Metatranscriptomes from diverse microbial communities: assessment of data reduction techniques for rigorous annotation

Background Metatranscriptome sequence data can contain highly redundant sequences from diverse populations of microbes and so data reduction techniques are often applied before taxonomic and functional annotation. For metagenomic data, it has been observed that the variable coverage and presence of closely related organisms can lead to fragmented assemblies containing chimeric contigs that may reduce the accuracy of downstream analyses and some advocate the use of alternate data reduction techniques. However, it is unclear how such data reduction techniques impact the annotation of metatranscriptome data and thus affect the interpretation of the results. Results To investigate the effect of such techniques on the annotation of metatranscriptome data we assess two commonly employed methods: clustering and de-novo assembly. To do this, we also developed an approach to simulate 454 and Illumina metatranscriptome data sets with varying degrees of taxonomic diversity. For the Illumina simulations, we found that a two-step approach of assembly followed by clustering of contigs and unassembled sequences produced the most accurate reflection of the real protein domain content of the sample. For the 454 simulations, the combined annotation of contigs and unassembled reads produced the most accurate protein domain annotations. Conclusions Based on these data we recommend that assembly be attempted, and that unassembled reads be included in the final annotation for metatranscriptome data, even from highly diverse environments as the resulting annotations should lead to a more accurate reflection of the transcriptional behaviour of the microbial population under investigation

Global discovery and characterization of small non-coding RNAs in marine microalgae

Background Marine phytoplankton are responsible for 50% of the CO2 that is fixed annually worldwide and contribute massively to other biogeochemical cycles in the oceans. Diatoms and coccolithophores play a significant role as the base of the marine food web and they sequester carbon due to their ability to form blooms and to biomineralise. To discover the presence and regulation of short non-coding RNAs (sRNAs) in these two important phytoplankton groups, we sequenced short RNA transcriptomes of two diatom species (Thalassiosira pseudonana, Fragilariopsis cylindrus) and validated them by Northern blots along with the coccolithophore Emiliania huxleyi. Results Despite an exhaustive search, we did not find canonical miRNAs in diatoms. The most prominent classes of sRNAs in diatoms were repeat-associated sRNAs and tRNA-derived sRNAs. The latter were also present in E. huxleyi. tRNA-derived sRNAs in diatoms were induced under important environmental stress conditions (iron and silicate limitation, oxidative stress, alkaline pH), and they were very abundant especially in the polar diatom F. cylindrus (20.7% of all sRNAs) even under optimal growth conditions. Conclusions This study provides first experimental evidence for the existence of short non-coding RNAs in marine microalgae. Our data suggest that canonical miRNAs are absent from diatoms. However, the group of tRNA-derived sRNAs seems to be very prominent in diatoms and coccolithophores and maybe used for acclimation to environmental conditions

Simon Moulton Hamlin Correspondence

Entries include a brief biography, a newspaper clipping, and a letter typed on 74th Congress stationery

The UEA sRNA Workbench (version 4.4): a comprehensive suite of tools for analyzing miRNAs and sRNAs

Motivation: RNA interference, a highly conserved regulatory mechanism, is mediated via small RNAs (sRNA). Recent technical advances enabled the analysis of larger, complex datasets and the investigation of microRNAs and the less known small interfering RNAs. However, the size and intricacy of current data requires a comprehensive set of tools, able to discriminate the patterns from the low-level, noise-like, variation; numerous and varied suggestions from the community represent an invaluable source of ideas for future tools, the ability of the community to contribute to this software is essential. Results: We present a new version of the UEA sRNA Workbench, reconfigured to allow an easy insertion of new tools/workflows. In its released form, it comprises of a suite of tools in a user-friendly environment, with enhanced capabilities for a comprehensive processing of sRNA-seq data e.g. tools for an accurate prediction of sRNA loci (CoLIde) and miRNA loci (miRCat2), as well as workflows to guide the users through common steps such as quality checking of the input data, normalization of abundances or detection of differential expression represent the first step in sRNA-seq analyses

miRCat2: Accurate prediction of plant and animal microRNAs from next-generation sequencing datasets

Motivation: MicroRNAs are a class of ∼21-22 nucleotide small RNAs which are excised from a stable hairpin-like secondary structure. They have important gene regulatory functions and are involved in many pathways including developmental timing, organogenesis and development in eukaryotes. There are several computational tools for miRNA detection from next-generation sequencing (NGS) datasets. However, many of these tools suffer from high false positive and false negative rates. Here we present a novel miRNA prediction algorithm, miRCat2. miRCat2 incorporates a new entropy-based approach to detect miRNA loci, which is designed to cope with the high sequencing depth of current NGS datasets. It has a user-friendly interface and produces graphical representations of the hairpin structure and plots depicting the alignment of sequences on the secondary structure. Results: We tested miRCat2 on a number of animal and plant datasets and present a comparative analysis with miRCat, miRDeep2, miRPlant and miReap. We also use mutants in the miRNA biogenesis pathway to evaluate the predictions of these tools. Results indicate that miRCat2 has an improved accuracy compared with other methods tested. Moreover, miRCat2 predicts several new miRNAs that are differentially expressed in wildtype versus mutants in the miRNA biogenesis pathway. Availability: miRCat2 is part of the UEA small RNA Workbench and is freely available from http://srnaworkbench.cmp.uea.ac.uk

Novel biosensor fabrication methodology based on processable conducting polyaniline nanoparticles

This work investigates polyaniline (PANI) nanoparticles, (synthesised using dodecylbenzenesulphonic acid (DBSA) as a dopant), as a novel, highly processable, non-diffusional mediating species in an enzyme biosensing application. These nanoparticles are readily dispersed in aqueous media which helps overcome some of the processability issues traditionally associated with polyaniline. Modification of screen-printed electrodes was readily achieved with these aqueous nanoparticle dispersions, where the nanoparticles were simply cast by a drop-coating method onto the surface. After suitable pH adjustment, it was shown that horseradish peroxidase (HRP) enzyme could be added to the dispersion, and cast simultaneously with the conducting polyaniline. This effective fabrication method involves no electrochemical steps, and as such is easily amenable to mass production. The feasibility of casting enzyme with polyaniline nanoparticles is demonstrated in this short communication. More accurate deposition of protein-containing inks onto screen-printed carbon working electrodes could in the future transfer the drop-coating protocol from manual deposition to largescale production by mechanical methods such as ink-jet printing

Electrical stimulation using conductive polymer polypyrrole promotes differentiation of human neural stem cells: a biocompatible platform for translational neural tissue engineering

Conductive polymers (CPs) are organic materials that hold great promise for biomedicine. Potential applications include in vitro or implantable electrodes for excitable cell recording and stimulation, and conductive scaffolds for cell support and tissue engineering. Here we demonstrate the utility of electroactive CP Polypyrrole (PPy) containing the anionic dopant dodecylbenzenesulfonate (DBS) to differentiate novel clinically relevant human neural stem cells (hNSCs). Electrical stimulation of PPy(DBS) induced hNSCs to predominantly β-III Tubulin (Tuj1) expressing neurons, with lower induction of glial fibrillary acidic protein (GFAP) expressing glial cells. In addition, stimulated cultures comprised nodes or clusters of neurons with longer neurites and greater branching than unstimulated cultures. Cell clusters showed a similar spatial distribution to regions of higher conductivity on the film surface. Our findings support the use of electrical stimulation to promote neuronal induction and the biocompatibility of PPy(DBS) with hNSCs, and opens up the possibility of identifying novel mechanisms of fate determination of differentiating human stem cells for advanced in vitro modelling, translational drug discovery and regenerative medicine

Wet-Spun Trojan Horse Cell Constructs for Engineering Muscle

Engineering of 3D regenerative skeletal muscle tissue constructs (skMTCs) using hydrogels containing muscle precursor cells (MPCs) is of potential benefit for repairing Volumetric Muscle Loss (VML) arising from trauma (e.g., road/industrial accident, war injury) or for restoration of functional muscle mass in disease (e.g., Muscular Dystrophy, muscle atrophy). Additive Biofabrication (AdBiofab) technologies make possible fabrication of 3D regenerative skMTCs that can be tailored to specific delivery requirements of VML or functional muscle restoration. Whilst 3D printing is useful for printing constructs of many tissue types, the necessity of a balanced compromise between cell type, required construct size and material/fabrication process cyto-compatibility can make the choice of 3D printing a secondary alternative to other biofabrication methods such as wet-spinning. Alternatively, wet-spinning is more amenable to formation of fibers rather than (small) layered 3D-Printed constructs. This study describes the fabrication of biosynthetic alginate fibers containing MPCs and their use for delivery of dystrophin-expressing cells to dystrophic muscle in the mdx mouse model of Duchenne Muscular Dystrophy (DMD) compared to poly(DL-lactic-co-glycolic acid) copolymer (PLA:PLGA) topically-seeded with myoblasts. In addition, this study introduces a novel method by which to create 3D layered wet-spun alginate skMTCs for bulk mass delivery of MPCs to VML lesions. As such, this work introduces the concept of “Trojan Horse” Fiber MTCs (TH-fMTCs) and 3d Mesh-MTCs (TH-mMTCs) for delivery of regenerative MPCs to diseased and damaged muscle, respectively

Antiepileptic effects of lacosamide loaded polymers implanted subdurally in GAERS

The current experiment investigated the ability of coaxial electrospun poly(D,L-lactide-co-glycolide) (PLGA) biodegradable polymer implants loaded with the antiepileptic drugs (AED) lacosamide to reduce seizures following implantation above the motor cortex in the Genetic Absence Epilepsy Rat from Strasbourg (GAERS). In this prospective, randomized, masked experiments, GAERS underwent surgery for implantation of skull electrodes (n = 6), skull electrodes and blank polymers (n = 6), or skull electrodes and lacosamide loaded polymers (n = 6). Thirty-minute electroencephalogram (EEG) recordings were started at day 7 after surgery and continued for eight weeks. The number of SWDs and mean duration of one SWD were compared week-by-week between the three groups. There was no difference in the number of SWDs between any of the groups. However, the mean duration of one SWD was significantly lower in the lacosamide polymer group for up to 7 weeks when compared to the control group (0.004 \u3c p \u3c 0.038). The mean duration of one seizure was also lower at weeks 3, 5, 6, and 7 when compared to the blank polymer group (p = 0.016, 0.037, 0.025, and 0.025, resp.). We have demonstrated that AED loaded PLGA polymer sheets implanted on the surface of the cortex could affect seizure activity in GAERS for a sustained period

Reevaluating the modernization hypothesis

We revisit and critically reevaluate the widely accepted modernization hypothesis which claims that per capita income causes the creation and the consolidation of democracy. Existing studies find support for this hypothesis because they fail to control for the presence of omitted variables. Controlling for these factors either by including country fixed effects in a linear model or by including parameterized random effects in a nonlinear double hazard model removes the correlation between income and the likelihood of transitions to and from democratic regimes. In addition, the estimated fixed effects from the linear model are related to historical factors that affect both the level of income per capita and the likelihood of democracy in a country. This evidence is consistent with the idea that events during critical historical junctures can lead to divergent political–economic development paths, some leading to prosperity and democracy, others to relative poverty and non-democracy