Simultaneous detection of 13 allergens in thermally processed food using targeted LC-MS/MS approach

Food allergy is a major concern for public health and food industries. Because of the large numbers of food ingredients to be tested, MS is considered an alternative to existing techniques in terms of high selectivity, sensitivity, and capability to analyze multiple allergens simultaneously. In this study, we developed the method for monitoring significant peptides derived from 13 food allergens (milk, eggs, cod, shrimp, lobster, almonds, brazil nuts, cashew nuts, hazelnuts, walnuts, peanuts, wheat, and soybeans) and evaluated it in thermally processed foods (bread, cookie, fried fish, and frozen pasta). To select significant peptides to monitor, we used a bioinformatics-based approach and experimental confirmatory analysis. It was demonstrated that the developed method could detect target food ingredients from thermally processed foods successfully

The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein-ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community

Kinetic Dissection of the Reaction of Human GDP-l-Fucose Synthase.

GDP-l-fucose is a universal sugar donor for the cellular biosynthesis of l-fucose-containing glycans. Its supply comes primarily from the reaction of GDP-l-fucose synthase (GFS), also known as GDP-4″-keto-6″-deoxy-d-mannose epimerase/reductase. GFS converts GDP-4″-keto-6″-deoxy-d-mannose by epimerization at both C-3″ and C-5″ followed by NADPH-dependent reduction of the carbonyl at C-4″. Here, we report kinetic and structural characterization of human GFS with the aim of dissecting the multistep pathway of the enzymatic reaction. Kinetic isotope effects due to [3″-2H] or [5″-2H] in GDP-4″-keto-6″-deoxy-d-mannose and [4S-2H] in NADPH were masked in the steady-state rate of the wild-type enzyme, indicating that the immediate catalytic steps were not rate-limiting for the overall reaction. An isotope effect, however, appeared with GFS variants defective in catalysis of an elementary step, when the isotope probe for that particular step was used in the reaction (C116S: C-3″ epimerization, [3″-2H]-substrate; Y143F: 4″-keto group reduction, [4S-2H]-NADPH). Evidence from steady-state and transient kinetic studies combined with reaction simulations revealed that GFS uses a random mechanism of substrate binding and product release, with the peculiarity that at saturating conditions of substrate and NADPH, the product dissociation happens from abortive GFS complexes with NADPH/GDP-l-fucose and NADP+/GDP-4″-keto-6″-deoxy-d-mannose and involves GDP-l-fucose release as the rate-determining step. GFS complex structures with NADP+ and NADP+/GDP suggest an induced-fit conformational change required to unbind the GDP moiety for dissociation from the enzyme as the molecular cause of the slow product release. Collectively, these results establish the basic kinetic framework of the GFS reaction, which is critical for understanding this important enzyme mechanistically and in its role as an inhibitor target to control glycan fucosylation in vivo

Assessing the function of chromatin modifying enzymes in medulloblastoma

Medulloblastoma is the most common pediatric brain tumor that arises during infancy and childhood and is a major cause of cancer related-morbidity and mortality in children. Recently, medulloblastomas are described as four distinct molecular subgroups (Wnt, sonic hedgehog, Group 3 and Group 4), which have distinct transcriptional, cytogenetic, and mutational spectra

Examining the role of chromatin modifying enzymes in medulloblastoma by utilizing a chemical library

Medulloblastoma (MB) is the most common paediatric brain tumor that arises during infancy and childhood and is a major cause of cancer related-morbidity and mortality in children. Recently, medulloblastomas are described as four distinct molecular subgroups (Wnt, sonic hedgehog, Group 3 and Group 4), which have distinct transcriptional, cytogenetic, and mutational spectra. Next-generation studies have revealed that adult medulloblastomas involve remarkably more somatic SNVs and indels than paediatric counterparts, suggesting that epigenetic deregulation might have a foremost role in the initiation and progression of paediatric medulloblastomas

Virtual fragment screening identification of a novel Quinoline-5,8-dicarboxylic acid derivative as selective JMJD3 inhibitor.

The quinoline-5,8 dicarboxylic acid scaffold has been identified by fragment-based approach as new potential lead compound for the development of JMJD3 inhibitors. Among them, compound 3 shows low micromolar inhibitory activity against JMJD3. The experimental evaluation of inhibition activity vs. related seven isoforms of JMJD3 highlighted an unprecedented selectivity towards the biological target of interest

Periostin expression in neoplastic and non-neoplastic diseases of bone and joint

Abstract Background Periostin is a matricellular protein that is expressed in bone and joint tissues. To determine the expression of periostin in primary bone tumours and to assess whether it plays a role in tumour progression, we carried out immunohistochemistry and ELISA for periostin in a range of neoplastic and non-neoplastic bone and joint lesions. Methods 140 formalin-fixed paraffin-embedded sections of bone tumours and tumour-like lesions were stained by an indirect immunoperoxidase technique with a polyclonal anti-periostin antibody. Periostin expression was also assessed in rheumatoid arthritis (RA) and non-inflammatory osteoarthritis (OA) synovium and synovial fluid immunohistochemistry and ELISA respectively. Results Periostin was most strongly expressed in osteoid/woven bone of neoplastic and non-neoplastic bone-forming lesions, including osteoblastoma, osteosarcoma, fibrous dysplasia, osteofibrous dysplasia, fracture callus and myositis ossificans, and mineralised chondroid matrix/woven bone in chondroblastoma and clear cell chondrosarcoma. Reactive host bone at the edge of growing tumours, particularly in areas of increased vascularity and fibrosis, also stained strongly for periostin. Vascular elements in RA synovium strongly expressed periostin, and synovial fluid levels of periostin were higher in RA than OA. Conclusions In keeping with its known role in modulating the synthesis of collagen and other extracellular matrix proteins in bone, strong periostin expression was noted in benign and malignant lesions forming an osteoid or osteoid-like matrix. Periostin was also noted in other bone tumours and was found in areas of reactive bone and increased vascularity at the edge of growing tumours, consistent with its involvement in tissue remodelling and angiogenesis associated with tumour progression

Advances and challenges in understanding histone demethylase biology

Within the last decade we have witnessed significant progress in the field of chromatin methylation, ranging from the discovery that chromatin methylation is reversible, to the identification of two classes of oxidative chromatin demethylases. Multiple genetic and cellular studies emphasize the role of members of the amine oxidase and 2-oxoglutarate oxygenase enzyme families involved in methyl-lysine in physiology and disease. Advances in understanding of the underlying biochemistry have resulted in development of first series of clinical inhibitors and tool compounds which continue to resolve and help understand the complex relationships between chromatin modification, control of gene expression and metabolic states

Linker histone H1.2 directs genome-wide chromatin association of the retinoblastoma tumor suppressor protein and facilitates its function

The retinoblastoma tumor suppressor protein pRb is a master regulator of cellular proliferation, principally through interaction with E2F and regulation of E2F target genes. Here, we describe the H1.2 linker histone as a major pRb interaction partner. We establish that H1.2 and pRb are found in a chromatin-bound complex on diverse E2F target genes. Interrogating the global influence of H1.2 on the genome-wide distribution of pRb indicated that the E2F target genes affected by H1.2 are functionally linked to cell-cycle control, consistent with the ability of H1.2 to hinder cell proliferation and the elevated levels of chromatin-bound H1-pRb complex, which occur in growth-arrested cells. Our results define a network of E2F target genes as susceptible to the regulatory influence of H1.2, where H1.2 augments global association of pRb with chromatin, enhances transcriptional repression by pRb, and facilitates pRb-dependent cell-cycle arrest