Identification of the Plasmodium falciparum rhoptry neck protein 5 (PfRON5)

In this study a gene for a drought stress-inducible putative membrane protein was cloned and characterised from root tissue of wild emmer wheat. Sequence analysis indicated that the protein is a member of the widespread but hitherto uncharacterised TMPIT (transmembrane protein inducible by TNF-α) family, so it was labelled TdicTMPIT1. Real-time RT-PCR showed that the TdicTMPIT1 gene is upregulated on drought stress in drought-tolerant wild emmer wheat, but not in a drought-sensitive accession or in cultivated durum wheat. The TdicTMPIT1 product was predicted to be a membrane protein with four transmembrane helices. The protein was expressed and analysed in Escherichia coli and Saccharomyces cerevisiae. Cellular localisation of the protein in the cell was also investigated using an eGFP-tagged form of the protein in S. cerevisiae. Results obtained by confocal laser microscopy indicated that the TdicTMPIT1 tagged with GFP was localised in a membraneous compartment. It is concluded that TdicTMPIT1 is a membrane protein associated with the drought stress response in wild emmer wheat, and so it may be useful for the improvement of modern wheat genotypes. Members of this protein family in other organisms are proposed also to be involved in stress responses.     

A liquid culture system for shoot proliferation and analysis of pharmaceutically active constituents of Catharanthus roseus (L.) G. Don

An efficient and cost effective micropropagation protocol using liquid medium was developed for Catharanthus roseus, a commercially important medicinal plant. Comparative analysis of shoot growth and proliferation in liquid Murashige and Skoog (MS) medium supplemented with different concentrations of cytokinins [6-Benzyladenine (BA), Kinetin (KN) and Thidiazuron (TDZ)] was conducted. Better response in terms of shoot proliferation, shoot diameter, number of leaves/shoot, number of branches/shoot, fresh weight and dry weight was observed in a liquid medium vis-à-vis solid medium. A sample of 20 ml of liquid medium supplemented with 5 ??M of BA was optimized for propagation of C. roseus by a liquid culture system. Among various concentrations of auxins tried, 1-Naphthaleneacetic acid (NAA) 5 ??M was found to be the best for root induction. Quantification of pharmaceutically important constituents (vincristine and vinblastine) and total alkaloid content of microshoots grown in solid and liquid medium as well as in vitro raised plants and mother plant was also conducted, hitherto unreported in this high-value medicinal plant. This work further lays the foundations for the shifting of plant production from small to commercial scale.     

Calpain-1 cleaves Rad21 to promote sister chromatid separation

Defining the mechanisms of chromosomal cohesion and dissolution of the cohesin complex from chromatids is important for understanding the chromosomal missegregation seen in many tumor cells. Here we report the identification of a novel cohesin-resolving protease and describe its role in chromosomal segregation. Sister chromatids are held together by cohesin, a multiprotein ring-like complex comprised of Rad21, Smc1, Smc3, and SA2 (or SA1). Cohesin is known to be removed from vertebrate chromosomes by two distinct mechanisms, namely, the prophase and anaphase pathways. First, PLK1-mediated phosphorylation of SA2 in prophase leads to release of cohesin from chromosome arms, leaving behind centromeric cohesins that continue to hold the sisters together. Then, at the onset of anaphase, activated separase cleaves the centromeric cohesin Rad21, thereby opening the cohesin ring and allowing the sister chromatids to separate. We report here that the calcium-dependent cysteine endopeptidase calpain-1 is a Rad21 peptidase and normally localizes to the interphase nuclei and chromatin. Calpain-1 cleaves Rad21 at L192, in a calcium-dependent manner. We further show that Rad21 cleavage by calpain-1 promotes separation of chromosome arms, which coincides with a calcium-induced partial loss of cohesin at several chromosomal loci. Engineered cleavage of Rad21 at the calpain-cleavable site without activation of calpain-1 can lead to a loss of sister chromatid cohesion. Collectively, our work reveals a novel function of calpain-1 and describes an additional pathway for sister chromatid separation in humans.     

Giant kelp (Macrocystis) fishery in Atacama (Northern Chile): biological basis for management of the integrifolia morph

In Bahia Chasco, Atacama, the integrifolia morph of Macrocystis forms one of the most important kelp forests in northern Chile. In order to determine effects of local harvesting policies, we evaluated the population dynamics of this resource in intact, frequently disturbed, and permanently and completely harvested areas. Recruitment, frond length, reproductive phenology and standing crop were assessed monthly. In intact areas, frond length and ratio of reproductive individuals were higher, but recruitment was poorly stimulated. On the other hand, complete harvest had an important effect on Macrocystis population dynamics. Whereas recruitment and growth were much higher after harvest events, reproductive phenology was lower. The harvest techniques with different frequencies practiced by Bahia Chasco fishermen were less harmful than complete harvest, and we conclude that current exploitation techniques applied in this location are not deleterious for the giant kelp beds. They even have favorable effects by renewing the population through stimulation of sexual reproduction, recruitment and growth of young individuals.     

Exome Sequencing Is an Efficient Tool for Variant Late-Infantile Neuronal Ceroid Lipofuscinosis Molecular Diagnosis

The neuronal ceroid-lipofuscinoses (NCL) is a group of neurodegenerative disorders characterized by epilepsy, visual failure, progressive mental and motor deterioration, myoclonus, dementia and reduced life expectancy. Classically, NCL-affected individuals have been classified into six categories, which have been mainly defined regarding the clinical onset of symptoms. However, some patients cannot be easily included in a specific group because of significant variation in the age of onset and disease progression. Molecular genetics has emerged in recent years as a useful tool for enhancing NCL subtype classification. Fourteen NCL genetic forms (CLN1 to CLN14) have been described to date. The variant late-infantile form of the disease has been linked to CLN5, CLN6, CLN7 (MFSD8) and CLN8 mutations. Despite advances in the diagnosis of neurodegenerative disorders mutations in these genes may cause similar phenotypes, which rends difficult accurate candidate gene selection for direct sequencing. Three siblings who were affected by variant late-infantile NCL are reported in the present study. We used whole-exome sequencing, direct sequencing and in silico approaches to identify the molecular basis of the disease. We identified the novel c.1219T>C (p.Trp407Arg) and c.1361T>C (p.Met454Thr) MFSD8 pathogenic mutations. Our results highlighted next generation sequencing as a novel and powerful methodological approach for the rapid determination of the molecular diagnosis of NCL. They also provide information regarding the phenotypic and molecular spectrum of CLN7 disease.     

A Panel of Trypanosoma brucei Strains Tagged with Blue and Red-Shifted Luciferases for Bioluminescent Imaging in Murine Infection Models

Background

Genetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients.

Methodology/Principal findings

We transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT.

Conclusions/Significance

We established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better kinetics of their substrate D-luciferin.     

Genome sequence of the Listia angolensis microsymbiont Microvirga lotononidis strain WSM3557T

Microvirga lotononidis is a recently described species of root-nodule bacteria that is an effective nitrogen- (N₂) fixing microsymbiont of the symbiotically specific African legume Listia angolensis (Welw. ex Bak.) B.-E. van Wyk & Boatwr. M. lotononidis possesses several properties that are unusual in root-nodule bacteria, including pigmentation and the ability to grow at temperatures of up to 45°C. Strain WSM3557^T is an aerobic, motile, Gram-negative, non-spore-forming rod isolated from a L. angolensis root nodule collected in Chipata, Zambia in 1963. This is the first report of a complete genome sequence for the genus Microvirga. Here we describe the features of Microvirga lotononidis strain WSM3557^T, together with genome sequence information and annotation. The 7,082,538 high-quality-draft genome is arranged in 18 scaffolds of 104 contigs, contains 6,956 protein-coding genes and 84 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

High quality draft genome sequence of Olivibacter sitiensis type strain (AW-6T), a diphenol degrader with genes involved in the catechol pathway

Olivibacter sitiensis Ntougias et al. 2007 is a member of the family Sphingobacteriaceae, phylum Bacteroidetes. Members of the genus Olivibacter are phylogenetically diverse and of significant interest. They occur in diverse habitats, such as rhizosphere and contaminated soils, viscous wastes, composts, biofilter clean-up facilities on contaminated sites and cave environments, and they are involved in the degradation of complex and toxic compounds. Here we describe the features of O. sitiensis AW-6^T, together with the permanent-draft genome sequence and annotation. The organism was sequenced under the Genomic Encyclopedia for Bacteria and Archaea (GEBA) project at the DOE Joint Genome Institute and is the first genome sequence of a species within the genus Olivibacter. The genome is 5,053,571 bp long and is comprised of 110 scaffolds with an average GC content of 44.61%. Of the 4,565 genes predicted, 4,501 were protein-coding genes and 64 were RNA genes. Most protein-coding genes (68.52%) were assigned to a putative function. The identification of 2-keto-4-pentenoate hydratase/2-oxohepta-3-ene-1,7-dioic acid hydratase-coding genes indicates involvement of this organism in the catechol catabolic pathway. In addition, genes encoding for β-1,4-xylanases and β-1,4-xylosidases reveal the xylanolytic action of O. sitiensis.     

Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2230 from Karijini National Park,Australia

Burkholderia sp. strain WSM2230 is an aerobic, motile, Gram-negative, non-spore-forming acid-tolerant rod isolated from acidic soil collected in 2001 from Karijini National Park, Western Australia, using Kennedia coccinea (Coral Vine) as a host. WSM2230 was initially effective in nitrogen-fixation with K. coccinea, but subsequently lost symbiotic competence. Here we describe the features of Burkholderia sp. strain WSM2230, together with genome sequence information and its annotation. The 6,309,801 bp high-quality-draft genome is arranged into 33 scaffolds of 33 contigs containing 5,590 protein-coding genes and 63 RNA-only encoding genes. The genome sequence of WSM2230 failed to identify nodulation genes and provides an explanation for the observed failure of the laboratory grown strain to nodulate. The genome of this strain is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.