Molecular analysis of Arabidopsis endosperm and embryo promoter trap lines: reporter-gene expression can result from T-DNA insertions in antisense orientation, in introns and in intergenic regions, in addition to sense insertion at the 5' end of genes

Random insertions of promoterless reporter genes in genomes are a common tool for identifying marker lines with tissue-specific expression patterns. Such lines are assumed to reflect the activity of endogenous promoters and should facilitate the cloning of genes expressed in the corresponding tissues. To identify genes active in seed organs, plant DNA flanking T-DNA insertions (T-DNAs) have been cloned in 16 Arabidopsis thaliana GUS-reporter lines. T-DNAs were found in proximal promoter regions, 5' UTR or intron with GUS in the same (sense) orientation as the tagged gene, but contrary to expectations also in inverted orientation in the 5' end of genes or in intergenic regions. RT-PCR, northern analysis, and data on expression patterns of tagged genes, compared with the expression pattern of the reporter lines, suggest that the expression pattern of a reporter gene will reflect the pattern of a tagged gene when inserted in sense orientation in the 5' UTR or intron. When inserted in the promoter region, the reporter-gene expression patterns may be restricted compared with the endogenous gene. Among the trapped genes, the previously described nitrate transporter gene AtNRT1.1, the cyclophilin gene ROC3, and the histone deacetylase gene AtHD2C were found. Reporter-gene expression when positioned in antisense orientation, for example, in the SLEEPY1 gene, is indicative of antisense expression of the tagged gene. For T-DNAs found in intergenic regions, it is suggested that the reporter gene is transcribed from cryptic promoters or promoters of as yet unannotated genes.     

The link between lymphocyte deficiency and autoimmunity: roles of endogenous T and B lymphocytes in tolerance

We demonstrate that transfer of OVA-specific DO11 CD4(+) T cells into mice that lack T and B cells and produce secreted OVA as an endogenous self-protein results in a severe systemic autoimmune reaction with skin inflammation, wasting, and death. The transferred DO11 T cells undergo massive expansion and produce IL-2 and IFN-gamma abundantly. Transfer of DO11 cells into OVA-expressing animals in which T cells are absent but B cells are present, leads to mild disease with no death. In this situation, the DO11 cells undergo similar expansion but show poor Th1 differentiation. This regulatory effect of B cells correlates with profound TCR down-regulation. If T cells are present, the DO11 cells fail to expand independent of B cells. These results suggest that both endogenous T and B lymphocytes control T cell tolerance induction and pathogenicity, but at different stages of an anti-self response. Although endogenous T cells prevent expansion and maintain homeostasis, endogenous B cells limit subsequent effector responses of autoreactive CD4(+) T cells.     

B7-independent inhibition of T cells by CTLA-4

CTLA-4 is an inhibitory molecule that regulates T cell expansion and differentiation. CTLA-4 binding to B7-1/B7-2 is believed to be crucial for its inhibitory signal both by competing for CD28 binding to the same ligands and aggregating CTLA-4 to deliver negative signals. In this study, we demonstrate that B7 binding is not essential for CTLA-4 activity. CTLA-4 knockout T cells are hyperresponsive compared with wild-type T cells in B7-free settings. Expression of a B7-nonbinding CTLA-4 mutant inhibited T cell proliferation, cytokine production, and TCR-mediated ERK activation in otherwise CTLA-4-deficient T cells. Finally, transgenic expression of the ligand-nonbinding CTLA-4 mutant delayed the lethal lymphoproliferation observed in CTLA-4-deficient mice. These results suggest that ligand binding is not essential for the CTLA-4 function and supports an essential role for CTLA-4 signaling during T cell activation.     

The core of bluetongue virus binds double-stranded RNA

Double-stranded RNA (dsRNA) viruses conceal their genome from the host to avoid triggering unfavorable cellular responses. The crystal structure of the core of one such virus, bluetongue virus, reveals an outer surface festooned with dsRNA. This may represent a deliberate strategy to sequester dsRNA released from damaged particles to prevent host cell shutoff.     

Detection and seasonal expression pattern of a pathogenesis-related protein (PR-10) in Douglas-fir (Pseudotsuga menziesii) tissues

Previously, a PR-10 protein Pin m III was described in western white pine. In this study, primers based on cDNA of Pin m III were utilized to obtain the genomic sequence of a Pin m III homologue – Pse m I – in Douglas-fir. A comparative analysis of a deduced amino acid sequence of Pin m III and Pse m I genes indicated about 80% similarity between the two protein sequences, and a consensus 20 amino acid sequence located around the p-loop sequence was used to synthesize a peptide of 20 amino acids. An antibody to this synthetic peptide was able to detect the Pse m I protein in Douglas-fir. The anti- Pse m I antibody was used in a western immunoblot to monitor seasonal variation of the Pse m I in Douglas-fir needles and its level was shown to increase with overwintering of Douglas-fir seedlings. However, unlike the Pin m III, there is no indication that the Pse m I is associated with frost hardiness. Analysis of infected Douglas-fir roots showed a possible trend to up-regulation of Pse m I by pathogens such as the laminated root rot fungus, Phellinus weirii . The expression of Pse m I protein in Douglas-fir seedlings is very low compared to the expression of Pin m III protein in western white pine seedlings. In addition, a light-harvesting complex I protein, PSI-F, was identified in Douglas-fir by N-terminal amino acid sequencing.     

ITPA Polymorphisms Are Associated with Hematological Side Effects during Antiviral Therapy for Chronic HCV Infection

Background/ObjectiveGenetic polymorphisms in the inosine triphosphatase (ITPA) gene have been associated with the protection from early ribavirin(RBV)-induced hemolytic anemia among patients with chronic hepatitis C virus (HCV) infection. The aim of the present study was to investigate the association between the functional ITPA variants and hematological side effects during antiviral therapy with pegylated interferon (PegIFN) and RBV.ResultsIn total, 213 patients were included. The predicted ITPase activity was normal among 152 (71%) patients; 61 (29%) patients had ITPase deficiency. By multivariable linear regression, RBV dose in mg per kilogram (Beta 0.09, 95%CI 0.04–0.13, p<0.001) and normal ITPase activity (Beta 0.89, 95%CI 0.64–1.14, p<0.001) were associated with more Hb decline at week 4 of treatment. Patients with normal ITPase activity underwent more dose adjustments of RBV than patients with ITPase deficiency (19(13%) vs 1(2%),p = 0.014) and received erythropoietin more frequently (12 (8%) vs 0 (0%),p = 0.024).ConclusionGenetic variants in the ITPA gene protected against RBV treatment-induced anemia among Caucasian patients with chronic HCV infection. Patients with normal ITPase activity underwent more dose reductions of RBV and received erythropoietin more frequently.     

Full-Genome Sequencing as a Basis for Molecular Epidemiology Studies of Bluetongue Virus in India

Since 1998 there have been significant changes in the global distribution of bluetongue virus (BTV). Ten previously exotic BTV serotypes have been detected in Europe, causing severe disease outbreaks in naïve ruminant populations. Previously exotic BTV serotypes were also identified in the USA, Israel, Australia and India. BTV is transmitted by biting midges (Culicoides spp.) and changes in the distribution of vector species, climate change, increased international travel and trade are thought to have contributed to these events. Thirteen BTV serotypes have been isolated in India since first reports of the disease in the country during 1964. Efficient methods for preparation of viral dsRNA and cDNA synthesis, have facilitated full-genome sequencing of BTV strains from the region. These studies introduce a new approach for BTV characterization, based on full-genome sequencing and phylogenetic analyses, facilitating the identification of BTV serotype, topotype and reassortant strains. Phylogenetic analyses show that most of the equivalent genome-segments of Indian BTV strains are closely related, clustering within a major eastern BTV ‘topotype’. However, genome-segment 5 (Seg-5) encoding NS1, from multiple post 1982 Indian isolates, originated from a western BTV topotype. All ten genome-segments of BTV-2 isolates (IND2003/01, IND2003/02 and IND2003/03) are closely related (>99% identity) to a South African BTV-2 vaccine-strain (western topotype). Similarly BTV-10 isolates (IND2003/06; IND2005/04) show >99% identity in all genome segments, to the prototype BTV-10 (CA-8) strain from the USA. These data suggest repeated introductions of western BTV field and/or vaccine-strains into India, potentially linked to animal or vector-insect movements, or unauthorised use of ‘live’ South African or American BTV-vaccines in the country. The data presented will help improve nucleic acid based diagnostics for Indian serotypes/topotypes, as part of control strategies.     

Effectiveness Study of Paromomycin IM Injection (PMIM) for the Treatment of Visceral Leishmaniasis (VL) in Bangladesh

Background

This study was conducted in Bangladeshi patients in an outpatient setting to support registration of Paromomycin Intramuscular Injection (PMIM) as a low-cost treatment option in Bangladesh.

Methodology

This Phase IIIb, open-label, multi-center, single-arm trial assessed the efficacy and safety of PMIM administered at 11 mg/kg (paromomycin base) intramuscularly once daily for 21 consecutive days to children and adults with VL in a rural outpatient setting in Bangladesh. Patients ≥5 and ≤55 years were eligible if they had signs and symptoms of VL (intermittent fever, weight loss/decreased appetite, and enlarged spleen), positive rK39 test, and were living in VL-endemic areas. Compliance was the percentage of enrolled patients who received 21 daily injections over no more than 22 days. Efficacy was evaluated by initial clinical response, defined as resolution of fever and reduction of splenomegaly at end of treatment, and final clinical response, defined as the absence of new clinical signs and symptoms of VL 6 months after end of treatment. Safety was assessed by evaluation of adverse events.

Principal Findings

A total of 120 subjects (49% pediatric) were enrolled. Treatment compliance was 98.3%. Initial clinical response in the Intent-to-Treat population was 98.3%, and final clinical response 6 months after end of treatment was 94.2%. Of the 119 subjects who received ≥1 dose of PMIM, 28.6% reported at least one adverse event. Injection site pain was the most commonly reported adverse event. Reversible renal impairment and/or hearing loss were reported in 2 subjects.

Conclusions/Significance

PMIM was an effective and safe treatment for VL in Bangladesh. The short treatment duration and lower cost of PMIM compared with other treatment options may make this drug a preferred treatment to be investigated as part of a combination therapy regimen. This study supports the registration of PMIM for use in government health facilities in Bangladesh.

Trial Registration

ClinicalTrials.gov identifier: NCT01328457     

Are Protected Areas Required to Maintain Functional Diversity in Human-Modified Landscapes?

The conversion of forest to agriculture across the world’s tropics, and the limited space for protected areas, has increased the need to identify effective conservation strategies in human-modified landscapes. Isolated trees are believed to conserve elements of ecological structure, providing micro-sites for conservation in matrix landscapes, and facilitating seed dispersal and forest restoration. Here we investigate the role of isolated Ficus trees, which are of critical importance to tropical forest ecosystems, in conserving frugivore composition and function in a human-modified landscape in Assam, India. We surveyed the frugivorous birds feeding at 122 isolated Ficus trees, 33 fruit trees, and 31 other large trees across a range of 32 km from the nearest intact forest. We found that Ficus trees attracted richer and more abundant assemblages of frugivores than the other tree categories. However, incidence function estimates revealed that forest specialist species decreased dramatically within the first kilometre of the forest edge. Despite this, species richness and functional diversity remained consistent across the human-modified landscape, as habitat generalists replaced forest-dependent frugivores, and accounted for most of the ecological function found in Ficus trees near the forest edge. We recommend that isolated Ficus trees are awarded greater conservation status, and suggest that their conservation can support ecologically functional networks of frugivorous bird communities.