract
Platelet microbicidal proteins (PMPs) are small, cationic peptides which possess potent microbicidal activities against common bloodstream pathogens, such as Staphylococcus aureus. We previously showed that S. aureus strains exhibiting resistance to thrombin-induced PMP (tPMP-1) in vitro have an enhanced capacity to cause human and experimental endocarditis (T. Wu, M. R. Yeaman, and A. S. Bayer, Antimicrob. Agents Chemother. 38:729-732, 1994; A. S. Bayer et al., Antimicrob. Agents Chemother. 42:3169-3172, 1998; V. K. Dhawan et al., Infect. Immun. 65:3293-3299, 1997). However, the mechanisms mediating tPMP-1 resistance in S. aureus are not fully delineated. The S. aureus cell membrane appears to be a principal target for the action of tPMP-1. To gain insight into the basis of tPMP-1 resistance, we compared several parameters of membrane structure and function in three tPMP-1-resistant (tPMP-1(r)) strains and their genetically related, tPMP-1-susceptible (tPMP-1(s)) counterpart strains. The tPMP-1(r) strains were derived by three distinct methods: transposon mutagenesis, serial passage in the presence of tPMP-1 in vitro, or carriage of a naturally occurring multiresistance plasmid (pSK1). All tPMP-1(r) strains were found to possess elevated levels of longer-chain, unsaturated membrane lipids, in comparison to their tPMP-1(s) counterparts. This was reflected in corresponding differences in cell membrane fluidity in the strain pairs, with tPMP-1(r) strains exhibiting significantly higher degrees of fluidity as assessed by fluorescence polarization. These data provide further support for the concept that specific alterations in the cytoplasmic membrane of S. aureus strains are associated with tPMP-1 resistance in vitro.