The polymodal ion channel transient receptor potential vanilloid 4 modulates calcium flux, spiking rate, and apoptosis of mouse retinal ganglion cells.

Sustained increase in intraocular pressure represents a major risk factor for eye disease, yet the cellular mechanisms of pressure transduction in the posterior eye are essentially unknown. Here we show that the mouse retina expresses mRNA and protein for the polymodal transient receptor potential vanilloid 4 (TRPV4) cation channel known to mediate osmotransduction and mechanotransduction. TRPV4 antibodies labeled perikarya, axons, and dendrites of retinal ganglion cells (RGCs) and intensely immunostained the optic nerve head. Müller glial cells, but not retinal astrocytes or microglia, also expressed TRPV4 immunoreactivity. The selective TRPV4 agonists 4α-PDD and GSK1016790A elevated [Ca2+]i in dissociated RGCs in a dose-dependent manner, whereas the TRPV1 agonist capsaicin had no effect on [Ca2+](RGC). Exposure to hypotonic stimulation evoked robust increases in [Ca2+](RGC). RGC responses to TRPV4-selective agonists and hypotonic stimulation were absent in Ca2+ -free saline and were antagonized by the nonselective TRP channel antagonists Ruthenium Red and gadolinium, but were unaffected by the TRPV1 antagonist capsazepine. TRPV4-selective agonists increased the spiking frequency recorded from intact retinas recorded with multielectrode arrays. Sustained exposure to TRPV4 agonists evoked dose-dependent apoptosis of RGCs. Our results demonstrate functional TRPV4 expression in RGCs and suggest that its activation mediates response to membrane stretch leading to elevated [Ca2+]i and augmented excitability. Excessive Ca2+ influx through TRPV4 predisposes RGCs to activation of Ca2+ -dependent proapoptotic signaling pathways, indicating that TRPV4 is a component of the response mechanism to pathological elevations of intraocular pressure.

3D reconstruction and analysis of wing deformation in free-flying dragonflies.

Insect wings demonstrate elaborate three-dimensional deformations and kinematics. These deformations are key to understanding many aspects of insect flight including aerodynamics, structural dynamics and control. In this paper, we propose a template-based subdivision surface reconstruction method that is capable of reconstructing the wing deformations and kinematics of free-flying insects based on the output of a high-speed camera system. The reconstruction method makes no rigid wing assumptions and allows for an arbitrary arrangement of marker points on the interior and edges of each wing. The resulting wing surfaces are projected back into image space and compared with expert segmentations to validate reconstruction accuracy. A least squares plane is then proposed as a universal reference to aid in making repeatable measurements of the reconstructed wing deformations. Using an Eastern pondhawk (Erythimus simplicicollis) dragonfly for demonstration, we quantify and visualize the wing twist and camber in both the chord-wise and span-wise directions, and discuss the implications of the results. In particular, a detailed analysis of the subtle deformation in the dragonfly's right hindwing suggests that the muscles near the wing root could be used to induce chord-wise camber in the portion of the wing nearest the specimen's body. We conclude by proposing a novel technique for modeling wing corrugation in the reconstructed flapping wings. In this method, displacement mapping is used to combine wing surface details measured from static wings with the reconstructed flapping wings, while not requiring any additional information be tracked in the high speed camera output.

Evaluation of Diagnostic and Triage Accuracy and Usability of a Symptom Checker in an Emergency Department: Observational Study.

BACKGROUND: Symptom checkers are clinical decision support apps for patients, used by tens of millions of people annually. They are designed to provide diagnostic and triage advice and assist users in seeking the appropriate level of care. Little evidence is available regarding their diagnostic and triage accuracy with direct use by patients for urgent conditions. OBJECTIVE: The aim of this study is to determine the diagnostic and triage accuracy and usability of a symptom checker in use by patients presenting to an emergency department (ED). METHODS: We recruited a convenience sample of English-speaking patients presenting for care in an urban ED. Each consenting patient used a leading symptom checker from Ada Health before the ED evaluation. Diagnostic accuracy was evaluated by comparing the symptom checker's diagnoses and those of 3 independent emergency physicians viewing the patient-entered symptom data, with the final diagnoses from the ED evaluation. The Ada diagnoses and triage were also critiqued by the independent physicians. The patients completed a usability survey based on the Technology Acceptance Model. RESULTS: A total of 40 (80%) of the 50 participants approached completed the symptom checker assessment and usability survey. Their mean age was 39.3 (SD 15.9; range 18-76) years, and they were 65% (26/40) female, 68% (27/40) White, 48% (19/40) Hispanic or Latino, and 13% (5/40) Black or African American. Some cases had missing data or a lack of a clear ED diagnosis; 75% (30/40) were included in the analysis of diagnosis, and 93% (37/40) for triage. The sensitivity for at least one of the final ED diagnoses by Ada (based on its top 5 diagnoses) was 70% (95% CI 54%-86%), close to the mean sensitivity for the 3 physicians (on their top 3 diagnoses) of 68.9%. The physicians rated the Ada triage decisions as 62% (23/37) fully agree and 24% (9/37) safe but too cautious. It was rated as unsafe and too risky in 22% (8/37) of cases by at least one physician, in 14% (5/37) of cases by at least two physicians, and in 5% (2/37) of cases by all 3 physicians. Usability was rated highly; participants agreed or strongly agreed with the 7 Technology Acceptance Model usability questions with a mean score of 84.6%, although "satisfaction" and "enjoyment" were rated low. CONCLUSIONS: This study provides preliminary evidence that a symptom checker can provide acceptable usability and diagnostic accuracy for patients with various urgent conditions. A total of 14% (5/37) of symptom checker triage recommendations were deemed unsafe and too risky by at least two physicians based on the symptoms recorded, similar to the results of studies on telephone and nurse triage. Larger studies are needed of diagnosis and triage performance with direct patient use in different clinical environments.

Receptive field center size decreases and firing properties mature in ON and OFF retinal ganglion cells after eye opening in the mouse.

Development of the mammalian visual system is not complete at birth but continues postnatally well after eye opening. Although numerous studies have revealed changes in the development of the thalamus and visual cortex during this time, less is known about the development of response properties of retinal ganglion cells (RGCs). Here, we mapped functional receptive fields of mouse RGCs using a Gaussian white noise checkerboard stimulus and a multielectrode array to record from retinas at eye opening, 3 days later, and 4 wk after birth, when visual responses are essentially mature. Over this time, the receptive field center size of ON and OFF RGC populations decreased. The average receptive field center size of ON RGCs was larger than that of OFF RGCs at eye opening, but they decreased to the same size in the adult. Firing properties were also immature at eye opening. RGCs had longer latencies, lower frequencies of firing, and lower sensitivity than in the adult. Hence, the dramatic maturation of the visual system during the first weeks of visual experience includes the retina.

What Counts?: A Qualitative Study of Adolescents' Lived Experience With Online Victimization and Cyberbullying.

OBJECTIVE: To inform development of cyberbullying interventions that are both accurate and meaningful to all adolescents, this qualitative analysis examines experiences of online peer victimization among a sample of predominately minority and low-income youth. METHODS: Adolescents ages 13 to 17 years who reported past-year cyberbullying on a previously validated survey were recruited from an urban pediatric clinic to complete semistructured interviews. Interview topics included definitions of cyberbullying, prior cyberbullying experiences, and strategies to reduce cyberbullying and its consequences. Interviews were audio-recorded and transcribed verbatim. Using thematic analysis, study team members applied both structural and emergent codes to transcripts. RESULTS: Saturation was reached after 23 interviews (mean age 14.8 years; 65% female, 47.8% Hispanic, 35% Black, 74% low socioeconomic status). Four main themes emerged: 1) Teens avoided the term "cyberbullying," due to its association with suicidality and severe depression; they preferentially described experiences (even those meeting criteria for repetition, power differential, etc.) as "online conflict". 2) In-person bullying categories (bully, victim, bully victim, bystander) apply to online conflict. Few identify purely as victims. 3) Cyberbullying is part of a larger continuum of peer violence, including physical fights and in-person bullying. 4) Teens want to help victims of cyberbullying; they desire more guidance in so doing. CONCLUSIONS: These youth rarely acknowledge presence of cyberbullying; instead, they describe online conflict as part of a larger spectrum of peer violence. Clinicians may consider prevention of a range of conflict-related behaviors (rather than focusing exclusively on cyberbullying), and may consider engaging adolescent bystanders in prevention of online conflict.

Pink1 and Parkin regulate Drosophila intestinal stem cell proliferation during stress and aging.

Intestinal stem cells (ISCs) maintain the midgut epithelium in Drosophila melanogaster Proper cellular turnover and tissue function rely on tightly regulated rates of ISC division and appropriate differentiation of daughter cells. However, aging and epithelial injury cause elevated ISC proliferation and decreased capacity for terminal differentiation of daughter enteroblasts (EBs). The mechanisms causing functional decline of stem cells with age remain elusive; however, recent findings suggest that stem cell metabolism plays an important role in the regulation of stem cell activity. Here, we investigate how alterations in mitochondrial homeostasis modulate stem cell behavior in vivo via RNA interference-mediated knockdown of factors involved in mitochondrial dynamics. ISC/EB-specific knockdown of the mitophagy-related genes Pink1 or Parkin suppresses the age-related loss of tissue homeostasis, despite dramatic changes in mitochondrial ultrastructure and mitochondrial damage in ISCs/EBs. Maintenance of tissue homeostasis upon reduction of Pink1 or Parkin appears to result from reduction of age- and stress-induced ISC proliferation, in part, through induction of ISC senescence. Our results indicate an uncoupling of cellular, tissue, and organismal aging through inhibition of ISC proliferation and provide insight into strategies used by stem cells to maintain tissue homeostasis despite severe damage to organelles.

Tricellular junctions regulate intestinal stem cell behaviour to maintain homeostasis.

Ageing results in loss of tissue homeostasis across taxa. In the intestine of Drosophila melanogaster, ageing is correlated with an increase in intestinal stem cell (ISC) proliferation, a block in terminal differentiation of progenitor cells, activation of inflammatory pathways, and increased intestinal permeability. However, causal relationships between these phenotypes remain unclear. Here, we demonstrate that ageing results in altered localization and expression of septate junction proteins in the posterior midgut, which is quite pronounced in differentiated enterocytes (ECs) at tricellular junctions (TCJs). Acute loss of the TCJ protein Gliotactin (Gli) in ECs results in increased ISC proliferation and a block in differentiation in intestines from young flies, demonstrating that compromised TCJ function is sufficient to alter ISC behaviour in a non-autonomous manner. Blocking the Jun N-terminal kinase signalling pathway is sufficient to suppress changes in ISC behaviour, but has no effect on loss of intestinal barrier function, as a consequence of Gli depletion. Our work demonstrates a pivotal link between TCJs, stem cell behaviour, and intestinal homeostasis and provides insights into causes of age-onset and gastrointestinal diseases.

Increased longevity mediated by yeast NDI1 expression in Drosophila intestinal stem and progenitor cells.

A functional decline in tissue stem cells and mitochondrial dysfunction have each been linked to aging and multiple aging-associated pathologies. However, the interplay between energy homeostasis, stem cells, and organismal aging remains poorly understood. Here, we report that expression of the single-subunit yeast alternative NADH dehydrogenase, ndi1, in Drosophila intestinal stem and progenitor cells delays the onset of multiple markers of intestinal aging and extends lifespan. In addition, expression of ndi1 in the intestine increases feeding behavior and results in organismal weight gain. Consistent with increased nutrient uptake, flies expressing ndi1 in the digestive tract display a systemic reduction in the activity of AMP-activated protein kinase (AMPK), a key cellular energy sensor. Together, these results demonstrate that ndi1 expression in the intestinal epithelium is an effective strategy to delay tissue and organismal aging.

Vortex visualization in ultra low Reynolds number insect flight.

We present the visual analysis of a biologically inspired CFD simulation of the deformable flapping wings of a dragonfly as it takes off and begins to maneuver, using vortex detection and integration-based flow lines. The additional seed placement and perceptual challenges introduced by having multiple dynamically deforming objects in the highly unsteady 3D flow domain are addressed. A brief overview of the high speed photogrammetry setup used to capture the dragonfly takeoff, parametric surfaces used for wing reconstruction, CFD solver and underlying flapping flight theory is presented to clarify the importance of several unsteady flight mechanisms, such as the leading edge vortex, that are captured visually. A novel interactive seed placement method is used to simplify the generation of seed curves that stay in the vicinity of relevant flow phenomena as they move with the flapping wings. This method allows a user to define and evaluate the quality of a seed's trajectory over time while working with a single time step. The seed curves are then used to place particles, streamlines and generalized streak lines. The novel concept of flowing seeds is also introduced in order to add visual context about the instantaneous vector fields surrounding smoothly animate streak lines. Tests show this method to be particularly effective at visually capturing vortices that move quickly or that exist for a very brief period of time. In addition, an automatic camera animation method is used to address occlusion issues caused when animating the immersed wing boundaries alongside many geometric flow lines. Each visualization method is presented at multiple time steps during the up-stroke and down-stroke to highlight the formation, attachment and shedding of the leading edge vortices in pairs of wings. Also, the visualizations show evidence of wake capture at stroke reversal which suggests the existence of previously unknown unsteady lift generation mechanisms that are unique to quad wing insects.

Missing optomotor head-turning reflex in the DBA/2J mouse.

PURPOSE: The optomotor reflex of DBA/2J (D2), DBA/2J-Gpnmb+ (D2-Gpnmb+), and C57BL/6J (B6) mouse strains was assayed, and the retinal ganglion cell (RGC) firing patterns, direction selectivity, vestibulomotor function and central vision was compared between the D2 and B6 mouse lines. METHODS: Intraocular pressure (IOP) measurements, real-time PCR, and immunohistochemical analysis were used to assess the time course of glaucomatous changes in D2 retinas. Behavioral analyses of optomotor head-turning reflex, visible platform Morris water maze and Rotarod measurements were conducted to test vision and vestibulomotor function. Electroretinogram (ERG) measurements were used to assay outer retinal function. The multielectrode array (MEA) technique was used to characterize RGC spiking and direction selectivity in D2 and B6 retinas. RESULTS: Progressive increase in IOP and loss of Brn3a signals in D2 animals were consistent with glaucoma progression starting after 6 months of age. D2 mice showed no response to visual stimulation that evoked robust optomotor responses in B6 mice at any age after eye opening. Spatial frequency threshold was also not measurable in the D2-Gpnmb+ strain control. ERG a- and b-waves, central vision, vestibulomotor function, the spiking properties of ON, OFF, ON-OFF, and direction-selective RGCs were normal in young D2 mice. CONCLUSIONS: The D2 strain is characterized by a lack of optomotor reflex before IOP elevation and RGC degeneration are observed. This behavioral deficit is D2 strain-specific, but is independent of retinal function and glaucoma. Caution is advised when using the optomotor reflex to follow glaucoma progression in D2 mice.

Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog.

In mammals, the PGC-1 transcriptional coactivators are key regulators of energy metabolism, including mitochondrial biogenesis and respiration, which have been implicated in numerous pathogenic conditions, including neurodegeneration and cardiomyopathy. Here, we show that overexpression of the Drosophila PGC-1 homolog (dPGC-1/spargel) is sufficient to increase mitochondrial activity. Moreover, tissue-specific overexpression of dPGC-1 in stem and progenitor cells within the digestive tract extends life span. Long-lived flies overexpressing dPGC-1 display a delay in the onset of aging-related changes in the intestine, leading to improved tissue homeostasis in old flies. Together, these results demonstrate that dPGC-1 can slow aging both at the level of cellular changes in an individual tissue and also at the organismal level by extending life span. Our findings point to the possibility that alterations in PGC-1 activity in high-turnover tissues, such as the intestine, may be an important determinant of longevity in mammals.

Effect of proinflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma on epithelial barrier function and matrix metalloproteinase-9 in Madin Darby canine kidney cells.

BACKGROUND: Elevated matrix metalloproteinase-9 production during inflammation may be deleterious to epithelial barrier function. Therefore we examined the effect of proinflammatory cytokines on the expression and regulation of matrix metalloproteinase-9 in a model renal epithelial cell system. Tight junctions limit diffusion between compartments and permit directional transport of solutes. Impairment of these junctional complexes by proteolysis may contribute to renal failure through loss of barrier function. METHODS: The renal epithelial cell model, MDCK cells were employed to examine metalloproteinase activity and mRNA expression. Epithelial barrier function was determined using paracellular flux studies. RESULTS: We found that matrix metalloproteinase-9 expression (MMP-9) and activity is markedly elevated in response to tumor necrosis factor-alpha exposure through a mitogen-activated protein kinase dependent pathway. The MMP-9 is predominately secreted into the apical compartment and elevated MMP-9 expression correlates with impaired cell barrier function that was restored using a specific inhibitor of MMP activity. Addition of recombinant MMP-9 to the apical compartment of MDCK cultures significantly elevated paracellular flux rate. CONCLUSIONS: We provide direct evidence for a MMP-9-mediated mechanism that produces junctional disruption. Collectively, these findings support the hypothesis that impaired epithelial barrier function due to activation of tissue/matrix degrading mechanisms occurs in response to specific inflammatory cues.