RELATIVE QUANTITATION OF PEPTIDES BY INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY

Currently, the detection/quantitation of biomolecules using Inductively Coupled Plasma Mass Spectrometry (ICR-MS) is focused on heteroatom containing (e.g. S. P, Se, Cu) compounds. This limits the scope of ICP-MS as a routine analytical tool in the bioanalytical sciences. To increase this scope novel strategies to incorporate elemental labels have been developed. Chemical derivatisation is one such strategy. Cyclic diethylenetriaminepentaacetic acid anhydride (cDTPA), a bifunctional chelating agent (BFCA), is reacted with N-terminal amines and is subsequently chelated with trivalent metals. This allows the detection of peptides that contained no heteroatom using liquid chromatography (LC)-ICP-MS. The derivatisation efficiency, optimised using LC-UV/vis detection, is approximately 99%. The derivatisation and chelation reactions products are characterised using LC-electrospray ionisation tandem mass spectrometry (ESI-MS-MS). The labelling procedure allowed the relative quantitation of peptides, by differential isotopic labelling. Two individually derivatised peptide samples are chelated with natural isotopic abundance and isotopically enriched 151Eu respectively. The Eu labelled peptides were combined and analysed by LC-ICP-MS. The resulting 153EU:151EU ratio, measured using the pseudo steady state approach for transient signals; is then used to calculate the original peptide proportions using a modified isotope dilution equation. The ICP-MS measured peptide ratio was within 2.8% of the theoretical peptide ratio. The absolute detection limit for the relative quantitation of Eu labelled bradykinin was 5 pmol, which is comparable to current ESI-MS methods. When cDTPA derivatisation was applied to a complex sample multiple by-products were observed in the LC-UV/vis chromatogram. However, the corresponding LC-ICP-MS chromatogram suggested that only singly derivatised peptides were chelated with the metal. To overcome by-product formation monoreactive BFCAs, namely isothiocyanate-benzyl- EDTA (SCN-Bz-EDTA) and isothiocyanate-benzyl-DTPA (SCN-Bz-DTPA). were evaluated as derivatisation reagents. Solid phase analytical derivatisation (SPAD) and solid phase extraction (SPE) were also evaluated for speed and ease of use. The monoreactive nature of SCN-Bz-EDTA and SCN-Bz-DTPA gave singly derivatised peptides when applied to complex samples. However, the resulting derivatisation efficiency was low and no significant improvement in efficiency was noted when SPAD was used. SCN-Bz-EDTA derivatised peptides could not be chelated to trivalent metals, whilst chelation occurred with SCN-Bz-DTPA derivatised peptides. Eu labelled peptides were isolated and selectively extracted by SPE for relative quantitation by LC-ICP-MS. However, due to the instability of the isothiocyanate reagent and instrumental effects the relative error on the measured peptide ratio was greater than 45% when compared to the theoretical ratio. Although the peptide ratio obtained using the SPE method agreed with the ratio from the non extracted sample

Identification and Parsing of SQL Query Command by Implementation of JSON

In today’s world mobile devices have become a necessity for many people. These devices have ability to keep in touch with family members and business partners. It is also used to share information in real time.But some time people don’t like to input text on a small mobile device which may lead to error, compared to text input via full-sized keyboard on desktop computer system. In this paper we are going to present how SQL database query can be input using voice and technically parsed using JSON data storage. Here we use Pocketsphinx for Android and Google voice API to convert speech to text and create database query by processing text which return SQL query by processing JSON

Complex Multifunctional Polymer/Carbon-Nanotube Composites

A methodology for developing complex multifunctional materials that consist of or contain polymer/carbon-nanotube composites has been conceived. As used here, "multifunctional" signifies having additional and/or enhanced physical properties that polymers or polymer-matrix composites would not ordinarily be expected to have. Such properties include useful amounts of electrical conductivity, increased thermal conductivity, and/or increased strength. In the present methodology, these properties are imparted to a given composite through the choice and processing of its polymeric and CNT constituents

MQTT and ROC Based Hybrid Robot as a Service (RaaS) Platform

Robots are rapidly evolving from factory work-horses to robot-companions. The future of robots will be as companions in the workplace functioning as interactive salespeople. In order to support this transition, it is important to combine service-oriented architecture and robotics. Service-oriented architecture and cloud computing have become dominant computing paradigms, and adding an RaaS (Robot as a Service) unit as a part of this system will help the companies manage and develop robots more efficiently. The major components of RaaS will be the integration of RMS (Robot Management System) and ROC (Robot Operation Center). As more and more robots are increasing in the service industry, the inter-robot communication is very critical. This communication can be achieved by ROC and the robots can be monitored remotely or locally via RMS. The RaaS platform will comply with all the standards of SOA (Service Oriented Architecture) like the development platform and execution unit, thereby creating a flexible and more development-friendly process

Antioxidative and physiological studies onColocasia esculentum in response to arsenic stress

The current study was undertaken to determine the effects of arsenic on Colocasia esculentum. Rhizomes were grown in pots containing 2.5 kg of garden soil with increasing concentration of arsenic. Arsenic  accumulation was more in shoots compared to roots at higher concentrations. High arsenic concentration  caused reduction in plant growth along with induction of few antioxidants. C. esculentum has a strong  antioxidative and physiological defense mechanism. Under arsenic stress, an increase in catalase, peoxidase,  few non-enzymatic antioxidants and an induction of few stress induced protein were observed, along with  some anatomical changes in roots. The increase in antioxidant stress enzyme activities in response to arsenic  exposure may be taken as evidence for an enhanced detoxification capacity of C. esculentum, a  herbaceous monocot plant, towards reactive oxygen species (and derivatives) that might be generated in the stressed plants.Key words: Arsenic, Colocasia esculentum, antioxidant, metal, root

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR DETERMINATION OF HYDROCHLOROTHIAZIDE, OLMESARTAN MEDOXOMIL AND THEIR RELATED SUBSTANCES IN COMBINED TABLET DOSAGE FORM

Objective: Development of RP-HPLC method for determination of Hydrochlorothiazide (HCTZ), Olmesartan medoxomil (OLM) and their related substances in combined tablet dosage form and validation of the developed method. Methods: Gradient mobile phase system was used for estimation of drug contents and their related substances. Mobile phase A contained the mixture of Acetonitrile and 15 mM Phosphate buffer (pH adjusted to 3.4 with orthophosphoric acid) in the ratio of 20:80. Mobile phase B contained the same mixture in the ratio of 80:20. Chromatographic separation was carried out at the mobile phase flow rate of 0.8 mL/min using C18 Phenomenax inplace of Enable (250 × 4.6 mm) 5 μm column and detection was made at 254 nm. Results: The linearity of developed method was tested in the range of 62.5-187.5 μg/mL for Hydrochlorothiazide, 100-300 μg/mL for Olmesartan medoxomil, 1-1.8 μg/mL for Hydrochlorothiazide. The % recovery was found to be 99.88-100.67 % (HCTZ), 99.14-99.91 % (OLM), 99.11-100.71% (HCTZ-IMP) and 98.13-100.83% (OLM-IMP). The assay of marketed formulation was found to be 99.78% (HCTZ) and 99.26% (OLM). Conclusion: A simple, precise and accurate RP-HPLC method was developed for determination of Hydrochlorothiazide, Olmesartan medoxomil and their related substances

SYNTHESIS AND ANTIMICROBIAL SCREENING OF SOME IMIDAZOLIDINE DERIVATIVES OF ISONICOTINAMIDE

New series of N (2-alkyl/aryl-5-oxo-imidazolidine-1-yl) isonicotinamide derivatives were synthesized by the reaction of Schiff base with amino acetic acid in the presence of 1:4 dioxane. Synthesized compounds were evaluated for their Anti-bacterial activity against Staphylococcus aureus and Echerichia coli, Antifungal activity against Candida Albicans and Anti-tubercular activity against Mycobacterium tuberculosis. Synthesized compounds show significant activity against bacterial, fungal and mycobacterium strains. Their structures were established on the basis of elemental analysis, IR, 1H NMR and Mass Spectral data

Synergistic Cytotoxic Effect of Busulfan and the PARP Inhibitor Veliparib in Myeloproliferative Neoplasms

ABSTRACT Patients with high-risk myeloproliferative neoplasms (MPNs), and in particular myelofibrosis (MF), can be cured only with allogeneic hematopoietic stem cell transplantation (HSCT). Because MPNs and JAK2V617F-mutated cells show genomic instability, stalled replication forks, and baseline DNA double-strand breaks, DNA repair inhibition with poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors represents a potential novel therapy. Because the alkylating agent busulfan is integral in conditioning regimens for HSCT and leads to stalled replication forks through DNA strand cross-linking, we hypothesized that PARP inhibition with veliparib in combination with busulfan may lead to synergistic cytotoxicity in MPN cells. We first treated 2 MPN cell lines harboring the JAK2V617F mutation (SET2 and HEL) with veliparib at increasing concentrations and measured cell proliferation. SET2 and HEL cells were relatively sensitive to veliparib (IC50 of 11.3 μM and 74.2 μM, respectively). We next treated cells with increasing doses of busulfan in combination with 4 μM veliparib and found that the busulfan IC50 decreased from 27 μM to 4 μM in SET2 cells and from 45.1 μM to 28.1 μM in HEL cells. The mean combination index was .55 for SET2 cells and .40 for HEL cells. Combination treatment of SET2 cells caused G2M arrest in 53% of cells, compared with 30% with veliparib alone and 35% with busulfan alone. G2M arrest was associated with activation of the ATR-Chk1 pathway, as shown by an immunofluorescence assay for phosphorylated Chk1 (p-Chk1). We then tested in vivo the effect of combined low doses of busulfan and veliparib in a JAK2V617F MPN-AML xenotransplant model. Vehicle- and veliparib-treated mice had similar median survival of 39 and 40 days, respectively. Combination treatment increased median survival from 47 days (busulfan alone) to 50 days (P = .02). Finally, we tested the combined effect of busulfan and veliparib on CD34+ cells obtained from the bone marrow or peripheral blood of 5 patients with JAK2V617F-mutated and 2 patients with CALR-mutated MF. MF cells treated with the combination of veliparib and busulfan showed reduced colony formation compared with busulfan alone (87% versus 68%; P = .001). In contrast, treatment of normal CD34+ cells with veliparib did not affect colony growth. Here we show that in vivo confirmation that treatment with the PARP-1 inhibitor veliparib and busulfan results in synergistic cytotoxicity in MPN cells. Our data provide the rationale for testing novel pretransplantation conditioning regimens with combinations of PARP-1 inhibition and reduced doses of alkylators, such as busulfan and melphalan, for high-risk MPNs or MPN-derived acute myelogenous leukemia