Genome evolution of a tertiary dinoflagellate plastid

The dinoflagellates have repeatedly replaced their ancestral peridinin-plastid by plastids derived from a variety of algal lineages ranging from green algae to diatoms. Here, we have characterized the genome of a dinoflagellate plastid of tertiary origin in order to understand the evolutionary processes that have shaped the organelle since it was acquired as a symbiont cell. To address this, the genome of the haptophyte-derived plastid in Karlodinium veneficum was analyzed by Sanger sequencing of library clones and 454 pyrosequencing of plastid enriched DNA fractions. The sequences were assembled into a single contig of 143 kb, encoding 70 proteins, 3 rRNAs and a nearly full set of tRNAs. Comparative genomics revealed massive rearrangements and gene losses compared to the haptophyte plastid; only a small fraction of the gene clusters usually found in haptophytes as well as other types of plastids are present in K. veneficum. Despite the reduced number of genes, the K. veneficum plastid genome has retained a large size due to expanded intergenic regions. Some of the plastid genes are highly diverged and may be pseudogenes or subject to RNA editing. Gene losses and rearrangements are also features of the genomes of the peridinin-containing plastids, apicomplexa and Chromera, suggesting that the evolutionary processes that once shaped these plastids have occurred at multiple independent occasions over the history of the Alveolata.     

Mode of action of CryIIA: a Bacillus thuringiensis delta-endotoxin

CryIIA is an effective insecticidal delta-endotoxin produced by several strains of Bacillus thuringiensis. Unlike CryI and CryIIIA-toxins that demonstrate some degree of saturable binding on the brush border of susceptible insects, neither saturable binding nor a saturable binding component was found for CryIIA on the midgut brush border of Helicoverpa zea. CryIIA did not dilute and block CryIA(c) binding, however, CryIA(c) effectively diluted CryIIA and stopped the initial binding of CryIIA to the brush border. These observations suggest that CryIIA and CryIA(c) toxins share a common component for binding on the midgut brush border. CryIIA formed voltage-dependent and not highly cation-selective channels in planar lipid bilayers unlike CryIA(c) and CryIIIA. Both CryIA(c) and CryIIA were stable in the digestive fluids of H. zea, but CryIIA was significantly less soluble than CryIA(c). Despite this difference in solubility, CryIIA arrested the feeding of third instar H. zea as rapidly as did CryIA(c), however, the onset of acute morbidity was delayed for CryIIA. Differences in solubility, binding, and ion channels formed by CryIIA toxin, resulted in reduced bioactivity against H. zea when compared with CryIA(c) but represent a unique mode of action among the delta endotoxins.     

The conservation of ground layer lichen communities in alvar grasslands and the relevance of substitution habitats

Semi-natural calcareous grasslands (alvars) are biodiversity hotspots in Northern Europe, particularly for herb layer plants. In the last century, traditional management has ceased, and the area of grasslands has declined due to extensive encroachment. We were interested in the drivers of ground layer (alias terricolous or epigeic) lichen communities. Our survey consisted of 86 habitat fragments in western Estonia, covering four types of historic alvar grasslands and three types of alvar-like habitats. We found that the ground lichen communities were primarily soil-type-specific, but were also affected by historic disturbances and land use change. In contrast to knowledge about herb layer communities, for which shrub encroachment has been shown to be main driver, the increased density of the herb layer and the reduced diversity of microhabitats were major drivers for the ground layer lichen community. These drivers caused a decrease in species richness, but only within the species of conservation value, and also led to a shift in the composition of lichen growth form from the dominance of squamulose and crustose towards fruticose lichens. We conclude that the traditional practice of restoring alvars by cutting shrubs is insufficient to maintain ground layer lichen biodiversity. Alvar maintenance practices should include grazing, which creates various small-scale ground disturbances and increases microhabitat heterogeneity. Alvar-like habitats originating from large-scale historic disturbances appeared to be suitable for calcicolous epigeic lichens, and can therefore be considered to be temporary substitution habitats, i.e. refugia for the regional species pool.     

An encapsulated Yersinia pseudotuberculosis is a highly efficient vaccine against pneumonic plague

Background

Plague is still a public health problem in the world and is re-emerging, but no efficient vaccine is available. We previously reported that oral inoculation of a live attenuated Yersinia pseudotuberculosis, the recent ancestor of Yersinia pestis, provided protection against bubonic plague. However, the strain poorly protected against pneumonic plague, the most deadly and contagious form of the disease, and was not genetically defined.

Methodology and Principal Findings

The sequenced Y. pseudotuberculosis IP32953 has been irreversibly attenuated by deletion of genes encoding three essential virulence factors. An encapsulated Y. pseudotuberculosis was generated by cloning the Y. pestis F1-encoding caf operon and expressing it in the attenuated strain. The new V674pF1 strain produced the F1 capsule in vitro and in vivo. Oral inoculation of V674pF1 allowed the colonization of the gut without lesions to Peyer''s patches and the spleen. Vaccination induced both humoral and cellular components of immunity, at the systemic (IgG and Th1 cells) and the mucosal levels (IgA and Th17 cells). A single oral dose conferred 100% protection against a lethal pneumonic plague challenge (33×LD₅₀ of the fully virulent Y. pestis CO92 strain) and 94% against a high challenge dose (3,300×LD₅₀). Both F1 and other Yersinia antigens were recognized and V674pF1 efficiently protected against a F1-negative Y. pestis.

Conclusions and Significance

The encapsulated Y. pseudotuberculosis V674pF1 is an efficient live oral vaccine against pneumonic plague, and could be developed for mass vaccination in tropical endemic areas to control pneumonic plague transmission and mortality.     

An ex-vivo Human Intestinal Model to Study Entamoeba histolytica Pathogenesis

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host''s pro-inflammatory cytokine secretion.     

Decreased expression of phospholamban is not associated with lower β-adrenergic activation in rat atria

The aim of the study was to find out whether low phospholamban level in atria as compared with ventricles is associated with differences in sarcoplasmic reticular Ca²⁺-uptake and contractile performance. Relationship between phospholamban and -adrenergic stimulation in rat left atria and papillary muscles were examined by means of contractile measurements, sarcoplasmic reticular oxalate-supported Ca²⁺-uptake, and Western blotting of phosphorylated phospholamban. Phosphoprotein determination after -adrenergic stimulation demonstrated that the levels of Ser¹⁶ and Thr¹⁷ phosphorylated phospholamban in atria remained at about one-third of that in ventricles. However, comparison of sarcoplasmic reticular Ca²⁺-uptake in control and isoproterenol perfused preparations demonstrated that the effect of -adrenergic stimulation on sarcoplasmic reticular Ca²⁺-uptake was stronger in atrial preparations. Moreover, atria responded to isoproterenol with much larger increases in developed tension, contractility and relaxation rates than papillary muscles. Thus, despite lower level of phospholamban, the -adrenergic activation of sarcoplasmic reticular Ca²⁺-uptake and contractile indices are higher in atria.     

CCR5 Haplotypes Influence HCV Serostatus in Caucasian Intravenous Drug Users

Background

Up to 90% HIV-1 positive intravenous drug users (IDUs) are co-infected with HCV. Although best recognized for its function as a major co-receptor for cell entry of HIV, CC chemokine receptor 5 (CCR5) has also been implicated in the pathogenesis of HCV infection. Here, we investigated whether CCR5 haplotypes influence HIV-1 and HCV seropositivity among 373 Caucasian IDUs from Estonia.

Methods

Of these IDUs, 56% and 44% were HIV and HCV seropositive, respectively, and 47% were coinfected. 500 blood donors seronegative for HIV and HCV were also evaluated. CCR5 haplotypes (HHA to HHG*2) were derived after genotyping nine CCR2–CCR5 polymorphisms. The association between CCR5 haplotypes with HIV and/or HCV seropositivity was determined using logistic regression analysis. Co-variates included in the models were length of intravenous drug use, HBV serostatus and copy number of CCL3L1, the gene encoding the most potent HIV-suppressive chemokine and ligand for CCR5.

Results

Compared to IDUs seronegative for both HCV and HIV (HCV−/HIV-), IDUs who were HCV+/HIV- and HCV+/HIV+were 92% and 82%, respectively, less likely to possess the CCR5-HHG*1 haplotype, after controlling for co-variates (P_adjusted = 1.89×10⁻⁴ and 0.003, respectively). This association was mostly due to subjects bearing the CCR5 HHE and HHG*1 haplotype pairs. Approximately 25% and<10% of HCV−/HIV- IDUs and HCV−/HIV- blood donors, respectively, possessed the HHE/HHG*1 genotype.

Conclusions

Our findings suggest that HHG*1-bearing CCR5 genotypes influence HCV seropositivity in a group of Caucasian IDUs.     

Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera

Background  

Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (mcy). mcy genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer.     

Comparative Analysis of the Effects of Neurotrophic Factors CDNF and GDNF in a Nonhuman Primate Model of Parkinson’s Disease

Cerebral dopamine neurotrophic factor (CDNF) belongs to a newly discovered family of evolutionarily conserved neurotrophic factors. We demonstrate for the first time a therapeutic effect of CDNF in a unilateral 6-hydroxydopamine (6-OHDA) lesion model of Parkinson’s disease in marmoset monkeys. Furthermore, we tested the impact of high chronic doses of human recombinant CDNF on unlesioned monkeys and analyzed the amino acid sequence of marmoset CDNF. The severity of 6-OHDA lesions and treatment effects were monitored in vivo using ¹²³I-FP-CIT (DaTSCAN) SPECT. Quantitative analysis of ¹²³I-FP-CIT SPECT showed a significant increase of dopamine transporter binding activity in lesioned animals treated with CDNF. Glial cell line-derived neurotrophic factor (GDNF), a well-characterized and potent neurotrophic factor for dopamine neurons, served as a control in a parallel comparison with CDNF. By contrast with CDNF, only single animals responded to the treatment with GDNF, but no statistical difference was observed in the GDNF group. However, increased numbers of tyrosine hydroxylase immunoreactive neurons, observed within the lesioned caudate nucleus of GDNF-treated animals, indicate a strong bioactive potential of GDNF.