A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.)

A construct suitable for genome-wide transfer-DNA (T-DNA) and subsequent transposon-based (Ds) gene trapping has been developed for use in rice (Oryza sativa). This T-DNA/Ds construct contains: Ds terminal sequences immediately inside T-DNA borders for subsequent Ds mobilization; promoterless green fluorescent protein (sgfpS65T) and beta-glucuronidase (uidA) reporter genes, each fused to an intron (from Arabidopsis GPA1 gene) to enable bidirectional gene trapping by T-DNA or Ds; an ampicillin resistance gene (bla) and a bacterial origin of replication (ori) to serve as the plasmid rescue system; an intron-containing hygromycin phosphotransferase gene (hph) as a selectable marker or Ds tracer; and an intron-containing barnase gene in the binary vector backbone (VB) to select against transformants carrying unwanted VB sequences. More than a threefold increase over previously reported reporter gene-based gene trapping efficiencies was observed in primary T-DNA/Ds transformant rice lines, returning an overall reporter gene expression frequency of 23%. Of the plant organs tested, 3.3-7.4% expressed either reporter at varying degrees of organ or tissue specificity. Approximately 70% of the right border (RB) flanking sequence tags (FSTs) retained 1-6 bp of the RB repeat and 30% of the left border (LB) FSTs retained 5-23 bp of the LB repeat. The remaining FSTs carried deletions of 2-84 bp inside the RB or 1-97 bp inside the LB. Transposition of Ds from the original T-DNA was evident in T-DNA/Ds callus lines super-transformed with a transposase gene (Ac) construct, as indicated by gene trap reporter activity and rescue of new FSTs in the resulting double transformant lines.     

The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides

In this study, we utilized potent antisense oligonucleotides to examine the role of two Bcl-2 family members found in human umbilical vein endothelial cells (HUVEC). The first, A1, is thought to be a TNF-alpha-inducible cytoprotective gene, and the second, Bcl-XL, is constitutively expressed. Inhibition of the constitutive levels of Bcl-XL caused 10-25% of the cell population to undergo apoptosis and increased the susceptibility of cells to treatment with low concentrations of staurosporin or ceramide. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2 prevented DNA fragmentation and DeltaYm loss caused by Bcl-XL inhibition or Bcl-XL inhibition combined with staurosporin. However, disruption of DeltaYm caused by Bcl-XL inhibition combined with ceramide treatment was not inhibited by benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2, although DNA fragmentation was completely prevented. Taken together, these results demonstrate a direct protective role for Bcl-XL under normal resting conditions and under low level apoptotic challenges to HUVEC. Furthermore, Bcl-XL protects cells from caspase-dependent and -independent mechanisms of DeltaYm disruption. In contrast to Bcl-XL, A1 inhibition did not show a marked effect on the susceptibility of HUVEC to undergo apoptosis in response to TNF-alpha, ceramide, or staurosporin. These results demonstrate that although A1 may be a cytoprotective gene induced by TNF-alpha, it is not primarily responsible for HUVEC resistance to this cytokine.     

Enzymatic activity and inhibition of the neurotoxic complex vipoxin from the venom of Vipera ammodytes meridionalis

Vipoxin from the venom of Vipera ammodytes meridionalis is an unique neurotoxic complex between a toxic phospholipase A2 and a highly homologous non-toxic protein inhibitor. It is an example of evolution of a catalytic and toxic function into inhibitory and non-toxic one. The activity of the V. ammodytes meridionalis toxin is 1.7 times higher than that of the closely related (92% sequence identity) neurotoxic complex RV4/RV7 from the venom of Vipera russelli formosensis The enhanced enzymatic activity of vipoxin is attributed to limited structural changes, in particular to the substitutions G54R and Q78K in the PLA2 subunit of the complex and to the T54R substitution in the inhibitor. Oleyloxyethylphosphocholine, aristolochic acid and vitamin E suppressed the enzymatic activity of vipoxin and its isolated PLA2 subunit. These compounds influence inflammatory processes in which PLA2 is implicated. The peptide Lys-Ala-Ile-Tyr-Ser, which is an integral part of the PLA2 components of the two neurotoxic complexes from V. ammodytes meridionalis and V. russelli formosensis (sequence 70-74) activated vipoxin increasing its PLA2 activity by 23%. This is in contrast to the inhibitory effect of the respective pentapeptides with 70-74 sequences on other group II PLA2s. Surprisingly, the same peptide inhibited 46% of the V. russelli formosensis PLA2 activity. The limited changes in the structure of the two highly homologous neurotoxins lead to considerable differences in their interaction with native peptides.     

Molecular-genetic analysis of two cases with retinoblastoma: Benefits for disease management

Effective counselling and management of retinoblastoma families using genetic information is presently practised in many parts of the world. We studied histopathological, chromosomal and molecular-genetic data of two retinoblastoma patients from India. The two patients, one with bilateral and the other with unilateral retinoblastoma, underwent complete ophthalmic examination, cytogenetic study, retinoblastoma gene (RB1) mutational analysis andRB1 promoter region methylation screening. In the bilateral retinoblastoma patient deletion of chromosome region 13q14 in peripheral blood lymphocytes and a hemizygous novel 8-bp deletion in exon 4 ofRB1 in tumour sample were observed. In the unilaterally affected patient CGA to TGA transition protein truncation mutations were observed in exons 8 and 14 ofRB1.     

The Enterococcus faecalis MSCRAMM ACE binds its ligand by the Collagen Hug model

We have determined the crystal structure of the ligand binding segment of the Enterococcus faecalis collagen binding MSCRAMM ACE (microbial surface components recognizing adhesive matrix molecules adhesin of collagen from enterococci). This segment is composed of two subdomains, N(1) and N(2), each adopting an IgG-like fold and forming a putative collagen binding surface at the interface between the two subdomains. This structure is very similar to that recently reported for CNA, the collagen binding MSCRAMM of Staphylococcus aureus, for which a unique ligand binding mechanism called the Collagen Hug was proposed. We suggest that ACE binds collagen by a similar mechanism and present the first biochemical evidence for this binding model. Replacing residues in the putative collagen binding trench of ACE N(2) with Ala residues affected collagen binding. A closed conformation of ACE stabilized by an engineered disulfide bond is unable to bind collagen. Finally, the importance of the residues in the N(2) extension in stabilizing the MSCRAMM-ligand complex is demonstrated by selected point and truncation mutations.     

Synthesis and biological evaluation of pyrazole linked benzothiazole-β-naphthol derivatives as topoisomerase I inhibitors with DNA binding ability

A series of new pyrazole linked benzothiazole-β-naphthol derivatives were designed and synthesized using a simple, efficient and ecofriendly route under catalyst-free conditions in good to excellent yields. These derivatives were evaluated for their cytotoxicity on selected human cancer cell lines. Among those, the derivatives 4j, 4k and 4l exhibited considerable cytotoxicity with IC₅₀ values ranging between 4.63 and 5.54?µM against human cervical cancer cells (HeLa). Structure activity relationship was elucidated by varying different substituents on benzothiazoles and pyrazoles. Further, flow cytometric analysis revealed that these derivatives induced cell cycle arrest in G2/M phase and spectroscopic studies such as UV–visible, fluorescence and circular dichroism studies showed that these derivatives exhibited good DNA binding affinity. Additionally, these derivatives can effectively inhibit the topoisomerase I activity. Viscosity studies and molecular docking studies demonstrated that the derivatives bind with the minor groove of the DNA.     

Noncovalent interaction of G-quadruplex DNA with acridine at low concentration monitored by MALDI-TOF mass spectrometry

The telomeric G-rich single-stranded DNA d(T(2)G(8)) can adopt in vitro G-quadruplex structure, even at low DNA concentration. Studies on stability of telomeric structures, has gained importance recently as the molecules, which can stabilize quadruplex structure, can inhibit cancer progression. In this study, G-quadruplex structure is formed by 1.0 mM NH(4)(I) ion. Stability of G-quadruplex complex is studied on interaction with acridine using CD and MALDI-TOF mass spectrometry. MALDI-TOF mass spectrometric experiments were carried out mainly to observe the noncovalent drug-DNA interactions at low concentration. From MALDI-TOF spectrum, it is identified that three ammonium ions are required for the formation of G-quadruplex structure and to provide stability to NH(4)(I)-G-quadruplex complex. With MALDI-TOF it is evident that two acridine molecules interact with NH(4)(I) G-quadruplex complex. CD studies, shows that stability of NH(4)(I) G-quadruplex, decreases and conformation change takes place on interaction with acridine. Interaction with drug reduces mostly due to transformation of G-quadruplex complex to single stranded DNA.     

Reprograming of gut microbiome energy metabolism by the FUT2 Crohn's disease risk polymorphism

Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. The H antigen is an oligosaccharide moiety that acts as both an attachment site and carbon source for intestinal bacteria. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn''s disease (CD). To characterize the effect of FUT2 polymorphism on the mucosal ecosystem, we profiled the microbiome, meta-proteome and meta-metabolome of 75 endoscopic lavage samples from the cecum and sigmoid of 39 healthy subjects (12 SeSe, 18 Sese and 9 sese). Imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism and glycan biosynthesis and metabolism-related pathways, and the depletion of amino-acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2^−/− genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. Therefore, the colonic microbiota of non-secretors is altered at both the compositional and functional levels, affecting the host mucosal state and potentially explaining the association of FUT2 genotype and CD susceptibility.     

Planning adaptation to climate change in fast-warming marine regions with seafood-dependent coastal communities

Many coastal communities rely on living marine resources for livelihoods and food security. These resources are commonly under stress from overfishing, pollution, coastal development and habitat degradation. Climate change is an additional stressor beginning to impact coastal systems and communities, but may also lead to opportunities for some species and the people they sustain. We describe the research approach for a multi-country project, focused on the southern hemisphere, designed to contribute to improving fishing community adaptation efforts by characterizing, assessing and predicting the future of coastal-marine food resources, and co-developing adaptation options through the provision and sharing of knowledge across fast-warming marine regions (i.e. marine ‘hotspots’). These hotspots represent natural laboratories for observing change and concomitant human adaptive responses, and for developing adaptation options and management strategies. Focusing on adaptation options and strategies for enhancing coastal resilience at the local level will contribute to capacity building and local empowerment in order to minimise negative outcomes and take advantage of opportunities arising from climate change. However, developing comparative approaches across regions that differ in political institutions, socio-economic community demographics, resource dependency and research capacity is challenging. Here, we describe physical, biological, social and governance tools to allow hotspot comparisons, and several methods to evaluate and enhance interactions within a multi-nation research team. Strong partnerships within and between the focal regions are critical to scientific and political support for development of effective approaches to reduce future vulnerability. Comparing these hotspot regions will enhance local adaptation responses and generate outcomes applicable to other regions.