The Protein Maker: an automated system for high-throughput parallel purification

The Protein Maker instrument addresses a critical bottleneck in structural genomics by allowing automated purification and buffer testing of multiple protein targets in parallel with a single instrument. Here, the use of this instrument to (i) purify multiple influenza-virus proteins in parallel for crystallization trials and (ii) identify optimal lysis-buffer conditions prior to large-scale protein purification is described

Hyaluronidase of Bloodsucking Insects and Its Enhancing Effect on Leishmania Infection in Mice

Hyaluronidases are enzymes degrading the extracellular matrix of vertebrates. Bloodsucking insects use them to cleave the skin of the host, enlarge the feeding lesion and acquire the blood meal. In addition, resulting fragments of extracellular matrix modulate local immune response of the host, which may positively affect transmission of vector-borne diseases, including leishmaniasis. Leishmaniases are diseases with a wide spectrum of clinical forms, from a relatively mild cutaneous affection to life-threatening visceral disease. Their causative agents, protozoans of the genus Leishmania, are transmitted by phlebotomine sand flies. Sand fly saliva was described to enhance Leishmania infection, but the information about molecules responsible for this exacerbating effect is still very limited. In the present work we demonstrated hyaluronidase activity in salivary glands of various Diptera and in fleas. In addition, we showed that hyaluronidase exacerbates Leishmania lesions in mice and propose that salivary hyaluronidase may facilitate the spread of other vector-borne microorganisms

Genomic organisation of the Mal d 1 gene cluster on linkage group 16 in apple

European populations exhibit progressive sensitisation to food allergens, and apples are one of the foods for which sensitisation is observed most frequently. Apple cultivars vary greatly in their allergenic characteristics, and a better understanding of the genetic basis of low allergenicity may therefore allow allergic individuals to increase their fruit intake. Mal d 1 is considered to be a major apple allergen, and this protein is encoded by the most complex allergen gene family. Not all Mal d 1 members are likely to be involved in allergenicity. Therefore, additional knowledge about the existence and characteristics of the different Mal d 1 genes is required. In the present study, we investigated the genomic organisation of the Mal d 1 gene cluster in linkage group 16 of apple through the sequencing of two bacterial artificial chromosome clones. The results provided new information on the composition of this family with respect to the number and orientation of functional and pseudogenes and their physical distances. The results were compared with the apple and peach genome sequences that have recently been made available. A broad analysis of the whole apple genome revealed the presence of new genes in this family, and a complete list of the observed Mal d 1 genes is supplied. Thus, this study provides an important contribution towards a better understanding of the genetics of the Mal d 1 family and establishes the basis for further research on allelic diversity among cultivars in relation to variation in allergenicity

TANGLE: Two-Level Support Vector Regression Approach for Protein Backbone Torsion Angle Prediction from Primary Sequences

Protein backbone torsion angles (Phi) and (Psi) involve two rotation angles rotating around the Cα-N bond (Phi) and the Cα-C bond (Psi). Due to the planarity of the linked rigid peptide bonds, these two angles can essentially determine the backbone geometry of proteins. Accordingly, the accurate prediction of protein backbone torsion angle from sequence information can assist the prediction of protein structures. In this study, we develop a new approach called TANGLE (Torsion ANGLE predictor) to predict the protein backbone torsion angles from amino acid sequences. TANGLE uses a two-level support vector regression approach to perform real-value torsion angle prediction using a variety of features derived from amino acid sequences, including the evolutionary profiles in the form of position-specific scoring matrices, predicted secondary structure, solvent accessibility and natively disordered region as well as other global sequence features. When evaluated based on a large benchmark dataset of 1,526 non-homologous proteins, the mean absolute errors (MAEs) of the Phi and Psi angle prediction are 27.8° and 44.6°, respectively, which are 1% and 3% respectively lower than that using one of the state-of-the-art prediction tools ANGLOR. Moreover, the prediction of TANGLE is significantly better than a random predictor that was built on the amino acid-specific basis, with the p-value<1.46e-147 and 7.97e-150, respectively by the Wilcoxon signed rank test. As a complementary approach to the current torsion angle prediction algorithms, TANGLE should prove useful in predicting protein structural properties and assisting protein fold recognition by applying the predicted torsion angles as useful restraints. TANGLE is freely accessible at http://sunflower.kuicr.kyoto-u.ac.jp/~sjn/TANGLE/

Salivary Gland Transcriptomes and Proteomes of Phlebotomus tobbi and Phlebotomus sergenti, Vectors of Leishmaniasis

Phlebotomine female sand flies require a blood meal for egg development, and it is during the blood feeding that pathogens can be transmitted to a host. Leishmania parasites are among these pathogens and can cause disfiguring cutaneous or even possibly fatal visceral disease. The Leishmania parasites are deposited into the bite wound along with the sand fly saliva. The components of the saliva have many pharmacologic and immune functions important in blood feeding and disease establishment. In this article, the authors identify and investigate the protein components of saliva of two important vectors of leishmaniasis, Phlebotomus tobbi and P. sergenti, by sequencing the transcriptomes of the salivary glands. We then compared the predicted protein sequences of these salivary proteins to those of other bloodsucking insects to elucidate the similarity in composition, structure, and enzymatic activity. Finally, this descriptive analysis of P. tobbi and P. sergenti transcriptomes can aid future research in identifying molecules for epidemiologic assays and in investigating sand fly-host interactions

Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5–40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins

The RESET project: constructing a European tephra lattice for refined synchronisation of environmental and archaeological events during the last c. 100 ka

This paper introduces the aims and scope of the RESET project (. RESponse of humans to abrupt Environmental Transitions), a programme of research funded by the Natural Environment Research Council (UK) between 2008 and 2013; it also provides the context and rationale for papers included in a special volume of Quaternary Science Reviews that report some of the project's findings. RESET examined the chronological and correlation methods employed to establish causal links between the timing of abrupt environmental transitions (AETs) on the one hand, and of human dispersal and development on the other, with a focus on the Middle and Upper Palaeolithic periods. The period of interest is the Last Glacial cycle and the early Holocene (c. 100-8 ka), during which time a number of pronounced AETs occurred. A long-running topic of debate is the degree to which human history in Europe and the Mediterranean region during the Palaeolithic was shaped by these AETs, but this has proved difficult to assess because of poor dating control. In an attempt to move the science forward, RESET examined the potential that tephra isochrons, and in particular non-visible ash layers (cryptotephras), might offer for synchronising palaeo-records with a greater degree of finesse. New tephrostratigraphical data generated by the project augment previously-established tephra frameworks for the region, and underpin a more evolved tephra 'lattice' that links palaeo-records between Greenland, the European mainland, sub-marine sequences in the Mediterranean and North Africa. The paper also outlines the significance of other contributions to this special volume: collectively, these illustrate how the lattice was constructed, how it links with cognate tephra research in Europe and elsewhere, and how the evidence of tephra isochrons is beginning to challenge long-held views about the impacts of environmental change on humans during the Palaeolithic. © 2015 Elsevier Ltd.RESET was funded through Consortium Grants awarded by the Natural Environment Research Council, UK, to a collaborating team drawn from four institutions: Royal Holloway University of London (grant reference NE/E015905/1), the Natural History Museum, London (NE/E015913/1), Oxford University (NE/E015670/1) and the University of Southampton, including the National Oceanography Centre (NE/01531X/1). The authors also wish to record their deep gratitude to four members of the scientific community who formed a consultative advisory panel during the lifetime of the RESET project: Professor Barbara Wohlfarth (Stockholm University), Professor Jørgen Peder Steffensen (Niels Bohr Institute, Copenhagen), Dr. Martin Street (Romisch-Germanisches Zentralmuseum, Neuwied) and Professor Clive Oppenheimer (Cambridge University). They provided excellent advice at key stages of the work, which we greatly valued. We also thank Jenny Kynaston (Geography Department, Royal Holloway) for construction of several of the figures in this paper, and Debbie Barrett (Elsevier) and Colin Murray Wallace (Editor-in-Chief, QSR) for their considerable assistance in the production of this special volume.Peer Reviewe

Progressive hemorrhage and myotoxicity induced by echis carinatus venom in murine model: neutralization by inhibitor cocktail of n,n,n `,n `-tetrakis (2-pyridylmethyl) ethane-1,2-diamine and silymarin

Viperbite is often associated with severe local toxicity, including progressive hemorrhage and myotoxicity, persistent even after the administration of anti-snake venom (ASV). In the recent past, investigations have revealed the orchestrated actions of Zn2+ metalloproteases (Zn(2+)MPs), phospholipase A(2)s (PLA(2)s) and hyaluronidases (HYs) in the onset and progression of local toxicity from the bitten site. As a consequence, venom researchers and medical practitioners are in deliberate quest of potent molecules alongside ASV to tackle the brutal local manifestations induced by aforesaid venom toxins. Based on these facts, we have demonstrated the protective efficacy of inhibitor cocktail containing equal ratios of N,N,N', N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and silymarin (SLN) against progressive local toxicity induced by Echis carinatus venom (ECV). In our previous study we have shown the inhibitory potentials of TPEN towards Zn(2+)MPs of ECV (IC50: 6.7 mu M). In this study we have evaluated in vitro inhibitory potentials of SLN towards PLA(2)s (IC50: 12.5 mu M) and HYs (IC50: 8 mu M) of ECV in addition to docking studies. Further, we have demonstrated the protection of ECV induced local toxicity with 10 mM inhibitor cocktail following 15, 30 min (for hemorrhage and myotoxicity); 60 min (for hemorrhage alone) of ECV injection in murine model. The histological examination of skin and thigh muscle sections taken out from the site of ECV injection substantiated the overall protection offered by inhibitor cocktail. In conclusion, the protective efficacy of inhibitor cocktail is of high interest and can be administered locally alongside ASV to treat severe local toxicity