Serum proteomics of glioma: methods and applications

The prognosis of patients with glioblastoma, the most malignant adult glial brain tumor, remains poor in spite of advances in treatment procedures, including surgical resection, irradiation and chemotherapy.Genetic heterogeneity of glioblastoma warrants extensive studies in order to gain a thorough understanding of the biology of this tumor. While there have been several studies of global transcript profiling of glioma with the identification of gene signatures for diagnosis and disease management, translation into clinics is yet to happen. Serum biomarkers have the potential to revolutionize the process of cancer diagnosis, grading, prognostication and treatment response monitoring. Besides having the advantage that serum can be obtained through a less invasive procedure, it contains molecules at an extraordinary dynamic range of ten orders of magnitude in terms of their concentrations. While the conventional methods, such as 2DE, have been in use for many years, the ability to identify the proteins through mass spectrometry techniques such as MALDI-TOF led to an explosion of interest in proteomics. Relatively new high-throughput proteomics methods such as SELDI-TOF and protein microarrays are expected to hasten the process of serum biomarker discovery. This review will highlight the recent advances in the proteomics platform in discovering serum biomarkers and the current status of glioma serum markers. We aim to provide the principles and potential of the latest proteomic approaches and their applications in the biomarker discovery process. Besides providing a comprehensive list of available serum biomarkers of glioma, we will also propose how these markers will revolutionize the clinical management of glioma patients.

Raman Spectroscopic Study of Serum Components

Early diagnosis of disease is important, because therapeutic intervention is most successful before it spread to the subject. The best health screenings method could be the blood test because the blood contains thousands of bio-molecules coming as by-products from the diseased part of the organism and would be non-invasive approach. The major limitation of this approach is the very low concentrations of the analytes need to be detected. Raman spectroscopy has been proven as one of the cutting edge technique applied in the field of histology, cytology and clinical chemistry. The primary obstacle of Raman spectroscopy is the low signal intensities. One of the promising approaches to overcome that is surface enhanced Raman spectroscopy (SERS) which has opened novel opportunities for chemical and biomedical analytics. Albumin is one of the most abundant proteins in blood, produced by liver. The state of albumin in serum determines the health of the liver and kidney. Serum albumin helps to transport many small molecules such as fatty acids, bilirubin, calcium, drugs through the blood. In this study, SERS is being used for the quantification and to understand of binding mechanism serum albumin.

Quantitative metabolic profiling of NMR spectral signatures of branched chain amino acids in blood serum

Branched Chain Amino Acids (BCAAs) are related to different aspects of diseases like pathogenesis, diagnosis and even prognosis. While in some diseases, levels of all the BCAAs are perturbed; in some cases, perturbation occurs in one or two while the rest remain unaltered. In case of ischemic heart disease, there is an enhanced level of plasma leucine and isoleucine but valine level remains unaltered. In `Hypervalinemia', valine is elevated in serum and urine, but not leucine and isoleucine. Therefore, identification of these metabolites and profiling of individual BCAA in a quantitative manner in body-fluid like blood plasma/serum have long been in demand. H-1 NMR resonances of the BCAAs overlap with each other which complicates quantification of individual BCAAs. Further, the situation is limited by the overlap of broad resonances of lipoprotein with the resonances of BCAAs. The widely used commercially available kits cannot differentially estimate the BCAAs. Here, we have achieved proper identification and characterization of these BCAAs in serum in a quantitative manner employing a Nuclear Magnetic Resonance-based technique namely T-2-edited Correlation Spectroscopy (COSY). This approach can easily be extended to other body fluids like bile, follicular fluids, saliva, etc.

An Eighteen Serum Cytokine Signature for Discriminating Glioma from Normal Healthy Individuals

Glioblastomas (GBM) are largely incurable as they diffusely infiltrate adjacent brain tissues and are difficult to diagnose at early stages. Biomarkers derived from serum, which can be obtained by minimally invasive procedures, may help in early diagnosis, prognosis and treatment monitoring. To develop a serum cytokine signature, we profiled 48 cytokines in sera derived from normal healthy individuals (n = 26) and different grades of glioma patients (n = 194). We divided the normal and grade IV glioma/GBM serum samples randomly into equal sized training and test sets. In the training set, the Prediction Analysis for Microarrays (PAM) identified a panel of 18 cytokines that could discriminate GBM sera fromnormal sera with maximum accuracy (95.40%) and minimum error (4.60%). The 18-cytokine signature obtained in the training set discriminated GBM sera from normal sera in the test set as well (accuracy 96.55%; error 3.45%). Interestingly, the 18-cytokine signature also differentiated grade II/Diffuse Astrocytoma (DA) and grade III/Anaplastic Astrocytoma (AA) sera from normal sera very efficiently (DA vs. normal-accuracy 96.00%, error 4.00%; AA vs. normal-accuracy 95.83%, error 4.17%). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using 18 cytokines resulted in the enrichment of two pathways, cytokine-cytokine receptor interaction and JAK-STAT pathways with high significance. Thus our study identified an 18-cytokine signature for distinguishing glioma sera fromnormal healthy individual sera and also demonstrated the importance of their differential abundance in glioma biology.

Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1 beta in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n = 27) and GBM (n = 28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p < 0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-Fc gamma RIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1 beta (IL1 beta) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1 beta neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies. Biological significance A complex antibody microarray based serum marker profiling identified a three-marker panel - LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1 beta which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma discrimination, identified IL1 beta as a potential candidate for developing targeted therapy. (C) 2015 Elsevier B.V. All rights reserved.

A role for nocturnal serum testosterone surge in regulating spermatogenesis in the adult non-human primate

Adult male bonnet monkeys exhibit nychthemeral rhythms in testosterone (T) secretion but the precise role of this heightened level of T secretion in regulating spermatogenesis is not known. Intranasal administration of microdoses (500 mu g or 250 mu g/day) of Norethisterone (IN-NET) to adult monkeys (n = 6) at 1600 h each day selectively and completely suppressed the nocturnal surge levels of serum T. Concomitant with this was a significant reduction (P<0.01) in serum LH but not FSH levels. DNA flow cytometric analysis of testicular biopsy tissue showed by week 10 of IN-NET treatment an arrest in meiotic transformation of primary spermatocytes (4C) to round/elongate (1C/HC) spermatids and by week 20 there was a complete absence of 4C, 1C and HC cells (with a relative accumulation in 2C cells). The accumulated meiotic (4C) cells at week 10 showed an increase (>80%, P<0.01) in coefficient of variation and a decrease in intensity of DNA-bound ethidium bromide fluorescence, parameters characteristic of degenerating 'apoptotic' subpopulation of germ cells. While two monkeys exhibited acute oligozoospermia 4 became azoospermic by 20 weeks of IN-NET treatment. A complete, qualitative reversal in the regressive changes in spermatogenesis and near-normal sperm output were apparent at the end of a 20-week recovery phase. These data demonstrate that prolonged, selective suppression of nocturnal surge levels of serum T secretion exerts a primary effect on meiosis in spermatogenesis leading to oligo/azoospermic status in adult bonnet monkeys.

Effect of pregnant mare serum gonadotropin on the induction and degradation of FSH and LH receptors in the granulosa cell of the immature rat

The relative induction of FSH and LH receptors in the granulosa cells of immature rat ovary by pregnant mare serum gonadotropin (PMSG) has been studied. A single injection of PMSG (15 IU) brought about a 3- and 12-fold increase in FSH and LH receptor concentration,respectively, in the granulosa cells. Maximal concentration was reached by 72 h but the receptor levels showed a sharp decline during the next 24–48 h. The kinetic properties of the newly formed FSH receptors were indistinguishable from the pre-existing ones. The induced FSH receptors were functional as demonstrated by an increase in the in vitro responsiveness of the cells to exogenous FSH in terms of progesterone production. Treatment of immature rats with cyanoketone, an inhibitor of Δ5,3β-hydroxysteroid dehydrogenase, prior to PMSG injection effectively reduced the PMSG-stimulated increase in the serum estradiol, uterine weight and LH receptors but had no effect on the FSH receptor induction. The ability of PMSG to induce gonadotropin receptors can be arrested at any given time by injecting its antibody, thereby suggesting a continuous need for the hormonal inducer. Estrogen in the absence of the primary inducer was unable to maintain the induced LH and FSH receptor concentration. Inhibition of prostaglandin synthesis using indomethacin also had no effect on either the induction or degradation of gonadotropin receptors. Administration of PMSG antiserum, 48 h after PMSG injection, brought about a rapid decline in the induced receptors over the next 24 h, with a rate constant and \iota 1/2 of 0.078 h−1 and 8.9 h for FSH receptors and 0.086 h−1 and 8.0 h for the LH receptors, respectively.

Optically active gossypol as a circular dichroism probe of interactions with serum albumins

The (+)-enantiomer of the polyphenolic binaphthyl gossypol, has been shown to be a useful CD probe of interactions with human and bovine serum albumin. (+)-Gossypol binds to albumin with same affinity as recemic (±)-gossypol, as shown by fluorescence quenching, and also displaces bilirubin from its albumin binding site. The CD characteristics of bound gossypol are different in the case of the two proteins.

Resolution of racemic gossypol and interaction of individual enantiomers with serum albumins and model peptides

Racemic gossypol has been resolved by HPLC separation of diastereomeric (−) norepinephrine adducts on a reverse-phase column. The binding constants for the interaction of the three gossypol forms (+, − and −) with human and bovine serum albumins have been determined by fluoresence quenching studies. The KD values demonstrate that all three forms bind equally effectively to the two proteins, suggesting an absence of chiral discrimination in albumin-gossypol interactions. Circular dichroism studies of (+)-gossypol binding to the model dibasic peptides, Boc-Lys-Pro-Aib-Lys-NHMe and gramicidin S, suggesting that distortions of binaphthyl geometry may occur only for specific orientations of interacting residues at the receptor site.

Studies on serum progesterone levels in Zebu × Holstein heifers during pre- and peripubertal periods

The peripheral serum progesterone levels of six normal Zebu × Holstein heifers (75% Holstein inheritance) during the prepubertal period (−150 days) were low (0.23 ± 0.06 ng/ml). They reached maximal values (0.73 ± 0.06 ng/ml) by −45 days (P<0.05 for progesterone values on −90th vs. −45th days) and thereafter decreased to the base level at the time of puberty. The mean (± SEM) age and body weight at puberty of these six heifers were 720 ± 20.70 days and 260.67 ± 8.82 kg, respectively. The serum progesterone levels remained low (0.38 ± 0.17 ng/ml) during early oestrus (up to 28 h) and gradually increased to 2.3 ± 0.84 ng/ml by the 15th day of the cycle.

Serum testosterone levels during the menstrual-cycle and early-pregnancy in the bonnet monkey (macaca-radiata)

Abstract is not available.

Fluorescence And Nmr-Studies Of The Binding Of Cholinergic Fluorescent-Probes To Horse Serum-Cholinesterase

The interaction of the cholinergic fluorescent probes, 1-(5-dimethyl-aminoaphthalene-1-sulfonamido) ethane-2-trimethylammonium perchlorate, 1-(5-dimethylaminonaphthalene-1-sulfonamido) pentane-5-trimethylammonium tartarate and 1-(5-dimethylaminonaphthalene-1-sulfonamido) decane-10- trimethylammonium tartarate with horse serum cholinesterase has been examined by fluorescence and n.m.r. methods. Fluorescence titrations show binding of the decane derivative to two sites on the protein whereas the lower homologs bind largely to one site. Active site inhibitors like curbamylcholine and decamethonium abolish binding of the decane derivative to the high affinity site. The inhibitors are largely without effect on the binding of the lower homologs. N.m.r. studies clearly establish immobilization of both ends of the molecule on binding in the case of the decane derivative, whereas in the lower homologs the dimethylamino group on the naphthalene ring is significantly more affected in the presence of enzyme. The probes are effective inhibitors of the enzyme with the decane derivative being two orders of magnitude more effective than its lower homologs. Based on the n.m.r., fluorescence and inhibition studies, a model for probe binding to the enzyme is advanced. It appears that the decane derivative binds with high affinity to the catalytic anionic site while the lower affinity site is assigned to a peripheral anionic site. The lower homologs probe only the peripheral site. A comparison of fluorescence, n.m.r. and inhibition studies with acetylcholinesterases from electric eel and bovine erythrocytes is presented.

Isolation and characterization of riboflavin-binding protein from pregnant-rat serum

A high-affinity riboflavin -binding protein was isolated and characterized for the first time from pregnant-rat sera by affinity chromatography on a lumiflavin-agarose column. The purified protein was homogeneous by the criteria of analytical polyacrylamide-gel disc electrophoresis, gel-filtration chromatography on Sephadex G-100 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It had a molecular weight of 90000+/-5000 and interacted with [14C]riboflavin with a 1:1 molar ratio with a dissociation constant (Kd) of 0.42 micron.

Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets

Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR-based diagnosis method by designing primers against a region that is unique to Salmonella enterica subsp. enterica serovar Typhi and Salmonella enterica subsp. enterica serovar Paratyphi A, corresponding to the STY0312 gene in S. Typhi and its homolog SPA2476 in S. Paratyphi A. An additional set of primers amplify another region in S. Typhi CT18 and S. Typhi Ty2 corresponding to the region between genes STY0313 to STY0316 but which is absent in S. Paratyphi A. The possibility of a false-negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella serovars as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying the region from clinical isolates of patients from various geographical locations in India, thereby showing that this region is potentially stable. These set of primers can also differentiate between S. Typhi CT18, S. Typhi Ty2, and S. Paratyphi A, which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivity of 95% compared to the Widal test which has a sensitivity of only 63%. As observed, in certain cases, the PCR assay was more sensitive than the blood culture test was, as the PCR-based detection could also detect dead bacteria.

Effect of human chorionic gonadotropin on serum levels of progesterone and estrogens in the pregnant bonnet monkev (Macaca radiata)

Administration of human chorionic gonadotropin to pregnant bonnet monkeys (Macaca radiata) at 55-60 days and 130-140 days of pregnancy resulted in a significant increase in serum progesterone levels. This effect could be observed even in lutectomized monkeys.However, no significant change in the serum estrogen level was noticed. These results suggest that although no chorionic gonadotropin is detectable in the serum after 35 days of pregnancy, the foetoplacental steroidogenic system is still responsive to exogenous gonadotropic stimulation.