Application of precise genome editing tools in the zebrafish model to elucidate protein function with regards to developmental phenotypes

Genetic engineering is a powerful tool for biologists and geneticists to study the natural world. In general, it allows for asking and answering precise gene or DNA level questions about biological systems. The drawback of genetic engineering is that many applications requires a deep knowledge of multiple disciplines from biology to chemistry, which can make it inaccessible. Here I give a brief history of the field and define four tenets of necessary actions to begin engineering DNA, and four classes of applications using genetic engineering. I present three papers that define an accessible method of genome editing using CRISPR/Cas called GeneWeld, and describe two series of tools pGTag and pPRISM that reduce the materials cost of beginning genetic engineering experiments. I describe use cases for each tool, and finally present an expansion of the methodology and tools to generate synthetic alleles and examine their function in living zebrafish. We expect that these tools and methods will increase the accessibility of genetic engineering using and enable new kinds of research questions

Angioplasty vs. Cryoplasty for the Treatment of Symptomatic Peripheral Vascular Disease: a Retrospective Review Looking at Comparative Outcomes

In 2007 it was reported that eight million Americans are affected by Peripheral Vascular Disease (PVD) and of these affected, around ten percent report being symptomatic (Mayo Clinic). With such a large percentage of Americans affected by this problem, many providers who care for these patients opt to treat with the least invasive option and then progress to more invasive means if necessary. To date, Percutaneous Transluminal Angioplasty (PTA) has been the preferred minimally invasive endovascular treatment option for symptomatic PVD. Though this option is preferred and used extensively, there are concerning short and long term potential consequence such as dissection and recoil of the arterial wall in the acute setting (Laird 2005), and, more importantly, re-stenosis of the affected area in the long term setting causing recurrent symptoms and the need for further treatment. There have been multiple reported results showing that the re-stenosis rates have been as high as 60% (Muradin 2001). Additionally, the Oxford trail showed that the quality of life in the long term setting was not improved and thus required additional therapy (Perkins 1996). Studies examining PTA procedures have shown that restenosis is caused by three processes: immediate elastic recoil, late vascular remodeling, and importantly myointimal hyperplasia (McCaslin 2007). Myo-intimal hyperplasia results from the balloon dilation of the vessel wall causing proliferation of the vascular smooth muscle cells in the intimal layer of the vessel. This proliferation results in increased mass of smooth muscle and thus a restriction of blood flow through the affected lumen. Due to the need for patency in the long term and the myointimal hyperplasia response of angioplasty, a technique building off of angioplasty called Cryoplasty is now offered. This method adds the dilation properties of angioplasty with an associated release of cold thermal energy to the arterial wall. The cold thermal energy is derived from the filling catheter containing nitrous oxide instead of the standard contrast and saline solution. The affect of this cooling process is thought to limit the variables such as myo-intimal hyperplasia and elastic recoil in the process of re-stenosis (Samson 2007). The success rate of cryoplasty has been promising with one study showing a 3-year patency rate of 75% (Laird 2006)

An Evaluation of Fitspiration Viewing and Exercise Behavior in College Students

Social media is widely used by college students, and is used for retrieving health information. “Fitspiration” (fitness and inspiration) pages are common on many popular social networking sites. Fitspiration pages have been previously associated with short term motivation for exercise and decreased body satisfaction. The purpose of this study was to explore ways in which viewing fitspiration pages may influence exercise behaviors for college students. Additionally, this study sought to explore if particular types of fitspiration pages can encourage increased exercise. This study utilized a cross-sectional survey approach and was distributed to college students at a large Midwestern university. About half of the students viewed fitspiration pages, and students who viewed fitness pages reported exercising more than those who did not. Students who viewed fitspiration pages also reported exercising for muscle-gain, enjoyment, fitness, reduced stress, and feeling better. CrossFit pages and professional fitness organization pages were associated with increased exercise. Students did not report feeling motivated by fitspiration. Additional research is needed to explore the relationships between fitspiration page viewing and fitness goals, and identify additional reasoning for viewing these pages

Transcriptional adaptation in caenorhabditis elegans

Transcriptional adaptation is a recently described phenomenon by which a mutation in one gene leads to the transcriptional modulation of related genes, termed adapting genes. At the molecular level, it has been proposed that the mutant mRNA, rather than the loss of protein function, activates this response. While several examples of transcriptional adaptation have been reported in zebrafish embryos and in mouse cell lines, it is not known whether this phenomenon is observed across metazoans. Here we report transcriptional adaptation in C. elegans, and find that this process requires factors involved in mutant mRNA decay, as in zebrafish and mouse. We further uncover a requirement for Argonaute proteins and Dicer, factors involved in small RNA maturation and transport into the nucleus. Altogether, these results provide evidence for transcriptional adaptation in C. elegans, a powerful model to further investigate underlying molecular mechanisms.publishedVersio

The Gene Sculpt Suite: a set of tools for genome editing

The discovery and development of DNA-editing nucleases (Zinc Finger Nucleases, TALENs, CRISPR/Cas systems) has given scientists the ability to precisely engineer or edit genomes as never before. Several different platforms, protocols and vectors for precision genome editing are now available, leading to the development of supporting web-based software. Here we present the Gene Sculpt Suite (GSS), which comprises three tools: (i) GTagHD, which automatically designs and generates oligonucleotides for use with the GeneWeld knock-in protocol; (ii) MEDJED, a machine learning method, which predicts the extent to which a double-stranded DNA break site will utilize the microhomology-mediated repair pathway; and (iii) MENTHU, a tool for identifying genomic locations likely to give rise to a single predominant microhomology-mediated end joining allele (PreMA) repair outcome. All tools in the GSS are freely available for download under the GPL v3.0 license and can be run locally on Windows, Mac and Linux systems capable of running R and/or Docker. The GSS is also freely available online at www.genesculpt.org

A worldwide phylogenetic classification of the Poaceae (Gramineae) III: An update

We present an updated worldwide phylogenetic classification of Poaceae with 11 783 species in 12 subfamilies, 7 supertribes, 54 tribes, 5 super subtribes, 109 subtribes, and 789 accepted genera. The subfamilies (in descending order based on the number of species) are Pooideae with 4126 species in 219 genera, 15 tribes, and 34 subtribes; Panicoideae with 3325 species in 242 genera, 14 tribes, and 24 subtribes; Bambusoideae with 1698 species in 136 genera, 3 tribes, and 19 subtribes; Chloridoideae with 1603 species in 121 genera, 5 tribes, and 30 subtribes; Aristidoideae with 367 species in three genera and one tribe; Danthonioideae with 292 species in 19 genera and 1 tribe; Micrairoideae with 192 species in nine genera and three tribes; Oryzoideae with 117 species in 19 genera, 4 tribes, and 2 subtribes; Arundinoideae with 36 species in 14 genera and 3 tribes; Pharoideae with 12 species in three genera and one tribe; Puelioideae with 11 species in two genera and two tribes; and the Anomochlooideae with four species in two genera and two tribes. Two new tribes and 22 new or resurrected subtribes are recognized. Forty-five new (28) and resurrected (17) genera are accepted, and 24 previously accepted genera are placed in synonymy. We also provide an updated list of all accepted genera including common synonyms, genus authors, number of species in each accepted genus, and subfamily affiliation. We propose Locajonoa, a new name and rank with a new combination, L. coerulescens. The following seven new combinations are made in Lorenzochloa: L. bomanii, L. henrardiana, L. mucronata, L. obtusa, L. orurensis, L. rigidiseta, and L. venusta.Fil: Soreng, Robert J.. National Museum of Natural History; Estados UnidosFil: Peterson, Paul M.. National Museum of Natural History; Estados UnidosFil: Zuloaga, Fernando Omar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica Darwinion. Academia Nacional de Ciencias Exactas, Físicas y Naturales. Instituto de Botánica Darwinion; ArgentinaFil: Romaschenko, Konstantin. National Museum of Natural History; Estados UnidosFil: Clark, Lynn G.. IOWA STATE UNIVERSITY (ISU);Fil: Teisher, Jordan K.. No especifíca;Fil: Gillespie, Lynn J.. No especifíca;Fil: Barberá, Patricia. No especifíca;Fil: Welker, Cassiano A. D.. No especifíca;Fil: Kellogg, Elizabeth A.. Donald Danforth Plant Science Center; Estados UnidosFil: Li, De Zhu. No especifíca;Fil: Davidse, Gerrit. No especifíca

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR compatible systems

Identification of Tsetse (Glossina spp.) using matrix-assisted laser desorption/ionisation time of flight mass spectrometry

Glossina (G.) spp. (Diptera: Glossinidae), known as tsetse flies, are vectors of African trypanosomes that cause sleeping sickness in humans and nagana in domestic livestock. Knowledge on tsetse distribution and accurate species identification help identify potential vector intervention sites. Morphological species identification of tsetse is challenging and sometimes not accurate. The matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF MS) technique, already standardised for microbial identification, could become a standard method for tsetse fly diagnostics. Therefore, a unique spectra reference database was created for five lab-reared species of riverine-, savannah- and forest- type tsetse flies and incorporated with the commercial Biotyper 3.0 database. The standard formic acid/acetonitrile extraction of male and female whole insects and their body parts (head, thorax, abdomen, wings and legs) was used to obtain the flies' proteins. The computed composite correlation index and cluster analysis revealed the suitability of any tsetse body part for a rapid taxonomical identification. Phyloproteomic analysis revealed that the peak patterns of G. brevipalpis differed greatly from the other tsetse. This outcome was comparable to previous theories that they might be considered as a sister group to other tsetse spp. Freshly extracted samples were found to be matched at the species level. However, sex differentiation proved to be less reliable. Similarly processed samples of the common house fly Musca domestica (Diptera: Muscidae; strain: Lei) did not yield any match with the tsetse reference database. The inclusion of additional strains of morphologically defined wild caught flies of known origin and the availability of large-scale mass spectrometry data could facilitate rapid tsetse species identification in the futur

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline