Differential frequency of NKG2C/<Emphasis Type="Italic">KLRC2</Emphasis> deletion in distinct African populations and susceptibility to Trachoma: a new method for imputation of <Emphasis Type="Italic">KLRC2</Emphasis> genotypes from SNP genotyping data

NKG2C is an activating receptor that is preferentially expressed on natural killer (NK) cells. The gene encoding NKG2C (killer cell lectin-like receptor C2, KLRC2) is present at different copy numbers in the genomes of different individuals. Deletion at the NKG2C locus was investigated in a case–control study of 1522 individuals indigenous to East- and West-Africa and the association with the ocular Chlamydia trachomatis infection and its sequelae was explored. The frequency of homozygous KLRC2 deletion was 13.7 % in Gambians and 4.7 % in Tanzanians. A significantly higher frequency of the deletion allele was found in West-Africans from the Gambia and Guinea-Bissau (36.2 % p = 2.105 × 10^?8, 26.8 % p = 0.050; respectively) in comparison to East-African Tanzanians where the frequency of the deletion is comparable to other human populations (20.9 %). We found no evidence for an association between the numbers of KLRC2 gene copies and the clinical manifestations of trachoma (follicular trachoma or conjunctival scarring). A new method for imputation of KLRC2 genotypes from single nucleotide polymorphism (SNP) data in 2621 individuals from the Gambia further confirmed these results. Our data suggest that NKG2C does not play a major role in trachomatous disease. We found that the deletion allele is present at different frequencies in different populations but the reason behind these differences is currently not understood. The new method offers the potential to use SNP arrays from genome wide association studies to study the frequency of KLRC2 deletion in other populations and its association with other diseases.     

Compensation and resistance to herbivory in seagrasses: induced responses to simulated consumption by fish

Herbivory can induce changes in plant traits that may involve both tolerance mechanisms that compensate for biomass loss and resistance traits that reduce herbivore preference. Seagrasses are marine vascular plants that possess many attributes that may favour tolerance and compensatory growth, and they are also defended with mechanisms of resistance such as toughness and secondary metabolites. We quantified phenotypic changes induced by herbivore damage on the temperate seagrass Posidonia oceanica in order to identify specific compensatory and resistance mechanisms in this plant, and to assess any potential trade-offs between these two strategies of defence. We simulated three natural levels of fish herbivory by repeatedly clipping seagrass leaves during the summer period of maximum herbivory. Compensatory responses were determined by measuring shoot-specific growth, photosynthetic rate, and the concentration of nitrogen and carbon resources in leaves and rhizomes. Induced resistance was determined by measuring the concentration of phenolic secondary metabolites and by assessing the long-term effects of continued clipping on herbivore feeding preferences using bioassays. Plants showed a significant ability to compensate for low and moderate losses of leaf biomass by increasing aboveground growth of damaged shoots, but this was not supported by an increase in photosynthetic capacity. Low levels of herbivory induced compensatory growth without any measurable effects on stored resources. In contrast, nitrogen reserves in the rhizomes played a crucial role in the plant’s ability to compensate and survive herbivore damage under moderate and high levels of herbivory, respectively. We found no evidence of inducibility of long-term resistance traits in response to herbivory. The concentration of phenolics decreased with increasing compensatory growth despite all treatments having similar carbon leaf content, suggesting reallocation of these compounds towards primary functions such as cell-wall construction.     

Population structure and phylogeography reveal pathways of colonization by a migratory marine reptile (Chelonia mydas) in the central and eastern Pacific

Climate, behavior, ecology, and oceanography shape patterns of biodiversity in marine faunas in the absence of obvious geographic barriers. Marine turtles are an example of highly migratory creatures with deep evolutionary lineages and complex life histories that span both terrestrial and marine environments. Previous studies have focused on the deep isolation of evolutionary lineages (>3 mya) through vicariance; however, little attention has been given to the pathways of colonization of the eastern Pacific and the processes that have shaped diversity within the most recent evolutionary time. We sequenced 770 bp of the mtDNA control region to examine the stock structure and phylogeography of 545 green turtles from eight different rookeries in the central and eastern Pacific. We found significant differentiation between the geographically separated nesting populations and identified five distinct stocks (F_ST = 0.08–0.44, P < 0.005). Central and eastern Pacific Chelonia mydas form a monophyletic group containing 3 subclades, with Hawaii more closely related to the eastern Pacific than western Pacific populations. The split between sampled central/eastern and western Pacific haplotypes was estimated at around 0.34 mya, suggesting that the Pacific region west of Hawaii has been a more formidable barrier to gene flow in C. mydas than the East Pacific Barrier. Our results suggest that the eastern Pacific was colonized from the western Pacific via the Central North Pacific and that the Revillagigedos Islands provided a stepping‐stone for radiation of green turtles from the Hawaiian Archipelago to the eastern Pacific. Our results fit with a broader paradigm that has been described for marine biodiversity, where oceanic islands, such as Hawaii and Revillagigedo, rather than being peripheral evolutionary “graveyards”, serve as sources and recipients of diversity and provide a mechanism for further radiation.     

A robust approach to estimate relative phytoplankton cell abundances from metagenomes

Phytoplankton account for >45% of global primary production, and have an enormous impact on aquatic food webs and on the entire Earth System. Their members are found among prokaryotes (cyanobacteria) and multiple eukaryotic lineages containing chloroplasts. Genetic surveys of phytoplankton communities generally consist of PCR amplification of bacterial (16S), nuclear (18S) and/or chloroplastic (16S) rRNA marker genes from DNA extracted from environmental samples. However, our appreciation of phytoplankton abundance or biomass is limited by PCR-amplification biases, rRNA gene copy number variations across taxa, and the fact that rRNA genes do not provide insights into metabolic traits such as photosynthesis. Here, we targeted the photosynthetic gene psbO from metagenomes to circumvent these limitations: the method is PCR-free, and the gene is universally and exclusively present in photosynthetic prokaryotes and eukaryotes, mainly in one copy per genome. We applied and validated this new strategy with the size-fractionated marine samples collected by Tara Oceans, and showed improved correlations with flow cytometry and microscopy than when based on rRNA genes. Furthermore, we revealed unexpected features of the ecology of these ecosystems, such as the high abundance of picocyanobacterial aggregates and symbionts in the ocean, and the decrease in relative abundance of phototrophs towards the larger size classes of marine dinoflagellates. To facilitate the incorporation of psbO in molecular-based surveys, we compiled a curated database of >18,000 unique sequences. Overall, psbO appears to be a promising new gene marker for molecular-based evaluations of entire phytoplankton communities.     

Screening of plant growth promoting Rhizobacteria isolated from sunflower (Helianthus annuus L.)

Background and Aims

This study was aimed at assessing the diversity of putatively diazotrophic rhizobacteria associated with sunflower (Helianthus annuus L.) cropped in the south of Brazil, and to examine key plant growth promotion (PGP) characteristics of the isolates for the purposes of increasing plant productivity.

Methods

299 strains were isolated from the roots and rhizosphere of sunflower cultivated in five different areas using N-free media. 16S rDNA PCR-RFLP and 16S rRNA partial sequencing were used for identification and the Shannon index was used to evaluate bacterial diversity. Production of siderophores and indolic compounds (ICs), as well phosphate solubilization activities of each isolate were also evaluated in vitro. On the basis of multiple PGP activities, eight isolates were selected and tested for their N-fixation ability, and their capacity as potential PGPR on sunflower plants was also assessed.

Results

All except three Gram-positive strains (phylum Actinobacteria) belonged to the Gram-negative Proteobacteria subgroups [Gamma (167), Beta (78), and Alpha (50)] and the family Flavobacteriaceae (1)]. Shannon indexes ranged from 0.96 to 2.13 between the five sampling sites. Enterobacter and Burkholderia were the predominant genera isolated from roots and rhizosphere, respectively. Producers of siderophores and ICs were widely found amongst the isolates, but only 19.8% of them solubilized phosphate. About 8% of the isolates exhibited all three PGP traits, and these mostly belonged to the genus Burkholderia. Four isolates were able to stimulate the growth of sunflower plants under gnotobiotic conditions.

Conclusions

Enterobacter and Burkholderia were the dominant rhizospheric bacterial genera associated with sunflower plants. Inoculation with isolates belonging to the genera Achromobacter, Chryseobacterium, Azospirillum, and Burkholderia had a stimulatory effect on plant growth.     

Simplifier: a web tool to eliminate redundant NGS contigs

Modern genomic sequencing technologies produce a large amount of data with reduced cost per base; however, this data consists of short reads. This reduction in the size of the reads, compared to those obtained with previous methodologies, presents new challenges, including a need for efficient algorithms for the assembly of genomes from short reads and for resolving repetitions. Additionally after abinitio assembly, curation of the hundreds or thousands of contigs generated by assemblers demands considerable time and computational resources. We developed Simplifier, a stand-alone software that selectively eliminates redundant sequences from the collection of contigs generated by ab initio assembly of genomes. Application of Simplifier to data generated by assembly of the genome of Corynebacterium pseudotuberculosis strain 258 reduced the number of contigs generated by ab initio methods from 8,004 to 5,272, a reduction of 34.14%; in addition, N50 increased from 1 kb to 1.5 kb. Processing the contigs of Escherichia coli DH10B with Simplifier reduced the mate-paired library 17.47% and the fragment library 23.91%. Simplifier removed redundant sequences from datasets produced by assemblers, thereby reducing the effort required for finalization of genome assembly in tests with data from Prokaryotic organisms.

Availability

Simplifier is available at http://www.genoma.ufpa.br/rramos/softwares/simplifier.xhtmlIt requires Sun jdk 6 or higher.     

Antiplasmodial activity study of angiotensin II via Ala scan analogs

Angiotensin II (AII) as well as analog peptides shows antimalarial activity against Plasmodium gallinaceum and Plasmodium falciparum, but the exact mechanism of action is still unknown. This work presents the solid‐phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide‐capped derivative and the evaluation of the antiplasmodial activity of these peptides against mature P. gallinaceum sporozoites. The Ala screening data indicates that the replacement of either the Ile⁵ or the His⁶ residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala⁶]‐AII (79%), and [Ala⁵]‐AII (75%). Analogs [Ala³]‐AII, [Ala¹]‐AII, and AII‐NH₂ showed antiplasmodial activity around 65%, whereas the activity of the [Ala⁸]‐AII, [Ala⁷]‐AII, [Ala⁴]‐AII, and [Ala²]‐AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a β‐fold conformation in different solutions. All AII analogs, except [Ala⁴]‐AII and [Ala⁸]‐AII, show contractile responses and interact with the AT₁ receptor, [Ala⁵]‐AII and [Ala⁶]‐AII. In conclusion, this approach is helpful to understand the contribution of each amino acid residue to the bioactivity of AII, opening new perspectives toward the design of new sporozoiticidal compounds. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

THE CALCAREOUS RESTING CYST OF PENTAPHARSODINIUM TYRRHENICUM COMB. NOV. (DINOPHYCEAE)1

While investigating dinoflagellate cyst assemblages in surface sediments of the Gulfs of Naples and Salerno (Mediterranean Sea), we found a new calcareous resting cyst. This cyst has a round to oval body surrounded by a thick mineral layer, which gives it the shape of a Napoleon hat, with a flat, oval face bearing the archeopyle and a convex keel on the opposite side. The cyst shape is variable in both natural samples and clonal cultures. The organic membrane underlying the calcareous covering is resistant to acetolysis, thus demonstrating the presence of sporopolleninlike material. The cyst germinated into a motile stage having the same morphological features and thecal plate pattern as Peridinium tyrrhenicum Balech. We believe the validity of the genus Pentapharsodinium Indelicato & Loeblich should be accepted. Based on the comparative examination of the species we collected and of a similar species, Pentapharsodinium trachodium Indelicato & Loeblich, we propose Pentapharsodinium tyrrhenicum as a new combination for Peridinium tyrrhenicum. The genus Pentapharsodinium also includes P. dalei Indelicato & Loeblich (= Peridinium faeroense Dale), which produces spiny, organic-walled cysts. The presence of species forming calcareous cysts and species producing noncalcareous cysts in the same genus raises questions about maintaining the family Calciodinellaceae. This family should only include calcareous cyst-forming peridinioids, in order to maintain a unified system of classification of fossil and recent dinoflagellates.     

Diagnostic epitope variability within Taenia solium 8 kDa antigen family: implications for cysticercosis immunodetection

To study diagnostic epitopes within the Taenia solium 8 kDa antigen family, six overlapping synthetic peptides from an 8 kDa family member (Ts8B2) were synthesized and evaluated by ELISA and MABA with sera from patients with neurocysticercosis (NCC), from infected pigs and from rabbits immunized with recombinant Ts8B2 protein. The pre-immune rabbit sera and the Ts8B2 recombinant protein served as negative and positive controls, respectively. A similar analysis was done with the already described antigenic peptides from another member of the 8 kDa family, highly similar to Ts8B2, the CyDA antigen. Surprisingly, neither the Ts8B2 peptides nor the CyDA peptides were recognized by infected human and porcine sera. However, the entire Ts8B2 recombinant, as well as amino and carboxy-terminal halves were recognized by the positive serum samples. The observed lack of recognition of linear Ts8B2 peptides suggests that the principal serological response to the Ts8B2 family is focused on conformational epitopes in contrast to the previously observed antigenicity of the CyDA peptides. This differential antigenicity of 8 kDa family peptides could be related with parasite antigenic variability. The fact that rabbits experimentally immunized with Ts8B2 did make anti-peptide antibodies to peptides Ts8B2-6 and CyDA-6, located in the carboxy-terminal region demonstrated that the Ts8B2 peptides are not intrinsically non-immunogenic.     

Phosphorylation of serine residues in the N-terminus modulates the activity of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana

ACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca(2+)-ATPase of Arabidopsis thaliana. Several phosphopeptides corresponding to portions of the regulatory N-terminus of ACA8 have been identified in phospho-proteomic studies. To mimic phosphorylation of the ACA8 N-terminus, each of the serines found to be phosphorylated in those studies (Ser19, Ser22, Ser27, Ser29, Ser57, and Ser99) has been mutated to aspartate. Mutants have been expressed in Saccharomyces cerevisiae and characterized: mutants S19D and S57D--and to a lesser extent also mutants S22D and S27D--are deregulated, as shown by their low activation by CaM and by tryptic cleavage of the N-terminus. The His-tagged N-termini of wild-type and mutant ACA8 (6His-(1)M-I(116)) were expressed in Escherichia coli, affinity-purified, and used to analyse the kinetics of CaM binding by surface plasmon resonance. All the analysed mutations affect the kinetics of interaction with CaM to some extent: in most cases, the altered kinetics result in marginal changes in affinity, with the exception of mutants S57D (K(D) ≈ 10-fold higher than wild-type ACA8) and S99D (K(D) about half that of wild-type ACA8). The ACA8 N-terminus is phosphorylated in vitro by two isoforms of A. thaliana calcium-dependent protein kinase (CPK1 and CPK16); phosphorylation of mutant 6His-(1)M-I(116) peptides shows that CPK16 is able to phosphorylate the ACA8 N-terminus at Ser19 and at Ser22. The possible physiological implications of the subtle modulation of ACA8 activity by phosphorylation of its N-terminus are discussed.