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The lack of success in clinical trials for HIV vaccines highlights the need to explore novel strategies for vaccine development. Research on highly exposed seronegative (HESN) HIV-resistant Kenyan female sex workers revealed naturally protective immunity is correlated with a focused immune response mediated by virus-specific CD8 T cells. Further studies indicated that the immune response is unconventionally focused on highly conserved sequences around HIV viral protease cleavage sites (VPCS). Thus, taking an unconventional approach to HIV vaccine development, we designed lipid nanoparticles loaded with mRNA that encodes multi-epitopes of VPCS (MEVPCS-mRNA LNP), a strategic design to boost antigen presentation by dendritic cells, promoting effective cellular immunity. Furthermore, we developed a novel cold-chain compatible mRNA LNP formulation, ensuring long-term stability and compatibility with cold-chain storage/transport, widening accessibility of mRNA LNP vaccine in low-income countries. The in-vivo mouse study demonstrated that the vaccinated group generated VPCS-specific CD8 memory T cells, both systemically and at mucosal sites of viral entry. The MEVPCS-mRNA LNP vaccine-induced CD8 T cell immunity closely resembled that of the HESN group and displayed a polyfunctional profile. Notably, it induced minimal to no activation of CD4 T cells. This proof-of-concept study underscores the potential of the MEVPCS-mRNA LNP vaccine in eliciting CD8 T cell memory specific to the highly conserved multiple VPCS, consequently having a broad coverage in human populations and limiting viral escape mutation. The MEVPCS-mRNA LNP vaccine holds promise as a candidate for an effective prophylactic HIV vaccine.

Chloroviruses are unusual among viruses infecting eukaryotic organisms in that they must, like bacteriophages, penetrate a rigid cell wall to initiate infection. Chlorovirus PBCV-1 infects its host, Chlorella variabilis NC64A by specifically binding to and degrading the cell wall of the host at the point of contact by a virus-packaged enzyme(s). However, PBCV-1 does not use any of the five previously characterized virus-encoded polysaccharide degrading enzymes to digest the Chlorella host cell wall during virus entry because none of the enzymes are packaged in the virion. A search for another PBCV-1-encoded and virion-associated protein identified protein A561L. The fourth domain of A561L is a 242 amino acid C-terminal domain, named A561L D4 , with cell wall degrading activity. An A561L D4 homolog was present in all 52 genomically sequenced chloroviruses, infecting four different algal hosts. A561L D4 degraded the cell walls of all four chlorovirus hosts, as well as several non-host Chlorella spp. Thus, A561L D4 was not cell-type specific. Finally, we discovered that exposure of highly purified PBCV-1 virions to A561L D4 increased the specific infectivity of PBCV-1 from about 25-30% of the particles forming plaques to almost 50%. We attribute this increase to removal of residual host receptor that attached to newly replicated viruses in the cell lysates.

Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K + ) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K + channel proteins consisting of 82 amino acids. The Kcv ATCV-1 protein has similarities to the family of two transmembrane domain K + channel proteins; it consists of two transmembrane α-helixes with a pore region in the middle, making it an ideal model for studying K + channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from Kcv ATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K + channels.

The unavailability of the reproductive structure and unpredictability of vegetative characters for the identification and phylogenetic study of bamboo prompted the application of molecular techniques for greater resolution and consensus. We first employed internal transcribed spacer (ITS1, 5.8S rRNA and ITS2) sequences to construct the phylogenetic tree of 21 tropical bamboo species. While the sequence alone could grossly reconstruct the traditional phylogeny amongst the 21-tropical species studied, some anomalies were encountered that prompted a further refinement of the phylogenetic analyses. Therefore, we integrated the secondary structure of the ITS sequences to derive individual sequence-structure matrix to gain more resolution on the phylogenetic reconstruction. The results showed that ITS sequence-structure is the reliable alternative to the conventional phenotypic method for the identification of bamboo species. The best-fit topology obtained by the sequence-structure based phylogeny over the sole sequence based one underscores closer clustering of all the studied Bambusa species (Sub-tribe Bambusinae), while Melocanna baccifera, which belongs to Sub-Tribe Melocanneae, disjointedly clustered as an out-group within the consensus phylogenetic tree. In this study, we demonstrated the dependability of the combined (ITS sequence+structure-based) approach over the only sequence-based analysis for phylogenetic relationship assessment of bamboo.

MicroRNAs (miRNAs) regulate numerous crucial biological processes in plants. However, information is limited on their involvement in the biosynthesis of specialized metabolites in plants, including Catharanthus roseus that produces a number of pharmaceutically valuable, bioactive terpenoid indole alkaloids (TIAs). Using small RNA-sequencing, we identified 181 conserved and 173 novel miRNAs (cro-miRNAs) in C. roseus seedlings. Genome-wide expression analysis revealed that a set of cro-miRNAs are differentially regulated in response to methyl jasmonate (MeJA). In silico target prediction identified 519 potential cro-miRNA targets that include several auxin response factors (ARFs). The presence of cleaved transcripts of miRNA-targeted ARFs in C. roseus cells was confirmed by Poly(A) Polymerase-Mediated Rapid Amplification of cDNA Ends (PPM-RACE). We showed that auxin (indole acetic acid, IAA) repressed the expression of key TIA pathway genes in C. roseus seedlings. Moreover, we demonstrated that a miRNA-regulated ARF, CrARF16, binds to the promoters of key TIA pathway genes and repress their expression. The C. roseus miRNAome reported here provides a comprehensive account of the cro-miRNA populations, as well as their abundance and expression profiles in response to MeJA. In addition, our findings underscore the importance of miRNAs in posttranscriptional control of the biosynthesis of specialized metabolites.

Removal of terminal buds (topping) and control of the formation of axillary shoots (suckers) are common agronomic practices that significantly impact the yield and quality of various crop plants. Application of chemicals (suckercides) to plants following topping is an effective method for sucker control. However, our current knowledge of the influence of topping, and subsequent suckercide applications, to gene expression is limited. We analyzed the differential gene expression using RNA-sequencing in tobacco (Nicotiana tabacum) that are topped, or treated after topping by two different suckercides, the contact-localized-systemic, Flupro(®) (FP), and contact, Off-Shoot-T(®). Among the differentially expressed genes (DEGs), 179 were identified as common to all three conditions. DEGs, largely related to wounding, phytohormone metabolism and secondary metabolite biosynthesis, exhibited significant upregulation following topping, and downregulation after suckercide treatments. DEGs related to photosynthetic processes were repressed following topping and suckercide treatments. Moreover, topping and FP-treatment affect the expression of auxin and cytokinin signaling pathway genes that are possibly involved in axillary shoot formation. Our results provide insights into the global change of plant gene expression in response to topping and suckercide treatments. The regulatory elements of topping-inducible genes are potentially useful for the development of a chemical-free sucker control system.

Oyster mushroom (Pleurotus sajor-caju) cultivated in the laboratory was studied for nutritional constituents, flavor components, antioxidant and antibacterial properties. Nutritional constituents estimated per 100 g dry weight (d.w.) include protein (29.3 g), carbohydrate (62.97 g), crude fat (0.91 g), ash (6.82 g) and crude fiber (12.3 g). Energy value of this mushroom was about 297.5 kcal/100 g d.w. Major mineral components estimated include Ca, Fe, and Mg with highest level of 505.0, 109.5 and 108.7 mg/100 g respectively. Methanolic extract containing significant amounts of phenols and flavonoids showed free radical scavenging potential and antibacterial activities against various spp. of Gram positive and Gram negative bacteria. Compounds responsible for antibacterial activities analyzed by GC-MS include β- Sistosterol, Cholestanol, 1,5-Dibenzoylnaphthalene and 1,2-Benzenedicarboxylic acid. Flavor components extracted by hot extraction method were found to be higher in number and concentration than the cold extraction method. The characteristic flavor component of mushroom i.e. 1-Octen-3-ol was better extracted by hot than the cold.

Background: Molecular markers allow rapid identification of biologically important germplasm/s having desired character. Previously we have reported a genotype specific molecular marker, Balco1128 [GenBank ID EU258678] of Bambusa balcooa containing an ORF (375 bp) having high similarity with receptor like cytoplasmic kinase of Arabidopsis and Oryza. Balco1128 was found to be associated only with bamboo genotypes endowed with high cellulose and low lignin contents of fibers. Under the above backdrop, it was necessitated to characterize this genetic marker for better understanding of its biological significance in context of superior quality fiber development., Results: The full length cDNA (3342 bp) of BbKst, a serine-threonine protein kinase was isolated from B. balcooa comprising of six LRR domains at the N-terminal end and a kinase domain at the C-terminal end. Bacteria-expressed BbKst-kinase domain (3339 bp long) showed Mg(2+) dependent kinase activity at pH 7.0, 28°C. Bioinformatics study followed by phospho-amino analysis further confirmed that BbKst-kinase belongs to the serine/threonine protein kinase family. Transcript analysis of the BbKst gene following RNA slot blot hybridization and qPCR revealed higher expression of BbKst during initiation and elongation stages of fiber development. Tissue specific expression studies showed much higher expression of BbKst transcript in stems and internodes of B. balcooa than in leaves and rhizomes. Southern analysis revealed single copy insertion of BbKst in most of the Agrobacterium mediated transgenic tobacco plants. Real-time PCR detected 150-200 fold enhanced expression of BbKst in different T1 tobacco lines than that of the vector transformed plants. Heterologous expression of BbKst under control of 35S promoter in transgenic tobacco showed high cellulose deposition in the xylem fibers. Number of xylary fibers was higher in transgenic T0 and T1 plants than that of empty-vector transformed tobacco plants offering enhanced mechanical strength to the transgenic plants, which was also substantiated by their strong upright phenotypes, significantly higher cellulose contents, flexibility coefficient, slenderness ratio, and lower Runkel ratio of the fibers., Conclusions: This finding clearly demonstrated that BbKst gene (GenBank ID JQ432560) encodes a serine/threonine protein kinase. BbKst induced higher cellulose deposition/synthesis in transgenic tobacco plants, an important attribute of fiber quality bestowing additional strength to the plant.

2-Mercaptobenzothiazolyl-(Z)-(2-aminothiazol-4-yl)-2-(tert-butoxycarbonyl) isopropoxyiminoacetate is used as supplementary additives in commercial-grade insecticides to compensate for the time factor needed for the actual pesticide chemical to start its action. This investigation describes the biodegradation of 2-mercaptobenzothiazolyl-(Z)-(2-aminothiazol-4-yl)-2-(tert-butoxycarbonyl) isopropoxyiminoacetate by Pseudomonas desmolyticum NCIM 2112. The biodegradation is influenced by other carbon and nitrogen sources and indicates that glucose and lactose are effective at 0.5% concentration whereas NaNO(3) and NaNO(2) at 0.05%. The percent degradation of 2-mercaptobenzothiazolyl-(Z)-(2-aminothiazol-4-yl)-2-(tert-butoxycarbonyl) isopropoxyiminoacetate was found to be 40%.The pH and temperature optima were found to be 7.0°C and 30°C, respectively. The effect on soil parameters was observed in treated soil and indicates remarkable decrease in soil fertility; the phytotoxicity indicates retarded growth and germination inhibition of treated seeds of Sorghum bicolor and Triticum aestivum. In paddy field the inhibition of germination of Oryza sativa was observed.

The present study reports the economic production of thermostable chitinase production from Oerskovia xanthineolytica NCIM 2839 by solid-state fermentation (SSF) technique and its application in fungal protoplasts formation. The Oerskovia xanthineolytica NCIM 2839 was found to produce thermostable chitinase 148 U g(-1) of solid substrate in SSF using wheat bran with colloidal chitin as base. Protoplasts of A. niger were formed by using crude chitinase produced in SSF and formed protoplasts were confirmed by using scanning electron microscopy. This is the simple and economical method for protoplast formation which makes it possible applications in strain improvement of various fungi by protoplasts fusion in Biotechnological industries.

Recently bamboo has gained reputation as a major resource of non-wood fiber. The present study was undertaken to generate information about fiber development process in bamboo (Bambusa balcooa) using PCR-based suppressive subtractive hybridization (PCR-SSH) technique, as molecular mechanism of its fiber development is yet to be explored. SSH was performed between cDNA isolated from leaf (as driver) and internodes (as tester) of B. balcooa which indicated up-regulation of 521 ESTs. Among these 41 were contigs and 65 ESTs were singletons. On the basis of BLASTX search 307 ESTs with known functions were classified into several functional categories including transport, metabolism, information, perception and response to stimuli while others were either non-significant (120) or hypothetical proteins (94). A total of 51 out of 307 functional ESTs were found fiber specific and their global distribution among different plant species like maize, rice, cotton and Arabidopsis ESTs were determined. Net distributions and differential expression patterns of 13 important B. balcooa fiber specific cDNAs among different internodes during bamboo development were studied using RNA slot-blot, semi-quantitative RT-PCR and real time PCR. In-situ localization of mRNA transcript for few selected bamboo fiber ESTs namely, V1Bb147 (protein kinase-like protein) and V1Bb88 (myb domain-containing protein) were detected using Confocal Laser Scanning Microscope. Transcript levels of these genes exhibited an orchestral turn-over during bamboo development, suggesting their close association with fiber development, an event associated with several metabolic and physiological changes. The results clearly suggest that these genes are involved in several concerted mechanisms involving Ca(+) signaling pathway, cell wall synthesis, hormone regulation, system maintaining cell turgor pressure and cytoskeleton synthesis pathway accountable for bamboo fiber development signifying fiber development as a complex but ordered metabolic process involving differential expression of large scale fiber associated genes. This is the first report on systematic analysis of genes involved in bamboo fiber development., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The thermophilic Bacillus licheniformis strain JS was isolated from a bed of mushrooms, Pleurotus sajor-caju. The organism could produce a novel, single-component, thermostable chitinase that was purified by ion-exchange chromatography using DEAE-cellulose in 7.64% yield and in an 8.1-fold enhancement in purity. Its molecular weight is 22kDa. The enzyme is a chitobiosidase, since the chitin hydrolysate is N(I),N(II)-diacetylchitobiose. The optimum temperature for enzyme activity is 55°C, and the optimum pH is 8.0. It was completely inhibited by Hg(2+) ions whereas Co(2+) ions served as an activator. The thermostability of this enzyme is important in the bioconversion of chitinous waste and for the production of chitooligosaccharides., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The mesophilic organism, Oerskovia xanthineolytica NCIM 2839, was adapted to grow at moderate thermophilic temperatures. At these elevated temperatures, it was found to produce two thermostable chitinases--C1 and C2. These were purified by ion exchange chromatography using DEAE cellulose. The chitinases C1 and C2 were found to be stable in a pH range from 3.0 to 9.0 with 7.5 and 8.0 being the optimum pH, respectively. The optimum temperatures of the activities of C1 and C2 were 50 and 55 degrees C, respectively. These were activated by Mn(++) and Cu(++)and inactivated by Hg(++). This is first report of an extracellular thermostable chitinase being produced by O. xanthineolytica NCIM 2839.

This paper attempts to evaluate the effectiveness of a keyword or descriptor in the title, from the viewpoint of document selection and information retrieval for scientists engaged in contraception research. A total of 2152 document titles recorded in the Index Medicus during 1973-75 was examined. Another set of 567 contraception titles published in the Index Medicus during 1963-65 was also studied to determine if there were any changes in the information content of the titles over a 10-year period. Employing the Montgomery-Swanson method, it was found that 92.2% of the documents from the later period and 88.0% of those from the earlier period had at least one "contraception term" in their titles. The data obtained in this study exceed those of published reports. A brief review of some of the previous studies dealing with the retrieval capabilities of titles in various branches of medical and life sciences is presented. It is concluded that the title search method is a very effective means of retrieving pertinent documents in the field of contraceptive technology. A trend of improvement in the information content of the scientific titles is also noted.