Automated detection of fluorescent cells in in-resin fluorescence sections for integrated light and electron microscopy.

Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high-resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via 'smart tracking'. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence.

Bilateral buphthalmia in a 4-month-old Texas longhorn steer.

Congenital ocular disease occurs uncommonly in cattle, with multiple abnormalities reported only sporadically in the literature. This report describes a case of anterior segment dysgenesis resulting in glaucoma in a 4-month-old Texas Longhorn steer. On clinical exam, bilateral buphthalmia was present and intraocular pressures exceeded 47 mm Hg in both eyes. On histopathologic examination, the iridocorneal angle and filtration apparatus were distorted due to collapse of the ciliary cleft and anterior displacement of the anterior portion of the ciliary body. No evidence of inflammation or other causes of glaucoma were recognized.

Effect of castration method on neurohormonal and electroencephalographic stress indicators in Holstein calves of different ages.

As public concern for food animal welfare increases, a need to validate objective pain assessment tools exists in order to formulate animal welfare policies and facilitate regulatory approval of compounds to alleviate pain in livestock in the United States. The aims of this study were (1) to compare the physiological response to pain induced by surgical and nonsurgical (band) castration in calves and (2) to elucidate age-related differences in pain response of calves subjected to different castration methods. Seventy-six Holstein bull calves were blocked by age (≤8-wk and ≥6-mo-old) and randomly assigned to 1 of 4 treatment groups: control (n=20), castration by banding (n=18), cut-and-clamp surgical castration (n=20), and cut-and-pull surgical castration (n=18). Measurements included electroencephalogram, heart rate variability, infrared thermography, electrodermal activity, and concentrations of serum cortisol and plasma substance P before, during, and within 20min following castration. Electroencephalogram recordings showed desynchronization for all treatments, consistent with increased arousal; yet the magnitude of desynchronization was greatest for 6-mo-old calves castrated by cut-and-clamp. Additionally, older calves in the cut-and-pull group showed greater desynchronization than younger calves in the same group. Based on the heart rate variability analysis, 6-mo-old calves in the control or cut-and-pull castration groups showed greater sympathetic tone than younger calves in the same treatment groups. Overall, younger calves showed lower electrodermal activity than older calves. Regardless of treatment, concentrations of cortisol and plasma substance P were greater in 6-mo-old calves relative to their younger counterparts, indicating a more robust response to all treatments in older calves. In summary, neurohormonal and electroencephalographic stress responses of calves to castration were age-specific. Castration by cut-and-clamp showed the most pronounced stress response in 6-mo-old calves. These findings provide evidence that support welfare policies recommending castration at an early age and the use of analgesic compounds at the time of surgical castration especially in older calves. However, the potential long-term negative consequences of early untreated pain must be considered and warrant further investigation.

Effect of nitrogen supplementation and zilpaterol-HCl on urea kinetics in steers consuming corn-based diets.

We studied effects of zilpaterol-HCl on steers consuming corn-based diets with nitrogen (N) supplementation provided by dried distillers grains with solubles (DDGS) or urea. Two sets of six steers (approximately 350 kg) were used in two replicates of similarly designed trials. Within each replicate, three steers were fed 60 mg/day of zilpaterol-HCl throughout the trial and three steers received no zilpaterol-HCl. Within zilpaterol treatment, three corn-based dietary N treatments were offered in Latin square designs: control (9.6% crude protein), urea (UREA; 12.4% crude protein) or DDGS (13.7% crude protein). Total feed intake was unexpectedly greater (p < 0.01) with zilpaterol feeding but was not affected by dietary N (p = 0.76). Nitrogen intake was greater (p < 0.01) when zilpaterol was fed and was greater (p < 0.05) for DDGS and UREA than for control. Despite greater N intake, zilpaterol did not affect urea entry rate (p = 0.80) or urea-N recycled to the gastrointestinal tract (GER; p = 0.94). As a percentage of N intake, urea entry rate (p = 0.19) tended to be less when zilpaterol was fed (91 vs. 123% of N intake), and GER was numerically (p = 0.34) less (72 vs. 92% of N intake). Microbial N flow was greater (p = 0.02) for zilpaterol than for control but did not differ (p = 0.78) among dietary N treatments. As a percentage of N intake, microbial N flow was unaffected by zilpaterol (p = 0.97), but was greater (p < 0.05) for control than DDGS or UREA. The lack of change in urea entry and GER in response to zilpaterol, despite greater N intake, as well as lower urea entry and GER when expressed as proportions of N intake provide some evidence that the amount of N available for urea production and recycling was reduced by zilpaterol.

Skimboarding: a new cause of water sport spinal cord injury.

STUDY DESIGN: Case series. OBJECTIVE: To present three cases of spinal cord injuries associated with skimboarding and to suggest aspects of the sport that may be associated with spinal cord injury. SETTING: Shepherd Center, Atlanta, GA, USA. Spinal cord injury rehabilitation facility. METHODS: Three males, aged 17-23 years, sustained cervical spine fractures resulting in tetraplegia after skimboarding accidents. RESULTS: The patients admitted from Florida hospitals presented with tetraplegia resulting from both incomplete and complete spinal cord injuries. The ASIA Impairment Scale (AIS) neurologic levels ranged from C3 to C5. All were injured by being thrown from the skimboard riding into the waves in shallow water. All patients required surgical stabilization and inpatient rehabilitation. CONCLUSIONS: Previous studies on skimboarding have demonstrated the risk of extremity fractures and soft tissue injuries. Spinal cord injury should be considered an additional risk associated with skimboarding, particularly as the sport has grown in popularity and become more 'extreme' in the maneuvers performed. Increased education and awareness about the potential risk of spinal cord injury are essential.

Standing balance of vehicle passengers: The effect of vehicle motion, task performance on post-drive balance.

BACKGROUND: Motion platforms and driving simulators have been shown to contribute to motion sickness and a short-term increase in standing postural sway. However, no studies to date have investigated how the motion of a passenger vehicle and the performance of a task during a drive on a closed test track affects post-drive standing balance. RESEARCH QUESTIONS: What are the effects of (1) a continuous, scripted drive on a closed test track, and (2) the performance of a handheld tablet-based task during the scripted drive, on post-drive standing balance? METHODS: Fifty adults (23 males, 27 females; 40.0 ± 20.6 yr) rode in the front passenger seat of a midsized sedan on a scripted drive. Participants were assigned to one of the acceleration levels (Low, Moderate) and completed both Task and No-Task test conditions, involving a visual-based task on a handheld tablet device. Before and after each scripted drive, participants completed two standing balance exercises: 1) feet tandem, eyes open, on firm support, and 2) feet together, eyes closed, on foam support. An inertial measurement unit (IMU) captured estimates of postural trunk sway. Root-mean-square (RMS) of angular position and velocity in the anteroposterior (A/P) and mediolateral (M/L) directions, and elliptical fit and path length of sway trajectory were computed. A nonparametric analysis was performed on the balance metrics. RESULTS: Exposure to a scripted drive in a vehicle affected participants' postural sway, especially after using a handheld device during the drive. M/L RMS sway velocity and path length increased for both exercises following the scripted drive with task. Additionally, M/L RMS sway increased for the more challenging balance exercise, during which participants stood with feet together on foam support with eyes closed. SIGNIFICANCE: This study is the first to explore balance following a scripted drive on a closed test track. Changes in post-drive balance introduces potential risks to vehicle passengers; concurrent performance of a task on a handheld device further increases the likelihood that post-drive balance will be negatively affected.

Riftia pachyptila Jones: Observations on the Vestimentiferan Worm from the Galapagos Rift.

The obturacular plume, composed of numerous tentacles, is suggested as a site for the uptake of molecular "food" by Riftia pachyptila (Pogonophora) from the Galápagos Rift; symbiotic bacteria are another possible source of nutrition. Differing organizations of the linings of the five major body cavities of Riftia demonstrate the inadequacy of "coelom" as a descriptive term.

Hemoglobin Kinetics of the Galapagos Rift Vent Tube Worm Riftia pachyptila Jones (Pogonophora; Vestimentifera).

Kinetics of the reactions of Riftia pachyptila hemoglobin with oxygen were followed spectrophotometrically by stopped-flow and laser flash photolysis techniques. The rate of oxygen dissociation increases eightfold over the range of 5 degrees to 20 degrees C (k = 2.2 sec(-1)at 10 degrees C). Oxygen recombination after flash photolysis was biphasic. The rates of both slow and fast phases of the reaction were independent of temperature from 0 degrees to 20 degrees C(k'fast = 7 x 10(6); k'slow = 1 x 16(6) liter mole (-1) sec(-1)). As the oxygen affinity is relatively temperature independent, analysis in terms of the two-state model of cooperativity requires that the conformational equilibrium constant L decrease by about 50-fold between 3 degrees and 15 degrees C.

Prokaryotic Cells in the Hydrothermal Vent Tube Worm Riftia pachyptila Jones: Possible Chemoautotrophic Symbionts.

The existence of a symbiotic association between vestimentiferan tube worms from deep-sea hydrothermal vents and chemoautotrophic sulfur-oxidizing prokaryotes, based on histological and enzymatic evidence, is suggested.

Involvement of platelet-endothelial cell adhesion molecule-1 in neutrophil recruitment in vivo.

During inflammation, neutrophils migrate from the vascular lumen into extravascular sites. In vitro assays have suggested that platelet-endothelial cell adhesion molecule-1 [PECAM-1 (CD31)], a member of the immunoglobulin superfamily, is required for the transmigration of neutrophils across endothelial monolayers. Antibody to human PECAM-1, which cross-reacts with rat PECAM-1, was found to block not only in vivo accumulation of rat neutrophils into the peritoneal cavity and the alveolar compartment of the lung but also neutrophil accumulation in human skin grafts transplanted onto immunodeficient mice. On the basis of these findings in three different models of inflammation, it appears that PECAM-1 is required for neutrophil transmigration in vivo and may thus be a potential therapeutic target.

Regulatory effects of endogenous interleukin-1 receptor antagonist protein in immunoglobulin G immune complex-induced lung injury.

IL-1 receptor antagonist (IL-1Ra) has regulatory effects on IL-1 activity both in vitro and in vivo. In the IgG immune complex model of lung injury in rats, exogenously administered human IL-1Ra suppressed neutrophil recruitment and ensuing lung injury. In this study, we sought to determine if endogenous rat IL-1Ra might regulate this lung-inflammatory response. By Northern blot analysis of lung mRNA and Western analysis of bronchoalveolar lavage (BAL) fluids, rat IL-1Ra expression was found to increase during development of inflammation in IgG immune complex-mediated alveolitis. By immunostaining, alveolar macrophages and recruited neutrophils were the apparent sources of IL-1Ra. In vivo blocking of endogenous IL-1Ra resulted in a 53% increase in lung vascular permeability and a 180% increase in BAL fluid neutrophils. In companion studies, a significant increase in IL-1beta was found, whereas no significant change in TNF-alpha activity was observed. Whereas the in vivo regulatory effects of IL-1R appear to be limited to IL-1beta, IL-10 regulates both IL-1beta and TNF-alpha in this model, reflected by a 48% increase in BAL IL-1beta in rats treated with anti-IL-10. These findings suggest that IL-1Ra is an intrinsic regulator of inflammatory injury after deposition of IgG immune complexes and that it regulates production of IL-1beta.

C5a-induced expression of P-selectin in endothelial cells.

Human umbilical vein endothelial cells have recently been shown to respond to C5a with increases in intracellular Ca2+, production of D-myo-inositol 1,4,5-triphosphate and superoxide anion generation. In the current studies, C5a had been found to cause in a time- and dose-dependent manner rapid expression of endothelial P-selectin, secretion of von Willebrand factor, and adhesiveness for human neutrophils. The effects of C5a in P-selectin expression and adhesiveness of neutrophils were similar to the effects of histamine and thrombin on endothelial cells. The adhesiveness of C5a-stimulated endothelium for neutrophils was blocked by anti-P-selectin, but not by antibodies to intercellular adhesion molecule 1, E-selectin, or CD18. A cell-based ELISA technique has confirmed upregulation of P-selectin in endothelial cells exposed to C5a. Binding of C5a to endothelial cells has been demonstrated, with molecules bound being approximately 10% of those binding to neutrophils. By a reverse transcriptase-PCR technique, endothelial cells have been shown to contain mRNA for the C5a receptor. These data suggest that C5a may be an important inflammatory mediator for the early adhesive interactions between neutrophils and endothelial cells in the acute inflammatory response.

Meta-analysis of the relative sensitivity of semi-natural vegetation species to ozone.

This study identified 83 species from existing publications suitable for inclusion in a database of sensitivity of species to ozone (OZOVEG database). An index, the relative sensitivity to ozone, was calculated for each species based on changes in biomass in order to test for species traits associated with ozone sensitivity. Meta-analysis of the ozone sensitivity data showed a wide inter-specific range in response to ozone. Some relationships in comparison to plant physiological and ecological characteristics were identified. Plants of the therophyte lifeform were particularly sensitive to ozone. Species with higher mature leaf N concentration were more sensitive to ozone than those with lower leaf N concentration. Some relationships between relative sensitivity to ozone and Ellenberg habitat requirements were also identified. In contrast, no relationships between relative sensitivity to ozone and mature leaf P concentration, Grime's CSR strategy, leaf longevity, flowering season, stomatal density and maximum altitude were found. The relative sensitivity of species and relationships with plant characteristics identified in this study could be used to predict sensitivity to ozone of untested species and communities.

Identifying ozone-sensitive communities of (semi-)natural vegetation suitable for mapping exceedance of critical levels.

Using published data on the responses of individual species to ozone, 54 EUNIS (European Nature Information System) level 4 communities with six or more ozone-sensitive species (%OS) and c. 20% or more species tested for ozone sensitivity, were identified as potentially ozone-sensitive. The largest number of these communities (23) was associated with Grasslands, with Heathland, scrub and tundra, and Mires, bogs and fens having the next highest representation at 11 and 8 level 4 communities each respectively. Within the grasslands classification, E4 (Alpine and sub-alpine grasslands), E5 (Woodland fringes and clearings) and E1 (Dry grasslands) were the most sensitive with 68.1, 51.6 and 48.6%OS respectively. It is feasible to map the land-cover for these and other communities at level 2, but it may not be currently possible to map the land-cover for all communities identified to be ozone-sensitive at levels 3 and 4.

Mineral Composition of Uroliths Obtained from Sheep and Goats with Obstructive Urolithiasis.

BACKGROUND: Knowledge of the mineral composition of the causative urolith is important to develop preventative strategies. Advances in analytic techniques have led to detection of urolith components not previously recognized. HYPOTHESIS/OBJECTIVES: The objectives of this study were to characterize uroliths in sheep and goats structurally and clinically. We hypothesized that amorphous magnesium calcium phosphate (AMCP) would be a naturally occurring urolith type in sheep and goats. ANIMALS: Forty-nine sheep and goats presenting for obstructive urolithiasis from June 15, 2014 through June 14, 2016 were reviewed along with the demographic data of all small ruminants admitted during that same period. METHODS: Medical records were reviewed for demographic and historical data, and 36 uroliths obtained from these cases during diagnostic or therapeutic procedures were analyzed by infrared spectroscopy to determine chemical composition. RESULTS: AMCP is a naturally occurring urolith type in obstructed male sheep and goats and was the most common urolith type in this study, where it occurred as a majority component with struvite (39% of uroliths) or as a pure component (11%). Pure struvite was found in 1 urolith (2%). Calcium carbonate was the second most frequent urolith with 31% of uroliths being pure calcium carbonate. CONCLUSIONS AND CLINICAL IMPORTANCE: This study demonstrates that uroliths, which appear consistent with struvite, could actually be calcium-containing AMCP. Urolith analysis is critical in determining mineral content of uroliths to guide dietary recommendations for prevention.

Complex hydrological controls on wet dune slacks: the importance of local variability.

Dune slacks are a species-rich habitat controlled largely by water chemistry and fluctuations in groundwater. Changes in water chemistry and water table level were analysed in 8 piezometers and 15 ephemeral surface water locations at a large UK dune system over a 12-month period. Total nitrogen concentrations in groundwater varied from 0.27-8.21 mg N L(-1), where dissolved organic nitrogen was dominant at the low nitrogen locations and nitrate was dominant at the high nitrogen locations. Principal components analysis of the water chemistry suggests at least four chemically distinct groundwater signatures. Water levels showed strong temporal heterogeneity. Comparisons of water levels with antecedent rainfall identified a component of year-round groundwater feed and differing seasonal responses overlain by a complex series of lags. In summer, there were lags of four, six and seven months with an additional rapid peaky response to daily rainfall with a one-day lag. In winter, water levels were strongly influenced by exogenous groundwater supply, but again exhibited multiple lags. This study shows that local variations in water chemistry and in hydrological regime can be more complicated than previously thought, with clear implications for optimum management of these high priority habitats for conservation.

Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation.

Current immunosuppressive/anti-inflammatory agents target the responding effector arm of the immune response and their nonspecific action increases the risk of infection and malignancy. These effects impact on their use in allogeneic haematopoietic cell transplantation and other forms of transplantation. Interventions that target activated dendritic cells (DCs) have the potential to suppress the induction of undesired immune responses (for example, graft versus host disease (GVHD) or transplant rejection) and to leave protective T-cell immune responses intact (for example, cytomegalovirus (CMV) immunity). We developed a human IgG1 monoclonal antibody (mAb), 3C12, specific for CD83, which is expressed on activated but not resting DC. The 3C12 mAb and an affinity improved version, 3C12C, depleted CD83(+) cells by CD16(+) NK cell-mediated antibody-dependent cellular cytotoxicity, and inhibited allogeneic T-cell proliferation in vitro. A single dose of 3C12C prevented human peripheral blood mononuclear cell-induced acute GVHD in SCID mouse recipients. The mAb 3C12C depleted CMRF-44(+)CD83(bright) activated DC but spared CD83(dim/-) DC in vivo. It reduced human T-cell activation in vivo and maintained the proportion of CD4(+) FoxP3(+) CD25(+) Treg cells and also viral-specific CD8(+) T cells. The anti-CD83 mAb, 3C12C, merits further evaluation as a new immunosuppressive agent in transplantation.

Pollination-Induced Ethylene in Carnation (Role of Stylar Ethylene in Corolla Senescence).

In carnation (Dianthus caryophyllus L. cv White Sim) cell to cell communication between the pollen and pistil induces ovary development and corolla senescence. The production of elevated ethylene by the style is the first measurable postpollination response. This is followed by a wave of ethylene production from the other floral organs. To investigate the regulation of ethylene biosynthesis in pollinated flowers we measured ethylene production and the expression of 1-aminocyclopropane-1-carboxylate synthase and 1-aminocyclopropane-1-carboxylate oxidase transcripts in individual floral organs after pollination. Ethylene production by pollinated styles can be defined temporally by three distinct peaks. By pollinating a single style from a multistyle gynoecium, it was determined that the unpollinated style produces ethylene that corresponds to the first and third peaks observed from a pollinated style. Inhibition of ethylene action in the pollinated style by diazocyclopentadiene treatment prevented both pollination-induced corolla senescence and ethylene production from the ovaries and petals. Treatment with diazocyclopentadiene decreased stylar ethylene production during the second peak and completely inhibited the third peak of ethylene in both pollinated and unpollinated styles. This later auto-catalytic ethylene in styles is likely responsible for pollination-induced corolla senescence and ovary development.

Pollination-Induced Ethylene in Carnation (Role of Pollen Tube Growth and Sexual Compatibility).

The pollen-pistil interactions that result in the stimulation of ethylene production and petal senescence in carnation (Dianthus caryophyllus L.) flowers were investigated. Pollination of White Sim flowers with Starlight pollen elicited an increase in ethylene production by styles, leading to increased petal ethylene and premature petal senescence. In contrast, pollination with 87-29G pollen led to an early increase in ethylene production, but this was not sustained, and did not lead to petal senescence. Both Starlight and 87-29G pollen germinated on White Sim stigmas and their tubes grew at similar rates, penetrating the length of the style. Crosses between Starlight and White Sim led to the production of viable seeds, whereas 87-29G pollen was infertile on White Sim flowers. Pollination of other carnations with 87-29G elicited ethylene production and petal senescence and led to the production of viable seeds. These results suggest that physical growth of pollen tubes is insufficient to elicit a sustained increase in ethylene production or to lead to the production of signals necessary for elicitation of petal ethylene production and senescence. Rather, the cell-cell recognition reactions leading to sexual compatibility in Dianthus appear to play a role in this interorgan signaling after pollination.

Leukocyte subsets and neutrophil function after short-term spaceflight.

Changes in leukocyte subpopulations and function after spaceflight have been observed but the mechanisms underlying these changes are not well defined. This study investigated the effects of short-term spaceflight (8-15 days) on circulating leukocyte subsets, stress hormones, immunoglobulin levels, and neutrophil function. At landing, a 1.5-fold increase in neutrophils was observed compared with preflight values; lymphocytes were slightly decreased, whereas the results were variable for monocytes. No significant changes were observed in plasma levels of immunoglobulins, cortisol, or adrenocorticotropic hormone. In contrast, urinary epinephrine, norepinephrine, and cortisol were significantly elevated at landing. Band neutrophils were observed in 9 of 16 astronauts. Neutrophil chemotactic assays showed a 10-fold decrease in the optimal dose response after landing. Neutrophil adhesion to endothelial cells was increased both before and after spaceflight. At landing, the expression of MAC-1 was significantly decreased while L-selectin was significantly increased. These functional alterations may be of clinical significance on long-duration space missions.