The use of different blood sample preparations in the development of biomarkers for the environmental monitoring of domestic farm animals

The application of biological effect monitoring for the detection of environmental chemical exposure in domestic animals is still in its infancy. This study investigated blood sample preparations in vitro for their use in biological effect monitoring. When peripheral blood mononuclear cells (PBMCs), isolated following the collection of multiple blood samples from sheep in the field, were cryopreserved and subsequently cultured for 24 hours a reduction in cell viability (<80%) was attributed to delays in the processing following collection. Alternative blood sample preparations using rat and sheep blood demonstrated that 3 to 5 hour incubations can be undertaken without significant alterations in the viability of the lymphocytes; however, a substantial reduction in viability was observed after 24 hours in frozen blood. Detectable levels of early and late apoptosis as well as increased levels of ROS were detectable in frozen sheep blood samples. The addition of ascorbic acid partly reversed this effect and reduced the loss in cell viability. The response of the rat and sheep blood sample preparations to genotoxic compounds ex vivo showed that EMS caused comparable dose-dependent genotoxic effects in all sample preparations (fresh and frozen) as detected by the Comet assay. In contrast, the effects of CdCl2 were dependent on the duration of exposure as well as the sample preparation. The analysis of leukocyte subsets in frozen sheep blood showed no alterations in the percentages of T and B lymphocytes but led to a major decrease in the percentage of granulocytes compared to those in the fresh samples. The percentages of IFN-γ and IL-4 but not IL-6 positive cells were comparable between fresh and frozen sheep blood after 4 hour stimulation with phorbol 12-myrisate 13-acetate and ionomycin (PMA+I). These results show that frozen blood gives comparable responses to fresh blood samples in the toxicological and immune assays used

Probing Convolutional Neural Networks for Event Reconstruction in {\gamma}-Ray Astronomy with Cherenkov Telescopes

A dramatic progress in the field of computer vision has been made in recent years by applying deep learning techniques. State-of-the-art performance in image recognition is thereby reached with Convolutional Neural Networks (CNNs). CNNs are a powerful class of artificial neural networks, characterized by requiring fewer connections and free parameters than traditional neural networks and exploiting spatial symmetries in the input data. Moreover, CNNs have the ability to automatically extract general characteristic features from data sets and create abstract data representations which can perform very robust predictions. This suggests that experiments using Cherenkov telescopes could harness these powerful machine learning algorithms to improve the analysis of particle-induced air-showers, where the properties of primary shower particles are reconstructed from shower images recorded by the telescopes. In this work, we present initial results of a CNN-based analysis for background rejection and shower reconstruction, utilizing simulation data from the H.E.S.S. experiment. We concentrate on supervised training methods and outline the influence of image sampling on the performance of the CNN-model predictions.Comment: 8 pages, 4 figures, Proceedings of the 35th International Cosmic Ray Conference (ICRC 2017), Busan, Kore

Listeriolysin S, a Novel Peptide Haemolysin Associated with a Subset of Lineage I Listeria monocytogenes

peer-reviewedStreptolysin S (SLS) is a bacteriocin-like haemolytic and cytotoxic virulence factor that plays a key role in the virulence of Group A Streptococcus (GAS), the causative agent of pharyngitis, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. Although it has long been thought that SLS and related peptides are produced by GAS and related streptococci only, there is evidence to suggest that a number of the most notorious Gram-positive pathogenic bacteria, including Listeria monocytogenes, Clostridium botulinum and Staphylococcus aureus, produce related peptides. The distribution of the L. monocytogenes cluster is particularly noteworthy in that it is found exclusively among a subset of lineage I strains; i.e., those responsible for the majority of outbreaks of listeriosis. Expression of these genes results in the production of a haemolytic and cytotoxic factor, designated Listeriolysin S, which contributes to virulence of the pathogen as assessed by murine- and human polymorphonuclear neutrophil–based studies. Thus, in the process of establishing the existence of an extended family of SLS-like modified virulence peptides (MVPs), the genetic basis for the enhanced virulence of a proportion of lineage I L. monocytogenes may have been revealed.Work is funded by the Irish Government under the National Development Plan, through a Science Foundation Ireland Investigator award to CH, PR and PC (06/IN.1/B98)

The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding

The validity of the identification and classification of human cancer using antibodies to detect biomarker proteins depends upon antibody specificity. Antibodies that bind to the tumour-suppressor protein p16INK4a are widely used for cancer diagnosis and research. In this study we examined the specificity of four commercially available anti-p16INK4a antibodies in four immunological applications. The antibodies H-156 and JC8 detected the same 16 kDa protein in western blot and immunoprecipitation tests, whereas the antibody F-12 did not detect any protein in western blot analysis or capture a protein that could be recognised by the H-156 antibody. In immunocytochemistry tests, the antibodies JC8 and H-156 detected a predominately cytoplasmic localised antigen, whose signal was depleted in p16INK4a siRNA experiments. F-12, in contrast, detected a predominately nuclear located antigen and there was no noticeable reduction in this signal after siRNA knockdown. Furthermore in immunohistochemistry tests, F-12 generated a different pattern of staining compared to the JC8 and E6H4 antibodies. These results demonstrate that three out of four commercially available p16INK4a antibodies are specific to, and indicate a mainly cytoplasmic localisation for, the p16INK4a protein. The F-12 antibody, which has been widely used in previous studies, gave different results to the other antibodies and did not demonstrate specificity to human p16INK4a. This work emphasizes the importance of the validation of commercial antibodies, aside to the previously reported use, for the full verification of immunoreaction specificity

The surface-associated exopolysaccharide of Bifidobacterium longum 35624 plays an essential role in dampening host proinflammatory responses and repressing local TH17 responses

The immune-modulating properties of certain bifidobacterial strains, such as Bifidobacterium longum subsp. longum 35624 (B. longum 35624), have been well described, although the strain-specific molecular characteristics associated with such immune-regulatory activity are not well defined. It has previously been demonstrated that B. longum 35624 produces a cell surface exopolysaccharide (sEPS), and in this study, we investigated the role played by this exopolysaccharide in influencing the host immune response. B. longum 35624 induced relatively low levels of cytokine secretion from human dendritic cells, whereas an isogenic exopolysaccharide-negative mutant derivative (termed sEPSneg) induced vastly more cytokines, including interleukin-17 (IL-17), and this response was reversed when exopolysaccharide production was restored in sEPSneg by genetic complementation. Administration of B. longum 35624 to mice of the T cell transfer colitis model prevented disease symptoms, whereas sEPSneg did not protect against the development of colitis, with associated enhanced recruitment of IL-17+ lymphocytes to the gut. Moreover, intranasal administration of sEPSneg also resulted in enhanced recruitment of IL-17+ lymphocytes to the murine lung. These data demonstrate that the particular exopolysaccharide produced by B. longum 35624 plays an essential role in dampening proinflammatory host responses to the strain and that loss of exopolysaccharide production results in the induction of local TH17 responses. IMPORTANCE: Particular gut commensals, such as B. longum 35624, are known to contribute positively to the development of mucosal immune cells, resulting in protection from inflammatory diseases. However, the molecular basis and mechanisms for these commensal-host interactions are poorly described. In this report, an exopolysaccharide was shown to be decisive in influencing the immune response to the bacterium. We generated an isogenic mutant unable to produce exopolysaccharide and observed that this mutation caused a dramatic change in the response of human immune cells in vitro. In addition, the use of mouse models confirmed that lack of exopolysaccharide production induces inflammatory responses to the bacterium. These results implicate the surface-associated exopolysaccharide of the B. longum 35624 cell envelope in the prevention of aberrant inflammatory responses

Cross-cultural effects on drivers' hazard perception

Hazard perception tests are used in several developed countries as part of the driver licensing curriculum, however little research has been done in developing countries where road safety is a primary concern. We conducted a cross-cultural hazard perception study to examine the transferability of hazard perception skills between Malaysia and the UK, using hazard clips filmed in both countries. The results showed that familiarity with both the driving environment and type of hazard facilitated drivers' ability to discriminate hazards in a timely manner, although overall drivers viewed and responded to hazards largely similarly regardless of origin. Visual strategies also appeared to be moderated mainly by the immediate driving environment rather than driver origin. Finally, Malaysian drivers required a higher threshold of danger than UK drivers before they would identify a situation as hazardous, possibly reflecting the more hazardous road environment in Malaysia. We suggest that hazard perception testing in developing countries requires a test where performance cannot be confounded with differing thresholds for hazardousness

The role of experience and advanced training on performance in a motorcycle simulator

Motorcyclists are over-represented in collision statistics. While many collisions may be the direct fault of another road user, a considerable number of fatalities and injuries are due to the actions of the rider. While increased riding experience may improve skills, advanced training courses may be required to evoke the safest riding behaviours. The current research assessed the impact of experience and advanced training on rider behaviour using a motorcycle simulator. Novice riders, experienced riders and riders with advanced training traversed a virtual world through varying speed limits and roadways of different curvature. Speed and lane position were monitored. In a comparison of 60 mph and 40 mph zones, advanced riders rode more slowly in the 40 mph zones, and had greater variation in lane position than the other two groups. In the 60 mph zones, both advanced and experienced riders had greater lane variation than novices. Across the whole ride, novices tended to position themselves closer to the kerb. In a second analysis across four classifications of curvature (straight, slight, medium, tight) advanced and experienced riders varied their lateral position more so than novices, though advanced riders had greater variation in lane position than even experienced riders in some conditions. The results suggest that experience and advanced training lead to changes in behaviour compared to novice riders which can be interpreted as having a potentially positive impact on road safety

Are situation awareness and decision-making in driving totally conscious processes? Results of a Hazard Prediction task

Detecting danger in the driving environment is an indispensable task to guarantee safety which depends on the driver's ability to predict upcoming hazards. But does correct prediction lead to an appropriate response? This study advances hazard perception research by investigating the link between successful prediction and response selection. Three groups of drivers (learners, novices and experienced drivers) were recruited, with novice and experienced drivers further split into offender and non-offender groups. Specifically, this works aims to develop an improved Spanish Hazard Prediction Test and to explore the differences in Situation Awareness, (SA: perception, comprehension and prediction) and Decision-Making ("DM") among learners, younger inexperienced and experienced drivers and between driving offenders and non-offenders. The contribution of the current work is not only theoretical; the Hazard Prediction Test is also a valid way to test Hazard Perception. The test, as well as being useful as part of the test for a driving license, could also serve a purpose in the renewal of licenses after a ban or as a way of training drivers. A sample of 121 participants watched a series of driving video clips that ended with a sudden occlusion prior to a hazard. They then answered questions to assess their SA ("What is the hazard?" "Where is it located?" "What happens next?") and "DM" ("What would you do in this situation?"). This alternative to the Hazard Perception Test demonstrates a satisfactory internal consistency (Alpha=0.750), with eleven videos achieving discrimination indices above 0.30. Learners performed significantly worse than experienced drivers when required to identify and locate the hazard. Interestingly, drivers were more accurate in answering the "DM" question than questions regarding SA, suggesting that drivers can choose an appropriate response manoeuvre without a totally conscious knowledge of the exact hazard

Hazard prediction discriminates between novice and experienced drivers

Typical hazard perception tests often confound multiple processes in their responses. The current study tested hazard prediction in isolation to assess whether this component can discriminate between novice and experienced drivers. A variant of the hazard perception test, based on the Situation Awareness Global Assessment Technique, found experienced drivers to outperform novices across three experiments suggesting that the act of predicting an imminent hazard is a crucial part of the hazard-perception process. Furthermore three additional hypotheses were tested in these experiments. First, performance was compared across clips of different length. There was marginal evidence that novice drivers' performance suffered with the longest clips, but experienced drivers’ performance did not, suggesting that experienced drivers find hazard prediction less effortful. Secondly, predictive accuracy was found to be dependent on the temporal proximity of visual precursors to the hazard. Thirdly the relationship between the hazard and its precursor was found to be important, with less obvious precursors improving the discrimination between novice and experience drivers. These findings demonstrate that a measure of hazard prediction, which is less confounded by the influence of risk appraisal than simple response time measures, can still discriminate between novice and experienced drivers. Application of this methodology under different conditions can produce insights into the underlying processes that may be at work, whilst also providing an alternative test of driver skill in relation to the detection of hazards

Bifidobacterium animalis AHC7 protects against pathogen-induced NF-κB activation in vivo

BACKGROUND: Bifidobacteria and lactobacilli are among the early and important colonizers of the gastrointestinal tract and are generally considered to be part of a normal, healthy microbiota. It is believed that specific strains within the microbiota can influence host immune-reactivity and may play a role in protection from infection and aberrant inflammatory activity. One such strain, Bifidobacterium animalis AHC7, has been previously shown to protect against Salmonella typhimurium infection in mice and helps resolve acute idiopathic diarrhea in dogs. The aim of this study was to investigate the potential molecular and cellular mechanisms underpinning the Bifidobacterium animalis AHC7 protective effect. RESULTS: Following 4 hours of infection with Salmonella typhimurium, NF-κB activation was significantly elevated in vivo in placebo and Enterococcus faecium-fed animals while Bifidobacterium animalis AHC7 consumption significantly attenuated the NF-κB response. In vitro anti-CD3/CD28 stimulated Peyer's patch cells secreted significantly less TNF-α and IFN-γ following Bifidobacterium animalis AHC7 consumption. Stimulated cells released more IL-12p70 but this difference did not reach statistical significance. No alteration in mucosal IL-6, IL-10 or MCP-1 levels were observed. No statistically significant change in the cytokine profile of mesenteric lymph node cells was noted. In vitro, Bifidobacterium animalis AHC7 was bound by dendritic cells and induced secretion of both IL-10 and IL-12p70. In addition, co-culture of CD4+ T cells with Bifidobacterium animalis AHC7-stimulated dendritic cells resulted in a significant increase in CD25+Foxp3+ T cell numbers. CONCLUSION: Bifidobacterium animalis AHC7 exerts an anti-inflammatory effect via the attenuation of pro-inflammatory transcription factor activation in response to an infectious insult associated with modulation of pro-inflammatory cytokine production within the mucosa. The cellular mechanism underpinning Bifidobacterium animalis AHC7 mediated attenuation of NF-κB activation may include recognition of the bacterium by dendritic cells and induction of CD25+Foxp3+ T cells