454-Pyrosequencing: A Molecular Battiscope for Freshwater Viral Ecology

Viruses, the most abundant biological entities on the planet, are capable of infecting organisms from all three branches of life, although the majority infect bacteria where the greatest degree of cellular diversity lies. However, the characterization and assessment of viral diversity in natural environments is only beginning to become a possibility. Through the development of a novel technique for the harvest of viral DNA and the application of 454 pyrosequencing, a snapshot of the diversity of the DNA viruses harvested from a standing pond on a cattle farm has been obtained. A high abundance of viral genotypes (785) were present within the virome. The absolute numbers of lambdoid and Shiga toxin (Stx) encoding phages detected suggested that the depth of sequencing had enabled recovery of only ca. 8% of the total virus population, numbers that agreed within less than an order of magnitude with predictions made by rarefaction analysis. The most abundant viral genotypes in the pond were bacteriophages (93.7%). The predominant viral genotypes infecting higher life forms found in association with the farm were pathogens that cause disease in cattle and humans, e.g. members of the Herpesviridae. The techniques and analysis described here provide a fresh approach to the monitoring of viral populations in the aquatic environment, with the potential to become integral to the development of risk analysis tools for monitoring the dissemination of viral agents of animal, plant and human diseases

Dexmedetomidine improves success of paediatric MRI sedation

OBJECTIVE: To improve success rates of children requiring sedation for MRI. METHODS: Audits of sedation success for children attending planned MRI using three different approaches: (1) National Institute for Health and Care Excellence (NICE) guidance (chloral hydrate if <15 kg and oral midazolam if ≥15 kg), (2) Chloral hydrate for all patients, (3) Chloral hydrate±intranasal dexmedetomidine if <15 kg and intranasal dexmedetomidine alone if ≥15 kg. RESULTS: 74 patients had 85 MRI scan attempts. Overall success rates were significantly higher when using intranasal dexmedetomidine compared with following NICE guidance (81% vs 52% p=0.017). Dexmedetomidine performed better than oral midazolam for the same indication (76% vs 33% p=0.026). The side effect profile for dexmedetomidine was as reported in larger studies. CONCLUSIONS: Intranasal dexmedetomidine is an effective alternative to oral midazolam for sedation for MRI and as a rescue medication where chloral hydrate has been ineffective

Differential Impacts of Online Delivery Methods on Student Learning: A Case Study in Biorenewables

In 2007, a Virtual Education Center for Biorenewable Resources was initiated that offered three distance education courses, one being Biorenewable Resources and Technology (BRT) 501 – Fundamentals of Biorenewable Resources and Technology, the subject of this study. The primary objective was to determine if course delivery method (video lecture format and the other in menu-driven auto-tutorial presentations (MDAP) deliv¬ered via Flash format), student major (agricultural and non-agricultural), and gender influence online student learning in BRT 501. We found that BRT 501 student performance was not significantly impacted by module delivery method. Students with agricultural majors were outperformed by students with non-agricultural majors, most of whom were engineering students, on the midterm and final exams, and course grade. Gender dif¬ferences seen on the biomass-module first-attempt total quiz score disappeared for the final total quiz score on that module

CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species

Identification of novel non-coding RNAs using profiles of short sequence reads from next generation sequencing data

<p>Abstract</p> <p>Background</p> <p>The increasing interest in small non-coding RNAs (ncRNAs) such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs) and recent advances in sequencing technology have yielded large numbers of short (18-32 nt) RNA sequences from different organisms, some of which are derived from small nucleolar RNAs (snoRNAs) and transfer RNAs (tRNAs). We observed that these short ncRNAs frequently cover the entire length of annotated snoRNAs or tRNAs, which suggests that other loci specifying similar ncRNAs can be identified by clusters of short RNA sequences.</p> <p>Results</p> <p>We combined publicly available datasets of tens of millions of short RNA sequence tags from <it>Drosophila melanogaster</it>, and mapped them to the <it>Drosophila </it>genome. Approximately 6 million perfectly mapping sequence tags were then assembled into 521,302 tag-contigs (TCs) based on tag overlap. Most transposon-derived sequences, exons and annotated miRNAs, tRNAs and snoRNAs are detected by TCs, which show distinct patterns of length and tag-depth for different categories. The typical length and tag-depth of snoRNA-derived TCs was used to predict 7 previously unrecognized box H/ACA and 26 box C/D snoRNA candidates. We also identified one snRNA candidate and 86 loci with a high number of tags that are yet to be annotated, 7 of which have a particular 18mer motif and are located in introns of genes involved in development. A subset of new snoRNA candidates and putative ncRNA candidates was verified by Northern blot.</p> <p>Conclusions</p> <p>In this study, we have introduced a new approach to identify new members of known classes of ncRNAs based on the features of TCs corresponding to known ncRNAs. A large number of the identified TCs are yet to be examined experimentally suggesting that many more novel ncRNAs remain to be discovered.</p

Red Galaxy Growth and the Halo Occupation Distribution

We have traced the past 7 Gyr of red galaxy stellar mass growth within dark matter halos. We have determined the halo occupation distribution, which describes how galaxies reside within dark matter halos, using the observed luminosity function and clustering of 40,696 0.2<z<1.0 red galaxies in Bootes. Half of 10^{11.9} Msun/h halos host a red central galaxy, and this fraction increases with increasing halo mass. We do not observe any evolution of the relationship between red galaxy stellar mass and host halo mass, although we expect both galaxy stellar masses and halo masses to evolve over cosmic time. We find that the stellar mass contained within the red population has doubled since z=1, with the stellar mass within red satellite galaxies tripling over this redshift range. In cluster mass halos most of the stellar mass resides within satellite galaxies and the intra-cluster light, with a minority of the stellar mass residing within central galaxies. The stellar masses of the most luminous red central galaxies are proportional to halo mass to the power of a third. We thus conclude that halo mergers do not always lead to rapid growth of central galaxies. While very massive halos often double in mass over the past 7 Gyr, the stellar masses of their central galaxies typically grow by only 30%.Comment: Accepted for publication in the ApJ. 34 pages, 22 Figures, 5 Table

Structural Models of Human eEF1A1 and eEF1A2 Reveal Two Distinct Surface Clusters of Sequence Variation and Potential Differences in Phosphorylation

BACKGROUND:Despite sharing 92% sequence identity, paralogous human translation elongation factor 1 alpha-1 (eEF1A1) and elongation factor 1 alpha-2 (eEF1A2) have different but overlapping functional profiles. This may reflect the differential requirements of the cell-types in which they are expressed and is consistent with complex roles for these proteins that extend beyond delivery of tRNA to the ribosome. METHODOLOGY/PRINCIPAL FINDINGS:To investigate the structural basis of these functional differences, we created and validated comparative three-dimensional (3-D) models of eEF1A1 and eEF1A2 on the basis of the crystal structure of homologous eEF1A from yeast. The spatial location of amino acid residues that vary between the two proteins was thereby pinpointed, and their surface electrostatic and lipophilic properties were compared. None of the variations amongst buried amino acid residues are judged likely to have a major structural effect on the protein fold, or to affect domain-domain interactions. Nearly all the variant surface-exposed amino acid residues lie on one face of the protein, in two proximal but distinct sub-clusters. The result of previously performed mutagenesis in yeast may be interpreted as confirming the importance of one of these clusters in actin-bundling and filament disorganization. Interestingly, some variant residues lie in close proximity to, and in a few cases show differences in interactions with, residues previously inferred to be directly involved in binding GTP/GDP, eEF1Balpha and aminoacyl-tRNA. Additional sequence-based predictions, in conjunction with the 3-D models, reveal likely differences in phosphorylation sites that could reconcile some of the functional differences between the two proteins. CONCLUSIONS:The revelation and putative functional assignment of two distinct sub-clusters on the surface of the protein models should enable rational site-directed mutagenesis, including homologous reverse-substitution experiments, to map surface binding patches onto these proteins. The predicted variant-specific phosphorylation sites also provide a basis for experimental verification by mutagenesis. The models provide a structural framework for interpretation of the resulting functional analysis

A simple vector system to improve performance and utilisation of recombinant antibodies

BACKGROUND: Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. RESULTS: We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs). Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3). The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3)) was investigated and found to be inferior to periplasmic expression in BL21 (DE3) cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner), bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule), or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and immunohistochemistry. CONCLUSION: Nine scFv expression vectors have been generated and tested. Three vectors showed utility for expression of functional scFv fragments. One vector, pSANG14-3F, produces scFv-alkaline phosphatase fusion molecules which offers a simple, convenient and sensitive way of determining the reactivity of recombinant antibody fragments in a variety of common assay systems

The effect of chronic high insulin exposure upon metabolic and myogenic markers in C2C12 skeletal muscle cells and myotubes.

Skeletal muscle is an insulin sensitive tissue and accounts for approximately 80% of post-prandial glucose disposal. This study describes the effects of insulin, delivered for 72 hours, to skeletal muscle myoblasts during differentiation or to skeletal muscle myotubes. After chronic treatment, cultures were acutely stimulated with insulin and analysed for total and phosphorylated Akt (Ser473), mRNA expression of metabolic and myogenic markers and insulin-stimulated glucose uptake. Skeletal muscle cells differentiated in the presence of insulin chronically, reduced acute insulin stimulated phosphorylation of Akt Ser473. In addition, there was a reduction in mRNA expression of Hexokinase II (HKII), GLUT4 and PGC-1α. Insulin-stimulated glucose uptake was attenuated when cells were differentiated in the presence of insulin. In contrast, myotubes exposed to chronic insulin showed no alterations in phosphorylation of Akt Ser473. Both HKII and GLUT4 mRNA expression were reduced by chronic exposure to insulin; while PGC-1α was not different between culture conditions and was increased by acute insulin stimulation. These data suggest that there are differential responses in insulin signalling, transcription and glucose uptake of skeletal muscle cells when cultured in either the presence of insulin during differentiation or in myotube cultures