A total of 460 kidney samples from wildlife (beavers, coyotes, deer, foxes, opossums, otters, raccoons, skunks) were obtained from road-kill and hunter/trapper donations in Ontario between January 2010 and November 2012. The objectives of the study were to detect Leptospira spp. by immunohistochemistry and polymerase chain reaction (PCR), to map presence of leptospires in wildlife relative to livestock and human populations, and to characterize positive samples by sequencing and comparison to leptospires known to affect domestic animals and humans. The proportion of samples that tested positive ranged from 0% to 42%, with the highest rates in skunks and raccoons. Leptospira spp. were present in kidneys of wildlife across Ontario, particularly in areas of high human density, and areas in which livestock populations are abundant. The PCR was too weak in most samples to permit genotyping and examination of the relationship between the leptospires found in this study and those affecting domestic animals and humans.
Local caspase activation at axonal branch points restricts arbor growth and synaptogenesis by interacting with Slit1a-Robo2 signaling in the central nervous system.
In addition to being critical for apoptosis, components of the apoptotic pathway, such as caspases, are involved in other physiological processes in many types of cells, including neurons. However, very little is known about their role in dynamic, nonphysically destructive processes, such as axonal arborization and synaptogenesis. We show that caspases were locally active in vivo at the branch points of young, dynamic retinal ganglion cell axonal arbors but not in the cell body or in stable mature arbors. Caspase activation, dependent on Caspase-3, Caspase-9, and p38 mitogen-activated protein kinase (MAPK), rapidly increased at branch points corresponding with branch tip addition. Time-lapse imaging revealed that knockdown of Caspase-3 and Caspase-9 led to more stable arbors and presynaptic sites. Genetic analysis showed that Caspase-3, Caspase-9, and p38 MAPK interacted with Slit1a-Robo2 signaling, suggesting that localized activation of caspases lie downstream of a ligand receptor system, acting as key promoters of axonal branch tip and synaptic dynamics to restrict arbor growth in vivo in the central nervous system.
Distal ulna fractures, especially styloid injuries, classically have not been repaired, and only recently have these injuries been considered important. Certain fracture patterns of the distal ulna contribute to distal radioulnar joint (DRUJ) incongruity and potential instability. Appropriate fixation of the distal ulna is frequently difficult for several reasons: (1) high incidence of osteoporois in the affected patient population, (2) proximity of the injury to articular surfaces, and (3) lack of a proper implant to treat these injuries. The 2.0-mm locking compression distal ulna plate (LC-DUP) is an anatomically contoured implant with a low profile and fixed angle that provides proper stability to treat injuries of the distal ulna. The plate was designed for the treatment of distal ulna fractures, but its success has led to an extension of its indications to be used in treating symptomatic basistyloid ulnar nonunions and in ulnar shortening osteotomy for ulnocarpal abutment syndrome. The authors' description of the techniques used for each indication as well as their perspectives in the treatment of distal ulna injuries are described in detail in this report.
distal ulna fracture; DUP; LC-DUP; distal ulna hook plate; ulnar styloid; ulnocarpal abutment syndrome; ulnar impaction syndrome; ulna nonunion; ulna fracture; ulnar shortening osteotomy
Four-corner fusion (4CF) is an accepted and regularly performed procedure when managing posttraumatic degenerative disorders in the wrist. This procedure consists of excision of the entire scaphoid in association with midcarpal fusion of the remaining four ulnar carpal bones (hamate, capitate, lunate, and triquetrum). In the majority of cases, the long-term outcome is a functional painless wrist. However, the exact procedure to best achieve a rapid solid bone union of the fusion mass without hardware complications remains controversial. The authors have developed a precise system to ensure precise positioning, firm fixation, and fusion at the midcarpal joint together with an early postoperative recovery, avoiding some of the issues reported with other implants used for 4CF. The described implant is a circular plate accommodating variable angle locking screws as well as compression screws that can firmly fix the plate to the carpal bones. The locking technology produces a very solid construct. A special reaming-distraction-compression guide has also been developed to both countersink the plate on the underlying carpal bone mass and allow distraction of the midcarpal joint for debridement and cancellous bone graft interposition. The features of the implant, its surgical technique, and a relevant case are described.
four-corner fusion; SLAC wrist; SNAC wrist; wrist
Axonal regeneration can occur within hours of injury, the first step being the formation of a new growth cone. For sensory and retinal axons, regenerative ability in vivo correlates with the potential to form a new growth cone after axotomy in vitro. We show that this ability to regenerate a new growth cone depends on local protein synthesis and degradation within the axon. Axotomy in vitro leads to a fourfold to sixfold increase in 3H-leucine incorporation in both neurones and axons, starting within 10 min and peaking 1 h after axotomy. Application of protein synthesis inhibitors (cycloheximide and anisomycin) to cut axons, including axons whose cell bodies were removed, or proteasome inhibitors (lactacystin and N-acetyl-Nor-Leu-Leu-Al) all result in a reduction in the proportion of transected axons able to reform growth cones. Similar inhibition of growth cone formation was observed on addition of target of rapamycin (TOR), p38 MAPK (mitogen-activated protein kinase), and caspase-3 inhibitors. Comparing retinal and sensory axons of different developmental stages, levels of ribosomal protein P0 and phosphorylated translation initiation factor are high in sensory axons, lower in embryonic axons, and absent in adult retinal axons. Conditioning lesions, which increase the regenerative ability of sensory axons, lead to increases in intra-axonal protein synthetic and degradative machinery both in vitro and in vivo. Collectively, these findings suggest that local protein synthesis and degradation, controlled by various TOR-, p38 MAPK-, and caspase-dependent pathways, underlie growth cone initiation after axotomy.
growth cone formation; axotomy; local protein synthesis; proteasome-mediated degradation; conditioning lesion; axon regeneration
A patient with a medial and posterior dislocation of the right sterno-clavicular (SC) joint and displacement of the trachea and brachiocephalic artery by the medial head of the clavicle underwent general anaesthetic in the operating theatre for an open reduction procedure. The surgeon initially attempted a closed reduction, but this required imaging to check SC alignment. The patient was transferred to an adjacent hybrid operating theatre for imaging. Cone beam computed tomography (CBCT) was performed, which successfully demonstrated a significant reduction in the dislocation of the SC joint. The trachea and brachiocephalic artery were no longer compressed or displaced. This case study demonstrates an alternative to the patient being transferred to the medical imaging department for multi-slice CT. It also describes a novel use of the hybrid operating theatre and its CBCT capabilities.
Cone beam CT; hybrid theatre; peri-operative; sterno-clavicular dislocation
Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ∼42 IsiA : Photosystem I in Synechococcus PCC 7942 and ∼12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b6f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes.
We studied the interactive effects of pCO2 and growth light on the coastal marine diatom Thalassiosira pseudonana CCMP 1335 growing under ambient and expected end-of-the-century pCO2 (750 ppmv), and a range of growth light from 30 to 380 µmol photons·m−2·s−1. Elevated pCO2 significantly stimulated the growth of T. pseudonana under sub-saturating growth light, but not under saturating to super-saturating growth light. Under ambient pCO2 susceptibility to photoinactivation of photosystem II (σi) increased with increasing growth rate, but cells growing under elevated pCO2 showed no dependence between growth rate and σi, so under high growth light cells under elevated pCO2 were less susceptible to photoinactivation of photosystem II, and thus incurred a lower running cost to maintain photosystem II function. Growth light altered the contents of RbcL (RUBISCO) and PsaC (PSI) protein subunits, and the ratios among the subunits, but there were only limited effects on these and other protein pools between cells grown under ambient and elevated pCO2.
DNA content and cell volume have both been hypothesized as controls on metabolic rate and other physiological traits. We use cultures of two cryptic species of Ditylum brightwellii (West) Grunow with an approximately two-fold difference in genome size and a small and large culture of each clone obtained by isolating small and large cells to compare the physiological consequences of size changes due to differences in DNA content and reduction in cell size following many generations of asexual reproduction. We quantified the growth rate, the functional absorption cross-section of photosystem II (PSII), susceptibility of PSII to photoinactivation, PSII repair capacity, and PSII reaction center proteins D1 (PsbA) and D2 (PsbD) for each culture at a range of irradiances. The species with the smaller genome has a higher growth rate and, when acclimated to growth-limiting irradiance, has higher PSII repair rate capacity, PSII functional optical absorption cross-section, and PsbA per unit protein, relative to the species with the larger genome. By contrast, cell division rates vary little within clonal cultures of the same species despite significant differences in average cell volume. Given the similarity in cell division rates within species, larger cells within species have a higher demand for biosynthetic reductant. As a consequence, larger cells within species have higher numbers of PSII per unit protein (PsbA), since PSII photochemically generates the reductant to support biosynthesis. These results suggest that DNA content, as opposed to cell volume, has a key role in setting the differences in maximum growth rate across diatom species of different size while PSII content and related photophysiological traits are influenced by both growth rate and cell size.
Severe neonatal hyperbilirubinemia continues to occur in healthy newborns. Recent guidelines have supported using transcutaneous devices in estimating bilirubin levels. Previous studies using these devices are limited.
Newborns requiring serum bilirubin level measurements before hospital discharge were recruited prospectively. The agreement between a transcutaneous bilirubin (TCB) and total serum bilirubin (TSB) level was assessed. Sensitivity analysis was conducted.
A total of 430 infants were enrolled. Correlation between the values was high (Pearson’s correlation coefficient 0.83; Lin’s concordance coefficient 0.81 [95% CI 0.77 to 0.84]; P<0.001). The mean (± SD) TSB level was 194±60 μmol/L. The TCB measurement tended to overestimate the value (mean difference 12.7), with wide 95% limits of agreement (−52 μmol/L to 77 μmol/L). Sensitivity and specificity analysis of TCB values allowed estimation of clinically important TSB levels.
The TCB correlated, but was imprecise in predicting TSB. TCB values can be used at the time of discharge to safely plan care for jaundiced infants if the limits of agreement are considered and clinical judgment is maintained.
Bilirubin; Hyperbilirubinemia; Jaundice; Newborn; Transcutaneous bilirubin measurement
Reef corals are heterotrophic coelenterates that achieve high productivity through their photosynthetic dinoflagellate symbionts. Excessive seawater temperature destabilises this symbiosis and causes corals to “bleach,” lowering their photosynthetic capacity. Bleaching poses a serious threat to the persistence of coral reefs on a global scale. Despite expanding research on the causes of bleaching, the mechanisms remain a subject of debate.
This study determined how light and food availability modulate the effects of temperature stress on photosynthesis in two reef coral species. We quantified the activities of Photosystem II, Photosystem I and whole chain electron transport under combinations of normal and stressful growth temperatures, moderate and high light levels and the presence or absence of feeding of the coral hosts. Our results show that PS1 function is comparatively robust against temperature stress in both species, whereas PS2 and whole chain electron transport are susceptible to temperature stress. In the symbiotic dinoflagellates of Stylophora pistillata the contents of chlorophyll and major photosynthetic complexes were primarily affected by food availability. In Turbinaria reniformis growth temperature was the dominant influence on the contents of the photosynthetic complexes. In both species feeding the host significantly protected photosynthetic function from high temperature stress.
Our findings support the photoinhibition model of coral bleaching and demonstrate that PS1 is not a major site for thermal damage during bleaching events. Feeding mitigates bleaching in two scleractinian corals, so that reef responses to temperature stresses will likely be influenced by the coinciding availabilities of prey for the host.
Regulated protein degradation via the ubiquitin-proteasome system (UPS) plays a central role in building synaptic connections, yet little is known about either which specific UPS components are involved or UPS targets in neurons. We report that inhibiting the UPS in developing Xenopus retinal ganglion cells (RGCs) with a dominant-negative ubiquitin mutant decreases terminal branching in the tectum but does not affect long-range navigation to the tectum. We identify Nedd4 as a prominently expressed E3 ligase in RGC axon growth cones and show that disrupting its function severely inhibits terminal branching. We further demonstrate that PTEN, a negative regulator of the PI3K pathway, is a key downstream target of Nedd4: not only does Nedd4 regulate PTEN levels in RGC growth cones, but also, the decrease of PTEN rescues the branching defect caused by Nedd4 inhibition. Together our data suggest that Nedd4-regulated PTEN is a key regulator of terminal arborization in vivo.
► The Ubiquitin-Proteasome System is required for terminal branching of retinal axons ► E3 ligase Nedd4 downregulates PTEN in retinal axon growth cones ► PTEN downregulation by Nedd4 promotes terminal branching of retinal axons
DEVBIO; MOLNEURO; SIGNALING
There are currently few studies describing the use of high-fidelity (hi-fi) simulation in teaching neonatal resuscitation. Traditionally, residents are certified in the neonatal resuscitation program (NRP) after successful completion of a multiple-choice written examination and demonstration of skills during a hands-on ‘mega-code’. In the present study, the use of a hi-fi simulation mannequin was compared with a standard plastic mannequin when teaching the megacode portion of the NRP.
In the present pilot study, 15 first-year residents were randomly assigned to demonstrate neonatal resuscitation knowledge, with either the hi-fi mannequin (SimBaby, Laerdal Medical Corporation, USA) or a traditional plastic mannequin (ALS Baby, Laerdal Medical Corporation, USA). A written evaluation was conducted before and after the intervention. Each pair of residents experienced the two scenarios. Video performance was then assessed and compared.
Residents randomly assigned to the hi-fi mannequin rated the experience higher (31±3.3 versus 27±3.5; P=0.026), and required less redirection from instructors during the megacode (scenario 1: 4.5±1.7 versus 15±6.9; P=0.015 and scenario 2: 1.8±1.3 versus 9.3±2.5; P=0.0009) than those who were randomly assigned to the plastic mannequin. Residents randomly assigned to the hi-fi mannequin did not have improved written scores or improved intubation times.
The present pilot study demonstrated that a hi-fi mannequin can be used as part of an educational program, such as the NRP. The use of this technology in neonatal resuscitation training is well-received by learners and may provide a more realistic model for training. Further work is required to clarify its role in task performance and team training.
High-fidelity simulation; Neonate; Resuscitation
We have investigated the earliest events in commitment of human epidermal keratinocytes to terminal differentiation. Phosphorylated Akt and caspase activation were detected in cells exiting the basal layer of the epidermis. Activation of Akt by retroviral transduction of primary cultures of human keratinocytes resulted in an increase in abortive clones founded by transit amplifying cells, while inhibition of the upstream kinase, PI3 kinase, inhibited suspension-induced terminal differentiation. Caspase inhibition also blocked differentiation, the primary mediator being caspase 8. Caspase activation was initiated by 2 hours in suspension, preceding the onset of expression of the terminal differentiation marker involucrin by several hours. Incubation of suspended cells with fibronectin or inhibition of PI3 kinase prevented caspase induction. At 2 hours in suspension, keratinocytes that had committed to terminal differentiation had increased side scatter, were 7AAD positive and annexin V negative; they exhibited loss of mitochondrial membrane potential and increased cardiolipin oxidation, but with no increase in reactive oxygen species. These properties indicate that the onset of terminal differentiation, while regulated by PI3 kinase and caspases, is not a classical apoptotic process.
epidermis; integrins; extracellular matrix
Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor α secretion. However, bone marrow-derived macrophages from tristetraprolin (TTP)-deficient mice were less sensitive than normal macrophages to this effect of p38 inhibitors, despite evidence for normal p38 activation in response to lipopolysaccharide. TTP is known to cause decreased stability of tumor necrosis factor α and granulocyte-macrophage colony-stimulating factor mRNAs after binding to an AU-rich element in their 3′ -untranslated regions. A recombinant TTP fusion protein could be phosphorylated by a recombinant p38 kinase in cell-free assays and was phosphorylated to the same extent by immunoprecipitated p38 derived from normal and TTP-deficient cells stimulated with lipopolysaccharide; in both cases, the enzyme activity was inhibited by the p38 inhibitors. TTP phosphorylation also was increased in intact macrophages after lipopolysaccharide stimulation, an effect that was blocked by the p38 inhibitors. Finally, TTP in mammalian cell extracts bound less well to an AU-rich element RNA probe than did the same amount of TTP following dephosphorylation. These results suggest that TTP may be a component of the signaling cascade, initiated by inflammatory stimuli and mediated in part by activation of p38, that ultimately leads to enhanced secretion of tumor necrosis factor α.
Prochlorococcus and Synechococcus picocyanobacteria are dominant contributors to marine primary production over large areas of the ocean. Phytoplankton cells are entrained in the water column and are thus often exposed to rapid changes in irradiance within the upper mixed layer of the ocean. An upward fluctuation in irradiance can result in photosystem II photoinactivation exceeding counteracting repair rates through protein turnover, thereby leading to net photoinhibition of primary productivity, and potentially cell death. Here we show that the effective cross-section for photosystem II photoinactivation is conserved across the picocyanobacteria, but that their photosystem II repair capacity and protein-specific photosystem II light capture are negatively correlated and vary widely across the strains. The differences in repair rate correspond to the light and nutrient conditions that characterize the site of origin of the Prochlorococcus and Synechococcus isolates, and determine the upward fluctuation in irradiance they can tolerate, indicating that photoinhibition due to transient high-light exposure influences their distribution in the ocean.
Severe hyperbilirubinemia is the most common cause of neonatal readmission to hospital in Canada even though, in the majority of cases, risk factors can be identified before discharge. Severe neonatal hyperbilirubinemia and kernicterus continue to be reported worldwide in otherwise healthy term infants. We conducted this study to estimate the incidence of severe neonatal hyperbilirubinemia in Canada and to determine underlying causes, improved knowledge of which would be valuable to help identify strategies for risk reduction.
Data on term infants 60 days of age and younger with unconjugated hyperbilirubinemia were collected prospectively through the Canadian Paediatric Surveillance Program from 2002 to 2004. Infants were included if they had a peak serum total bilirubin level of more than 425 μmol/L or underwent an exchange transfusion. Infants with rhesus iso-immunization or who were born at less than 36 weeks' gestation were excluded.
Of 367 cases reported, 258 were confirmed to be severe neonatal hyperbilirubinemia, for an estimated incidence of 1 in 2480 live births. Causes were identified in 93 cases and included ABO incompatibility (n = 48), glucose-6-phosphate dehydrogenase deficiency (n = 20), other antibody incompatibility (n = 12) and hereditary spherocytosis (n = 7). The mean peak bilirubin level reported was 471 μmol/L (standard deviation [SD] 76 μmol/L, range 156– 841 μmol/L). Fifty-seven infants (22.1%) underwent an exchange transfusion. A total of 185 infants (71.7%) were readmitted to hospital, 121 (65.4%) of them within 5 days of age.
Severe neonatal hyperbilirubinemia continues to occur frequently in Canada. In the majority of cases, the underlying cause was not identified. The high readmission rate within days after initial discharge indicates a need for a more thorough assessment of newborn infants and consideration of strategies to identify at-risk newborns, such as predischarge measurement of serum bilirubin levels.
We evaluated a rapid antigen-capture assay (VecTest) for detection of West Nile virus in oropharyngeal and cloacal swabs, collected at necropsy from owls (N = 93) and raptors (N = 27). Sensitivity was 93.5%–95.2% for northern owl species but <42.9% for all other species. Specificity was 100% for owls and 85.7% for raptors.
West Nile virus; Owl; Raptor; Bird; VecTest; Antigen-capture; PCR; diagnosis; test; Ontario; dispatch
Cyanobacteria are ecologically important photosynthetic prokaryotes that also serve as popular model organisms for studies of photosynthesis and gene regulation. Both molecular and ecological studies of cyanobacteria benefit from real-time information on photosynthesis and acclimation. Monitoring in vivo chlorophyll fluorescence can provide noninvasive measures of photosynthetic physiology in a wide range of cyanobacteria and cyanolichens and requires only small samples. Cyanobacterial fluorescence patterns are distinct from those of plants, because of key structural and functional properties of cyanobacteria. These include significant fluorescence emission from the light-harvesting phycobiliproteins; large and rapid changes in fluorescence yield (state transitions) which depend on metabolic and environmental conditions; and flexible, overlapping respiratory and photosynthetic electron transport chains. The fluorescence parameters FV/FM, FV′/FM′,qp,qN, NPQ, and φPS II were originally developed to extract information from the fluorescence signals of higher plants. In this review, we consider how the special properties of cyanobacteria can be accommodated and used to extract biologically useful information from cyanobacterial in vivo chlorophyll fluorescence signals. We describe how the pattern of fluorescence yield versus light intensity can be used to predict the acclimated light level for a cyanobacterial population, giving information valuable for both laboratory and field studies of acclimation processes. The size of the change in fluorescence yield during dark-to-light transitions can provide information on respiration and the iron status of the cyanobacteria. Finally, fluorescence parameters can be used to estimate the electron transport rate at the acclimated growth light intensity.
A blood culture cohort study investigating issues related to isolation of coagulase-negative staphylococci (CoNS) and other skin microflora is reported. Data were collected over 12 weeks to determine the incidence of significant CoNS bacteremia versus that of pseudobacteremia (contaminants) and to evaluate drug therapy in patients with cultures positive for CoNS. In addition, the effectiveness of 0.2% chlorine peroxide as a bactericidal disinfectant was compared to that of 10% providone iodine. A total of 3,276 cultures of blood from 1,433 patients were evaluated in the study. Eighty-nine cultures were positive for skin flora, with 81 of 89 (91%) involving CoNS. The incidence of significant CoNS bacteremia was 20 of 81 (24.7%), that of indeterminate bacteremia was 10 of 81 (12.3%), and that of contamination was 59 of 81 (72.8%). The incidence of significant bacteremia involving CoNS was double the 10 to 12% rate based on previous estimations at our institutions. In tests with the two bactericidal disinfectants, 22 of 1,639 cultures (1.3%) in the chlorine peroxide group versus 37 of 1,637 (2.3%) in the providone iodine group were considered contaminated (P = 0.065). Rates of contamination for venipuncture versus catheter collection were not significantly different (P = 0.46). The overall contamination rate was 59 of 3,276 (1.8%), which is consistent with the lower end of published quality assurance benchmark standards. The low rate was believed to be due to the professional phlebotomy staff in our institutions. There was excellent agreement between retrospective analysis by reviewers, when formal criteria were used, and the attending physicians’ intuitive clinical impressions in the classification of significant bloodstream infections (100% agreement) or contamination (95% agreement). However, physicians still used antimicrobial agents to treat nearly one-half of the patients with contaminated blood cultures, with vancomycin being misused in 34% of patients. In addition, 10% of patients with significant bacteremia were treated with inappropriate agents. There were no significant adverse events or prolonged hospital stays due to the unnecessary use of vancomycin; however, the additional costs of treating patients whose cultures contained CoNS contaminants was estimated to be $1,000 per patient. Measures to limit the unnecessary use of vancomycin (and other agents) are important.