Specific Protein Patterns Research Articles

Prospecting Specific Protein Patterns for High Body Mass Index (BMI), Metabolic Syndrome and Type 2 Diabetes in Saliva and Blood Plasma From a Brazilian Population.

Obesity and its associated metabolic disorders, such as T2DM and MeS, are a growing public health problem worldwide. Our goal was the identification of protein patterns that are uniquely characteristic of higher BMI, MeS, and T2DM in a Brazilian population. Saliva and plasma proteomes, clinical parameters were analyzed in a population from the state of Rio de Janeiro, Brazil, a mixed-race population. Volunteers were sorted by their BMI into normal (n = 29), overweight (n = 25), and obese (n = 15) and were compared with individuals with MeS (n = 23) and T2DM (n = 11). The Random Forest (RF) predictive model revealed that three clinical variables, BMI, HOMA-IR, and fasting blood glucose, are most important for predicting MeS and T2DM. A total of six plasmatic proteins (ABCD4, LDB1, PDZ, podoplanin, lipirin-alpha-3, and WRS) and six salivary proteins (hemoglobin subunit beta, POTEE, T cell receptor alpha variable 9-2, lactotransferrin, cystatin-S, carbonic anhydrase 6), are enhanced in T2DM and in MeS. Our data revealed similar alterations in protein composition across individuals with abnormal weight gain, T2DM, and MeS. This finding confirms the close link between these conditions at the molecular level in the studied population, potentially enhancing our understanding of these diseases and paving the way for the development of novel diagnostic tools.

BIOM-25. IDENTIFICATION OF PLASMA-DERIVED PREDICTIVE BIOMARKERS INCLUDING SERPINA3 FOR GLIOMA AND GLIOBLASTOMA

Abstract Advancement in the diagnosis and surveillance of gliomas has been limited posing both diagnostic and therapeutic challenges. Definitive diagnosis, distinction of progression versus pseudoprogression and prognosis assessment still depend on invasive neurosurgical biopsy procedure. Minimally-invasive plasma sampling could minimize the risks associated with invasive tissue sampling. Therefore, we aimed to identify specific plasma protein patterns of low- and high-grade glioma patients, compared to healthy individuals and investigated whether different plasma-derived protein signatures were associated with survival in glioma patients. Plasma samples were from patients with glioma grades I to IV collected during tumor removal and from healthy controls donating blood were processed for Liquid Chromatography Mass Spectrometry (LC/MS) proteomics after depletion of the 14 most abundant plasma proteins, in order to specifically detect low-abundant tumor-derived proteins. Overall, 646 proteins were measured across 104 plasma samples from glioma patients and 57 plasmas from the healthy cohort. PCA showed 2 clusters of samples, one containing low- and high-grade glioma samples and the other containing healthy and some grade IV (GBM) plasma samples. Interestingly, a spatial analysis of tumor location by MRI associated the GBM samples separated from healthy samples with subventricular vicinity. 26 differentially expressed proteins (DEPs) were identified discriminating between glioma and healthy samples and 30 DEPs discriminating GBM and healthy samples. The top most overexpressed proteins in gliomas vs controls were SERPINA3, F13A1, and TKT. Multivariate analysis identified SERPINA3, PPBP and MYH9 as strong discriminators of healthy vs GBM but also of low-grade vs GBM, indicating that these proteins may represent specific plasma biomarkers for GBM. SERPINA3, MYL1 and CLU were associated with poor survival in GBM. In conclusion, we describe sets of plasma derived proteins, in particular SERPINA3, as predictive biomarkers that could be used to assess tumor progression allowing patient-centered treatment options.

Machine Learning on Large-Scale Proteomics Data Identifies Tissue and Cell-Type Specific Proteins.

Using data from 183 public human data sets from PRIDE, a machine learning model was trained to identify tissue and cell-type specific protein patterns. PRIDE projects were searched with ionbot and tissue/cell type annotation was manually added. Data from physiological samples were used to train a Random Forest model on protein abundances to classify samples into tissues and cell types. Subsequently, a one-vs-all classification and feature importance were used to analyze the most discriminating protein abundances per class. Based on protein abundance alone, the model was able to predict tissues with 98% accuracy, and cell types with 99% accuracy. The F-scores describe a clear view on tissue-specific proteins and tissue-specific protein expression patterns. In-depth feature analysis shows slight confusion between physiologically similar tissues, demonstrating the capacity of the algorithm to detect biologically relevant patterns. These results can in turn inform downstream uses, from identification of the tissue of origin of proteins in complex samples such as liquid biopsies, to studying the proteome of tissue-like samples such as organoids and cell lines.

HIP1R and vimentin immunohistochemistry predict 1p/19q status in IDH-mutant glioma.

IDH-mutant gliomas are separate based on the codeletion of the chromosomal arms 1p and 19q into oligodendrogliomas IDH-mutant 1p/19q-codeleted and astrocytomas IDH-mutant. While nuclear loss of ATRX expression excludes 1p/19q codeletion, its limited sensitivity prohibits to conclude on 1p/19q status in tumors with retained nuclear ATRX expression. Employing mass spectrometry based proteomic analysis in a discovery series containing 35 fresh frozen and 72 formalin fixed and paraffin embedded tumors with established IDH and 1p/19q status, potential biomarkers were discovered. Subsequent validation immunohistochemistry was conducted on two independent series (together 77 oligodendrogliomas IDH-mutant 1p/19q-codeleted and 92 astrocytomas IDH-mutant). We detected highly specific protein patterns distinguishing oligodendroglioma and astrocytoma. In these patterns, high HIP1R and low vimentin levels were observed in oligodendroglioma while low HIP1R and high vimentin levels occurred in astrocytoma. Immunohistochemistry for HIP1R and vimentin expression in 35 cases from the FFPE discovery series confirmed these findings. Blinded evaluation of the validation cohorts predicted the 1p/19q status with a positive and negative predictive value as well as an accuracy of 100% in the first cohort and with a positive predictive value of 83%; negative predictive value of 100% and an accuracy of 92% in the second cohort. Nuclear ATRX loss as marker for astrocytoma increased the sensitivity to 96% and the specificity to 100%. We demonstrate that immunohistochemistry for HIP1R, vimentin, and ATRX predict 1p/19q status with 100% specificity and 95% sensitivity and therefore, constitutes a simple and inexpensive approach to the classification of IDH-mutant glioma.

Identification and Visualization of Protein Expression in Whole Mouse Embryos by Immunofluorescence.

Mouse embryo studies are pivotal for the understanding of early development. Analysis of the spatial and temporal changes of protein expression during development of a mouse embryo allows us to identify the genetic basis of errors of development in animal disease models. Immunofluorescence is a powerful technique to study the localization and variation in expression pattern of specific proteins in cells, tissues, and organs. Detecting the antigens with their specific antibodies labeled with fluorescent probes allows visualization of proteins at the cellular level. Here, we provide the optimized protocol of immunostaining whole mouse embryos at embryonic stages E7.5 to E11.5.

Honey botanical origin and honey-specific protein pattern: Characterization of some European honeys

Honey adulteration generates low quality products on the market. The study aimed to find a simple, specific and less time-consuming method than standard melissopalynology only, for monitoring honey botanical and geographical origin. In this study 42 honey samples from different sources were examined for their botanical origin by using melissopalynology and their specific protein patterns by electrophoresis on SDS polyacrylamide gels (SDS-PAGE). The melissopalynological analysis consisted of counting all melliferous pollen, non-melliferous pollen and honeydew elements to identify the species of pollen present in each sample. The honey samples had predominant pollen from Apiaceae, Boraginaceae, Brassicaceae, Compositae H, Fabaceae (Trifolium sp.) and Fagaceae family. From the Fagaceae family, the most important species was Castanea sativa Mill., while the Boraginaceae was represented by Echium sp. and Myosotis sp. SDS-PAGE showed that the different origin honeys shared protein bands between 45 and 85 kDa (animal origin proteins, i.e. major royal jelly proteins, enzymes) and that specific proteins (presumably plant origin proteins) can be attributed to individual honey types. This study shows that the combination of melissopalynology and SDS-PAGE is a useful tool for modern discrimination between different kinds of honey, even without performing specific protein identification.

Protein profile of fiber types in human skeletal muscle: a single-fiber proteomics study

BackgroundHuman skeletal muscle is composed of three major fiber types, referred to as type 1, 2A, and 2X fibers. This heterogeneous cellular composition complicates the interpretation of studies based on whole skeletal muscle lysate. A single-fiber proteomics approach is required to obtain a fiber-type resolved quantitative information on skeletal muscle pathophysiology.MethodsSingle fibers were dissected from vastus lateralis muscle biopsies of young adult males and processed for mass spectrometry-based single-fiber proteomics. We provide and analyze a resource dataset based on relatively pure fibers, containing at least 80% of either MYH7 (marker of slow type 1 fibers), MYH2 (marker of fast 2A fibers), or MYH1 (marker of fast 2X fibers).ResultsIn a dataset of more than 3800 proteins detected by single-fiber proteomics, we selected 404 proteins showing a statistically significant difference among fiber types. We identified numerous type 1 or 2X fiber type–specific protein markers, defined as proteins present at 3-fold or higher levels in these compared to other fiber types. In contrast, we could detect only two 2A-specific protein markers in addition to MYH2. We observed three other major patterns: proteins showing a differential distribution according to the sequence 1 > 2A > 2X or 2X > 2A > 1 and type 2–specific proteins expressed in 2A and 2X fibers at levels 3 times greater than in type 1 fibers. In addition to precisely quantifying known fiber type–specific protein patterns, our study revealed several novel features of fiber type specificity, including the selective enrichment of components of the dystrophin and integrin complexes, as well as microtubular proteins, in type 2X fibers. The fiber type–specific distribution of some selected proteins revealed by proteomics was validated by immunofluorescence analyses with specific antibodies.ConclusionWe here show that numerous muscle proteins, including proteins whose function is unknown, are selectively enriched in specific fiber types, pointing to potential implications in muscle pathophysiology. This reinforces the notion that single-fiber proteomics, together with recently developed approaches to single-cell proteomics, will be instrumental to explore and quantify muscle cell heterogeneity.

The molecular pathway triggered by zirconia in endothelial cells involves epigenetic control

The requirement to achieve natural looking restorations is one of the most challenging aspects in dentistry. Although zirconia has provided new opportunities for achieving superior aesthetics and physicochemical outcomes, very little has been achieved for its cellular and molecular performance, especially considering angiogenesis and osteogenesis. As angiogenesis is a secondary event and concomitant to osteogenesis, an indirect effect of dental implant on endothelial cells could be the release of active molecules such as those already reported affecting osteoblasts. To better address this issue, we challenged human endothelial cells (HUVECs) with zirconia-conditioned medium up to 72 h to allow analysis specific gene expression and protein pattern of mediators of epigenetic machinery in full. Our data shows involvement of zirconia in triggering intracellular signaling through MAPK-ERK activation, leading the signal to activate histone deacetylase HDAC6 likely with concomitant well-modulated DNA methylation profile by DNMTs and TETs. These signaling pathways seem to culminate in cytoskeleton rearrangement of endothelial cells, an important prerequisite to cell migration expected in angiogenesis. Collectively, this study demonstrates for the first time epigenetic-related molecular mechanism involved in endothelial cells responding to zirconia, revealing a repertoire of signaling molecules capable of executing the reprogramming process of gene expression, which are necessary to drive cell proliferation, migration, and consequently angiogenesis. This set of data can further studies using gene editing approaches to better elucidate functional roles.

Does the Use of the "Proseek® Multiplex Oncology I Panel" on Peritoneal Fluid Allow a Better Insight in the Pathophysiology of Endometriosis, and in Particular Deep-Infiltrating Endometriosis?

Endometriosis appears to share certain cancer-related processes, such as cell attachment, invasion, proliferation and neovascularization, some of which can also be found in other healthy tissues. In order to better understand the altered milieu of the peritoneal cavity, while acknowledging the reported similarities between endometriosis and neoplastic processes, we applied a multiplex oncology panel to search for specific biomarker signatures in the peritoneal fluid of women with endometriosis, women with deep-infiltrating endometriosis (DIE), as well as controls. In total, 84 patients were included in our study, 53 women with endometriosis and 31 controls. Ninety-two proteins were measured in prospectively collected peritoneal fluid (PF) samples, using the “Proseek® Multiplex Oncology I Panel”. We first compared patients with endometriosis versus controls, and in a second step, DIE versus endometriosis patients without DIE. Out of the 92 analyzed proteins, few showed significant differences between the groups. In patients with endometriosis, ICOS ligand, Endothelial growth factor, E-selectin, Receptor tyrosine-protein kinase erbB-2, Interleukin-6 receptor alpha, Vascular endothelial growth factor receptor 2, Fms-related tyrosine kinase 3 ligand, C-X-C motif chemokine 10, Epididymal secretory protein E4 and Folate receptor-alpha were decreased, while Interleukin-6 and Interleukin-8 were increased compared to controls. Looking at patients with DIE, we found Chemokine ligand 19, Stem cell factor, Vascular endothelial growth factor D, Interleukin-6 receptor alpha and Melanoma inhibitory activity to be increased compared to endometriosis patients without DIE. We have shown a distinct regulation of the immune response, angiogenesis, cell proliferation, cell adhesion and inhibition of apoptosis in PF of patients with endometriosis compared to controls. The specific protein pattern in the PF of DIE patients provides new evidence that DIE represents a unique entity of extrauterine endometriosis with enhanced angiogenetic and pro-proliferative features.

Comprehensive Proteomic Profiling of Pressure Ulcers in Patients with Spinal Cord Injury Identifies a Specific Protein Pattern of Pathology.

Objective: Severe pressure ulcers (PUs) do not respond to conservative wound therapy and need surgical repair. To better understand the pathogenesis and to advance on new therapeutic options, we focused on the proteomic analysis of PU, which offers substantial opportunities to identify significant changes in protein abundance during the course of PU formation in an unbiased manner.Approach: To better define the protein pattern of this pathology, we performed a proteomic approach in which we compare severe PU tissue from spinal cord injury (SCI) patients with control tissue from the same patients.Results: We found 76 proteins with difference in abundance. Of these, 10 proteins were verified as proteins that define the pathology: antithrombin-III, alpha-1-antitrypsin, kininogen-1, alpha-2-macroglobulin, fibronectin, apolipoprotein A-I, collagen alpha-1 (XII) chain, haptoglobin, apolipoprotein B-100, and complement factor B.Innovation: This is the first study to analyze differential abundance protein of PU tissue from SCI patients using high-throughput protein identification and quantification by tandem mass tags followed by liquid chromatography tandem mass spectrometry.Conclusion: Differential abundance proteins are mainly involved in tissue regeneration. These proteins might be considered as future therapeutic options to enhance the physiological response and permit cellular repair of damaged tissue.

Theme 5 Human cell biology and pathology

Background: The delay from onset of the first symptoms to a definite ALS diagnosis depends also on the elusiveness of the initial clinical manifestations. The lack of disease-specific biomarkers to detect early pathology when ALS is supposed complicates the situation. This latency reduces the therapeutic time frame, in which neuron-rescuing strategies exert their greatest chance to work. Various biomarkers are currently promised, but none of them are specific enough to allow monitoring of disease progression. This, as well as the heterogeneity of the disease concerning clinical onset pattern and survival rates, makes difficult the correct stratification of patients into clinical trials, masking the potential positive outcome in some patients.Objective: Our main objective is to establish and test an early diagnostic tool based on microscopic immune cell monitoring of ALS patients' blood samples by using the Toponome Imaging System (TIS).Methods: TIS is based on automatically controlled microscopic device involving conjugated dye-tag incubation, protein-tag-dye-imaging, and tag-dye bleaching (1). This leads to the collection of at least 21 cycle images of fixated peripheral blood mononuclear cells (PBMCs) isolated from freshly drawn blood of ALS patients and healthy "control" donors. Resulting data sets contain combinatorial molecular information about the spatial protein network, called toponome. The PBMC toponome architectures are quantitatively analyzed as a threshold-binary code with 1 = protein is present and 0 = protein is absent.Results: Preliminary screening data of PBMCs from 4 ALS patients reveal a subpopulation of lymphocytes expressing a specific surface protein pattern, called "ALS toponome". These aberrant T cells could not be found in blood samples of controls. We observe that the number of these cells correlate with the ALS progression rate of patients, supporting the conclusion that these cells may be causal for the disease.Discussion and conclusion: Although these findings open up a potential strategy to detect early ALS disease and to monitor disease progression, a statistical analysis with many more patients, as well as data based differentiation to other neurodegenerative diseases, is mandatory. A clinical trial initiated by our faceALS foundation with at least 60 patients classified in three subsets (1. control, 2. ALS, and 3. Multiple Sclerosis (MS)) and in close cooperation with leading ALS centres in Germany is still in progress. The detection of specific and/or aberrant immune cells in blood samples of ALS patients may provide a key to understand disease onset and progression, could be used for the "staging" of disease, and contribute to effective therapy options.

Dynamic urinary proteomic analysis in a Walker 256 intracerebral tumor model.

BackgroundPatients with primary and metastatic brain cancer have an extremely poor prognosis, mostly due to the late diagnosis of disease. Urine, which lacks homeostatic mechanisms, is an ideal biomarker source that accumulates early and highly sensitive changes to provide information about the early stage of disease.MethodsA rat model mimicking the local tumor growth process in the brain was established with intracerebral Walker 256 (W256) cell injection. Urine samples were collected on days 3, 5, and 8 after injection, and then analyzed by liquid chromatography coupled with tandem mass spectrometry.ResultsIn the intracerebral W256 model, no obvious clinical manifestations or abnormal magnetic resonance imaging (MRI) signals were found on days 3 or 5; at these time points, 9 proteins were changed significantly in the urine of all eight tumor rats. On day 8, when tumors were detected by MRI, 25 differential proteins were identified, including 10 that have been reported to be closely related to brain metastasis or primary tumors. The differential urinary proteome was compared with those from the subcutaneous W256 model and the intracerebral C6 model. Few differential proteins overlapped, and specific differential protein patterns were observed among the three models.ConclusionsThese findings demonstrate that early changes in the urine proteome can be detected in the intracerebral W256 model. The urinary proteome can reflect the difference when tumor cells with different growth characteristics are inoculated into the brain and when identical tumor cells are inoculated into different areas, specifically, the subcutis and the brain.

Plasma Proteome Profiling Reveals Dynamics of Inflammatory and Lipid Homeostasis Markers after Roux-En-Y Gastric Bypass Surgery.

Obesity-related diseases affect half of the global population, and bariatric surgery is one of the few interventions with long-lasting weight loss and cardio-metabolic effects. Here, we investigated the effect of Roux-en-Y gastric bypass surgery on the plasma proteome, hypothesizing that specific proteins or protein patterns may serve as key mediators and markers of the metabolic response. We performed mass spectrometry (MS)-based proteomics on two longitudinal studies encompassing 47 morbidly obese patients, generating quantitative information on more than 1,700 proteins. A global correlation matrix incorporating about 200,000 relationships revealed functional connections between proteins and assigned them to physiological processes. The main classes of significantly altered proteins were markers of systemic inflammation and those involved in lipid metabolism. Our data highlight robust correlative and anti-correlative behaviors of circulating proteins to each other and to clinical parameters. A group of inflammation-related proteins showed distinct inverse relationships to proteins consistently associated with insulin sensitivity.

Differences in secretome in culture media when comparing blastocysts and arrested embryos using multiplex proximity assay

Objectives: The aim of this study was to assess different patterns of the human embryo secretome analysed as protein levels in culture media. Furthermore, analyses to correlate protein levels with quality and timing to development of human embryos were performed.Material and methods: Human day-2 cryopreserved embryos were cultured for four days in an EmbryoScope® with a time-lapse camera, and embryo quality was evaluated retrospectively. After culture, the media were collected and relative levels of secreted proteins were analysed using Proseek Multiplex Assays. Protein levels were evaluated in relation to timing to development and the ability to form a blastocyst.Results: Specific patterns of timing of development of blastocysts were found, where a difference in time to start of cavitation was found between high- and low-quality blastocysts. There appeared to be a correlation between specific protein patterns and successful formation of morulae and blastocysts. Embryos developing into blastocysts had higher levels of EMMPRIN than arrested embryos, and levels of caspase-3 were lower in high- versus low-quality blastocysts. Also, higher levels of VEGF-A, IL-6, and EMMPRIN correlated with shorter times to morula formation.Conclusions: The secretome and timing to development differ in embryos forming blastocysts and those that become arrested, and in high- versus low-quality blastocysts. The levels of certain proteins also correlate to specific times to development.

Clinical proteomic analysis of scrub typhus infection

BackgroundScrub typhus is an acute and febrile infectious disease caused by the Gram-negative α-proteobacterium Orientia tsutsugamushi from the family Rickettsiaceae that is widely distributed in Northern, Southern and Eastern Asia. In the present study, we analysed the serum proteome of scrub typhus patients to investigate specific clinical protein patterns in an attempt to explain pathophysiology and discover potential biomarkers of infection.MethodsSerum samples were collected from three patients (before and after treatment with antibiotics) and three healthy subjects. One-dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry was performed to identify differentially abundant proteins using quantitative proteomic approaches. Bioinformatic analysis was then performed using Ingenuity Pathway Analysis.ResultsProteomic analysis identified 236 serum proteins, of which 32 were differentially expressed in normal subjects, naive scrub typhus patients and patients treated with antibiotics. Comparative bioinformatic analysis of the identified proteins revealed up-regulation of proteins involved in immune responses, especially complement system, following infection with O. tsutsugamushi, and normal expression was largely rescued by antibiotic treatment.ConclusionsThis is the first proteomic study of clinical serum samples from scrub typhus patients. Proteomic analysis identified changes in protein expression upon infection with O. tsutsugamushi and following antibiotic treatment. Our results provide valuable information for further investigation of scrub typhus therapy and diagnosis.

Genus specific protein patterns of viruses

In the generation of rising and re-emerging viral infections, diagnostics and its allied fields have a predominant function to play in combating the diseases. Enormous quantity of the molecular sequence data handy in the public domain has the practicable to contribute in a important way in the improvement of novel diagnostic tools. One of the perquisites for such a learn about is the identification of signature sequences i.e., small stretches of protein/nucleotide sequences that are special to a given family/genus/organism. There exist numerous resources in the public area archiving signature sequences of proteins based on sequence identity/ similarity. However, these resources do not take into account the taxonomic fact which has a extensive position to play in viral diagnostics. The present find out about is an effort to explicitly take into account the taxonomic information and thereby derive genus-specific signature sequences of viral proteins. The preliminary facts for obtaining patterns viz., multiple sequence alignment (MSA) is received from VirGen database. An in-house developed perl script is used to derive the patterns from the MSA. The patterns are then validated by using search against the non-redundant protein sequence database at NCBI, thereby enabling the computation of their sensitivity and specificity. Such a validation requires datasets pertaining to true-positives and true-negatives. True-positive dataset is acquired from the taxonomy database at NCBI by formulating an Entrez query such that the whole variety of species belonging to a given genus is retrieved. The true-negative dataset constituted of any protein sequence that belongs to genus different than the one in question. Of the 262 proteins belonging to 19 families (RNA viruses) in VirGen, patterns should be detected for 125 proteins, all of which actually uncommon true-positives and false-positive sequences. These patterns when mapped onto their corresponding 3D constructions (25 unique entries of Protein Data Bank) are located to be part of essential useful regions like energetic website and dimerisation interface. The unique viral signature sequences/peptides consequently obtained have applications not only in detection assays and as therapeutics but also can serve as putative targets for viral vaccines. Viruses have induced some of the most dramatic and deadly sickness pandemics in human history. Before it was declared to be eradicated in 1980, smallpox, a rather contagious human disorder caused by means of the Variola virus, killed 300 to five hundred million humans international in the 20th century alone. The 1918–1919 “Spanish flu” pandemic infected roughly one-third of the world's human populace and brought on an estimated 50 to a hundred million deaths. In the past half century, deadly disorder outbreaks brought about through novel viruses of animal origin—Nipah virus in Malaysia, Hendra virus in Australia, hantavirus in the United States, Ebola virus in Africa, along with HIV (human immunodeficiency virus), various influenza subtypes, and the SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome) coronaviruses—have underscored the urgency of grasp elements influencing viral disease emergence and spread. The world's modern main infectious killer, HIV, has prompted an estimated 36 million deaths since the first cases were pronounced in 1981. In 2012, more than two million people were newly contaminated with the virus, and 1.6 million died of HIV/AIDS. In 2009, a novel swine-origin H1N1 strain of influenza A hastily unfold to over 213 countries in the first declared pandemic of the 21st century. And now, on August 8, 2014, the World Health Organization (WHO) Director-General Margaret Chan declared the Ebola outbreak in West Africa a “public fitness emergency of worldwide concern,” triggering powers below the 2005 International Health Regulations (IHR). The IHR require countries to increase national preparedness capacities, such as the obligation to file internationally huge events, habits surveillance, and exercising public fitness powers, while balancing human rights and global trade.

Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples

A method we previously developed has been applied to the determination of the glycosylation pattern of specific proteins in biological samples. Six proteins (alpha-1-antitrypsin, transferrin, haptoglobin, C1 inhibitor, alpha-1 acid glycoprotein, and immunoglobulin G) were studied in serum samples from five individuals and cerebrospinal fluid (CSF) samples from three individuals, to investigate the expected normal distribution of glycosylation patterns and to assess whether this methodology can be used to discriminate between samples from different individuals. For serum samples, the differences were shown to be small, while much larger differences were found for the CSF samples, with a greater number of glycoforms present. This can be linked to the occurrence of differential glycosylation in proteins expressed in the brain compared with proteins expressed elsewhere in the body. The developed method could distinguish differences in the glycosylation pattern of specific proteins in the individual samples, which was not reflected in the glycan content of total CSF. This is the first time that the glycoforms of several of these proteins have been investigated in CSF.

Proteome Analysis of Human Perilymph Using an Intraoperative Sampling Method.

The knowledge about the etiology and pathophysiology of sensorineural hearing loss (SNHL) is still very limited. This study aims at the improvement of understanding different types of SNHL by proteome analysis of human perilymph. Sampling of perilymph was established during inner ear surgeries (cochlear implantation, vestibular schwannoma surgeries), and safety of the sampling method was determined by checking hearing threshold with pure-tone audiometry postoperatively. An in-depth shot-gun proteomics approach was performed to identify cochlear proteins and the individual proteome in perilymph of patients. This method enables the identification and quantification of protein composition of perilymph. The proteome of 41 collected perilymph samples with volumes of 1-12 μL was analyzed by data-dependent acquisition, resulting in overall 878 detected protein groups. At least 203 protein groups were solely identified in perilymph, not in reference samples (serum, cerebrospinal fluid), displaying a specific protein pattern for perilymph. Samples were grouped by patient's age and surgery type, leading to the identification of some proteins specific to particular subgroups. Proteins with different abundances between different sample groups were subjected to classification by gene ontology annotations. The identified proteins might serve as biomarkers to develop tools for noninvasive inner ear diagnostics and to elucidate molecular profiles of SNHL.

Poly Acrylamide Gel Electrophoresis of Serum Protein, Application to Kala-azar Patients

Sera samples were collected from 60 children aged 4-60 months, all were clinically and serologically proven cases of visceral leishmaniasis, as well as from 10 healthy children, all were seronegative with no history of parasitic infection who serve as a control during the study. Serum total protein and albumin were measured and compared between the control and visceral leishmaniasis patients. Serum protein profiles have been investigated using the conventional sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS-PAGE). Serum of control group showed the specific protein pattern with five protein bands, while serum protein profile in visceral leishmaniasis patients revealed three groups of electrophoretic banding patterns. , 50% showed twelve bands, 36.66% of the patients showed nine bands and 13.33% showed ten bands. At least four of these bands were found to be common among the infected groups which may be of diagnostic value and required further investigations. The three different electrophoretic patterns groups may be correlated with the previous epidemiological observations in respect of different clinical presentation of the disease and different response the chemotherapeutic agents at many endemic areas around the world.
 Key words: Protein band, protein patterns, serum protein, sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE), visceral leishmaniasis.

Performance of HBsAg quantification assays for detection of Hepatitis B virus genotypes and diagnostic escape-variants in clinical samples

BackgroundThe impact of hepatitis B virus (HBV) genomic variability on the measurement of HBsAg level has been poorly evaluated. ObjectiveThis study was designed to compare the performance of all the available assays measuring HBsAg level in this setting. Study designA large selection of wild type HBV genotypes (n=184) and HBsAg strains harboring mutations in the S gene (n=81) from clinical samples was studied with three HBsAg quantification assays: Architect HBsAg (Abbott), LiaisonXL Murex HBsAg Quant (DiaSorin) and the Elecsys HBsAgII (Roche). ResultsThe overall percentage of positive results was 99.2% for Abbott, 98.9% for DiaSorin and 98.1% for Roche. Abbott and Roche assays provided an excellent concordance in HBsAg quantification (global mean bias of −0.006 logIU/mL). By contrast, DiaSorin underestimated HBsAg level with values 0.112 logIU/ml and 0.103 logIU/ml lower than Abbott and Roche, respectively. By contrast, DiaSorin slightly over quantified gtC (2.5% over the expected value) while Abbott provided values 6.2% lower than expected and 16.2% lower than what observed for the other genotypes. HBsAg quantitative assays were influenced by HBs protein substitutions irrespective to the genotype but no specific protein pattern that would particularly impair the quantification by one technique has been identified. However, Roche seemed to be particularly impacted by substitutions at 145 residue: 75% of under quantified samples carried a substituted 145 residue. ConclusionThis head-to-head comparison indicates a good correlation between all current systems used to quantify HBsAg but clearly shows an influence of both the genotype and the presence of “a” determinant variants in the absolute quantification of HBsAg. While these discrepancies may not translate into major clinical consequence, they may explain an absence of detection of weak concentration of HBsAg on some systems.