Effect of substrate thermal resistance on space-domain microchannel

In recent years, Fluorescent Melting Curve Analysis (FMCA) has become an almost ubiquitous feature of commercial quantitative PCR (qPCR) thermal cyclers. Here a micro-fluidic device is presented capable of performing FMCA within a microchannel. The device consists of modular thermally conductive blocks which can sandwich a microfluidic substrate. Opposing ends of the blocks are held at differing temperatures and a linear thermal gradient is generated along the microfluidic channel. Fluorescent measurements taken from a sample as it passes along the micro-fluidic channel permits fluorescent melting curves to be generated. In this study we measure DNA melting temperature from two plasmid fragments. The effects of flow velocity and ramp-rate are investigated, and measured melting curves are compared to those acquired from a commercially available PCR thermocycler

Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., “MTMRs”). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder

How to Educate Entrepreneurs?

Entrepreneurship education has two purposes: To improve students’ entrepreneurial skills and to provide impetus to those suited to entrepreneurship while discouraging the rest. While entrepreneurship education helps students to make a vocational decision its effects may conflict for those not suited to entrepreneurship. This study shows that vocational and the skill formation effects of entrepreneurship education can be identified empirically by drawing on the Theory of Planned Behavior. This is embedded in a structural equation model which we estimate and test using a robust 2SLS estimator. We find that the attitudinal factors posited by the Theory of Planned Behavior are positively correlated with students’ entrepreneurial intentions. While conflicting effects of vocational and skill directed course content are observed in some individuals, overall these types of content are complements. This finding contradicts previous results in the literature. We reconcile the conflicting findings and discuss implications for the design of entrepreneurship courses

Genome Analysis of the Domestic Dog (Korean Jindo) by Massively Parallel Sequencing

Although pioneering sequencing projects have shed light on the boxer and poodle genomes, a number of challenges need to be met before the sequencing and annotation of the dog genome can be considered complete. Here, we present the DNA sequence of the Jindo dog genome, sequenced to 45-fold average coverage using Illumina massively parallel sequencing technology. A comparison of the sequence to the reference boxer genome led to the identification of 4 675 437 single nucleotide polymorphisms (SNPs, including 3 346 058 novel SNPs), 71 642 indels and 8131 structural variations. Of these, 339 non-synonymous SNPs and 3 indels are located within coding sequences (CDS). In particular, 3 non-synonymous SNPs and a 26-bp deletion occur in the TCOF1 locus, implying that the difference observed in cranial facial morphology between Jindo and boxer dogs might be influenced by those variations. Through the annotation of the Jindo olfactory receptor gene family, we found 2 unique olfactory receptor genes and 236 olfactory receptor genes harbouring non-synonymous homozygous SNPs that are likely to affect smelling capability. In addition, we determined the DNA sequence of the Jindo dog mitochondrial genome and identified Jindo dog-specific mtDNA genotypes. This Jindo genome data upgrade our understanding of dog genomic architecture and will be a very valuable resource for investigating not only dog genetics and genomics but also human and dog disease genetics and comparative genomics

The effectiveness of public health interventions to reduce the health impact of climate change:a systematic review of systematic reviews

Climate change is likely to be one of the most important threats to public health in the coming years. Yet despite the large number of papers considering the health impact of climate change, few have considered what public health interventions may be of most value in reducing the disease burden. We aimed to evaluate the effectiveness of public health interventions to reduce the disease burden of high priority climate sensitive diseases

ngs_backbone: a pipeline for read cleaning, mapping and SNP calling using Next Generation Sequence

Background: The possibilities offered by next generation sequencing (NGS) platforms are revolutionizing biotechnological laboratories. Moreover, the combination of NGS sequencing and affordable high-throughput genotyping technologies is facilitating the rapid discovery and use of SNPs in non-model species. However, this abundance of sequences and polymorphisms creates new software needs. To fulfill these needs, we have developed a powerful, yet easy-to-use application. Results: The ngs_backbone software is a parallel pipeline capable of analyzing Sanger, 454, Illumina and SOLiD (Sequencing by Oligonucleotide Ligation and Detection) sequence reads. Its main supported analyses are: read cleaning, transcriptome assembly and annotation, read mapping and single nucleotide polymorphism (SNP) calling and selection. In order to build a truly useful tool, the software development was paired with a laboratory experiment. All public tomato Sanger EST reads plus 14.2 million Illumina reads were employed to test the tool and predict polymorphism in tomato. The cleaned reads were mapped to the SGN tomato transcriptome obtaining a coverage of 4.2 for Sanger and 8.5 for Illumina. 23,360 single nucleotide variations (SNVs) were predicted. A total of 76 SNVs were experimentally validated, and 85% were found to be real. Conclusions: ngs_backbone is a new software package capable of analyzing sequences produced by NGS technologies and predicting SNVs with great accuracy. In our tomato example, we created a highly polymorphic collection of SNVs that will be a useful resource for tomato researchers and breeders. The software developed along with its documentation is freely available under the AGPL license and can be downloaded from http://bioinf. comav.upv.es/ngs_backbone/ or http://github.com/JoseBlanca/franklin.Blanca Postigo, JM.; Pascual Bañuls, L.; Ziarsolo Areitioaurtena, P.; Nuez Viñals, F.; Cañizares Sales, J. (2011). Ngs_backbone: a pipeline for read cleaning, mapping and SNP calling using Next Generation Sequence. BMC Genomics. 12:1-8. doi:10.1186/1471-2164-12-285S1812Metzker ML: Sequencing technologies - the next generation. Nature Reviews Genetics. 2010, 11 (1): 31-46. 10.1038/nrg2626.454 sequencing. [ http://www.454.com/ ]Illumina Inc. [ http://www.illumina.com/ ]Flicek P, Birney E: Sense from sequence reads: methods for alignment and assembly (vol 6, pg S6, 2009). Nature Methods. 2010, 7 (6): 479-479.Chevreux B, Pfisterer T, Drescher B, Driesel AJ, Muller WEG, Wetter T, Suhai S: Using the miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs. Genome Research. 2004, 14 (6): 1147-1159. 10.1101/gr.1917404.Li H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009, 25 (14): 1754-1760. 10.1093/bioinformatics/btp324.Langmead B, Trapnell C, Pop M, Salzberg SL: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology. 2009, 10 (3):Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data P: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009, 25 (16): 2078-2079. 10.1093/bioinformatics/btp352.1000 Genomes. A deep Catalog of Human Genetic Variation. [ http://1000genomes.org/wiki/doku.php?id=1000_genomes:analysis:vcf4.0 ]The seqanswers internet forum. [ http://seqanswers.com/ ]Blankenberg D, Taylor J, Schenck I, He JB, Zhang Y, Ghent M, Veeraraghavan N, Albert I, Miller W, Makova KD, Ross CH, Nekrutenko A: A framework for collaborative analysis of ENCODE data: Making large-scale analyses biologist-friendly. Genome Research. 2007, 17 (6): 960-964. 10.1101/gr.5578007.CloVR Automated Sequence Analysis from Your Desktop. [ http://clovr.org/ ]Papanicolaou A, Stierli R, Ffrench-Constant RH, Heckel DG: Next generation transcriptomes for next generation genomes using est2assembly. Bmc Bioinformatics. 2009, 10:Applied Biosystems by life technologies. [ http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing.html ]Wall PK, Leebens-Mack J, Chanderbali AS, Barakat A, Wolcott E, Liang HY, Landherr L, Tomsho LP, Hu Y, Carlson JE, Ma H, Schuster SC, Soltis DE, Soltis PS, Altman N, dePamphilis CW: Comparison of next generation sequencing technologies for transcriptome characterization. Bmc Genomics. 2009, 10:Murchison EP, Tovar C, Hsu A, Bender HS, Kheradpour P, Rebbeck CA, Obendorf D, Conlan C, Bahlo M, Blizzard CA, Pyecroft S, Kreiss A, Kellis M, Stark A, Harkins TT, Marshall Graves JA, Woods GM, Hanon GJ, Papenfuss AT: The Tasmanian Devil Transcriptome Reveals Schwann Cell Origins of a Clonally Transmissible Cancer. Science. 2010, 327 (5961): 84-87. 10.1126/science.1180616.Parchman TL, Geist KS, Grahnen JA, Benkman CW, Buerkle CA: Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery. Bmc Genomics. 2010, 11:Babik W, Stuglik M, Qi W, Kuenzli M, Kuduk K, Koteja P, Radwan J: Heart transcriptome of the bank vole (Myodes glareolus): towards understanding the evolutionary variation in metabolic rate. BMC Genomics. 2010, 11: 390-10.1186/1471-2164-11-390.Miller JC, Tanksley SD: RFLP analysis of phylogenetic-relationships and genetic-variation in the genus Lycopersicon. Theoretical and Applied Genetics. 1990, 80 (4): 437-448.Williams CE, Stclair DA: Phenetic relationships and levels of variability detected by restriction-fragment-length-polymorphism and random amplified polymorphic DNA analysis of cultivated and wild accessions of Lycopersicon-esculentum. Genome. 1993, 36 (3): 619-630. 10.1139/g93-083.Rick CM: Tomato, Lycopersicon esculentum (Solanaceae). Evolution of crop plants. Edited by: Simmonds NW. 1976, London: Longman Group, 268-273.Labate JA, Baldo AM: Tomato SNP discovery by EST mining and resequencing. Molecular Breeding. 2005, 16 (4): 343-349. 10.1007/s11032-005-1911-5.Yano K, Watanabe M, Yamamoto N, Maeda F, Tsugane T, Shibata D: Expressed sequence tags (EST) database of a miniature tomato cultivar, Micro-Tom. Plant and Cell Physiology. 2005, 46: S139-S139.Jimenez-Gomez JM, Maloof JN: Sequence diversity in three tomato species: SNPs, markers, and molecular evolution. Bmc Plant Biology. 2009, 9:Yang WC, Bai XD, Kabelka E, Eaton C, Kamoun S, van der Knaap E, Francis D: Discovery of single nucleotide polymorphisms in Lycopersicon esculentum by computer aided analysis of expressed sequence tags. Molecular Breeding. 2004, 14 (1): 21-34.Van Deynze A, Stoffel K, Buell CR, Kozik A, Liu J, van der Knaap E, Francis D: Diversity in conserved genes in tomato. Bmc Genomics. 2007, 8:Sim SC, Robbins MD, Chilcott C, Zhu T, Francis DM: Oligonucleotide array discovery of polymorphisms in cultivated tomato (Solanum lycopersicum L.) reveals patterns of SNP variation associated with breeding. Bmc Genomics. 2009, 10:Bioinformatics at COMAV. [ http://bioinf.comav.upv.es/ngs_backbone/index.html ]Broad institute. [ http://www.broadinstitute.org/igv ]Bioinformatics at COMAV. [ http://bioinf.comav.upv.es/ngs_backbone/install.html ]Github social coding. [ http://github.com/JoseBlanca/franklin ]Chou HH, Holmes MH: DNA sequence quality trimming and vector removal. Bioinformatics. 2001, 17 (12): 1093-1104. 10.1093/bioinformatics/17.12.1093.Picard. [ http://picard.sourceforge.net/index.shtml ]McKenna A, Hanna M, Banks E, Sivachenko A, Citulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Research. 2010, 20: 1297-1303. 10.1101/gr.107524.110.Sol Genomics Network. [ ftp://ftp.solgenomics.net/ ]NCBI Genbank. [ http://www.ncbi.nlm.nih.gov/genbank/ ]Gundry CN, Vandersteen JG, Reed GH, Pryor RJ, Chen J, Wittwer CT: Amplicon melting analysis with labeled primers: A closed-tube method for differentiating homozygotes and heterozygotes. Clinical Chemistry. 2003, 49 (3): 396-406. 10.1373/49.3.396

Small firms and patenting revisited

In order to observe a patent application at the firm level two conditions need to be met: new products need to be of patentable quality, which depends both on the degree of novelty of innovations and on the total number (portfolio) of innovations; and the benefits of patents need to be higher than the costs of owning them. Analyzing the patent propensity of small and large UK firms using a novel innovation-level survey (the SIPU survey) linked to Community Innovation Survey data we find that when we consider the whole innovation portfolio smaller firms do patent less than larger firms. However, using data on individual innovations, we find that smaller firms are no less likely to patent any specific innovation than larger firms. We argue that size differences in the probability to patent relate primarily to the ‘portfolio effect’, i.e. larger firms generate more innovations than smaller firms and therefore are more likely to create one or more which are patentable. As for the decision to patent a patentable innovation, we find that cost barriers, more than issues of innovation quality or enforceability, deter small firms from patenting specific innovations. Measures to address the costs of patenting for smaller firms – perhaps by considering patents as eligible costs for R&D tax credits – and/or subsidizing SMEs’ participation in IP litigation schemes may both encourage patent use by smaller firms

DNMT3A-Coordinated Splicing Governs the Stem State Switch Towards Differentiation in Embryonic and Haematopoietic Stem Cells

Upon stimulation by extrinsic stimuli, stem cells initiate a programme that enables differentiation or self-renewal. Disruption of the stem state exit has catastrophic consequences for embryogenesis and can lead to cancer. While some elements of this stem state switch are known, major regulatory mechanisms remain unclear. Here we show that this switch involves a global increase in splicing efficiency coordinated by DNA methyltransferase 3α (DNMT3A), an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and haematopoietic stem cells depends on messenger RNA processing, influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukaemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem state towards differentiation

PLoS Genet

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., "MTMRs"). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder

Lack of EGFR-activating mutations in European patients with triple-negative breast cancer could emphasise geographic and ethnic variations in breast cancer mutation profiles

INTRODUCTION: Triple-negative breast cancers (TNBCs) are characterised by lack of expression of hormone receptors and epidermal growth factor receptor 2 (HER-2). As they frequently express epidermal growth factor receptors (EGFRs), anti-EGFR therapies are currently assessed for this breast cancer subtype as an alternative to treatments that target HER-2 or hormone receptors. Recently, EGFR-activating mutations have been reported in TNBC specimens in an East Asian population. Because variations in the frequency of EGFR-activating mutations in East Asians and other patients with lung cancer have been described, we evaluated the EGFR mutational profile in tumour samples from European patients with TNBC. METHODS: We selected from a DNA tumour bank 229 DNA samples isolated from frozen, histologically proven and macrodissected invasive TNBC specimens from European patients. PCR and high-resolution melting (HRM) analyses were used to detect mutations in exons 19 and 21 of EGFR. The results were then confirmed by bidirectional sequencing of all samples. RESULTS: HRM analysis allowed the detection of three EGFR exon 21 mutations, but no exon 19 mutations. There was 100% concordance between the HRM and sequencing results. The three patients with EGFR exon 21 abnormal HRM profiles harboured the rare R836R SNP, but no EGFR-activating mutation was identified. CONCLUSIONS: This study highlights variations in the prevalence of EGFR mutations in TNBC. These variations have crucial implications for the design of clinical trials involving anti-EGFR treatments in TNBC and for identifying the potential target population