Subcellular Localization of the Non-Structural Proteins 3C and 3CD of the Honeybee Virus Deformed Wing Virus

Apis mellifera L., the European honeybee, is a crucial pollinator of many important agricultural crops in the United States. Recently, honeybee colonies have been affected by Colony Collapse Disorder (CCD), a disorder in which the colony fails due to the disappearance of a key functional group of worker bees. Though no direct causalrelationship has been confirmed, hives that experience CCD have been shown to have a high incidence of Deformed Wing Virus (DWV), a common honeybee virus. While the genome sequence and gene-order of DWV has been analyzed fairly recently, few other studies have been performed to understand the molecular characterization of the virus.Since little is known about where DWV proteins localize in infected host cells, the objective of this project was to determine the subcellular localization of two of the important non-structural proteins that are encoded in the DWV genome. This project focused on the protein 3C, an autocatalytic protease which cleaves itself from a longer polyprotein and helps to cut all of the other proteins apart from one another so that they can become functional, and 3D, the RNA-dependent RNA polymerase (RdRp) which is critical for replication of the virus because it copies the viral genome. By tagging nested constructs containing these two proteins and tracking where they localized in living cells, this study aimed to better understand the replication of DWV and to elicit possible targetsfor further research on how to control the virus. Since DWV is a picorna-like virus, distantly related to human viruses such as polio, and picornavirus non-structural proteins aggregate at cellular membranes during viral replication, the major hypothesis was that the 3C and 3CD proteins would localize at cellular organelle membranes as well. Using confocal microscopy, both proteins were found to localize in the cytoplasm, but the 3CDprotein was found to be mostly diffuse cytoplasmic, and the 3C protein was found to localize more specifically on membranous structures just outside of the nucleus.

Detection of Internal Defects in Concrete Members Using Global Vibration Characteristics

Rock-pocket and honeycomb defects impair overall stiffness, accelerate aging, reduce service life, and cause structural problems in hardened concrete members. Traditional methods for detecting such deficient volumes involve visual observations or localized nondestructive methods, which are labor-intensive, time-consuming, highly sensitive to test conditions, and require knowledge of and accessibility to defect locations. The authors propose a vibration response-based nondestructive technique that combines experimental and numerical methodologies for use in identifying the location and severity of internal defects of concrete members. The experimental component entails collecting mode shape curvatures from laboratory beam specimens with size-controlled rock pocket and honeycomb defects, and the numerical component entails simulating beam vibration response through a finite element (FE) model parameterized with three defect-identifying variables indicating location (x, coordinate along the beam length) and severity of damage (alpha, stiffness reduction and beta, mass reduction). Defects are detected by comparing the FE model predictions to experimental measurements and inferring the low number of defect-identifying variables. This method is particularly well-suited for rapid and cost-effective quality assurance for precast concrete members and for inspecting concrete members with simple geometric forms.

Effects Of The Flash Welding Process On Mechanical And Microstructural Properties Of Structural Steel Joints Assessed Using Dest

ASTM A529 carbon¿manganese steel angle specimens were joined by flash butt welding and the effects of varying process parameter settings on the resulting welds were investigated. The weld metal and heat affected zones were examined and tested using tensile testing, ultrasonic scanning, Rockwell hardness testing, optical microscopy, and scanning electron microscopy with energy dispersive spectroscopy in order to quantify the effect of process variables on weld quality. Statistical analysis of experimental tensile and ultrasonic scanning data highlighted the sensitivity of weld strength and the presence of weld zone inclusions and interfacial defects to the process factors of upset current, flashing time duration, and upset dimension. Subsequent microstructural analysis revealed various phases within the weld and heat affected zone, including acicular ferrite, Widmanstätten or side-plate ferrite, and grain boundary ferrite. Inspection of the fracture surfaces of multiple tensile specimens, with scanning electron microscopy, displayed evidence of brittle cleavage fracture within the weld zone for certain factor combinations. Test results also indicated that hardness was increased in the weld zone for all specimens, which can be attributed to the extensive deformation of the upset operation. The significance of weld process factor levels on microstructure, fracture characteristics, and weld zone strength was analyzed. The relationships between significant flash welding process variables and weld quality metrics as applied to ASTM A529-Grade 50 steel angle were formalized in empirical process models.

Performance Tuning Non-Uniform Sampling for Sensitivity Enhancement of Signal-Limited Biological NMR

Non-uniform sampling (NUS) has been established as a route to obtaining true sensitivity enhancements when recording indirect dimensions of decaying signals in the same total experimental time as traditional uniform incrementation of the indirect evolution period. Theory and experiments have shown that NUS can yield up to two-fold improvements in the intrinsic signal-to-noise ratio (SNR) of each dimension, while even conservative protocols can yield 20-40 % improvements in the intrinsic SNR of NMR data. Applications of biological NMR that can benefit from these improvements are emerging, and in this work we develop some practical aspects of applying NUS nD-NMR to studies that approach the traditional detection limit of nD-NMR spectroscopy. Conditions for obtaining high NUS sensitivity enhancements are considered here in the context of enabling H-1,N-15-HSQC experiments on natural abundance protein samples and H-1,C-13-HMBC experiments on a challenging natural product. Through systematic studies we arrive at more precise guidelines to contrast sensitivity enhancements with reduced line shape constraints, and report an alternative sampling density based on a quarter-wave sinusoidal distribution that returns the highest fidelity we have seen to date in line shapes obtained by maximum entropy processing of non-uniformly sampled data.