214 research outputs found

    Requirement of a Phage-Induced 5'-Exonuclease for the Expression of Late Genes of Bacteriophage T5

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    Isolation of galactosyltransferase from human milk and the determination of its N-terminal amino acid sequence

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    Galactosyltransferase (EC 2.4.1.22), purified to homogeneity from human milk by affinity chromatography, had an apparent molecular weight of 53,000 as determined by denaturing polyacrylamide gel electrophoresis. Subtration of the estimated contribution of the oligosaccharide portion of the molecule leaves a Mr of 47,000. An N-terminal amino acid sequence analysis of the isolated protein revealed a sequence similar to that found near the 5' end of a cDNA clone isolated by Shaper et al (11), which encodes a 35,500 molecular weight protein. Either the molecular weight of galactosyltransferase, has been overestimated, or a discrepancy exists between the actual molecular weight of galactosyltransferase and that predicted by the bovine cDNA clone isolated by Shaper et al (11).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26099/1/0000175.pd

    Isolation of a cDNA coding for human galactosyltransferase

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    Human milk galactosyltransferase (EC 2.4.1.22) was purified to homogeneity using affinity chromatography. Edman degradation was used to determine the amino acid sequences of eight peptide fragments isolated from the purified enzyme. A 60-mer "optimal" oligonucleotide probe that corresponded to the amino acid sequence of one of the galactosyltransferase peptide fragments was constructed and used to screen a [lambda]gt10 cDNA library. Two hybridization-positive recombinant phages, each with a 1.7 Kbp insert, were detected among 3 x 106 recombinant [lambda]gt10 phages. Sequencing of one of the cDNA inserts revealed a 783 bp galactosyltransferase coding sequence. The remainder of the sequence corresponded to the 3'-region of the mRNA downstream from the termination codon.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26068/1/0000142.pd

    A generative approach for image-based modeling of tumor growth

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    22nd International Conference, IPMI 2011, Kloster Irsee, Germany, July 3-8, 2011. ProceedingsExtensive imaging is routinely used in brain tumor patients to monitor the state of the disease and to evaluate therapeutic options. A large number of multi-modal and multi-temporal image volumes is acquired in standard clinical cases, requiring new approaches for comprehensive integration of information from different image sources and different time points. In this work we propose a joint generative model of tumor growth and of image observation that naturally handles multi-modal and longitudinal data. We use the model for analyzing imaging data in patients with glioma. The tumor growth model is based on a reaction-diffusion framework. Model personalization relies only on a forward model for the growth process and on image likelihood. We take advantage of an adaptive sparse grid approximation for efficient inference via Markov Chain Monte Carlo sampling. The approach can be used for integrating information from different multi-modal imaging protocols and can easily be adapted to other tumor growth models.German Academy of Sciences Leopoldina (Fellowship Programme LPDS 2009-10)Academy of Finland (133611)National Institutes of Health (U.S.) (NIBIB NAMIC U54-EB005149)National Institutes of Health (U.S.) (NCRR NAC P41- RR13218)National Institutes of Health (U.S.) (NINDS R01-NS051826)National Institutes of Health (U.S.) (NIH R01-NS052585)National Institutes of Health (U.S.) (NIH R01-EB006758)National Institutes of Health (U.S.) (NIH R01-EB009051)National Institutes of Health (U.S.) (NIH P41-RR014075)National Science Foundation (U.S.) (CAREER Award 0642971

    Genomic, Proteomic and Physiological Characterization of a T5-like Bacteriophage for Control of Shiga Toxin-Producing Escherichia coli O157:H7

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    Despite multiple control measures, Escherichia coli O157:H7 (STEC O157:H7) continues to be responsible for many food borne outbreaks in North America and elsewhere. Bacteriophage therapy may prove useful for controlling this pathogen in the host, their environment and food. Bacteriophage vB_EcoS_AKFV33 (AKFV33), a T5-like phage of Siphoviridae lysed common phage types of STEC O157:H7 and not non-O157 E. coli. Moreover, STEC O157:H7 isolated from the same feedlot pen from which the phage was obtained, were highly susceptible to AKFV33. Adsorption rate constant and burst size were estimated to be 9.31×10−9 ml/min and 350 PFU/infected cell, respectively. The genome of AKVF33 was 108,853 bp (38.95% G+C), containing 160 open reading frames (ORFs), 22 tRNA genes and 32 strong promoters recognized by host RNA polymerase. Of 12 ORFs without homologues to T5-like phages, 7 predicted novel proteins while others exhibited low identity (<60%) to proteins in the National Centre for Biotechnology Information database. AKVF33 also lacked the L-shaped tail fiber protein typical of T5, but was predicted to have tail fibers comprised of 2 novel proteins with low identity (37–41%) to tail fibers of E. coli phage phiEco32 of Podoviridae, a putative side tail fiber protein of a prophage from E. coli IAI39 and a conserved domain protein of E. coli MS196-1. The receptor-binding tail protein (pb5) shared an overall identify of 29–72% to that of other T5-like phages, with no region coding for more than 6 amino acids in common. Proteomic analysis identified 4 structural proteins corresponding to the capsid, major tail, tail fiber and pore-forming tail tip (pb2). The genome of AKFV33 lacked regions coding for known virulence factors, integration-related proteins or antibiotic resistance determinants. Phage AKFV33 is a unique, highly lytic STEC O157:H7-specific T5-like phage that may have considerable potential as a pre- and post-harvest biocontrol agent

    Altered microRNA expression in frontotemporal lobar degeneration with TDP-43 pathology caused by progranulin mutations

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    <p>Abstract</p> <p>Background</p> <p>Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative disorder that can be triggered through genetic or sporadic mechanisms. MicroRNAs (miRNAs) have become a major therapeutic focus as their pervasive expression and powerful regulatory roles in disease pathogenesis become increasingly apparent. Here we examine the role of miRNAs in FTLD patients with TAR DNA-binding protein 43 pathology (FTLD-TDP) caused by genetic mutations in the progranulin (<it>PGRN</it>) gene.</p> <p>Results</p> <p>Using miRNA array profiling, we identified the 20 miRNAs that showed greatest evidence (unadjusted P < 0.05) of dysregulation in frontal cortex of eight FTLD-TDP patients carrying <it>PGRN </it>mutations when compared to 32 FTLD-TDP patients with no apparent genetic abnormalities. Quantitative real-time PCR (qRT-PCR) analyses provided technical validation of the differential expression for 9 of the 20 miRNAs in frontal cortex. Additional qRT-PCR analyses showed that 5 out of 9 miRNAs (miR-922, miR-516a-3p, miR-571, miR-548b-5p, and miR-548c-5p) were also significantly dysregulated (unadjusted P < 0.05) in cerebellar tissue samples of <it>PGRN </it>mutation carriers, consistent with a systemic reduction in PGRN levels. We developed a list of gene targets for the 5 candidate miRNAs and found 18 genes dysregulated in a reported FTLD mRNA study to exhibit anti-correlated miRNA-mRNA patterns in affected cortex and cerebellar tissue. Among the targets is brain-specific angiogenesis inhibitor 3, which was recently identified as an important player in synapse biology.</p> <p>Conclusions</p> <p>Our study suggests that miRNAs may contribute to the pathogenesis of FTLD-TDP caused by <it>PGRN </it>mutations and provides new insight into potential future therapeutic options.</p

    Transcriptional and Post-Transcriptional Regulation of SPAST, the Gene Most Frequently Mutated in Hereditary Spastic Paraplegia

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    Hereditary spastic paraplegias (HSPs) comprise a group of neurodegenerative disorders that are characterized by progressive spasticity of the lower extremities, due to axonal degeneration in the corticospinal motor tracts. HSPs are genetically heterogeneous and show autosomal dominant inheritance in ∼70–80% of cases, with additional cases being recessive or X-linked. The most common type of HSP is SPG4 with mutations in the SPAST gene, encoding spastin, which occurs in 40% of dominantly inherited cases and in ∼10% of sporadic cases. Both loss-of-function and dominant-negative mutation mechanisms have been described for SPG4, suggesting that precise or stoichiometric levels of spastin are necessary for biological function. Therefore, we hypothesized that regulatory mechanisms controlling expression of SPAST are important determinants of spastin biology, and if altered, could contribute to the development and progression of the disease. To examine the transcriptional and post-transcriptional regulation of SPAST, we used molecular phylogenetic methods to identify conserved sequences for putative transcription factor binding sites and miRNA targeting motifs in the SPAST promoter and 3′-UTR, respectively. By a variety of molecular methods, we demonstrate that SPAST transcription is positively regulated by NRF1 and SOX11. Furthermore, we show that miR-96 and miR-182 negatively regulate SPAST by effects on mRNA stability and protein level. These transcriptional and miRNA regulatory mechanisms provide new functional targets for mutation screening and therapeutic targeting in HSP

    High Resolution Sharp Computational Methods for Elliptic and Parabolic Problems in Complex Geometries

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