Ascertaining clonal fidelity of micropropagated plants of Dendrocalamus hamiltonii Nees et Arn. ex Munro using molecular markers

Dendrocalamus hamiltonii is a giant, evergreen, clumping, multipurpose bamboo with strong culms which are mainly used for construction, handicrafts and fuel. The tender shoots are also used as food. Overexploitation of existing natural stocks coupled with harvesting of culms before seed formation, a long flowering cycle, irregular and poor seed production, short seed viability, seed sterility, limited availability of offsets and rhizomes and seasonal dependence are some of the major bottlenecks in conventional propagation of this species. Therefore, alternative methods like micropropagation can fill the gap in demand and supply of true-to-type planting material. Recently, our micropropagation protocol for rapid multiplication of D. hamiltonii through axillary bud proliferation using nodal explants from mature culms was standardized, and more than 3,000 plants were transferred to the field. However, somaclonal variations are known to appear in the in vitro-derived clones due to culture-induced stresses. Therefore, the present investigation was conducted to ascertain the effect of the length of in vitro culture age on clonal fidelity of regenerated plants using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. The genomic DNA samples (i.e. mother plant, in vitro-raised shoots from the 3rd to 30th passage, and in vitro-raised plants transferred to the field) were subjected to PCR amplification using 90 primer combinations (25 each of RAPD, ISSR and SSR, and 15 AFLP primer combinations) of which 76 (23 RAPD, 24 ISSR, 21 SSR and 8 AFLP) markers showed amplified DNA fragments. The 23 RAPD primers produced 162 distinct amplified DNA fragments from 2 (OPE-5) to 16 (OPE-16) fragments per primer, while 24 ISSR primers produced 181 distinct amplified DNA fragments with an average of 7.5 fragments per primer. The number of bands generated by SSR primers varied from 3 (RM-7 and RM-240) to 14 (RM-44), and the eight combinations of AFLP primers produced 369 distinct and scorable amplified DNA fragments with an average of 46.1 fragments per primer. Appearance of monomorphic bands with all the tested primer combinations confirmed the true-to-type nature of the in vitro clones of D. hamiltonii and hence the suitability of the developed micropropagation protocol for commercial-scale plant production.     

Microarray analysis of miRNA expression profiles following whole body irradiation in a mouse model

Abstract

Context: Accidental exposure to life-threatening radiation in a nuclear event is a major concern; there is an enormous need for identifying biomarkers for radiation biodosimetry to triage populations and treat critically exposed individuals.

Objective: To identify dose-differentiating miRNA signatures from whole blood samples of whole body irradiated mice.

Methods: Mice were whole body irradiated with X-rays (2?Gy–15?Gy); blood was collected at various time-points post-exposure; total RNA was isolated; miRNA microarrays were performed; miRNAs differentially expressed in irradiated vs. unirradiated controls were identified; feature extraction and classification models were applied to predict dose-differentiating miRNA signature.

Results: We observed a time and dose responsive alteration in the expression levels of miRNAs. Maximum number of miRNAs were altered at 24-h and 48-h time-points post-irradiation. A 23-miRNA signature was identified using feature selection algorithms and classifier models. An inverse correlation in the expression level changes of miR-17 members, and their targets were observed in whole body irradiated mice and non-human primates.

Conclusion: Whole blood-based miRNA expression signatures might be used for predicting radiation exposures in a mass casualty nuclear incident.     

Structural and Functional Studies of a Phosphatidic Acid-Binding Antifungal Plant Defensin MtDef4: Identification of an RGFRRR Motif Governing Fungal Cell Entry

MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β₂ and β₃ strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.     

Highly Specific Culture-Independent Detection of YGNGV Motif-Containing Pediocin-Producing Strains

A novel method based on (1) initial microbiological screening and (2) a highly specific PCR is described for selection of strains expressing YGNGV motif-containing pediocin. Initial screening is carried out using spot on the lawn assay for selection of acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity producing strains. This is followed by highly specific PCR for amplification of 406-bp fragment using forward primer: 5′-tggccaatatcattggtggt-3′ targeting signal peptide sequence of pediocin structural gene and reverse primer: 5′-ctactaacgcttggctggca-3′ encoding N-terminus of immunity gene. The assay was validated with Pediococcus pentosaceus NCDC273 and Pediococcus acidilactici NCDC252 using (1) digestion of amplified 406-bp fragment with HindIII restriction enzyme-producing two restriction fragments of expected sizes (227 and 179 bp), (2) nucleotide sequencing of 406-bp fragment from both strains found these pediocins identical to pediocin PA-1/AcH and (3) identification of both pediocins as pediocin PA-1 at protein level using RP-HPLC. The assay was used for screening six strains (3 pediococci, 2 lactobacilli and an Enterococcus faecium) producing acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity. This resulted in the detection of three new strains (P. pentosaceus NCDC35, E. faecium NCDC124 and Lactobacillus plantarum NCDC20) producing YGNGV motif-containing pediocins.     

miReader: Discovering Novel miRNAs in Species without Sequenced Genome

Along with computational approaches, NGS led technologies have caused a major impact upon the discoveries made in the area of miRNA biology, including novel miRNAs identification. However, to this date all microRNA discovery tools compulsorily depend upon the availability of reference or genomic sequences. Here, for the first time a novel approach, miReader, has been introduced which could discover novel miRNAs without any dependence upon genomic/reference sequences. The approach used NGS read data to build highly accurate miRNA models, molded through a Multi-boosting algorithm with Best-First Tree as its base classifier. It was comprehensively tested over large amount of experimental data from wide range of species including human, plants, nematode, zebrafish and fruit fly, performing consistently with >90% accuracy. Using the same tool over Illumina read data for Miscanthus, a plant whose genome is not sequenced; the study reported 21 novel mature miRNA duplex candidates. Considering the fact that miRNA discovery requires handling of high throughput data, the entire approach has been implemented in a standalone parallel architecture. This work is expected to cause a positive impact over the area of miRNA discovery in majority of species, where genomic sequence availability would not be a compulsion any more.     

The N276 Glycosylation Site Is Required for HIV-1 Neutralization by the CD4 Binding Site Specific HJ16 Monoclonal Antibody

Immunogen design for HIV-1 vaccines could be based on epitope identification of naturally occurring neutralizing antibodies in infected patients. A tier 2 neutralizing monoclonal antibody (mAb), HJ16 recognizes a new epitope in the CD4 binding site (CD4bs) region that only partially overlaps with the b12 epitope. We aimed to identify the critical binding site by resistance induction in a sensitive primary CRF02_AG strain. In four independent dose-escalation studies, the N276D mutation was consistently the only alteration found and it was confirmed to be responsible for resistance to HJ16 by site-directed mutagenesis in envelopes (envs) of the homologous CRF02_AG, as well as of a subtype A and a subtype C primary isolate. This mutation removes an N-linked glycosylation site. The effect of N276D was very selective, as it failed to confer resistance to a range of other entry inhibitors. Remarkably, sensitivity to the CD4bs VRC01 and VRC03 mAbs was increased in the N276D mutated viruses. These data indicate that binding of the CD4bs specific HJ16 mAb critically depends on the interaction with the N276-glycan, thus indicating that HJ16 is the first glycan dependent CD4bs-specific mAb.     

Modeling the dynamics of Hepatitis C virus with combined antiviral drug therapy: Interferon and Ribavirin

A mathematical modeling of hepatitis C virus (HCV) dynamics and antiviral therapy has been presented in this paper. The proposed model, which involves four coupled ordinary differential equations, describes the interaction of target cells (hepatocytes), infected cells, infectious virions and non-infectious virions. The model takes into consideration the addition of ribavirin to interferon therapy and explains the dynamics regarding a biphasic and triphasic decline of viral load in the model. A critical drug efficacy parameter has been defined and it is shown that for an efficacy above this critical value, HCV is eradicated whereas for efficacy lower this critical value, a new steady state for infectious virions is reached, which is lower than the previous steady state value.     

An Anionic Phospholipid Enables the Hydrophobic Surfactant Proteins to Alter Spontaneous Curvature

The hydrophobic surfactant proteins, SP-B and SP-C, greatly accelerate the adsorption of the surfactant lipids to an air/water interface. Previous studies of factors that affect curvature suggest that vesicles may adsorb via a rate-limiting structure with prominent negative curvature, in which the hydrophilic face of the lipid leaflets is concave. To determine if SP-B and SP-C might promote adsorption by inducing negative curvature, we used small-angle x-ray scattering to test whether the physiological mixture of the two proteins affects the radius of cylindrical monolayers in the inverse hexagonal phase. With dioleoyl phosphatidylethanolamine alone, the proteins had no effect on the hexagonal lattice constant, suggesting that the proteins fail to insert into the cylindrical monolayers. The surfactant lipids also contain ∼10% anionic phospholipids, which might allow incorporation of the cationic proteins. With 10% of the anionic dioleoyl phosphatidylglycerol added to dioleoyl phosphatidylethanolamine, the proteins induced a dose-related decrease in the hexagonal lattice constant. At 30°C, the reduction reached a maximum of 8% relative to the lipids alone at ∼1% (w/w) protein. Variation of NaCl concentration tested whether the effect of the protein represented a strictly electrostatic effect that screening by electrolyte would eliminate. With concentrations up to 3 M NaCl, the dose-related change in the hexagonal lattice constant decreased but persisted. Measurements at different hydrations determined the location of the pivotal plane and proved that the change in the lattice constant produced by the proteins resulted from a shift in spontaneous curvature. These results provide the most direct evidence yet that the surfactant proteins can induce negative curvature in lipid leaflets. This finding supports the model in which the proteins promote adsorption by facilitating the formation of a negatively curved, rate-limiting structure.