Structural and molecular basis of interaction of HCV non-structural protein 5A with human casein kinase 1 alpha and PKR

Background: Interaction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1 alpha (ck1 alpha) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV. Results: Serine 232 of NS5A is known to be phosphorylated by human ck1 alpha. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1 alpha has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1 alpha has been identified from the model and these are found to be conserved well in the ck1 family. ck1 alpha - substrate peptide complex has also been used to understand the structural basis of association between ck1 alpha and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available. Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified. Conclusions: The substrate interacting residues in ck1 alpha have been identified using the structural model of kinase substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional significance and nature of interaction of interferon sensitivity determining region and variable region 3 of NS5A in different genotypes with PKR which was experimentally shown are also supported by the findings of evolutionary trace analysis. Designing inhibitors to prevent this interaction could enable the HCV genotype 1 infected patients respond well to interferon therapy.

Fully complex-valued ELM classifiers for human action recognition

In this paper, we present a fast learning neural network classifier for human action recognition. The proposed classifier is a fully complex-valued neural network with a single hidden layer. The neurons in the hidden layer employ the fully complex-valued hyperbolic secant as an activation function. The parameters of the hidden layer are chosen randomly and the output weights are estimated analytically as a minimum norm least square solution to a set of linear equations. The fast leaning fully complex-valued neural classifier is used for recognizing human actions accurately. Optical flow-based features extracted from the video sequences are utilized to recognize 10 different human actions. The feature vectors are computationally simple first order statistics of the optical flow vectors, obtained from coarse to fine rectangular patches centered around the object. The results indicate the superior performance of the complex-valued neural classifier for action recognition. The superior performance of the complex neural network for action recognition stems from the fact that motion, by nature, consists of two components, one along each of the axes.

A novel adeno-associated virus serotype (AAV)-8 capsid mutant improves human coagulation factor IX expression in vivo

Recombinant AAV-8 vectors have shown significant promise for hepatic gene therapy of hemophilia B. However, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses. We hypothesized that AAV8 during its intracellular trafficking, are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery and modification of specific serine/threonine kinase or ubiquitination targets on AAV8 capsid (Fig.1A) may improve its transduction efficiency. To test this, point mutations at specific serine (S)/threonine (T) > alanine (A) or lysine (K)>arginine (R) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant(S276A, S501A, S671A, T251A and K137R) capsids were evaluated for their liver transduction efficiency at a dose of 5 X 1010 vgs/ animal in C57BL/6 mice in vivo. The best performing mutant was found to be the K137R vector in terms of either the gene expression (46-fold) or the vector copy numbers in the hepatocytes (22-fold) compared to WT-AAV8 (Fig.1B). The K137R-AAV8 vector that showed significantly decreased ubiquitination of the viral capsid had reduced activation of markers of innate immune response [IL-6, IL-12, tumor necrosis factor α, Kupffer cells and TLR-9]. In addition, animals injected with the K137R mutant also demonstrated decreased (2-fold) levels of cross-neutralizing antibodies when compared to animals that received the WT-AAV8 vector. To study further the utility of the novel AAV8-K137R mutant in a therapeutic setting, we delivered human coagulation factor IX (h.FIX) under the control of liver specific promoters (LP1 or hAAT) at two different doses (2.5x10^10 and 1x10^11 vgs per mouse) in 8-12 weeks old male C57BL/6 mice. As can be seen in Fig.1C/D, the circulating levels of h.FIX were higher in all the K137R-AAV8 treated groups as compared to the WT-AAV8 treated groups either at 2 weeks (62% vs 37% for hAAT constructs and 47% vs 21% for LP1 constructs) or 4 weeks (78% vs 56% for hAAT constructs and 64% vs 30% for LP1 constructs) post hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel vector for potential gene therapy of hemophilia B.

Remarkable visible light-triggered cytotoxicity of mitochondria targeting mixed-ligand cobalt(III) complexes of curcumin and phenanthroline bases binding to human serum albumin

Six new mixed-ligand cobalt(III) complexes of formulation Co(N-N)(2)(O-O)](ClO4)(2) (1-6), where N-N is a N,N-donor phenanthroline base, namely, 1,10-phenanthroline (phen in 1, 2), dipyrido3,2-d:2',3'-f] quinoxaline (dpq in 3, 4), and dipyrido3,2-a:2',3'-c]phenazine (dppz in 5, 6), O-O is acetylacetonate (acac in 1, 3, 5) or curcumin (bis(4-hydroxy-3-methoxyphenyl)-1,6-diene-3,5-dione, cur in 2, 4, 6), have been synthesized and characterized. The X-ray crystal structures of complex 1 (as PF6- salt, 1a) and 3 show distorted octahedral geometries formed by the CoN4O2 core. The complexes 1, 3 and 5 having the simple acac ligand are prepared as control species to understand the role of curcumin. The optimized geometries and the frontier orbitals of the curcumin complexes 2, 4, and 6 are obtained from the DFT calculations. The complexes 2, 4, and 6 having the photoactive curcumin moiety display an absorption band in the visible region near 420 nm and show remarkable photocytotoxicity in HeLa cancer cells with respective IC50 values of 7.4 mu M, 5.1 mu M and 1.6 mu M while being much less toxic in dark. MTT assay using complex 6 shows that it is not significantly photocytotoxic to MCF-10A normal cells. The control complexes having the acac ligand are non-toxic both in the presence and absence of light. The cell death is apoptotic in nature and triggered by the photogeneration of reactive oxygen species. Fluorescence imaging experiments on HeLa cells reveals that complex 6 accumulated primarily inside the mitochondria. Human serum albumin (HSA) binding experiments show that the complexes bind HSA with good affinity, but 6 binds with the highest affinity, with a K-b value of 9.8 x 10(5) M-1. Thus, complex 6 with its negligible toxicity in the dark and in normal cells but remarkable toxicity in visible light holds significant photochemotherapeutic potential.

Human Action Recognition in Compressed Domain using PBL-McRBFN Approach

Large variations in human actions lead to major challenges in computer vision research. Several algorithms are designed to solve the challenges. Algorithms that stand apart, help in solving the challenge in addition to performing faster and efficient manner. In this paper, we propose a human cognition inspired projection based learning for person-independent human action recognition in the H.264/AVC compressed domain and demonstrate a PBL-McRBEN based approach to help take the machine learning algorithms to the next level. Here, we use gradient image based feature extraction process where the motion vectors and quantization parameters are extracted and these are studied temporally to form several Group of Pictures (GoP). The GoP is then considered individually for two different bench mark data sets and the results are classified using person independent human action recognition. The functional relationship is studied using Projection Based Learning algorithm of the Meta-cognitive Radial Basis Function Network (PBL-McRBFN) which has a cognitive and meta-cognitive component. The cognitive component is a radial basis function network while the Meta-Cognitive Component(MCC) employs self regulation. The McC emulates human cognition like learning to achieve better performance. Performance of the proposed approach can handle sparse information in compressed video domain and provides more accuracy than other pixel domain counterparts. Performance of the feature extraction process achieved more than 90% accuracy using the PTIL-McRBFN which catalyzes the speed of the proposed high speed action recognition algorithm. We have conducted twenty random trials to find the performance in GoP. The results are also compared with other well known classifiers in machine learning literature.

Human action recognition in H.264/AVC compressed domain using meta-cognitive radial basis function network

In this paper, we propose a H.264/AVC compressed domain human action recognition system with projection based metacognitive learning classifier (PBL-McRBFN). The features are extracted from the quantization parameters and the motion vectors of the compressed video stream for a time window and used as input to the classifier. Since compressed domain analysis is done with noisy, sparse compression parameters, it is a huge challenge to achieve performance comparable to pixel domain analysis. On the positive side, compressed domain allows rapid analysis of videos compared to pixel level analysis. The classification results are analyzed for different values of Group of Pictures (GOP) parameter, time window including full videos. The functional relationship between the features and action labels are established using PBL-McRBFN with a cognitive and meta-cognitive component. The cognitive component is a radial basis function, while the meta-cognitive component employs self-regulation to achieve better performance in subject independent action recognition task. The proposed approach is faster and shows comparable performance with respect to the state-of-the-art pixel domain counterparts. It employs partial decoding, which rules out the complexity of full decoding, and minimizes computational load and memory usage. This results in reduced hardware utilization and increased speed of classification. The results are compared with two benchmark datasets and show more than 90% accuracy using the PBL-McRBFN. The performance for various GOP parameters and group of frames are obtained with twenty random trials and compared with other well-known classifiers in machine learning literature. (C) 2015 Elsevier B.V. All rights reserved.

On the detection of human influence in extreme precipitation over India

Climate change is expected to influence extreme precipitation which in turn might affect risks of pluvial flooding. Recent studies on extreme rainfall over India vary in their definition of extremes, scales of analyses and conclusions about nature of changes in such extremes. Fingerprint-based detection and attribution (D&A) offer a formal way of investigating the presence of anthropogenic signals in hydroclimatic observations. There have been recent efforts to quantify human effects in the components of the hydrologic cycle at large scales, including precipitation extremes. This study conducts a D&A analysis on precipitation extremes over India, considering both univariate and multivariate fingerprints, using a standardized probability-based index (SPI) from annual maximum one-day (RX1D) and five-day accumulated (RX5D) rainfall. The pattern-correlation based fingerprint method is used for the D&A analysis. Transformation of annual extreme values to SPI and subsequent interpolation to coarser grids are carried out to facilitate comparison between observations and model simulations. Our results show that in spite of employing these methods to address scale and physical processes mismatch between observed and model simulated extremes, attributing changes in regional extreme precipitation to anthropogenic climate change is difficult. At very high (95%) confidence, no signals are detected for RX1D, while for the RX5D and multivariate cases only the anthropogenic (ANT) signal is detected, though the fingerprints are in general found to be noisy. The findings indicate that model simulations may underestimate regional climate system responses to increasing human forcings for extremes, and though anthropogenic factors may have a role to play in causing changes in extreme precipitation, their detection is difficult at regional scales and not statistically significant. (C) 2015 Elsevier B.V. All rights reserved.

Combined Effect of Cryogel Matrix and Temperature-Reversible Soluble-Insoluble Polymer for the Development of in Vitro Human Liver Tissue

Hepatic cell culture on a three-dimensional (3D) matrix or as a hepatosphere appears to be a promising in vitro biomimetic system for liver tissue engineering applications. In this study, we have combined the concept of a 3D scaffold and a spheroid culture to develop an in vitro model to engineer liver tissue for drug screening. We have evaluated the potential of poly(ethylene glycol)-alginate-gelatin (PAG) cryogel matrix for in vitro culture of human liver cell lines. The synthesized cryogel matrix has a flow rate of 7 mL/min and water uptake capacity of 94% that enables easy nutrient transportation in the in vitro cell culture. Youngs modulus of 2.4 kPa and viscoelastic property determine the soft and elastic nature of synthesized cryogel. Biocompatibility of PAG cryogel was evaluated through MTT assay of HepG2 and Huh-7 cells on matrices. The proliferation and functionality of the liver cells were enhanced by culturing hepatic cells as spheroids (hepatospheres) on the PAG cryogel using temperature-reversible soluble-insoluble polymer, poly(N-isopropylacrylamide) (PNIPAAm). Pore size of the cryogel above 100 mu m modulated spheroid size that can prevent hypoxia condition within the spheroid culture. Both the hepatic cells have shown a significant difference (P < 0.05) in terms of cell number and functionality when cultured with PNIPAAm. After 10 days of culture using 0.05% PNIPAAm, the cell number increased by 11- and 7-fold in case of HepG2 and Huh-7 cells, respectively. Similarly, after 10 days of hepatic spheroids culture on PAG cryogel, the albumin production, urea secretion, and CYP450 activity were significantly higher in case of culture with PNIPAAm. The developed tissue mass on the PAG cryogel in the presence of PNIPAAm possess polarity, which was confirmed using F-actin staining and by presence of intercellular bile canalicular lumen. The developed cryogel matrix supports liver cells proliferation and functionality and therefore can be used for in vitro and in vivo drug testing.