''Studies on the malpighian tubules of Locusta migratoria miqratorioides (R + F), with particular reference to the role of Na(^+)-K(^+) activated ATPase in fluid secretion

The presence of a Na(^+)-K(^+) activated, Mg(^2+) dependent ATPase (E.C. 3.6.1.3.) has been demonstrated in microsomal preparations from Malpighian tubules and hindgutof Locusta miqratoria miqratorioides (R+F) and the conditions for optimal activity determined. The pH optimum was 7.5 and the temperature optima 45ºC for Na(^+)- K(^+)ATPase whilst Mg(^2+)ATPase activity was still rising at 50ºC. Activation energies for the Na(^+)- K(^+) ATPase were 121.6 - (^+) 4.5 and 59.8 (^+)-2.7 Kjoules Mole(^-1) between 6.3-21ºC and 21-42ºC respectively. The Mg(^2+) ATPase had activation energies of 95.8 (^+)- 1.9 and 51.7 - 3.9 Kjoules Mole (-1) over the same ranges. Maximal activation of the Nat(^+)- K(^+)ATPase occured at an ATP : Mg(^2+) ratio of 1:1.3, and at l00mM Na(^+): 20mM K(^+). The Na(^+)- K(^+) ATPase was inhibited by ouabain with a pI(_50) value of 6.1. The Km of Na(^+)-ATPase was 0.15 (^+)- 0.01 mMole 1(^-1) with no significant difference between values obtained at 20ºC, 30ºC and 40ºC. The values for Vmax were 113.6(^+)-42, 213 (^+)- 53 and 373 (^+)- 109 for these temperatures respectively. Physiological studies on fluid secretion by Malpighian tubules in vitro have shown secretion was inhibited by ouabain at concentrations greater than 10(^-6)M, and also in the absence of Na(^+) or K(^+) ions. This strongly suggested the .involvement of Na- K ATPase in secretion which was further implicated by polarographic studies showing a corresponding 35% reduction in oxygen uptake in the presence of ouabain, or absence of K(^+). Similarly the trans-wall potential has been shown to fall in these circumstances. The optimum temperature for secretion was 40ºC, the process having an activation energy of 58.307 Kjoules Mole(^-1) between 5-40ºC. Rate of secretion was inversely proportional to osmotic concentration, and the urine was hyperosmotic to the bathing medium by 22.9 + 2.6m. Osmoles. Fluid secretion was stimulated by cyclic AMP (3 x 10(^-4)M), but 5HT had no effect. Potassium has been shown to be concentrated in the urine, but as electrophysiological studies revealed that the trans- wall potential did not correspond with the values for K(^+) predicted by NERNST, the K(^+) movement must have resulted from active transport. Ultrastructural studies using both scanning and transmission electron microscopy in normal and ouabain Ringer solutions revealed that ouabain had no effect on structure. The results were discussed in terms of the relationship between cell structure, fluid secretion and Na(^+)-K(^+)ATPase activity

Recommendations for dealing with waste contaminated with Ebola virus: a Hazard Analysis of Critical Control Points approach

Objective To assess, within communities experiencing Ebola virus outbreaks, the risks associated with the disposal of human waste and to generate recommendations for mitigating such risks. Methods A team with expertise in the Hazard Analysis of Critical Control Points framework identified waste products from the care of individuals with Ebola virus disease and constructed, tested and confirmed flow diagrams showing the creation of such products. After listing potential hazards associated with each step in each flow diagram, the team conducted a hazard analysis, determined critical control points and made recommendations to mitigate the transmission risks at each control point. Findings The collection, transportation, cleaning and shared use of blood-soiled fomites and the shared use of latrines contaminated with blood or bloodied faeces appeared to be associated with particularly high levels of risk of Ebola virus transmission. More moderate levels of risk were associated with the collection and transportation of material contaminated with bodily fluids other than blood, shared use of latrines soiled with such fluids, the cleaning and shared use of fomites soiled with such fluids, and the contamination of the environment during the collection and transportation of blood-contaminated waste. Conclusion The risk of the waste-related transmission of Ebola virus could be reduced by the use of full personal protective equipment, appropriate hand hygiene and an appropriate disinfectant after careful cleaning. Use of the Hazard Analysis of Critical Control Points framework could facilitate rapid responses to outbreaks of emerging infectious disease

Controlled Dephasing of Electrons by Non-Gaussian Shot Noise

In a 'controlled dephasing' experiment [1-3], an interferometer loses its coherence due to entanglement with a controlled quantum system ('which path' detector). In experiments that were conducted thus far in mesoscopic systems only partial dephasing was achieved. This was due to weak interactions between many detector electrons and the interfering electron, resulting in a Gaussian phase randomizing process [4-10]. Here, we report the opposite extreme: a complete destruction of the interference via strong phase randomization only by a few electrons in the detector. The realization was based on interfering edge channels (in the integer quantum Hall effect regime, filling factor 2) in a Mach-Zehnder electronic interferometer, with an inner edge channel serving as a detector. Unexpectedly, the visibility quenched in a periodic lobe-type form as the detector current increased; namely, it periodically decreased as the detector current, and thus the detector's efficiency, increased. Moreover, the visibility had a V-shape dependence on the partitioning of the detector current, and not the expected dependence on the second moment of the shot noise, T(1-T), with T the partitioning. We ascribe these unexpected features to the strong detector-interferometer coupling, allowing only 1-3 electrons in the detector to fully dephase the interfering electron. Consequently, in this work we explored the non-Gaussian nature of noise [11], namely, the direct effect of the shot noise full counting statistics [12-15].Comment: 14 pages, 4 figure

Hazard Analysis of Critical Control Points Assessment as a Tool to Respond to Emerging Infectious Disease Outbreaks

Highly pathogenic avian influenza virus (HPAI) strain H5N1 has had direct and indirect economic impacts arising from direct mortality and control programmes in over 50 countries reporting poultry outbreaks. HPAI H5N1 is now reported as the most widespread and expensive zoonotic disease recorded and continues to pose a global health threat. The aim of this research was to assess the potential of utilising Hazard Analysis of Critical Control Points (HACCP) assessments in providing a framework for a rapid response to emerging infectious disease outbreaks. This novel approach applies a scientific process, widely used in food production systems, to assess risks related to a specific emerging health threat within a known zoonotic disease hotspot. We conducted a HACCP assessment for HPAI viruses within Vietnam’s domestic poultry trade and relate our findings to the existing literature. Our HACCP assessment identified poultry flock isolation, transportation, slaughter, preparation and consumption as critical control points for Vietnam’s domestic poultry trade. Introduction of the preventative measures highlighted through this HACCP evaluation would reduce the risks posed by HPAI viruses and pressure on the national economy. We conclude that this HACCP assessment provides compelling evidence for the future potential that HACCP analyses could play in initiating a rapid response to emerging infectious diseases

Mapping the spatiotemporal dynamics of calcium signaling in cellular neural networks using optical flow

An optical flow gradient algorithm was applied to spontaneously forming net- works of neurons and glia in culture imaged by fluorescence optical microscopy in order to map functional calcium signaling with single pixel resolution. Optical flow estimates the direction and speed of motion of objects in an image between subsequent frames in a recorded digital sequence of images (i.e. a movie). Computed vector field outputs by the algorithm were able to track the spatiotemporal dynamics of calcium signaling pat- terns. We begin by briefly reviewing the mathematics of the optical flow algorithm, and then describe how to solve for the displacement vectors and how to measure their reliability. We then compare computed flow vectors with manually estimated vectors for the progression of a calcium signal recorded from representative astrocyte cultures. Finally, we applied the algorithm to preparations of primary astrocytes and hippocampal neurons and to the rMC-1 Muller glial cell line in order to illustrate the capability of the algorithm for capturing different types of spatiotemporal calcium activity. We discuss the imaging requirements, parameter selection and threshold selection for reliable measurements, and offer perspectives on uses of the vector data.Comment: 23 pages, 5 figures. Peer reviewed accepted version in press in Annals of Biomedical Engineerin

Deficiency in origin licensing proteins impairs cilia formation: implications for the aetiology of meier-gorlin syndrome

Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA-mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency

The microaerophilic microbiota of de-novo paediatric inflammatory bowel disease: the BISCUIT study

<p>Introduction: Children presenting for the first time with inflammatory bowel disease (IBD) offer a unique opportunity to study aetiological agents before the confounders of treatment. Microaerophilic bacteria can exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. We set out to study these and other microaerophilic bacteria in de-novo paediatric IBD.</p> <p>Patients and Methods: 100 children undergoing colonoscopy were recruited including 44 treatment naïve de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis.</p> <p>Results: 129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates). Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae). No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312).</p> <p>Conclusions: This study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon.</p&gt

Identification of Class I HLA T Cell Control Epitopes for West Nile Virus

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity. © 2013 Kaabinejadian et al

Financial considerations in the conduct of multi-centre randomised controlled trials: evidence from a qualitative study.

National Coordinating Centre for Research Methodology; Medical Research Council, UK Department of Health; Chief Scientist OfficeNot peer reviewedPublisher PD