Thermal Optimization of Growth and Quality in Protein Crystals

Experimental evidence suggests that larger and higher quality crystals can be attained in the microgravity of space; however, the effect of growth rate on protein crystal quality is not well documented. This research is the first step towards providing strategies to grow crystals under constant rates of growth. Controlling growth rates at a constant value allows for direct one-to-one comparison of results obtained in microgravity and on earth. The overall goal of the project was to control supersaturation at a constant value during protein crystal growth by varying temperature in a predetermined manner. Applying appropriate theory requires knowledge of specific physicochemical properties of the protein solution including the effect of supersaturation on growth rates and the effect of temperature on protein solubility. Such measurements typically require gram quantities of protein and many months of data acquisition. A second goal of the project applied microcalorimetry for the rapid determination of these physicochemical properties using a minimum amount of protein. These two goals were successfully implemented on hen egg-white lysozyme. Results of these studies are described in the attached reprints

Characterization of seed nuclei in glucagon aggregation using light scattering methods and field-flow fractionation

<p>Abstract</p> <p>Background</p> <p>Glucagon is a peptide hormone with many uses as a therapeutic agent, including the emergency treatment of hypoglycemia. Physical instability of glucagon in solution leads to problems with the manufacture, formulation, and delivery of this pharmaceutical product. Glucagon has been shown to aggregate and form fibrils and gels <it>in vitro</it>. Small oligomeric precursors serve to initiate and nucleate the aggregation process. In this study, these initial aggregates, or seed nuclei, are characterized in bulk solution using light scattering methods and field-flow fractionation.</p> <p>Results</p> <p>High molecular weight aggregates of glucagon were detected in otherwise monomeric solutions using light scattering techniques. These aggregates were detected upon initial mixing of glucagon powder in dilute HCl and NaOH. In the pharmaceutically relevant case of acidic glucagon, the removal of aggregates by filtration significantly slowed the aggregation process. Field-flow fractionation was used to separate aggregates from monomeric glucagon and determine relative mass. The molar mass of the large aggregates was shown to grow appreciably over time as the glucagon solutions gelled.</p> <p>Conclusion</p> <p>The results of this study indicate that initial glucagon solutions are predominantly monomeric, but contain small quantities of large aggregates. These results suggest that the initial aggregates are seed nuclei, or intermediates which catalyze the aggregation process, even at low concentrations.</p

Constructing Fluorogenic Bacillus Spores (F-Spores) via Hydrophobic Decoration of Coat Proteins

Background: Bacterial spores are protected by a coat consisting of about 60 different proteins assembled as a biochemically complex structure with intriguing morphological and mechanical properties. Historically, the coat has been considered a static structure providing rigidity and mainly acting as a sieve to exclude exogenous large toxic molecules, such as lytic enzymes. Over recent years, however, new information about the coat’s architecture and function have emerged from experiments using innovative tools such as automated scanning microscopy, and high resolution atomic force microscopy. Principal Findings: Using thin-section electron microscopy, we found that the coat of Bacillus spores has topologically specific proteins forming a layer that is identifiable because it spontaneously becomes decorated with hydrophobic fluorogenic probes from the milieu. Moreover, spores with decorated coat proteins (termed F-spores) have the unexpected attribute of responding to external germination signals by generating intense fluorescence. Fluorescence data from diverse experimental designs, including F-spores constructed from five different Bacilli species, indicated that the fluorogenic ability of F-spores is under control of a putative germination-dependent mechanism. Conclusions: This work uncovers a novel attribute of spore-coat proteins that we exploited to decorate a specific layer imparting germination-dependent fluorogenicity to F-spores. We expect that F-spores will provide a model system to gai

FIRST CRYOMODULE TEST AT AMTF HALL FOR THE EUROPEAN X-RAY FREE ELECTRON LASER (XFEL)

Abstract The Accelerator Module Test Facility (AMTF) at DESY in Hamburg is dedicated to the tests of RF cavities and accelerating cryomodules for the European X-ray Free Electron Laser (XFEL). The AMTF hall is equipped with two vertical cryostats, which are used for RF cavities testing and three test benches that will be used for tests of the accelerating cryomodules. Recently, the first cryomodule teststand (XATB3) was commissioned and the first XFEL cryomodule (XM-2) was tested by team of physicists, engineers and technicians from The Henry

Outcomes of truncal vascular injuries in children.

BACKGROUND: Pediatric truncal vascular injuries occur infrequently and have a reported mortality rate of 30% to 50%. This report examines the demographics, mechanisms of injury, associated trauma, and outcome of patients presenting for the past 10 years at a single institution with truncal vascular injuries. METHODS: A retrospective review (1997-2006) of a pediatric trauma registry at a single institution was undertaken. RESULTS: Seventy-five truncal vascular injuries occurred in 57 patients (age, 12 +/- 3 years); the injury mechanisms were penetrating in 37%. Concomitant injuries occurred with 76%, 62%, and 43% of abdominal, thoracic, and neck vascular injuries, respectively. Nonvascular complications occurred more frequently in patients with abdominal vascular injuries who were hemodynamically unstable on presentation. All patients with thoracic vascular injuries presenting with hemodynamic instability died. In patients with neck vascular injuries, 1 of 2 patients who were hemodynamically unstable died, compared to 1 of 12 patients who died in those who presented hemodynamically stable. Overall survival was 75%. CONCLUSIONS: Survival and complications of pediatric truncal vascular injury are related to hemodynamic status at the time of presentation. Associated injuries are higher with trauma involving the abdomen

Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish

Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies

History, Commemoration, and Belief: Abraham Lincoln in American Memory, 1945-2001

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91765/1/Schuman-History_Commemoration_Belief.pd

Changes of the human gut microbiome induced by a fermented milk product

The gut microbiota (GM) consists of resident commensals and transient microbes conveyed by the diet but little is known about the role of the latter on GM homeostasis. Here we show, by a conjunction of quantitative metagenomics, in silico genome reconstruction and metabolic modeling, that consumption of a fermented milk product containing dairy starters and Bifidobacterium animalis potentiates colonic short chain fatty acids production and decreases abundance of a pathobiont Bilophila wadsworthia compared to a milk product in subjects with irritable bowel syndrome (IBS, n = 28). The GM changes parallel improvement of IBS state, suggesting a role of the fermented milk bacteria in gut homeostasis. Our data challenge the view that microbes ingested with food have little impact on the human GM functioning and rather provide support for beneficial health effects

Rejuvenation of Spent Media via Supported Emulsion Liquid Membranes

The overall goal of this project is to maximize the reuseability of spent fermentation media. Supported emulsion liquid membrane separation, a highly efficient extraction technique, is used to remove inhibitory byproducts during fermentation; thus, improving the yield while reducing the need for fresh water. The key objectives of this study are: Develop an emulsion liquid membrane system targeting low molecular weight organic acids which has minimal toxicity on a variety of microbial systems; Conduct mass transfer studies to allow proper modeling and design of a supported emulsion liquid membrane system; Investigate the effect of gravity on emulsion coalescence within the membrane unit; Access the effect of water re-use on fermentation yields in a model microbial system; Develop a perfusion-type fermentor utilizing a supported emulsion liquid membrane system to control inhibitory fermentation byproducts; Work for the coming year will focus on the determination of toxicity of various solvents, selection of the emulsifying agents, as well as characterizing the mass transfer of hollow-fiber contactors