Healthy Human Serum Research Articles

The IL-6-neutralizing sIL-6R-sgp130 buffer system is disturbed in patients with type 2 diabetes.

Serum levels of interleukin-6 (IL-6) are increased in patients with type 2 diabetes (T2D). IL-6 exerts its pleiotropic effects via the IL-6 α-receptor (IL-6R), which exists in membrane-bound and soluble (sIL-6R) forms and activates cells via the β-receptor glycoprotein 130 (gp130). The nonsynonymous single-nucleotide polymorphism (SNP) rs2228145 (Asp358Ala) within the IL6R locus is associated with T2D. The aim of this study was to determine whether sIL-6R in combination with soluble gp130 (sgp130) is able to form an IL-6-neutralizing buffer in healthy subjects and whether this is disturbed in T2D. We found that sIL-6R-sgp130 indeed forms an IL-6-neutralizing buffer in the serum of healthy humans, whose capacity is controlled by the SNP of the IL-6R. Circulating sIL-6R-sgp130 levels were lower in T2D subjects (P < 0.001), whereas IL-6 was high and inversely correlated with sIL-6R (r = -0.57, P < 0.001), indicating a severe disturbance of the buffer. This phenomenon is also observed in sex- and age-matched patients with both T2D and atherosclerosis but not in patients with atherosclerosis alone. In conclusion, sIL-6R and sgp130 serum levels were significantly lower in T2D patients compared with healthy subjects or atherosclerosis patients, although IL-6 levels were high. These data suggest that disturbance of the protective buffer may be closely associated with T2D pathophysiology.

PS972 THE AML SECRETOME AND T CELL FUNCTION: MODULATION OF AMG 330‐MEDIATED T CELL PROLIFERATION AND CYTOTOXICITY

Background:T cell recruiting antibody constructs are a novel strategy to target chemorefractory acute myeloid leukemia (AML) cells. A CD33 specific BiTE® antibody construct, AMG 330, is currently in evaluation in a first in human phase I trial with promising tolerability and anti‐leukemic activity in relapsed / refractory AML (Ravandi et al, ASH 2018). We have developed a long‐term culture system for AML patient samples and showed substantial activity of AMG 330 redirected autologous residual T cells against AML cells in 76 % of patient samples (Krupka et al, Blood 2014; Krupka et al, Leukemia 2016). Having observed suboptimal response in the remaining samples, we conducted this study to investigate potential mechanisms leading to decreased sensitivity of AML cells to BiTE® recruited T cell activity.Aims:We hypothesized that AML cells secrete humoral inhibitory factors that influence T cell function and thereby modulate AMG 330‐mediated cytotoxicity. Hence, we analyzed the influence of AML patient plasma (PtP) derived from bone marrow (BM) on T cell proliferation and AMG 330‐mediated cytotoxicity against AML cell lines.Methods:T cell proliferation assays were performed using CFSE‐labeled healthy donor (HD) T cells stimulated with CD3/CD28 beads and IL‐2 and analyzed by flow cytometry (day 3). AML PtP were collected from BM (n = 16) at time of initial diagnosis. HD T cells were cultured in either 20 % of PtP, FCS or healthy human serum (HS). To test for a dominant negative factor within PtP, mixing experiments of FCS and PtP were performed (ratio: 4:0 ‐ 4:4). In addition, conditioned medium (CM) of three different AML cell lines (HL60, OCI‐AML3, KG1a) was analyzed on its modulation of T cell function. To dissect the relevance of cell‐cell contact vs the AML secretome, transwell assays with HD T cells and AML cell lines were performed.Results:We observed that AML PtP negatively impacted T cell proliferation of HD in vitro which was not observed in the presence of FCS or HS (mean % T cell proliferation PtP: 9.4 (n = 16), FCS: 74.0 (n = 16), HS: 35.5 (n = 11)). T cell proliferation was still reduced after additional supplementation of 20 % FCS in 4 of 11 AML samples (mean % T cell proliferation PtP+FCS: 40.2, FCS: 75.9). Affirmatively, CM of the cell lines HL60 and OCI‐AML3 negatively impacted T cell proliferation (mean % T cell proliferation CM of HL60: 11.5 (n = 12), OCI‐AML3: 34.7 (n = 9), KG1a: 57.2 (n = 6), control: 54.6 (n = 12)). Reduced T cell proliferation and AMG 330‐mediated cytotoxicity was also observed in the presence of all three AML cell lines within the transwell assays (mean % T cell proliferation HL60: 2.6, OCI‐AML3: 15.4, KG1a: 28.4, control: 40.0; % specific cytotoxicity HL60: 21.9, OCI‐AML3: 45.6, KG1a: 56.4, control: 69.9; n = 10). This was not due to serum deprivation as gamma‐irradiated AML cell lines (n = 6) still significantly reduced T cell proliferation and AMG 330‐mediated cytotoxicity.Summary/Conclusion:We conclude that AML patient plasma samples contain soluble factors which may inhibit T cell functions to a varying degree. By using an in vitro model, we could demonstrate that this was mediated through soluble factors secreted by AML cells. Analysis of AML patient plasma (n = 35) and healthy BM plasma (n = 5) by Proximity Extension Assay technology (Immuno‐Oncology Panel, Olink) revealed significant differences in plasma levels of 66 of 92 protein biomarkers. We are in the process to dissect their relevance in ongoing experiments. We aim to identify inhibitory factors that might be targetable and suitable for combinatorial approaches to enhance T cell‐based immunotherapy in AML.

Simultaneous determination of nine trace concentration angiotensin peptides in human serum using ultra high performance liquid chromatography with tandem mass spectrometry with sephadex LH-20 gel solid-phase extraction.

The renin-angiotensin system is a highly complex enzymatic system consisting of multiple peptide hormones, enzymes, and receptors. Here, an assay to simultaneously quantify eight angiotensin peptides and bradykinin in human serum was developed and validated, using ultra high performance liquid chromatography coupled with tandem mass spectrometry. A pre-concentration method of Sephadex LH-20 gel solid-phase extraction was first applied for analysis of angiotensin peptides from serum sample. The triple quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The analytical time was within 5min, much raising the analysis efficiency. Limits of detection ranged from 0.9 to 1.3pg/mL, and displayed the same level of sensitivity compared with radioimmunoassay. The method was successfully applied to 22 healthy human serum samples, giving the concentrations of angiotensin I, angiotensin II, angiotensin III, angiotensin IV, angiotensin 1-9, angiotensin 1-7, angiotensin 1-5, Asn1 ,Val5 -Angiotensin II, and bradykinin for reference. This novel metabolic profile study of vasoactive peptides based on gel solid-phase extraction concentration provided not only an accurate quantitative assay of the serum concentrations, but also a promising methodology for evaluating the diagnostic values of the various peptides.

Identification of preexisting adaptive immunity to Cas9 proteins in humans.

The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR-Cas9 system moves toward clinical trials.

A proteome-wide screen of Campylobacter jejuni using protein microarrays identifies novel and conformational antigens.

Campylobacter jejuni (C. jejuni) is a foodborne intestinal pathogen and major cause of gastroenteritis worldwide. C. jejuni proteins that are immunogenic have been sought for their potential use in the development of biomarkers, diagnostic assays, or subunit vaccines for humans or livestock. To identify new immunogenic C. jejuni proteins, we used a native protein microarray approach. A protein chip, with over 1400 individually purified GST-tagged C. jejuni proteins, representing over 86% of the proteome, was constructed to screen for antibody titers present in test sera raised against whole C. jejuni cells. Dual detection of GST signals was incorporated as a way of normalizing the variation of protein concentrations contributing to the antibody staining intensities. We detected strong signals to 102 C. jejuni antigens. In addition to antigens recognized by antiserum raised against C. jejuni, parallel experiments were conducted to identify antigens cross-reactive to antiserum raised against various serotypes of E. coli or Salmonella or to healthy human sera. This led to the identification of 34 antigens specifically recognized by the C. jejuni antiserum, only four of which were previously known. The chip approach also allowed identification of conformational antigens. We demonstrate in the case of Cj1621 that antigen signals are lost to denaturing conditions commonly used in other approaches to identify immunogens. Antigens identified in this study include those possessing sequence features indicative of cell surface localization, as well as those that do not. Together, our results indicate that the unbiased chip-based screen can help reveal the full repertoire of host antibodies against microbial proteomes.

Investigation of a low cost tapered plastic fiber optic biosensor based on manipulation of colloidal gold nanoparticles

We demonstrate a low cost Tapered Fiber Optic Biosensor (TFOBS) based on manipulation of colloidal gold nanoparticle in the evanescent field of tapered fiber. The sensor consists of a biconical tapered plastic optical fiber with colloidal gold nanoparticles coated on the tapered region. The strength of evanescent field absorption as a function of gold nanoparticle size is evaluated using Finite Difference Time Domain simulation. Simulation results suggest that in order to achieve maximum absorption in visible range of wavelength, the size of the gold nanoparticle should be in the range 3–4 nm. Gold nanoparticle of desired size is then synthesized and coated on the tapered region to fabricate a TFOBS. It is found experimentally that coating of gold nanoparticles enhances the fraction of light coming out to the cladding surface. This is explained as a consequence of local surface plasmonic resonance. The TFOBS is then tested for its sensing application to detect Bovine Serum Albumin. Level of detection of the proposed sensor is calculated and is found as 0.326gm/dL. As normal albumin level in healthy human blood serum is between 3.5 to 5.5gm/dL, the proposed TFOBS can be used as a low cost accurate albumin sensor.

Determination of Some Human Enzymes Activities in Presence of Titanium Oxide Nanoparticles

Titanium oxide nanoparticles (TiO2NPs) are the most common particles that have industrial, commercial, medial and antibacterial applications. On the other hand, they have toxic properties within some concentrations that show the ability to inhibit some human enzymes. In this study a diluted solutions of TiO2NPs were prepared by weighting(0.08 gm)with deionized water as(10−1, 10−2, 10−3, 10−4, 10−5) with low conductivity to avoid interaction with other ions. Our study aimed to estimate the effect of different concentration of TiO2NPs on liver enzymesaspartate transaminase (GOT), alanine transaminase (GPT), alkaline phosphatase(ALP) and Amylase in human serum by using a healthy human serum with a mixture of enzyme solution in absence and presence of diluted solution of TiO2NPs. The results showed that different TiO2NPs concentrations used had inhibitory effect on all the enzymes.

Synthesis and Antibacterial Activity of Some New Derivatives Containing Thiazole moiety and Study of Their Effects on MAO Enzyme Activity (In vitro)

The study includes synthesis and characterization of several heterocyclic systems such as 1, 2, 3 triazoles(5), some pyrazole derivatives(8a-c)and(12a, b), thaizolo(10) and chalcone derivatives(11a, b) by reaction of 4-nitro aniline with appropriate reagents. All synthesized derivatives containing thaizole moiety. These derivatives were characterized by IR, 1HNMR and mass spectroscopy. Newly synthesized compounds were in vitro screened against several bacterial species as well as S.aureus E.coli. effect of the prepared compounds were studied on monoaminooxidase (MAO) in healthy human serum. The results showed that all compounds cause competitive inhibition with enzyme except (2, 7b, 10, 11a, 12b) cause uncompetitive inhibition and (6, 8a, 8c) caused mixed inhibition.

Cloning, expression & evaluation of potential immunogenic recombinant capsid premembrane protein of West Nile virus.

Background & objectives:West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and in areas where multiple flaviviruses are endemic. Diagnosis of WNV infection is primarily based on serodiagnosis, followed by virus isolation and identification. The aim of this study was to develop and evaluate a highly sensitive and specific immunoglobulin M (IgM) ELISA using the recombinant CprM protein (rWNV-CprM) for rapid, early and accurate diagnosis of WNV.Methods:The gene coding for the CprM protein of WNV was cloned and expressed in pET 28a vector followed by purification. An indirect IgM microplate ELISA using purified rWNV-CprM protein was optimized having no cross-reactivity with healthy human serum and serum samples obtained from patients with dengue and Japanese encephalitis viruses infection.Results:The comparative evaluation of this rWNV-CprM protein-specific IgM ELISA with plaque reduction neutralization test using 105 blood samples collected from patients suspected to have acute WNV infection revealed 98 per cent concordance with sensitivity and specificity of 100 and 97 per cent, respectively.Interpretation & conclusions:The recombinant CprM protein-based WNV-specific ELISA reported in this study may be useful for rapid screening of large numbers of blood samples in endemic areas during outbreaks.

Healthy human serum N-glycan profiling reveals the influence of ethnic variation on the identified cancer-relevant glycan biomarkers.

BackgroundMost glycomics studies have focused on understanding disease mechanisms and proposing serum markers for various diseases, yet the influence of ethnic variation on the identified glyco-biomarker remains poorly addressed. This study aimed to investigate the inter-ethnic serum N-glycan variation among US origin control, Japanese, Indian, and Ethiopian healthy volunteers.MethodsHuman serum from 54 healthy subjects of various ethnicity and 11 Japanese hepatocellular carcinoma (HCC) patients were included in the study. We employed a comprehensive glycoblotting-assisted MALDI-TOF/MS-based quantitative analysis of serum N-glycome and fluorescence HPLC-based quantification of sialic acid species. Data representing serum N-glycan or sialic acid levels were compared among the ethnic groups using SPSS software.ResultsTotal of 51 N-glycans released from whole serum glycoproteins could be reproducibly quantified within which 33 glycoforms were detected in all ethnicities. The remaining N-glycans were detected weakly but exclusively either in the Ethiopians (13 glycans) or in all the other ethnic groups (5 glycans). Highest abundance (p < 0.001) of high mannose, core-fucosylated, hyperbranched/hypersialylated N-glycans was demonstrated in Ethiopians. In contrast, only one glycan (m/z 2118) significantly differed among all ethnicities being highest in Indians and lowest in Ethiopians. Glycan abundance trend in Ethiopians was generally close to that of Japanese HCC patients. Glycotyping analysis further revealed ethnic-based disparities mainly in the branched and sialylated structures. Surprisingly, some of the glycoforms greatly elevated in the Ethiopian subjects have been identified as serum biomarkers of various cancers. Sialic acid level was significantly increased primarily in Ethiopians, compared to the other ethnicities.ConclusionThe study revealed ethnic-specific differences in healthy human serum N-glycome with highest abundance of most glycoforms in the Ethiopian ethnicity. The results strongly emphasized the need to consider ethnicity matching for accurate glyco-biomarker identification. Further large-scale study employing various ethnic compositions is needed to verify the current result.

Rapid, Reproducible, Quantifiable NMR Metabolomics: Methanol and Methanol: Chloroform Precipitation for Removal of Macromolecules in Serum and Whole Blood.

Background: Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1d-1H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. Methods: A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. Results: In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at −80 °C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. Conclusions: In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.

Abstract LB-350: IgG4 expressing B cells associate with poor survival in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is an aggressive form of breast cancer with high levels of lymphocyte infiltration. Although high levels of infiltrating B cells are present in TNBC, their role in progression of this disease is unclear. IgG4 is one of four subclasses of IgG and is the least abundant in healthy human serum. IgG4 has been found at higher percentages in both tissue and serum in cases of chronic inflammation, which promotes class switching to IgG4. Expression and secretion of IgG4 by B cells may be non-beneficial and potentially harmful in the cancer setting. Unlike the other subclasses of IgG, IgG4 does not activate complement dependent cytotoxicity and has a lower affinity for FcγR, thus resulting in a poor ability to engage immune effector cells and activate antibody-depended cell-mediated cytotoxicity. Additionally, IgG4 can compete with other IgG subclasses by binding to antigens and can sequestering IgG through Fc-Fc interactions, thus impairing potential anti-tumor antibody-mediated responses. As IgG4 expressing B cells have been shown to be associated with poor outcomes in other aggressive cancers such melanoma, glioblastoma and pancreatic cancer, we sought to determine the expression of IgG4 in TNBC. We performed immunohistological staining of IgG4 in paraffin-embedded tissue microarrays from 75 treatment-naive TNBC specimens obtained from women who underwent surgical resection of their tumors at the Helen F Graham Cancer Center and Research Institute from 2006-2007. We found that 69 % of TNBC cases contain IgG4+ cell infiltration. Moreover, the presence of IgG4+ cells is significantly correlated with poorer progression free (p&amp;lt;0.0003) and overall survival (p&amp;lt;0.0001) as determined by a Log-rank (Mantel-Cox) test. These findings indicate the presence of an immunosuppressive B cell population. Further study is needed to characterize these cells and to determine the significance of IgG4+ tumor infiltrating B cells, and their potential immunosuppressive role in the progression of TNBC. Citation Format: Nicole Flynn, Ugochukwu Amadi, Rajasekharan Somasundaram, Jennifer Sims-Mourtada. IgG4 expressing B cells associate with poor survival in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-350.

Circulating exosomes contain protein biomarkers of metastatic non-small-cell lung cancer.

The present study aimed to investigate the overall changes in exosomal proteomes in metastatic and non‐metastatic non‐small‐cell lung cancers (NSCLC) and healthy human serum samples, and evaluate the potential of serum exosomal biomarkers to predict NSCLC metastasis. Tandem mass tags combined with multidimensional liquid chromatography and mass spectrometry analysis were used for screening the proteomic profiles of serum samples. Quantitative proteome, significant pathway, and functional categories of patients with metastatic and non‐metastatic NSCLC and healthy donors were investigated. In total, 552 proteins of the 628 protein groups identified were quantified. Bioinformatics analysis indicated that quantifiable proteins were mainly involved in multiple biological functions, metastasis‐related pathways. Moreover, lipopolysaccharide‐binding proteins (LBP) in the exosomes were found to be well distinguished between patients with metastatic and patients with non‐metastatic NSCLC. Area under the curve (AUC) was 0.803 with a sensitivity of 83.1% and a specificity of 67% (P < .0001). Circulating LBP were also well distinguishable between metastatic and non‐metastatic NSCLC, the AUC was 0.683 with a sensitivity of 79.5% and a specificity of 47.2% (P = .005). This novel study provided a reference proteome map for metastatic NSCLC. Patients with metastatic and non‐metastatic NSCLC differed in exosome‐related proteins in the serum. LBP might be promising and effective candidates of metastatic NSCLC.

The potential atheroprotective role of plant MIR156a as a repressor of monocyte recruitment on inflamed human endothelial cells

MicroRNAs have become the spotlight of the biological community for more than a decade, but we are only now beginning to understand their functions. The detection of stably expressed endogenous microRNAs in human blood suggests that these circulating miRNAs can mediate intercellular communication. Our previous study reported the surprising finding that exogenous rice MIR168a could regulate liver low-density lipoprotein receptor adapter protein 1 (LDLRAP1) gene expression in mice. Here, we show that plant MIR156a, which is abundantly expressed in dietary green veggies, also stably presents in healthy human serum. Compared with age-matched individuals, decreased levels of MIR156a are observed both in serum and blood vessel of cardiovascular disease (CVD) patients. In vitro studies demonstrate that MIR156a can directly target the junction adhesion molecule-A (JAM-A), which is up-regulated in atherosclerotic lesions from CVD patients. Functional studies show that ectopic expression of MIR156a in human aortic endothelial cells reduces inflammatory cytokine-induced monocytes adhesion by suppressing JAM-A. These findings offer a novel vasoprotective molecular mechanism of green veggies through plant microRNAs.

GC-MS Based Metabolomics and NMR Spectroscopy Investigation of Food Intake Biomarkers for Milk and Cheese in Serum of Healthy Humans.

The identification and validation of food intake biomarkers (FIBs) in human biofluids is a key objective for the evaluation of dietary intake. We report here the analysis of the GC-MS and 1H-NMR metabolomes of serum samples from a randomized cross-over study in 11 healthy volunteers having consumed isocaloric amounts of milk, cheese, and a soy drink as non-dairy alternative. Serum was collected at baseline, postprandially up to 6 h, and 24 h after consumption. A multivariate analysis of the untargeted serum metabolomes, combined with a targeted analysis of candidate FIBs previously reported in urine samples from the same study, identified galactitol, galactonate, and galactono-1,5-lactone (milk), 3-phenyllactic acid (cheese), and pinitol (soy drink) as candidate FIBs for these products. Serum metabolites not previously identified in the urine samples, e.g., 3-hydroxyisobutyrate after cheese intake, were detected. Finally, an analysis of the postprandial behavior of candidate FIBs, in particular the dairy fatty acids pentadecanoic acid and heptadecanoic acid, revealed specific kinetic patterns of relevance to their detection in future validation studies. Taken together, promising candidate FIBs for dairy intake appear to be lactose and metabolites thereof, for lactose-containing products, and microbial metabolites derived from amino acids, for fermented dairy products such as cheese.

A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells.

The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00-1.34) and 0.97 (0.00-1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.

Highly Porous Metal-Free Graphitic Carbon Derived from Metal-Organic Framework for Profiling of N-Linked Glycans.

In this work, a highly efficient profiling of N-linked glycans was achieved by a facile and eco-friendly synthesized highly porous metal-free carbon material. The metal-free carbon was derived from a well-defined nanorod zinc metal-organic framework via the metal removal under a high-temperature carbonization, which exhibited a highly specific surface area of 1700 m2/g. After further oxidation, the oxidized metal-free carbon was applied to the selective isolation of N-linked glycans from complex biological samples due to the strong interaction between carbon and glycan as well as the size-exclusion mechanism. Twenty six N-linked glycans could be identified from the digest of a standard glycoprotein ovalbumin at a concentration of 0.01 μg/μL, and the detection limit of glycans could be down to 1 ng/μL with 21 N-linked glycans identified. When the mass ratio of the interfering protein bovine serum albumin vs a standard ovalbumin digest is up to 500:1, there were 24 N-glycans confidentially identified. From a real complex sample of a healthy human serum, there were 43 N-linked glycans identified after the enrichment of oxidized metal-free carbon. In a word, the metal-free carbon is opening up new prospect for the high-throughput identification of glycan.

Establishment of a Multivariate Analysis Based on Photonic Crystal Hydrogel Bead Arrays and Its Application for Detecting Platelet-specific Antibodies

ABSTRACTPhotonic crystals are nanomaterials with unique structural coloration, and they have been used in multiple analyses, gene function analyses, and nanoarray biosensors. Here, we used agarose hydrogel microcarriers with characteristic reflection peak codes to improve the biocompatibility of the material and the efficiency of detection, which may be helpful for further clinical applications. The paper has two parts: condition optimization and product application. Goat antihuman immune globulin G, human immune globulin G, and fluorescein isothiocyanate-labeled goat antihuman immune globulin G are commonly used materials in double-antibody sandwich techniques, which are used for optimization in the application of advanced composite materials. Goat antihuman immune globulin G was immobilized on the surfaces of photonic crystal hydrogel beads through chemical bonding. When the target immune globulin G was captured, the fluorescence-labeled antibody could combine with photonic crystal hydrogel beads. The reaction conditions were optimized using the gray-scale value of fluorescent intensities. Photonic crystal hydrogel beads with different reflection spectra were coated with purified platelet glycoprotein IIb/IIIa and glycoprotein Ib/IX. A total of 64 clinical samples including sera from patients with immune thrombocytopenia (n = 32) and healthy human sera (n = 32) were tested using photonic crystal hydrogel beads and monoclonal antibody immobilization of platelet antigen (MAIPA). Photonic crystal hydrogel bead array reaction conditions were established for multiple analysis, showing similar sensitivity and specificity with MAIPA in testing different platelet antibodies and shortening the analysis time. The features of these composite materials make them suitable for potential applications in clinical screening assays.

Quantitative analysis of protocadherin 10 promoter methylation in peripheral blood of patients with gastric cancer by real-time and methylation specific polymerase techniques and its significance

Objective To investigate protocadherin 10 (PCDH10) methylation at promoter 5’-cytosine-phosphoric acid-guanine motif (CpG) island as a tool for non-invasive diagnosis and prognosis evaluation of primary gastric cancer. Methods Real-time fluorescent quantitative methylation specific polymerase chain reaction (Real-time MSP) technique was used for detection of PCDH10 promoter methylation in both tissue and serum specimens from 202 gastric cancer (GC), 52 gastric epithelial dysplasia, and 68 chronic gastritis, as well as 120 healthy human serum specimens. The relationship between PCDH10 methylation with clinicopathological parameters and prognosis was analyzed. Results The incidence of PCDH10 methylation in gastric cancer tissues (80.2%, 162/202) was significantly higher than that in paired adjacent tissues (26.7%, 54/202), gastric epithelial dysplasia (23.1%, 12/52) and chronic gastritis specimens (14.7%, 10/68) (all P=0.000). Detectable rate of PCDH10 methylation in serum specimens was in good agreement with that in the paired tumor tissues, with the Aζ value being 0.862 (P=0.000). The level of PCDH10 methylation in serum was closely correlated to age at diagnosis (P=0.018), H. pylori (Hp) infection (P=0.000), tumor size (P=0.000), tumor differentiation (P=0.002), lymphatic invasion (P=0.000), venous invasion (P=0.007), invasion depth (P=0.000), lymphatic metastasis (P=0.000), distant metastasis (P=0.015), clinical stages (P=0.000) and poor prognosis (P=0.000), but not with gender (P=0.435), tumor site (P=0.458) and growth pattern (P=0.435). Conclusion PCDH10 methylation plays an important role in the tumorigenesis and development of gastric cancer. Key words: Gastric cancer; Protocadherin 10; DNA methylation; Early diagnosis; Prognosis

Comparison of the extraction and determination of serum exosome and miRNA in serum and the detection of miR-27a-3p in serum exosome of ALS patients.

Amyotrophic Lateral Sclerosis (ALS) is a muscle-bone degenerative disease, which lacks a specific index for diagnosis. In our previous studies, we found that exosomes mediated the interaction mechanism between muscle and bone at the cellular level, and myoblast exosomes can transfer miR-27a-3p to promote osteoblast mineralization. Therefore, we suppose that the expression of miR-27a-3p in the serum exosomes of ALS patients also changes. In this study, we used healthy human serum as a sample to find out the conditions and methods for extraction and detection. Then through comparison of the expression of miR-27a-3p in the serum exosomes of 10 ALS patients and healthy subjects, we found that in the ALS patients miR-27a-3p was down-regulated, and may be involved in the development of ALS, and therefore has potential as a reference for the diagnosis of ALS in the clinic.