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

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

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

Requirement of Cognate CD4+ T-Cell Recognition for the Regulation of Allospecific CTL by Human CD4+CD127−CD25+FOXP3+ Cells Generated in MLR

Although immunoregulation of alloreactive human CTLs has been described, the direct influence of CD4+ Tregs on CD8+ cytotoxicity and the interactive mechanisms have not been well clarified. Therefore, human CD4+CD127−CD25+FOXP3+ Tregs were generated in MLR, immunoselected and their allospecific regulatory functions and associated mechanisms were then tested using modified 51Chromium release assays (Micro-CML), MLRs and CFSE-based multi-fluorochrome flow cytometry proliferation assays. It was observed that increased numbers of CD4+CD127−CD25+FOXP3+ cells were generated after a 7 day MLR. After immunoselection for CD4+CD127−CD25+ cells, they were designated as MLR-Tregs. When added as third component modulators, MLR-Tregs inhibited the alloreactive proliferation of autologous PBMC in a concentration dependent manner. The inhibition was quasi-antigen specific, in that the inhibition was non-specific at higher MLR-Treg modulator doses, but non-specificity disappeared with lower numbers at which specific inhibition was still significant. When tested in micro-CML assays CTL inhibition occurred with PBMC and purified CD8+ responders. However, antigen specificity of CTL inhibition was observed only with unpurified PBMC responders and not with purified CD8+ responders or even with CD8+ responders plus Non-T “APC”. However, allospecificity of CTL regulation was restored when autologous purified CD4+ T cells were added to the CD8+ responders. Proliferation of CD8+ cells was suppressed by MLR-Tregs in the presence or absence of IL-2. Inhibition by MLR-Tregs was mediated through down-regulation of intracellular perforin, granzyme B and membrane-bound CD25 molecules on the responding CD8+ cells. Therefore, it was concluded that human CD4+CD127−CD25+FOXP3+ MLR-Tregs down-regulate alloreactive cytotoxic responses. Regulatory allospecificity, however, requires the presence of cognate responding CD4+ T cells. CD8+ CTL regulatory mechanisms include impaired proliferation, reduced expression of cytolytic molecules and CD25+ activation epitopes

Differentiation of regulatory myeloid and T-cells from adult human hematopoietic stem cells after allogeneic stimulation

IntroductionDonor hematopoietic stem cell (DHSC) infusions are increasingly being studied in transplant patients for tolerance induction.MethodsTo analyze the fate of infused DHSCs in patients, we developed an in vitro culture system utilizing CD34+DHSCs stimulated with irradiated allogeneic cells in cytokine supplemented medium long-term.ResultsFlow cytometric analyses revealed loss of the CD34 marker and an increase in CD33+ myeloid and CD3+ T-cell proportion by 10.4% and 72.7%, respectively, after 21 days in culture. T-cells primarily expressed TcR-αβ and were of both CD4+ and CD8+ subsets. Approximately 80% of CD3+ T cells lacked expression of the co-stimulatory receptor CD28. The CD4+ compartment was predominated by CD4+CD25+CD127-FOXP3+ Tregs (>50% CD4+CD127- compartment) with <1% of all leukocytes exhibiting a CD4+CD127+ phenotype. Molecular analyses for T-cell receptor excision circles showed recent and increased numbers of TcR rearrangements in generated T cells over time suggesting de novo differentiation from DHSCs. CD33+ myeloid cells mostly expressed HLA-DR, but lacked expression of co-stimulatory receptors CD80 and CD83. When studied as modulators in primary mixed lymphocyte reactions where the cells used to stimulate the DHSC were used as responders, the DHSC-lines and their purified CD8+, CD4+, CD33+ and linage negative subsets inhibited the responses in a dose-dependent and non-specific fashion. The CD8+ cell-mediated inhibition was due to direct lysis of responder cells.DiscussionExtrapolation of these results into the clinical situation would suggest that DHSC infusions into transplant recipients may generate multiple subsets of donor “chimeric” cells and promote recipient Treg development that could regulate the anti-donor immune response in the periphery. These studies have also indicated that T cell maturation can occur in vitro in response to allogeneic stimulation without the pre-requisite of a thymic-like environment or NOTCH signaling stimulatory cell line