Roles of carboxyl-terminal and farnesylated residues in the functions of the large hepatitis delta antigen

The large hepatitis delta antigen (HDAg-L) mediates hepatitis delta virus (HDV) assembly and inhibits HDV RNA replication. Farnesylation of the cysteine residue within the HDAg-L carboxyl terminus is required for both functions. Here, HDAg-L proteins from different HDV genotypes and genotype chimeric proteins were analyzed for their ability to incorporate into virus-like particles (VLPs). Observed differences in efficiency of VLP incorporation could be attributed to genotype-specific differences within the HDAg-L carboxyl terminus. Using a novel assay to quantify the extent of HDAg-L farnesylation, we found that genotype 3 HDAg-L was inefficiently farnesylated when expressed in the absence of the small hepatitis delta antigen (HDAg-S). However, as the intracellular ratio of HDAg-S to HDAg-L was increased, so too was the extent of HDAg-L farnesylation for all three genotypes. Single point mutations within the carboxyl terminus of HDAg-L were screened, and three mutants that severely inhibited assembly without affecting farnesylation were identified. The observed assembly defects persisted under conditions where the mutants were known to have access to the site of VLP assembly. Therefore, the corresponding residues within the wild-type protein are likely required for direct interaction with viral envelope proteins. Finally, it was observed that when HDAg-S was artificially myristoylated, it could efficiently inhibit HDV RNA replication. Hence, a general association with membranes enables HDAg to inhibit replication. In contrast, although myristoylated HDAg-S was incorporated into VLPs far more efficiently than HDAg-S or nonfarnesylated HDAg-L, it was incorporated far less efficiently than wild-type HDAg-L; thus, farnesylation was required for efficient assembly.     

Improving conformational searches by geometric screening

MOTIVATION: Conformational searches in molecular docking are a time-consuming process with wide range of applications. Favorable conformations of the ligands that successfully bind with receptors are sought to form stable ligand-receptor complexes. Usually a large number of conformations are generated and their binding energies are examined. We propose adding a geometric screening phase before an energy minimization procedure so that only conformations that geometrically fit in the binding site will be prompted for energy calculation. RESULTS: Geometric screening can drastically reduce the number of conformations to be examined from millions (or higher) to thousands (or lower). The method can also handle cases when there are more variables than geometric constraints. An early-stage implementation is able to finish the geometric filtering of conformations for molecules with up to nine variables in 1 min. To the best of our knowledge, this is the first time such results are reported deterministically. CONTACT: mzhang@mdanderson.org.     

A monolithic silicon based integrated signal generation and detection system for monitoring DNA hybridisation

The aim of this work was to develop an integrated solution to DNA hybridisation monitoring for diagnostics on a monolithic silicon platform. A fabrication process was developed incorporating a gold initiation electrode patterned directly onto a PIN photodiode detector. Patterned interdigitated type electrodes exhibited the smallest reduction in photodiode sensitivity, therefore these were chosen as the ECL initiator design. A novel DNA hybridisation assay was developed based on the displacement of a partially mismatched complementary strand by a perfectly matched labelled complementary strand. Pre-hybridised thiolated oligonucleotide and unlabelled 25% mismatched oligonucleotide were assembled on the gold initiation electrode. On addition of the labelled perfectly complementary oligonucleotide, the mismatched strands were displaced and a signal was generated. The sensitivity of the photodiode to light emitted at 620 nm, the ruthenium emission wavelength, was determined and subsequently, the diode current response to light generated by flow addition of ruthenium solution was found to be measurable to a concentration of 10 fM. Pre-hybridised duplex DNA, consisting of thiolated oligonucleotide and ruthenium labelled complementary oligonucleotide, was assembled on the gold initiation electrode. The difference between the current measured during flow of buffer and the ECL co-reactant TPA was three orders of magnitude, indicating that DNA assembled on the surface comprised sufficient ruthenium to generate a measurable signal. Finally, the displacement of unlabelled partial mismatch oligonucleotide from the sensor surface was monitored on addition of the ruthenium labelled perfectly complementary oligonucleotide in TPA flow and the measured photodiode current response was up to 50 times greater.     

Prioritizing multiple-use landscapes for conservation: methods for large multi-species planning problems

Across large parts of the world, wildlife has to coexist with human activity in highly modified and fragmented landscapes. Combining concepts from population viability analysis and spatial reserve design, this study develops efficient quantitative methods for identifying conservation core areas at large, even national or continental scales. The proposed methods emphasize long-term population persistence, are applicable to both fragmented and natural landscape structures, and produce a hierarchical zonation of regional conservation priority. The methods are applied to both observational data for threatened butterflies at the scale of Britain and modelled probability of occurrence surfaces for indicator species in part of Australia. In both cases, priority landscapes important for conservation management are identified.     

Diversity within cyanobacterial mat communities in variable salinity meltwater ponds of McMurdo Ice Shelf, Antarctica

This study investigated the diversity of cyanobacterial mat communities of three meltwater ponds--Fresh, Orange and Salt Ponds, south of Bratina Island, McMurdo Ice Shelf, Antarctica. A combined morphological and genetic approach using clone libraries was used to investigate the influence of salinity on cyanobacterial diversity within these ecosystems without prior cultivation or isolation of cyanobacteria. We were able to identify 22 phylotypes belonging to Phormidium sp., Oscillatoria sp. and Lyngbya sp. In addition, we identified Antarctic Nostoc sp., Nodularia sp. and Anabaena sp. from the clone libraries. Fresh (17 phylotypes) and Orange (nine phylotypes) Ponds showed a similar diversity in contrast to that of the hypersaline Salt Pond (five phylotypes), where the diversity within cyanobacterial mats was reduced. Using the comparison of identified phylotypes with existing Antarctic sequence data, it was possible to gain further insight into the different levels of distribution of phylotypes identified in the investigated cyanobacterial mat communities of McMurdo Ice Shelf.     

Characterization of a new syndrome that associates craniosynostosis, delayed fontanel closure, parietal foramina, imperforate anus, and skin eruption: CDAGS

We describe the clinical characterization, molecular analyses, and genetic mapping of a distinct genetic condition characterized by craniosynostosis, delayed closure of the fontanel, cranial defects, clavicular hypoplasia, anal and genitourinary malformations, and skin eruption. We have identified seven patients with this phenotype in four families from different geographic regions and ethnic backgrounds. This is an autosomal recessive condition that brings together apparently opposing pathophysiologic and developmental processes, including accelerated suture closure and delayed ossification. Selected candidate genes--including RUNX2, CBFB, MSX2, ALX4, TWIST1, and RECQL4--were screened for mutations, by direct sequencing of their coding regions, and for microdeletions, by fluorescent in situ hybridization. No mutations or microdeletions were detected in any of the genes analyzed. A genomewide screen yielded the maximum estimated LOD score of +2.38 for markers D22S283 and D22S274 on chromosome 22q12-q13. We hypothesize that the gene defect in this condition causes novel context-dependent dysregulation of multiple signaling pathways, including RUNX2, during osteoblast differentiation and craniofacial morphogenesis.     

Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein

Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5-7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5-7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was apparently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity.     

Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs [corrected

This paper describes a protocol to encapsulate cells in sub-millimeter-sized cylindrical collagen modules and to assemble these modules into a tissue-engineered construct within a continuous loop flow circuit. Modules are fabricated by gelling a solution of collagen, that contains suspended cells, within the lumen of a small-bore polyethylene tube. The tubing is then cut into short lengths using an automated cutter and gently vortexed to remove the cell-containing collagen modules from the tubing lumen. Modules are then randomly assembled into a modular construct by pipetting a suspension of modules into a larger tube that is positioned within a continuous flow circuit. A range of cylinder aspect ratios are achievable; therefore, this method could potentially be used to create short discs or, alternatively, long threads of soft gels, with or without encapsulated cells, for a variety of tissue-engineering applications. Module fabrication requires 1 d and assembly of a modular construct requires 2 h.     

Optimization and use of peptide mass measurement accuracy in shotgun proteomics

Mass spectrometers that provide high mass accuracy such as FT-ICR instruments are increasingly used in proteomic studies. Although the importance of accurately determined molecular masses for the identification of biomolecules is generally accepted, its role in the analysis of shotgun proteomic data has not been thoroughly studied. To gain insight into this role, we used a hybrid linear quadrupole ion trap/FT-ICR (LTQ FT) mass spectrometer for LC-MS/MS analysis of a highly complex peptide mixture derived from a fraction of the yeast proteome. We applied three data-dependent MS/MS acquisition methods. The FT-ICR part of the hybrid mass spectrometer was either not exploited, used only for survey MS scans, or also used for acquiring selected ion monitoring scans to optimize mass accuracy. MS/MS data were assigned with the SEQUEST algorithm, and peptide identifications were validated by estimating the number of incorrect assignments using the composite target/decoy database search strategy. We developed a simple mass calibration strategy exploiting polydimethylcyclosiloxane background ions as calibrant ions. This strategy allowed us to substantially improve mass accuracy without reducing the number of MS/MS spectra acquired in an LC-MS/MS run. The benefits of high mass accuracy were greatest for assigning MS/MS spectra with low signal-to-noise ratios and for assigning phosphopeptides. Confident peptide identification rates from these data sets could be doubled by the use of mass accuracy information. It was also shown that improving mass accuracy at a cost to the MS/MS acquisition rate substantially lowered the sensitivity of LC-MS/MS analyses. The use of FT-ICR selected ion monitoring scans to maximize mass accuracy reduced the number of protein identifications by 40%.     

Seasonal home ranges and fidelity to breeding sites among ringed seals

Population structure and patterns of habitat use among ringed seals (Phoca hispida) are poorly known, in part because seasonal movements have not been adequately documented. We monitored the movements of 98 ringed seals in the Beaufort and Chukchi seas between 1990 and 2006 using three forms of telemetry. In the winter—spring period (when the seals were occupying shorefast ice), we used radio and ultra-sonic tags to track movements above and below the ice, respectively. We used satellite-linked transmitters in summer and fall (when the seals ranged away from their winter sites) to track at-sea movements. In the shorefast ice habitat, the home ranges of 27 adult males ranged from <1 to 13.9 km² (median = 0.628) while the home ranges of 28 adult females ranged from <1 to 27.9 km² (median = 0.652). The 3-dimensional volumes used by 9 seals tracked acoustically under the ice averaged 0.07 (SD = 0.04) km³ for subadults and adult males and 0.13 (SD = 0.04) km³ for adult females. Three of the radio-tracked seals and 9 tracked by satellite ranged up to 1,800 km from their winter/spring home ranges in summer but returned to the same small (1–2 km²) sites during the ice-bound months in the following year. The restricted movements of ringed seals during the ice-bound season—including the breeding season—limits their foraging activities for most of the year and may minimize gene flow within the species.