Proteases in malaria parasites - a phylogenomic perspective

Malaria continues to be one of the most devastating global health problems due to the high morbidity and mortality it causes in endemic regions. The search for new antimalarial targets is of high priority because of the increasing prevalence of drug resistance in malaria parasites. Malarial proteases constitute a class of promising therapeutic targets as they play important roles in the parasite life cycle and it is possible to design and screen for specific protease inhibitors. In this mini-review, we provide a phylogenomic overview of malarial proteases. An evolutionary perspective on the origin and divergence of these proteases will provide insights into the adaptive mechanisms of parasite growth, development, infection, and pathogenesis.B.     

Multigene analyses of bilaterian animals corroborate the monophyly of Ecdysozoa, Lophotrochozoa, and Protostomia

Almost a decade ago, a new phylogeny of bilaterian animals was inferred from small-subunit ribosomal RNA (rRNA) that claimed the monophyly of two major groups of protostome animals: Ecdysozoa (e.g., arthropods, nematodes, onychophorans, and tardigrades) and Lophotrochozoa (e.g., annelids, molluscs, platyhelminths, brachiopods, and rotifers). However, it received little additional support. In fact, several multigene analyses strongly argued against this new phylogeny. These latter studies were based on a large amount of sequence data and therefore showed an apparently strong statistical support. Yet, they covered only a few taxa (those for which complete genomes were available), making systematic artifacts of tree reconstruction more probable. Here we expand this sparse taxonomic sampling and analyze a large data set (146 genes, 35,371 positions) from a diverse sample of animals (35 species). Our study demonstrates that the incongruences observed between rRNA and multigene analyses were indeed due to long-branch attraction artifacts, illustrating the enormous impact of systematic biases on phylogenomic studies. A refined analysis of our data set excluding the most biased genes provides strong support in favor of the new animal phylogeny and in addition suggests that urochordates are more closely related to vertebrates than are cephalochordates. These findings have important implications for the interpretation of morphological and genomic data.     

The complete mitochondrial genome sequence of the little egret (Egretta garzetta)

Many phylogenetic questions in the Ciconiiformes remain unresolved and complete mitogenome data are urgently needed for further molecular investigation. In this work, we determined the complete mitogenome sequence of the little egret (Egretta garzetta). The genome was 17,361 bp in length and the gene organization was typical of other avian mtDNA. In protein-coding genes (PCGs), a C insertion was found in ND3, and COIII and ND4 terminated with incomplete stop codons (T). tRNA-Val and tRNA-Ser (AGY) were unable to fold into canonical cloverleaf secondary structures because they had lost the DHU arms. Long repetitive sequences consisting of five types of tandem repeats were found at the 3′ end of Domain III in the control region. A phylogenetic analysis of 11 species of Ciconiiformes was done using complete mitogenome data and 12 PCGs. The tree topologies obtained with these two strategies were identical, which strongly confirmed the monophyly of Ardeidae, Threskiorothidae and Ciconiidae. The phylogenetic analysis also revealed that Egretta was more closely related to Ardea than to Nycticorax in the Ardeidae, and Platalea was more closely related to Threskiornis than to Nipponia in the Threskiornithidae. These findings contribute to our understanding of the phylogenetic relationships of Ciconiiformes based on complete mitogenome data.     

Co-option of an anteroposterior head axis patterning system for proximodistal patterning of appendages in early bilaterian evolution

The enormous diversity of extant animal forms is a testament to the power of evolution, and much of this diversity has been achieved through the emergence of novel morphological traits. The origin of novel morphological traits is an extremely important issue in biology, and a frequent source of this novelty is co-option of pre-existing genetic systems for new purposes (Carroll et al., 2008). Appendages, such as limbs, fins and antennae, are structures common to many animal body plans which must have arisen at least once, and probably multiple times, in lineages which lacked appendages. We provide evidence that appendage proximodistal patterning genes are expressed in similar registers in the anterior embryonic neurectoderm of Drosophila melanogaster and Saccoglossus kowalevskii (a hemichordate). These results, in concert with existing expression data from a variety of other animals suggest that a pre-existing genetic system for anteroposterior head patterning was co-opted for patterning of the proximodistal axis of appendages of bilaterian animals.     

The complete mitochondrial genome sequence of the black-capped capuchin (Cebus apella)

The phylogenetic relationships of primates have been extensively investigated, but key issues remain unresolved. Complete mitochondrial genome (mitogenome) data have many advantages in phylogenetic analyses, but such data are available for only 46 primate species. In this work, we determined the complete mitogenome sequence of the black-capped capuchin (Cebus apella). The genome was 16,538 bp in size and consisted of 13 protein-coding genes, 22 tRNAs, two rRNAs and a control region. The genome organization, nucleotide composition and codon usage did not differ significantly from those of other primates. The control region contained several distinct repeat motifs, including a putative termination-associated sequence (TAS) and several conserved sequence blocks (CSB-F, E, D, C, B and 1). Among the protein-coding genes, the COII gene had lower nonsynonymous and synonymous substitutions rates while the ATP8 and ND4 genes had higher rates. A phylogenetic analysis using Maximum likelihood and Bayesian methods and the complete mitogenome data for platyrrhine species confirmed the basal position of the Callicebinae and the sister relationship between Atelinae and Cebidae, as well as the sister relationship between Aotinae (Aotus) and Cebinae (Cebus/Saimiri) in Cebidae. These conclusions agreed with the most recent molecular phylogenetic investigations on primates. This work provides a framework for the use of complete mitogenome information in phylogenetic analyses of the Platyrrhini and primates in general.     

Assessing visual requirements for social context-dependent activation of the songbird song system

Social context has been shown to have a profound influence on brain activation in a wide range of vertebrate species. Best studied in songbirds, when males sing undirected song, the level of neural activity and expression of immediate early genes (IEGs) in several song nuclei is dramatically higher or lower than when they sing directed song to other birds, particularly females. This differential social context-dependent activation is independent of auditory input and is not simply dependent on the motor act of singing. These findings suggested that the critical sensory modality driving social context-dependent differences in the brain could be visual cues. Here, we tested this hypothesis by examining IEG activation in song nuclei in hemispheres to which visual input was normal or blocked. We found that covering one eye blocked visually induced IEG expression throughout both contralateral visual pathways of the brain, and reduced activation of the contralateral ventral tegmental area, a non-visual midbrain motivation-related area affected by social context. However, blocking visual input had no effect on the social context-dependent activation of the contralateral song nuclei during female-directed singing. Our findings suggest that individual sensory modalities are not direct driving forces for the social context differences in song nuclei during singing. Rather, these social context differences in brain activation appear to depend more on the general sense that another individual is present.     

Theoretical evidence for the functional benefit of root cortical aerenchyma in soils with low phosphorus availability

Background and Aims

The formation of root cortical aerenchyma (RCA) reduces root respiration and nutrient content by converting living tissue to air volume. It was hypothesized that RCA increases soil resource acquisition by reducing the metabolic and phosphorus cost of soil exploration.

Methods

To test the quantitative logic of the hypothesis, SimRoot, a functional–structural plant model with emphasis on root architecture and nutrient acquisition, was employed. Sensitivity analyses for the effects of RCA on the initial 40 d of growth of maize (Zea mays) and common bean (Phaseolus vulgaris) were conducted in soils with varying degrees of phosphorus availability. With reference to future climates, the benefit of having RCA in high CO₂ environments was simulated.

Key Results

The model shows that RCA may increase the growth of plants faced with suboptimal phosphorus availability up to 70 % for maize and 14 % for bean after 40 d of growth. Maximum increases were obtained at low phosphorus availability (3 µm). Remobilization of phosphorus from dying cells had a larger effect on plant growth than reduced root respiration. The benefit of both these functions was additive and increased over time. Larger benefits may be expected for mature plants. Sensitivity analysis for light-use efficiency showed that the benefit of having RCA is relatively stable, suggesting that elevated CO₂ in future climates will not significantly effect the benefits of having RCA.

Conclusions

The results support the hypothesis that RCA is an adaptive trait for phosphorus acquisition by remobilizing phosphorus from the root cortex and reducing the metabolic costs of soil exploration. The benefit of having RCA in low-phosphorus soils is larger for maize than for bean, as maize is more sensitive to low phosphorus availability while it has a more ‘expensive’ root system. Genetic variation in RCA may be useful for breeding phosphorus-efficient crop cultivars, which is important for improving global food security.     

Susceptibility of experimental animals to reinfection with Clonorchis sinensis

The present study observed the resistance to reinfection with Clonorchis sinensis in various experimental animals including mice, guinea pigs, rabbits, and dogs, as well as rats and hamsters. The resistance rates to reinfection in rats, mice, hamsters, guinea pigs, rabbits, and dogs were 79.7%, 58.0%, -12.6%, 54.8%, 62.6%, and 6.0%, respectively. Worms recovered from reinfected rats and mice were immature, and significantly smaller than those from the primarily infected (P < 0.01), whereas those from other animals were fully matured to adults. These findings indicate that the protective response against reinfection with C. sinensis is prominent in rats and mice, and that they may be a good animal model to investigate the mechanism of resistance to reinfection with C. sinensis.     

Inoculation with arbuscular mycorrhizal fungi suppresses initiation of haustoria in the root hemiparasite Pedicularis tricolor

Background and Aims

Plant parasitism and arbuscular mycorrhizal (AM) associations have many parallels and share a number of regulatory pathways. Despite a rapid increase in investigations addressing the roles of AM fungi in regulating interactions between parasitic plants and their hosts, few studies have tested the effect of AM fungi on the initiation and differentiation of haustoria, the parasite-specific structures exclusively responsible for host attachment and nutrient transfer. In this study, we tested the influence of AM fungi on haustorium formation in a root hemiparasitic plant.

Methods

Using a facultative root hemiparasitic species (Pedicularis tricolor) with the potential to form AM associations, the effects of inoculation were tested with two AM fungal species, Glomus mosseae and Glomus intraradices, on haustorium initiation in P. tricolor grown alone or with Hordeum vulgare ‘Fleet’ (barley) as the host plant. This study consisted of two greenhouse pot experiments.

Key Results

Both AM fungal species dramatically suppressed intraspecific haustorium initiation in P. tricolor at a very low colonization level. The suppression over-rode inductive effects of the parasite''s host plant on haustoria production and caused significant growth depression of P. tricolor.

Conclusions

AM fungi had strong and direct suppressive effects on haustorium formation in the root hemiparasite. The significant role of AM fungi in haustorium initiation of parasitic plants was demonstrated for the first time. This study provides new clues for the regulation of haustorium formation and a route to development of new biocontrol strategies in management of parasitic weeds.     

Auxin increases the hydrogen peroxide (H2O2) concentration in tomato (Solanum lycopersicum) root tips while inhibiting root growth

Background and Aims

The hormone auxin and reactive oxygen species (ROS) regulate root elongation, but the interactions between the two pathways are not well understood. The aim of this study was to investigate how auxin interacts with ROS in regulating root elongation in tomato, Solanum lycopersicum.

Methods

Wild-type and auxin-resistant mutant, diageotropica (dgt), of tomato (S. lycopersicum ‘Ailsa Craig’) were characterized in terms of root apical meristem and elongation zone histology, expression of the cell-cycle marker gene Sl-CycB1;1, accumulation of ROS, response to auxin and hydrogen peroxide (H₂O₂), and expression of ROS-related mRNAs.

Key Results

The dgt mutant exhibited histological defects in the root apical meristem and elongation zone and displayed a constitutively increased level of hydrogen peroxide (H₂O₂) in the root tip, part of which was detected in the apoplast. Treatments of wild-type with auxin increased the H₂O₂ concentration in the root tip in a dose-dependent manner. Auxin and H₂O₂ elicited similar inhibition of cell elongation while bringing forth differential responses in terms of meristem length and number of cells in the elongation zone. Auxin treatments affected the expression of mRNAs of ROS-scavenging enzymes and less significantly mRNAs related to antioxidant level. The dgt mutation resulted in resistance to both auxin and H₂O₂ and affected profoundly the expression of mRNAs related to antioxidant level.

Conclusions

The results indicate that auxin regulates the level of H₂O₂ in the root tip, so increasing the auxin level triggers accumulation of H₂O₂ leading to inhibition of root cell elongation and root growth. The dgt mutation affects this pathway by reducing the auxin responsiveness of tissues and by disrupting the H₂O₂ homeostasis in the root tip.     

Continuous,high-resolution biospeckle imaging reveals a discrete zone of activity at the root apex that responds to contact with obstacles

Background and Aims

Shining a laser onto biological material produces light speckles termed biospeckles. Patterns of biospeckle activity reflect changes in cell biochemistry, developmental processes and responses to the environment. The aim of this work was to develop methods to investigate the biospeckle activity in roots and to characterize the distribution of its intensity and response to thigmostimuli.

Methods

Biospeckle activity in roots of Zea mays, and also Jatropha curcas and Citrus limonia, was imaged live and in situ using a portable laser and a digital microscope with a spatial resolution of 10 μm per pixel and the ability to capture images every 0·080 s. A procedure incorporating a Fujii algorithm, image restoration using median and Gaussian filters, image segmentation using maximum-entropy threshold methods and the extraction of features using a tracing algorithm followed by spline fitting were developed to obtain quantitative information from images of biospeckle activity. A wavelet transform algorithm was used for spectral decomposition of biospeckle activity and generalized additive models were used to attribute statistical significance to changes in patterns of biospeckle activity.

Key Results

The intensity of biospeckle activity was greatest close to the root apex. Higher frequencies (3–6 Hz) contributed most to the total intensity of biospeckle activity. When a root encountered an obstacle, the intensity of biospeckle activity decreased abruptly throughout the root system. The response became attenuated with repeated thigmostimuli.

Conclusions

The data suggest that at least one component of root biospeckle activity resulted from a biological process, which is located in the zone of cell division and responds to thigmostimuli. However, neither individual cell division events nor root elongation is likely to be responsible for the patterns of biospeckle activity.     

Cnidarian and bilaterian promoters can direct GFP expression in transfected hydra

Complete sexual development is not easily amenable to experimentation in hydra. Therefore, the analysis of gene function and gene regulation requires the introduction of exogenous DNA in a large number of cells of the hydra polyps and the significant expression of reporter constructs in these cells. We present here the procedure whereby we coupled DNA injection into the gastric cavity to electroporation of the whole animal in order to efficiently transfect hydra polyps. We could detect GFP fluorescence in both endodermal and ectodermal cell layers of live animals and in epithelial as well as interstitial cell types of dissociated hydra. In addition, we could confirm GFP protein expression by showing colocalisation between GFP fluorescence and anti-GFP immunofluorescence. Finally, when a FLAG epitope was inserted in-frame with the GFP coding sequence, GFP fluorescence also colocalised with anti-FLAG immunofluorescence. This GFP expression in hydra cells was directed by various promoters, either homologous, like the hydra homeobox cnox-2 gene promoter, or heterologous, like the two nematode ribosomal protein S5 and L28 gene promoters, and the chicken beta-actin gene promoter. This strategy provides new tools for dissecting developmental molecular mechanisms in hydra; more specifically, the genetic regulations that take place in endodermal cells at the time budding or regeneration is initiated.     

Composite Cucurbita pepo plants with transgenic roots as a tool to study root development

Background and Aims

In most plant species, initiation of lateral root primordia occurs above the elongation zone. However, in cucurbits and some other species, lateral root primordia initiation and development takes place in the apical meristem of the parental root. Composite transgenic plants obtained by Agrobacterium rhizogenes-mediated transformation are known as a suitable model to study root development. The aim of the present study was to establish this transformation technique for squash.

Methods

The auxin-responsive promoter DR5 was cloned into the binary vectors pKGW-RR-MGW and pMDC162-GFP. Incorporation of 5-ethynyl-2′-deoxyuridine (EdU) was used to evaluate the presence of DNA-synthesizing cells in the hypocotyl of squash seedlings to find out whether they were suitable for infection. Two A. rhizogenes strains, R1000 and MSU440, were used. Roots containing the respective constructs were selected based on DsRED1 or green fluorescent protein (GFP) fluorescence, and DR5::Egfp-gusA or DR5::gusA insertion, respectively, was verified by PCR. Distribution of the response to auxin was visualized by GFP fluorescence or β-glucuronidase (GUS) activity staining and confirmed by immunolocalization of GFP and GUS proteins, respectively.

Key Results

Based on the distribution of EdU-labelled cells, it was determined that 6-day-old squash seedlings were suited for inoculation by A. rhizogenes since their root pericycle and the adjacent layers contain enough proliferating cells. Agrobacterium rhizogenes R1000 proved to be the most virulent strain on squash seedlings. Squash roots containing the respective constructs did not exhibit the hairy root phenotype and were morphologically and structurally similar to wild-type roots.

Conclusions

The auxin response pattern in the root apex of squash resembled that in arabidopsis roots. Composite squash plants obtained by A. rhizogenes-mediated transformation are a good tool for the investigation of root apical meristem development and root branching.     

质体系统发育基因组解析旋花科系统发育关系

旋花科是一个世界广布的类群,具有丰富的形态特征和重要的经济价值。然而,目前该科主要分支或族间的系统发育关系问题一直未解决。为解析旋花科内系统发育关系,该研究代表性选取旋花科内8个族40个物种,基于质体全基因组数据,使用最大似然法和贝叶斯推论进行系统发育分析。结果表明：(1)旋花科质体基因组均为四分体结构,质体基因组大小为113 273～164 112 bp,蛋白质编码基因数目为66～79个。(2)基于五种DNA矩阵(即WCG、CDS、LSC、IR、SSC)的系统发育分析结果显示,WCG矩阵和CDS矩阵的拓扑结构基本一致,仅少数分支的支持率略有差异;LSC矩阵和WCG矩阵的拓扑结构差异在于菟丝子族、马蹄金族和盐帚花族的系统位置;AU检验和SH检验结果显示,WCG矩阵和SSC矩阵与IR矩阵的拓扑结构有显著冲突。(3)所有系统发育分析结果均显示,菟丝子属和马蹄金族都包括在旋花亚科内,应处理为族等级。(4)基于WCG矩阵和CDS矩阵较好地解决了旋花科8个族之间的系统发育关系,即心被藤族和丁公藤族聚为一支,最先从旋花亚科分化出来,随后是菟丝子族,剩下的5个族分成2个分支。(5)系统发育基因组分析...     

A phylogenomic investigation into the origin of metazoa

The evolution of multicellular animals (Metazoa) from theirunicellular ancestors was a key transition that was accompaniedby the emergence and diversification of gene families associatedwith multicellularity. To clarify the timing and order of specificevents in this transition, we conducted expressed sequence tagsurveys on 4 putative protistan relatives of Metazoa includingthe choanoflagellate Monosiga ovata, the ichthyosporeans Sphaeroformaarctica and Amoebidium parasiticum, and the amoeba Capsasporaowczarzaki, and 2 members of Amoebozoa, Acanthamoeba castellaniiand Mastigamoeba balamuthi. We find that homologs of genes involvedin metazoan multicellularity exist in several of these unicellularorganisms, including 1 encoding a membrane-associated guanylatekinase with an inverted arrangement of protein-protein interactiondomains (MAGI) in Capsaspora. In Metazoa, MAGI regulates tightjunctions involved in cell-cell communication. By phylogenomicanalyses of genes encoded in nuclear and mitochondrial genomes,we show that the choanoflagellates are the closest relativesof the Metazoa, followed by the Capsaspora and Ichthyosporealineages, although the branching order between the latter 2groups remains unclear. Understanding the function of "metazoan-specific"proteins we have identified in these protists will clarify theevolutionary steps that led to the emergence of the Metazoa.     

Ultrastructural aspects of the 'statocyst' of Xenoturbella (Deuterostomia) cast doubt on its function as a georeceptor

The "statocyst" in the enigmatic worm Xenoturbella is a structure containing motile flagellated cells. It is situated inside the subepidermal membrane complex (between epidermis and muscular layers) in the anterior end of the body. The motile cells contain a lipophilic refractile body ("statolith"), and a series of vesicles from small dense core vesicles presumably formed from the refractile body to large vesicles with dense aggregates of filamentous tubules that become exocytized through secretion. It is unlikely that the statocyst is a georeceptor (true statocyst); maybe it has an endocrine function.     

Ray Meta: scalable de novo metagenome assembly and profiling

Voluminous parallel sequencing datasets, especially metagenomic experiments, require distributed computing for de novo assembly and taxonomic profiling. Ray Meta is a massively distributed metagenome assembler that is coupled with Ray Communities, which profiles microbiomes based on uniquely-colored k-mers. It can accurately assemble and profile a three billion read metagenomic experiment representing 1,000 bacterial genomes of uneven proportions in 15 hours with 1,024 processor cores, using only 1.5 GB per core. The software will facilitate the processing of large and complex datasets, and will help in generating biological insights for specific environments. Ray Meta is open source and available at http://denovoassembler.sf.net.