NMR structure shows that the SARS-unique domain contains a macrodomain fold

The NMR structure of a central segment of the previously annotated "SARS-unique domain" (SUD-M; "middle of the SARS-unique domain") in the SARS coronavirus (SARS-CoV) non-structural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3-residues 528-648, and there is a flexibly extended N-terminal tail with the residues 513-527 and a C-terminal flexible tail of residues 649-651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527-651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly-A and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1''-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows 3D structure homology with several helicases and NTP-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection.

Interaction of severe acute respiratory syndrome-coronavirus and NL63 coronavirus spike proteins with angiotensin converting enzyme-2

Although in different groups, the coronaviruses severe acute respiratory syndrome-coronavirus (SARS-CoV) and NL63 use the same receptor, angiotensin converting enzyme (ACE)-2, for entry into the host cell. Despite this common receptor, the consequence of entry is very different; severe respiratory distress in the case of SARS-CoV but frequently only a mild respiratory infection for NL63. Using a wholly recombinant system, we have investigated the ability of each virus receptor-binding protein, spike or S protein, to bind to ACE-2 in solution and on the cell surface. In both assays, we find that the NL63 S protein has a weaker interaction with ACE-2 than the SARS-CoV S protein, particularly in solution binding, but the residues required for contact are similar. We also confirm that the ACE-2-binding site of NL63 S lies between residues 190 and 739. A lower-affinity interaction with ACE-2 might partly explain the different pathological consequences of infection by SARS-CoV and NL63.

Ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the N-terminal domain of N protein

Conserved among all coronaviruses are four structural proteins: the matrix (M), small envelope (E), and spike (S) proteins that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in the lumen. The N-terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding, while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C terminus of the N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17 A (monoclinic) and at 1.85 A (cubic), respectively, resolved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core, is oriented similarly to that in the IBV N-NTD, and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggests a common mode of RNA recognition, but they probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs suggests that they use different modes of both RNA recognition and oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

Nuclear magnetic resonance structure of the N-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus

This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four beta-strands and two alpha-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.

Knowledge transfer processes in PFI/PPP: critical success factors

Successful knowledge transfer is an important process which requires continuous improvement in today’s knowledge-intensive economy. However, improving knowledge transfer processes represents a challenge for construction practitioners due to the complexity of knowledge acquisition, codification and sharing. Although knowledge transfer is context based, understanding the critical success factors can lead to improvements in the transfer process. This paper seeks to identify and evaluate the most significant critical factors for improving knowledge transfer processes in Public Private Partnerships/Private Finance Initiatives (PPP/PFI) projects. Drawing upon a questionnaire survey of 52 construction firms located in the UK, data is analysed using Severity Index (SI) and Coefficient of Variation (COV), to examine and identify these factors in PPP/PFI schemes. The findings suggest that a supportive leadership, participation/commitment from the relevant parties, and good communication between the relevant parties are crucial to improving knowledge transfer processes in PFI schemes. Practitioners, managers and researchers can use the findings to efficiently design performance measures for analysing and improving knowledge transfer processes.

Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles

Coronaviruses (CoV), like other positive-stranded RNA viruses, redirect and rearrange host cell membranes for use as part of the viral genome replication and transcription machinery. Specifically, coronaviruses induce the formation of double-membrane vesicles in infected cells. Although these double-membrane vesicles have been well characterized, the mechanism behind their formation remains unclear, including which viral proteins are responsible. Here, we use transfection of plasmid constructs encoding full-length versions of the three transmembrane-containing nonstructural proteins (nsps) of the severe acute respiratory syndrome (SARS) coronavirus to examine the ability of each to induce double-membrane vesicles in tissue culture. nsp3 has membrane disordering and proliferation ability, both in its full-length form and in a C-terminal-truncated form. nsp3 and nsp4 working together have the ability to pair membranes. nsp6 has membrane proliferation ability as well, inducing perinuclear vesicles localized around the microtubule organizing center. Together, nsp3, nsp4, and nsp6 have the ability to induce double-membrane vesicles that are similar to those observed in SARS coronavirus-infected cells. This activity appears to require the full-length form of nsp3 for action, as double-membrane vesicles were not seen in cells coexpressing the C-terminal truncation nsp3 with nsp4 and nsp6. IMPORTANCE Although the majority of infections caused by coronaviruses in humans are relatively mild, the SARS outbreak of 2002 to 2003 and the emergence of the human coronavirus Middle Eastern respiratory syndrome (MERS-CoV) in 2012 highlight the ability of these viruses to cause severe pathology and fatality. Insight into the molecular biology of how coronaviruses take over the host cell is critical for a full understanding of any known and possible future outbreaks caused by these viruses. Additionally, since membrane rearrangement is a tactic used by all known positive-sense single-stranded RNA viruses, this work adds to that body of knowledge and may prove beneficial in the development of future therapies not only for human coronavirus infections but for other pathogens as well.

Atlas of coronavirus replicase structure

The international response to SARS-CoV has produced an outstanding number of protein structures in a very short time. This review summarizes the findings of functional and structural studies including those derived from cryoelectron microscopy, small angle X-ray scattering, NMR spectroscopy, and X-ray crystallography, and incorporates bioinformatics predictions where no structural data is available. Structures that shed light on the function and biological roles of the proteins in viral replication and pathogenesis are highlighted. The high percentage of novel protein folds identified among SARS-CoV proteins is discussed.

Genetic diversity and specialisation of Eudarluca caricis on some graminaceous Puccinia species

Eudarluca caricis is a common hyperparasite of rusts. A total of 100 cultures were isolated from six Puccinia species or forms growing on 10 species of British grasses at two sites approximately 3 km apart. 82 isolates collected in 2005 were partially sequenced at the ITS locus, and amplified fragment length polymorphism profiles generated for 86 isolates from 2005 and 12 from 2007. Partial ITS sequences of most isolates grouped closely, in a clade with previously reported graminaceous Puccinia isolates and a number of Melampsora isolates. A second clade was very distinct and contained mostly isolates from P. poarum on Poa trivialis. All isolates had distinct AFLP haplotypes. The P. poarum isolates were very distinct from isolates collected from other rusts at the same site. Isolates from P. brachypodii f. sp. arrehenatheri growing on Arrhenatherum elatius in 2005 and 2007 at the same location were distinct (P < 0.001). Isolates from each rust or grass in one year and site were more similar than expected from overall variation between isolates (P<0.001). Isolates from P. coronata on different grasses clustered together (with isolates from P. brachypodii f. sp. poae-nemoralis), suggesting partial host rust specialisation in E. caricis.

The proteome of the infectious bronchitis virus Beau-R Virion

Infectious bronchitis is a highly contagious respiratory disease of poultry caused by the coronavirus IBV. It was thought that coronavirus virions were composed of three major viral structural proteins, until investigations of other coronaviruses showed that coronavirus virions also include viral non-structural and group specific proteins as well as host cell proteins. To study the proteome of IBV virions, virus was grown in embryonated chicken eggs and purified by sucrose gradient ultracentrifugation and analysed by mass spectrometry proteomic. Analysis of three preparations of purified IBV yielded the three expected structural proteins plus thirty-five additional virion-associated host proteins. Virion-associated host proteins had a diverse range of functional attributions, being involved in cytoskeleton formation, RNA binding and protein folding pathways. Some of these proteins were unique to this study, whilst others were found to be orthologous to proteins identified in SARS-CoV virions, and also virions from a number of other RNA and DNA viruses. Together these results demonstrate that coronaviruses have the capacity to incorporate a substantial variety of host protein, which may have implications for the disease process.

D.A. Pennebaker: Interviews

This wide-ranging and insightful collection of interviews with D. A. Pennebaker (b. 1925) spans the prolific career of this pioneer of observational cinema. From the 1950s to the present day, D. A. Pennebaker has made documentary films that have revealed the world of politics, celebrity culture, and the music industry. Following his early collaborations with Robert Drew on a number of works for television, his feature-length portrait of Bob Dylan on tour in England in 1965 (the landmark film Dont Look Back) established so-called direct cinema as a form capable of achieving broad theatrical release. With Monterey Pop, Pennebaker inaugurated the popular mode of rock concert film (or "rockumentary"), a style of filmmaking he has expanded on through a number of films, including Ziggy Stardust and the Spiders from Mars and Depeche Mode: 101. Pennebaker has always regarded collaboration as an integral part of his filmmaking methods. His long-running collaboration with Richard Leacock and subsequently his work with Chris Hegedus have enriched his approach and, in the process, have instituted collaboration as a working practice integral to American direct cinema. His other collaborations, in particular, with Jean-Luc Godard and Norman Mailer, resulted in innovative combinations of observational techniques and fictional aesthetics. Such films as The War Room, which was about the 1992 Democratic primaries and was nominated for an Academy Award, and the 2009 Kings of Pastry continue to explore the capacities of observational documentary. In 2012 Pennebaker was the first documentary filmmaker to be awarded an Academy Honorary Award by the Academy of Motion Picture Arts and Sciences.

Middle East respiratory syndrome coronavirus antibody reactors among camels in Dubai, United Arab Emirates, in 2005

We tested, using a low starting dilution, sequential serum samples from dromedary camels, sheep and horses collected in Dubai from February/April to October of 2005 and from dromedary camels for export/import testing between Canada and USA in 2000-2001. Using a standard Middle East respiratory syndrome coronavirus (MERS-CoV) neutralization test, serial sera from three sheep and three horses were all negative while sera from 9 of 11 dromedary camels from Dubai were positive for antibodies supported by similar results in a MERS-CoV recombinant partial spike protein antibody ELISA. The two negative Dubai camels were both dromedary calves and remained negative over the 5 months studied. The six dromedary samples from USA and Canada were negative in both tests. These results support the recent findings that infection with MERS-CoV or a closely related virus is not a new occurrence in camels in the Middle East. Therefore, interactions of MERS-CoV at the human-animal interface may have been ongoing for several, perhaps many, years and by inference, a widespread pandemic may be less likely unless significant evolution of the virus allow accelerated infection and spread potential in the human population.

Remoção de matéria orgânica, de nutrientes e de coliformes no processo anaeróbio em dois estágios (reator compartimentado seguido de reator UASB) para o tratamento de águas residuárias de suinocultura

Funda��ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Concepção e estudo de um biofiltro para tratamento de compostos orgânicos voláteis COVs

In the last decade, biological purification of gaseous waste has become an important alternative to many conventional methods of exhaust air treatment. More recently, biofiltration has proved to be an effective and inexpensive method for the treatment of air contaminated with volatile organic compounds (VOCs). A biofilter consists in a reactor packed with a porous solid bed material, where the microorganisms are fixed. During the biofiltration process, polluted air is transported through the biofilter medium where the contaminant is degraded. Within the biofilm, the pollutants in the waste gases are energy and carbon sources for microbial metabolism and are transformed into CO2, water and biomass. The bed material should be characterized by satisfactory mechanical and physical properties as structure, void fraction, specific area and flow resistance. The aim of this research was the biofilter construction and study of the biological degradation of ethanol and toluene, as well as the modeling of the process. Luffa cylindrica is a brazilian fiber that was used as the filtering material of the present work. The parameters and conditions studied were: composition of nutrients solution; effect of microflorae strains, namely Pseudomanas putida and Rhodococcus rhodochrous; waste gas composition; air flow rate; and inlet load of VOCs. The biofilter operated in diffusion regime and the best results for remotion capacity were obtained when a microorganisms consortion of Pseudomanas putida and Rhodococcus rhodochrous,were used, with a gas flow rate of 1 m3.h-1 and molar ratio nitrogene/phosphore N/P=2 in the nutrients solution. The maximum remotion capacity for ethanol was around 90 g.m-3.h-1 and 50 g.m-3.h-1 to toluene. It was proved that toluene has inhibitory effect on the ethanol remotion When the two VOCs were present in the same waste gas, there was a decrease of 40% in ethanol remotion capacity. Luffa cylindrica does not present considerable pressure drop. Ottengraf and van Lith models were used to represent the results obtained for ethanol and toluene, respectively. The application of the transient model indicated a satisfactory approximation between the experimental results obtained for ethanol and toluene vapors biofiltration and the ones predicted it

Evaluation of development traits of beef buffalo in Brazil using multivariate analysis

The buffalo population in Brazil increased about 12.9% between 1998 and 2003, to 2.8 million head, evidencing the importance of this species for the country. The objective this work was evaluation of animal growth using multivariate analysis. The data were from 2,944 water buffalo from 10 herds raised in pasture conditions in Brazil. Principal components and genetic distances were estimated using proc PRINCOMP and proc CANDISC in SAS (SAS Inst. Inc. Cary, NC, USA). Variables analyzed were birth weight (BW), age at weaning (AW), weaning weight (WT), weight adjusted to 205 d (W205), total gain between BW and WT (TG), daily gain between BW and WT (DG), weight adjusted to 365 d (W365), total gain between WT and W365 (TG3), daily gain between WT and W365 (TGD3), weight adjusted to 550 d (W550) and weight adjusted to 730 d (W730). Means and standard deviations for each variable were 39.4 +/- 3.2 kg, 225.6 +/- 38.8 d, 209.4 +/- 39.4 kg, 195.4 +/- 30.2 kg, 157.4 +/- 32.0 kg, 0.77 +/- 0.16 kg/d, 282.0 +/- 43.5 kg, 73.9 +/- 33.9 kg, 0.53 +/- 0.21 kg/d, 406.8 +/- 67.9 kg, and 468.2 +/- 70.6 kg, respectively. The eigenvalues to four first principal components were 5.29, 2.54, 1.66, 1.01, and justify 48%, 23%, 15% and 9%, respectively, with a total cumulative 95%. We created an index using the first principal component which is Y. 0.0552 BW + 0.0438 AW + 0.3142 WT + 0.3549 W205 + 0.3426 TG + 0.3426 DG + 0.4070 W365- 0.1531 TG3 - 0.2059 TGD3 - 0.3833 W550 - 0.3966 W730. This index accounted for 48% the variation in the correlation matrix. This principal component emphasizes early growth of the animal. Estimates the pair-wise squared distances between herds, D2(i vertical bar j)= ((x) over bar (i)-(x) over bar (j))' cov(-1)((x) over bar (i)-(x) over bar (j)), using with basis the average of weight of animals, showed the largest distance between herds eight (Murrah: DF) and seven (Murrah: Amazon) and the closest distance between herds one (Mediterranean - RS) and five (Jafarabadi - SP).

Adapta��ão transcultural Portugal-Brasil do Inventário de Burnout de Maslach para estudantes

OBJETIVO: Realizar a adapta��ão transcultural da versão em português do Inventário de Burnout de Maslach para estudantes e investigar sua confiabilidade, validade e invariância transcultural. MÉTODOS: A valida��ão de face envolveu participa��ão de equipe multidisciplinar. Foi realizada valida��ão de conteúdo. A versão em português foi preenchida em 2009, pela internet, por 958 estudantes universitários brasileiros e 556 portugueses da zona urbana. Realizou-se análise fatorial confirmatória utilizando-se como índices de ajustamento o χ²/df, o comparative fit index (CFI), goodness of fit index (GFI) e o root mean square error of approximation (RMSEA). Para verifica��ão da estabilidade da solução fatorial conforme a versão original em inglês, realizou-se valida��ão cruzada em 2/3 da amostra total e replicada no 1/3 restante. A validade convergente foi estimada pela variância extra��da média e confiabilidade composta. Avaliou-se a validade discriminante e a consistência interna foi estimada pelo coeficiente alfa de Cronbach. A validade concorrente foi estimada por análise correlacional da versão em português e dos escores médios do Inventário de Burnout de Copenhague; a divergente foi comparada à Escala de Depressão de Beck. Foi avaliada a invariância do modelo entre a amostra brasileira e a portuguesa. RESULTADOS: O modelo trifatorial de Exaustão, Descrença e Eficácia apresentou ajustamento adequado (χ²/df = 8,498; CFI = 0,916; GFI = 0,902; RMSEA = 0,086). A estrutura fatorial foi estável (λ: χ²dif = 11,383, p = 0,50; Cov: χ²dif = 6,479, p = 0,372; Resíduos: χ²dif = 21,514, p = 0,121). Observou-se adequada validade convergente (VEM = 0,45;0,64, CC = 0,82;0,88), discriminante (ρ² = 0,06;0,33) e consistência interna (α = 0,83;0,88). A validade concorrente da versão em português com o Inventário de Copenhague foi adequada (r = 0,21;0,74). A avalia��ão da validade divergente do instrumento foi prejudicada pela aproxima��ão do conceito teórico das dimensões Exaustão e Descrença da versão em português com a Escala de Beck. Não se observou invariância do instrumento entre as amostras brasileiras e portuguesas (λ:χ²dif = 84,768, p < 0,001; Cov: χ²dif = 129,206, p < 0,001; Resíduos: χ²dif = 518,760, p < 0,001). CONCLUSÕES: A versão em português do Inventário de Burnout de Maslach para estudantes apresentou adequada confiabilidade e validade, mas sua estrutura fatorial não foi invariante entre os pa��ses, apontando ausência de estabilidade transcultural.