All-Serotype Dengue Virus Detection through Multilayered Origami-Based Paper/Polymer Microfluidics.

The dengue virus (DENV) infection commonly triggers threatening seasonal outbreaks all around the globe (estimated yearly infections are in the order of 100 million, combining all the viral serotypes), testifying the need for early detection to facilitate disease management and patient recovery. The laboratory-based testing procedures for detecting DENV infection early enough are challenged by the need of resourced settings that result in inevitable cost penalty and unwarranted delay in obtaining the test results due to distance-related factors with respect to the patient's location. Recognizing that the introduction of alternative extreme point-of-care technologies for early detection may potentially mitigate this challenge largely, we develop here a multiplex paper/polymer-based detection strip that interfaces with an all-in-one simple portable device, synchronizing the pipeline of nucleic acid isolation, isothermal amplification, and colorimetric analytics as well as readout for detecting all the four serotypes of dengue viruses in around 30 min from about 50 µL of human blood serum with high specificity and sensitivity. Aligned with the mandatory guidelines of the World Health Organization, the ultralow-cost test is ideal for dissemination at different community centers via a user-friendly device interface, not only as a critical surveillance measure in recognizing the potential cocirculation of the infection across regions that are hyperendemic for all four DENV serotypes but also for facilitating effective monitoring of patients infected by any one of the particular viral serotypes as well as timely administration of life-saving measures on need.

Detection of Aspergillus flavus in Wheat Grains Using Anti-mannoprotein (MP1) and Spore Protein Polyclonal Antibodies.

Cell wall mannoprotein (MP1) gene of an aflatoxigenic strain of Aspergillus flavus, isolated from stored wheat grains, was cloned and sequenced. MP1 protein was expressed in E. coli in soluble form and purified. Polyclonal antibodies were raised against recombinant MP1 protein and inactivated spores of this fungus in rabbit and purified by ammonium sulphate precipitation, Protein A sepharose and antigen affinity chromatography. The minimum concentration of purified mycelial or spore proteins that could be detected by ELISA was determined as 100 ng using 2 µg of these antibodies. The anti-MP1 antibody was found more sensitive than anti-spore protein antibody. Western blot and immunofluorescence analysis showed reactivity of these antibodies to various proteins (30 to 200 kDa) distributed throughout the surface of mycelia and spore of A. flavus. Cross-reactivity of these antibodies was detected with fungi belonging to different Aspergillus, Rhizopus and Alternaria species out of fourteen different fungal species tested. In fungal contaminated wheat grains, these antibodies could detect presence of as low as 1 µg mycelia or 103 spores per gram of wheat grains using ELISA. The results suggest that the developed antibodies could be successfully applied for the detection of predominant fungal infestation in stored wheat grains.

Response of a Zn(II)-based metal-organic coordination polymer towards trivalent metal ions (Al3+, Fe3+ and Cr3+) probed by spectroscopic methods.

A new zinc-based two-dimensional coordination polymer, [Zn(5-AIP)(Ald-4)]·H2O (5-AIP = 5-amino isophthalate, Ald-4 = aldrithiol-4), 1, has been synthesized at room temperature by the layer diffusion technique. Single-crystal X-ray diffraction analysis of 1 showed a two-dimensional bilayer structure. An aqueous suspension of 1 upon excitation at 300 nm displayed an intense blue emission at 403 nm. The luminescence spectra were interestingly responsive and selective to Al3+, Cr3+ and Fe3+ ions even in the presence of other interfering ions. The calculated detection limits for Al3+, Cr3+ and Fe3+ were 0.35 µM ([triple bond, length as m-dash]8.43 ppb), 0.46 µM ([triple bond, length as m-dash]22.6 ppb) and 0.30 µM ([triple bond, length as m-dash]15.85 ppb), respectively. Notably, with the cumulative addition of Al3+ ions, the luminescence intensity at 403 nm decreased steadily with a gradual red shift up to 427 nm. Afterward, this red shifted peak showed a turn-on effect upon further addition of Al3+ ions. On the other hand, for Cr3+ and Fe3+ ions, there was only drastic luminescence quenching and a large red shift up to 434 nm. This indicated the formation of a complex between 1 and these metal ions, which was also supported by the UV-Visible absorption spectra of 1 that showed the appearance of a new band at 280 nm in the presence of these three metal ions. The FTIR spectra revealed that these ions interacted with the carboxylate oxygen atom of 5-AIP and the nitrogen atom of the Ald-4 ligand in the structure. The luminescence lifetime decay analysis manifested that a charge-transfer type complex was formed between 1 and Cr3+ and Fe3+ ions that resulted in huge luminescence quenching due to the efficient charge transfer involving the vacant d-orbitals, whereas for Al3+ ions having no vacant d-orbital, turn-on of luminescence occurred because of the increased rigidity of 1 upon complexation.

Molecular characterization of Antheraea mylitta arylphorin gene and its encoded protein.

Antheraea mylitta arylphorin protein was extracted from the silk gland of fifth instar larvae and purified by ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. The N-terminal sequencing of ten amino acids (NH2-SVVHPPHHEV-COOH) showed similarity with Antheraea pernyi arylphorin. Based on N-terminal and C-terminal A. pernyi arylphorin sequences, primers were designed, and A. mylitta arylphorin cDNA was cloned by RT-PCR from silk gland mRNA. Sequencing of complete cDNA including 25 nucleotides at 5' UTR (obtained by 5' RACE) showed that it consisted of an ORF of 2115 nucleotides which could encode a protein of 704 amino acids (predominantly aromatic residues) having molecular weight 83 kDa. Homology modelling was done using A. pernyi arylphorin as a template. Cloned arylphorin cDNA was expressed in E. coli and recombinant His-tagged protein was purified by Ni-NTA affinity chromatography. Analysis of tissue-specific expression of arylphorin by real-time PCR showed maximum expression in the fat body followed by silk gland and integument. 5' flanking region (759 bp) of arylphorin gene was amplified by inverse PCR and the full length gene (5359 nucleotides) containing five exons and four introns was cloned from the A. mylitta genomic DNA and sequenced. Polyclonal antibody was raised against purified arylphorin and more native arylphorin protein (500 kDa) was purified from the fat body by antibody affinity chromatography. Study of mitogenic effect of native and chymotrypsin hydrolysate of arylphorin on different insect cell lines showed that arylphorin could be used as serum substitute for in vitro cultivation of insect cells.

Delineation of crosstalk between HSP27 and MMP-2/MMP-9: A synergistic therapeutic avenue for glioblastoma management.

BACKGROUND: Epithelial to mesenchymal transition (EMT) and extracellular matrix (ECM) remodeling, are the two elemental processes promoting glioblastoma (GBM). In the present work we propose a mechanistic modelling of GBM and in process establish a hypothesis elucidating critical crosstalk between heat shock proteins (HSPs) and matrix metalloproteinases (MMPs) with synergistic upregulation of EMT-like process and ECM remodeling. METHODS: The interaction and the precise binding site between the HSP and MMP proteins was assayed computationally, in-vitro and in GBM clinical samples. RESULTS: A positive crosstalk of HSP27 with MMP-2 and MMP-9 was established in both GBM patient tissues and cell-lines. This association was found to be of prime significance for ECM remodeling and promotion of EMT-like characteristics. In-silico predictions revealed 3 plausible interaction sites of HSP27 interacting with MMP-2 and MMP-9. Site-directed mutagenesis followed by in-vitro immunoprecipitation assay (IP) with 3 mutated recombinant HSP27, confirmed an interface stretch containing residues 29-40 of HSP27 to be a common interaction site for both MMP-2 and MMP-9. This was further validated with in-vitro IP of truncated (sans AA 29-40) recombinant HSP27 with MMP-2 and MMP-9. CONCLUSION: The association of HSP27 with MMP-2 and MMP-9 proteins along with the identified interacting stretch has the potential to contribute towards drug development to inhibit GBM infiltration and migration. GENERAL SIGNIFICANCE: Current findings provide a novel therapeutic target for GBM opening a new horizon in the field of GBM management.

Elucidation of the mechanism of disulfide exchange between staphylococcal thioredoxin2 and thioredoxin reductase2: A structural insight.

The redox homeostasis of cytoplasm is maintained by a series of disulfide exchange reactions mediated by proteins belonging to the thioredoxin superfamily. Thioredoxin and thioredoxin reductase, being the major members of the family, play a key role in oxidative stress response of Staphylococcus aureus. In this report, we have identified and characterised an active thioredoxin system of the mentioned pathogen. Crystal structure of thioredoxin2 (SaTrx2) in its reduced form reveals that it contains the conserved redox active WCXXC motif and a thioredoxin fold. Thioredoxin reductase2 (SaTR2) is a flavoprotein and consists of two Rossmann folds as the binding sites for FAD and NADPH. Crystal structure of the SaTR2 holoenzyme shows that the protein consists of two domains and the catalytic site comprises of an intramolecular disulfide bond formed between two sequentially distal cysteine residues. Biophysical and biochemical studies unveil that SaTrx2 and SaTR2 can physically interact in solution and in the course of sustaining the redox equilibrium, the latter reduces the former. Molecular docking has been performed to illustrate the interface formed between SaTrx2 and SaTR2 during the disulfide exchange reaction.

Serum-based diagnostic prediction of oral submucous fibrosis using FTIR spectrometry.

Oral submucous fibrosis (OSF) is found to have the highest malignant potentiality among all other pre-cancerous lesions. However, its detection prior to tissue biopsy can be challenging in clinics. Moreover, biopsy examination is invasive and painful. Hence, there is an urgent need of new technology that facilitates accurate diagnostic prediction of OSF prior to biopsy. Here, we used FTIR spectroscopy coupled with chemometric techniques to distinguish the serum metabolic signatures of OSF patients (n=30) and healthy controls (n=30). Serum biochemical analyses have been performed to further support the FTIR findings. Absorbance intensities of 45 infrared wavenumbers differed significantly between OSF and normal serum FTIR spectra representing alterations in carbohydrates, proteins, lipids and nucleic acids. Nineteen prominent significant wavenumbers (P≤0.001) at 1020, 1025, 1035, 1039, 1045, 1078, 1055, 1100, 1117, 1122, 1151, 1169, 1243, 1313, 1398, 1453, 1544, 1650 and 1725cm-1 provided excellent segregation of OSF spectra from normal using multivariate statistical techniques. These findings provided essential information on the metabolic features of blood serum of OSF patients and established that FTIR spectroscopy coupled with chemometric analysis can be potentially useful in the rapid and accurate preoperative screening/diagnosis of OSF.

Molecular insights into RNA-binding properties of Escherichia coli-expressed RNA-dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus.

Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is responsible for morbidity of the Indian non-mulberry silkworm, A. mylitta. AmCPV belongs to the family Reoviridae and has 11 double-stranded (ds) RNA genome segments (S1-S11). Segment 2 (S2) encodes a 123-kDa polypeptide with RNA-dependent RNA polymerase (RdRp) activity. To examine the RNA-binding properties of the viral polymerase, the full-length RdRp and its three domains (N-terminal, polymerase and C-terminal domains) were expressed in Escherichia coli BL21 (DE3) cells with hexahistidine and trigger factor tag fused consecutively at its amino terminus, and the soluble fusion proteins were purified. The purified full-length polymerase specifically bound to the 3' untranslated region (3'-UTR) of a viral plus-sense (+) strand RNA with strong affinity regardless of the salt concentrations, but the isolated polymerase domain of the enzyme exhibited poor RNA-binding ability. Further, the RdRp recognition signals were found to be different from the cis-acting signals that promote minus-sense (-) strand RNA synthesis, because different internal regions of the 3'-UTR of the (+) strand RNA did not effectively compete out the binding of RdRp to the intact 3'-UTR of the (+) strand RNA, but all of these RNA molecules could serve as templates for (-) strand RNA synthesis by the polymerase.

Molecular genetic approaches for environmental stress tolerant crop plants: Progress and prospects.

Global food security is threatened by the severe environmental conditions that have reduced the worldwide crop yield. Plants possess inherent mechanisms to cope with the initial stress phase but to ensure their survival through harsh climate, the intervention of genetic engineering is desirable. Elucidation of genetic loci and deciphering the underlying mechanisms that confer tolerance to plants against stressful conditions followed by its successful introgression into elite, high-yielding crop varieties can be an effective way to engineer the crops for increasing productivity. This review provides an overview about the effects of abiotic and biotic stresses on crop plants and the use of genetic engineering approach to cope with these environmental stresses for a sustainable agriculture. Major patents in the field of plant stress tolerance in the last five years have also been summarized.

Molecular Genetic Approaches for Environmental Stress Tolerant Crop Plants: Progress and Prospects.

BACKGROUND: Global food security is threatened by the severe environmental conditions that have reduced the worldwide crop yield. Plants possess inherent mechanisms to cope with the initial stress phase but to ensure their survival through harsh climate, the intervention of genetic engineering is desirable. OBJECTIVE: We present a comprehensive review on the progress made in the field of developing environmental stress tolerant crops and the prospects that can be undertaken for achieving it. METHODS: We review the effects of abiotic and biotic stresses on crop plants, and the use of different molecular genetic approaches to cope with these environmental stresses for establishment of sustainable agriculture. The various strategies employed in different crops have also been discussed. We also summarized the major patents in the field of plant stress tolerance that have been granted in the last five years. RESULTS: On the basis of these analyses, we propose that genetic engineering of crops is the preferred approach over the traditional methods for yielding healthier and viable agriculture in response to the different stressful environments. The wild progenitors of cultivated crop species can prove to be highly potential genetic resources in this regard and can be exploited to produce better crops that are relatively tolerant towards various environmental stresses. CONCLUSION: Thus, elucidation of genetic loci and deciphering the underlying mechanisms that confer tolerance to plants against stressful conditions followed by its successful introgression into elite, high-yielding crop varieties can be an effective way to engineer the crops for sustainable agriculture.

Protection of human γB-crystallin from UV-induced damage by epigallocatechin gallate: spectroscopic and docking studies.

The transparency of the human eye lens depends on the solubility and stability of the structural proteins of the eye lens, the crystallins. Although the mechanism of cataract formation is still unclear, it is believed to involve protein misfolding and/or aggregation of proteins due to the influence of several external factors such as ultraviolet (UV) radiation, low pH, temperature and exposure to chemical agents. In this article, we report the study of UV induced photo-damage (under oxidative stress) of recombinant human γB-crystallin in vitro in the presence of the major green tea polyphenol, (-)-epigallocatechin gallate (EGCG). We have shown that EGCG has the ability to protect human γB-crystallin from oxidative stress-induced photo-damage.

Structural elucidation of the NADP(H) phosphatase activity of staphylococcal dual-specific IMPase/NADP(H) phosphatase.

NADP(H)/NAD(H) homeostasis has long been identified to play a pivotal role in the mitigation of reactive oxygen stress (ROS) in the intracellular milieu and is therefore critical for the progression and pathogenesis of many diseases. NAD(H) kinases and NADP(H) phosphatases are two key players in this pathway. Despite structural evidence demonstrating the existence and mode of action of NAD(H) kinases, the specific annotation and the mode of action of NADP(H) phosphatases remains obscure. Here, structural evidence supporting the alternative role of inositol monophosphatase (IMPase) as an NADP(H) phosphatase is reported. Crystal structures of staphylococcal dual-specific IMPase/NADP(H) phosphatase (SaIMPase-I) in complex with the substrates D-myo-inositol-1-phosphate and NADP(+) have been solved. The structure of the SaIMPase-I-Ca(2+)-NADP(+) ternary complex reveals the catalytic mode of action of NADP(H) phosphatase. Moreover, structures of SaIMPase-I-Ca(2+)-substrate complexes have reinforced the earlier proposal that the length of the active-site-distant helix α4 and its preceding loop are the predisposing factors for the promiscuous substrate specificity of SaIMPase-I. Altogether, the evidence presented suggests that IMPase-family enzymes with a shorter α4 helix could be potential candidates for previously unreported NADP(H) phosphatase activity.

Functional insights from molecular modeling, docking, and dynamics study of a cypoviral RNA dependent RNA polymerase.

Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) contains 11 double stranded RNA genome segments and infects tasar silkworm A. mylitta. RNA-dependent RNA polymerase (RdRp) is reported as a key enzyme responsible for propagation of the virus in the host cell but its structure function relationship still remains elusive. Here a computational approach has been taken to compare sequence and secondary structure of AmCPV RdRp with other viral RdRps to identify consensus motifs. Then a reliable pairwise sequence alignment of AmCPV RdRp with its closest sequence structure homologue λ3 RdRp is done to predict three dimensional structure of AmCPV RdRp. After comparing with other structurally known viral RdRps, important sequence and/or structural features involved in substrate entry or binding, polymerase reaction and the product release events have been identified. A conserved RNA pentanucleotide (5'-AGAGC-3') at the 3'-end of virus genome is predicted as cis-acting signal for RNA synthesis and its docking and simulation study along with the model of AmCPV RdRp has allowed to predict mode of template binding by the viral polymerase. It is found that template RNA enters into the catalytic center through nine sequence-independent and two sequence-dependent interactions with the specific amino acid residues. However, number of sequence dependent interactions remains almost same during 10 nano-second simulation time while total number of interactions decreases. Further, docking of N(7)-methyl-GpppG (mRNA cap) on the model as well as prediction of RNA secondary structure has shown the template entry process in the active site. These findings have led to postulate the mechanism of RNA-dependent RNA polymerization process by AmCPV RdRp. To our knowledge, this is the first report to evaluate structure function relationship of a cypoviral RdRp.

Genome segment 4 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes RNA triphosphatase and methyltransferases.

Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) genome segment S4 showed that it consists of 3410 nt with a single ORF of 1110 aa which could encode a protein of ~127 kDa (p127). Bioinformatics analysis showed the presence of a 5' RNA triphosphatase (RTPase) domain (LRDR), a S-adenosyl-l-methionine (SAM)-binding (GxGxG) motif and the KDKE tetrad of 2'-O-methyltransferase (MTase), which suggested that S4 may encode RTPase and MTase. The ORF of S4 was expressed in Escherichia coli as a His-tagged fusion protein and purified by nickel-nitrilotriacetic acid affinity chromatography. Biochemical analysis of recombinant p127 showed its RTPase as well as SAM-dependent guanine N(7)-and ribose 2'-O-MTase activities. A MTase assay using in vitro transcribed AmCPV S2 RNA having a 5' G*pppG end showed that guanine N(7) methylation occurred prior to the ribose 2'-O methylation to yield a m(7)GpppG/m(7)GpppGm RNA cap. Mutagenesis of the SAM-binding (GxGxG) motif (G831A) completely abolished N(7)- and 2'-O-MTase activities, indicating the importance of these residues for capping. From the kinetic analysis, the Km values of N(7)-MTase for SAM and RNA were calculated as 4.41 and 0.39 µM, respectively. These results suggested that AmCPV S4-encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5' end of viral RNA.

Genome segment 5 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes a bona fide guanylyltransferase.

BACKGROUND: Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects non mulberry Indian silk worm, Antheraea mylitta, and contains eleven segmented double stranded RNA in its genome (S1-S11). Some of its genome segments (S1-S3, and S6-S11) have been previously characterized but genome segment encoding the viral guanylyltransferase which helps in RNA capping has not been characterized. RESULTS: In this study genome segment 5 (S5) of AmCPV was converted to cDNA, cloned and sequenced. S5 consisted of 2180 nucleotides, with one long ORF of 1818 nucleotides and could encode a protein of 606 amino acids with molecular mass of ~65 kDa (p65). Bioinformatics analysis showed presence of KLRS and HxnH motifs as observed in some other reoviral guanylyltransferase and suggests that S5 may encodes viral guanylyltransferase. The ORF of S5 was expressed in E. coli as 65 kDa his tagged fusion protein, purified through Ni-NTA chromatography and polyclonal antibody was raised. Immunoblot analysis of virion particles with the purified antibody showed specific immunoreactive band and suggests p65 as a viral structural protein. Functional analysis showed that recombinant p65 possesses guanylyltransferase activity, and transfers GMP moiety to the 5' diphosphate (A/G) ended viral RNA after the formation of p65-GMP complex for capping. Kinetic analysis showed K(m) of this enzyme for GTP and RNA was 34.24 uM and 98.35 nM, respectively. Site directed mutagenesis at K21A in KLRS motif, and H93A or H105A in HxnH motif completely abolished the autoguanylylation activity and indicates importance of these residues at these sites. Thermodynamic analysis showed p65-GTP interaction was primarily driven by enthalpy (ΔH = -399.1 ± 4.1 kJ/mol) whereas the p65-RNA interaction by favorable entropy (0.043 ± 0.0049 kJ/ mol). CONCLUSION: Viral capping enzymes play a critical role in the post transcriptional or post replication modification in case of RNA virus. Our results of cloning, sequencing and functional analysis of AmCPV S5 indicates that S5 encoded p65 through its guanylyltransferase activity can transfer guanine residue to the 5' end of viral RNA for capping. Further studies will help to understand complete capping process of cypoviral RNA during viral replication within the viral capsid.

Complete genome sequence of Enterobacter sp. IIT-BT 08: A potential microbial strain for high rate hydrogen production.

Enterobacter sp. IIT-BT 08 belongs to Phylum: Proteobacteria, Class: Gammaproteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae. The organism was isolated from the leaves of a local plant near the Kharagpur railway station, Kharagpur, West Bengal, India. It has been extensively studied for fermentative hydrogen production because of its high hydrogen yield. For further enhancement of hydrogen production by strain development, complete genome sequence analysis was carried out. Sequence analysis revealed that the genome was linear, 4.67 Mbp long and had a GC content of 56.01%. The genome properties encode 4,393 protein-coding and 179 RNA genes. Additionally, a putative pathway of hydrogen production was suggested based on the presence of formate hydrogen lyase complex and other related genes identified in the genome. Thus, in the present study we describe the specific properties of the organism and the generation, annotation and analysis of its genome sequence as well as discuss the putative pathway of hydrogen production by this organism.

Kinetic analysis, expression pattern, and production of a recombinant fungal protease inhibitor of tasar silkworm Antheraea mylitta.

Antheraea mylitta, a tasar silk-producing insect of Saturniidae family, expresses a fungal protease inhibitor named as A. mylitta fungal protease inhibitor-1 (AmFPI-1). AmFPI-1 inhibits alkaline protease of Aspergillus oryzae but its mechanism of action is not known. To understand the mode of inhibition of AmFPI-1 against the fungal protease, it was purified from the hemolymph of A. mylitta larvae and inhibitory activity against A. oryzae protease was studied. Kinetic analysis of purified AmFPI-1 on alkaline protease of A. oryzae showed that AmFPI-1 acts as a canonical-type competitive inhibitor with equilibrium dissociation constant (K ( i )) of 60 nM. Expression of AmFPI-1 in different body tissues of fifth instar A. mylitta larvae was determined by real-time PCR, and the highest expression was observed in fat body followed by integument, silk gland, and gut, indicating that AmFPI-1 has pleiotropic functions including protection from invading fungi. The cDNA of AmFPI-1 was expressed in Escherichia coli, and recombinant His-tagged fusion protein was purified by Ni-NTA chromatography. Recombinant AmFPI-1 showed inhibitory activity against A. oryzae protease and suggested its use in various biological applications to prevent proteolysis.

Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from Staphylococcus aureus MSSA476.

The gene product of the sas2203 ORF of Staphylococcus aureus MSSA476 encodes a 30 kDa molecular-weight protein with a high sequence resemblance (29% identity) to tetrameric inositol monophosphatase from Thermotoga maritima. The protein was cloned, expressed, purified to homogeneity and crystallized. Crystals appeared in several conditions and good diffraction-quality crystals were obtained from 0.2 M Li(2)SO(4), 20% PEG 3350, 0.1 M HEPES pH 7.0 using the sitting-drop vapour-diffusion method. A complete diffraction data set was collected to 2.6 Šresolution using a Rigaku MicroMax-007 HF Cu Kα X-ray generator and a Rigaku R-AXIS IV(++) detector. The diffraction data were consistent with the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.98, b = 68.35, c = 143.79 Å, α = ß = γ = 90°, and the crystal contained two molecules in the asymmetric unit.

Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus.

Genome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123 kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123 kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37 degrees C at pH 6.0 in the presence of 3 mM MgCl(2). Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents.

Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an atypical two-cysteine peroxiredoxin (SAOUHSC_01822) from Staphylococcus aureus NCTC 8325.

An atypical two-cysteine peroxidase, SAOUHSC_01822, from the virulent Staphylococcus aureus strain NCTC 8325 plays a major role in the response of the bacterium to oxidative stress. The protein was cloned, expressed, purified to homogeneity and crystallized. The protein was crystallized from 2 M ammonium sulfate, 0.1 M Na HEPES pH 7, 2%(v/v) PEG 400. A complete diffraction data set was collected to 2.3 angstrom resolution using a Rigaku MicroMax HF007 Cu K alpha X-ray generator and a Rigaku R-AXIS IV(+)(+) detector. The crystals belonged to space group P2(1), with unit-cell parameters a = 43.50, b = 149.35, c = 73.73 angstrom, beta = 104.4 degrees, and contained four molecules in the asymmetric unit.