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A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro , in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.

Background Salmonid fishes exhibit high levels of phenotypic and ecological variation and are thus ideal model systems for studying evolutionary processes of adaptive divergence and speciation. Furthermore, salmonids are of major interest in fisheries, aquaculture, and conservation research. Improving understanding of the genetic mechanisms underlying traits in these species would significantly progress research in these fields. Here we generate high quality de novo transcriptomes for four salmonid species: Atlantic salmon ( Salmo salar ), brown trout ( Salmo trutta ), Arctic charr ( Salvelinus alpinus ), and European whitefish ( Coregonus lavaretus ). All species except Atlantic salmon have no reference genome publicly available and few if any genomic studies to date. Results We used paired-end RNA-seq on Illumina to generate high coverage sequencing of multiple individuals, yielding between 180 and 210 M reads per species. After initial assembly, strict filtering was used to remove duplicated, redundant, and low confidence transcripts. The final assemblies consisted of 36,505 protein-coding transcripts for Atlantic salmon, 35,736 for brown trout, 33,126 for Arctic charr, and 33,697 for European whitefish and are made publicly available. Assembly completeness was assessed using three approaches, all of which supported high quality of the assemblies: 1) ~78% of Actinopterygian single-copy orthologs were successfully captured in our assemblies, 2) orthogroup inference identified high overlap in the protein sequences present across all four species (40% shared across all four and 84% shared by at least two), and 3) comparison with the published Atlantic salmon genome suggests that our assemblies represent well covered (~98%) protein-coding transcriptomes. Thorough comparison of the generated assemblies found that 84-90% of transcripts in each assembly were orthologous with at least one of the other three species. We also identified 34-37% of transcripts in each assembly as paralogs. We further compare completeness and annotation statistics of our new assemblies to available related species. Conclusion New, high-confidence protein-coding transcriptomes were generated for four ecologically and economically important species of salmonids. This offers a high quality pipeline for such complex genomes, represents a valuable contribution to the existing genomic resources for these species and provides robust tools for future investigation of gene expression and sequence evolution in these and other salmonid species.

Deep sequencing was used to bring high resolution to the human cytomegalovirus (HCMV) transcriptome at the stage when infectious virion production is under way, and major findings were confirmed by extensive experimentation using conventional techniques. The majority (65.1%) of polyadenylated viral RNA transcription is committed to producing four noncoding transcripts (RNA2.7, RNA1.2, RNA4.9, and RNA5.0) that do not substantially overlap designated protein-coding regions. Additional noncoding RNAs that are transcribed antisense to protein-coding regions map throughout the genome and account for 8.7% of transcription from these regions. RNA splicing is more common than recognized previously, which was evidenced by the identification of 229 potential donor and 132 acceptor sites, and it affects 58 protein-coding genes. The great majority (94) of 96 splice junctions most abundantly represented in the deep-sequencing data was confirmed by RT-PCR or RACE or supported by involvement in alternative splicing. Alternative splicing is frequent and particularly evident in four genes (RL8A, UL74A, UL124, and UL150A) that are transcribed by splicing from any one of many upstream exons. The analysis also resulted in the annotation of four previously unrecognized protein-coding regions (RL8A, RL9A, UL150A, and US33A), and expression of the UL150A protein was shown in the context of HCMV infection. The overall conclusion, that HCMV transcription is complex and multifaceted, has implications for the potential sophistication of virus functionality during infection. The study also illustrates the key contribution that deep sequencing can make to the genomics of nuclear DNA viruses.

Niche-adaptation of a bacterial pathogen hinges on the ability to recognize the complexity of signals from the environment and integrate that information with the regulation of genes critical for infection. Here we report the transcriptome of the attaching and effacing pathogen Citrobacter rodentium during infection of its natural murine host. Pathogen gene expression in vivo was heavily biased towards the virulence factor repertoire and was found to be co-ordinated uniquely in response to the host. Concordantly, we identified the host-specific induction of a metabolic pathway that overlapped with the regulation of virulence. The essential type 3 secretion system and an associated suite of distinct effectors were found to be modulated co-ordinately through a unique mechanism involving metabolism of microbiota-derived 1,2-propanediol, which dictated the ability to colonize the host effectively. This study provides novel insights into how host-specific metabolic adaptation acts as a cue to fine-tune virulence. Infection of mice with Citrobacter rodentium is a common model of infection with attaching-and-effacing pathogens. Here, Connolly et al. analyse the transcriptome of C . rodentium during mouse infection, showing host-induced coordinated upregulation of virulence factors and 1,2-propanediol metabolism.

UV-B radiation in sunlight has diverse effects on humans, animals, plants, and microorganisms. UV-B can cause damage to molecules and cells, and consequently organisms need to protect against and repair UV damage to survive in sunlight. In plants, low nondamaging levels of UV-B stimulate transcription of genes involved in UV-protective responses. However, remarkably little is known about the underlying mechanisms of UV-B perception and signal transduction. Here we report that Arabidopsis UV RESISTANCE LOCUS 8 (UVR8) is a UV-B-specific signaling component that orchestrates expression of a range of genes with vital UV-protective functions. Moreover, we show that UVR8 regulates expression of the transcription factor HY5 specifically when the plant is exposed to UV-B. We demonstrate that HY5 is a key effector of the UVR8 pathway, and that it is required for survival under UV-B radiation. UVR8 has sequence similarity to the eukaryotic guanine nucleotide exchange factor RCC1, but we found that it has little exchange activity. However, UVR8, like RCC1, is located principally in the nucleus and associates with chromatin via histones. Chromatin immunoprecipitation showed that UVR8 associates with chromatin in the HY5 promoter region, providing a mechanistic basis for its involvement in regulating transcription. We conclude that UVR8 defines a UV-B-specific signaling pathway in plants that orchestrates the protective gene expression responses to UV-B required for plant survival in sunlight.

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites' genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme's active site, consistent with the fact that the resistant line continues to produce the enzyme's product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself.

Cyanobacteria are photosynthetic prokaryotes being developed as sustainable platforms that use renewable resources (light, water, and air) for diverse applications in energy, food, environment, and medicine. Despite the attractive promise that cyanobacteria offer to industrial biotechnology, slow growth rates pose a major challenge in processes which typically require large amounts of biomass and are often toxic to the cells. Two-stage cultivation strategies are an attractive solution to prevent any undesired growth inhibition by de-coupling biomass accumulation (stage I) and the industrial process (stage II). In cyanobacteria, two-stage strategies involve costly transfer methods between stages I and II, and little work has been focussed on using the distinct growth and stationary phases of batch cultures to autoregulate stage transition. In the present study, we identified and characterised a growth phase-specific promoter, which can serve as an auto-inducible switch to regulate two-stage bioprocesses in cyanobacteria. First, growth phase-specific genes were identified from a new RNAseq dataset comparing two growth phases and six nutrient conditions in Synechocystis sp. PCC 6803, including two new transcriptomes for low Mg and low K. A type II NADH dehydrogenase ( ndbA ) showed robust induction when the cultures transitioned from exponential to stationary phase growth. Behaviour of a 600-bp promoter sequence (PndbA600) was then characterised in detail following the expression of PndbA600:GFP in Synechococcus sp. PCC 7002. Culture density and growth media analyses showed that PndbA600 activation was not dependent on increases in culture density per se but on N availability and on another activating factor present in the spent media of stationary phase cultures (Factor X). PndbA600 deactivation was dependent on the changes in culture density and in either N availability or Factor X. Electron transport inhibition studies revealed a photosynthesis-specific enhancement of active PndbA600 levels. Our findings are summarised in a model describing the environmental regulation of PndbA600, which can now inform the rational design of two-stage industrial processes in cyanobacteria.

Metabolic reprogramming is a key hallmark of cancer, but less is known about metabolic plasticity of the same tumor at different sites. Here, we investigated the metabolic adaptation of leukemia in two different microenvironments, the bone marrow and the central nervous system (CNS). We identified a metabolic signature of fatty acid synthesis in CNS leukemia, highlighting stearoyl-CoA desaturase (SCD) as a key player. In vivo SCD overexpression increases CNS disease, whereas genetic or pharmacological inhibition of SCD decreases CNS load. Overall, we demonstrated that leukemic cells dynamically rewire metabolic pathways to suit local conditions and that targeting these adaptations can be exploited therapeutically.Gottlieb and colleagues show that stearoyl-CoA desaturase promotes metabolic adaptation of acute lymphoblastic leukemia cells to the central nervous system microenvironment, revealing a potential site-specific metabolic vulnerability of this disease.

Background Outer membrane proteins (OMPs) of Pasteurella multocida have various functions related to virulence and pathogenesis and represent important targets for vaccine development. Various bioinformatic algorithms can predict outer membrane localization and discriminate OMPs by structure or function. The designation of a confident prediction framework by integrating different predictors followed by consensus prediction, results integration and manual confirmation will improve the prediction of the outer membrane proteome. Results In the present study, we used 10 different predictors classified into three groups (subcellular localization, transmembrane β-barrel protein and lipoprotein predictors) to identify putative OMPs from two available P. multocida genomes: those of avian strain Pm70 and porcine non-toxigenic strain 3480. Predicted proteins in each group were filtered by optimized criteria for consensus prediction: at least two positive predictions for the subcellular localization predictors, three for the transmembrane β-barrel protein predictors and one for the lipoprotein predictors. The consensus predicted proteins were integrated from each group into a single list of proteins. We further incorporated a manual confirmation step including a public database search against PubMed and sequence analyses, e.g. sequence and structural homology, conserved motifs/domains, functional prediction, and protein-protein interactions to enhance the confidence of prediction. As a result, we were able to confidently predict 98 putative OMPs from the avian strain genome and 107 OMPs from the porcine strain genome with 83% overlap between the two genomes. Conclusions The bioinformatic framework developed in this study has increased the number of putative OMPs identified in P. multocida and allowed these OMPs to be identified with a higher degree of confidence. Our approach can be applied to investigate the outer membrane proteomes of other Gram-negative bacteria.

Background Comparative analysis of tissue-specific transcriptomes is a powerful technique to uncover tissue functions. Our FlyAtlas.org provides authoritative gene expression levels for multiple tissues of Drosophila melanogaster (1). Although the main use of such resources is single gene lookup, there is the potential for powerful meta-analysis to address questions that could not easily be framed otherwise. Here, we illustrate the power of data-mining of FlyAtlas data by comparing epithelial transcriptomes to identify a core set of highly-expressed genes, across the four major epithelial tissues (salivary glands, Malpighian tubules, midgut and hindgut) of both adults and larvae. Method Parallel hypothesis-led and hypothesis-free approaches were adopted to identify core genes that underpin insect epithelial function. In the former, gene lists were created from transport processes identified in the literature, and their expression profiles mapped from the flyatlas.org online dataset. In the latter, gene enrichment lists were prepared for each epithelium, and genes (both transport related and unrelated) consistently enriched in transporting epithelia identified. Results A key set of transport genes, comprising V-ATPases, cation exchangers, aquaporins, potassium and chloride channels, and carbonic anhydrase, was found to be highly enriched across the epithelial tissues, compared with the whole fly. Additionally, a further set of genes that had not been predicted to have epithelial roles, were co-expressed with the core transporters, extending our view of what makes a transporting epithelium work. Further insights were obtained by studying the genes uniquely overexpressed in each epithelium; for example, the salivary gland expresses lipases, the midgut organic solute transporters, the tubules specialize for purine metabolism and the hindgut overexpresses still unknown genes. Conclusion Taken together, these data provide a unique insight into epithelial function in this key model insect, and a framework for comparison with other species. They also provide a methodology for function-led datamining of FlyAtlas.org and other multi-tissue expression datasets.