Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes

Type IV collagen, which is present in all metazoan, exists as a family of six homologous alpha(IV) chains, alpha1-alpha6, in mammals. The six chains assemble into three different triple helical protomers and self-associate as three distinct networks. The network underlies all epithelia as a component of basement membranes, which play important roles in cell adhesion, growth, differentiation, tissue repair and molecular ultrafiltration. The specificity of both protomer and network assembly is governed by amino acid sequences of the C-terminal noncollagenous (NC1) domain of each chain. In this study, the structural basis for protomer and network assembly was investigated by determining the crystal structure of the ubiquitous [(alpha1)(2).alpha2](2) NC1 hexamer of bovine lens capsule basement membrane at 2.0 A resolution. The NC1 monomer folds into a novel tertiary structure. The (alpha1)(2).alpha2 trimer is organized through the unique three-dimensional domain swapping interactions. The differences in the primary sequences of the hypervariable region manifest in different secondary structures, which determine the chain specificity at the monomer-monomer interfaces. The trimer-trimer interface is stabilized by the extensive hydrophobic and hydrophilic interactions without a need for disulfide cross-linking.     

Dynamical correlation of non-stationary signals in time domain—A comparative study

In a thorough study, the multitaper (MTM) and the extended continuous wavelet-transform (CWT) coherence-analysis methods were compared in terms of there application in determining the dynamics from the electroencephalogram (EEG) and electromyogram (EMG) signals of patients with Parkinsonian tremor. The main aim of the study in a biological point of view is to analyze whether the basic tremor frequency and its “first harmonic” frequency of Parkinsonian tremor are really harmonically related or are in fact distinct processes.The extension of the CWT is achieved by using a Morlet wavelet as the analysis window with an adjustable relative bandwidth which gives the flexibility in setting a desired frequency resolution. In order to obtain a perspective view of the two methods, they were applied to two different model signals to determine their actual threshold in detecting short-lived changes in the analysis of non-stationary signals and to determine their noise thresholds by adding external noise to the signals to test the reduction in coherence to be not merely due to the random fluctuations in stochastic signals. Beyond applying an autoregressive 2nd-order and a coupled van der Pol model system, however, also true EEG and EMG data from five Parkinson patients were used. The results were compared in terms of the time and frequency resolutions of these two methods, and it was determined that the multitaper method was able to detect reduction in power and coherence as short as 1 s. The extended CWT analysis only revealed gaps that were longer than 3 s.The time gaps in the coherence indicate the loss of connection between the cortex and muscle during the respective time intervals. This more accurate analysis of the MTM was also seen in the dynamical EEG–EMG coherence at the tremor frequency and its “first harmonic” of Parkinsonian patients.In terms of our “biological” aim, this shows distinct prevalence of the corticomuscular coupling at those frequencies over time. Applying this method to biological data reveals important aspects about their dynamics, e.g., the distinct dynamics between basic frequency and “first harmonic” frequency over time in Parkinsonian tremor.     

ZnO nanoparticles augment ALT,AST, ALP and LDH expressions in C2C12 cells

The present study aimed to investigate the effect of ZnO nanoparticles on alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) enzyme expressions in C2C12 cells. ZnO nanoparticles are widely used in the several cosmetic lotions and other biomedical products. Several studies report on ZnO nanoparticle mediated cytotoxicity. However, there are no reports on the effect of ZnO nanoparticles on ALT, AST, ALP and LDH enzyme expressions in C2C12 cells. A cytotoxicity assay was carried out to determine the effect of ZnO nanoparticles (1–5 mg/ml) on C2C12 cell viability at 48 and 72 h. ZnO nanoparticles increased ALT, AST, ALP and LDH enzyme mRNA expression and their activities in C2C12 cells. In conclusion, the present study showed that ZnO nanoparticles increased these enzyme activities and its mRNA expression in C2C12 cells in a dose-dependent manner.     

Molecular characterization of a truncated antigen (Wb14) of SXP-1 of Wuchereria bancrofti from four endemic regions in India

Wb14 of Wuchereria bancrofti, an orthologue of Brugia malayiSXP-1 and W. bancrofti SXP-1, was amplified from genomic DNA of W. bancrofti microfilaria collected from four distant geographical locations in India viz., Vellore, Bhubaneshwar, Pondicherry and Sevagram. The gene was sub-cloned in a prokaryotic vector pRSET and expressed in Escherichia coli as a truncated protein (∼23 kDa). The nucleotide sequence of the gene is 98% similar to that of WbSXP-1 and is found to be intron-less. However, the analysis and comparison of the derived amino acid sequence with WbSXP-1 showed that Wb14 is truncated at amino acid position 153. The distribution of the two genes in the studied four geographical locations indicated that WbSXP-1 is prevalent only in parasite samples from Sevagram while Wb14 is present in parasites from all the other locations. Only a limited polymorphism was observed in both the genes among the parasites from different geographical locations.     

An efficient randomized algorithm for contact-based NMR backbone resonance assignment

MOTIVATION: Backbone resonance assignment is a critical bottleneck in studies of protein structure, dynamics and interactions by nuclear magnetic resonance (NMR) spectroscopy. A minimalist approach to assignment, which we call 'contact-based', seeks to dramatically reduce experimental time and expense by replacing the standard suite of through-bond experiments with the through-space (nuclear Overhauser enhancement spectroscopy, NOESY) experiment. In the contact-based approach, spectral data are represented in a graph with vertices for putative residues (of unknown relation to the primary sequence) and edges for hypothesized NOESY interactions, such that observed spectral peaks could be explained if the residues were 'close enough'. Due to experimental ambiguity, several incorrect edges can be hypothesized for each spectral peak. An assignment is derived by identifying consistent patterns of edges (e.g. for alpha-helices and beta-sheets) within a graph and by mapping the vertices to the primary sequence. The key algorithmic challenge is to be able to uncover these patterns even when they are obscured by significant noise. RESULTS: This paper develops, analyzes and applies a novel algorithm for the identification of polytopes representing consistent patterns of edges in a corrupted NOESY graph. Our randomized algorithm aggregates simplices into polytopes and fixes inconsistencies with simple local modifications, called rotations, that maintain most of the structure already uncovered. In characterizing the effects of experimental noise, we employ an NMR-specific random graph model in proving that our algorithm gives optimal performance in expected polynomial time, even when the input graph is significantly corrupted. We confirm this analysis in simulation studies with graphs corrupted by up to 500% noise. Finally, we demonstrate the practical application of the algorithm on several experimental beta-sheet datasets. Our approach is able to eliminate a large majority of noise edges and to uncover large consistent sets of interactions. AVAILABILITY: Our algorithm has been implemented in the platform-independent Python code. The software can be freely obtained for academic use by request from the authors.     

Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating

Coxsackievirus A7 (CAV7) is a rarely detected and poorly characterized serotype of the Enterovirus species Human enterovirus A (HEV-A) within the Picornaviridae family. The CAV7-USSR strain has caused polio-like epidemics and was originally thought to represent the fourth poliovirus type, but later evidence linked this strain to the CAV7-Parker prototype. Another isolate, CAV7-275/58, was also serologically similar to Parker but was noninfectious in a mouse model. Sequencing of the genomic region encoding the capsid proteins of the USSR and 275/58 strains and subsequent comparison with the corresponding amino acid sequences of the Parker strain revealed that the Parker and USSR strains are nearly identical, while the 275/58 strain is more distant. Using electron cryomicroscopy and three-dimensional image reconstruction, the structures of the CAV7-USSR virion and empty capsid were resolved to 8.2-Å and 6.1-Å resolutions, respectively. This is one of the first detailed structural analyses of the HEV-A species. Using homology modeling, reconstruction segmentation, and flexible fitting, we constructed a pseudoatomic T = 1 (pseudo T = 3) model incorporating the three major capsid proteins (VP1 to VP3), addressed the conformational changes of the capsid and its constituent viral proteins occurring during RNA release, and mapped the capsid proteins'' variable regions to the structure. During uncoating, VP4 and RNA are released analogously to poliovirus 1, the interfaces of VP2 and VP3 are rearranged, and VP1 rotates. Variable regions in the capsid proteins were predicted to map mainly to the surface of VP1 and are thus likely to affect the tropism and pathogenicity of CAV7.