BK virus large T and VP-1 expression in infected human renal allografts

Objective. We investigated the expression of early and late phase BK virus (BKV) proteins and their interactions with host cell proteins in renal allografts, with ongoing polyomavirus associated nephropathy (PVAN), and correlated this with the nuclear and cell morphology. Methods. Frozen sections from three patients with renal allografts (two biopsies, one explant) with PVAN were analysed by indirect immunofluorescence using BKV specific anti-polyoma large T-antigen and anti-VP-1 antibodies, as well as anti-p53, anti-Ki67, anti-caspase-3, anti-bcl2 and anti-cytokeratin 22 antibodies. Nuclear morphology and size were estimated by DNA Hoechst staining. Results. In infected tubular cells the early and late phases of infection could be distinguished according to expression of large T-antigen or VP-1. The early phase revealed almost normal nuclear proportions, whereas in later phases nuclear size increased about 2 to 3 fold. Expression of large T-antigen was strongly associated with accumulation of p53 in the nucleus, accompanied by the activation of the cell cycle associated cell protein Ki67. In contrast, expression of BKV VP1 correlated only weakly with p53. Virus dependent cell lysis was due to necrosis, since neither caspase 3 nor nuclear nor cytoskeleton changes indicated apoptosis. Conclusion. In our selected patients with PVAN a clear distinction between early and late phases was possible, according to the protein expression patterns of BKV markers. Striking nuclear enlargement is only present in the late phase of infection. In the inflammatory setting of PVAN, BKV dependent effects appear to be mediated by the inhibition of p53, resulting in the activation of the cell cycle. We assume that in PVAN similar BKV mechanisms are operative as in certain in vitro system

CCMpred-fast and precise prediction of protein residue-residue contacts from correlated mutations.

Motivation: Recent breakthroughs in protein residue-residue contact prediction have made reliable de novo prediction of protein structures possible. The key was to apply statistical methods that can distinguish direct couplings between pairs of columns in a multiple sequence alignment from merely correlated pairs, i.e. to separate direct from indirect effects. Two classes of such methods exist, either relying on regularized inversion of the covariance matrix or on pseudo-likelihood maximization (PLM). Although PLM-based methods offer clearly higher precision, available tools are not sufficiently optimized and are written in interpreted languages that introduce additional overheads. This impedes the runtime and large-scale contact prediction for larger protein families, multi-domain proteins and protein-protein interactions. Results: Here we introduce CCMpred, our performance-optimized PLM implementation in C and CUDA C. Using graphics cards in the price range of current six-core processors, CCMpred can predict contacts for typical alignments 35-113 times faster and with the same precision as the most accurate published methods. For users without a CUDA-capable graphics card, CCMpred can also run in a CPU mode that is still 4-14 times faster. Thanks to our speed-ups (http://dictionary.cambridge.org/dictionary/british/speed-up) contacts for typical protein families can be predicted in 15-60s on a consumer-grade GPU and 1-6min on a six-core CPU

Evolutionary coupling methods in de novo protein structure prediction

An understanding of protein tertiary structure is important for both basic and translational research, for example to understand molecular mechanisms, engineer new or optimized catalysts, or formulate new cures. Protein tertiary structures are typically determined experimentally, a time-consuming process with average costs in the hundred thousands of US dollars for determining a single protein structure. Consequently, there is much interest in using computational methods for driving down the cost of obtaining new structures. While great successes have been made in transferring structural information from already structurally solved homologous proteins, the sensitivity improvements of methods for detecting homologous proteins have plateaued in recent years and homology-based Protein structure prediction is ultimately limited by the availability of a suitable template that must be determined experimentally. De novo protein structure prediction could theoretically use physical models to determine the native conformation of a protein without Prior structural information but in practice, such approaches are limited by the computational costs of evaluating expensive energy functions for many different points in an enormous search space. An old idea in protein bioinformatics is to use the compensatory mutations observed due to the evolutionary pressure of maintaining a protein fold to predict which residue pairs in a protein structures are interacting in the folded structure. If such interactions can be reliably predicted, they can be used to constrain the search space of de novo protein structure prediction sufficiently so that the lowest-energy conformation can be found. Through recent improvements in the accuracy of such residue-residue interaction predictors, Protein domain structures of typical size could be predicted in a blinded experiment for the first time in 2011. However, the new class of methods is still limited in its applicability in that methods are sensitive to false-positive predictions of interactions and can only provide reliable predictions with low false-positive rates for Protein families that have a high number of homologous sequences. This work aims to improve residue-residue contact predictions by improving the underlying mathematical models in a Bayesian framework. By explicitly modelling noise effects inherent in the underlying data and including priors to reflect the nature of residue-residue interactions, an attempt is made to reduce random and systematic errors inherent in contact prediction to make protein de novo structure prediction widely applicable

BK virus large T and VP-1 expression in infected human renal allografts

Objective. We investigated the expression of early and late phase BK virus (BKV) proteins and their interactions with host cell proteins in renal allografts, with ongoing polyomavirus associated nephropathy (PVAN), and correlated this with the nuclear and cell morphology

C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques

Background. Detection of C4d along peritubular capillaries (PTC) in renal allograft biopsies is an independent prognostic marker of poor long-term graft survival. It is typically associated with circulating donor-specific antibodies. Since only little information is available on the best technique to stain C4d, we compared the two methods most often used for detecting C4d in renal allograft specimens. Methods. We investigated the expression of C4d along PTC in 64 renal allograft biopsies using a monoclonal antibody (Quidel) and immunofluorescence for frozen (F-IF) and a polyclonal antibody (Biomedica) and immunohistochemistry for formalin-fixed and paraffin-embedded (P-IHC) tissue samples. We compared the staining extent (diffuse, focal, minimal, no staining) in frozen and paraffin sections and evaluated the intra- and inter-observer concordance rates using kappa statistics. In addition, we determined the inter-observer concordance in 240 paraffin-embedded biopsies of a multi-centre study. Results. The inter- and intra-investigator concordance rate (κ = 0.9) of analysing the C4d expression by F-IF was excellent. In contrast, the detection of C4d by P-IHC demonstrated a substantially lower prevalence and extent of C4d expression with a lower intra- and inter-observer concordance rate (κ = 0.3). Only 69% of diffuse and 13% of focal C4d-expressing cases were in line classified by F-IF and P-IHC. On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by P-IHC than by F-IF. The inter-observer concordance rate in paraffin of the 64 renal biopsies and the multi-centre study was good, but not perfect (κ = 0.57 or 0.67). Conclusions. C4d staining determined on frozen tissue samples using F-IF with a monoclonal antibody appears to be better suited for diagnostic as well as research purposes. Future studies should correlate C4d staining patterns with circulating donor-specific antibodie

Variations in metamorphic grade in metapelites in transects across the Quetico Subprovince north of Thunder Bay, Ontario

The Quetico subprovince is a northeast-southwest striking linear belt of migmatites, gneisses, and metasedimentary rocks. These Archean rocks form part of the southern Superior Province- This study involves an examination of variations in metamorphic grade along cross-strike transects in an area north of Thunder Bay, Ontario. The rocks of the Quetico subprovince include metasedimentary rocks with well preserved primary structures, knotted schists, gneisses, migmatites, and anatectic granitic rocks. Metamorphic porphyroblasts include muscovite, biotite, garnet, staurolite, cordierite, andalusite, and sillimanite. Chemical analyses of garnets, geothermobarometry, and mineral assemblage data were used to determine variations in metamorphic grade in transects across the subprovince. Mineral assemblages characteristic of low to high grade metamorphism are exposed along an across-strike transect. Metamorphic grade rises gradually from low grade (521°C) to high grade (714°C) northwards along Highway 527. North of the peak conditions, the grade drops off sharply. Garnet-biotite geothermometry confirms this pattern. Maximum pressure reached in the study area is approximately 5 kbar. The model proposed to account for the distribution of metamorphic assemblages and minerals involves transpression of the Quetico accretionary prism between the Wabigoon volcanic cratonic margin to the north and the docking Wawa volcanic complex to the south. Buckling and folding of the sedimentary rocks was accompanied by thrusting. Erosion has exposed high grade migmatitic and anatectic rocks within the Quetico fold belt which developed as a result of thermal relaxation of depressed isotherms. The boundaries between metavolcanic and metasedimentary terranes are structurally complex. Boundaries may be best described as geometrically complex zones up to several kilometres in extent in which various rock types representative of the adjacent terranes have been folded, faulted, and intruded

Oberflächencharakterisierung von III-V MOCVD-Filmen aus heterozyklischen Single Source Precursoren

Oberflächencharakterisierung von III-V MOCVD-Filmen aus heterozyklischen Single Source Precursoren In vielen mikro- und optoelektronischen Bauteilen finden Antimon-haltige Verbindungshalbleiter Verwendung. Insbesondere ist GaSb geeignet für Bauelemente, die im mittleren IR-Bereich arbeiten. Neben den klassischen Herstellungsverfahren gewinnt die chemische Gasphasenabscheidung (CVD) in der Industrie eine immer größere Bedeutung. Jedoch ist der klassische CVD-Ansatz, bei dem von einem Precursormolekül für Ga und einem anderen für Sb (Dual Source Ansatz) in diesem Fall problematisch, da ungünstige Prozessparameter schnell zur Abscheidung nicht-stöchiometrischer Filme und zu Kohlenstoffkontaminationen führen können. Eine Alternative stellt der sog. Single Source Precursor Ansatz dar, bei dem das gewünschte Material bereits in der richtigen Stöchiometrie vorliegt. In der vorliegenden Arbeit wurden vollständig alkyl-substituierte Single Source Precursoren zur Abscheidung von GaSb-Filmen auf Si(001)-Wafern untersucht. Das Sublimationsverhalten der verwendeten Precursoren wurde massenspektrometrisch untersucht. Die hergestellten Filme wurden oberflächensensitiv mittels Elektronenspektroskopie (AES, S-XPS) hinsichtlich ihrer chemischen Zusammensetzung und mittels Rastersondenmikroskopie (AFM) hinsichtlich ihrer Morphologie untersucht. Der Precursor (tBu2 Ga Sb Et2)2 hat sich als zur Herstellung von stöchiometrischen GaSb-Filmen geeignet herausgestellt. Der Vorteil dieser Precursorsubstanz ist seine geringere Toxizität im Vergleich zu Trialkylgallanen oder Stiban, sowie seine einfachere Lagerung, da es sich um einen kristallinen Feststoff handelt. Allerdings ist die Synthese recht aufwändig und für die Herstellung von GaSb-Filmen ist auf Grund des niedrigen Dampfdruckes der Precursorsubstanz ein Vakuum nötig

Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease

Background. Toll-like receptors (TLR) recognize a variety of ligands, including pathogen-associated molecular patterns and link innate and adaptive immunity. Individual receptors can be up-regulated during infection and inflammation. We examined the expression of selected TLRs at the protein level in various types of renal disease. Methods. Frozen sections of renal biopsies were stained with monoclonal antibodies to TLR-2, -4 and -9. Results. Up-regulation of the three TLRs studied was seen, although the extent was modest. TLR-2- and -4-positive cells belonged to the population of infiltrating inflammatory cells; only in the case of TLR-9 were intrinsic glomerular cells positive in polyoma virus infection and haemolytic uraemic syndrome (HUS). Conclusions. Evidence for the involvement of the three TLRs tested in a variety of human renal diseases was found. These findings add to our understanding of the role of the innate immune system in kidney diseas