Fluorescent Protein Biosensor for Use in Parkinson\u27s Research

Purinergic signaling is a type of extracellular communication that occurs between cells, mediated by adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine. In Parkinson’s Disease, purinergic signaling is disrupted, which contributes to neurodegeneration. In order to monitor this change in cell-to-cell signaling, there is a need for the development of a fluorescent protein (FP) biosensor to study the changes in the concentration of the signaling molecule ATP and its decomposition bioproduct ADP. This summer a genetically encoded ADP sensor that measures changes in ADP concentration was developed. This sensor utilizes Forster Resonance Energy Transfer (FRET) which is a sensing technique that is based on the energy transfer from a donor FP to an acceptor FP. Since this transfer is distance dependent, a change in the sensing domain allows for detection of ADP concentration through changes in fluorescence emission. To develop this FRET based sensor, we are utilizing a cyan-yellow FP pair, as well as a non-fluorescent protein that binds to ADP. Using traditional cloning methods, a small library of ADP sensors from five different versions of both the cyan and yellow proteins was created. This library was screened in E. coli cultures using a method developed to optimize an ATP-sensor. The cloning for this sensor has been confirmed and the library is being tested for sensors responsive to changing concentrations of ADP. With confirmation of a responsive sensor, this sensor design will be validated, allowing for further optimization of this biosensor for the study of purinergic signaling and neurodegeneration

Plate-based transfection and culturing technique for genetic manipulation of Plasmodium falciparum

Genetic manipulation of malaria parasites remains an inefficient, time-consuming and resource-intensive process. Presented here is a set of methods for 96-well plate-based transfection and culture that improve the efficiency of genetic manipulation of Plasmodium falciparum. Compared to standard protocols plate-based transfection requires 20-fold less DNA, transient transfection efficiency achieved is approximately seven-fold higher, whilst stable transfection success rate is above 90%. Furthermore the utility of this set of protocols to generate a knockout of the PfRH3 pseudogene, screened by whole-cell PCR, is demonstrated. The methods and tools presented here will facilitate genome-scale genetic manipulation of P. falciparum

Oxygen Deficient Metabolism in Organs: A Link to Combustion Science

In an attempt to better understand and model transport of oxygen, O2, from capillaries to living cells in surrounding tissue, the group combustion (O2 deficient) concept from the field of combustion science in engineering is applied to the biological field of microvascular O2 transport from capillaries to cells immersed in interstitial fluid (IF). The conventional Krogh model represents typical biological models, considering tissue cylinder with uniform oxygen source/sink term (US) (m''', g/s/cm^3) and O2 transport from capillary on axis (COA) towards the surface; engineering models consider cylinders with O2 supplied from the surface of cylinder (COS); in addition, they present i) transport (diffusion) and ii) kinetics limited sink rates and profiles for O2. Diffusion limitation causes m''' to be proportional to local O2 concentration. Thus, the present work modifies COS engineering models for COA cases and considers only diffusion limited transport of O2 to metabolic cells from IF. O2 profiles and resulting specific metabolic rates, SMRs (W/g), are generated for four models: I) COA with oxygen dependent consumption source term (O2) (COA-O2), II) COA-US, III) COS-O2, and IV) COS-US. In order to validate the current approach, the model results are verified with the following different types of experimental data: A) If SMRs (mq W/g) are given by the allometric law, kbmk^kq=am for organ k, then COS models under limiting conditions suggest -1/3<bk<0 where bk=0 for small organs following isometric law and -1/3 for large organs while COA models suggest -2/3<bk<0 under similar conditions. Most experimental data for vital organs yield -0.27<bk<0 which suggests better correlation with COS models. B) Measured capillary-IF interface pressure of O2 (pO2,cap-IF) for rat mesentery system as given by Tsai et. al. is about 38 mmHg while predicted pO2,cap-IF is 54.5, 38.1, 55.3 and 46.2 mmHg for COA-O2, COS-O2, COA-US and COS-US respectively. C) Further, SMR for the average human liver is about 0.01 W/g as given by Wang et. al. (2010) while the COA-O2, COS-O2, COA-US and COS-US models yield 0.006, 0.032, 0.003 and 0.022 W/g respectively. COS models are of the same order magnitude as the experimental value while COS-US model is closest

An Evaluation of Trapping Efforts to Capture Bobcats, Coyotes, and Red Fox

Wildlife biologists are often involved in efforts to capture free-ranging felids and canids. The objective of these efforts is usually to remove individuals causing unwanted or excessive predation, or to obtain study animals. The most common method used to capture carnivores includes some type of leg-hold trap. Numerous references provide information on the technique of leg-hold trapping (Taylor 1971, Musgrove and Blair 1979); however few reports include an evaluation of these methods

Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice

The FDA approved drug rapamycin increases lifespan in rodents and delays age-related dysfunction in rodents and humans. Nevertheless, important questions remain regarding the optimal dose, duration, and mechanisms of action in the context of healthy aging. Here we show that 3 months of rapamycin treatment is sufficient to increase life expectancy by up to 60% and improve measures of healthspan in middle-aged mice. This transient treatment is also associated with a remodeling of the microbiome, including dramatically increased prevalence of segmented filamentous bacteria in the small intestine. We also define a dose in female mice that does not extend lifespan, but is associated with a striking shift in cancer prevalence toward aggressive hematopoietic cancers and away from non-hematopoietic malignancies. These data suggest that a short-term rapamycin treatment late in life has persistent effects that can robustly delay aging, influence cancer prevalence, and modulate the microbiome.P30 AG013280 - NIA NIH HHS; T32 AG000057 - NIA NIH HH

Fracture Blisters

Fracture blisters are a relatively uncommon complication of fractures in locations of the body, such as the ankle, wrist elbow and foot, where skin adheres tightly to bone with little subcutaneous fat cushioning. The blister that results resembles that of a second degree burn

Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems

USING MODELING TO PREDICT THE EFFECTS OF AUTOMATION ON MEDEVAC PILOT COGNITIVE WORKLOAD

The Holistic Situational Awareness - Decision Making (HSA-DM) program is researching ways to aid pilots via avionics essential to the Future Vertical Lift (FVL) rotor-wing platform. As pilots manage the new avionics that FVL will bring to the battlefield, automation assistance will be essential. This study’s goal is to determine to what extent automation reduces pilot cognitive workload particularly when performing communication tasks. The quantitative analysis is based on cognitive walkthroughs with active-duty helicopter pilots. Pilot interviews were also conducted to assess how tasks are completed, and more importantly, to ascertain the cognitive workload associated with those tasks. This information is implemented into computer models of a routine helicopter flight to accurately predict pilot workload during a mission. These models also predict which tasks would add the most value to pilots and FVL if automated mission tasks were implemented. The research indicates that by automating communication tasks for the pilot and copilot, workload is reduced to an optimal level. Based on these findings, monitor radio nets, adjust volume, input channel, select channel, and send JVMF messages should be automated. In addition, this analysis establishes a cost-effective, valid, and repeatable framework for future workload studies on automated tasks in FVL.Major, United States Army ReserveMajor, United States ArmyCaptain, United States ArmyMajor, United States ArmyMajor, United States ArmyApproved for public release. Distribution is unlimited

Trematodes of the Great Barrier Reef, Australia: emerging patterns of diversity and richness in coral reef fishes

The Great Barrier Reef holds the richest array of marine life found anywhere in Australia, including a diverse and fascinating parasite fauna. Members of one group, the trematodes, occur as sexually mature adult worms in almost all Great Barrier Reef bony fish species. Although the first reports of these parasites were made 100 years ago, the fauna has been studied systematically for only the last 25 years. When the fauna was last reviewed in 1994 there were 94 species known from the Great Barrier Reef and it was predicted that there might be 2,270 in total. There are now 326 species reported for the region, suggesting that we are in a much improved position to make an accurate prediction of true trematode richness. Here we review the current state of knowledge of the fauna and the ways in which our understanding of this fascinating group is changing. Our best estimate of the true richness is now a range, 1,100–1,800 species. However there remains considerable scope for even these figures to be incorrect given that fewer than one-third of the fish species of the region have been examined for trematodes. Our goal is a comprehensive characterisation of this fauna, and we outline what work needs to be done to achieve this and discuss whether this goal is practically achievable or philosophically justifiable

Stab wound leading to sub lingual haematoma

We report a case of a sublingual haematoma and proptosis of the right eye as a result of penetrative injury to the suprazygomatic supratemporal fossa. The case was seen in the accident and emergency department at the Royal London Hospital (RLH). The case was referred to the anaesthetic and oral and maxillofacial unit, following neurosurgery and ophthalmology assessments. The case highlights the need for a multi disciplinary approach to dealing with penetrative wounds in the craniofacial region. It is essential that all healthcare professionals have excellent working knowledge of the anatomy of the orbit and its association with anatomical planes in the head and neck. (1) When diagnosing orbital compartment syndrome (also known as retrobulbar haemorrhage), radiographic findings should always be correlated to clinical signs and symptoms. Management of such a condition should be tailored to the individual. In this case medical management alone of 80mg of dexamethasone and 500mg of Mannitol was appropriate to enable a full recovery