A single dose of SAAVI MVA-C reboosts rhesus macaques after more than 3 years post DNA-MVA prime-boost vaccination

We have previously reported induction of robust immune responses in rhesus macaques following a prime boost immunization with candidate HIV-1 vaccines, SAAVI DNA-C (DNA) and SAAVI MVA-C (MVA). These vaccines are already in clinical evaluation. In the current study, we investigated whether re-boosting these animals with a single MVA inoculation after more than 3 years was sufficient to restore previous magnitudes of HIV-specific immune responses

The Yeast Resource Center Public Image Repository: A large database of fluorescence microscopy images

<p>Abstract</p> <p>Background</p> <p>There is increasing interest in the development of computational methods to analyze fluorescent microscopy images and enable automated large-scale analysis of the subcellular localization of proteins. Determining the subcellular localization is an integral part of identifying a protein's function, and the application of bioinformatics to this problem provides a valuable tool for the annotation of proteomes. Training and validating algorithms used in image analysis research typically rely on large sets of image data, and would benefit from a large, well-annotated and highly-available database of images and associated metadata.</p> <p>Description</p> <p>The Yeast Resource Center Public Image Repository (YRC PIR) is a large database of images depicting the subcellular localization and colocalization of proteins. Designed especially for computational biologists who need large numbers of images, the YRC PIR contains 532,182 TIFF images from nearly 85,000 separate experiments and their associated experimental data. All images and associated data are searchable, and the results browsable, through an intuitive web interface. Search results, experiments, individual images or the entire dataset may be downloaded as standards-compliant OME-TIFF data.</p> <p>Conclusions</p> <p>The YRC PIR is a powerful resource for researchers to find, view, and download many images and associated metadata depicting the subcellular localization and colocalization of proteins, or classes of proteins, in a standards-compliant format. The YRC PIR is freely available at <url>http://images.yeastrc.org/</url>.</p

High-throughput assay for determining enantiomeric excess of chiral diols, amino alcohols, and amines and for direct asymmetric reaction screening

Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters. The method is based on dynamic self-assembly of commercially available chiral amines, 2-formylphenylboronic acid, and chiral diols in acetonitrile to form fluorescent diastereomeric complexes. Each analyte enantiomer engenders a diastereomer with distinct fluorescence wavelength/intensity originating from enantiopure fluorescent ligands. In this assay, enantiomers of amines and amine derivatives assemble with diol-type ligands containing a binaphthol moiety (BINOL and VANOL), whereas diol enantiomers form complexes with the enantiopure amine-type fluorescent ligand tryptophanol. The differential fluorescence is utilized to determine the amount of each enantiomer in the mixture with an error of &lt;1% e.e. This method enables high-throughput real-time evaluation of enantiomeric/diastereomeric excess (e.e./d.e.) and product yield of crude asymmetric reaction products. The procedure comprises high-throughput liquid dispensing of three components into 384-well plates and recording of fluorescence using an automated plate reader. The approach enables scaling up the screening of combinatorial libraries and, together with parallel synthesis, creates a robust platform for discovering chiral catalysts or auxiliaries for asymmetric transformations and chiral drug development. The procedure takes ~4–6 h and requires 10–20 ng of substrate per well. Our fluorescence-based assay offers distinct advantages over existing methods because it is not sensitive to the presence of common additives/impurities or unreacted/incompletely utilized reagents or catalysts.</p

Analysis of Two Novel Midgut-Specific Promoters Driving Transgene Expression in Anopheles stephensi Mosquitoes

Background: Tissue-specific promoters controlling the expression of transgenes in Anopheles mosquitoes represent a valuable tool both for studying the interaction between these malaria vectors and the Plasmodium parasites they transmit and for novel malaria control strategies based on developing Plasmodium-refractory mosquitoes by expressing anti-parasitic genes. With this aim we have studied the promoter regions of two genes from the most important malaria vector, Anopheles gambiae, whose expression is strongly induced upon blood feeding. Results: We analysed the A. gambiae Antryp1 and G12 genes, which we have shown to be midgut-specific and maximally expressed at 24 hours post-bloodmeal (PBM). Antryp1, required for bloodmeal digestion, encodes one member of a family of 7 trypsin genes. The G12 gene, of unknown function, was previously identified in our laboratory in a screen for genes induced in response to a bloodmeal. We fused 1.1 kb of the upstream regions containing the putative promoter of these genes to reporter genes and transformed these into the Indian malaria vector A. stephensi to see if we could recapitulate the expression pattern of the endogenous genes. Both the Antryp1 and G12 upstream regions were able to drive femalepredominant, midgut-specific expression in transgenic mosquitoes. Expression of the Antryp1-driven reporter in transgenic A. stephensi lines was low, undetectable by northern blot analysis, and failed to fully match the induction kinetics of the endogenous Antryp1 gene in A. gambiae. This incomplete conservation of expression suggests either subtle differences i

Age and gender differences in disabling foot pain using different definitions of the manchester foot pain and disability index

Extent: 9p.Background: The Manchester Foot Pain and Disability Index (MFPDI) has been used to determine the prevalence of disabling foot pain in several studies, however there is some debate as to which case definition is most appropriate. The objective of this study was to explore age and gender differences in the proportion of people with disabling foot pain using three different case definitions of the MFPDI and for each individual MFPDI item. Methods: A random sample of 223 participants aged 27 to 90 years (88 males and 135 females) from the North West Adelaide Health Study, who reported having pain, aching or stiffness in either of their feet on most days in the last month, completed the MFPDI by telephone interview. The proportion of people with disabling foot pain was determined using three definitions: (i) Definition A-at least one of the 17 items documented on at least some days in the last month; (ii) Definition B-at least one of the 17 items documented on most/every day(s) in the last month, and; (iii) Definition C-at least one of the ten functional limitation items documented on most/every day(s) in the last month. Cross-tabulations and chi-squared statistics were used to explore differences in responses to the MFPDI items according to age and gender. Results: The proportion of people with disabling foot pain according to each definition was as follows: Definition A (100%), Definition B (95.1%) and Definition C (77.6%). Definition C was most sensitive to age and gender differences. Exploration of individual MFPDI items indicated that age significantly affected both the pain intensity and functional limitation items, with younger people more likely to report their foot pain being worse in the morning, and older people more likely to report functional limitations. Although gender did not influence responses to the personal appearance items, women were more likely report functional limitations than men. Conclusions: Definition C of the MFPDI is more sensitive to age and gender differences in the proportion of people with disabling foot pain, and would therefore seem to be the most appropriate case definition to use in epidemiological studies involving a broad age range of participants.Hylton B Menz, Tiffany K Gill, Anne W Taylor and Catherine L Hil

Insights into the Mechanism of Bovine CD38/NAD+Glycohydrolase from the X-Ray Structures of Its Michaelis Complex and Covalently-Trapped Intermediates

Bovine CD38/NAD+glycohydrolase (bCD38) catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2′-fluorinated analogs of NAD+. Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1′ of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2′-OH of the substrate NAD+. Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism

Fin-Tail Coordination during Escape and Predatory Behavior in Larval Zebrafish

Larval zebrafish innately perform a suite of behaviors that are tightly linked to their evolutionary past, notably escape from threatening stimuli and pursuit and capture of prey. These behaviors have been carefully examined in the past, but mostly with regard to the movements of the trunk and tail of the larvae. Here, we employ kinematics analyses to describe the movements of the pectoral fins during escape and predatory behavior. In accord with previous studies, we find roles for the pectoral fins in slow swimming and immediately after striking prey. We find novel roles for the pectoral fins in long-latency, but not in short-latency C-bends. We also observe fin movements that occur during orienting J-turns and S-starts that drive high-velocity predatory strikes. Finally, we find that the use of pectoral fins following a predatory strike is scaled to the velocity of the strike, supporting a role for the fins in braking. The implications of these results for central control of coordinated movements are discussed, and we hope that these results will provide baselines for future analyses of cross-body coordination using mutants, morphants, and transgenic approaches

Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability

Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu–Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de-novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized

Long Distance Dispersal and Connectivity in Amphi-Atlantic Corals at Regional and Basin Scales

Among Atlantic scleractinian corals, species diversity is highest in the Caribbean, but low diversity and high endemism are observed in various peripheral populations in central and eastern Atlantic islands and along the coasts of Brazil and West Africa. The degree of connectivity between these distantly separated populations is of interest because it provides insight into processes at both evolutionary and ecological time scales, such as speciation, recruitment dynamics and the persistence of coral populations. To assess connectivity in broadly distributed coral species of the Atlantic, DNA sequence data from two nuclear markers were obtained for six coral species spanning their distributional ranges. At basin-wide scales, significant differentiation was generally observed among populations in the Caribbean, Brazil and West Africa. Concordance of patterns in connectivity among co-distributed taxa indicates that extrinsic barriers, such as the Amazon freshwater plume or long stretches of open ocean, restrict dispersal of coral larvae from region to region. Within regions, dispersal ability appears to be influenced by aspects of reproduction and life history. Two broadcasting species, Siderastrea siderea and Montastraea cavernosa, were able to maintain gene flow among populations separated by as much as 1,200 km along the coast of Brazil. In contrast, brooding species, such as Favia gravida and Siderastrea radians, had more restricted gene flow along the Brazilian coast