Fast interaction dynamics of G-quadruplex and RGG-rich peptides unveiled in zero-mode waveguides

G-quadruplexes (GQs), a non-canonical form of DNA, are receiving a huge interest as target sites for potential applications in antiviral and anticancer drug treatments. The biological functions of GQs can be controlled by specifically binding proteins known as GQs binding proteins. Some of the GQs binding proteins contain an arginine and glycine-rich sequence known as RGG peptide. Despite the important role of RGG, the GQs-RGG interaction remains poorly understood. By single molecule measurements, the interaction dynamics can be determined in principle. However, the RGG–GQs interaction occurs at micromolar concentrations, making conventional single-molecule experiments impossible with a diffraction-limited confocal microscope. Here, we use a 120 nm zero-mode waveguide (ZMW) nanoaperture to overcome the diffraction limit. The combination of dual-color fluorescence cross-correlation spectroscopy (FCCS) with FRET is used to unveil the interaction dynamics and measure the association and dissociation rates. Our data show that the RGG–GQs interaction is predominantly driven by electrostatics but that a specific affinity between the RGG sequence and the GQs structure is preserved. The single molecule approach at micromolar concentration is the key to improve our understanding of GQs function and develop its therapeutic applications by screening a large library of GQs-targeting peptides and proteins.     

Pyrophosphatase, Peroxidase and Polyphenoloxidase Activities during Leaf Development and Senescence

Inorganic pyrophosphatase, peroxidase, and polyphenoloxidase activities were studied as the function of leaf insertion level in eight monocotyledonous and eight dicotyledonous species. Alkaline inorganic pyrophosphatase shows a declining activity toward the end of senescence whereas no regular drift in either peroxidase or polyphenoloxidase activities was noticed during senescence of attached leaves. In the primary leaves of rice, peroxidase and polyphenoloxidase activities were high in the senescent leaves and there exists a correlation between chlorophyll content and peroxidase activity though not with polyphenoloxidase activity. Upon detachment leaves exhibit increasing peroxidase and polyphenoloxidase activities with time. The distribution of the enzyme activities during senescence of attached leaves is suggested to be species-specific, and an increase in peroxidase and polyphenoloxidase activities cannot be taken as an indicator of leaf senescence.     

Proteomic analysis of zygote and ookinete stages of the avian malaria parasite Plasmodium gallinaceum delineates the homologous proteomes of the lethal human malaria parasite Plasmodium falciparum

Delineation of the complement of proteins comprising the zygote and ookinete, the early developmental stages of Plasmodium within the mosquito midgut, is fundamental to understand initial molecular parasite-vector interactions. The published proteome of Plasmodium falciparum does not include analysis of the zygote/ookinete stages, nor does that of P. berghei include the zygote stage or secreted proteins. P. gallinaceum zygote, ookinete, and ookinete-secreted/released protein samples were prepared and subjected to Multidimensional protein identification technology (MudPIT). Peptides of P. gallinaceum zygote, ookinete, and ookinete-secreted proteins were identified by MS/MS, mapped to ORFs (> 50 amino acids) in the extent P. gallinaceum whole genome sequence, and then matched to homologous ORFs in P. falciparum. A total of 966 P. falciparum ORFs encoding orthologous proteins were identified; just over 40% of these predicted proteins were found to be hypothetical. A majority of putative proteins with predicted secretory signal peptides or transmembrane domains were hypothetical proteins. This analysis provides a more comprehensive view of the hitherto unknown proteome of the early mosquito midgut stages of P. falciparum. The results underpin more robust study of Plasmodium-mosquito midgut interactions, fundamental to the development of novel strategies of blocking malaria transmission.     

Use of essential oil as botanical-pesticide against post harvest spoilage in Malus pumilo fruit

During antifungal screening of the essentialoils of some angiospermic plants, oil ofCymbopogon flexuosus showed potentbioactivity against dominant post harvestfungal pathogens. The minimum bioactiveconcentrations with fungicidal action of theoil was found to be 0.2 µl ml^–1 for Alternaria alternata, 0.4 µlml^–1 for Aspergillus flavus, A.fumigatus, A. niger, A. parasiticus,Cladosporium cladosporioides, Colletotrichumcapsici, C. falcatum, Curvularia lunata,Fusarium cerealis, F. culmorum, F. oxysporum,F. udum, Gloeosporium fructigenum, Penicilliumexpansum, P. italicum, P. implicatum, P. digitatum,P. minio-luteum, P. variable,and 0.5 µl ml^–1 forBotrytis cinerea, Helminthosporium oryzae, H.maydis, Phoma violacea, Rhizopus nigricans.The oil exhibited potency against heavy doses(30 mycelial disc, each of 5 mm in diameter) ofinoculum at 1.0 µl ml^–1 concentrations.The bioactivity of the oil was thermostable upto 100°C and lasted up to 48 months.The oil preparation did not exhibit anyphytotoxic effect on the fruit skins ofMalus pumilo up to 50 µlml^–1 concentrations. In vivo trials of theoil as a fungicidal spray on Malus pumilofor checking the rotting of fruits, it showedthat 20 µl ml^–1 concentration controls100% infection by pre-inoculation treatment,while in post-inoculation treatment, 30 µlml^–1 concentration of fungicidal spraywas required for the 100% control of rotting.The fungicidal spray was found to be costeffective (INR 15/l), has long shelf life (48month) and was devoid of any adverse effects.Therefore, it can be used as a potential sourceof sustainable eco-friendly botanicalpesticide, after successfulcompletion of wide range trials.     

Use of Long-Range Repetitive Element Polymorphism-PCR To Differentiate Bacillus anthracis Strains

The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105 B. anthracis strains of diverse geographical origins. All B. anthracis strains produced fingerprints comprising seven to eight bands, referred to as “skeleton” bands, while one to three “diagnostic” bands differentiated between B. anthracis strains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracis strains.     

The crucial role and regulations of miRNAs in zebrafish development

To comprehend the events during developmental biology, fundamental knowledge about the basic machinery of regulation is a prerequisite. MicroRNA (miRNAs) act as regulators in most of the biological processes and recently, it has been concluded that miRNAs can act as modulatory factors even during developmental process from lower to higher animal. Zebrafish, because of its favorable attributes like tiny size, transparent embryo, and rapid external embryonic development, has gained a preferable status among all other available experimental animal models. Currently, zebrafish is being utilized for experimental studies related to stem cells, regenerative molecular medicine as well drug discovery. Therefore, it is important to understand precisely about the various miRNAs that controls developmental biology of this vertebrate model. In here, we have discussed about the miRNA-controlled zebrafish developmental stages with a special emphasis on different miRNA families such as miR-430, miR-200, and miR-133. Moreover, we have also reviewed the role of various miRNAs during embryonic and vascular development stages of zebrafish. In addition, efforts have been made to summarize the involvement of miRNAs in the development of different body parts such as the brain, eye, heart, muscle, and fin, etc. In each section, we have tried to fulfill the gaps of zebrafish developmental biology with the help of available knowledge of miRNA research. We hope that precise knowledge about the miRNA-regulated developmental stages of zebrafish may further help the researchers to efficiently utilize this vertebrate model for experimental purpose.     

Salicylic acid triggers genotoxic adaptation to methyl mercuric chloride and ethyl methane sulfonate, but not to maleic hydrazide in root meristem cells of Allium cepa L

Salicylic acid (SA), 0.01 mM, a signalling phytohormone, was tested for induction of adaptive response against genotoxicity of methyl mercuric chloride (MMCl), 0.013 mM; ethylmethane sulfonate (EMS), 2.5 mM, or maleic hydrazide (MH), 5 mM, in root meristem cells of Allium cepa. Induction of adaptive response to EMS by hydrogen peroxide (H2O2), 1 mM, and yet another secondary signal molecule was tested for comparison. Assessed by the incidence of mitoses with spindle and/or chromosome aberration and micronucleus, the findings provided evidence that SA-conditioning triggered adaptive response against the genotoxic-challenges of MMCl and EMS, but failed to do so against MH. H2O2, which is known to induce adaptive response to MMCl and MH, failed to induce the same against EMS in the present study. The findings pointed to the possible role of signal transduction in the SA-induced adaptive response to genotoxic stress that perhaps ruled out an involvement of H2O2.