Protein Spots Research Articles

Prediction of o-glycosylation pathways in Mycobacterial immunogenic proteins

Aims and objectives: There is an emergence need to identifying Mycobacterial proteins of value to diagnostic assays and vaccine formulations. Glycosylation is an important post-translational modification of proteins and affects more than half of all the proteins in a cell. O-linked glycosylation refers to the event in which a carbohydrate is covalently linked to the hydroxyl group of serine or threonine. This modification influences a number of properties of proteins including proteolytic resistance, solubility, immunological properties and ligand binding. Host cells can bind to Mycobacterial cell wall carbohydrates via a class of surface localized or secreted proteins known as lectins, and these interactions strongly contribute to bacterial adhesion and uptake, and are also associated with the capability of Mtb to survive. This study represents the attempt to predict o-glycosylation pathways of immunogenic TB proteins as potential therapeutic strategies against TB. Methods: A Mycobacterium tuberculosis culture filtrate enriched with mannose-containing proteins that were purified by conA-Affinity chromatography. One and two dimensional gel electrophoresis carried out by standard methods. Gel staining procedures are based on the periodic Acid-Schiff reaction. The Protein spots of interest were excised from the gel and then analysis by MALDI Mass Spectrometry. GlycoPP software, Version 1.0, was used for glycosylation prediction and protein sequences. Results: Protein bands or spots were excised from the gel and analyzed by mass spectrometry. These proteins have O-glycosite residue and a glycan attached covalently and enzymatically at amide or hydroxyl group which includes Rv0475, Rv3367, Rv3699, Rv0251c, Rv2558 and Rv1284. Rv0475 has 17 Potential O-Linked Glycosylated Sites but 6 sites of Potential Glycosylated. Rv3367 has 67 Potential O-Linked Glycosylated Sites and 55 sites of Potential Glycosylated. Additionally, Rv3699 has 26 Potential O-Linked Glycosylated Sites and 10 sites Potential Glycosylated. Conclusions: This study may be shown the role of glycoprotein's in Mycobacterium pathogenesis, disease development, and its implications in drug development. All of these proteins have several putative antigenic epitopes and induces an immune response especially production of antibodies. In addition, Rv3367as a member of the Mycobacterium tuberculosis PE family and PGRS subfamily, gly-rich proteins have carbohydrate moieties and also are known to play a role in several pathological processes. Because of the importance of M.tuberculosis immunogenic antigens, the availability of the secretion and glycosylation of MTB proteins is a tool that will allow a deeper understanding of role these proteins play in the pathogenesis of disease and provide the basis for more sensitive and discriminative clinical diagnostic tests and improving to formulate new Mycobacterial vaccines.

Identification and Immunological Characterization of Somatic Proteins from Adults of Toxocara cati by Proteomics Technique.

Background:Toxocara cati is considered as one of the main etiological agents of toxocariasis with global and regional importance. As there is no information on proteomics of T. cati, herein, we reported the results obtained by proteomic analysis of somatic proteins extract, using a mass spectrometry (LC–MS/MS) approach.Methods:Somatic extract fractions were separated by two-dimensional SDS-PAGE and were electro blotted on to PVDF membranes for immunoblot analysis, then collected the immunogenic spots which response of antibodies of the paratenic hosts (mice) to the antigens (Mashhad, 2017), and analyzed by LC–MS/MS. The LC-MS/MS data were analyzed by Mascot database, Taxonomy Toxocara, and common contaminants, in Omics Center, Biotechnology Medical University of Graz (Austria, 2018).Result:The protein spots were isolated between 15–140 kDa ranges using 3–10 non-linear IPG strips and Brilliant Blue Coomassie. Ten proteins were characterized as immunogenic proteins, seven of them were identified and three of them were unknown proteins.Conclusion:This study provided additional information about the somatic antigens of T. cati, which can lead to the development of new strategies for novel immuno-modulators, drug targets, subunit vaccines and immunodiagnostic kits for toxocariasis.

Correlation of 24-hour urinary protein excretion and spot urine protein-creatinine ratio in women with preeclampsia: A cross sectional study

Objective: To examine the correlation between spot urine PCR and 24-hour urine protein excretion in patients being evaluated for preeclampsia. Material and Methods: A cross sectional study was conducted on 100 patients at the Department of Obstetrics and Gynecology at Chalmeda Anand Rao Institute of Medical Sciences, Karimnagar, Telangana State for a period from December 2019 to August 2020, with inclusion of singleton viable pregnancy, Gestational age between 24 and 38 weeks, Confirmed by last menstrual period and early ultrasound. Result: Among 100 patient’s patients having mean age of 25 ± 3.266 years, among the patients 19% percent of the patients had past history of preeclampsia. Mean gestational age for the patients was 32.39 ± 3.42 weeks. Study showed 62 % of moderate urinary protein levels. There was significant correlation between spot PCR and 24 hour urine protein collection (spearman’s rho=0.7, P<0.0001). Conclusion: Urine protein: creatine ratio is one of the important investigation in hypertensive disorder of the pregnancy. Spot Urine protein: creatine ratio can reliably be taken as a simple, accurate, and convenient replacement for tedious, time consuming 24 hour urine protein estimation, especially in countries like India where hospitals are flooded with a large number of in-patients. Therefore Urine protein: creatine ratio estimation should be introduced and adopted in practice in testing for proteinuria.

Analysis of Two-Dimensional Gel Electrophoresis Map of Methicillin-Resistant &amp;lt;i&amp;gt;Staphylococcus aureus&amp;lt;/i&amp;gt; Treated with Acetone Extract from &amp;lt;i&amp;gt;Canarium odontophyllum&amp;lt;/i&amp;gt; Miq. Leaves

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a global health concern that has caused severe health threats over the past decade. Leaves extract of C. odontophyllum has been proven previously as an anti MRSA agent. Proteomics provide a technique that used to analyze the differential of protein expression profile between untreated and treated MRSA with subinhibitory concentrations of acetone extract from C. odontophyllum leaves. This study aims to determine the optimum parameter for analysis of protein expression profile using two-dimension gels electrophoresis (2-DE) for MRSA protein after treatment with acetone extract from C. odontophyllum leaves. Comparison of the Protein Expression Profile (PEP) between the untreated and treated MRSA was analyzed using PDQuest software. The optimum condition for MRSA protein treated with acetone extract from C. odontophyllum leaves to produce the best resolution with greater spot distribution was as follows: 100 μg volume of MRSA protein that loaded after passive rehydration then was run until reaching 25 kVrhs during IEF using 17 cm IPG strip within ranges of pH 4 - 7. Analysis of protein expression from the 2-DE gel map shows that 9 protein spots up-regulated and 41 protein spots were down-regulated with more than 2-fold differences (p C. odontophyllum leave may provide an insight into the antimicrobial mechanism, which could lead to the identification of target protein for future novel therapeutic development against MRSA infections.

Study on specific proteins involved in articular cartilage regeneration activity induced by decalcified bone transplantation.

BackgroundThrough a comprehensive analysis of the joint synovial fluid produced in the process of rabbit articular cartilage regeneration, the role and characteristics of knee synovial fluid in the process of decalcified bone transplantation-induced articular cartilage regeneration were explored.MethodsTwenty New Zealand white rabbits (approximately 2.5 kg in weight) were selected, and bilateral distal femoral bones from two randomly selected rabbits were extracted. After decalcification, the bones were cut into 2 mm × 4 cm long decalcified bone strips. Meanwhile, the other 18 rabbits were randomly divided into three groups: the test group (8 rabbits), the positive control group (6 rabbits), and the blank group (4 rabbits). In the test group, the decalcified bone joint was transplanted into the rabbits at the articular cartilage defect; in the positive control group, the articular cartilage defect of the rabbits were treated and put aside; in the blank group, no rabbits were treated. On the day of transplantation, and on the 4th, 8th, 12th, and 16th weeks after transplantation, the joint synovial fluid of each group was taken for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, and related database verification and identification, and compared with the positive control group and the blank group.ResultsUsing 2D-PAGE to separate various proteins in the synovial fluid of the rabbit knee joints, it was found that there were differential protein spots in the test group compared with the blank group and the positive control group. After conducting a comparative search and query in the UniProt database, through comprehensive analysis, it was finally found that three proteins with molecular weights of 23,429.4, 57,431.4, and 26,071.1 that may be related to the regeneration of articular cartilage appeared in the test group.ConclusionsIn the process of inducing the regeneration of articular cartilage using decalcified bone transplantation, knee joint synovial fluid produced specific proteins, which may play an important role in the regeneration of articular cartilage. These findings may offer novel ideas in laying a foundation for the in-depth study of articular cartilage regeneration.

Identification of Cytoplasmic and Conserved Hypothetical Mycobacterium tuberculosis proteins based on mass spectrometry and iedb analysis resource as potent biomarkers for therapeutic strategies or valuable new TB vaccine

Aims and objectives: Tuberculosis (TB) remains a major challenge to public health. The disease burden caused by TB is falling globally, in all WHO regions, and in most countries, but not fast enough to reach the first, 2020, milestones of the End TB Strategy. By 2020, the TB incidence rate needs to be falling at 4–5% per year, and the proportion of people with Tuberculosis who die from the disease needs to fall to 10%. These deaths are in part due to late or missed diagnosis and could be prevented with appropriate treatment. The aim of this study was to identify new relevant cytoplasmic and conserved hypothetical protein biomarkers as TB diagnostic or therapeutic targets. Methods: TB Protein contents were prepared by detergent phase separation method. Two dimensional gel electrophoresis (2DE) was performed using the Ettan IPGphor 3 isoelecteric focusing (GE Healthcare). Comprehensive genomic and proteomic data were retrieved from MycoBrowser portal (http://www.mycobrowser.epfl.ch) followed by matrix assisted laser desorption ionization time-of flight Mass Spectrometry. The predictive of the MHC I peptide binding is carried out by the IEDB analysis resource (http://www.iedb.org). Results: At least six purified proteins with assigned putative functions were determined that corresponded to aldehyde dehydrogenase (Rv0147), iron regulated H-NS (Rv3597c), translocase SecE2 (Rv0379), methyltransferase (Rv3699), adenosylmethionin (Rv1392) and conserved hypothetical protein of Rv0443. Within the category of cytoplasm, membrane and conserved hypothetical patterns, six identified proteins were shown high expression during exponential growth in middlebrook 7H9 broth media. The MHC I peptide binding predictions of these protein spots on the SDS page gels were valued by the Immune Epitope Database tools. The IEDB-AR prediction result is given based on IC50nM value and percentile grad. Principally, a lower score, specified higher affinity. Peptides with IC50 estimation &lt; 50 nM are regarded as high affinity. Conclusions: The output scores of the predicting peptides have been shown strong binding for each of identified peptides. Rank of the predicted output affinity of Rv0443 compared to the two others, Rv0147 and Rv0379, presumably leads to stronger binding affinity. The conserved hypothetical proteins, Rv3699 and Rv0443 displayed significant homology with several other TB proteins. These proteins can also be included in TB vaccine constructs or subunit vaccine mixtures combined with a potent adjuvant. We considered these protein profiles to represent reputed biomarker as a new TB vaccine or therapeutic strategies against TB.

The Alterations in Methylene Blue/Light-Treated Frozen Plasma Proteins Revealed by Proteomics

Introduction: The aim of this study was to investigate the modified proteins in methylene blue/light-treated frozen plasma (MB-FP) compared with fresh frozen plasma (FFP) in order to gain a better application of MB/light-treated plasma in clinic transfusion. Methods: MB-FP and FFP were collected from Changchun central blood station, and a trichloroacetic acid/acetone precipitation method was used to remove albumin for the enrichment of lower abundance proteins. The plasma protein in MB-FP and FFP were separated using two-dimensional gel electrophoresis (2-DE) and the differentially expressed protein spots were analyzed using mass spectrometry. Finally, the differentially expressed proteins were tested using Western blot and enzyme-linked immunosorbent assay (ELISA). Results: Approximately 14 differentially expressed protein spots were detected in the MB-FP, and FFP was chosen as the control. After 2-DE comparison analysis and mass spectrometry, 8 significantly differentially expressed protein spots were identified, corresponding to 6 different proteins, including complement C1r subcomponent (C1R), inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4), keratin, type II cytoskeletal 1 (KRT1), hemopexin (HPX), fibrinogen gamma chain (FGG), and transthyretin (TTR). Western blot showed no significant difference in the expression level of KRT1 between MB-FP and FFP (p > 0.05). Both Western blot and ELISA indicated that the level of HPX was significantly higher in FFP than in MB-FP (p < 0.05). Conclusion: This comparative proteomics study revealed that some significantly modified proteins occur in MB-FP, such as C1R, ITI-H4, KRT1, HPX, FGG, and TTR. Our findings provide more theoretical data for using MB-FP in transfusion medicine. However, the relevance of the data for the transfusion of methylene blue/light-treated plasma remains unclear. The exact modification of these proteins and the effects of these modified proteins on their functions and their effects in clinical plasma infusion need to be further studied.

Differentiating Two Closely Related Alexandrium Species Using Comparative Quantitative Proteomics.

Alexandrium minutum and Alexandrium tamutum are two closely related harmful algal bloom (HAB)-causing species with different toxicity. Using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics and two-dimensional differential gel electrophoresis (2D-DIGE), a comprehensive characterization of the proteomes of A. minutum and A. tamutum was performed to identify the cellular and molecular underpinnings for the dissimilarity between these two species. A total of 1436 proteins and 420 protein spots were identified using iTRAQ-based proteomics and 2D-DIGE, respectively. Both methods revealed little difference (10–12%) between the proteomes of A. minutum and A. tamutum, highlighting that these organisms follow similar cellular and biological processes at the exponential stage. Toxin biosynthetic enzymes were present in both organisms. However, the gonyautoxin-producing A. minutum showed higher levels of osmotic growth proteins, Zn-dependent alcohol dehydrogenase and type-I polyketide synthase compared to the non-toxic A. tamutum. Further, A. tamutum had increased S-adenosylmethionine transferase that may potentially have a negative feedback mechanism to toxin biosynthesis. The complementary proteomics approach provided insights into the biochemistry of these two closely related HAB-causing organisms. The identified proteins are potential biomarkers for organismal toxicity and could be explored for environmental monitoring.

Ultraviolet-B Radiation Stress-Induced Toxicity and Alterations in Proteome of Deinococcus radiodurans

Deinococcus radiodurans is a polyextremophilic bacterium capable to survive and grow at high doses of ionizing radiation. Besides resistance to ionizing radiation, the bacterium is also resistant to toxic chemicals and desiccation. This study deals with the effects of non-ionizing radiation (ultraviolet-B) on survival, alterations in proteomic profile, and gene expression in D. radiodurans. Exposure of culture to UV-B caused decrease in the percentage survival with increasing duration, complete killing occurred after 16 h. D. radiodurans also showed enhancement in the generation of reactive oxygen species and activities of antioxidative enzymes. Separation of proteins by 2-dimensional gel electrophoresis revealed major changes in number and abundance of different proteins. Twenty-eight differentially abundant protein spots were identified by MALDI-TOF MS/MS analysis and divided into 8 groups including unknown proteins. Gene expression of a few identified proteins was also analyzed employing qRT-PCR, which showed differential expression corresponding to the respective proteins. In silico analysis of certain hypothetical proteins (HPs) suggested that these are novel and as yet not reported from D. radiodurans subjected to UV-B stress. These HPs may prove useful in future studies especially for assessing their significance in the adaptation and management of stress responses against UV-B stress.

Identification of novel fibronectin-binding proteins by 2D-far Western blot in atypical enteropathogenic Escherichia coli serotype O55:H7

Atypical enteropathogenic Escherichia coli (aEPEC) is a subgroup of EPEC, which is one of the major pathogens responsible for fatal diarrhoea in children. Compared with typical EPEC (tEPEC), aEPEC lack an EAF (EPEC adherence factor) plasmid (pEAF), which encodes a series of virulence-associated genes. The extracellular matrix (ECM) component of human cells has been reported to be an important element in the interaction between host and bacterial pathogens. In this research, a 2D-Far Western blot method was performed to identifiy the bacterial proteins that could bind to fibronectin, one of the most common constituents of ECM. A total of 17 protein spots were identified, including 4 outer membrane proteins (OMPs), namely, OmpC, OmpD, OmpX and LamB. In vitro studies were used to determine whether these OMPs were involved in the adherence process. Through indirect immunofluorescence assays, four OMPs could be observed on the surfaces of host cells. After incubating the cells with the recombinant proteins, the adhesion rate of the O55:H7 isolate was decreased. Furthermore, the deletion of OmpX and LamB can also decrease the adhesion rate of WT. Taken together, a high-throughput screening method for host ECM-binding proteins based on 2D Far-Western blot was established, and four outer membrane proteins identified by this method were found to be involved in the adherence process.

Physiological and Differential Proteomic Analyses of Imitation Drought Stress Response in Sorghum bicolor Root at the Seedling Stage.

Drought is one of the most important constraints on the growth and productivity of many crops, including sorghum. However, as a primary sensing organ, the plant root response to drought has not been well documented at the proteomic level. In the present study, we compared physiological alteration and differential accumulation of proteins in the roots of sorghum (Sorghum bicolor) inbred line BT×623 response to Polyethylene Glycol (PEG)-induced drought stress at the seedling stage. Drought stress (up to 24 h after PEG treatment) resulted in increased accumulation of reactive oxygen species (ROS) and subsequent lipid peroxidation. The proline content was increased in drought-stressed plants. The physiological mechanism of sorghum root response to drought was attributed to the elimination of harmful free radicals and to the alleviation of oxidative stress via the synergistic action of antioxidant enzymes, such as superoxide dismutase, peroxidase, and polyphenol oxidase. The high-resolution proteome map demonstrated significant variations in about 65 protein spots detected on Coomassie Brilliant Blue-stained 2-DE gels. Of these, 52 protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) representing 49 unique proteins; the levels of 43 protein spots were increased, and 22 were decreased under drought condition. The proteins identified in this study are involved in a variety of cellular functions, including carbohydrate and energy metabolism, antioxidant and defense response, protein synthesis/processing/degradation, transcriptional regulation, amino acid biosynthesis, and nitrogen metabolism, which contribute jointly to the molecular mechanism of outstanding drought tolerance in sorghum plants. Analysis of protein expression patterns and physiological analysis revealed that proteins associated with changes in energy usage; osmotic adjustment; ROS scavenging; and protein synthesis, processing, and proteolysis play important roles in maintaining root growth under drought stress. This study provides new insight for better understanding of the molecular basis of drought stress responses, aiming to improve plant drought tolerance for enhanced yield.

Immunoproteomics approach revealed elevated autoantibody levels against ANXA1 in early stage gallbladder carcinoma

BackgroundEarly diagnosis is important for the timely treatment of gallbladder carcinoma (GBC) patients and may lead to increased survival outcomes. Here, we have applied serological proteome analysis (SERPA), an immunoproteomics approach, for the detection of ‘tumor-associated antigens (TAAs) that elicit humoral response’ in early stage GBC patients.MethodsTotal protein from pooled tumor tissue of GBC patients (n = 7) was resolved by two-dimensional gel electrophoresis (2-DE) followed by immunoblotting using pooled blood plasma from healthy volunteers (n = 11) or gallstone disease (GSD) cases (n = 11) or early stage GBC (Stage I and II) (n = 5) or GBC stage IIIA (n = 9). 2-D gel and immunoblot images were acquired and analyzed using PDQuest software to identify immunoreactive spots in GBC cases in comparison to controls. Proteins from immunoreactive spots were identified by liquid chromatography- tandem mass spectrometric analysis (LC-MS/MS). Autoantibody levels for two of the functionally relevant proteins were investigated in individual plasma samples (52 cases and 89 controls) by dot blot assay using recombinant proteins.ResultsImage analysis using PDQuest software identified 25 protein spots with significantly high or specific immunoreactivity in GBC cases. Mass spectrometric analysis of 8 corresponding protein spots showing intense immunoreactivity (based on densitometric analysis) in early stage GBC or GBC stage IIIA cases led to the identification of 27 proteins. Some of the identified proteins include ANXA1, HSPD1, CA1, CA2, ALDOA and CTSD. Among the two proteins, namely ANXA1 and HSPD1 verified using a cohort of samples, significantly elevated autoantibody levels against ANXA1 were observed in early stage GBC cases in comparison to healthy volunteers or GSD cases (unpaired t-test, p < 0.05). Receiver operating characteristic (ROC) curve analysis for ANXA1 showed an Area under the Curve (AUC) of 0.69, with 41.7% sensitivity against a specificity of 89.9% for early stage GBC. IHC analysis for ANXA1 protein showed ‘high’ expression levels in 72% of GBC cases whereas all the controls showed ‘low’ expression levels.ConclusionsThe study suggests that the ANXA1 autoantibody levels against ANXA1 may be potentially employed for early stage detection of GBC patients. Other proteins could also be explored and verified in a large cohort of clinical samples.

Identification of novel substrates of a disintegrin and metalloprotease 17 by specific labeling of surface proteins

A disintegrin and metalloprotease 17 (ADAM17) catalyzes the cleavage and release of the ectodomains of its substrates at the cell surface in a process termed ectodomain shedding. However, not all ADAM17 substrates have been identified. Here, we used cell surface protein-specific labeling and proteomic approaches to detect and identify ADAM17 substrates. HeLa cell surface proteins were labeled with a fluorescent dye and cultured with or without TAPI-2, an ADAM17 inhibitor. Labeled proteins released into the culture medium were detected by 2-dimensional gel electrophoresis (2DE). Protein spots showing decreased intensity in response to TAPI-2 were selected as substrates of ADAM17 or their binding proteins, and identified by mass spectrometry. ADAM17 knockdown was preformed to examine the behavior of identified proteins. Of 347 proteins detected by 2DE, 49 showed lower intensity in TAPI-2 (+) than in TAPI-2 (-) samples (p < 0.05), and were considered as candidate substrates of ADAM17. Mass spectrometric analysis of 14 protein spots showing >50% decreased intensity identified clusterin as a novel ADAM17 substrate, in addition to known substrates such as desmoglein-2. Western blot analysis showed that ADAM17 knockdown decreased the levels of clusterin fragments cleaved and released from the cell surface. The results identified clusterin as a novel ADAM17 substrate. The method used to identify clusterin could be used to identify the substrates of other sheddases involved in ectodomain shedding.

Comparative proteomic analysis of oil palm (Elaeis guineensis Jacq.) during early fruit development

To gain insights on protein changes in fruit setting and growth in oil palm, a comparative proteomic approach was undertaken to study proteome changes during its early development. The variations in the proteome at five early developmental stages were investigated via a gel-based proteomic technique. A total of 129 variant proteins were determined using mass spectrometric analysis, resulting in 80 identifications. The majority of the identified protein species were classified as energy and metabolism, stress response/defence and cell structure during early oil palm development representing potential candidates for the control of final fruit size and composition. Seven prominent protein species were then characterised using real-time polymerase chain reaction to validate the mRNA expression against the protein abundant profiles. Transcript and protein profiles were parallel across the developmental stages, but divergent expression was observed in one protein spot, indicative of possible post-transcriptional events. Our results revealed protein changes in early oil palm fruit development provide valuable information in the understanding of fruit growth and metabolism during early stages that may contribute towards improving agronomic traits. Biological significanceTwo-dimensional gel electrophoresis coupled with mass spectrometry approach was used in this study to identify differentially expressed proteins during early oil palm fruit development. A total of 80 protein spots with significant change in abundance were successfully identified and selected genes were analysed using real time PCR to validate their expression. The dynamic changes in oil palm fruit proteome during early development were mostly active in primary and energy metabolism, stress responses, cell structure and protein metabolism. This study reveals the physiological processes during early oil palm fruit development and provides a reference proteome for further improvements in fruit quality traits.

Identification and differential expression of serotransferrin and apolipoprotein A-I in the plasma of HIV-1 patients treated with first-line antiretroviral therapy

BackgroundPlasma proteins are known to interfere the drug metabolism during therapy. As limited information is available regarding the role of plasma proteins in HIV drug resistance during ART in HIV/AIDS patients, the present study aimed to identify and characterize the differentially expressed plasma proteins in the drug resistant and drug respondent groups of HIV-1 infected patients with > 6 years of first line ART.MethodsFour-drug resistant (treatment failure) and four-drug respondent (treatment responder) patients were selected for plasma proteomic analysis based on viral load and drug resistance associated mutations from a cohort study designed on the first line ART patients who were enrolled in the antiretroviral therapy center, Sarojini Naidu Medical College, Agra, India from December 2009 to November 2016.After depleting high abundant proteins, plasma proteins were resolved using two-dimensional gel electrophoresis on IPG strips, pH range of 3–10. Spots were selected in the gel based on the density of staining which was common in the drug resistant and drug respondent groups separately. The fold change of each spot was calculated using image-J. Each protein spot was identified using the matrix assisted laser desorption/ionization-time of flight/time of flight (MALDI-TOF/TOF) after tryptic digestion. Peptide peaks were identified through flex analysis version 3.3, and a search against a protein data base using the internal Mascot. Gene ontology study was completed through STRING v.11 and Panther15.0.ResultsOut of eight spots from 2D gel samples analyzed by MALDITOF/TOF, two proteins were found to have significant score (> 56) after Flex analysis. These two proteins were identified to be apolipoprotein A1 and serotransferrin. The fold change expression of these two proteins were analyzed in drug resistant and drug respondent group. Apolipoprotein-A1 and serotransferrin were observed to be expressed 1.76 and 1.13-fold more respectively in drug respondent group compared to drug resistant group. The gene ontology analysis revealed the involvement of these two proteins in various important physiological processes.ConclusionApolipoprotein A-I and serotransferrin were found to be expressed more in drug respondent group compared to drug resistant group.

Molecular drug targets for scabies: amedicinal chemistry perspective.

Sarcoptes scabiei is a causative organism for scabies that affects an estimated global population of 300million and remains a disease of significant concern. Recently, a number of potential drug targets were identified for scabies, including hydrolytic enzymes, inactivated paralogues of hydrolytic enzymes, inhibitors of host proteolytic enzymes and other proteins of interest. These discoveries remain confined to academic laboratories and institutions, failing to attract interest from researchers in commercial drug development. Here, we summarize the latest developments in the scabies mite biology andthe drug targets that were subsequently identified, and we propose several peptide and nonpeptide ligands targeting the hot spots for protein-protein interactions. We also identify gaps in the development of ligands as inhibitors or modulators of these macromolecules.

Can Serum Nitrosoproteome Predict Longevity of Aged Women?

Aging is characterized by increase in reactive oxygen (ROS) and nitrogen (RNS) species, key factors of cardiac failure and disuse-induced muscle atrophy. This study focused on serum nitroproteome as a trait of longevity by adopting two complementary gel-based techniques: two-dimensional differential in gel electrophoresis (2-D DIGE) and Nitro-DIGE coupled with mass spectrometry of albumin-depleted serum of aged (A, n = 15) and centenarian (C, n = 15) versus young females (Y, n = 15). Results indicate spots differently expressed in A and C compared to Y and spots changed in A vs. C. Nitro-DIGE revealed nitrosated protein spots in A and C compared to Y and spots changed in A vs. C only (p-value < 0.01). Nitro-proteoforms of alpha-1-antitripsin (SERPINA1), alpha-1-antichimotripsin (SERPINA3), ceruloplasmin (CP), 13 proteoforms of haptoglobin (HP), and inactive glycosyltransferase 25 family member 3 (CERCAM) increased in A vs. Y and C. Conversely, nitrosation levels decreased in C vs. Y and A, for immunoglobulin light chain 1 (IGLC1), serotransferrin (TF), transthyretin (TTR), and vitamin D-binding protein (VDBP). Immunoblottings of alcohol dehydrogenase 5/S-nitrosoglutathione reductase (ADH5/GSNOR) and thioredoxin reductase 1 (TRXR1) indicated lower levels of ADH5 in A vs. Y and C, whereas TRXR1 decreased in A and C in comparison to Y. In conclusion, the study identified putative markers in C of healthy aging and high levels of ADH5/GSNOR that can sustain the denitrosylase activity, promoting longevity.

Differential proteome analysis of the leaves of lead hyperaccumulator, Rhoeo discolor (L. Her.) Hance.

The present study investigated Rhoeo discolor (L. Her.) Hance for its ability to accumulate Pb, which is of relevance to phytoremediation applications. Based on this analysis, plants were found to accumulate greater than 10 mg/g (0.1%) of dry weight Pb in the shoots, which classifies the plant a Pb hyperaccumulator. Further, changes in the leaf proteome profiles in response to Pb stress were investigated. Wild-type plants were subjected to a high concentration of Pb(NO3 )2 , and the levels of Pb that accumulated in different plant tissues were determined using atomic absorption spectrophotometry. Using 2D-difference gel electrophoresis, 181 protein spots were detected to be differentially abundant in response to Pb stress and selected spots exhibiting the strongest differential abundance suggested an impairment of the photosynthetic apparatus of the plant under Pb stress. Subsequently, a more extensive, proteome wide analysis utilizing label-free quantitation further identified a predominant decrease in protein levels and a significant effect on the nuclear proteome, as well as photosynthesis, carbon fixation and metabolism, providing insight into the Pb tolerance of this system in a potential phytoremediation context.

Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the β-Subunit of Acid Ceramidase.

A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency. Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively. Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung > heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the β-subunit of aCDase, which consists of α- and β-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ~56 kDa and ~13 kDa and the β-subunit at ~43 kDa, the purified SM deacylase was detectable only by the antibody to the β-subunit at ~43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading β-subunit that evokes the ceramide deficiency in AD skin.

Exploiting the potential of 2D DIGE and 2DE immunoblotting for comparative analysis of crude extract of Trichinella britovi and Trichinella spiralis muscle larvae proteomes

The Trichinella genus poses an interesting puzzle for researchers, having diverged very early in the evolution of the nematodes. The Trichinella spiralis proteome is a cosmopolitan and well-studied model of Trichinella; however, Trichinella britovi also circulates in the sylvatic environment and both species infect humans, resulting in the development of trichinellosis. Few experiments have examined the proteins belonging to the T. britovi proteome. The aim of the present study was to compare the protein expression profiles of crude extracts of T. spiralis and T. britovi muscle larvae using a highly-sensitive two-dimensional differential in-gel electrophoresis (2D DIGE) technique coupled with 2DE immunoblotting. Selected immunoreactive protein spots were then identified by liquid chromatography coupled with mass spectrometry analysis (LC–MS/MS), and their function in Trichinella and the host-parasite interaction was determined by gene ontology analysis. Spots common to both T. spiralis and T. britovi, spots with different expressions between the two and spots specific to each species were labelled with different cyanine dyes. In total, 196 protein spots were found in both proteomes; of these 165 were common, 23 expressed exclusively in T. spiralis and 8 in T. britovi. A comparative analysis of volume ratio values with Melanie software showed that among the common spots, nine demonstrated higher expression in T. spiralis, and 17 in T. britovi. LC–MS/MS analysis of 11 selected spots identified 41 proteins with potential antigenic characteristics: 26 were specific for T. spiralis, six for T. britovi, and eight were found in both proteomes. Gene Ontology analysis showed that the identified T. spiralis proteins possess hydrolytic endopeptidase, endonuclease and transferase activities. Similarly, most of the T. britovi proteins possess catalytic activities, such as lyase, hydrolase, isomerase and peptidase activity. The applied 2D DIGE technique visualized Trichinella spp. protein spots with different molecular weights or isoelectric point values, as well as those with different expression levels. The identified immunoreactive proteins participate in multiple processes associated with host muscle cell invasion and larval adaptation to the host environment. Their reactivity with the host immune system makes them possible candidates for the development of a novel trichinellosis diagnostic test or vaccine against helminthiasis caused by T. spiralis or T. britovi.