The impact of engineered design constraints upon bacteriophage T7 evolution

Since the establishment of molecular biology until the present, a shift has taken place by which engineering of biology has increased in its scope, power, and influence. Synthetic biology is the latest term that advances the overall goal of engineering control of biological systems. Enabled by the recent progress in the ability to read and write nucleic acids, synthetic biology stands out in its desire for standardization of parts, processes, assays, and even organisms. This standardization has led to multiple successful outcomes which justify its utility. However, this standardization via synthetic biology is imbued with an implicit hubris: it may be that certain aspects of biological phenomena are too complex to lend themselves to systemization. Engineering biology, unlike other engineering disciplines, is not obviously governed by a simple series of equations but, rather, united by one universal theme: evolution happens. Because of this, much work has been done in order to study not just how evolution works and but also into controlling, accelerating, and biasing it in a laboratory setting. The former work can be generally classified as “evolutionary biology” and the latter called “directed evolution”. In this dissertation, work is presented which blurs the line between evolutionary biology and directed evolution yielding unanticipated outcomes and new methods of biological control. Three projects are presented in which evolution of bacteriophage T7 is studied in response to rationally engineered design constraints. In the first, the predictability of bacteriophage T7 promoter evolution in response to a novel RNA polymerase is determined. In the second, the evolution of the entirety of the transcriptional apparatus of bacteriophage T7 is studied in the context of an evolutionarily adapted bacteriophage population. Finally, the construction, exploration, and optimization of a designed bacteriophage T7 life-cycling system reliant on proteases are described. Together, this work provides evidence that although the ability to rationally design biology is important, various ways to rationally control and direct evolution offer a complementary strategy to the systematization of synthetic biology.Cellular and Molecular Biolog

Active Control of Transition Using the Lorentz Force

A new concept and technique has been developed to directly control boundary-layer transition and turbulence. Near-wall vertical motions are directly suppressed through the application of Lorentz force. Current (j) and magnetic (b) fields are applied parallel to the boundary and normal to each other to produce a Lorentz force (j x B) normal to the boundary. This approach is called magnetic turbulence control (MTC). Experiments have been performed on flat-plate transitional and turbulent boundary layers in water seeded with a weak electrolyte

ETV2 and VEZF1 interaction and regulation of the hematoendothelial lineage during embryogenesis

Ets variant 2 (Etv2), a member of the Ets factor family, has an essential role in the formation of endothelial and hematopoietic cell lineages during embryonic development. The functional role of ETS transcription factors is, in part, dependent on the interacting proteins. There are relatively few studies exploring the coordinated interplay between ETV2 and its interacting proteins that regulate mesodermal lineage determination. In order to identify novel ETV2 interacting partners, a yeast two-hybrid analysis was performed and the C2H2 zinc finger transcription factor VEZF1 (vascular endothelial zinc finger 1) was identified as a binding factor, which was specifically expressed within the endothelium during vascular development. To confirm this interaction, co-immunoprecipitation and GST pull down assays demonstrated the direct interaction between ETV2 and VEZF1. During embryoid body differentiation, Etv2 achieved its peak expression at day 3.0 followed by rapid downregulation, on the other hand Vezf1 expression increased through day 6 of EB differentiation. We have previously shown that ETV2 potently activated Flt1 gene transcription. Using a Flt1 promoter-luciferase reporter assay, we demonstrated that VEZF1 co-activated the Flt1 promoter. Electrophoretic mobility shift assay and Chromatin immunoprecipitation established VEZF1 binding to the Flt1 promoter. Vezf1 knockout embryonic stem cells had downregulation of hematoendothelial marker genes when undergoing embryoid body mediated mesodermal differentiation whereas overexpression of VEZF1 induced the expression of hematoendothelial genes during differentiation. These current studies provide insight into the co-regulation of the hemato-endothelial lineage development via a co-operative interaction between ETV2 and VEZF1

Mooring Lines Reliability Analysis Case Study: Fso Abherka,

Floating Storage and Offloading (FSO) demands good designs. One of the design is FSO’s mooring lines. The mooring lines of the FSO would face various environmental conditions, so that they are required to have good reliability. FSO Abherka, installed in the Madura stratit, has dimensions of LOA 242.90 m, LPP 232 m, Breadth 41.6 m, Height 19.7 m, Draft 12,894 m, and a deadweight of 85829 tons. The strait has a water depth of 100 m with contour variations of ± 2 m. Each four cluster of mooring lines of FSO Abherka has three mooring ropes. This study analyzed the strength of mooring lines in intact and damaged conditions using DNV software Genie and DNV HydroD. This study models a hydrodynamic FSO. This study also measured the reliability of the mooring lines from 2 cases: 1 intact and one damage from the most dangerous conditions. This study used the mean Value First Order Second Moment method to find the reliability of these mooring lines. Based on the reliability calculation set by DNV according to DNV-OS E301, the mooring lines design meet the established reliability criteri

Effects of pre-meal whey protein consumption on acute food intake and energy balance over a 48-hour period

The effects of pre-meal whey protein consumption on acute food intake and subsequent energy balance measured over 48-h was investigated in males of healthy-weight (HW) or living with overweight and obesity (OV/OB). On two separate trial days, following a controlled breakfast (09:00) and lunch (13:00), 12 HW and 12 OV/OB males consumed either whey protein (20 g) or flavoured water beverages (16:40), and ad libitum test meal (17:00). A controlled 48-h assessment of energy intake and expenditure was used to determine any compensatory behaviour. Test meal energy intake reduced 15.9 % in HW (P = 0.003), and 17.8 % in OV/OB (P = 0.005) following whey protein, compared to placebo. We report no between-group differences and no changes in compensatory behaviour. A small dose of whey protein reduces energy intake at the next meal, without upregulating compensatory behaviours in both HW and OV/OB males. However, chronic effects on body composition and weight loss remain to be elucidated

3-Phenyl-6-(2-pyrid­yl)-1,2,4,5-tetra­zine

The title compound, C13H9N5, is the first asymmetric diaryl-1,2,4,5-tetra­zine to be crystallographically characterized. We have been inter­ested in this motif for incorporation into supra­molecular assemblies based on coordination chemistry. The solid state structure shows a centrosymmetric mol­ecule, forcing a positional disorder of the terminal phenyl and pyridyl rings. The mol­ecule is completely planar, unusual for aromatic rings with N atoms in adjacent ortho positions. The stacking observed is very common in diaryl­tetra­zines and is dominated by π stacking [centroid-to-centroid distance between the tetrazine ring and the aromatic ring of an adjacent molecule is 3.6 Å, perpendicular (centroid-to-plane) distance of about 3.3 Å]

Formation of submarine lava channel textures : insights from laboratory simulations

Author Posting. © American Geophysical Union, 2006. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research 111 (2006): B03104, doi:10.1029/2005JB003796.Laboratory simulations using polyethylene glycol (PEG) extruded at a constant rate and temperature into a tank with a uniform basal slope and filled with a cold sucrose solution generate channels that are defined by stationary levees and mobile flow interiors. These laboratory channels consistently display the following surface textures in the channel: smooth, folded, lineated, and chaotic. In the simulations, we can observe specific local conditions including flow rate, position within the channel, and time that combine to develop each texture. The textures in PEG flows form due to relative differences in shear forces between the PEG crust and the underlying liquid wax. Minimal shear forces form smooth crust, whereas folded crust forms when the shear is sufficiently high to cause ductile deformation. Brittle deformation of solid PEG creates a chaotic texture, and lineated crust results from shear forces along the channel-levee margin. We observe similar textures in submarine lava channels with sources at or near the Axial Summit Trough of the East Pacific Rise between 9° and 10°N. We mapped the surface textures of nine submarine lava channels using high-resolution digital images collected during camera tows. These textural maps, along with observations of the formation of similar features in analog flows, reveal important information about the mechanisms occurring across the channel during emplacement, including relative flow velocity and shear stress.The cruise was funded by a grant to WHOI from the National Science Foundation (NSF) OCE-9819261, with additional funding provided by WHOI thorough the Vetlesen Foundation. The PEG experiments were funded by NSF OCE-0425073 in a grant to Tracy Gregg

Neurological, Cognitive, and Psychological Findings Among Survivors of Ebola Virus Disease From the 1995 Ebola Outbreak in Kikwit, Democratic Republic of Congo: A Cross-sectional Study.

BackgroundClinical sequelae of Ebola virus disease (EVD) have not been described more than 3 years postoutbreak. We examined survivors and close contacts from the 1995 Ebola outbreak in Kikwit, Democratic Republic of Congo (DRC), and determined prevalence of abnormal neurological, cognitive, and psychological findings and their association with EVD survivorship.MethodsFrom August to September 2017, we conducted a cross-sectional study in Kikwit, DRC. Over 2 decades after the EVD outbreak, we recruited EVD survivors and close contacts from the outbreak to undergo physical examination and culturally adapted versions of the Folstein mini-mental status exam (MMSE) and Goldberg anxiety and depression scale (GADS). We estimated the strength of relationships between EVD survivorship and health outcomes using linear regression models by comparing survivors versus close contacts, adjusting for age, sex, educational level, marital status, and healthcare worker status.ResultsWe enrolled 20 EVD survivors and 187 close contacts. Among the 20 EVD survivors, 4 (20%) reported at least 1 abnormal neurological symptom, and 3 (15%) had an abnormal neurological examination. Among the 187 close contacts, 14 (11%) reported at least 1 abnormal neurologic symptom, and 9 (5%) had an abnormal neurological examination. EVD survivors had lower mean MMSE and higher mean GADS scores as compared to close contacts (MMSE: adjusted coefficient: -1.85; 95% confidence interval [CI]: -3.63, -0.07; GADS: adjusted coefficient: 3.91; 95% CI: 1.76, 6.04).ConclusionsEVD survivors can have lower cognitive scores and more symptoms of depression and anxiety than close contacts more than 2 decades after Ebola virus outbreaks