Transfer of grain softness from 5U-5A wheat-Aegilops triuncialis substitution line to bread wheat through induced homeologous pairing

Journal of Plant Biochemistry and Biotechnology - Bread wheat sustains genes for grain softness on “Ha” locus of short arm of 5D chromosome. Pina-D1 and Pinb-D1 alleles of...     

A micropropagation protocol for <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl. from root explants

The aim of this study was to develop a new micropropagation system for Cassia angustifolia Vahl., an important medicinal legume using root explant as starting material. Root explants taken from 30-day-old aseptic seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators: 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Organogenic nodular calli obtained on MS + TDZ (1.0 μM) were transferred to shoot regeneration medium supplemented with different cytokinins (BA, Kn or TDZ) either alone or in combination with auxin:indole-3-acetic acid or α-naphthalene acetic acid. Maximum shoot regeneration frequency (90%) was obtained on MS + BA (2.5 μM) + NAA (0.6 μM) wherein a maximum of 42.76 ± 1.47 shoot buds per explant were induced with a maximum conversion rate of 35.63 ± 0.75 shoots per explant and average shoot length of 5.43 ± 0.20 cm. Elongated microshoots were successfully rooted under ex vitro conditions by pulse treatment in 200 μM of indole-3-butyric acid for half an hour. Microshoots were rooted, acclimatized and hardened off simultaneously in sterilized soilrite inside the growth room and then established in pots containing sterilized soil and manure (1:1) and grown under greenhouse condition with 90% survival rate. The histological sections at different developmental stages of shoot buds revealed the organization of nodular meristematic zone leading to the orientation and differentiation of shoot buds in large number and thereafter conversion into healthy shoots.     

Diversity and dynamics of free-living and particle-associated Betaproteobacteria and Actinobacteria in relation to phytoplankton and zooplankton communities

The diversity of attached and free-living Actinobacteria and Betaproteobacteria, based on 16S rRNA gene sequences, was investigated in a mesotrophic lake during two periods of contrasting phytoplankton dominance. Comparison analyses showed a phylogenetic difference between attached and free-living communities for the two bacterial groups. For Betaproteobacteria, the betaI clade was detected at all sampling dates in free-living and attached bacterial communities and was the dominant clade contributing to 57.8% of the total retrieved operational taxonomic units (OTUs). For Actinobacteria, the acIV cluster was found to be dominant, followed by acI contributing to 45% and 25% of the total retrieved OTUs, respectively. This study allows the determination of eight new putative clades among the Betaproteobacteria termed lbI-lbVIII and a new putative clade named acLBI belonging to the Actinobacteria. The seasonal dynamics of phytoplankton and zooplankton communities have been reflected as changes in distinct bacterial phylotypes for both attached and free-living communities. For attached communities, relationships were observed between Actinobacteria and Chrysophyceae, and between Betaproteobacteria and Dinophyceae and Chlorophyceae biomass. On the other hand, within free-living communities, few actinobacterial clades were found to be dependent on either nutrients or phytoplankton communities, whereas Betaproteobacteria were mainly associated with biological parameters (i.e. phytoplankton and copepod communities).     

Methylthioadenosine/S-adenosylhomocysteine nucleosidase, a critical enzyme for bacterial metabolism

The importance of methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase in bacteria has started to be appreciated only in the past decade. A comprehensive analysis of its various roles here demonstrates that it is an integral component of the activated methyl cycle, which recycles adenine and methionine through S-adenosylmethionine (SAM)-mediated methylation reactions, and also produces the universal quorum-sensing signal, autoinducer-2 (AI-2). SAM is also essential for synthesis of polyamines, N-acylhomoserine lactone (autoinducer-1), and production of vitamins and other biomolecules formed by SAM radical reactions. MTA, SAH and 5'-deoxyadenosine (5'dADO) are product inhibitors of these reactions, and are substrates of MTA/SAH nucleosidase, underscoring its importance in a wide array of metabolic reactions. Inhibition of this enzyme by certain substrate analogues also limits synthesis of autoinducers and hence causes reduction in biofilm formation and may attenuate virulence. Interestingly, the inhibitors of MTA/SAH nucleosidase are very effective against the Lyme disease causing spirochaete, Borrelia burgdorferi, which uniquely expresses three homologous functional enzymes. These results indicate that inhibition of this enzyme can affect growth of different bacteria by affecting different mechanisms. Therefore, new inhibitors are currently being explored for development of potential novel broad-spectrum antimicrobials.     

Ultrasensitive signal-on DNA biosensor based on nicking endonuclease assisted electrochemistry signal amplification

Combining the advantages of signal-on strategy and nicking endonuclease assisted electrochemistry signal amplification (NEAESA), a new sensitive and signal-on electrochemical DNA biosensor for the sequence specific DNA detection based on NEAESA has been developed for the first time. A Hairpin-shape probe (HP), containing the target DNA recognition sequence, is thiol-modified at 5' end and immobilized on gold electrode via Au-S bonding. Subsequently, the HP modified electrode is hybridized with target DNA to form a duplex. Then the nicking endonuclease is added and nicks the HP strand in the duplex. After nicking, 3'-ferrocene (Fc)-labeled part complementary probe (Fc-PCP) is introduced on the electrode surface by hybridizing with the thiol-modified HP fragment, which results in the generation of electrochemical signal. Hence, the DNA biosensor is constructed successfully. The present DNA biosensor shows a wide linear range of 5.0×10(-13)-5.0×10(-8)M for detecting target DNA, with a low detection limit of 0.167pM. The proposed strategy does not require any amplifying labels (enzymes, DNAzymes, nanoparticles, etc.) for biorecognition events, which avoids false-positive results to occur frequently. Moreover, the strategy has the benefits of simple preparation, convenient operation, good selectivity, and high sensitivity. With the advantages mentioned above, this simple and sensitive strategy has the potential to be integrated in portable, low cost and simplified devices for diagnostic applications.     

Prolonged (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells in macaques

Although phosphoantigen-specific Vgamma2Vdelta2 T cells appear to play a role in antimicrobial and anticancer immunity, mucosal immune responses and effector functions of these gammadelta T cells during infection or phospholigand treatment remain poorly characterized. In this study, we demonstrate that the microbial phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment of macaques induced a prolonged major expansion of circulating Vgamma2Vdelta2 T cells that expressed CD8 and produced cytotoxic perforin during their peak expansion. Interestingly, HMBPP-activated Vgamma2Vdelta2 T cells underwent an extraordinary pulmonary accumulation, which lasted for 3-4 mo, although circulating Vgamma2Vdelta2 T cells had returned to baseline levels weeks prior. The Vgamma2Vdelta2 T cells that accumulated in the lung following HMBPP/IL-2 cotreatment displayed an effector memory phenotype, as follows: CCR5+CCR7-CD45RA-CD27+ and were able to re-recognize phosphoantigen and produce copious amounts of IFN-gamma up to 15 wk after treatment. Furthermore, the capacity of massively expanded Vgamma2Vdelta2 T cells to produce cytokines in vivo coincided with an increase in numbers of CD4+ and CD8+ alphabeta T cells after HMBPP/IL-2 cotreatment as well as substantial perforin expression by CD3+Vgamma2- T cells. Thus, the prolonged HMBPP-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells may confer immunotherapeutics against infectious diseases and cancers.     

Differences in cell morphology, lipid and apo B secretory capacity in caco-2 cells following long term treatment with saturated and monounsaturated fatty acids

The suitability of the caco-2 cell line as a model for studying the long term impact of dietary fatty acids on intestinal lipid handling and chylomicron production was examined. Chronic supplementation of caco-2 cells with palmitic acid (PA) resulted in a lower triacylglycerol secretion than oleic acid (OA). This was coupled with a detrimental effect of PA, but not OA, on transepithelial electrical resistance (TER) measurements, suggesting a loss of structural integrity across the cell monolayer. Addition of OA reversed the adverse effects of PA and stearic acid on TER and increased the ability of cells to synthesise and accumulate lipid, but did not normalise the secretion of lipids by caco-2 cells. Increasing amounts of OA and decreasing amounts of PA in the incubation media markedly improved the ability of cells to synthesise apolipoprotein B and secrete lipids. Real time RT-PCR revealed a down regulation of genes involved in lipoprotein synthesis following PA than OA. Electron microscopy showed adverse effects of PA on cellular morphology consistent with immature enterocytes such as stunted microvilli and poor tight junction formation. In conclusion, previously reported differences in lipoprotein secretion by caco-2 cells supplemented with saturated fatty acids (SFA) and OA may partly reflect early cytotoxic effects of SFA on cellular integrity and function.     

HIV-1 Vpr potently induces programmed cell death in the CNS in vivo

The human immunodeficiency virus type I (HIV-1) accessory protein Vpr has been associated with the induction of programmed cell death (apoptosis) and cell-cycle arrest. Studies have shown the apoptotic effect of Vpr on primary and established cell lines and on diverse tissues including the central nervous system (CNS) in vitro. However, the relevance of the effect of Vpr observed in vitro to HIV-1 neuropathogenesis in vivo, remains unknown. Due to the narrow host range of HIV-1 infection, no animal model is currently available. This has prompted us to consider a small animal model to evaluate the effects of Vpr on CNS in vivo through surrogate viruses expressing HIV-1Vpr. A single round of replication competent viral vectors, expressing Vpr, were used to investigate the apoptosis-inducing capabilities of HIV-1Vpr in vivo. Viral particles pseudotyped with VSV-G or N2c envelopes were generated from spleen necrosis virus (SNV) and HIV-1-based vectors to transduce CNS cells. The in vitro studies have demonstrated that Vpr generated by SNV vectors had less apoptotic effects on CNS cells compared with Vpr expressed by HIV-1 vectors. The in vivo study has suggested that viral particles, expressing Vpr generated by HIV-1-based vectors, when delivered through the ventricle, caused loss of neurons and dendritic processes in the cortical region. The apoptotic effect was extended beyond the cortical region and affected the hippocampus neurons, the lining of the choroids plexus, and the cerebellum. However, the effect of Vpr, when delivered through the cortex, showed neuronal damage only around the site of injection. Interestingly, the number of apoptotic neurons were significantly higher with HIV-1 vectors expressing Vpr than by the SNV vectors. This may be due to the differences in the proteins expressed by these viral vectors. These results suggest that Vpr induces apoptosis in CNS cells in vitro and in vivo. To our knowledge, this is the first study to investigate the apoptosis-inducing capabilities of HIV-1Vpr in vivo in neonatal mice. We propose that this, in expensive animal model, may be of value to design-targeted neuroprotective therapeutics.