Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus

<p>Abstract</p> <p>Background</p> <p><it>Antheraea mylitta </it>cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of <it>Reoviridae </it>family, infects Indian non-mulberry silkworm, <it>Antheraea mylitta</it>, and contains 11 segmented double stranded RNA (S1-S11) in its genome. Some of its genome segments (S2 and S6-S11) have been previously characterized but genome segments encoding viral capsid have not been characterized.</p> <p>Results</p> <p>In this study genome segments 1 (S1) and 3 (S3) of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of ~141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of ~137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related <it>cypoviruses </it>like <it>Bombyx mori </it>CPV (BmCPV), <it>Lymantria dispar </it>CPV (LdCPV), and <it>Dendrolimus punctatus </it>CPV (DpCPV). The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in <it>Escherichia coli </it>M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs) by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein.</p> <p>Conclusion</p> <p>Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3 encoded viral structural proteins can self assemble to form viral outer capsid and S1 encoded protein remains associated with it as inner capsid to maintain the stability. Further studies will help to understand the molecular mechanism of capsid formation during cypovirus replication.</p

Curd-Peptide Based Novel Hydrogel Inhibits Biofilm Formation, Quorum Sensing, Swimming Mortility of Multi-Antibiotic Resistant Clinical Isolates and Accelerates Wound Healing Activity

The search for a bioactive natural antibacterial agent with wound healing properties is a common practice for the development of new-generation molecules. Antimicrobial peptides are a good alternative to antibiotics and easy-to-form hydrogels under self-assembled conditions without pH adjustment. With this in mind, the peptide pool was extracted from a formulated curd composed of a blend of probiotic bacteria such as Streptococcus thermophilus, Lactobacillus casei, and Bifidobacterium bifidum at an optimized ratio of 7:1:2. The water content of curd was collected by the drainage column, centrifuged, filtered through a 0.45-μM filter, and used for hydrogel preparation. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis confirmed the presence of peptide pool in the extracted water. The prepared hydrogel was freeze dried, and its effect on biofilm formation, swarming mortality, antimicrobials, wound healing, and biocompatibility was subsequently verified. Transmission electron microscope (TEM) and scanning electron microscope (SEM) images revealed the fibrous network of peptides after self-assembly with non-polar n-hexane solvent and a porous structure after drying, respectively. The observed biocompatibility, antimicrobial activity, and strong wound healing activity of the developed curd-based hydrogel have opened a new platform for antibacterial ointment formulation

Analysis of Transcripts Expressed in One-Day-Old Larvae and Fifth Instar Silk Glands of Tasar Silkworm, Antheraea mylitta

Antheraea mylitta is one of the wild nonmulberry silkworms, which produces tasar silk. An EST project has been undertaken to understand the gene expression profile of A. mylitta silk gland. Two cDNA libraries, one from the whole bodies of one-day-old larvae and the other from the silkglands of fifth instar larvae, were constructed and sequenced. A total of 2476 good-quality ESTs (1239 clones) were obtained and grouped into 648 clusters containing 390 contigs and 258 singletons to represent 467 potential unigenes. Forty-five sequences contained putative coding region, and represented potentially novel genes. Among the 648 clusters, 241 were categorized according to Gene Ontology hierarchy and showed presence of several silk and immune-related genes. The A. mylitta ESTs have been organized into a freely available online database “AmyBASE”. These data provide an initial insight into the A. mylitta transcriptome and help to understand the molecular mechanism of silk protein production in a Lepidopteran species

Identification of RAPD and SCAR markers associated with yield traits in the Indian tropical tasar silkworm Antheraea mylitta drury

Abstract The tropical tasar silkworm, Antheraea mylitta, is a semi-domesticated vanya silk-producing insect of high economic importance. To date, no molecular marker associated with cocoon and shell weights has been identified in this species. In this report, we identified a randomly amplified polymorphic DNA (RAPD) marker and examined its inheritance, and also developed a stable diagnostic sequence-characterized amplified region (SCAR) marker. Silkworms were divided into groups with high (HCSW) and low (LCSW) cocoon and shell weights, and the F 2 progeny of a cross between these two groups were obtained. DNA from these silkworms was screened by PCR using 34 random primers and the resulting RAPD fragments were used for cluster analysis and discriminant function analysis (DFA). The clustering pattern in a UPGMA-based dendogram and DFA clearly distinguished the HCSW and LCSW groups. Multiple regression analysis identified five markers associated with cocoon and shell weights. The marker OPW16 905 bp showed the most significant association with cocoon and shell weights, and its inheritance was confirmed in F 2 progeny. Cloning and sequencing of this 905 bp fragment showed 88% identity between its 134 nucleotides and the Bmc-1/Yamato-like retroposon of A. mylitta. This marker was further converted into a diagnostic SCAR marker (SCOPW 16 826 bp ). The SCAR marker developed here may be useful in identifying the right parental stock of tasar silkworms for high cocoon and shell weights in breeding programs designed to enhance the productivity of tasar silk

Screening of antimicrobial peptides from hemolymph extract of tasar silkworm Antheraea mylitta against urinary tract and wound infecting multidrug-resistant bacteria

Antimicrobial peptides (AMPs) are an evolutionarily conserved component of the innate immune response and they were found among all classes of life forms. In the present study AMPs were extracted from the hemolymph of Antheraea mylitta and fractionated by High Performance Liquid Chromatography (HPLC). Antimicrobial activity was tested against three clinically isolated multidrug-resistant (MDR) bacteria, such as urinary tract infecting Escherichia coli, wound infecting Pseudomonas aeruginosa and Bacillus pumilus. Fraction I (comprised of three different peptides of varying mass) did not inhibit the growth of any of these clinical isolates, whereas, fraction III inhibited the growth of B. pumilus without affecting the growth of gram-negative isolates. Fraction II exhibited bactericidal effects against P. aeruginosa and E. coli, whereas, B. pumilus was not susceptible. Scanning electron microscopic study revealed that serious structural alterations of cell morphology and disruption of the outer membrane, that facilitates the release of cytoplasmic content through holes and channels in E. coli, treated with this isolated peptide. Our results indicate that the peptide from the isolated fraction could be used as potent alternative antimicrobial compounds for the treatment of MDR E. coli andP. aeruginosa infections

Iron oxide nanoparticle assisted purification and mass spectrometry based proteolytic mapping of intact CD4⁺T cells from human blood

Iron oxide nanoparticles have been used for many years as clinical applications. We have developed a rapid immunoaffinity isolation method of CD4⁺T cells from a mixed cell population of human blood using iron oxide nanoparticles. Anti CD4-antibody has been attached to iron oxide nanoparticles after its surface modification. The antibody tagged iron oxide nanoparticle beads are simply incubated with the mixed cell population of human blood and CD4⁺T cells are purified using an external magnetic field. The purification level was checked by fluorescence microscopy and flow cytometry. The purified CD4⁺T cells were digested with trypsin with different time periods and the products were analyzed by MALDI-TOF mass spectrometry, without further fractionation or purification, to obtain its proteome pattern. A database search showed a number of peptide masses matched specific to T-cell peptide masses. These results indicate that iron oxide nanoparticles are useful for CD4⁺T cell purification, and mass spectrometry based proteolytic fingerprint is simple and swift for identifying putative surface biomarkers from the whole cell surfaces

Urea Stibamine: An Improved Method of Preparation and its Antileishmanial Activity

Urea stibamine, developed by Brahmachari (1) as an effective antileishmanial drug, was used extensively during the second and third decades of the present century for the treatment of visceral leishmaniasis (Kala-azar). The drug was responsible for saving the lives of countless patients in eastern India (2), although the chemical nature of urea stibamine was never thoroughly understood. Ghosh et al. (3) reported that the value of the antimony content varied from 20 to 43% in different samples; therefore the toxicity test was performed batchwise. The chemical reaction involved in the preparation of the drug was also not clear (2). The present paper deals with an improved method for preparation of urea stibamine, its chromatographic and spectroscopic properties, toxicity, and antileishmanial activity

Carbon nanofiber reinforced nonmulberry silk protein fibroin nanobiocomposite for tissue engineering applications

Natural silk protein fibroin based biomaterial are exploited extensively in tissue engineering due to their aqueous preparation, slow biodegradability, mechanical stability, low immunogenicity, dielectric properties, tunable properties, sufficient and easy availability. Carbon nanofibers are reported for their conductivity, mechanical strength and as delivery vehicle of small molecules. Limited evidence about their cytocompatibility and their poor dispersibility are the key issues for them to be used as successful biomaterials. In this study, carbon nanofiber is functionalized and dispersed using the green aqueous-based method within the regenerated nonmulberry (tropical tasar: Antheraea mylitta) silk fibroin (AmF), which contains inherent – R-G-D- sequences. Carbon Nanofiber (CNF) reinforced silk films are fabricated using solvent evaporation technique. Different biophysical characterizations and cytocompatibility of the composite matrices are assessed. The investigations show that the presence of the nanofiber greatly influence the property of the composite films in terms of excellent conductivity (up to 6.4 × 10–6 Mho cm, which is 3 orders of magnitude of pure AmF matrix), and superior tensile modulus (up to 1423 MPa, which is 12.5 times more elastic than AmF matrix). The composite matrices (composed of up to 1 mg of CNF per mL of 2% AmF) also support better fibroblast cell growth and proliferation. The fibroin-carbon nanofiber matrices can lead to a novel multifunctional biomaterial platform, which will support conductive as well as load bearing tissue (such as, muscle, bone, and nerve tissue) regenerations

Proteomics view of a Rhizobium isolate response to arsenite [As(III)] stress

The genus Rhizobium colonizes in leguminous plants by symbiotic relationship and enriches soil nitrogen through the formation of the root nodule. Legumes are the appropriate crops for the recovery of marginal lands and they can easily grow in adverse climatic condition. Rhizobium sp. VMA301 was isolated from the root nodules of V. mungo , grown in arsenic contaminated field. The LC50 value of arsenite for VMA301 was found to be 1.8 mM. Sixteen differentially expressed proteins were identified using RP-HPLC and MALDI ToF mass spectrometry from arsenite induced whole cell lysate soluble proteins. It is also found that nine proteins were up-regulated and seven proteins were down-regulated in comparison to the control group (cells grown without arsenite). These differential protein expressions mitigate the toxic effect of arsenite and stimulate the detoxification process