BRAF mutation testing for patients diagnosed with stage III or stage IV melanoma: practical guidance for the Australian setting.

Targeted therapy (BRAF inhibitor plus MEK inhibitor) is now among the possible treatment options for patients with BRAF mutation-positive stage III or stage IV melanoma. This makes prompt BRAF mutation testing an important step in the management of patients diagnosed with stage III or IV melanoma; one that can help better ensure that the optimal choice of systemic treatment is initiated with minimal delay. This article offers guidance about when and how BRAF mutation testing should be conducted when patients are diagnosed with melanoma in Australia. Notably, it recommends that pathologists reflexively order BRAF mutation testing whenever a patient is found to have American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) stage III or IV melanoma (i.e., any metastatic spread beyond the primary tumour) and that patient's BRAF mutation status is hitherto unknown, even if BRAF mutation testing has not been specifically requested by the treating clinician (in Australia, Medicare-subsidised BRAFV600 mutation testing does not need to be requested by the treating clinician). When performed in centres with appropriate expertise and experience, immunohistochemistry (IHC) using the anti-BRAF V600E monoclonal antibody (VE1) can be a highly sensitive and specific means of detecting BRAFV600E mutations, and may be used as a rapid and relatively inexpensive initial screening test. However, VE1 immunostaining can be technically challenging and difficult to interpret, particularly in heavily pigmented tumours; melanomas with weak, moderate or focal BRAFV600E immunostaining should be regarded as equivocal. It must also be remembered that other activating BRAFV600 mutations (including BRAFV600K), which account for ∼10-20% of BRAFV600 mutations, are not detected with currently available IHC antibodies. For these reasons, if available and practicable, we recommend that DNA-based BRAF mutation testing always be performed, regardless of whether IHC-based testing is also conducted. Advice about tissue/specimen selection for BRAF mutation testing of patients diagnosed with stage III or IV melanoma is also offered in this article; and potential pitfalls when interpreting BRAF mutation tests are highlighted.

The sensor region of the ubiquitous cytosolic sensor kinase, PdtaS, contains PAS and GAF domain sensing modules.

Two-component systems, a sensor histidine kinase (HK) and a response regulator (RR), are ubiquitous signaling systems that allow prokaryotes to respond to external challenges. HKs normally have sensing modules and highly conserved cytosolic histidine kinase and ATPase domains. The interaction between the activated phosphohistidine and the cognate RR allows an external signal to be passed from the exterior of gram-positive bacteria (GPB) to the cytoplasm. Orthologs of the PdtaS/PdtaR regulatory system, found in most GPB phyla, are unusual in two respects. The HK is not membrane anchored, and the RR acts at the level of transcriptional antitermination. The structure of the complete sensor region of the cytosolic HK, PdtaS, from Mycobacterium tuberculosis consists of closely linked GAF and PAS domains. The structure and sequence analysis suggest that the PdtaS/PdtaR regulatory system is structurally equivalent to the EutW/EutV system regulating ethanolamine catabolism in some phyla but that the effector for the PAS domain is not ethanolamine in the Actinobacteria.

On the routine use of soft X-rays in macromolecular crystallography. Part V. Molecular replacement and anomalous scattering.

Currently, about two thirds of all new macromolecular structures are determined by molecular replacement. In general the method works reliably, but it reaches its limits when the search model differs too much from the target structure in terms of coordinate deviations or completeness. Since anomalously scattering substructures are better conserved than the overall structure, these substructures and the corresponding anomalous intensity differences can be utilized to enhance the performance of molecular-replacement approaches. It is demonstrated that the combined and concomitant use of structure-factor amplitudes and anomalous differences constitutes a promising approach to push the limits of molecular replacement and to make more structures amenable to structure solution by this technique.

Structure and genetic analysis of the arterivirus nonstructural protein 7alpha.

Arterivirus replicase polyproteins are cleaved into at least 13 mature nonstructural proteins (nsps), and in particular the nsp5-to-nsp8 region is subject to a complex processing cascade. The function of the largest subunit from this region, nsp7, which is further cleaved into nsp7α and nsp7ß, is unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the solution structure of nsp7α of equine arteritis virus, revealing an interesting unique fold for this protein but thereby providing little clue to its possible functions. Nevertheless, structure-based reverse genetics studies established the importance of nsp7/nsp7α for viral RNA synthesis, thus providing a basis for future studies.

Additional phase information from UV damage of selenomethionine labelled proteins.

Currently, selenium is the most widely used phasing vehicle for experimental phasing, either by single anomalous scattering or multiple-wavelength anomalous dispersion (MAD) procedures. The use of the single isomorphous replacement anomalous scattering (SIRAS) phasing procedure with selenomethionine containing proteins is not so commonly used, as it requires isomorphous native data. Here it is demonstrated that isomorphous differences can be measured from intensity changes measured from a selenium labelled protein crystal before and after UV exposure. These can be coupled with the anomalous signal from the dataset collected at the selenium absorption edge to obtain SIRAS phases in a UV-RIPAS phasing experiment. The phasing procedure for two selenomethionine proteins, the feruloyl esterase module of xylanase 10B from Clostridium thermocellum and the Mycobacterium tuberculosis chorismate synthase, have been investigated using datasets collected near the absorption edge of selenium before and after UV radiation. The utility of UV radiation in measuring radiation damage data for isomorphous differences is highlighted and it is shown that, after such measurements, the UV-RIPAS procedure yields comparable phase sets with those obtained from the conventional MAD procedure. The results presented are encouraging for the development of alternative phasing approaches for selenomethionine proteins in difficult cases.

Structural characterization of the multidomain regulatory protein Rv1364c from Mycobacterium tuberculosis.

The open reading frame rv1364c of Mycobacterium tuberculosis, which regulates the stress-dependent σ factor, σ(F), has been analyzed structurally and functionally. Rv1364c contains domains with sequence similarity to the RsbP/RsbW/RsbV regulatory system of the stress-response σ factor of Bacillus subtilis. Rv1364c contains, sequentially, a PAS domain (which shows sequence similarity to the PAS domain of the B. subtilis RsbP protein), an active phosphatase domain, a kinase (anti-σ(F) like) domain and a C-terminal anti-σ(F) antagonist like domain. The crystal structures of two PAS domain constructs (at 2.3 and 1.6 Å) and a phosphatase/kinase dual domain construct (at 2.6 Å) are described. The PAS domain is shown to bind palmitic acid but to have 100 times greater affinity for palmitoleic acid. The full-length protein can exist in solution as both monomer and dimer. We speculate that a switch between monomer and dimer, possibly resulting from fatty acid binding, affects the accessibility of the serine of the C-terminal, anti-σ(F) antagonist domain for dephosphorylation by the phosphatase domain thus indirectly altering the availability of σ(F).

Single isomorphous replacement phasing of selenomethionine-containing proteins using UV-induced radiation damage.

The most commonly used heavy-atom derivative, selenium, requires the use of a tunable beamline to access the Se K edge for experimental phasing using anomalous diffraction methods, whereas X-ray diffraction experiments for selenium-specific ultraviolet radiation-damage-induced phasing can be performed on fixed-wavelength beamlines or even using in-house X-ray sources. Several nonredundant X-ray diffraction data sets were collected from three different selenomethionine (Mse) derivatized protein crystals at energies far below the absorption edge before and after exposing the crystal to ultraviolet (UV) radiation using 266 nm lasers of flux density 1.7 × 10¹5 photons s⁻¹â€…mm⁻² for 10-50 min. A detailed analysis revealed that significant changes in diffracted intensities were induced by ultraviolet irradiation whilst retaining crystal isomorphism. These intensity changes allowed the crystal structures to be solved by the radiation-damage-induced phasing (RIP) technique. Inspection of the crystal structures and electron-density maps demonstrated that covalent bonds between selenium and carbon at all sites located in the core of the proteins or in a hydrophobic environment were much more susceptible to UV radiation-induced cleavage than other bonds typically present in Mse proteins. The rapid UV radiation-induced bond cleavage opens a reliable new paradigm for phasing when no tunable X-ray source is available. The behaviour of Mse derivatives of various proteins provides novel insights and an initial basis for understanding the mechanism of selenium-specific UV radiation damage.

Resonance assignment of nsp7α from arterivirus.

The (1)H, (15)N and (13)C resonance assignment of nsp7α, a non-structural protein of unknown function from the equine arteritis virus, is reported.

Papain-like protease 1 from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates.

Coronaviruses encode two classes of cysteine proteases, which have narrow substrate specificities and either a chymotrypsin- or papain-like fold. These enzymes mediate the processing of the two precursor polyproteins of the viral replicase and are also thought to modulate host cell functions to facilitate infection. The papain-like protease 1 (PL1(pro)) domain is present in nonstructural protein 3 (nsp3) of alphacoronaviruses and subgroup 2a betacoronaviruses. It participates in the proteolytic processing of the N-terminal region of the replicase polyproteins in a manner that varies among different coronaviruses and remains poorly understood. Here we report the first structural and biochemical characterization of a purified coronavirus PL1(pro) domain, that of transmissible gastroenteritis virus (TGEV). Its tertiary structure is compared with that of severe acute respiratory syndrome (SARS) coronavirus PL2(pro), a downstream paralog that is conserved in the nsp3's of all coronaviruses. We identify both conserved and unique structural features likely controlling the interaction of PL1(pro) with cofactors and substrates, including the tentative mapping of substrate pocket residues. The purified recombinant TGEV PL1(pro) was shown to cleave a peptide mimicking the cognate nsp2|nsp3 cleavage site. Like its PL2(pro) paralogs from several coronaviruses, TGEV PL1(pro) was also found to have deubiquitinating activity in an in vitro cleavage assay, implicating it in counteracting ubiquitin-regulated host cell pathways, likely including innate immune responses. In combination with the prior characterization of PL2(pro) from other alphacoronaviruses, e.g., human coronaviruses 229E and NL63, our results unequivocally establish that these viruses employ two PL(pro)s with overlapping specificities toward both viral and cellular substrates.

Insights into the role of the non-structural protein 2 (NS2) in Bluetongue virus morphogenesis.

Bluetongue virus (BTV) non-structural protein 2 (NS2) belongs to a class of highly conserved proteins found in Orbiviruses of the Reoviridae family. NS2 forms large multimeric complexes and localizes to cytoplasmic inclusions in infected cells. Due to its ability to bind single-stranded RNA (ssRNA), it has been suggested that the protein participates in the selection and sequestration of the 10 different BTV-ssRNA segments, prior to their encapsidation and conversion into the BTV double-stranded RNA (dsRNA) genome. Recent advances in understanding how BTV-NS2 is organized and functions are largely inferred from structural studies. The X-ray crystal structure of the N-terminal domain of NS2 suggests that the full-length protein could assemble as homomultimers of maximally 10-11 subunits. The crystallographic structural information combined with small-angle X-ray scattering experiments on the C-terminal domain as well as negative-stain electron microscopy on the full-length protein give us a first glimpse of how the two protein domains associate and function. Herein, we survey biochemical and recent structural investigations on NS2 important to the understanding of the molecular events underlying the process of BTV morphogenesis. We also present a phylogenetic analysis of the NS2 sequences.

Antiviral strategies to control calicivirus infections.

Caliciviridae are human or non-human pathogenic viruses with a high diversity. Some members of the Caliciviridae, i.e. human pathogenic norovirus or rabbit hemorrhagic disease virus (RHDV), are worldwide emerging pathogens. The norovirus is the major cause of viral gastroenteritis worldwide, accounting for about 85% of the outbreaks in Europe between 1995 and 2000. In the United States, 25 million cases of infection are reported each year. Since its emergence in 1984 as an agent of fatal hemorrhagic diseases in rabbits, RHDV has killed millions of rabbits and has been dispersed to all of the inhabitable continents. In view of their successful and apparently increasing emergence, the development of antiviral strategies to control infections due to these viral pathogens has now become an important issue in medicine and veterinary medicine. Antiviral strategies have to be based on an understanding of the epidemiology, transmission, clinical symptoms, viral replication and immunity to infection resulting from infection by these viruses. Here, we provide an overview of the mechanisms underlying calicivirus infection, focusing on the molecular aspects of replication in the host cell. Recent experimental data generated through an international collaboration on structural biology, virology and drug design within the European consortium VIZIER is also presented. Based on this analysis, we propose antiviral strategies that may significantly impact on the epidemiological characteristics of these highly successful viral pathogens.

Crystallization and preliminary crystallographic analysis of the measles virus hemagglutinin in complex with the CD46 receptor.

The measles virus (MV) hemagglutinin (MV-H) mediates the attachment of MV particles to cell-surface receptors for entry into host cells. MV uses two receptors for attachment to host cells: the complement-control protein CD46 and the signalling lymphocyte activation molecule (SLAM). The MV-H glycoprotein from an Edmonston MV variant and the MV-binding fragment of the CD46 receptor were overproduced in mammalian cells and used to crystallize an MV-H-CD46 complex. Well diffracting crystals containing two complexes in the asymmetric unit were obtained and the structure of the complex was solved by the molecular-replacement method.

Structure of the X (ADRP) domain of nsp3 from feline coronavirus.

The structure of the X (or ADRP) domain of a pathogenic variant of feline coronavirus (FCoV) has been determined in tetragonal and cubic crystal forms to 3.1 and 2.2 A resolution, respectively. In the tetragonal crystal form, glycerol-3-phosphate was observed in the ADP-ribose-binding site. Both crystal forms contained large solvent channels and had a solvent content of higher than 70%. Only very weak binding of this domain to ADP-ribose was detected in vitro. However, the structure with ADP-ribose bound was determined in the cubic crystal form at 3.9 A resolution. The structure of the FCoV X domain had the expected macro-domain fold and is the first structure of this domain from a coronavirus belonging to subgroup 1a.

Fatty acid- and retinoid-binding proteins have distinct binding pockets for the two types of cargo.

Parasitic nematodes cause serious diseases in humans, animals, and plants. They have limited lipid metabolism and are reliant on lipid-binding proteins to acquire these metabolites from their hosts. Several structurally novel families of lipid-binding proteins in nematodes have been described, including the fatty acid- and retinoid-binding protein family (FAR). In Caenorhabditis elegans, used as a model for studying parasitic nematodes, eight C. elegans FAR proteins have been described. The crystal structure of C. elegans FAR-7 is the first structure of a FAR protein, and it exhibits a novel fold. It differs radically from the mammalian fatty acid-binding proteins and has two ligand binding pockets joined by a surface groove. The first can accommodate the aliphatic chain of fatty acids, whereas the second can accommodate the bulkier retinoids. In addition to demonstrating lipid binding by fluorescence spectroscopy, we present evidence that retinol binding is positively regulated by casein kinase II phosphorylation at a conserved site near the bottom of the second pocket. far-7::GFP (green fluorescent protein) expression shows that it is localized in the head hypodermal syncytia and the excretory cell but that this localization changes under starvation conditions. In conclusion, our study provides the basic structural and functional information for investigation of inhibitors of lipid binding by FAR proteins.

On the combination of molecular replacement and single-wavelength anomalous diffraction phasing for automated structure determination.

A combination of molecular replacement and single-wavelength anomalous diffraction phasing has been incorporated into the automated structure-determination platform Auto-Rickshaw. The complete MRSAD procedure includes molecular replacement, model refinement, experimental phasing, phase improvement and automated model building. The improvement over the standard SAD or MR approaches is illustrated by ten test cases taken from the JCSG diffraction data-set database. Poor MR or SAD phases with phase errors larger than 70 degrees can be improved using the described procedure and a large fraction of the model can be determined in a purely automatic manner from X-ray data extending to better than 2.6 A resolution.

Structure of the C-terminal domain of nsp4 from feline coronavirus.

Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26-31 kb) encodes 15-16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication-transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain ( approximately 100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 A resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4(3). The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly alpha-helical content displaying a unique fold that could be engaged in protein-protein interactions.

Applied large eddy simulation.

Large eddy simulation (LES) is now seen more and more as a viable alternative to current industrial practice, usually based on problem-specific Reynolds-averaged Navier-Stokes (RANS) methods. Access to detailed flow physics is attractive to industry, especially in an environment in which computer modelling is bound to play an ever increasing role. However, the improvement in accuracy and flow detail has substantial cost. This has so far prevented wider industrial use of LES. The purpose of the applied LES discussion meeting was to address questions regarding what is achievable and what is not, given the current technology and knowledge, for an industrial practitioner who is interested in using LES. The use of LES was explored in an application-centred context between diverse fields. The general flow-governing equation form was explored along with various LES models. The errors occurring in LES were analysed. Also, the hybridization of RANS and LES was considered. The importance of modelling relative to boundary conditions, problem definition and other more mundane aspects were examined. It was to an extent concluded that for LES to make most rapid industrial impact, pragmatic hybrid use of LES, implicit LES and RANS elements will probably be needed. Added to this further, highly industrial sector model parametrizations will be required with clear thought on the key target design parameter(s). The combination of good numerical modelling expertise, a sound understanding of turbulence, along with artistry, pragmatism and the use of recent developments in computer science should dramatically add impetus to the industrial uptake of LES. In the light of the numerous technical challenges that remain it appears that for some time to come LES will have echoes of the high levels of technical knowledge required for safe use of RANS but with much greater fidelity.

Developing large eddy simulation for turbomachinery applications.

For jets, large eddy resolving simulations are compared for a range of numerical schemes with no subgrid scale (SGS) model and for a range of SGS models with the same scheme. There is little variation in results for the different SGS models, and it is shown that, for schemes which tend towards having dissipative elements, the SGS model can be abandoned, giving what can be termed numerical large eddy simulation (NLES). More complex geometries are investigated, including coaxial and chevron nozzle jets. A near-wall Reynolds-averaged Navier-Stokes (RANS) model is used to cover over streak-like structures that cannot be resolved. Compressor and turbine flows are also successfully computed using a similar NLES-RANS strategy. Upstream of the compressor leading edge, the RANS layer is helpful in preventing premature separation. Capturing the correct flow over the turbine is particularly challenging, but nonetheless the RANS layer is helpful. In relation to the SGS model, for the flows considered, evidence suggests issues such as inflow conditions, problem definition and transition are more influential.

Structural and biophysical characterization of the proteins interacting with the herpes simplex virus 1 origin of replication.

The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8DeltaC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8DeltaC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (Kd approximately 6 nM) followed by a second binding event (Kd approximately 0.8 nM). It is also shown that the stoichiometry of the ternary UL9ct-ICP8DeltaC-dsDNA15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.