Grish C. Varshney

Immunosensors for pesticide analysis: antibody production and sensor development.

ABSTRACT:  Immunosensors, a type of affinity biosensor, are based on the binding interactions between an immobilized biomolecule (antibody/antigen) on the electronic transducer surface with the analyte of interest (antigen/antibody), resulting in a detectable signal. The sensor system takes advantage of the high selectivity provided by the molecular recognition characteristic of an antibody, which binds reversibly with a specific antigen. This review article presents the current status of immunosensors, highlighting their potential benefits and limitations for pesticide analysis. The basic criteria for generating specific antibodies against low-molecular-mass pesticides, which are usually nonimmunogenic in nature, are briefly discussed. The article also describes the fundamentals of important transducer technologies and their use in immunosensor development.

Human MUC4 mucin induces ultra-structural changes and tumorigenicity in pancreatic cancer cells.

MUC4 is a type-1 transmembrane glycoprotein and is overexpressed in many carcinomas. It is a heterodimeric protein of 930 kDa, composed of a mucin-type subunit, MUC4α, and a membrane-bound growth factor-like subunit, MUC4β. MUC4 mRNA contains unique 5′ and 3′ coding sequences along with a large variable number of tandem repeat (VNTR) domain of 7–19 kb. A direct association of MUC4 overexpression has been established with the degree of invasiveness and poor prognosis of pancreatic cancer. To understand the precise role of MUC4 in pancreatic cancer, we engineered a MUC4 complementary DNA construct, mini-MUC4, whose deduced protein (320 kDa) is comparable with that of wild-type MUC4 (930 kDa) but represents only 10% of VNTR. Stable ectopic expression of mini-MUC4 in two human pancreatic cancer cell lines, Panc1 and MiaPaCa, showed that MUC4 minigene expression follows a biosynthesis and localisation pattern similar to the wild-type MUC4. Expression of MUC4 resulted in increased growth, motility, and invasiveness of the pancreatic cancer cells in vitro. Ultra-structural examination of MUC4-transfected cells showed the presence of increased number and size of mitochondria. The MUC4-expressing cells also demonstrated an enhanced tumorigenicity in an orthotopic xenograft nude mice model, further supporting a direct role of MUC4 in inducing the cancer properties. In conclusion, our results suggest that MUC4 promotes tumorigenicity and is directly involved in growth and survival of the cancer cells.

IFN-|[gamma]|-induced expression of MUC4 in pancreatic cancer cells is mediated by STAT-1 upregulation: a novel mechanism for IFN-|[gamma]| response

MUC4 is a transmembrane mucin, which is aberrantly expressed in pancreatic adenocarcinoma with no detectable expression in the normal pancreas. Here, we present a novel mechanism of IFN-γ-induced expression of MUC4 in pancreatic cancer cells. Our studies highlight the upregulation of STAT-1 as a basis for MUC4 induction and demonstrate that its activation and upregulation by IFN-γ are two distinct, albeit temporally integrated, signalling events that drive the selective induction of IRF-1 and MUC4, respectively, within a single cell system. The profile of interferon regulatory factor (IRF)-1 gene induction by IFN-γ is consistent with its rapid transactivation by phospho-Y701-STAT-1. In contrast, the induction of the MUC4 mucin gene expression is relatively delayed, and occurs only in response to an increase in STAT-1 expression. A progressive binding of STAT-1 to various γ-interferon-activated sequences (GAS) in the MUC4 promoter is observed in chromatin immunoprecipitation assay, indicating its direct association. Stimulation of STAT-1 expression by double-stranded polynucleotides or ectopic expression is shown to induce MUC4 expression, without Y701 phosphorylation of STAT-1. This effect is abrogated by short interfering RNA (siRNA)-mediated inhibition of STAT-1 expression, supporting further the relevance of STAT-1 in MUC4 regulation. In conclusion, our findings identify a novel mechanism for MUC4 regulation in pancreatic cancer cells and unfold new perspectives on the foundation of IFN-γ-dependent gene regulation.

Hemolytik: a database of experimentally determined hemolytic and non-hemolytic peptides

Hemolytik (http://crdd.osdd.net/raghava/hemolytik/) is a manually curated database of experimentally determined hemolytic and non-hemolytic peptides. Data were compiled from a large number of published research articles and various databases like Antimicrobial Peptide Database, Collection of Anti-microbial Peptides, Dragon Antimicrobial Peptide Database and Swiss-Prot. The current release of Hemolytik database contains ∼3000 entries that include ∼2000 unique peptides whose hemolytic activities were evaluated on erythrocytes isolated from as many as 17 different sources. Each entry in Hemolytik provides comprehensive information about a peptide, like its name, sequence, origin, reported function, property such as chirality, types (linear and cyclic), end modifications as well as details pertaining to its hemolytic activity. In addition, tertiary structure of each peptide has been predicted, and secondary structure states have been assigned. To facilitate the scientific community, a user-friendly interface has been developed with various tools for data searching and analysis. We hope, Hemolytik will be useful for researchers working in the field of designing therapeutic peptides.

HaptenDB: a comprehensive database of haptens, carrier proteins and anti-hapten antibodies

UNLABELLED The key requirement for successful immunochemical assay is the availability of antibodies with high specificity and desired affinity. Small molecules, when used as haptens, are not immunogenic. However, on conjugating with carrier molecule they elicit antibody response. The production of anti-hapten antibodies of desired specificity largely depends on the hapten design (preserving greatly the chemical structure and spatial conformation of target compound), selection of the appropriate carrier protein and the conjugation method. This manuscript describes a curated database HaptenDB, where information is collected from published literature and web resources. The current version of the database has 2021 entries for 1087 haptens and 25 carrier proteins, where each entry provides comprehensive details about (1) nature of the hapten, (2) 2D and 3D structures of haptens, (3) carrier proteins, (4) coupling method, (5) method of anti-hapten antibody production, (6) assay method (used for characterization) and (7) specificities of antibodies. The current version of HaptenDB covers a wide array of haptens including pesticides, herbicides, insecticides, drugs, vitamins, steroids, hormones, toxins, dyes, explosives, etc. It provides internal and external links to various databases/resources to obtain further information about the nature of haptens, carriers and respective antibodies. For structure similarity comparison of haptens, the database also integrates tools like JME Editor and JMOL for sketching, displaying and manipulating hapten 2D/3D structures online. So the database would be of great help in identifying functional group(s) in smaller molecules using antibodies as well as for the development of immunodiagnostics/therapeutics by providing data and procedures available so far for the generation of specific or cross-reactive antibodies. AVAILABILITY HaptenDB is available on http://www.imtech.res.in/raghava/haptendb/ and http://bioinformatics.uams.edu/raghava/haptendb/ (Mirror site).

Truncated hemoglobin, HbN, is post-translationally modified in Mycobacterium tuberculosis and modulates host-pathogen interactions during intracellular infection

Background: Mycobacterium tuberculosis HbN detoxifies nitric oxide and protects its host under nitrosative stress. Results: The HbN remains glycosylated and membrane-localized in M. tuberculosis and modulates host-pathogen interactions. Conclusion: The HbN facilitates intracellular infection and cell survival by evading the immune system of the host. Significance: This study unravels new knowledge about function(s) of HbN in biology and pathogenesis of M. tuberculosis. Mycobacterium tuberculosis (Mtb) is a phenomenally successful human pathogen having evolved mechanisms that allow it to survive within the hazardous environment of macrophages and establish long term, persistent infection in the host against the control of cell-mediated immunity. One such mechanism is mediated by the truncated hemoglobin, HbN, of Mtb that displays a potent O2-dependent nitric oxide dioxygenase activity and protects its host from the toxicity of macrophage-generated nitric oxide (NO). Here we demonstrate for the first time that HbN is post-translationally modified by glycosylation in Mtb and remains localized on the cell membrane and the cell wall. The glycan linkage in the HbN was identified as mannose. The elevated expression of HbN in Mtb and M. smegmatis facilitated their entry within the macrophages as compared with isogenic control cells, and mutation in the glycan linkage of HbN disrupted this effect. Additionally, HbN-expressing cells exhibited higher survival within the THP-1 and mouse peritoneal macrophages, simultaneously increasing the intracellular level of proinflammatory cytokines IL-6 and TNF-α and suppressing the expression of co-stimulatory surface markers CD80 and CD86. These results, thus, suggest the involvement of HbN in modulating the host-pathogen interactions and immune system of the host apart from protecting the bacilli from nitrosative stress inside the activated macrophages, consequently driving cells toward increased infectivity and intracellular survival.

ParaPep: a web resource for experimentally validated antiparasitic peptide sequences and their structures

ParaPep is a repository of antiparasitic peptides, which provides comprehensive information related to experimentally validated antiparasitic peptide sequences and their structures. The data were collected and compiled from published research papers, patents and from various databases. The current release of ParaPep holds 863 entries among which 519 are unique peptides. In addition to peptides having natural amino acids, ParaPep also consists of peptides having d-amino acids and chemically modified residues. In ParaPep, most of the peptides have been evaluated for growth inhibition of various species of Plasmodium, Leishmania and Trypanosoma. We have provided comprehensive information about these peptides that include peptide sequence, chemical modifications, stereochemistry, antiparasitic activity, origin, nature of peptide, assay types, type of parasite, mode of action and hemolytic activity. Structures of peptides consisting of natural, as well as modified amino acids have been determined using state-of-the-art software, PEPstr. To facilitate users, various user-friendly web tools, for data fetching, analysis and browsing, have been integrated. We hope that ParaPep will be advantageous in designing therapeutic peptides against parasitic diseases. Database URL: http://crdd.osdd.net/raghava/parapep/

Identification of Proteins Secreted by Malaria Parasite into Erythrocyte using SVM and PSSM profiles

BackgroundMalaria parasite secretes various proteins in infected RBC for its growth and survival. Thus identification of these secretory proteins is important for developing vaccine/drug against malaria. The existing motif-based methods have got limited success due to lack of universal motif in all secretory proteins of malaria parasite.ResultsIn this study a systematic attempt has been made to develop a general method for predicting secretory proteins of malaria parasite. All models were trained and tested on a non-redundant dataset of 252 secretory and 252 non-secretory proteins. We developed SVM models and achieved maximum MCC 0.72 with 85.65% accuracy and MCC 0.74 with 86.45% accuracy using amino acid and dipeptide composition respectively. SVM models were developed using split-amino acid and split-dipeptide composition and achieved maximum MCC 0.74 with 86.40% accuracy and MCC 0.77 with accuracy 88.22% respectively. In this study, for the first time PSSM profiles obtained from PSI-BLAST, have been used for predicting secretory proteins. We achieved maximum MCC 0.86 with 92.66% accuracy using PSSM based SVM model. All models developed in this study were evaluated using 5-fold cross-validation technique.ConclusionThis study demonstrates that secretory proteins have different residue composition than non-secretory proteins. Thus, it is possible to predict secretory proteins from its residue composition-using machine learning technique. The multiple sequence alignment provides more information than sequence itself. Thus performance of method based on PSSM profile is more accurate than method based on sequence composition. A web server PSEApred has been developed for predicting secretory proteins of malaria parasites,the URL can be found in the Availability and requirements section.

Herceptin Resistance Database for Understanding Mechanism of Resistance in Breast Cancer Patients

Monoclonal antibody Trastuzumab/Herceptin is considered as frontline therapy for Her2-positive breast cancer patients. However, it is not effective against several patients due to acquired or de novo resistance. In last one decade, several assays have been performed to understand the mechanism of Herceptin resistance with/without supplementary drugs. This manuscript describes a database HerceptinR, developed for understanding the mechanism of resistance at genetic level. HerceptinR maintains information about 2500 assays performed against various breast cancer cell lines (BCCs), for improving sensitivity of Herceptin with or without supplementary drugs. In order to understand Herceptin resistance at genetic level, we integrated genomic data of BCCs that include expression, mutations and copy number variations in different cell lines. HerceptinR will play a vital role in i) designing biomarkers to identify patients eligible for Herceptin treatment and ii) identification of appropriate supplementary drug for a particular patient. HerceptinR is available at http://crdd.osdd.net/raghava/herceptinr/.

Secretory nucleoside diphosphate kinases from both intra- and extracellular pathogenic bacteria are functionally indistinguishable.

Nucleoside diphosphate kinase (NDK), responsible for the maintenance of NTP pools, is an ATP-utilizing enzyme secreted by different pathogens. We found that NDK from Salmonella enterica serovar Typhimurium (S. Typhimurium) is also secretory in nature. Secretory NDK is known to play a crucial role in the survival of pathogenic microbes within host cells through their interaction with extracellular ATP. To elucidate this aspect, we assessed the contribution of secretory products containing NDK from intracellular (Mycobacterium tuberculosis and S. Typhimurium) and extracellular (Vibrio cholerae) pathogens to the process of ATP-induced J774 mouse macrophage cell lysis by monitoring lactate dehydrogenase (LDH) release in the culture medium. Compared with an untreated control, our results demonstrate that S. Typhimurium secretory products caused a greater than twofold decrease in LDH release from J774 macrophage cells treated with ATP. Furthermore, the secretory products from an ndk-deleted strain of S. Typhimurium did not display such behaviour. Contrary to this observation, the secretory products containing NDK of V. cholerae were found to be cytotoxic to J774 cells. At the amino acid level, the sequences of both the NDKs (S. Typhimurium and V. cholerae) exhibited 65 % identity, and their biochemical characteristics (autophosphorylation and phosphotransfer activities) were indistinguishable. However, to our surprise, the secretory product of an ndk-deleted strain of S. Typhimurium, when complemented with V. cholerae ndk, was able to prevent ATP-induced cytolysis. Taken together, our results unambiguously imply that the intrinsic properties of secretory NDKs are identical in intra- and extracellular pathogens, irrespective of their mode of manifestation.