Penthetria heteroptera, we propose that local orientation of small hairs (microtrichia) reflects a balance of three nonexclusive strategies: (1) preventing water from becoming stuck in intervenous grooves (microtrichia point upslope), (2) shedding water off the wing as readily as possible (microtrichia point towards the nearest edge), and, (3) shedding water away from the body . We present evidence for all three and show that local microtrichial orientation is seldom determined by any one factor. We develop a mathematical model that employs factor-specific weighting values determined via optimization. Our predictions are tested against the orientation of microtrichia randomly sampled from a P. heteroptera specimen. Using the best-fit weighting parameters, the model displays a median residual of 20°; no residual is greater than 46°. The model also reproduces qualitative aspects of microtrichial orientation, such as bifurcation midway between veins and convergence toward peaks. This strong correspondence between modelled and observed orientation supports the role of microtrichia as directional antiwetting devices and highlights the importance of considering both function and wing geometry to explain the organization of natural microstructure arrays.Microstructures on insect wings can promote directional drop shedding, and the local orientation of these structures is expected to facilitate drop removal. However, microstructures may exhibit very different orientations at different locations on the wing. Using the march fly  Many insects display dense arrays of micro or nanostructures that promote antiwetting on wing surfaces. These structures can take on a variety of forms, such as bumps, asters, scales and hairs –4. Hairset al. [Morpho aega butterfly wing (Hubner 1982) prevented drops pushed against the grain from detaching, but promoted drop shedding along the grain. Prakash and Bush [Biological microstructures often have a nonrandom orientation, which can promote shedding of water drops and contaminants in one direction over another. Zheng et al.  showed tHubner 192 preventDrosophila spp. [et al. [While microstructures on insect wings may have a nonrandom direction locally, they often display variation in orientation across the wing surface. The developmental and genetic basis of microtrichial orientation has been explored in ila spp. –12; howe [et al.  noted muIf microtrichia  form directional wetting surfaces, then we would expect variation in orientation to promote efficient shedding of water and contaminants from the wing. One strategy would be to locally orient the array towards the nearest edge, thereby promoting quick drop shedding. There are, however, two main problems with this strategy. First, shedding drops from the wing to the body would be counterproductive in terms of load reduction. Second, grooves between veins on highly corrugated surfaces could potentially trap drops, and drops may shed off the wing more easily if water were kept out of these grooves, regardless of their distance to a wing edge.Penthetria heteroptera, Say 1823). Next we demonstrate an association between large scale topography  and local microtrichial orientation in this same species. We also demonstrate that microtrichia orient towards the nearest edge as well as distally overall, though no one factor is responsible for all the variation observed. Finally, we develop a simple mathematical model that captures these three effects and demonstrate that this model can reproduce the majority of variation of microtrichial orientation observed on the dipteran wing.We therefore propose that local microstructure orientation on insect wings is a result of balancing three strategies that sometimes compete: (1) preventing water from becoming stuck in intervenous grooves, (2) shedding water off the wing quickly and (3) shedding water away from the body. First, we establish microtrichia as antiwetting structures in a large dipteran (Penthetria heteroptera (Diptera: family Bibionidae) were collected September 9th 2012 from private land near Lloyd Creek Natural Reserve, Alberta, Canada, after obtaining permission from the land owner. The collection of specimens complied with all provincial and national regulations, and did not involve endangered or protected species. Samples were preserved in a freezer at -80°C until use. When removing wings from specimens, the experimenter was careful to seize the wing only near the wing base.Adult P. heteroptera female was imaged (For scanning electron microscope (SEM) imaging, wings were gently placed, dorsal-side up, on a pin-type stub with double-sided carbon tape. Wings were gently pressed flat against the tape with a soft and flat plastic implement. The wing and stub were sputter coated with gold and imaged using a JEOL 6301F Field Emission Scanning Electron Microscope. One pair of wings from a s imaged .μL distilled water drops were produced from a 2 mL syringe with a regular bevel, 27 gauge needle by slowly applying manual pressure to the plunger until a water drop detached from the needle tip. The syringe was held securely in place by a stand. Drop contact angle on the wing was measured in ImageJ from profile view photos of static drops taken with a Canon PowerShot Elph 300HS camera with an attached Bushnel 8 × 20 mm monocular.Double-sided tape was placed flat on a microscope slide. Wings were removed from a preserved specimen, placed dorsal-side-up on the tape, and gently flattened with a soft plastic implement. 5 For drop rolloff angle measurements, 10 wings from five males were flattened on microscope slides and positioned on a Newport GON65-U goniometric stage, such that the torsional axis was oriented downslope. Distilled water drops produced using the same method as above were placed in the center of the level wings. Pictures were taken at 1° increments until the drop was observed to roll off the wing or the maximum inclination of 10° was reached. This process was repeated a minimum of four more times in both the distal and proximal tilting directions. Three wings (two right and one left) were rejected from the analysis because the cuticle was folded over near the wing centre, creating a strong pinning point.RFR) in the proximal vs. distal directions was computed fromβp and βd are the rolloff angles in the proximal and distal directions, respectively.For the remaining wings, images of drops were analyzed and the drop rolloff angle for a particular trial was taken as the lowest angle at which the drop contact line was observed to advance downslope compared to its starting position on the wing. If depinning had not occurred by the 10° mark, the rolloff angle was set at 11°. Drop rolloff angles in the distal and proximal directions respectively were taken as a weighted average of mean rolloff angle per wing in a given direction. The weights were chosen as the reciprocal of the standard error of the mean rolloff angle in a particular direction for a given wing. The ratio of force of retention (Wings flattened on microscope slides were imaged with a Nikon Coolpix 8400 camera attached to an Olympus SZX16 microscope at 10× magnification. This same setup was used to image wing sections (described below). The experimenter was careful to keep the microtrichia of the upper surface in focus, and the underside microtrichia out of focus. Images from the same wing were stitched together in Photoshop (v. CS2) to form a complete compound image of each wing see .The orientations of microtrichia were assessed relative to the torsional axis of the wing see . The torμm) were centred at random coordinates on the wing determined by a pseudo-random number generator. Quadrat size was chosen so as to minimize quadrat-edge effects while easing data collection. The orientation of each microtrichium was determined from the orientation of the line connecting the centre of the hair base to the hair tip, and was measured relative to the horizontal axis of the image in ImageJ. If the centre of a hair base lay within the quadrat, that microtrichium was included in the quadrat sample; otherwise it was excluded. Hairs on veins were not sampled. The local orientation at the location of each quadrat centre was taken as the arithmetic mean orientation of all hairs within the quadrat.A composite image of the upper wing surface was rotated so that the image horizontal axis was parallel to the torsional axis. 24 quadrats 75 × 75 px . Any obvious wax portions that remained were removed manually in Photoshop. If there was any ambiguity as to whether an object was wax or cuticle, that feature was untouched. The experimenter was careful to ensure that no wing features, including microtrichia and setae, were excluded by this procedure. Using the sections as a guide, wing veins were assessed as local topographic maxima or minima.W1 and W2. W1 and W2 represent weighting factors for an upslope microtrichial orientation and an orientation to the nearest-edge, respectively; these parameters are discussed in depth in the “Modelling” section below. For optimization, values of 0 ≤ W1 ≤ 2 and 0 μm ≤ W2 ≤ 1493 μm were tested, with resolutions of 0.001 in W1 and 0.75 μm in W2. The modelled microtrichial orientation at the location of each quadrat centre was compared against the observed average orientation of the corresponding quadrat, and the sum-square-residual (SSR) between modelled and observed orientation was computed for each combination of W1 and W2. The optimal combination of W1 and W2 was taken as the pair of values that minimized the SSR relative to observations. For the optimization procedure, as well as statistical analyses involving nearest-edge orientation, a single quadrat whose centre lay outside the wing was not used, as the model would then be searching for orientations of microtrichia outside the wing surface. Data is available as supplemental files using a comma-delimited format . Model optimization was also performed in MATLAB to determine the best-fit values of d format –S4 FilesP. heteroptera female. Microtrichia and setae . Therefore, microtrichia would be the primary antiwetting structure on the insect wing surface.To establish the role of microtrichia as antiwetting structures, measurements of static drop contact angles were performed on flat portions of the wing. Contact angles were measured to be 150±2° (mean ± standard error). Such a high contact angle cannot be produced by purely chemical means on insect cuticle, and is only realizable by virtue of cuticular microstructures , supportvs. proximal directions, and was found to be 5.8 ± 0.5° and 8.5 ± 0.4° respectively . The ratio in the force of retention in the proximal vs. distal directions , and 7% in Episyrphus balteatus (De Geer 1776). Hairs viewed on the sections in To establish the relationship between microtrichial orientation and wing topography, chordwise wing sections were examined . Maximume.g. A, B, and H. The orientation of microtrichia in the anal region of the wing  is ambiguous, suggesting that this area does not have a preferred upslope orientation. Because this region is more flexible than others, there is no static depression in which drops may become stuck; thus, an upslope orientation may be less important here. However, because the sections of This upslope trend was not observed everywhere. Any portion of the sections in ridge peaks) were identified. The direction from the centre of each quadrat to the nearest point along a ridge peak was used as a proxy for the local upslope direction. In 16 of 24 quadrats, the microtrichia pointed more upslope than downslope on average. Under a binomial distribution, there is only an 8% chance that this or an even stronger upslope bias would be observed from randomly oriented microtrichia in a random sample of 24 quadrats. Thus it is most likely that the overall upslope bias is a real effect. Because not every hair pointed upslope, however, additional factors are at play in determining the local microstructure orientation.Based on the sections in r = 0.52 and p = 0.01 from a least-squares linear fit). However, the least-squares linear fit exhibits R2 = 0.27, indicating that distance to the nearest edge explains only a small portion of the observed variation in microtrichial orientation.Hairs close to the wing margin (or edge) were observed to orient normal to the margin; another factor that appears important in determining local orientation. In 18 of 23 quadrats, the microtrichia pointed, on average, more toward the nearest edge than away from it. There is only a 0.5% chance that, in a random sample of 23 quadrats, an equal or greater proportion displaying edge orientation would be observed if microtrichia were randomly oriented. Thus, the bias towards the nearest edge appears to be another real effect. As quadrat distance to the nearest edge increases, so too does the residual between the angle of orientation to the nearest edge and the angle of microtrichial orientation (D. melanogaster (Meigen 1830) [et al. [M. aega and that drops roll most easily away from the body, suggesting that a distal orientation of microstructures may reduce the likelihood of drop shedding onto the body. Of the 462 microtrichia sampled on the P. heteroptera wing, 461 pointed more distally than proximally. This indicates a very strong bias for microtrichia to point away from the body, preventing drops from shedding onto the body from the wing. The only hair that oriented proximally exhibited ϕ = −87.5°; a very small proximal bias. In this case, the hair was close to a ridge peak, and the upslope vector pointed proximally.A distal bias in microtrichial orientation has been noted in en 1830)  and can P. heteroptera wing displays 47% greater drop adhesion in the proximal compared to distal direction. Greater adhesion in the proximal direction is expected given the observed distal bias, and supports the role of microtrichia as directional antiwetting devices. However, it does not definitively exclude potential contributions by other features (e.g. the greater density of veins near the base).The The analysis of sections and sampled quadrats shows strong upslope, nearest-edge, and distal biases; however, few hairs pointed precisely upslope, precisely to the nearest edge or precisely distally. We seek a simple mathematical description of how these factors counterbalance in determining the local orientation observed in each quadrat, which could be used to predict local microtrichial orientation anywhere on the wing, and could even be extended to other species. This model is established in the next section.W1 is the unitless weighting factor for the upslope bias.Our mathematical model denotes hair direction as a unit vector in two dimensions  is the displacement vector to the nearest edge (with units of μm) and W2 is the weighting factor for the edge-weighting term , indicating that the model explains 64% of the observed variance in local orientation.Despite these artifacts, e.g. by reducing W2 in the anal area).The similarity between model and observation highlights the explanatory power of our model in relating microstructure orientation to antiwetting function and large-scale wing morphology. However, there is room for improvement. First, we noted a prediction of divergent orientation that was not observed in the anal area . This waP. heteroptera; such an approach would facilitate construction of a significantly more detailed and complex model, and is beyond the scope of this study. Adding to the difficulty of accurate measurement, the “upslope direction” may also have limited functional consequence in the anal area, as here the wing is highly compliant and the nearest peak is a relatively large distance away , but the gradient may actually point in a different direction. Micro Computed Tomography (micro-CT) techniques have been used to create detailed three-dimensional reconstructions of large insect wings , howevernce away . The upsW1 and W2 in Finally, the weighting factors Although opportunities for improvements remain, the simple model implemented in the present study was able to capture, both quantitatively and qualitatively, most aspects of local microtrichial orientation in this species. By employing constant rather than variable weighting values, our model possesses appealing simplicity and a reduced number of variables. The ability to predict local microstructural orientation accurately based on higher-order wing morphology and functional arguments supports the role of microtrichia as directional antiwetting devices. This ability also highlights local variation in orientation as a balance between different, and sometimes competing, antiwetting strategies.upslope orientation), (2) shedding water off the wing as quickly as possible (nearest-edge orientation), and (3) shedding water away from the body . We determined the relative importance of these adaptive pressures in the dipteran Penthetria heteroptera through optimization of weighting factors compared to observed local orientation sampled on the dorsal side of the wing. The model explained the majority of the variation of orientation observed when the optimal weights were applied, which supports the role played by microtrichia as directional antiwetting devices. The simple model that we have presented can be readily translated to other insects with directional microstructures, and underscores the importance of considering both function and local wing geometry to explain the organization of natural microstructure arrays.Previous studies have demonstrated that microstructures on insect wings promote directional drop shedding, but exhibit complex variation in orientation throughout the wing. We have proposed a simple two-dimensional mathematical model to explain local variation in microstructure orientation as a balance between differing adaptive pressures in terms of antiwetting: (1) keeping water raised above the surface and preventing pinning in grooves Click here for additional data file.S1 FileFor the case of the ridge peaks, each ridge starts at a starting node (denoted by “1” in the starting node column) and ends at the coordinate immediately prior to the next starting node. Pixel coordinates refer to (CSV)Click here for additional data file.S2 FileQuadrats are 75 px by 75 px. Pixel coordinates refer to (CSV)Click here for additional data file.S3 Fileϕ are standard angles measured from the image positive X axis, according to a cartesian coordinate system where Y is vertical in the image (see Quadrats are numbered as in (CSV)Click here for additional data file.S4 Fileϕ are standard angles measured from the image positive X axis, according to a cartesian coordinate system where Y is vertical in the image (see Quadrats are numbered as in (CSV)Click here for additional data file.


Amblyglyphidodon curacao, we quantified inter- and intra-individual variation in behavioural and kinematic components of escape performance during repeated presentations of a stimulus at 15 min intervals. Individual maximum escape performance was repeatable through time, but there was considerable variation in the magnitude of responses both among and within fish. We found no evidence of habituation or fatigue due to repeated stimulations, suggesting that fish can be stimulated multiple times to ensure that an accurate estimate of maximum escape performance is obtained.Fast-start escape responses are critical behaviours used by fishes during predator-prey encounters and some interactions with hetero- and conspecifics. In experimental studies, escape responses are often measured once per individual and considered representative of maximum performance. However, few studies have compared variability and repeatability in escape performances within and among individuals. Using the tropical damselfish  Summary: Quantification of the inter- and intra-individual variation in behavioural and kinematic components of escape performance of the tropical damselfish Amblyglyphidodon curacao found no evidence of habituation or fatigue due to repeated stimulations. Survival following an encounter with a predator depends heavily on the prey's escape performance. To escape attacking predators, many fish species perform sudden accelerations called fast-start escape responses . Fast-stDomenici, 2010aUnlike the first two stages of the fast-start escape response that generate most of the acceleration, stage 3 can be highly variable and involves continuous swimming, coasting or breaking . TherefoThe fast-start escape response of fishes has long been considered a stereotyped behaviour, most likely because of its very short duration and critical role in predator evasion. Stereotyped behaviours are defined as exhibiting little variation across trials under a given set of conditions . HoweverAmblyglyphidodon curacao, the staghorn damselfish (Teleostei: Pomacentridae). Other than for escaping predators, A. curacao commonly employs fast-starts to terminate interactions with cheating cleaner fish, Labroides dimidiatus  above 0.20 for all escape response components (A. curacao had consistently high latencies (>200 ms), indicative of non-Mauthner-cell escapes . Response latency was also the only escape component measured that varied significantly more among than within individuals: the CV among individuals fell outside the 95% CI of the mean CV within individuals  . With the exception of response latency, which varied considerably among individuals (see previous paragraph), the variation in escape performance observed among trials in the same fish  was similar to that observed among different individuals: the 95% CI of the mean CV within individuals overlapped the CV among individuals  . This re P≤0.01)  and sugg P≤0.01) . Importaividuals . Given tDesc, Umax, and Amax, . Although a very short time interval (5 s) between stimulus presentations causes a reduction in the magnitude of escape responses   .Table 1A. curacao. The magnitude of this variability was similar within and among individuals. Although performance measures are repeatable, we recommend performing several stimulations per individual when possible to improve the accuracy of maximum escape performance estimates. The number of repetitions should be positively related to the magnitude of variation within individuals  at the Lizard Island Research Station  in Queensland, Australia, during September 2014. Fish were caught on reefs surrounding Lizard Island using barrier nets and housed in flow-through aquaria with water transported directly from the reef. Fish were given a 10 cm diameter PVC pipe as shelter and were fed daily with commercial fish flakes up to 24 h prior to the experiments. Research was conducted with approval from the Great Barrier Reef Marine Park Authority (G37047.1) and the Queensland Animal Ethics Committee (CA 2014/06/780).Experiments were performed on 14 −1) to maintain a stable temperature  and high oxygen levels (>90% saturation). Each experimental tank was illuminated with two 500 W work lights placed at opposite ends of the tank. The water depth was maintained at 20 cm to minimize fish displacement in the vertical plane but allow full extension of the anal and dorsal fins. A mechano-acoustic stimulus was used to initiate escape responses. Two plastic cylinders with a tapered tip  were suspended 70 cm above the water surface on either side of the tank, 13 cm from the edge . The experimenter was shielded behind a black tarp where the camera could be operated and the electromagnet switched off. A mirror was placed under each plastic pipe to record the exact timing of when the stimulus hit the water surface.Fast-start trials were conducted in two large grey circular tanks  with flow-through seawater (6 litres minEach fish's centre of mass (CoM) was marked dorsally with a piece of reflective tape placed on each side of the dorsal fin . A singlDesc) (distance covered by the CoM in 42 ms), maximum velocity (Umax), and maximum acceleration (Amax); (3) stage 1 turning rate, calculated as stage 1 turning angle divided by stage 1 duration (with stage 1 turning angle defined as the angle between the linear body segment running between the CoM and the tip of the snout at the beginning of the response and the same body segment at the end of stage 1). We used a five-point quadratic polynomial regression  and initial distance from the stimulus (distance between the CoM and the stimulus) to check for an influence of these variables on escape performance.n=14), (2) Desc (n=12), (3) Umax (n=12), (4) Amax (n=12), and (5) turning rate (n=12). Two fish that had consistently weak responses and long latencies were excluded from the kinematic analyses. Fish size (TL), distance to the stimulus and body angle relative to the stimulus were included as covariates in the models. Fish ID was specified as a random factor to account for the non-independence of measurements on the same individual through time. Homoscedasticity and normality of residuals were checked with plots of residuals versus fitted values and qqplots of residuals. Response latency was log transformed to meet model assumptions. We calculated the marginal R2  and conditional R2  following We used general linear mixed-effects models (LMM) to test the effect of trial order (accumulated fatigue and/or habituation to repeated presentations of the stimulus) on five measures of escape performance: (1) escape latency  Umax, (4) Amax, and (5) turning rate were chosen from the five escape responses for each fish, and these two values were compared for each individual using a Pearson correlation. Intraclass correlation coefficients are presented in supplementary material Table S2. We also calculated the coefficient of variation  of these five performance measures for each fish, across all five stimulus presentations. The CV is a unitless measure of dispersion relative to the mean, which allows comparing the variability of different variables , which represents variation among individuals, taking into account all five stimulus presentations.We tested repeatability following ariables . We thenhttp://dx.doi.org/10.6084/m9.figshare.1421988).Analyses were done in R 3.1.2 . Data ar


Sixty randomly selected DM1 patients (14.5 ± 3.2 years) and 30 healthy (13.5 ± 2.8 years) volunteers were enrolled in the study. Our results indicate that three monocyte subsets are distinguishable in the groups of young diabetic patients and the healthy, similarly to in the blood of adults. DM1 patients were characterized by higher values of apoptotic monocytes than the healthy. The manipulation with drugs inhibiting TNF-R1 expression diminished the pool of CD16+ apoptotic monocytes. Infliximab reduced the apoptotic CD16− cells. In conclusion, diabetes mellitus type 1 is associated with greater apoptosis of three monocyte subsets which may contribute to the development of microvascular complications. TNF-α modifiers appear to ameliorate monocyte apoptosis. They may be useful for controlling excessive monocyte apoptosis in diabetic patients.Diabetes mellitus type 1 is associated with an enhanced apoptosis of different cells and tissues, accelerating occurrence of diabetic microvascular complications. The aim of our study was to determine spontaneous apoptotic potential of the monocyte subsets in juvenile-onset complication-free diabetes mellitus type 1 and to compare them with the corresponding values of the healthy. Moreover, we wanted to assess effects of TNF-R1 blocking agents and those of general TNF- One of the manifestations of this is prolonged survival of immune B cells and both CD4+ and CD8+ T lymphocyte subsets [α-induced apoptosis than those from control animals [Diabetes mellitus type 1 (DM1) is associated with defects in TNF- subsets . On the  subsets –5. The a animals . Neutrop animals , 8.κB transcription factor, which is physiologically bound to the inhibiting Iκ-Bα subunit. NOD mice, therefore, do not show degradation of the Iκ-Bα subunit, which permanently suppresses the NFκ-B-dependent transcription of cell survival-promoting proteins. Thus, stimulation of lymphocytes by TNF-α preferentially triggers the proapoptotic pathway [Molecular studies in NOD mice have revealed several genetic defects that alter the immune function within the MHC genomic region. A point mutation has been localized in the shared promoter-enhancer region of genes encoding LMP2 and TAP-1 proteins, which negatively regulates the transcription of these genes. This mutation impairs proteolytic cleavage and activation of the NF- pathway , 9, 10.κ-Bα subunit. A single amino acid substitution defect was found to prevent the NFκ-B-dependent activation. Such negative regulation of the NFκ-B transcription factor hampers transcription of the prosurvival and antiapoptotic proteins [In humans, the genetic defects that are important for altered apoptosis are distinct from those in NOD mice. An important role has been ascribed to a mutation identified in a gene-coding small ubiquitin-like modifier 4 (SUMO4) . The SUMproteins , 12. Anaproteins .α may be through the proapoptotic TNF-R1-dependent path [Many more molecular defects are expected in DM1, but these have not yet been detailed. Different malfunctioning proteins have already been found in the TNF-R2 signaling pathway, resulting in inefficacies in the TNF-R2 pathway which is important for cell survival and cell proliferation. Thus, in the case of nonactive TNF-R2 receptors, the only way of signaling by TNF-ent path . All theα, the chief inflammatory mediator of microangiopathy, triggers a sequence of biological events that culminate in apoptosis of vascular endothelial cells [α on the expression of adhesive molecules on both endothelial cells and leukocytes. Adhering inflammatory cells produce an array of angiogenic, inflammatory, and fibrogenic factors that promote endothelial cell-junction breakdown, blood-retinal barrier loss, and injury and apoptotic death of retinal endothelium and pericytes [It has been recognized that enhanced apoptosis underlies retinopathy and nephropathy, the late diabetic microvascular complications , 5. TNF-al cells . The exaericytes –16. Longericytes . Similarericytes . A largeericytes .+CD16+ monocytes comprise about 10% of the whole CD14+ monocyte population. They are enriched in genes associated with the differentiation processes for an antiproliferative and proapoptotic state. The CD16+ subsets are expanded in different kinds of inflammatory disease, such as rheumatoid arthritis, Crohn's disease, HIV, hepatitis, severe asthma, coronary artery disease, end-stage renal disease, sarcoidosis, tuberculosis, and stroke [ In vitro experiments on whole blood cell cultures have revealed that the CD16+ monocytes may be generated by TNF-α treatment to approximately 30% of the total monocytes. In the blood of septic patients, the number of these cells correlated with the blood levels of TNF-α [+ cells as compared to those originating from classical monocytes [+ monocytes produce chemokines that favour their migration to the vascular wall [α, releasing this cytokine preferentially on the layer of endothelial cells and not spilling it over to the blood stream [Monocytes appear to be a heterogeneous population. A subset of monocytes, the so called “nonclassical” CD14d stroke –22. In vof TNF-α . The denonocytes . CD16+ mlar wall . Thus, td stream .++CD16− cells; it constitutes the main monocyte pool, comprising about 85% of the whole monocyte population. They are mobile cells and may be rapidly recruited from the blood to the infected tissues, where they undergo activation and transform into the CD14+CD16+ forms. When stimulated, these cells produce a set of inflammatory mediators including IL-8, CCL-2, CCL-3, IL-6, TNF-α, NO, and ROS [The second monocyte subset is the “classical” CD14 and ROS . The chaAlthough the distinction between monocyte subsets has been demonstrated in different diseases and in healthy volunteers, there are no data to support the notion that such differentiation also exists among healthy juveniles and DM1 patients. Moreover, bearing in mind the general apoptotic defect in DM1, it would be interesting to characterize the spontaneous apoptotic potential of these monocyte subsets, if it exists. To date no data has appeared concerning the spontaneous apoptotic capacity of monocyte subsets in DM1.++CD16− and CD14+CD16+ monocyte subsets in juvenile-onset complication-free diabetes mellitus type 1 and to compare them with the corresponding values in the healthy age- and sex-matched control group. Next, we planned to enumerate the spontaneously apoptotic monocytes within both subsets in the DM1 patients and to compare the results with the values for the healthy subjects. Moreover, since the data imply that the proapoptotic potential of a DM1 patient's cells may be dependent on an overactive TNF-R1 pathway, it would be interesting to determine the expression of TNF-R1 receptors on monocytes and then to manipulate this with TNF-R1 blocking or enhancing agents so that its effect on monocyte apoptosis may be observed. Finally, we wanted to assess the effect of infliximab, which is a chimeric monoclonal antibody against TNF-α with a murine variable region and a human immunoglobulin constant region, on the apoptosis of monocytes of DM1 patients and healthy controls.The aim of our study therefore was to provide quantitative data on CD14The group examined consisted of 60 randomly selected children and adolescents aged 14.5 ± 3.2 years (28 boys and 32 girls) with long-standing DM1  from the Diabetology Outpatient Clinic at the Medical University of Gdańsk, Poland. The diagnosis of DM1 was made in accordance with the American Diabetes Association criteria [1c was measured by an immunoturbidometric method using the Unimate 3 set  with a normal range of 3.0–6.0%. Fasting glucose was measured by an enzymatic test . Urinary albumin excretion was expressed as the average of three 24-hour collections obtained during the six months prior to enrolment in the study. Microalbuminuria was defined as albumin excretion within the range of 30–299 mg/24 hours in at least two out of three urine samples. Urinary albumin excretion was measured by the immunoturbidometric assay using Tina-quant .HbA5/well. Fc receptors were blocked with 1 μg of human IgG/105 cells for 15 minutes at room temperature.Peripheral blood mononuclear cells were obtained by density gradient centrifugation on the Ficoll/Isopaque . Isolated peripheral blood mononuclear cells were placed on 24-well plates in RPMI 1640 containing 5% FCS  at a density of 1 × 10μL of infliximab . The third set was incubated with one of the following substances: anti-TNF-R1 mAb (clone H398) 10 μg/mL , anti-TNF-R2 mAb (clone M1) 10 μg/mL , 16 μM, sodium salicylate 10 mM, and 4-bromophenacyl bromide (BPB) . Plates were incubated for 24 h at 37°C with 5% CO2. After incubation plates were kept for 5 minutes on ice.Samples from each patient were divided into three series. The first was left without additives and the second was kept with 50 μL of 5% RPMI and the following mAbs were added: 4 μL anti-CD14 PerCP  , 3 μL anti-CD16 APC-Cy7 or FITC  , and 5 μL anti-TNF-R1 PE  . Cells of the healthy volunteer group were divided into only two parts, those without a modifier and those with infliximab.The cell suspension was transferred to cytometric tubes. The tubes were rinsed three times with cold PBS and then centrifuged and the supernatants removed. The sedimented cells were suspended in 200 μL of Annexin V Binding Buffer , supplemented with 3 μL of Annexin-FITC . Plates were incubated for 15 minutes in the dark at room temperature. After incubation the tubes were filled with 400 μL of Annexin V Binding Buffer and read on a cytometer. The measurements were performed using a LSRII flow cytometer  and equipped with a solid state Coherent Sapphire blue laser , a solid state UV Laser-light wave X-cyte laser , and a helium-neon gas laser . The expression of cell surface markers was assessed after gating on live cells determined by scatter characteristics.Parallel with specific staining fluorochrome-conjugated isotypic controls were run. Plates were incubated for 30 minutes in the dark on ice and were then centrifuged and the supernatant was discarded. After being washed with PBS, the cellular pellet was suspended in 70 Data were analyzed using the FACS Diva 6.0 Software . The region containing monocytes was generated on the basis of their forward versus right-angle light scatters. Typically, 10,000 events were acquired in this region. CD14 monocytes were assessed using an SSC/CD14 dot plot. The monocyte subsets were analyzed from dot plots representing CD14 versus CD16 staining. The threshold level for the fluorescence of positive cells was set for each sample from a difference between curves obtained from the specific mAb and isotype control mAb staining. TNF-R1-positive cells were analyzed in the CD14PerCP and CD16 FITC gates.P < 0.05 and two-sided tests were performed as the standard.The results were analyzed using Statistica Version 10 . Continuous variables were tested for normality using the Kolmogorov-Smirnov test. Normally distributed variables were analyzed with the ANOVA test. For comparison of the skew-distributed variables the nonparametric Kruskal-Wallis ANOVA test was applied. The significance level was set at ++CD16− and CD14+CD16+ cells , an irreversible inhibitor of cyclooxygenase (Cox) activity, significantly reduced the percentage of CD16+ apoptotic monocytes. Anti-TNF-R2 antagonistic mAb had no significant effect on the parameters examined (data are not presented here) (It appeared that anti-TNF-R1 antagonistic mAb significantly decreased the percentage of apoptotic cells only within the CD16ed here) .− and CD16+ monocyte subsets by more than 90%. Our results indicate therefore that the TNF-R1 receptor transduces the apoptotic signal in the monocytes of DM1 patients. It is easy to manipulate the apoptotic ability of diabetic monocytes through TNF-R1 modifiers (Bromophenacyl bromide (BPB), an agent potentiating apoptosis, increased the percentage of the apoptotic cells in both CD16odifiers .α drug, may also control the apoptotic potential of monocytes. The effect of infliximab was analyzed on the TNF-R1+ apoptotic monocytes in the DM1 patients and the healthy controls. + Annexin V-positive monocytes.The next stage was to pose the question of whether infliximab, an approved anti-TNFα and hinders its access to the TNF-R1 and TNF-R2 receptors. Its effect on DM1 patients' monocytes has not been known. In order to learn whether infliximab may influence the apoptotic CD16− and CD16+ monocytes, peripheral blood mononuclear cells were incubated in a culture medium for 24 hours either without additives or with 50 μL of infliximab. After incubation the mAb against CD14, CD16, and TNF-R1 were applied together with Annexin V for visualization of the TNF-R1+ apoptotic monocytes. The effect of infliximab on the DM1 patients' monocytes is presented in Infliximab binds and neutralizes the soluble and transmembrane forms of TNF-−TNF-R1+ apoptotic monocytes while having no effect on the corresponding values of the CD16+TNF-R1 apoptotic cells /Apo2 protein, which is responsible for transduction of the TNF-dependent apoptotic signal, simultaneously generates enhanced expression of adhesion molecule ICAM-1 on endothelium in conjunction with enhanced expression of its ligand, the CD18 molecule, on blood cells [While it is obvious that apoptosis of different cells and tissues poses a serious risk of injury to the organs targeted and of functional failure in these organs, not much is known about the role of apoptotic monocytes in DM1. It can, however, be deduced that apoptotic monocytes cause serious deterioration of the target organs of late diabetic complications. These cells may be causally related to the accelerated development of retinopathy and nephropathy. Apoptotic monocytes may directly stimulate endothelial cell activation and enhance adhesion of blood cells to endothelium. This has been documented in human vascular endothelial cellsod cells . The (TRod cells . In thisod cells . At the od cells . Greaterα receptors initiate the apoptotic process, the technique of Annexin V binding was used for enumeration of the apoptotic cells. This technique detects early apoptotic events and enables the TNF-R receptors to be identified on cells in initial phase of apoptosis. Our results therefore indicate that the proapoptotic potential of a DM1 patient's monocytes is dependent on the TNF-R1 pathway. In this paper, we presented that manipulation with drugs that inhibit TNF-R1 expression [+ monocytes undergoing apoptosis, while CD16− monocytes appear nonreactive towards TNF-R1 inhibitors. On the other hand, augmentation of apoptosis by PBP [α modifiers may be useful for controlling excessive monocyte apoptosis in diabetic patients. A reduction in CD16+ apoptotic monocytes may be achieved by means of such widely used TNF-R1 blockers as sodium salicylate.In order to determine which of the TNF-pression –35 may ds by PBP  reached + monocytes in juvenile-onset complication-free DM1 patients during a five-year observation period, during which the percentage and absolute number of the CD14+CD16+ monocytes doubled [We have recently documented an expansion of the CD16 doubled . The preα, suggesting that this cytokine plays an important role in the expansion of the CD16+ monocyte pool. From experiments conducted in vitro with monocytes, TNF-α-mediated apoptosis of these cells appeared to be regulated by paracrine signaling. TNF-α binds to the TNF-R1 receptors, triggering the apoptotic signal. When apoptosis starts the TNF-R1 receptors are still on the monocyte surface, but later the TNF-α-TNF-R1 complexes become internalized and the number of TNF-R1-positive monocytes diminishes [Our previous study indicated that the size of the nonclassical monocyte subset correlated with the blood level of TNF-minishes .+ apoptotic monocytes, while infliximab may be appropriate for the reduction of apoptotic CD16− cells. The two approaches complement each other.Since an enlarged pool of apoptotic monocytes in DM1 patients increases the danger of leukostasis in the small vessels of the target organs of microangiopathy, it would be desirable to reduce the number of these cells in these patients. Our results suggest that TNF-R1 inhibitors such as sodium salicylate and its derivatives may be useful for eliminating the CD16Our experiment with sodium salicylate as TNF-R1-suppressing factor indicates that this widely used anti-inflammatory agent may confer benefits on diabetic patients. Hitherto this substance has not been recommended in DM1 therapy. However, previously published results for NOD mice indicate that sodium salicylate derivatives are effective in protection from retinopathy, which is in keeping with our data .Infliximab has, to our knowledge, not been used in experimental therapy for DM1 patients. However, a recent clinical study indicates the beneficial effect of Etanercept, the other general TNF-inhibitor, on the clinical manifestation of DM1 . Moreoveα and by this means moderates the degree of inflammation. Infliximab causes the TNF-R2 receptors to be shed from monocytes and stimulates interleukin-10 secretion in monocyte cultures in vitro [In our study, infliximab appeared to moderate apoptosis of monocytes in juvenile DM1 patients. It has been known to neutralize soluble and membrane TNF-in vitro . This inin vitro . Moreovein vitro  and selein vitro . Its putα activity in type 1 diabetes mellitus should be undertaken in complication-free phase of the disease. The study done by group of researches analyzing monocytes in patients with advanced diabetes type 1 and type 2 showed that in patients with late diabetic complications prevails the anti-inflammatory profile with an enhanced IL-10 blood level and CD16+ monocytes are at lower frequency as compared with patients without complications. This paper suggests also that the disappearance of CD16+ monocytes from blood is associated with their transformation into macrophages [α modifiers.It is important to realize that modification of TNF-rophages . Macrophα agonist [In conclusion, diabetes mellitus type 1 is associated with greater apoptosis of monocyte subsets which may contribute to the development of microvascular complications. TNF-R1 modifiers, together with infliximab, appear to ameliorate monocyte apoptosis and should be further examined for their assistance in the treatment of diabetes mellitus as protective agents against late diabetic microvascular complications. They should join other experimental forms of treatment currently under consideration for preventing capillary leukostasis, such as treatment with statins , a PPARα agonist , or anti agonist .


Eye-tracking technology has been used to measure human cognitive processes and has the potential to improve the usability of health information technology (HIT). However, it is still unclear how the eye-tracking method can be integrated with other traditional usability methodologies to achieve its full potential.The objective of this study was to report on HIT evaluation studies that have used eye-tracker technology, and to envision the potential use of eye-tracking technology in future research.We used four reference databases to initially identify 5248 related papers, which resulted in only 9 articles that met our inclusion criteria.Eye-tracking technology was useful in finding usability problems in many ways, but is still in its infancy for HIT usability evaluation. Limited types of HITs have been evaluated by eye trackers, and there has been a lack of evaluation research in natural settings.More research should be done in natural settings to discover the real contextual-based usability problems of clinical and mobile HITs using eye-tracking technology with more standardized methodologies and guidance. Health information technology (HIT) systems are promising tools for improving quality, patient safety, and efficiency in health care systems -4. This Eye-tracking technology has been used to measure cognitive processes since the 1970s . HoweverTwo important measurements of eye-tracking technology are fixation and saccade . FixatioEye-tracking data can be integrated, synthesized, and visualized using software suites, such as commercially available analysis tools. Different types of visualizations, such as heat maps and gaze plots, communicate different types of information . A heat Based on the International Organization for Standardization (ISO) Standard, usability is the extent to which users can achieve a goal effectively, efficiently, and with satisfaction . Due to We conducted a systematic online database search to identify articles published before September 2014 that were relevant to the aims of this study. Articles were included as indexed in four reference databases: Medline, Web of Science, ScienceDirect, and PsycINFO. Broad keyword searches were used to identify relevant articles in each database. Each initial search focused on one of three key components: (1) a word or phrase related to usability evaluation, (2) a word or phrase related to HIT, or (3) a word or phrase related eye-tracker technology.Keywords related to usability evaluation included usability testing, user experience, user test, user-centered design, system design, interface design, and interaction design. Keywords related to HIT included health IT, health informatics, health technology, medical technology, eHealth, telemedicine, communication tools, educational technology, decision support technology, health app, and wearable technology. Keywords related to eye-tracker technology included eye-tracking technology, eye tracker, Tobii, Sensomotoric Instruments, eye movements, gaze, eye fixation, and saccade. We also identified potentially eligible articles by manual literature searches, by examining article reference lists and by searching in Google Scholar.We initially defined the scope of the review by determining inclusion and exclusion criteria. Papers were included if they contained all of the following: (1) the research used eye-tracking technology as a data collection tool, (2) the research evaluated an HIT with users, and (3) the research explicitly mentioned the improvement of HIT usability based on the eye-tracking data. Papers were excluded if they (1) were not in English, (2) were published 10 or more years ago , (3) did not evaluate an HIT, (4) focused on technologies not related to health care, (5) used eye-tracker technology for some purpose other than data collection , or (6) did not mention any indications of the system usability based on the eye-tracker data.Based on the methods-description approach, we analyzed the selected papers that met the inclusion criteria . Key artA total of 5248 papers were found by using the search terms and databases described above. Of these, 1888 papers were removed due to duplication. After reviewing the titles and abstracts based on inclusion and exclusion criteria, we eliminated another 3351 papers. This resulted in a total of 9 papers remaining for this review see . An overAll selected papers discussed user evaluation of a type of HIT using eye-tracking technology as a data collection tool. Of the 9 papers, 3 of them 33%) mainly discussed a usability evaluation of an HIT using eye-tracker technology % mainly ,40. Of tWe identified different research questions that are answered by eye-tracker technology in the selected papers. The first question that can be answered by eye-tracker technology is whether the user experience and performance using an HIT has been improved based on the eye-gaze patterns, which primarily reflects the system effectiveness, efficiency, and user satisfaction -36,40,41We identified different types of HITs in the selected papers. In terms of functionality, the technologies included online health information website interfaces -40, surgWe identified different experimental apparatuses of the user tests. Researchers evaluated HITs in the forms of developed computer website/application ,34,38,40Out of the 9 papers, 2 of them (22%) reported a failure to collect eye-tracking data during the usability test ,39. Of tWe identified three basic eye-tracker measurements in our selected papers. The measurements included fixation duration ,34,38-40The selected papers also showed other usability evaluation methods that are combined with eye-tracker data to explore usability problems in HIT systems. The methods include the System Usability Scale (SUS) ,35,36, tThe purpose of this literature review was to examine usability evaluations of any type of HIT using eye-tracking technology. This review also aimed to identify the research gap and potential uses of eye-tracker technology in future HIT research. This review was conducted based on the inclusion and exclusion criteria specified in the Methods section. Based on the results, we determined that, although eye trackers provide rich data for the improvement of HIT systems, the use of eye trackers for usability evaluation of HITs is still in its infancy, as only 9 papers were found that fit within the inclusion criteria.We organized the results into five main questions: (1) What research questions are answered by eye-tracking technology?, (2) What types of health care information technologies were evaluated using an eye tracker?, (3) What was the experimental apparatus of usability evaluation?, (4) What did the eye trackers measure and how was data analyzed?, and (5) How was eye-tracker technology combined with other usability methods?Papers that were included in this review had different purposes and research goals. The types of HITs evaluated were limited, resonating with our finding that the use of eye trackers for the evaluation of health IT is in an early stage. However, eye trackers are becoming a promising tool for usability studies, as demonstrated by the increasing number of research studies in recent years. We also found that researchers used various means of data collection and analysis using eye trackers. On the one hand, this demonstrates the rich variety of data that can be captured by eye trackers and the flexibility of interpretation of eye-tracker data. On the other hand, it shows the lack of a consensus on how to conduct user evaluation of HITs using eye trackers at this stage. In addition, we found that eye-tracking technology, as a part of usability evaluation methodology, was supplemented by other traditional methods. Generally, eye-tracking data can reveal the patterns of user difficulties when completing tasks using HIT, while other supplemental inquiries are used to unfold the reasons behind those patterns. Therefore, eye-tracking technology has to integrate with other techniques, as most physiology measurements do, because eye-tracking technology alone cannot tell the entire story.The reviewed papers reflected different research questions that were answered by eye-tracking technology. Of the 9 papers, 6 of them (67%) were directly related to the system usability, focusing on the efficiency, effectiveness, and satisfaction when completing tasks with a specific HIT. Of these, 1 paper was related to the examination of a gap between visual and cognitive process. For example, a user missed information because he/she did not pay attention to it, even if eye-tracking technology suggested that the user had seen that information . AnotherThe reviewed papers involved five different types of HITs, including 3 out of 9 papers (33%) evaluating health information website interfaces, 3 papers (33%) evaluating decision support systems, 1 paper (11%) evaluating a surgical interface for physicians, 1 paper (11%) evaluating a computerized provider order entry system for physicians, and 1 paper (11%) evaluating a symptom and quality-of-life information system for physicians. The reviewed papers involved three different types of users, including 3 papers (33%) for general public health IT, 2 papers (22%) for patients, and 4 papers (44%) for physicians. Thus far, eye trackers have been used most often to evaluate health information website interfaces. This indicates that evaluating a website interface using eye-tracker analysis may provide rich theoretical guidance and reveal available practices that researchers can refer to ,43. MoreHowever, there is much potential for eye-tracker technology to be applied to other types of health IT as well. One particular aspect of health IT that lacks usability research using eye-tracker technology is electronic health record (EHR) systems. EHR systems have helped to revitalize physician and nursing practice, and have the potential for positive impact on clinical processes in terms of efficiency, productivity, and patient safety . Health Another gap exists in the application of eye-tracking technology to usability studies of novel consumer HITs. Health apps and devices are becoming prevalent in the market. Devices such as the iPad, iPhone, iPod Touch, and Apple Watch have been the target devices to provide a richer and more convenient user experience of health care information technology . WearablIn terms of the prototypes that were evaluated in the reviewed papers, a majority of them  evaluated developed computer programs that have already been adopted in health care systems. Of these papers, 2 of them evaluated screenshots of the real websites, and 2 of them evaluated usability using a simulated prototype in high fidelity. Unlike many other usability techniques, such as the formative usability evaluation approach, that are primarily introduced in the early phase of the user-centered design process, we found almost all the papers evaluated HITs at a very late phase or even after implementation, as a summative approach. The benefit of doing a summative usability evaluation is that researchers are able to create an approximation of the actual use scenario of HITs. Compared with low-fidelity, nonfunctional prototypes, such late-phase testing is more likely to uncover real usability problems . HoweverUnfortunately, at this point there has been no usability evaluation conducted using eye-tracking technology in real settings. All of the reviewed papers conducted user studies in a meeting room or a usability lab. Possible reasons for this gap could be the mobility limitations of eye-tracker technology, the possible intrusion of such technology on work, technical difficulties, and the calibration process of the eye-tracking equipment. Nearly half of the papers  used eye trackers that were embedded within a computer screen, which are impossible to move into real settings. Of the 9 papers, 2 of them (22%) used head-mounted eye trackers, which are easy to move but intrusive to the health care provider’s work if the evaluation is conducted in the field, negatively influencing the work in that time-sensitive environment. Moreover, such head-mounted trackers are more likely to have technological difficulties, which risk accurate data collection. Of the 9 papers, 3 of them (33%) used mobile eye trackers, which are probably the best equipment to be incorporated into field research in the real HIT setting. However, the calibration process may add additional steps to the already complex workload of nurses or physicians. Moreover, it is unlikely that a health care provider will stay in one place for a long period of time, and their movements will disrupt the calibration . DespiteWe found that the eye-tracker measurements in the reviewed papers were mainly fixation and saccade, which supports the finding by Poole and Ball . More thQualitative analysis is becoming prevalent partly because of the improvement of software suites, making the analysis easier and less intensive. Of the 9 papers, 3 of them (33%) translated the data into qualitative visualization, such as heat maps and gaze plots. While statistical analysis is powerful in comparing completion time and errors, it is only part of the usability evaluation. For a full usability evaluation, we believe the qualitative data in visualization can illustrate more usability problems. Using a heat map, it is easy to determine if specific content is usable or not. Using a gaze plot, it is possible to determine if users follow an efficient and predetermined route when searching for specific information on the interface.However, we found that the interpretation of such visualizations lacked scientific guidance based on an established theoretical method, so interpretations tended to seem arbitrary and subjective. At this point, researchers are struggling to find a theory or a commonly used procedure to guide the interpretation of heat maps and gaze plots. Therefore, we expect that in the future a more structured system of interpretation will be developed for heat maps and gaze plots.With the visualization of eye-tracker data, researchers can identify the areas of an interface that have created difficulties in participants’ minds. However, based solely on the eye-tracker data, there is no way to understand the precise cognitive reasons behind a participant’s eye-gaze patterns. For example, there might be many possibilities for an eye fixation, such as fatigue, distraction, confusion, and engagement ,60. TherIt is interesting that there are three different think-aloud methods used in the reviewed papers: concurrent think aloud, RTA, and a hybrid of both. The concurrent think-aloud method is the traditional method widely accepted and applied by usability evaluation researchers. It is a method that asks participants to verbalize their thoughts while interacting with the system . HoweverAnother reviewed paper used a hybrid of concurrent think-aloud and RTA methods . The resAlthough eye tracking is a promising technology, the application of eye-tracking technology to health IT usability evaluation is still in its infancy, with limited theoretical guidance and practice. Therefore, we reviewed papers that were related to usability evaluations of HIT using an eye tracker, to understand the current state, identify the gaps, and envision future research. There is no doubt that eye-tracker technology would be able to provide valuable data if well-integrated with other traditional usability evaluation methodologies. However, the lack of field research of clinical and mobile HITs in natural settings is a huge gap that needs to be filled. Scientific guidance is also needed for the interpretation of eye-tracking visualizations. Eye trackers can play a significant role in the future of usability evaluations of HIT if they are used effectively and correctly.


Treatment of the organ of Corti with TNF-α can induce hair cell damage. However, the resulting morphological changes have not been systematically examined. In the present study, cochlear organotypic cultures from neonatal mice were treated with various concentrations and durations of TNF-α to induce inflammatory responses. Confocal microscopy was used to evaluate the condition of hair cells and supporting cells following immunohistochemical staining. In addition, the ultrastructure of the stereocilia bundle, hair cells, and supporting cells were examined by scanning and transmission electron microscopy. TNF-α treatment resulted in a fusion and loss of stereocilia bundles in hair cells, swelling of mitochondria, and vacuolation and degranulation of the endoplasmic reticulum. Disruption of tight junctions between hair cells and supporting cells was also observed at high concentrations. Hair cell loss was preceded by apoptosis of Deiters’ and pillar cells. Taken together, these findings detail the morphological changes in the organ of Corti after TNF-α treatment, and provide an  Inflammation plays a critical role in the pathogenesis of many types of sensorineural hearing loss and tinnitus. Inflammatory responses occur in the inner ear in a variety of pathologic conditions, such as noise-induced hearing loss , 2, and TNF-α is considered the prominent primary upstream mediator of the inflammatory response , for whiIn vitro, excessive levels of TNF-α increase the expression of apoptosis-related genes and induce cochlear cell death [Intracochlear perfusion of TNF-α increases hearing threshold, whereas blockade of TNF-α significantly reduces post-meningitic hearing loss and cochlear injury . Perfusill death , 12. Thell death .Incochlear organotypic cultures, TNF-α ototoxicity to hair cells is dose-dependent . HoweverThe care and use of animals were in accordance with the published code of practice for the use of animals in research by the Chinese National Health and Medical Research Council and were approved by the Beijing Ethics Committee for Animal Usage (China).2. The culture medium was replaced with 2 mL fresh culture medium with or without various concentrations of TNF-α the following day, and replaced every 2 d.Postnatal-day 3 CD-1 mouse pups were euthanized by decapitation and the cochleae were dissected out. The basilar membrane together with the organ of Corti and spiral ganglion neurons was isolated as described elsewhere . A drop n ≥ 6 per condition). A stock solution of TNF-α , which dilutions were made in sterile, distilled water, was added to the culture medium for final concentrations of 0.5, 5.0, and 20.0 μg/mL. In the control group, we added the same volume of sterile, distilled water to the SFM. Control and TNF-α-treated groups were harvested for analysis after 2, 4, or 8 d.For the dose-time-dependent studies, the cochleae were randomly divided into 12 groups  in phosphate-buffered saline (PBS) at 4°C overnight. After washing with PBS, the explants were treated with 0.3% Triton X-100 in PBS for 30 min, and then blocked using 0.1% bovine serum albumin and 10% normal goat or donkey serum for 1 h at room temperature. We used primary antibodies against myosin 7a (diluted 1:200)  and cleaved caspase-3 (diluted 1:200) . Alexa Fluor 488-conjugated phalloidin (diluted 1:300)  was used to label F-actin. After rinsing in PBS three times, Alexa Fluor 488-, 568- or 647-conjugated (diluted 1:300) (Invitrogen) secondary antibodies were used along with DAPI (diluted 1:500)  for nuclear visualization. The specimens were mounted on glass slides with Fluromount-G . Images were acquired using epifluorescence  with a 60 × objective lens or by confocal microscopy  with a 63 × objective and analyzed with Photoshop CS4 .2, washed in phosphate buffer (PB), and then post fixed for 10 min with 1% OsO4 in the same buffer and washed. The tissues were dehydrated through an ethanol series, critical-point dried by CO2  and sputter-coated with platinum. The samples were examined on a field-emission scanning electron microscope .Organotypic cultures were fixed for 15 min with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, containing 2 mM CaCl4 in the same buffer, samples were dehydrated through a graded ethanol series and embedded in Spurr’s epoxy resin via propylene oxide. Ultra-thin sections were stained with uranyl acetate and lead citrate and mounted on grids. Grids were examined using a transmission electron microscope .The explants were fixed for 2 h with 2.5% glutaraldehyde in 0.1 M PB (pH 7.4) at room temperature, then washed in phosphate buffer. After post fixation for 1 h with 1% OsOThe number of hair cells was counted in 166.7-μm intervals along the entire length of the cochlea from base to apex under an epifluorescence microscope, and the loss of hair cells was calculated as a percent for each interval. A cochleogram was constructed showing the percentage hair cell loss as a function of the distance from the apex of the cochlea [Alexa Fluor 488-conjugated phalloidin was used to label stereocilia bundles and cuticular plates. Hair cell loss was counted if both the stereocilia and the cuticular plate were absent. All quantitative analyses were independently performed in a blind fashion by two investigators, and both sets of data were averaged.t-test, and p < 0.05 was considered to be statistically significant.Data were analyzed and converted into cochleogram using GraphPad Prism 5 . The data were shown as the mean ± standard deviation. Differences were evaluated using the Student’s The effects of various concentrations of TNF-α on organ of Corti viability were examined at different time points. TNF-α induced ototoxicity to the stereocilia bundle was assessed by phalloidin labeling within a series of confocal images from the middle turn of the cochlea (approximately 60% of the distance from the apex) with the focal plane along the height of the stereocilia bundle. Three rows of OHCs and one row of inner hair cells (IHCs) were visible in control groups cultured for 2, 4, or 8 d . The aliHair cells presented an increasingly disorderly arrangement after 4 d with 0.5, 5.0, and 20.0 μg/mL TNF-α . Some OHTEM revealed orderly arranged bundles with the central filaments inserted straightly into the cuticular plate in the control group after 2 d in culture . In compTEM of control OHCs cultured for 2 d revealed healthy mitochondria with normal outer, inner, and cristal membranes . All theEndoplasmic reticulum and their ribosomal components could be clearly seen by TEM in hair cells cultured for 2 d , arrow. In control cultures, the tight junction region formed between adjacent cell membranes was separated by intercellular space , arrow. SEM also revealed that TNF-α treatment (20 μg/mL for 4 d) severely truncated OHC stereocilia bundles Fig  and 3D. Cleaved caspase-3 was detected by immunohistochemistry in cultures after treatment for 4 d with 20 μg/mL TNF-α . In partCleaved caspase-3 immunostaining was also detected in OHCs at the apical turn of the organ of Corti after 8 d of treatment with 20 μg/mL TNF-α . In the This study describes the TNF-α-induced morphologic and ultrastructural changes in the organ of Corti. The findings demonstrate dose- and time-dependent damage, that was most pronounced in the basal turn area of the explants, consistent with the results by Haake et al. . The incStereocilia disorganization was the initial sign of TNF-α-induced damage and fusion. Hair cell stereocilia are apical membrane protrusions comprised of uniformly polarized actin filament bundles. The actin paracrystal is tapered at the base of the bundles, and the majority of filaments terminate in close positions to the plasma membrane, leaving only the central filaments to insert in the cuticular plate. The stereocilia bundle is vulnerable to mechanical and ototoxic drug insults because of its delicate microarchitecture and membrane proteins, including ion channels, chemoreceptors, and cell adhesion molecules , 19. TheIt is not entirely clear whether the initial damage is a direct result of TNF-α ototoxicity or secondary to the changes in mitochondria, such as enlargement, vacuolation and fission, and/or the endoplasmic reticulum, including edema and degranulation. Mitochondria are a major target of inflammation-associated injury, and TNF-α can induce suppression of complexes I and IV and pyruvate dehydrogenase activities  and causBCL gene family, which are one of the determining factors for anti-apoptosis. Moreover, it is possible that OHCs may contain more TNF receptors than IHCs do. All these possible mechanisms may explain why IHCs are more resilient to insults than OHCs.We show that TNF-α treatment resulted in loss of approximately 40% of OHCs in the basal turn. Although IHC damage (such as damage in the stereocilia bundles) was also observed, the damage to OHCs was more extensive and severe, consistent with other findings , 25. AltTNF receptor super family 1a gene (TNFRSF1a) and the TNFRSF1b gene are expressed in both IHCs and OHCs, but the expression is very close to the baseline intensity level [Supporting cells serve important functions in the development, survival, death, phagocytosis, and regeneration of other cell types within the inner ear . Howeverty level . These tConfocal microscopy revealed a loss of stereocilia and the cuticular plate with TNF-α treatment. Hair cell loss can occur by sub-laminal and extrusion , 29, forIn our experiments, treatment of cochlear explants with a high concentration of TNF-α (20 μg/mL) for eight days led to approximately 40% loss of OHCs. In contrast, Haake et al.  found thCompared with aminoglycoside-induced ototoxicity , treatmein vivo experiments [in vivo is much lower than the concentrations used in our study. Moreover, culture conditions are different from the in vivo environment. However, we choose the in vitro model in order to more precisely control the concentrations and exposure duration to investigate TNF-α-induced ototoxicity. Therefore, extensive hair and supporting cell death was observed in our study. Nevertheless, with this model, we were able to investigate and detail the mechanism and pattern of proinflammatory cytokine-induced cochlear damage.Previous work has indicated that TNF-α causes minimal hair cell death at concentrations used eriments . We acknin vitro model of proinflammatory-induced ototoxicity.In summary, our study for the first time demonstrates the ultrastructural changes of organ of Corti after TNF-α treatment. TNF-α induces apoptosis of hair cells and supporting cells through caspase signaling. Treatment with high concentration and long duration of TNF-α results in a moderate degree of loss to hair cells. The present study provides an


The goal of the International Journal of Behavioral Nutrition and Physical Activity (IJBNPA) is to be the leading diet and physical activity journal. To achieve this aim we embrace and publish a number of different research designs from small, but in depth, qualitative studies to large scale cohort studies. IJBNPA prioritises research based on randomised controlled trials (RCTs), systematic reviews , and well conducted observational studies that expand knowledge and understanding of the area. IJBNPA will also consider and publish other study designs that are of sufficient quality such as strong or ground-breaking methodological papers, rigorous qualitative studies, debate papers and commentaries. However, due to the demands on the journal, we publish pilot studies only in exceptional circumstances and we do not publish protocol papers or letters to the editors. The goal of this editorial is to highlight to our readers and authors the process by which we identify which papers to review and publish along with our editorial priorities. The first issue of IJBNPA was published in 2004, since when the number of publications has increased steadily to its current level of around 160 articles per year. However, the number of submissions has also risen dramatically and we are expecting in excess of 700 submissions for 2016. The rise in the number of submissions and the quality of the papers is a testament to work of authors, reviewers and editors. It does, however, mean that editorial decisions about which papers to send for peer review, which to accept, and which to reject are becoming increasingly more challenging. Journal editing and reviewing is a “volunteer activity” and there is a limit to the number of papers for which we can find high quality reviewers. As an editing team we are therefore faced with daily decisions about which papers should be published and we use two fundamental criteria to inform our decisions: novelty and methodological rigour and a paper has to meet both criteria to be considered for peer review. In terms of novelty, editors are asked to consider if the paper represents a new contribution to the evidence. While we view replication of findings to be an essential component of research, there has to be an upper limit on the number of times a study is replicated. After several studies have been conducted on the same topic it is more likely that a systematic review, rather than another small empirical study, will advance understanding. We therefore ask authors to justify clearly in their cover letter the uniqueness of their study and its key contribution to evidence.Methodological rigour is essential for ensuring that results are reproducible and answer the question posed . Thus, wA key goal of the editing team is for IJBNPA to set the agenda for diet and physical activity research. To achieve this we hope to become a home for both empirical papers and thought provoking debate papers. A debate paper provides the opportunity for authors to present conflicting views  or synthThe goal of the IJBNPA editorial team is to publish the highest quality studies to advance the science of behaviour change in relation to diet, physical activity and related behaviours. As part of this goal we will publish papers in a variety of formats, including debate papers, which have the potential to initiate discussion and challenge the current orthodoxy.


Tumor necrosis factor (TNF) α is a major proinflammatory cytokine involved in the immune response in inflammatory bowel disease (IBD). Anti-TNF drugs such as infliximab and adalimumab are used to treat IBD; however, approximately 30% of patients do not respond to treatment. Individual genetic differences could contribute to lack of efficacy. Genetic studies have tried to uncover the factors underlying differences in response, however, knowledge remains limited, and the results obtained should be validated, so that pharmacogenetic information can be applied in clinical practice. In this review, we gather current knowledge in the pharmacogenetics of anti-TNF drugs in patients with IBD. We observed a connection between the major genes described as possible predictors of response to anti-TNF drugs in IBD and the cytokines and molecules involved in the T helper (Th) 17 pathway. TL. TLTLR9,response  [23]. Hofindings .et al. found mRNA expression and serum levels of IL17 to be increased in patients with IBD [IL17A had a poorer response to treatment with anti-TNF drugs [Fujino with IBD  and suggNF drugs .Finally, INFγ is also produced by Th1 and regulates Th17 cells . The SNPIn conclusion, most of the genes associated with response to anti-TNF drugs in patients with IBD are associated with the Th17 pathway. However, data are limited, and further research is necessary to increase our knowledge of the Th17 process and understand its implication in response to anti-TNF drugs.


Tumor necrosis factor alpha (TNF-?) is an important pro-inflammatory cytokine associated with a variety of ocular diseases. The currently available TNF-? inhibitors are etanercept, infliximab, adalimumab, golimumab, and certolizumab. Experimental and clinical studies on the intravitreal use of these agents have been reported with etanercept, infliximab, and adalimumab: etanercept has shown limited efficacy in scarce reports; infliximab has been associated with local safety concerns but appears to benefit certain cases; adalimumab has shown no efficacy in cases of age-related macular degeneration (AMD) or diabetic macular edema (DME), but the combination with bevacizumab may be effective in refractory cases of macular diseases. Further preclinical and clinical studies are warranted in order to be able to obtain a more robust conclusion on the use of intravitreal TNF-? inhibitors. Inflammation has been implicated as one of the pivotal mediators of retinal injury in the pathogenesis of most sight-threatening ocular diseases including uveitis, diabetic retinopathy and diabetic macular edema (DME), and atrophic and neovascular age-related macular degeneration (AMD) ,2]. Thu. Thu2]. Tumor necrosis factor alpha (TNF-?) is an important pro-inflammatory cytokine with pleiotropic functions synthesized mainly by T lymphocytes and macrophages and to a lesser extent by neutrophils and mast cells . It playThe currently available TNF-? inhibitors are etanercept, infliximab, adalimumab, golimumab, and certolizumab. Etanercept  is a TNF receptor-IgG fusion protein that mimics the inhibitory effects of naturally occurring soluble TNF receptors that is injected subcutaneously. Infliximab  is a mouse-human chimeric antibody that neutralizes the biological activity of TNF-? by high-affinity binding to the soluble and transmembrane forms of TNF-?, therefore preventing the effective binding of TNF-? with its receptors. It is administered intravenously. Adalimumab  is a fully human monoclonal antibody that also binds selectively all forms of TNF-?. It is also injected subcutaneously. Golimumab  is a fully human monoclonal antibody against TNF-? that is administered via a subcutaneous injection. Certolizumab  is a monoclonal antibody that combines the Fab fragment of the TNF antibody with polyethylene glycol that is delivered subcutaneously. Table Reported risks of the systemic administration (intravenous or subcutaneous) of TNF-? inhibitors include fatal blood disorders, secondary infections, and reactivation of latent infections, tumorigenesis, drug-induced lupus, or demyelinating central nervous system disorders among others. These potentially severe adverse events led to the investigation by ophthalmologists of alternative administration routes minimizing these risks but preserving the efficacy of the drugs. The possibility of performing intravitreal injection of TNF-? inhibitors could fulfill both needs . HoweverThe purposes of the present review are to analyze the current published evidence with regard to intravitreal injection of TNF-? inhibitors and to summarize the outcomes with this novel therapeutic approach.A systematic review of all the peer-reviewed articles indexed in PubMed was performed. A comprehensive search of the literature was conducted using the online biomedical search engine PubMed. Search terms included the following: intravitreal, etanercept, infliximab, adalimumab, golimumab, certolizumab, tumor necrosis factor inhibitors. No publication date limit was applied, thus including all the available reports. Preclinical experimental models, clinical case reports, pilot studies, and case series were reviewed independently for the intravitreal use of etanercept, infliximab, and adalimumab. Relevant articles cited in papers retrieved from PubMed were also reviewed. No preclinical or clinical experience with intraocular administration of golimumab or certolizumab was found.Experimental animal models showed that intravitreal injections of etanercept up to 2.5 mg may be well tolerated without significant toxic effects on the retina ,13]..13].Clinical experience has been limited to a small pilot study that included seven patients with refractory DME . FollowiExperimental animal models showed that intravitreal injections of infliximab were tolerated up to 2.0 mg. However, doses higher than 3.3 mg may be potentially toxic to the retina -20]. Al. Al20]. Beer et al. suggested that infliximab may be suitable for compounding and could be a cost-effective intravitreal medication for use in clinical practice . A summaTheodossiadis and colleagues treated three patients with exudative AMD unresponsive to ranibizumab with two injections of 2 mg of infliximab given 2 months apart . They reFarvardin et al. reported positive both short- and long-term (6 months) outcomes after intravitreal infliximab 1.5 mg/0.15 mL in ten eyes of seven patients with chronic persistent noninfectious uveitis without any ocular adverse event, showing a visual mean visual improvement from 1.37 logMAR to 0.67 logMAR 1 month after the injection, which was not sustained by month 6 due to recurrence of macular edema and vitreous haze ,30]..30].Markomichelakis et al. reported that a single intravitreal injection of infliximab 1.0 mg/0.05 mL controlled intraocular inflammation in 15 cases with relapsing posterior uveitis associated with Behçet's disease .Wu et al. found that a single intravitreal injection of infliximab 1.0 mg/0.10 mL achieved excellent outcomes after 6 months, without any intraocular inflammation, in seven cases of refractory pseudophakic cystoid macular edema .Experimental animal models have shown that intravitreal injections of adalimumab were tolerated up to 5.0 mg without electroretinographic or histological alterations . HoweverThe Pan-American Collaborative Retina Study Group published their clinical experience with intravitreal injections of adalimumab for patients with refractory DME and partially responsive neovascular AMD to VEGF inhibitors. Intravitreal injections of adalimumab 2.0 mg/0.08 mL did not provide any anatomical or functional benefit in five cases of DME nonresponsive to conventional therapies and four cases of neovascular AMD. No ocular side effects were reported by the authors following intravitreal injections of adalimumab ,27]..27].The possible role of intravitreal adalimumab in combination with anti-angiogenic agents (bevacizumab) has also been analyzed by these authors. In their case series of seven eyes of five patients with macular edema of various etiologies including partially responsive neovascular AMD, a clinically relevant response was achieved after combination intravitreal treatment with adalimumab 2 mg/0.08 mL and bevacizumab 1.25 mg/0.05 mL .Overall, the literature review of all published manuscripts regarding the use of intravitreal TNF-? inhibitors reveals the following conclusions: 1) intravitreal injections of etanercept may not be useful in the management of ocular diseases, 2) intravitreal injections of infliximab may potentially be related to severe intraocular inflammation, 3) intravitreal injections of infliximab do not appear to benefit cases of refractory DME or partially responsive neovascular AMD, 4) intravitreal injections of infliximab may benefit cases of persistent noninfectious posterior uveitis and refractory pseudophakic cystoid macular edema, 5) intravitreal injections of adalimumab do not appear to benefit cases of DME or neovascular AMD, 6) intravitreal combination of adalimumab and bevacizumab may be effective in the management of cases with partially responsive neovascular AMD and macular edema of various etiologies, and 7) no preclinical or clinical experience with intravitreal golimumab or certolizumab has yet been published.It is important to emphasize that all the reviewed reports include small samples, variable endpoints and follow-up periods, and heterogeneity in the doses administered, the selection criteria, and the patient population -11]. Th. Th11]. AMD: age-related macular degenerationDME: diabetic macular edemaTNF: tumor necrosis factorVEGF: vascular endothelial growth factorThe authors declare that they have no competing interests.All authors have made substantial contributions to the conception and design, acquisition of data, or analysis and interpretation of data; have been involved in drafting the manuscript or revising it critically for important intellectual content; have given final approval of the version to be published; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.


This study aimed to evaluate the clinical significance of cysteine-rich 61 (Cyr-61/CCN1) and cyclooxygenase-2 (COX-2), and further explored their combined prognostic significance in gastric cancer.This retrospective study examined the expressions of Cyr-61 and COX-2 in 82 surgically removed gastric cancer specimens and 43 non-tumor gastric mucosa specimens by immunohistochemical staining to identify the abnormal expression of Cyr-61 or COX-2 in gastric cancer. Crude survival curves were constructed by the Kaplan-Meier method and Cox proportional hazards regression analysis was performed to confirm the prognostic roles of Cyr-61/COX-2 as well as sex and histological grade.p < 0.001) and COX-2 (p = 0.001) were both significantly up-regulated in gastric cancer samples compared with non-tumor gastric mucosa samples. The high expression of Cyr-61 or COX-2 was associated with invasion, lymph node metastasis, distant metastases, poor histological differentiation, advanced TNM stage and lower 5-year survival rate . Both Cyr-61 and COX-2 high expressions  was associated the higher risk of death during 5 years follow up than single Cyr-61 high expression  or COX-2 high expression .The expressions of Cyr-61 (Cyr-61 and COX-2 expressions are associated with the progression of gastric cancer. Additionally, combined expressions of Cyr-61 and COX-2 has a higher prognostic value than single expression. Gastric cancer is one of the most prevalent and the second leading cause of malignant diseases worldwide . In spitCysteine-rich 61 (Cyr-61/CCN1) is a member of the connective tissue growth factor family (CCN), which plays important roles in cell proliferation, adhesion, migration, angiogenesis and apoptosis. Studies in recent years have revealed the link between Cyr-61 expression and multiple malignant tumors such as lung cancer , breast Recent studies have shown that COX-2 highly expressed in esophageal cancer , colorecThis retrospective study was approved by the Ethics Committee in the China Medical University, which conforms to the provisions of the World Medical Association’s Declaration of Helsinki in 2013 . GastricImmunohistochemical staining of biopsy specimens surgically collected from primary gastric cancer tissues as well as non-tumor gastric mucosa specimens was carried out following manufacturer’s instruction  . ParaffiPositive staining of Cyr-61 and COX-2 was determined by the detection of brown particles in the cytoplasm under a binocular light microscope . Staining results were divided into the following category based on percentage of positive cells in total cells: < 25 % as low expression and ≥ 25 % as high expression . Based oP values of less than 0.05 were considered statistically significant.Data analysis was performed by using SPSS 17.0 software . Expression of Cyr-61 and COX-2 between gastric carcinoma tissues and non-tumor tissues as well as clinicopathologic features between low and high expressions of Cyr-61/COX-2 groups were compared by Fisher test. Crude survival curves were constructed by the Kaplan-Meier method and survival rates between low and high expression of Cyr-61/COX-2 groups were compared by the log-rank test. To confirm the prognostic roles of Cyr-61 and COX-2, cox proportional hazards regression analysis with a enter method was used to calculate the hazard ratio (HR) with adjustment by sex and histological grade in multivariate analysis. A total of 82 cancer patients were enrolled in our study, including 58 (70.73 %) males and 24 (29.27 %) females. The average age of the whole cohort was 55.61 years, with a range from 25 to 79 years. Of the 82 patients, 31 cases were well differentiated and 51 were poorly differentiated. The number of these patients with T1-T4 stage of gastric cancers was 4, 24, 25 and 29, respectively; the number of patients with TNM stage I-IV was 19, 13, 25 and 25, respectively. Fifty-six cases had lymph node metastasis and 23 had distant metastasis. Finally, a total of 55 patients  were completely followed-up. These 55 patients were aged 25–79 years (average age of 67.64 years).p = 0.008). In addition, COX-2 expression levels had a similar trend with the expression of Cyr-61  , lymph node metastasis (p = 0.001), distant metastases (p = 0.045), poor histological differentiation (p = 0.031), and advanced TNM staging (p = 0.009). However, the expression level of Cyr-61 was not related to the diameter of tumor (p = 0.190), or gender (p = 0.752) (Table p = 0.003), invasion depth (p < 0.001), lymph node metastasis (p < 0.001), distant metastases (p = 0.017), poor tissue differentiation (p = 0.015), and advanced TNM staging (p < 0.001), but not the gender of patients (p = 0.971) , Cyr-61 expression (p < 0.001), COX-2 expression (p < 0.001), and the combined expressions of Cyr-61 and COX-2 (p = 0.001) were all significantly associated with the survival of gastric cancer patients.To confirm the prognostic role of Cyr-61 or COX-2, the Cox proportional hazards regression models were adopted. Meanwhile, the other factors  were also analyzed for exploring additional prognostic factors. The univariate analyses demonstrated that histological grade (p = 0.008), COX-2 expression (p = 0.039), and the combined expressions of Cyr-61 and COX-2 (p = 0.003) were prognostic factors independent of gender and histological grade  in this study; however, the conclusions may be more convincing if we supplemented the data of Western blot or in situ hybridization. Fourth, because immunohistochemical figures cannot be exactly distinguish cell types, we did not describe the cell types of COX2 and Cyr-61expressions. Lastly, there is no mechanism study in this study, further investigations for the downstream signaling pathways of Cyr-61/COX-2 and their interaction in gastric cancer are necessary.In conclusion, both Cyr-61 and COX-2 expression are associated with the progression of gastric cancer. Meanwhile, prognostic role of Cyr-61 and COX-2 is confirmed in this study. Moreover, combined expression of Cyr-61 and COX-2 has higher prognostic value than single expression for the survival of gastric cancer patients.CI, confidence interval; COX-2, cyclooxygenase-2; Cyr-61/CCN1, cysteine-rich 61; DAB, diaminobenzidine; HR, hazard ratio; TNM, tumor node metastasis