Remarkable Effect of Chalcogen Substitution on an Enzyme Mimetic for Deiodination of Thyroid Hormones

Iodothyronine deiodinases are selenoenzymes which regulate the thyroid hormone homeostasis by catalyzing the regioselective deiodination of thyroxine (T4). Synthetic deiodinase mimetics are important not only to understand the mechanism of enzyme catalysis, but also to develop therapeutic agents as abnormal thyroid hormone levels have implications in different diseases, such as hypoxia, myocardial infarction, critical illness, neuronal ischemia, tissue injury, and cancer. Described herein is that the replacement of sulfur/selenium atoms in a series of deiodinase mimetics by tellurium remarkably alters the reactivity as well as regioselectivity toward T4. The tellurium compounds reported in this paper represent the first examples of deiodinase mimetics which mediate sequential deiodination of T4 to produce all the hormone derivatives including T0 under physiologically relevant conditions.

Action Recognition from Motion Capture Data using Meta-cognitive RBF Network Classifier

Action recognition plays an important role in various applications, including smart homes and personal assistive robotics. In this paper, we propose an algorithm for recognizing human actions using motion capture action data. Motion capture data provides accurate three dimensional positions of joints which constitute the human skeleton. We model the movement of the skeletal joints temporally in order to classify the action. The skeleton in each frame of an action sequence is represented as a 129 dimensional vector, of which each component is a 31) angle made by each joint with a fixed point on the skeleton. Finally, the video is represented as a histogram over a codebook obtained from all action sequences. Along with this, the temporal variance of the skeletal joints is used as additional feature. The actions are classified using Meta-Cognitive Radial Basis Function Network (McRBFN) and its Projection Based Learning (PBL) algorithm. We achieve over 97% recognition accuracy on the widely used Berkeley Multimodal Human Action Database (MHAD).

A carboxy terminal domain of the L protein of rinderpest virus possesses RNA triphosphatase activity - The first enzyme in the viral mRNA capping pathway

The large protein L of negative-sense RNA viruses is a multifunctional protein involved in transcription and replication of genomic RNA. It also possesses enzymatic activities involved in capping and methylation of viral mRNAs. The pathway for mRNA capping followed by the L protein of the viruses in the Morbillivirus genus has not been established, although it has been speculated that these viruses may follow the unconventional capping pathway as has been shown for some viruses of Rhabdoviridae family. We had earlier shown that the large protein L of Rinderpest virus expressed as recombinant L-P complex in insect cells as well as the ribonucleoprotein complex from purified virus possesses RNA triphosphatase (RTPase) and guanylyltransferase activities, in addition to RNA dependent RNA polymerase activity. In the present work, we demonstrate that RTPase as well as nucleoside triphosphatase (NTPase) activities are exhibited by a subdomain of the L protein in the C terminal region (a.a. 1640 1840). The RTPase activity depends absolutely on a divalent cation, either magnesium or manganese. Both the RTPase and NTPase activities of the protein show dual metal specificity. Two mutant proteins having alanine mutations in the glutamic acid residues in motif-A of the RTPase domain did not show RTPase activity, while exhibiting reduced NTPase activity suggesting overlapping active sites for the two enzymatic functions. The RTPase and NTPase activities of the L subdomain resemble those of the Vaccinia capping enzyme D1 and the baculovirus LEF4 proteins. (C) 2015 Elsevier Inc. All rights reserved.

An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea

We have recently reported significant association of non-polio enteroviruses (NPEVs) with acute and persistent diarrhea (18-21% of total diarrheal cases), and non-diarrheal Increased Frequency of Bowel Movements (IFoBM-ND) (about 29% of the NPEV infections) in children and that the NPEV-associated diarrhea was as significant as rotavirus diarrhea. However, their diarrhea-causing potential is yet to be demonstrated in an animal model system. Since the determination of virus titers by the traditional plaque assay takes 4-7 days, there is a need for development of a rapid method for virus titer determination to facilitate active clinical research on enterovirus-associated diarrhea. The goal of this study is to develop a cell-based rapid detection and enumeration method and to demonstrate the diarrhea-inducing potential of purified and characterized non-polio enteroviruses, which were isolated from diarrheic children. Here we describe generation of monoclonal and polyclonal antibodies against purified strains belonging to different serotypes, and development of an enzyme-linked immuno focus assay (ELIFA) for detection and enumeration of live NPEV particles in clinical and purified virus samples, and a newborn mouse model for NPEV diarrhea. Plaque-purified NPVEs, belonging to different serotypes, isolated from children with diarrhea, were grown in cell culture and purified by isopycnic CsCl density gradient centrifugation. By ELIFA, NPEVs could be detected and enumerated within 12 h post-infection. Our results demonstrated that Coxsackievirus B1 (CVB1) and CVB5 strains, isolated from diarrheic children, induced severe diarrhea in orally-inoculated 9-12 day-old mouse pups, fulfilling Koch's postulates. The methods described here would facilitate studies on NPEV-associated gastrointestinal disease. (C) 2015 Elsevier B.V. All rights reserved.

Novel pppGpp binding site at the C-terminal region of the Rel enzyme from Mycobacterium smegmatis

Mycobacterium tuberculosis elicits the stringent response under unfavorable growth conditions, such as those encountered by the pathogen inside the host. The hallmark of this response is production of guanosine tetra-and pentaphosphates, collectively termed (p)ppGpp, which have pleiotropic effects on the bacterial physiology. As the stringent response is connected to survival under stress, it is now being targeted for developing inhibitors against bacterial persistence. The Rel enzyme in mycobacteria has two catalytic domains at its N-terminus that are involved in the synthesis and hydrolysis of (p)ppGpp, respectively. However, the function of the C-terminal region of the protein remained unknown. Here, we have identified a binding site for pppGpp in the C-terminal region of Rel. The binding affinity of pppGpp was quantified by isothermal titration calorimetry. The binding site was determined by crosslinking using the nucleotide analog azido-pppGpp, and examining the crosslink product by mass spectrometry. Additionally, mutations in the Rel protein were created to confirm the site of pppGpp binding by isothermal titration calorimetry. These mutants showed increased pppGpp synthesis and reduced hydrolytic activity. We believe that binding of pppGpp to Rel provides a feedback mechanism that allows the protein to detect and adjust the (p)ppGpp level in the cell. Our work suggests that such sites should also be considered while designing inhibitors to target the stringent response.

Structural insights into N-terminal to C-terminal interactions and implications for thermostability of a (beta/alpha)(8)-triosephosphate isomerase barrel enzyme

Although several factors have been suggested to contribute to thermostability, the stabilization strategies used by proteins are still enigmatic. Studies on a recombinant xylanase from Bacilllus sp. NG-27 (RBSX), which has the ubiquitous (beta/alpha)(8)-triosephosphate isomerase barrel fold, showed that just a single mutation, V1L, although not located in any secondary structural element, markedly enhanced the stability from 70 degrees C to 75 degrees C without loss of catalytic activity. Conversely, the V1A mutation at the same position decreased the stability of the enzyme from 70 degrees C to 68 degrees C. To gain structural insights into how a single extreme N-terminus mutation can markedly influence the thermostability of the enzyme, we determined the crystal structure of RBSX and the two mutants. On the basis of computational analysis of their crystal structures, including residue interaction networks, we established a link between N-terminal to C-terminal contacts and RBSX thermostability. Our study reveals that augmenting N-terminal to C-terminal noncovalent interactions is associated with enhancement of the stability of the enzyme. In addition, we discuss several lines of evidence supporting a connection between N-terminal to C-terminal noncovalent interactions and protein stability in different proteins. We propose that the strategy of mutations at the termini could be exploited with a view to modulate stability without compromising enzymatic activity, or in general, protein function in diverse folds where N and C termini are in close proximity. Database The coordinates of RBSX, V1A and V1L have been deposited in the PDB database under the accession numbers 4QCE, 4QCF, and 4QDM, respectively

Probing the role of highly conserved residues in triosephosphate isomerase-analysis of site specific mutants at positions 64 and 75 in the Plasmodial enzyme

Highly conserved residues in enzymes are often found to be clustered close to active sites, suggesting that functional constraints dictate the nature of amino acid residues accommodated at these sites. Using the Plasmodiumfalciparum triosephosphate isomerase (PfTIM) enzyme () as a template, we have examined the effects of mutations at positions 64 and 75, which are not directly involved in the proton transfer cycle. Thr (T) occurring at position 75 is completely conserved, whereas only Gln (Q) and Glu (E) are accommodated at position 64. Biophysical and kinetic data are reported for four T75 (T75S/V/C/N) and two Q64 (Q64N/E) mutants. The dimeric structure is weakened in the Q64E and Q64N mutants, whereas dimer integrity is unimpaired in all four T75 mutants. Measurement of the concentration dependence of enzyme activity permits an estimate of K-d values for dimer dissociation (Q64N=73.79.2nm and Q64E=44.6 +/- 8.4nm). The T75S/V/C mutants have activities comparable to the wild-type enzyme, whereas a fourfold drop is observed for T75N. All four T75 mutants show a dramatic fall in activity between 35 degrees C and 45 degrees C. Crystal structure determination of the T75S/V/N mutants provides insights into the variations in local interactions, with the T75N mutant showing the largest changes. Hydrogen-bond interactions determine dimer stability restricting the choice of residues at position 64 to Gln (Q) and Glu (E). At position 75, the overwhelming preference for Thr (T) may be dictated by the imperative of maintaining temperature stability of enzyme activity.

Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase

Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins has not been studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to the identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein-tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys-224. This residue is situated in a lid region that covers the enzyme's active site. Consequently, acetylation and succinylation negatively regulate the activity of PtpB.

Covalent interlocking of glucose oxidase and peroxidase in the voids of paper: enzyme-polymer ``spider webs''

A modular, general method for trapping enzymes within the voids of paper, without chemical activation of cellulose, is reported. Glucose oxidase and peroxidase were crosslinked with poly(acrylic acid) via carbodiimide chemistry, producing 3-dimensional networks interlocked in cellulose fibers. Interlocking prevented enzyme activity loss and enhanced the washability and stability.

No More HF: Teflon-Assisted Ultrafast Removal of Silica to Generate High-Surface-Area Mesostructured Carbon for Enhanced CO2 Capture and Supercapacitor Performance

An innovative technique to obtain high-surface-area mesostructured carbon (2545m(2)g(-1)) with significant microporosity uses Teflon as the silica template removal agent. This method not only shortens synthesis time by combining silica removal and carbonization in a single step, but also assists in ultrafast removal of the template (in 10min) with complete elimination of toxic HF usage. The obtained carbon material (JNC-1) displays excellent CO2 capture ability (ca. 26.2wt% at 0 degrees C under 0.88bar CO2 pressure), which is twice that of CMK-3 obtained by the HF etching method (13.0wt%). JNC-1 demonstrated higher H-2 adsorption capacity (2.8wt%) compared to CMK-3 (1.2wt%) at -196 degrees C under 1.0bar H-2 pressure. The bimodal pore architecture of JNC-1 led to superior supercapacitor performance, with a specific capacitance of 292Fg(-1) and 182Fg(-1) at a drain rate of 1Ag(-1) and 50Ag(-1), respectively, in 1m H2SO4 compared to CMK-3 and activated carbon.

Vacancy-Engineered Nanoceria: Enzyme Mimetic Hotspots for the Degradation of Nerve Agents

Organophosphorus-based nerve agents, such as paraoxon, parathion, and malathion, inhibit acetylcholinesterase, which results in paralysis, respiratory failure, and death. Bacteria are known to use the enzyme phosphotriesterase (PTE) to break down these compounds. In this work, we designed vacancy-engineered nanoceria (VE CeO2 NPs) as PTE mimetic hotspots for the rapid degradation of nerve agents. We observed that the hydrolytic effect of the nano-material is due to the synergistic activity between both Ce3+ and Ce4+ ions located in the active site-like hotspots. Furthermore, the catalysis by nanoceria overcomes the product inhibition generally observed for PTE and small molecule-based PTE mimetics.

Effect of solvents on the enzyme mediated degradation of copolymers

The biodegradation of polycaprolactone (PCL), polylactic acid (PLA), polyglycolide (PGA) and their copolymers, poly (lactide-co-glycolide) and poly (D, L-lactide-co-caprolactone) (PLCL) was investigated. The influence of different solvents on the degradation of these polymers at 37 degrees C in the presence of two different lipases namely Novozym 435 and the free lipase of porcine pancreas was investigated. The rate coefficients for the polymer degradation and enzyme deactivation were determined using continuous distribution kinetics. Among the homopolymers, the degradation of PGA was nearly an order of magnitude lower than that for PCL and PLA. The overall rate coefficients of the copolymers were higher than their respective homopolymers. Thus, PLCL degraded faster than either PCL or PLA. The degradation was highly dependent on the viscosity of the solvent used with the highest degradation observed in acetone. The degradation of the polymers in acetone was nearly twice that observed in dimethyl sulfoxide indicating that the degradation decreases with increase in the solvent viscosity. The degradation of the polymers in water-solvent mixtures indicated an optimal water content of 2.5 wt% of water.

No More HF: Teflon-Assisted Ultrafast Removal of Silica to Generate High-Surface-Area Mesostructured Carbon for Enhanced CO2 Capture and Supercapacitor Performance

An innovative technique to obtain high-surface-area mesostructured carbon (2545m(2)g(-1)) with significant microporosity uses Teflon as the silica template removal agent. This method not only shortens synthesis time by combining silica removal and carbonization in a single step, but also assists in ultrafast removal of the template (in 10min) with complete elimination of toxic HF usage. The obtained carbon material (JNC-1) displays excellent CO2 capture ability (ca. 26.2wt% at 0 degrees C under 0.88bar CO2 pressure), which is twice that of CMK-3 obtained by the HF etching method (13.0wt%). JNC-1 demonstrated higher H-2 adsorption capacity (2.8wt%) compared to CMK-3 (1.2wt%) at -196 degrees C under 1.0bar H-2 pressure. The bimodal pore architecture of JNC-1 led to superior supercapacitor performance, with a specific capacitance of 292Fg(-1) and 182Fg(-1) at a drain rate of 1Ag(-1) and 50Ag(-1), respectively, in 1m H2SO4 compared to CMK-3 and activated carbon.

Vacancy-Engineered Nanoceria: Enzyme Mimetic Hotspots for the Degradation of Nerve Agents

Organophosphorus-based nerve agents, such as paraoxon, parathion, and malathion, inhibit acetylcholinesterase, which results in paralysis, respiratory failure, and death. Bacteria are known to use the enzyme phosphotriesterase (PTE) to break down these compounds. In this work, we designed vacancy-engineered nanoceria (VE CeO2 NPs) as PTE mimetic hotspots for the rapid degradation of nerve agents. We observed that the hydrolytic effect of the nano-material is due to the synergistic activity between both Ce3+ and Ce4+ ions located in the active site-like hotspots. Furthermore, the catalysis by nanoceria overcomes the product inhibition generally observed for PTE and small molecule-based PTE mimetics.

Effect of solvents on the enzyme mediated degradation of copolymers

The biodegradation of polycaprolactone (PCL), polylactic acid (PLA), polyglycolide (PGA) and their copolymers, poly (lactide-co-glycolide) and poly (D, L-lactide-co-caprolactone) (PLCL) was investigated. The influence of different solvents on the degradation of these polymers at 37 degrees C in the presence of two different lipases namely Novozym 435 and the free lipase of porcine pancreas was investigated. The rate coefficients for the polymer degradation and enzyme deactivation were determined using continuous distribution kinetics. Among the homopolymers, the degradation of PGA was nearly an order of magnitude lower than that for PCL and PLA. The overall rate coefficients of the copolymers were higher than their respective homopolymers. Thus, PLCL degraded faster than either PCL or PLA. The degradation was highly dependent on the viscosity of the solvent used with the highest degradation observed in acetone. The degradation of the polymers in acetone was nearly twice that observed in dimethyl sulfoxide indicating that the degradation decreases with increase in the solvent viscosity. The degradation of the polymers in water-solvent mixtures indicated an optimal water content of 2.5 wt% of water.