Reconstruction of the repetitive antifreeze glycoprotein genomic loci in the cold-water gadids Boreogadus saida and Microgadus tomcod.

Abstract Antifreeze glycoproteins (AFGPs) are a novel evolutionary innovation in members of the northern cod fish family (Gadidae), crucial in preventing death from inoculative freezing by environmental ice in their frigid Arctic and sub-Arctic habitats. However, the genomic origin and molecular mechanism of evolution of this novel life-saving adaptive genetic trait remained to be definitively determined. To this end, we constructed large insert genomic DNA BAC (bacterial artificial chromosome) libraries for two AFGP-bearing gadids, the high-Arctic polar cod Boreogadus saida and the cold-temperate Atlantic tomcod Microgadus tomcod, to isolate and sequence their AFGP genomic regions for fine resolution evolutionary analyses. The BAC library construction encountered poor cloning efficiency initially, which we resolved by pretreating the agarose-embedded erythrocyte DNA with a cationic detergent, a method that may be of general use to BAC cloning for teleost species and/or where erythrocytes are the source of input DNA. The polar cod BAC library encompassed 92,160 clones with an average insert size of 94.7 kbp, and the Atlantic tomcod library contained 73,728 clones with an average insert size of 89.6 kbp. The genome sizes of B. saida and M. tomcod were estimated by cell flow cytometry to be 836 Mbp and 645 Mbp respectively, thus their BAC libraries have approximately 10- and 9.7-fold genome coverage respectively. The inclusiveness and depth of coverage were empirically confirmed by screening the libraries with three housekeeping genes. The BAC clones that mapped to the AFGP genomic loci of the two gadids were then isolated by screening the BAC libraries with gadid AFGP gene probes. Eight minimal tiling path (MTP) clones were identified for B. saida, sequenced, and assembled. The B. saida AFGP locus reconstruction produced both haplotypes, and the locus comprises three distinct AFGP gene clusters, containing a total of 16 AFGP genes and spanning a combined distance of 512 kbp. The M. tomcod AFGP locus is much smaller at approximately 80 kbp, and contains only three AFGP genes. Fluorescent in situ hybridization with an AFGP gene probe showed the AFGP locus in both species occupies a single chromosomal location. The large AFGP locus with its high gene dosage in B. saida is consistent with its chronically freezing high Arctic habitats, while the small gene family in M. tomcod correlates with its milder habitats in lower latitudes. The results from this study provided the data for fine resolution sequence analyses that would yield insight into the molecular mechanisms and history of gadid AFGP gene evolution driven by northern hemisphere glaciation

Phonological and syntactic phrasing in Bemba relatives

Tone as a distinctive feature used to differentiate not only words but also clause types, is a characteristic feature of Bantu languages. In this paper we show that Bemba relatives can be marked with a low tone in place of a segmental relative marker. We treat this low tone as a morpheme rather than as just triggering a change in tone pattern that can then be related to relativization. The low tone strategy of relativization, which imposes a restrictive reading of relatives, manifests a phonological phrasing that requires the head noun to be phrased together with the relative clause that it modifies as opposed to non-restrictives where this is not the case. The paper shows that the resultant phonological phrasing favours a head-raising analysis of relativization where the head noun is considered to be inside CP. Despite the syntactic use of the relative tonal morpheme we see that it is also subject to purely phonological constraints that results in its being unable to be used to mark headless relatives. This paper therefore highlights the phonology-syntax connection and shows that phonology can directly inform syntactic analyses. © Walter de Gruyter

Statistical Workflow for Feature Selection in Human Metabolomics Data.

High-throughput metabolomics investigations, when conducted in large human cohorts, represent a potentially powerful tool for elucidating the biochemical diversity underlying human health and disease. Large-scale metabolomics data sources, generated using either targeted or nontargeted platforms, are becoming more common. Appropriate statistical analysis of these complex high-dimensional data will be critical for extracting meaningful results from such large-scale human metabolomics studies. Therefore, we consider the statistical analytical approaches that have been employed in prior human metabolomics studies. Based on the lessons learned and collective experience to date in the field, we offer a step-by-step framework for pursuing statistical analyses of cohort-based human metabolomics data, with a focus on feature selection. We discuss the range of options and approaches that may be employed at each stage of data management, analysis, and interpretation and offer guidance on the analytical decisions that need to be considered over the course of implementing a data analysis workflow. Certain pervasive analytical challenges facing the field warrant ongoing focused research. Addressing these challenges, particularly those related to analyzing human metabolomics data, will allow for more standardization of as well as advances in how research in the field is practiced. In turn, such major analytical advances will lead to substantial improvements in the overall contributions of human metabolomics investigations

Double Field Inflation

We present an inflationary universe model which utilizes two coupled real scalar fields. The inflation field $\phi$ experiences a first order phase transition and its potential dominates the energy density of the Universe during the inflationary epoch. This field $\phi$ is initially trapped in its metastable minimum and must tunnel through a potential barrier to reach the true vacuum. The second auxiliary field $\psi$ couples to the inflaton field and serves as a catalyst to provide an abrupt end to the inflationary epoch; i.e., the $\psi$ field produces a time-dependent nucleation rate for bubbles of true $\phi$ vacuum. In this model, we find that bubbles of true vacuum can indeed percolate and we argue that thermalization of the interiors can more easily take place. The required degree of flatness (i.e., the fine tuning) in the potential of the $\psi$ field is comparable to that of other models which invoke slowly rolling fields. Pseudo Nambu-Goldstone bosons may naturally provide the flat potential for the rolling field.Comment: 18 pages, 2 figures, This early paper is being placed on the archive to make it more easily accessible in light of recent interest in reviving tunneling inflationary models and as its results are used in an accompanying submissio

Interstellar and Circumstellar Optical & Ultraviolet Lines Towards SN1998S

We have observed SN1998S which exploded in NGC3877, with the UES at the WHT and with the E230M echelle of STIS aboard HST. Both data sets were obtained at two seperate epochs. From our own Galaxy we detect interstellar absorption lines of CaII, FeII, MgI, and probably MnII from the edge of the HVC Complex M. We derive gas-phase abundances which are very similar to warm disk clouds in the local ISM, which we believe argues against the HVC material having an extragalactic origin. At the velocity of NGC3877 we detect interstellar MgI, MgII, MnII, CaII, & NaI. Surprisingly, one component is seen to increase by a factor of ~1 dex in N(NaI) and N(MgI) between the two epochs over which the data were taken. Unusually, our data also show narrow Balmer, HeI, and metastable FeII P-Cygni profiles, with a narrow absorption component superimposed on the bottom of the profile's absorption trough. Both the broad and narrow components of the optical lines are seen to increase substantially in strength between the two epochs. Most of the low-ionization absorption can be understood in terms of gas co-rotating with the disk of NGC 3877, providing the SN is at the back of an HI disk with a similar thickness to that of our own Galaxy. However, the variable absorption components, and the classic P-Cygni emission profiles, most likely arise in slow-moving circumstellar outflows originating from the red supergiant progenitor of SN1998S. [Abridged.]Comment: Accepted by ApJ, 26 pages including 9 figure

Comparative Effectiveness of Carotid Endarterectomy vs Initial Medical Therapy in Patients With Asymptomatic Carotid Stenosis

Importance Carotid endarterectomy (CEA) among asymptomatic patients involves a trade-off between a higher short-term perioperative risk in exchange for a lower long-term risk of stroke. The clinical benefit observed in randomized clinical trials (RCTs) may not extend to real-world practice. Objective To examine whether early intervention (CEA) was superior to initial medical therapy in real-world practice in preventing fatal and nonfatal strokes among patients with asymptomatic carotid stenosis. Design, Setting, and Participants This comparative effectiveness study was conducted from August 28, 2018, to March 2, 2020, using the Corporate Data Warehouse, Suicide Data Repository, and other databases of the US Department of Veterans Affairs. Data analyzed were those of veterans of the US Armed Forces aged 65 years or older who received carotid imaging between January 1, 2005, and December 31, 2009. Patients without a carotid imaging report, those with carotid stenosis of less than 50% or hemodynamically insignificant stenosis, and those with a history of stroke or transient ischemic attack in the 6 months before index imaging were excluded. A cohort of patients who received initial medical therapy and a cohort of similar patients who received CEA were constructed and followed up for 5 years. The target trial method was used to compute weighted Kaplan-Meier curves and estimate the risk of fatal and nonfatal strokes in each cohort in the pragmatic sample across 5 years of follow-up. This analysis was repeated after restricting the sample to patients who met RCT inclusion criteria. Cumulative incidence functions for fatal and nonfatal strokes were estimated, accounting for nonstroke deaths as competing risks in both the pragmatic and RCT-like samples. Exposures Receipt of CEA vs initial medical therapy. Main Outcomes and Measures Fatal and nonfatal strokes. Results Of the total 5221 patients, 2712 (51.9%; mean [SD] age, 73.6 [6.0] years; 2678 men [98.8%]) received CEA and 2509 (48.1%; mean [SD] age, 73.6 [6.0] years; 2479 men [98.8%]) received initial medical therapy within 1 year after the index carotid imaging. The observed rate of stroke or death (perioperative complications) within 30 days in the CEA cohort was 2.5% (95% CI, 2.0%-3.1%). The 5-year risk of fatal and nonfatal strokes was lower among patients randomized to CEA compared with patients randomized to initial medical therapy (5.6% vs 7.8%; risk difference, −2.3%; 95% CI, −4.0% to −0.3%). In an analysis that incorporated the competing risk of death, the risk difference between the 2 cohorts was lower and not statistically significant (risk difference, −0.8%; 95% CI, −2.1% to 0.5%). Among patients who met RCT inclusion criteria, the 5-year risk of fatal and nonfatal strokes was 5.5% (95% CI, 4.5%-6.5%) among patients randomized to CEA and was 7.6% (95% CI, 5.7%-9.5%) among those randomized to initial medical therapy (risk difference, −2.1%; 95% CI, −4.4% to −0.2%). Accounting for competing risks resulted in a risk difference of −0.9% (95% CI, −2.9% to 0.7%) that was not statistically significant. Conclusions and Relevance This study found that the absolute reduction in the risk of fatal and nonfatal strokes associated with early CEA was less than half the risk difference in trials from 20 years ago and was no longer statistically significant when the competing risk of nonstroke deaths was accounted for in the analysis. Given the nonnegligible perioperative 30-day risks and the improvements in stroke prevention, medical therapy may be an acceptable therapeutic strategy

Efficacy of BET bromodomain inhibition in Kras-mutant non-small cell lung cancer

PurposeAmplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that BET bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven NSCLC with BET inhibition.Experimental DesignWe performed functional assays to evaluate the effects of JQ1 in genetically defined NSCLC cells lines harboring KRAS and/or LKB1 mutations. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. Effects of bromodomain inhibition on transcriptional pathways were explored and validated by expression analysis.ResultsWhile JQ1 is broadly active in NSCLC cells, activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knock-down of LKB1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1.ConclusionBromodomain inhibition comprises a promising therapeutic strategy for KRAS mutant NSCLC with wild-type LKB1, via inhibition of MYC function. Clinical studies of BET bromodomain inhibitors in aggressive NSCLC will be actively pursued

Unique reporter-based sensor platforms to monitor signalling in cells

Introduction: In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. <p/>Methods: Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. <p/>Findings: We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. <p/>Conclusions: We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters

Linguistics

Contains table of contents for Section 4, an introduction and abstracts for eleven doctoral dissertations