Stable germ line transformation of a leafy vegetable crop amaranth (Amaranthus tricolor L.) mediated by Agrobacterium tumefaciens

We have optimized a procedure for genetic transformation of a major leafy vegetable crop, Amaranthus tricolor L., using epicotyl explant co-cultivation with Agrobacterium tumefaciens. Two disarmed A. tumefaciens strains EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT harboring the neomycin phosphotransferase II gene (nptII) and the β-glucuronidase gene (gus), were evaluated as vector systems. The former displayed a higher transforming efficiency. Several key factors influencing the transformation events were optimized. The highest percentage of transformed shoots (24.24%) was achieved using hand-pricked epicotyl explants, a 10-min infection period, with 100 μM acetosyringone-pretreated Agrobacterium culture corresponding to OD₆₀₀???0.6 and diluted to 10⁹ cells ml^?1, followed by 4 d co-cultivation in the regeneration medium. Putative transformed explants capable of forming shoots were selected on medium supplemented with 75 μg?ml^?1 kanamycin, and transient as well as stable glucuronidase expression was determined by histochemical analysis. From a total of 48 selected shoot lines derived from independent transformation events with epicotyl explants co-cultivated with EHA 105, 32 showed positive PCR amplification for both the nptII and gus genes. Germ line transformation and transgene stability were evident in progeny of primary transformed plants (T₀). Among T₁ seedlings of 12 selected transgenic plant lines, kanamycin-resistant and kanamycin-sensitive seedlings segregated in a ratio typical of the Mendelian monohybrid pattern (3:1) as verified by the chi-square (χ ²) test. Southern hybridization of genomic DNA from kanamycin-resistant T₁ transgenic segregants to an nptII probe substantiated stable integration of the transgene. Neomycin phosphotransferase (NPTII) activity was detected in leaf protein extracts of selected T₁ transgenic plants, thereby confirming stable expression of the nptII gene.     

KINETIC STUDIES OF L-ASPARAGINASE FROM Penicillium digitatum

L-Asparaginase is an enzyme used in the treatment of acute lymphoblastic leukemia and other related malignancies. Its further use includes reduction of asparagine concentration in food products, which may lead to formation of acrylamide. Currently bacterial asparaginase is produced at industrial scale, but the enzyme isolated from bacterial origin is often associated with adverse reactions. These side effects require development of asparaginase from alternative sources. In the present study, Penicillium digitatum was explored for the production of extracellular L-asparaginase using modified Czapek–Dox media. The enzyme was purified about 60.95-fold and then kinetic study showed that the Km value of the enzyme was 1 × 10^?5 M. The optimum pH and temperature for the enzyme were 7.0 and 30°C, respectively. The optimum incubation period for L-asparaginase was 15 min. This work concludes that this enzyme can be a suitable candidate due to its strong kinetic properties, and further research can usher into development of asparaginase formulation from fungal origin with less adverse effects.     

Synthesis of Cholesteryl Supports and Phosphoramidite for Automated DNA Synthesis of Triple-Helix Forming Oligonucleotides (Tfos)

Abstract

DMT-Cholesteryl succinylamino solid supports (CPG, loading: 33 μmole/gram and TentaGel loading: 152 μmole/gram) and DMT-cholesteryl phosphoramidite were prepared for use in automated DNA synthesis of cholesteryl modified TFOs in the synthesis scales from 0.2 to 300 μmole. The modified TFOs were found to have a 5 to 50 fold increase in their uptake properties.     

Physicomechanical Characterization and Optimization of EDTA–mPEG and Avicel®–EDTA–mPEG In Situ Melt Dispersion Mini-Pellets

The purpose of this study was to develop a physicomechanically customizable oral metal chelatory in situ hot melt dispersion mini-pellet entity which could be utilized within a binary drug delivery system. Avicel^® RC/CL type R-591 was included within the in situ hot melt dispersion mini-pellet formulations to determine the physicomechanical effect this compound would have on the mini-pellet formulations. The physicomechanical properties of the hot melt in situ mini-pellet formulations were mathematically fitting to regression curves. Physicomechanical adjustment of the in situ hot melt dispersion mini-pellet formulations could be mathematically predicted with the derived regression curve equations. The addition of Avicel^® RC/CL type R-591 increased the physicomechanical properties such as matrix hardness and increased total disintegration of the in situ hot melt dispersion mini-pellet formulations. The utilization of a physicomechanically customizable oral metal chelatory in situ hot melt dispersion mini-pellet entity within a binary drug delivery system would to achieve a synergistically enhance the activity of a drug-carrying entity or a permeation enhancing entity within a single drug delivery unit. The experimental results indicated that weights of the pellets that achieved optimal hardness ranged between 35 and 45 mg. The melt–dispersion formulations disintegrated within shorter time periods and maintained higher ethylenediaminetetraacetic acid (EDTA) concentrations whereas melt–dispersion formulations which included Avicel^® had superior physicomechanical properties. Disintegration times ranged between 1,000 s for melt–dispersions containing EDTA and methyloxy polyethylene glycol 2000 (mPEG) only, to >6,000 s for melt–dispersions comprising EDTA, mPEG, and Avicel^®.     

A Review of Polymeric Refabrication Techniques to Modify Polymer Properties for Biomedical and Drug Delivery Applications

Polymers are extensively used in the pharmaceutical and medical field because of their unique and phenomenal properties that they display. They are capable of demonstrating drug delivery properties that are smart and novel, such properties that are not achievable by employing the conventional excipients. Appropriately, polymeric refabrication remains at the forefront of process technology development in an endeavor to produce more useful pharmaceutical and medical products because of the multitudes of smart properties that can be attained through the alteration of polymers. Small alterations to a polymer by either addition, subtraction, self-reaction, or cross reaction with other entities have the capability of generating polymers with properties that are at the level to enable the creation of novel pharmaceutical and medical products. Properties such as stimuli-responsiveness, site targeting, and chronotherapeutics are no longer figures of imaginations but have become a reality through utilizing processes of polymer refabrication. This article has sought to review the different techniques that have been employed in polymeric refabrication to produce superior products in the pharmaceutical and medical disciplines. Techniques such as grafting, blending, interpenetrating polymers networks, and synthesis of polymer complexes will be viewed from a pharmaceutical and medical perspective along with their synthetic process required to attain these products. In addition to this, each process will be evaluated according to its salient features, impeding features, and the role they play in improving current medical devices and procedures.     

Proteomic profiling of serum samples from chikungunya-infected patients provides insights into host response

Background

Chikungunya is a highly debilitating febrile illness caused by Chikungunya virus, a single-stranded RNA virus, which is transmitted by Aedes aegypti or Aedes albopictus mosquito species. The pathogenesis and host responses in individuals infected with the chikungunya virus are not well understood at the molecular level. We carried out proteomic profiling of serum samples from chikungunya patients in order to identify molecules associated with the host response to infection by this virus.

Results

Proteomic profiling of serum obtained from the infected individuals resulted in identification of 569 proteins. Of these, 63 proteins were found to be differentially expressed (≥ 2-fold) in patient as compared to control sera. These differentially expressed proteins were involved in various processes such as lipid metabolism, immune response, transport, signal transduction and apoptosis.

Conclusions

This is the first report providing a global proteomic profile of serum samples from individuals infected with the chikungunya virus. Our data provide an insight into the proteins that are involved as host response factors during an infection. These proteins include clusterin, apolipoproteins and S100A family of proteins.     

Utility of Host Delivered RNAi of Two FMRF Amide Like Peptides,flp-14 and flp-18, for the Management of Root Knot Nematode,Meloidogyne incognita

Root knot nematode, Meloidogyne incognita, is an obligate sedentary endoparasite that infects a large number of crop species and causes substantial yield losses. Non-chemical based control strategies for these nematodes are gaining importance. In the present study, we have demonstrated the significance of two FMRFamide like peptide genes (flp-14 and flp-18) for infection and development of resistance to M. incognita through host-derived RNAi. The study demonstrated both in vitro and in planta validation of RNAi-induced silencing of the two genes cloned from J2 stage of M. incognita. In vitro silencing of both the genes interfered with nematode migration towards the host roots and subsequent invasion into the roots. Transgenic tobacco lines were developed with RNAi constructs of flp-14 and flp-18 and evaluated against M. incognita. The transformed plants did not show any visible phenotypic variations suggesting the absence of any off-target effects. Bioefficacy studies with deliberate challenging of M. incognita resulted in 50-80% reduction in infection and multiplication confirming the silencing effect. We have provided evidence for in vitro and in planta silencing of the genes by expression analysis using qRT-PCR. Thus the identified genes and the strategy can be used as a potential tool for the control of M. incognita. This is the first ever report that has revealed the utility of host delivered RNAi of flps to control M. incognita. The strategy can also be extended to other crops and nematodes.     

Theoretical investigation on the kinetics and branching ratio of the gas phase reaction of sevoflurane with Cl atom

The present work deals with the theoretical investigation on the Cl initiated H-atom abstraction reaction of sevoflurane, (CF₃)₂CHOCH₂F. A dual-level procedure has been adopted for studying the kinetics of the reaction. Geometrical optimization and frequency calculation were performed at DFT(BHandHLYP)/6-311G(d,p) while single-point energy calculation was made at CCSD(T)/6-311G(d,p) level of theory. The intrinsic reaction coordinate (IRC) calculation has also been performed to confirm the smooth transition from the reactant to product through the respective transition state. The rate constants were calculated using conventional transition state theory (TST). It has been found that 99 % of the reaction proceeded via the H-atom abstraction from the –CH₂F end of the sevoflurane. The rate constant of the dominant path is found to be 1.13 × 10^?13 cm³ molecule^?1 s^?1. This is in excellent agreement with the reported experimental rate constant of 1.10 × 10^?13 cm³ molecule^?1 s^?1 obtained by relative rate method using FTIR/Smog chamber and LP/LIF techniques.     

405 nm-Excited Fluorescence Spectra of the Juice of the Lemon (Citrus x Limon)

A 405 nm diode laser is used to excite fluorescence of juices of raw and ripe lemons. Emission bands appear approximately at 520 nm and 670 nm. Fluorescence intensity ratio F520/F670 is determined for the two stages. Variation in the fluorescence intensity ratio is observed during the process of ripening or growth of the fruit. Time-resolved spectra at this excitation wavelength reveal two decay times at both the stages at the emission wavelength of 520 nm, and two decay times at the raw stage and one decay time at the ripe stage at 670 nm.