High-Throughput Sequencing, Characterization and Detection of New and Conserved Cucumber miRNAs

Micro RNAS (miRNAs) are a class of endogenous small non coding RNAs involved in the post-transcriptional regulation of gene expression. In plants, a great number of conserved and specific miRNAs, mainly arising from model species, have been identified to date. However less is known about the diversity of these regulatory RNAs in vegetal species with agricultural and/or horticultural importance

Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in Buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tisischemia could contribute to the development of the tissue necrosis seen in BU lesions

Beyond the Gene

This paper is a response to the increasing difficulty biologists find in agreeing upon a definition of the gene, and indeed, the increasing disarray in which that concept finds itself. After briefly reviewing these problems, we propose an alternative to both the concept and the word gene—an alternative that, like the gene, is intended to capture the essence of inheritance, but which is both richer and more expressive. It is also clearer in its separation of what the organism statically is (what it tangibly inherits) and what it dynamically does (its functionality and behavior). Our proposal of a genetic functor, or genitor, is a sweeping extension of the classical genotype/phenotype paradigm, yet it appears to be faithful to the findings of contemporary biology, encompassing many of the recently emerging—and surprisingly complex—links between structure and functionality

Optimizing a Massive Parallel Sequencing Workflow for Quantitative miRNA Expression Analysis

BACKGROUND: Massive Parallel Sequencing methods (MPS) can extend and improve the knowledge obtained by conventional microarray technology, both for mRNAs and short non-coding RNAs, e.g. miRNAs. The processing methods used to extract and interpret the information are an important aspect of dealing with the vast amounts of data generated from short read sequencing. Although the number of computational tools for MPS data analysis is constantly growing, their strengths and weaknesses as part of a complex analytical pipe-line have not yet been well investigated. PRIMARY FINDINGS: A benchmark MPS miRNA dataset, resembling a situation in which miRNAs are spiked in biological replication experiments was assembled by merging a publicly available MPS spike-in miRNAs data set with MPS data derived from healthy donor peripheral blood mononuclear cells. Using this data set we observed that short reads counts estimation is strongly under estimated in case of duplicates miRNAs, if whole genome is used as reference. Furthermore, the sensitivity of miRNAs detection is strongly dependent by the primary tool used in the analysis. Within the six aligners tested, specifically devoted to miRNA detection, SHRiMP and MicroRazerS show the highest sensitivity. Differential expression estimation is quite efficient. Within the five tools investigated, two of them (DESseq, baySeq) show a very good specificity and sensitivity in the detection of differential expression. CONCLUSIONS: The results provided by our analysis allow the definition of a clear and simple analytical optimized workflow for miRNAs digital quantitative analysis

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Identification of drought-responsive microRNAs in Medicago truncatula by genome-wide high-throughput sequencing

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are small, endogenous RNAs that play important regulatory roles in development and stress response in plants by negatively affecting gene expression post-transcriptionally. Identification of miRNAs at the global genome-level by high-throughout sequencing is essential to functionally characterize miRNAs in plants. Drought is one of the common environmental stresses limiting plant growth and development. To understand the role of miRNAs in response of plants to drought stress, drought-responsive miRNAs were identified by high-throughput sequencing in a legume model plant, <it>Medicago truncatula</it>.</p> <p>Results</p> <p>Two hundreds eighty three and 293 known miRNAs were identified from the control and drought stress libraries, respectively. In addition, 238 potential candidate miRNAs were identified, and among them 14 new miRNAs and 15 new members of known miRNA families whose complementary miRNA*s were also detected. Both high-throughput sequencing and RT-qPCR confirmed that 22 members of 4 miRNA families were up-regulated and 10 members of 6 miRNA families were down-regulated in response to drought stress. Among the 29 new miRNAs/new members of known miRNA families, 8 miRNAs were responsive to drought stress with both 4 miRNAs being up- and down-regulated, respectively. The known and predicted targets of the drought-responsive miRNAs were found to be involved in diverse cellular processes in plants, including development, transcription, protein degradation, detoxification, nutrient status and cross adaptation.</p> <p>Conclusions</p> <p>We identified 32 known members of 10 miRNA families and 8 new miRNAs/new members of known miRNA families that were responsive to drought stress by high-throughput sequencing of small RNAs from <it>M. truncatula</it>. These findings are of importance for our understanding of the roles played by miRNAs in response of plants to abiotic stress in general and drought stress in particular.</p

Cerebral malaria: insights from host-parasite protein-protein interactions

<p>Abstract</p> <p>Background</p> <p>Cerebral malaria is a form of human malaria wherein <it>Plasmodium falciparum</it>-infected red blood cells adhere to the blood capillaries in the brain, potentially leading to coma and death. Interactions between parasite and host proteins are important in understanding the pathogenesis of this deadly form of malaria. It is, therefore, necessary to study available protein-protein interactions to identify lesser known interactions that could throw light on key events of cerebral malaria.</p> <p>Methods</p> <p>Sequestration, haemostasis dysfunction, systemic inflammation and neuronal damage are key processes of cerebral malaria. Key events were identified from literature as being crucial to these processes. An integrated interactome was created using available experimental and predicted datasets as well as from literature. Interactions from this interactome were filtered based on Gene Ontology and tissue-specific annotations, and further analysed for relevance to the key events.</p> <p>Results</p> <p>PfEMP1 presentation, platelet activation and astrocyte dysfunction were identified as the key events influencing the disease. 48896 host-parasite along with other host-parasite, host-host and parasite-parasite protein-protein interactions obtained from a disease-specific corpus were combined to form an integrated interactome. Filtering of the interactome resulted in five host-parasite PPI, six parasite-parasite and two host-host PPI. The analysis of these interactions revealed the potential significance of apolipoproteins and temperature/Hsp expression on efficient PfEMP1 presentation; role of MSP-1 in platelet activation; effect of parasite proteins in TGF-β regulation and the role of albumin in astrocyte dysfunction.</p> <p>Conclusions</p> <p>This work links key host-parasite, parasite-parasite and host-host protein-protein interactions to key processes of cerebral malaria and generates hypotheses for disease pathogenesis based on a filtered interaction dataset. These hypotheses provide novel and significant insights to cerebral malaria.</p

Long-term follow-up and treatment in nine boys with X-linked creatine transporter defect

The creatine transporter (CRTR) defect is a recently discovered cause of X-linked intellectual disability for which treatment options have been explored. Creatine monotherapy has not proved effective, and the effect of treatment with L-arginine is still controversial. Nine boys between 8 months and 10 years old with molecularly confirmed CRTR defect were followed with repeated 1H-MRS and neuropsychological assessments during 4–6 years of combination treatment with creatine monohydrate, L-arginine, and glycine. Treatment did not lead to a significant increase in cerebral creatine content as observed with H1-MRS. After an initial improvement in locomotor and personal-social IQ subscales, no lasting clinical improvement was recorded. Additionally, we noticed an age-related decline in IQ subscales in boys affected with the CRTR defect

Identification and characterization of 27 conserved microRNAs in citrus

MicroRNAs (miRNAs) are a class of non-protein-coding small RNAs. Considering the conservation of many miRNA genes in different plant genomes, the identification of miRNAs from non-model organisms is both practicable and instrumental in addressing miRNA-guided gene regulation. Citrus is an important staple fruit tree, and publicly available expressed sequence tag (EST) database for citrus are increasing. However, until now, little has been known about miRNA in citrus. In this study, 27 known miRNAs from Arabidopsis were searched against citrus EST databases for miRNA precursors, of which 13 searched precursor sequences could form fold-back structures similar with those of Arabidopsis. The ubiquitous expression of those 13 citrus microRNAs and other 13 potential citrus miRNAs could be detected in citrus leaf, young shoot, flower, fruit and root by northern blotting, and some of them showed differential expression in different tissues. Based on the fact that miRNAs exhibit perfect or nearly perfect complementarity with their target sequences, a total of 41 potential targets were identified for 15 citrus miRNAs. The majority of the targets are transcription factors that play important roles in citrus development, including leaf, shoot, and root development. Additionally, some other target genes appear to play roles in diverse physiological processes. Four target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate. Overall, this study in the identification and characterization of miRNAs in citrus can initiate further study on citrus miRNA regulation mechanisms, and it can help us to know more about the important roles of miRNAs in citrus

Physiological traits of the symbiotic bacterium Teredinibacter turnerae isolated from the mangrove shipworm Neoteredo reynei

Nutrition in the Teredinidae family of wood-boring mollusks is sustained by cellulolytic/nitrogen fixing symbiotic bacteria of the Teredinibacter clade. The mangrove Teredinidae Neoteredo reynei is popularly used in the treatment of infectious diseases in the north of Brazil. In the present work, the symbionts of N. reynei, which are strictly confined to the host's gills, were conclusively identified as Teredinibacter turnerae. Symbiont variants obtained in vitro were able to grow using casein as the sole carbon/nitrogen source and under reduced concentrations of NaCl. Furthermore, cellulose consumption in T. turnerae was clearly reduced under low salt concentrations. As a point of interest, we hereby report first hand that T. turnerae in fact exerts antibiotic activity. Furthermore, this activity was also affected by NaCl concentration. Finally, T. turnerae was able to inhibit the growth of Gram-negative and Gram-positive bacteria, this including strains of Sphingomonas sp., Stenotrophomonas maltophilia, Bacillus cereus and Staphylococcus sciuri. Our findings introduce new points of view on the ecology of T. turnerae, and suggest new biotechnological applications for this marine bacterium