Phospholipase D1 produces phosphatidic acid at sites of secretory vesicle docking and fusion.

Phospholipase D1 (PLD1) activity is essential for the stimulated exocytosis of secretory vesicles where it acts as a lipid-modifying enzyme to produces phosphatidic acid (PA). PLD1 localizes to the plasma membrane and secretory vesicles, and PLD1 inhibition or knockdowns reduce the rate of fusion. However, temporal data resolving when and where PLD1 and PA are required during exocytosis is lacking. In this work, PLD1 and production of PA are measured during the trafficking, docking, and fusion of secretory vesicles in PC12 cells. Using fluorescently tagged PLD1 and a PA-binding protein, cells were imaged using TIRF microscopy to monitor the presence of PLD1 and the formation of PA throughout the stages of exocytosis. Single docking and fusion events were imaged to measure the recruitment of PLD1 and the formation of PA. PLD1 is present on mobile, docking, and fusing vesicles and also colocalizes with Syx1a clusters. Treatment of cells with PLD inhibitors significantly reduces fusion, but not PLD1 localization to secretory vesicles. Inhibitors also alter the formation of PA; when PLD1 is active, PA slowly accumulates on docked vesicles. During fusion, PA is reduced in cells treated with PLD1 inhibitors, indicating that PLD1 produces PA during exocytosis.

Management of vagus nerve stimulation therapy in the peri-operative period: Guidelines from the Association of Anaesthetists: Guidelines from the Association of Anaesthetists.

Vagus nerve stimulation is a well-established treatment option for patients with drug-resistant epilepsy and has an expanding range of other clinical indications. Side effects of vagus nerve stimulation therapy include: cough; voice changes; vocal cord adduction; rarely, obstructive sleep apnoea; and arrhythmia. Patients with implanted vagus nerve stimulation devices may present for unrelated surgery and critical care to clinicians who are unfamiliar with their function and safe management. These guidelines have been formulated by multidisciplinary consensus based on case reports, case series and expert opinion to support clinicians in the management of patients with these devices. The aim is to provide specific guidance on the management of vagus nerve stimulation devices in the following scenarios: the peri-operative period; peripartum period; during critical illness; and in the MRI suite. Patients should be aware of the importance of carrying their personal vagus nerve stimulation device magnet with them at all times to facilitate urgent device deactivation if necessary. We advise that it is generally safer to formally deactivate vagus nerve stimulation devices before general and spinal anaesthesia. During periods of critical illness associated with haemodynamic instability, we also advise cessation of vagus nerve stimulation and early consultation with neurology services.

Impacts of microgravity on amino acid metabolism during spaceflight.

Spaceflight exerts an extreme and unique influence on human physiology as astronauts are subjected to long-term or short-term exposure to microgravity. During spaceflight, a multitude of physiological changes, including the loss of skeletal muscle mass, bone resorption, oxidative stress, and impaired blood flow, occur, which can affect astronaut health and the likelihood of mission success. In vivo and in vitro metabolite studies suggest that amino acids are among the most affected nutrients and metabolites by microgravity (a weightless condition due to very weak gravitational forces). Moreover, exposure to microgravity alters gut microbial composition, immune function, musculoskeletal health, and consequently amino acid metabolism. Appropriate knowledge of daily protein consumption, with a focus on specific functional amino acids, may offer insight into potential combative and/or therapeutic effects of amino acid consumption in astronauts and space travelers. This will further aid in the successful development of long-term manned space mission and permanent space habitats.

Exosome secretion kinetics are controlled by temperature.

When multivesicular endosomes (MVEs) fuse with the plasma membrane, exosomes are released into the extracellular space where they can affect other cells. The ability of exosomes to regulate cells nearby or further away depends on whether they remain attached to the secreting cell membrane. The regulation and kinetics of exosome secretion are not well characterized, but probes for directly imaging single MVE fusion events have allowed for visualization of the fusion and release process. In particular, the design of an exosome marker with a pH-sensitive dye in the middle of the tetraspanin protein CD63 has facilitated studies of individual MVE fusion events. Using TIRF microscopy, single fusion events were measured in A549 cells held at 23-37°C and events were identified using an automated detection algorithm. Stable docking precedes fusion almost always and a decrease in temperature was accompanied by decrease in the rate of content loss and in the frequency of fusion events. The loss of CD63-pHluorin fluorescence was measured at fusion sites and fit with a single or double exponential decay, with most events requiring two components and a plateau because the loss of fluorescence was typically incomplete. To interpret the kinetics, fusion events were simulated as a localized release of tethered/untethered exosomes coupled with the membrane diffusion of CD63. The experimentally observed decay required three components in the simulation: 1) free exosomes, 2) CD63 membrane diffusion from the endosomal membrane into the plasma membrane, and 3) tethered exosomes. Modeling with slow diffusion of the tethered exosomes (0.0015-0.004 µm2/s) accurately fits the experimental data for all temperatures. However, simulating with immobile tethers or the absence of tethers fails to replicate the data. Our model suggests that exosome release from the fusion site is incomplete due to postfusion, membrane attachment.

Phosphatidic Acid Accumulates at Areas of Curvature in Tubulated Lipid Bilayers and Liposomes.

Phosphatidic acid (PA) is a signaling lipid that is produced enzymatically from phosphatidylcholine (PC), lysophosphatidic acid, or diacylglycerol. Compared to PC, PA lacks a choline moiety on the headgroup, making the headgroup smaller than that of PC and PA, and PA has a net negative charge. Unlike the cylindrical geometry of PC, PA, with its small headgroup relative to the two fatty acid tails, is proposed to support negatively curved membranes. Thus, PA is thought to play a role in a variety of biological processes that involve bending membranes, such as the formation of intraluminal vesicles in multivesicular bodies and membrane fusion. Using supported tubulated lipid bilayers (STuBs), the extent to which PA localizes to curved membranes was determined. STuBs were created via liposome deposition with varying concentrations of NaCl (500 mM to 1 M) on glass to form supported bilayers with connected tubules. The location of fluorescently labeled lipids relative to tubules was determined by imaging with total internal reflection or confocal fluorescence microscopy. The accumulation of various forms of PA (with acyl chains of 16:0-6:0, 16:0-12:0, 18:1-12:0) were compared to PC and the headgroup labeled phosphatidylethanolamine (PE), a lipid that has been shown to accumulate at regions of curvature. PA and PE accumulated more at tubules and led to the formation of more tubules than PC. Using large unilamellar liposomes in a dye-quenching assay, the location of the headgroup labeled PE was determined to be mostly on the outer, positively curved leaflet, whereas the tail labeled PA was located more on the inner, negatively curved leaflet. This study demonstrates that PA localizes to regions of negative curvature in liposomes and supports the formation of curved, tubulated membranes. This is one way that PA could be involved with curvature formation during a variety of cell processes.

School choice increases racial segregation even when parents do not care about race.

This research examines how school choice impacts school segregation. Specifically, this work demonstrates that even if parents do not take the racial demographics of schools into account, preference differences between Black and White parents for other school attributes can still result in segregation. These preference differences stem from motivational differences in pursuit of social status. Given that the de facto US racial hierarchy assigns Black people to a lower social status, Black parents are more motivated to seek schools that signal that they can improve their children's status. Simulations of parental school decisions at scale show that preference differences under an unmitigated school-choice policy lead to more segregated schools, impacting more than half a million US children for every 3-percentage-point increase in school-choice availability. In contrast, if Black and White parents have similar preferences, unmitigated school choice would reduce racial segregation. This research may inform public policy concerning school choice and school segregation.

Drones can reliably, accurately and with high levels of precision, collect large volume water samples and physio-chemical data from lakes.

The rapid development and application of drone technology has included water sampling and collection of physiochemical data from lakes. Previous research has demonstrated the significant potential of drones to play a future pivotal role in the collection of such data from lakes that fulfil requirements of large-scale monitoring programmes. However, currently the utilisation of drone technology for water quality monitoring is hindered by a number of important limitations: i) the low rate of successful sample captured; ii) the relatively low volume of water sample retrieved for analyses of multiple water chemistry parameters; and critically iii) differences between water chemistry parameters when using a drone versus samples collected by boat. Here we present results comparing the water chemistry results of a large number of parameters (pH, dissolved oxygen concentration, temperature, conductivity, alkalinity, hardness, true colour, chloride, silica, ammonia, total oxidised nitrogen, nitrite, nitrate, ortho-phosphate, total phosphorous and chlorophyll) sampled via drone with samples collected by boat in a number of lakes. The drone water sampling method used here is the first to collect a sufficiently large volume of water to meet the monitoring requirements of large scale water monitoring programmes, 2 L, at a 100% success rate and most crucially, with water chemistry variables that are not significantly different to those taken using traditional boat water sampling. This study therefore shows that drone technology can be utilised to collect water chemistry data and samples from lakes in a reliable, more rapid and cost effective manner than traditional sampling using boats, that is safer for personnel and poses less of a biosecurity risk.

Body camera footage leads to lower judgments of intent than dash camera footage.

Police departments use body-worn cameras (body cams) and dashboard cameras (dash cams) to monitor the activity of police officers in the field. Video from these cameras informs review of police conduct in disputed circumstances, often with the goal of determining an officer's intent. Eight experiments (N = 2,119) reveal that body cam video of an incident results in lower observer judgments of intentionality than dash cam video of the same incident, an effect documented with both scripted videos and real police videos. This effect was due, in part, to variation in the visual salience of the focal actor: the body cam wearer is typically less visually salient when depicted in body versus dash cam video, which corresponds with lower observer intentionality judgments. In showing how visual salience of the focal actor may introduce unique effects on observer judgment, this research establishes an empirical platform that may inform public policy regarding surveillance of police conduct.

Canis Caveat (Beware of the Dog) - Septic Shock Due To Capnocytophaga Canimorsus Contracted From A Dog Bite

We describe the case of a 61-year-old immunocompetent male who developed septic shock and multiorgan failure due to Capnocytophaga canimorsus (C. canimorsus) bloodstream infection, sustained from a dog bite. Unusually, this patient developed acute liver failure and splenic infarction in addition to many of the better-known clinical sequelae of C. canimorsus infection.

Clinical and Molecular Phenotypes of Low-Penetrance Variants of NLRP3: Diagnostic and Therapeutic Challenges.

OBJECTIVE: Cryopyrin-associated periodic syndromes (CAPS) result from gain-of-function mutations in the NLRP3 gene, which causes excessive release of interleukin-1ß (IL-1ß) and systemic inflammation. While pathogenetic NLRP3 variant phenotypes are well-characterized, low-penetrance NLRP3 variants represent a significant clinical challenge. The aims of this study were to determine the clinical phenotype, the in vitro biologic phenotype, and the effect of anti-IL-1 treatment in patients with low-penetrance NLRP3 variants. METHODS: A multicenter study of consecutive symptomatic patients with low-penetrance NLRP3 variants recruited from 7 centers between May 2012 and May 2013 was performed. The observed findings were transferred into a study database, from which they were extracted for analysis. Controls were patients with a known pathogenetic NLRP3 variant. Clinical presentation and CAPS markers of inflammation were captured. Functional assays of inflammasome activation, including caspase 1 activity, NF-κB release, cell death, and IL-1ß release, were performed. Treatment effects of IL-1 were determined. Comparisons between low-penetrance and pathogenetic NLRP3 variants were performed. RESULTS: The study included 45 patients, 21 of which were female (47%); 26 of the patients (58%) were children. NLRP3 low-penetrance variants identified in the patients were Q703K (n = 19), R488K (n = 6), and V198M (n = 20). In the controls, 28 had pathogenetic NLRP3 variants. Patients with low-penetrance NLRP3 variants had significantly more fever (76%) and gastrointestinal symptoms (73%); eye disease, hearing loss, and renal involvement were less common. Functional inflammasome testing identified an intermediate phenotype in low-penetrance NLRP3 variants as compared to wild-type and pathogenetic NLRP3 variants. All treated patients responded to IL-1 inhibition, with complete response documented in 50% of patients. CONCLUSION: Patients with low-penetrance NLRP3 variants display a distinct clinical phenotype and an intermediate biologic phenotype, including IL-1ß and non-IL-1ß-mediated inflammatory pathway activation.

A 25-year longitudinal study of the natural course of aging after spinal cord injury.

STUDY DESIGN: Longitudinal; Survey. OBJECTIVE: The purpose of this study was to investigate the natural course of changes in activity patterns, health indicators, life satisfaction, and adjustment over 25-year period among people with spinal cord injury (SCI) in the USA. SETTING: The preliminary data were collected from a Midwestern United States university hospital of the USA, whereas the follow-up data were collected at a large Southeastern United States rehabilitation hospital. METHOD: The Life Situation Questionnaire was used to identify changes in education/employment, activities, medical treatments, adjustment, and life satisfaction. RESULTS: Adjustment scores, satisfaction with employment, satisfaction with finances, years of education, and employment indicators significantly improved over time. In contrast, satisfaction with sex life, satisfaction with health, and then number of weekly visitors significantly decreased and the number of nonroutine medical visits and days hospitalized within 2 years prior to the study significantly increased over the 25-year period. CONCLUSION: Given the mixed pattern of favorable and unfavorable changes, the findings challenge the assumption that aging will inevitably be associated with the overall decline in outcomes and quality of life.

Time-lapse video reveals immediate heterogeneity and heritable damage among human ileocecal carcinoma HCT-8 cells treated with raltitrexed (ZD1694).

BACKGROUND: Cellular heterogeneity in drug response has important clinical implications, and is believed to develop over many generations during clonal evolution in human tumors. The purpose of this study was to determine the level of heterogeneity exhibited by sister cells soon after their birth. METHODS: Human ileocecal carcinoma cells (HCT-8) were followed up to 11 days in vitro after a 2-h exposure to 1 microM raltitrexed (IC(95)) in a time-lapse video system. RESULTS: Over five experiments, 414 cells were followed after exposure to raltitrexed. Immediate sterility occurred in 74% of treated cells. Only 6% of cells could produce more than two generations of offspring, and heterogeneity in drug response was seen. Comparing sister cells < 24 h old, the more proliferative sibling produced up to 73 times more offspring, with a median ratio of 9.0 (control median = 1.19). Offspring of prolific drug-treated cells had a decreased probability of division (68% compared with 92%) and an increased average interdivision time (19.0 h compared with 15.1 h). CONCLUSIONS: Short-term exposure to raltitrexed resulted in increased interdivision times and production of sterile offspring extending seven generations. Cellular heterogeneity (difference in proliferation potential comparing drug-treated sister cells) was evident without a period of clonal evolution.

MR imaging of the pleura and chest wall.

Evaluation of pleural and chest-wall disease begins with a chest radiograph. In many cases, further evaluation of the chest wall and pleura requires cross-sectional imaging with CT scan or MR imaging. MR imaging is reserved for specific cases of tumors, infections, pleural effusions, and masses. The superior soft-tissue contrast of MR imaging is useful in evaluating the extent of infections and tumors involving the pleura and chest wall. The multiplanar capability of MR imaging aids in the evaluation of chest wall and pleural abnormalities, particularly in the apical regions.

Bronchoperitoneal fistula secondary to chronic Klebsiella pneumoniae subphrenic abscess.

We treated a case of bronchoperitoneal fistula secondary to a Klebsiella pneumoniae subphrenic abscess. This fistulous communication and the surgical procedure used to treat it are described.

Topical contrast-enhanced CT and MR dacryocystography: imaging the lacrimal drainage apparatus of healthy volunteers.

OBJECTIVE: The purpose of this study was to evaluate the feasibility of administering topical contrast material during helical CT dacryocystography and topical saline solution during MR dacryocystography to reveal the lacrimal drainage apparatus. SUBJECTS AND METHODS: Fourteen healthy volunteers underwent helical CT dacryocystography, MR dacryocystography, or both. Eight of the 14 subjects underwent both techniques; three subjects underwent MR dacryocystography, and three subjects underwent CT dacryocystography. Images were evaluated by two radiologists for degree of visualization of components of the lacrimal drainage apparatus. Each volunteer was questioned about the relative discomfort of contrast material and saline solution administration. RESULTS: The lacrimal drainage system was seen on both CT dacryocystography and MR dacryocystography. CT dacryocystography allowed two-dimensional and three-dimensional reconstructions on which adjacent bone anatomy could be seen. The MR dacryocystography two-dimensional reconstructions and maximum intensity projections also showed the drainage apparatus. However, smaller drainage structures were more consistently seen on CT dacryocystography than on MR dacryocystography. Saline solution was more comfortable than contrast material. CONCLUSION: CT dacryocystography and MR dacryocystography reproducibly and non-invasively revealed the lacrimal drainage apparatus and allowed a better physiologic examination than cannulation dacryocystography. MR dacryocystography can be performed without administration of ionizing radiation or contrast material, but this technique cannot show adjacent bone anatomy and less consistently showed the smaller drainage structures than CT dacryocystography.

Imaging of lung cancer: old and new.

Lung cancer is the leading cause of cancer death in the United States. Lung cancer is frequently encountered by the radiologist, whether the lung cancer is detected on a chest radiograph obtained in a symptomatic patient, or is an incidental finding. The radiologic workup of pulmonary lesions suspected of being lung carcinoma has evolved as new technology has become available. Current imaging modalities which are useful in the workup of suspected lung cancers include plain radiography, computed tomography, magnetic resonance imaging, with the recent addition of positron emission tomography and endoscopic ultrasound. The following article discusses the merits of these imaging modalities and their role in the workup of patients with lung cancer.

Aggressive surgical management of sternoclavicular joint infections.

BACKGROUND: Although the sternoclavicular joint is an unusual site for infection, thoracic surgeons may preferentially be called on to coordinate management of cases refractory to antibiotic therapy because of the anatomic relationship of this joint to major vascular structures. METHODS: Since 1994 we have surgically managed nine sternoclavicular joint infections in eight patients. Associated medical problems were frequent and included diabetes mellitus (n = 2), end-stage renal disease (n = 2), hematologic disorders (n = 2), and multiple joints affected by sepsis (n = 4). Open joint exploration with drainage and débridement with the use of general anesthesia was performed in four patients. The remaining four patients (one with bilateral sternoclavicular joint infections) had computed tomographic evidence of diffuse joint and surrounding bone destruction with infection extending into mediastinal soft tissues. Surgical therapy for these five joint infections involved en bloc resection of the sternoclavicular joint with an ipsilateral pectoralis major muscle covering the bony defect. RESULTS: There were two deaths unrelated to the surgical procedure. After a mean follow-up of 20 months, the remaining six survivors (seven joints) have complete healing with no apparent limitation in the range of motion even after en bloc resection. CONCLUSIONS: Most cases of early sternoclavicular joint infections will respond to conservative measures. However, when radiographic evidence of infection beyond the sternoclavicular joint is present, en bloc resection, although seemingly aggressive, results in immediate eradication of all infection with negligible functional morbidity. Prolonged antibiotic therapy or continued local drainage procedures appear to have little value in these cases, adding only to patient care costs and the potential sequelae of chronic infections.

Pulmonary aspergillosis: a spectrum of disease.

Although many species of the fungus Aspergillus have been identified, the most common human pathogen is A. fumigatus, which has a worldwide distribution. Although any organ may become infected, pulmonary aspergillosis is the most common manifestation. The spectrum of pulmonary aspergillosis includes saprophytic aspergillomas, allergic bronchopulmonary aspergillosis, chronic necrotizing aspergillosis, and invasive aspergillosis. Immune status of the host, and the presence of underlying lung disease are important in determining the type of pulmonary involvement. Thus, the radiographic findings are variable. This article reviews the various manifestations of pulmonary aspergillosis, including the immune status of the patient, the presence of underlying lung disease, and the radiographic appearance of the different entities.