Hypoxia Modulates Platelet Purinergic Signalling Pathways.

BACKGROUND: Hypoxia resulting from ascent to high-altitude or pathological states at sea level is known to increase platelet reactivity. Previous work from our group has suggested that this may be adenosine diphosphate (ADP)-specific. Given the clinical importance of drugs targeting ADP pathways, research into the impact of hypoxia on platelet ADP pathways is highly important. METHODS: Optimul aggregometry was performed on plasma from 29 lowland residents ascending to 4,700 m, allowing systematic assessment of platelet reactivity in response to several platelet agonists. Aggregometry was also performed in response to ADP in the presence of inhibitors of the two main ADP receptors, P2Y1 and P2Y12 (MRS2500 and cangrelor, respectively). Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a key determinant of platelet aggregation, was analysed using the VASPFix assay. RESULTS: Hypobaric hypoxia significantly reduced the ability of a fixed concentration of cangrelor to inhibit ADP-induced aggregation and increased basal VASP phosphorylation. However, in the absence of P2Y receptor inhibitors, we did not find evidence of increased platelet sensitivity to any of the agonists tested and found reduced sensitivity to thrombin receptor-activating peptide-6 amide. CONCLUSION: Our results provide evidence of increased P2Y1 receptor activity at high altitude and suggest down-regulation of the P2Y12 pathway through increased VASP phosphorylation. These changes in ADP pathway activity are of potential therapeutic significance to high-altitude sojourners and hypoxic sea level patients prescribed platelet inhibitors and warrant further investigation.

Coblation® intracapsular tonsillectomy (tonsillotomy) in children: A prospective study of 500 consecutive cases with long-term follow-up.

OBJECTIVES: Intracapsular tonsillectomy (tonsillotomy) has been used internationally, mainly in the management of obstructive sleep apnoea, rather than recurrent tonsillitis, with few published data evaluating its use for this latter indication. We present long-term prospective data from 500 paediatric cases undergoing Coblation® intracapsular tonsillectomy, for both obstructive and infective indications. DESIGN: Prospective case series, March 2013-January 2016, all with completed follow-up. SETTING: Tertiary paediatric otolaryngological practice. PARTICIPANTS: A total of 500 consecutive patients (6 months to 18 years, mean 5.1 years) undergoing Coblation® intracapsular tonsillectomy (with or without adenoidectomy), for obstructive and/ or infective indications, almost exclusively under the care of the senior author (DJT). MAIN OUTCOME MEASURES: Validated parent-reported T-14 tonsil symptom questionnaires were used in all cases pre- and postoperatively, including in the long term. Parents also recorded duration of analgesia, time to return to school, any complications and whether they would recommend the procedure. RESULTS: With a mean follow-up 7.4 months, symptom control has been excellent (mean total T-14 score (/70) 31.01 preoperatively, 2.68 postoperatively, P<.0000001), with similar trends for obstructive and infective domains. Two small secondary haemorrhages required readmission and observation only (0.4%); otherwise, no complications, delayed discharges or readmissions occurred; 12/500 (2.4%) have since undergone revision tonsil surgery, 10 for obstructive and 2 for infective symptoms, the majority in very young children, with revision adenoidectomy at the same time. More than 99% of parents would recommend the surgery. CONCLUSIONS: Our experience of this technique has been very positive, with excellent control of both obstructive and infective symptoms, and exceptionally low rates of complications. Further work will be required to allow conclusive demonstration of its advantages over extracapsular tonsillectomy.

Generation of attosecond electron packets via conical surface plasmon electron acceleration.

We present a method for the generation of high kinetic energy attosecond electron packets via magnetostatic and aperture filtering of conical surface plasmon (SP) accelerated electrons. The conical SP waves are excited by coupling an ultrafast radially polarized laser beam to a conical silica lens coated with an Ag film. Electromagnetic and particle tracking models are employed to characterize the ultrafast electron packets.

Terahertz Plasmonic Field-Induced Conductivity Modulation in Gold.

We report the observation of terahertz (THz) electric field induced conductivity modulation in sub-wavelength gold plasmonic media. Through all-THz pump-probe time-resolved transmission spectroscopy, we demonstrate that the presence of induced surface charges influences near-field mediated light propagation. The phenomenon is ascribed to the enhanced metal conductivity due to enhanced surface density of conduction electrons. The surface induced charge dynamics are revealed via phase-dependent time-resolved signatures. The phenomenon is a prelude to a wide class of ultrafast active THz plasmonic devices and paves the way for plasmonic field effects devices, similar to semiconductor ones.

Electron acceleration and kinetic energy tailoring via ultrafast terahertz fields.

We propose a mechanism for tuning the kinetic energy of surface plasmon generated electron pulses through control of the time delay between a pair of externally applied terahertz pulses. Varying the time delay results in translation, compression, and broadening of the kinetic energy spectrum of the generated electron pulse. We also observe that the electrons' kinetic energy dependence on the carrier envelope phase of the surface plasmon is preserved under the influence of a terahertz electric field.

The restoration of Loch Leven, Scotland, UK.

This paper reviews the progress made towards the restoration of Loch Leven, the largest lake in lowland Scotland, over the last 20 years. In particular, the importance of direct regulation and of setting water quality objectives and targets is examined. Various means of engaging with stakeholders and the general public are also considered. Success criteria and catchment management initiatives are described and briefly reviewed.

The impact of fine sediment accumulation on the survival of incubating salmon progeny: implications for sediment management.

This paper draws on results from a recent research programme on the impact of fine sediment transport through catchments to present a case for the development of new approaches to improving the quality of salmonid spawning and incubation habitats. To aid the development of these programmes, this paper summarises the mechanisms by which fine sediment accumulation influences the availability of oxygen (O2) to incubating salmon embryos. The results of the investigation indicate that incubation success is inhibited by: (i) the impact of fine sediment accumulation on gravel permeability and, subsequently, the rate of passage of oxygenated water through the incubation environment; (ii) reduced intragravel O2 concentrations that occur when O2 consuming material infiltrates spawning and incubation gravels; and (iii) the impact of fine particles (clay) on the exchange of O2 across the egg membrane. It is concluded that current granular measures of spawning and incubation habitat quality do not satisfactorily describe the complexity of factors influencing incubation success. Furthermore, an assessment of the trends in fine sediment infiltration indicates that only a small proportion of the total suspended sediment load infiltrates spawning and incubation gravels. This casts doubt over the ability of current catchment-based land use management strategies to adequately reduce fine sediment inputs.

Leg patterning driven by proximal-distal interactions and EGFR signaling.

wingless and decapentaplegic signaling establishes the proximal-distal axis of Drosophila legs by activating the expression of genes such as Distalless and dachshund in broad proximal-distal domains during early leg development. However, here we show that wingless and decapentaplegic are not required throughout all of proximal-distal development. The tarsus, which has been proposed to be an ancestral structure, is instead defined by the activity of Distalless, dachshund, and a distal gradient of epidermal growth factor receptor (EGFR)-Ras signaling. Our results uncover a mechanism for appendage patterning directed by genes expressed in proximal-distal domains and possibly conserved in other arthropods and vertebrates.

Rapsynoid/partner of inscuteable controls asymmetric division of larval neuroblasts in Drosophila.

Asymmetric cell division generates daughter cells with different developmental fates. In Drosophila neuroblasts, asymmetric divisions are characterized by (1) a difference in size between the two daughter cells and (2) an asymmetric distribution of cell fate determinants, including Prospero and Numb, between the two daughter cells. In embryonic neuroblasts, the asymmetric localization of cell fate determinants is under the control of the protein Inscuteable (Insc), which is itself localized asymmetrically as an apical crescent. Here, we describe a new Drosophila protein, Rapsynoid (Raps), which interacts in a two-hybrid assay with the signal transduction protein Galpha(i). We show that Raps is localized asymmetrically in dividing larval neuroblasts and colocalizes with Insc. Moreover, in raps mutants, the asymmetric divisions of neuroblasts are altered: (1) Insc is no longer asymmetrically localized in the dividing neuroblast; and (2) the neuroblast division produces two daughter cells of similar sizes. However, the morphologically symmetrical divisions of raps neuroblasts still lead to daughter cells with different fates, as shown by differences in gene expression. Our data show that Raps is a novel protein involved in the control of asymmetric divisions of neuroblasts.

Complexity- and risk-adjusted model for measuring surgical outcome.

BACKGROUND: Although currently available surgical scoring systems have good outcome predictive power, their use is often limited by complexity and their non-dynamic nature. The aim of this study was to develop and test a risk adjustment for general surgical audit which is both simple and dynamic, while preserving a high predictive power for surgical morbidity. METHODS: Twelve easily measured, well defined prognostic variables for morbidity were identified from the Otago Surgical Audit data collection form and stratified into suitable categories. Logistic regression was used to adjust for confounding between factors, identifying risk factors with the strongest prognostic value for the outcome of severe and intermediate complications. The resulting model was tested by back-validation and validation. RESULTS: The derived risk adjustment included all 12 variables. Adjusted odds ratios for all variables were markedly lower than unadjusted values. After logistic regression, the strongest predictors of postoperative morbidity were duration of operation, operation category, inpatient status and organ system in which the procedure was carried out. The area under the receiver operating characteristic curve was 0.86. CONCLUSION: A simple dynamic model for surgical morbidity has been developed which is comparable to previously published surgical scoring systems in terms of predictive power. This risk adjustment tool can be incorporated into the existing audit system, enabling comparison of surgical unit performance.

loco encodes an RGS protein required for Drosophila glial differentiation.

In Drosophila, glial cell development depends on the gene glial cells missing (gcm). gcm activates the expression of other transcription factors such as pointed and repo, which control subsequent glial differentiation. In order to better understand glial cell differentiation, we have screened for genes whose expression in glial cells depends on the activity of pointed. Using an enhancer trap approach, we have identified loco as such a gene. loco is expressed in most lateral CNS glial cells throughout development. Embryos lacking loco function have an normal overall morphology, but fail to hatch. Ultrastructural analysis of homozygous mutant loco embryos reveals a severe glial cell differentiation defect. Mutant glial cells fail to properly ensheath longitudinal axon tracts and do not form the normal glial-glial cell contacts, resulting in a disruption of the blood-brain barrier. Hypomorphic loco alleles were isolated following an EMS mutagenesis. Rare escapers eclose which show impaired locomotor capabilities. loco encodes the first two known Drosophila members of the family of Regulators of G-protein signalling (RGS) proteins, known to interact with the alpha subunits of G-proteins. loco specifically interacts with the Drosophila alphai-subunit. Strikingly, the interaction is not confined to the RGS domain. This interaction and the coexpression of LOCO and Galphai suggests a function of G-protein signalling for glial cell development.

Postoperative adhesive small bowel obstruction: the resources impacts.

AIM: To assess the resource implications of managing small bowel obstruction, which is common and has diverse causes and outcomes. METHOD: A retrospective study of 332 patients documented to have postoperative, adhesive small bowel obstruction, from 1988 to 1996, was carried out. Complications and resources used were recorded and costs were determined. Results. There were 207 females and 125 males, with a median age of 63 years. There were 374 hospital admissions, in 121 (32.4%) of which no surgical operation was performed. Patients had a median hospital stay of eight days. The overall median cost for a patient who had no operation was NZ$1 039 (minimum $94, maximum $13 262), compared to NZ$7 630 (minimum $2 038, maximum $135 173) for a patient who had an operation. Postoperative adhesive small bowel obstruction accounted for 1.3% of all admissions, 59.2% of all cases of bowel obstruction, 65.2% of all admissions with small bowel conditions, 73.5% of laparotomies for bowel obstruction and 4.1% of all laparotomies. Sixty-eight patients (20.5%) developed a total of 102 complications and there were eight deaths (2.4%). CONCLUSION: Postoperative adhesive small bowel obstruction is a common condition, which is associated with a substantial morbidity and workload. The treatment of these conditions has significant health care costs. Most are emergencies and the costs of their management are very variable.

The alcohol dehydrogenase gene is nested in the outspread locus of Drosophila melanogaster.

This report describes the structure and expression of the outspread (osp) gene of Drosophila melanogaster. Previous work showed that chromosomal breakpoints associated with mutations of the osp locus map to both sides of the alcohol dehydrogenase gene (Adh), suggesting that Adh and the adjacent gene Adhr are nested in osp. We extended a chromosomal walk and mapped additional osp mutations to define the maximum molecular limit of osp as 119 kb. We identified a 6-kb transcript that hybridizes to osp region DNA and is altered or absent in osp mutants. Accumulation of this RNA peaks during embryonic and pupal periods. The osp cDNAs comprise two distinct classes based on alternative splicing patterns. The 5' end of the longest cDNA was extended by PCR amplification. When hybridized to the osp walk, the 5' extension verifies that Adh and Adhr are nested in osp and shows that osp has a transcription unit of > or = 74 kb. In situ hybridization shows that osp is expressed both maternally and zygotically. In the ovary, osp is transcribed in nurse cells and localized in the oocyte. In embryos, expression is most abundant in the developing visceral and somatic musculature.

The role of homeotic genes in the specification of the Drosophila gonad.

BACKGROUND: In Drosophila, the gonads are composed of two cell populations: the germ line, derived from the pole cells, and a somatic component, derived from the mesoderm of abdominal segments 5-8. Formation of the gonad requires the function of a specific homeotic gene, abdominal-A (abd-A). Other genes of the bithorax complex, Ultrabithorax (Ubx) or Abdominal-B (Abd-B), cannot substitute for this requirement when abd-A is removed. RESULTS: We show here that, in embryos lacking the entire bithorax complex, experimentally induced expression of either ABD-A or UBX protein in the mesoderm will rescue the expression of a gonad-specific marker, 412 RNA. Ubiquitous expression of these homeotic proteins within the mesoderm results in the formation of ectopic gonad tissue anterior to the normal location of the gonads. In the absence of any endogenous bithorax-complex gene expression, however, mesoderm expressing gonad markers still condenses preferentially in the posterior segments of the abdomen, even in the absence of pole cells. CONCLUSIONS: The specific requirement for abd-A and not Ubx in gonad development does not reflect differences in the properties of the proteins that these genes encode, but presumably reflects differences in their regulation. In normal development, the restriction of gonad formation to the posterior abdomen does not depend on the overlap of abd-A and Abd-B expression, but must depend on the regulation of abd-A and Ubx in the sub-population of the mesoderm that forms the gonad. Factors other than homeotic gene expression provide some cues that direct gonadal mesoderm to condense in the correct location.

Dissecting the temporal requirements for homeotic gene function.

Homeotic genes confer identity to the different segments of Drosophila. These genes are expressed in many cell types over long periods of time. To determine when the homeotic genes are required for specific developmental events we have expressed the Ultrabithorax, abdominal-A and Abdominal-Bm proteins at different times during development using the GAL4 targeting technique. We find that early transient homeotic gene expression has no lasting effects on the differentiation of the larval epidermis, but it switches the fate of other cell types irreversibly (e.g. the spiracle primordia). We describe one cell type in the peripheral nervous system that makes sequential, independent responses to homeotic gene expression. We also provide evidence that supports the hypothesis of in vivo competition between the bithorax complex proteins for the regulation of their down-stream targets.

Homeotic genes autonomously specify one aspect of pattern in the Drosophila mesoderm.

Transplantation and ablation experiments have led to the generalization that in insects the mesoderm is naive, and that pattern is imposed upon it by the ectoderm. This has been demonstrated directly by mosaic analysis for the case of one muscle in Drosophila. The unique character of this muscle depends on the activity of sex-determining and homeotic genes, not in the muscle itself, but in the nerve that innervates it. Indirect evidence suggests, however, that homeotic genes specify some aspects of mesoderm patterning autonomously. Homeotic genes are expressed in the mesoderm, and are regulated in a segment-specific pattern analogous to, but different from, that seen in the ectoderm. Moreover, the effects of homeotic mutations on the muscles do not always mirror transformations seen in the epidermis. Here we examine this problem directly, by expressing homeotic genes ectopically in the mesoderm without altering their expression in the overlying ectoderm. We find that the pattern of adult muscle precursor cells characteristic of the thorax can be converted to that seen in the abdomen by expressing the homeotic gene abdominal-A specifically in the mesoderm.

Detection of peptidases in Trypanosoma cruzi epimastigotes using chromogenic and fluorogenic substrates.

Detergent extracts of Trypanosoma cruzi epimastigotes catalysed the hydrolysis of a range of amino-acyl and peptidyl p-nitro-anilides and aminomethylcoumarins. At least three enzymes were detected that cleave Z-Phe-Arg-MCA. Two of these were optimally active at alkaline pH, the other at pH 4.0. Of the two enzymes with alkaline pH optima, one was a cysteine peptidase and was unable to cleave Bz-Arg-MCA readily, whilst the other cleaved Bz-Arg-MCA and was inhibited by diisopropyl fluorophosphate. The acidic enzyme was similar to cathespin L of other eukaryotes with respect to its pH profile, substrate-specificity and inhibitor-sensitivity. Evidence was presented that epimastigotes contain a cysteine-type dipeptidyl aminopeptidase, one or more aminopeptidases, and a serine peptidase that cleaves Boc-Ala-Ala-pNA. Digitonin solubilization of the activities from cells supports the hypothesis that the cathespin L-like enzyme and the dipeptidyl aminopeptidase are lysosomal, whilst the Bz-Arg-MCA hydrolase, the aminopeptidases and the Boc-Ala-Ala-pNA serine peptidase are cytosolic.

Electrophoretic detection of Trypanosoma cruzi peptidases.

Peptidases of Trypanosoma cruzi epimastigotes were examined by polyacrylamide gel electrophoresis in gels containing gelatin as peptidase substrate. Mini-gels were far superior to large gels in their sensitivity of peptidase detection. Patterns of peptidases were similar between different strains of T. cruzi, although some inter-strain heterogeneity was found. In strain Y, at least five peptidases were detected: four of these enzymes were shown to be cysteine-type peptidases with acidic pH optima. The other peptidase was a 60-kDa membrane-associated peptidase that was sensitive to o-phenanthroline; it was tentatively characterised as a metallopeptidase, and was optimally active at alkaline pH. This membrane-associated peptidase was conserved between strains of T. cruzi.