Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells.

Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.

"Forwards, Not Backwards": How the U.S. Supreme Court May Save the Plight of Individuals with Mental Disabilities.

When federal district court Judge Carlton Reeves penned his opinion in U.S. v. Mississippi,1 the case that seemed poised to overhaul Mississippi's suffering mental health system, he began with the story of Ms. Melanie Worsham, a mental health patient, also a certified peer support specialist. Ms. Worsham works to help those like herself who suffer with lifelong serious mental illness (SMI) to "overcome the obstacles that might be getting in their way of living the life they want to live." She also assists those with SMI by aiding in "navigating the system, to find resources, and then just being moral support."2.

3D printed transwell-integrated nose-on-chip model to evaluate effects of air flow-induced mechanical stresses on mucous secretion.

While there are many chip models that simulate the air-tissue interface of the respiratory system, only a few represent the upper respiratory system. These chips are restricted to unidirectional flow patterns that are not comparable to the highly dynamic and variable flow patterns found in the native nasal cavity. Here we describe the development of a tunable nose-on-chip device that mimics the air-mucosa interface and is coupled to an air delivery system that simulates natural breathing patterns through the generation of bi-directional air flow. Additionally, we employ computational modeling to demonstrate how the device design can be tuned to replicate desired mechanical characteristics within specific regions of the human nasal cavity. We also demonstrate how to culture human nasal epithelial cell line RPMI 2650 within the lab-on-chip (LOC) device. Lastly, Alcian Blue histological staining was performed to label mucin proteins, which play important roles in mucous secretion. Our results revealed that dynamic flow conditions can increase mucous secretion for RPMI 2650 cells, when compared to no flow, or stationary, conditions.

Colorism and classism confounded: Perceptions of discrimination in Latin America.

Despite competing narratives of mestizaje (race-mixing) emphasizing class discrimination and social movements highlighting the existence of racial discrimination in Latin America, little work has examined the overlap of class and color in people's understandings of discrimination. This study moves beyond the color/class binary by examining perceptions of only class, only color, and both class and color discrimination (dual discrimination). I also examine whether individuals have difficulty attributing the causes of discrimination by expanding upon the social psychological concept of attributional ambiguity. Using nationally representative data from the 2010 LAPOP's Americas Barometer survey, I find that color-based explanations have not replaced class-based explanations. Instead, both class and color appear to be part of schemas drawn upon by individuals to understand the unfavorable treatment they perceive-in line with scholarship showing both class disadvantage and color conjointly influence the stratification systems of Latin America. There is also suggestive evidence that individuals may have trouble disentangling the causes of the discrimination they perceive.

Acceptance and Commitment Therapy versus Cognitive Behavior Therapy for Children With Anxiety: Outcomes of a Randomized Controlled Trial.

Acceptance and Commitment Therapy (ACT) has a growing empirical base in the treatment of anxiety among adults and children with other concerns. This study reports on the main outcomes of a randomized controlled trial of ACT and traditional cognitive behavioral therapy (CBT) in children with a Diagnostic and Statistical Manual of Mental Disorders (4th ed.) anxiety disorder. Participants were 193 children from urban Sydney, Australia, who were block-randomized to a 10-week group-based program of ACT or CBT or a 10-week waitlist control (WLC). Completers included 157 children (ACT = 54, CBT = 57, WLC = 46; M = 11 years, SD = 2.76; 78% Caucasian, 58% female). Pretreatment, posttreatment, and 3 months posttreatment assessments included clinician/self/parent-reported measures of anxiety, quality of life (QOL; anxiety interference, psychosocial and physical health-related QOL), and acceptance/defusion outcomes. Completer and intention-to-treat analyses revealed that ACT and CBT were both superior to WLC across outcomes, reflecting statistically and clinically significant differences, with gains maintained at 3 months posttreatment. Both completer and intention-to-treat analyses found ACT and CBT to produce similar outcomes. There was some support for ACT having greater effect sizes for QOL outcomes but not for avoidance/fusion. Although this study does not suggest that ACT is equivalent to CBT or should be adopted in its place, it does provide evidence that ACT might be another empirically supported treatment option for anxious youth. Further research is needed to replicate these findings.

MRI compatible MS2 nanoparticles designed to cross the blood-brain-barrier: providing a path towards tinnitus treatment.

Fundamental challenges of targeting specific brain regions for treatment using pharmacotherapeutic nanoparticle (NP) carriers include circumventing the blood-brain-barrier (BBB) and tracking delivery. Angiopep-2 (AP2) has been shown to facilitate the transport of large macromolecules and synthetic nanoparticles across the BBB. Thus, conjugation of AP2 to an MS2 bacteriophage based NP should also permit transport across the BBB. We have fabricated and tested a novel MS2 capsid-based NP conjugated to the ligand AP2. The reaction efficiency was determined to be over 70%, with up to two angiopep-2 conjugated per MS2 capsid protein. When linked with a porphyrin ring, manganese (Mn2+) remained stable within MS2 and was MRI detectable. Nanoparticles were introduced intracerebroventricularly or systemically. Systemic delivery yielded dose dependent, non-toxic accumulation of NPs in the midbrain. Design of a multifunctional MRI compatible NP platform provides a significant step forward for the diagnosis and treatment of intractable brain conditions, such as tinnitus.

The Americleft Speech Project: A Training and Reliability Study.

OBJECTIVE: To describe the results of two reliability studies and to assess the effect of training on interrater reliability scores. DESIGN: The first study (1) examined interrater and intrarater reliability scores (weighted and unweighted kappas) and (2) compared interrater reliability scores before and after training on the use of the Cleft Audit Protocol for Speech-Augmented (CAPS-A) with British English-speaking children. The second study examined interrater and intrarater reliability on a modified version of the CAPS-A (CAPS-A Americleft Modification) with American and Canadian English-speaking children. Finally, comparisons were made between the interrater and intrarater reliability scores obtained for Study 1 and Study 2. PARTICIPANTS: The participants were speech-language pathologists from the Americleft Speech Project. RESULTS: In Study 1, interrater reliability scores improved for 6 of the 13 parameters following training on the CAPS-A protocol. Comparison of the reliability results for the two studies indicated lower scores for Study 2 compared with Study 1. However, this appeared to be an artifact of the kappa statistic that occurred due to insufficient variability in the reliability samples for Study 2. When percent agreement scores were also calculated, the ratings appeared similar across Study 1 and Study 2. CONCLUSION: The findings of this study suggested that improvements in interrater reliability could be obtained following a program of systematic training. However, improvements were not uniform across all parameters. Acceptable levels of reliability were achieved for those parameters most important for evaluation of velopharyngeal function.

Disease progression in iridocorneal angle tissues of BMP2-induced ocular hypertensive mice with optical coherence tomography.

PURPOSE: The goal of the present study was to test for the first time whether glaucomatous-like disease progression in a mouse can be assessed morphologically and functionally with spectral domain optical coherence tomography (SD-OCT). METHODS: We monitored progressive changes in conventional outflow tissues of living mice overexpressing human bone morphogenetic protein 2 (BMP2), a model for glaucoma. Intraocular pressure (IOP) and outflow tissue morphology/Young's modulus were followed in mice for 36 days with rebound tonometry and SD-OCT, respectively. Results were compared to standard histological methods. Outflow facility was calculated from flow measurements with direct cannulation of anterior chambers subjected to three sequential pressure steps. RESULTS: Overexpression of BMP2 significantly elevated IOP in a biphasic manner over time compared to mice that overexpressed green fluorescent protein in outflow cells and naïve controls. SD-OCT revealed changes in outflow tissues overexpressing BMP2 that corresponded with the timing of the IOP phases and decreased outflow facility. In the first phase, the angle was open, but the trabecular meshwork and the cornea were thickened. OCT detected increased trabecular meshwork stiffness after provocative IOP challenges of the BMP2 eyes, which corresponded to increased collagen deposition with transmission electron microscopy. In contrast, the angle was closed in the second phase. IOP elevation over 36 days due to BMP2 overexpression resulted in significant retinal ganglion cell and axon loss. CONCLUSIONS: Although not a feasible open-angle glaucoma model, the BMP2 mice were useful for demonstrating the utility of SD-OCT in following disease progression and differentiating between two forms of ocular pathology over time that resulted in ocular hypertension.

Empty chairs at the dinner table: Black-white disparities in exposure to household member deaths.

As a result of Black-White inequities in life expectancy, recent research has indicated that Black individuals are disproportionately exposed to the deaths of multiple family members compared to White individuals. Black individuals are also more likely to live in coresident households-that is, households that extend beyond the nuclear family. However, it is unclear the degree to which this population may be disproportionately exposed to the loss of deaths marked by the geographic closeness of the household. In this study, I use data from the Panel Study of Income Dynamics to provide the first nationally representative estimates of Black-White disparities in exposure to household member deaths. I find that Black people are significantly more likely than White individuals to have experienced the death of a household member. Based on these findings, I argue the dual inequities of racial disparities in life expectancy and racial disparities in coresidence are an overlooked, salient source of racial disparities in exposure to death. By illuminating a broader range of network sources that contribute to racial inequities in exposure to death, this study sets forth a new conceptual unit of analysis-that of the household-to investigate the intergenerational reproduction of inequality in health and socioeconomic status due to exposure to death.

Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2.

Glioblastoma (GBM) is a malignant brain tumor with uncontrolled invasive growth. Here, we demonstrate how GBM cells usurp guidance receptor Plexin-B2 to gain biomechanical plasticity for polarized migration through confined space. Using live-cell imaging to track GBM cells negotiating microchannels, we reveal active endocytosis at cell front and filamentous actin assembly at rear to propel GBM cells through constrictions. These two processes are interconnected and governed by Plexin-B2 that orchestrates cortical actin and membrane tension, shown by biomechanical assays. Molecular dynamics simulations predict that balanced membrane and actin tension are required for optimal migratory velocity and consistency. Furthermore, Plexin-B2 mechanosensitive function requires a bendable extracellular ring structure and affects membrane internalization, permeability, phospholipid composition, as well as inner membrane surface charge. Together, our studies unveil a key element of membrane tension and mechanoelectrical coupling via Plexin-B2 that enables GBM cells to adapt to physical constraints and achieve polarized confined migration.

Autophagy deficiency protects against ocular hypertension and neurodegeneration in experimental and spontanous glaucoma mouse models.

Glaucoma is a group of diseases that leads to chronic degeneration of retinal ganglion cell (RGC) axons and progressive loss of RGCs, resulting in vision loss. While aging and elevated intraocular pressure (IOP) have been identified as the main contributing factors to glaucoma, the molecular mechanisms and signaling pathways triggering RGC death and axonal degeneration are not fully understood. Previous studies in our laboratory found that overactivation of autophagy in DBA/2J::GFP-LC3 mice led to RGC death and optic nerve degeneration with glaucomatous IOP elevation. We found similar findings in aging GFP-LC3 mice subjected to chronic IOP elevation. Here, we further investigated the impact of autophagy deficiency on autophagy-deficient DBA/2J-Atg4bko and DBA/2J-Atg4b+/- mice, generated in our laboratory via CRISPR/Cas9 technology; as well as in Atg4bko mice subjected to the experimental TGFß2 chronic ocular hypertensive model. Our data shows that, in contrast to DBA/2J and DBA/2J-Atg4b+/- littermates, DBA/2J-Atg4bko mice do not develop glaucomatous IOP elevation. Atg4b deficiency also protected against glaucomatous IOP elevation in the experimental TGFß2 chronic ocular hypertensive model. Atg4 deletion did not compromise RGC or optic nerve survival in Atg4bko mice. Moreover, our results indicate a protective role of autophagy deficiency against RGC death and ON atrophy in the hypertensive DBA/2J-Atg4b+/- mice. Together, our data suggests a pathogenic role of autophagy activation in ocular hypertension and glaucoma.

Shear stress induces autophagy in Schlemm's canal cells via primary cilia-mediated SMAD2/3 signaling pathway.

The Schlemm's canal (SC) is a circular, lymphatic-like vessel located at the limbus of the eye that participates in the regulation of aqueous humor drainage to control intraocular pressure (IOP). Circumferential flow of aqueous humor within the SC lumen generates shear stress, which regulates SC cell behaviour. Using biochemical analysis and real-time live cell imaging techniques, we have investigated the activation of autophagy in SC cells by shear stress. We report, for the first time, the primary cilium (PC)-dependent activation of autophagy in SC cells in response to shear stress. Moreover, we identified PC-dependent shear stress-induced autophagy to be positively regulated by phosphorylation of SMAD2 in its linker and C-terminal regions. Additionally, SMAD2/3 signaling was found to transcriptionally activate LC3B, ATG5 and ATG7 in SC cells. Intriguingly, concomitant to SMAD2-dependent activation of autophagy, we also report here the activation of mTOR pathway, a classical autophagy inhibitor, in SC cells by shear stress. mTOR activation was found to also be dependent on the PC. Moreover, pharmacological inhibition of class I PI3K increased phosphorylation of SMAD2 at the linker and activated autophagy. Together, our data indicates an interplay between PI3K and SMAD2/3 signaling pathways in the regulation of PC-dependent shear stress-induced autophagy in SC cells.

Biting Innovations of Mosquito-Based Biomaterials and Medical Devices.

Mosquitoes are commonly viewed as pests and deadly predators by humans. Despite this perception, investigations of their survival-based behaviors, select anatomical features, and biological composition have led to the creation of several beneficial technologies for medical applications. In this review, we briefly explore these mosquito-based innovations by discussing how unique characteristics and behaviors of mosquitoes drive the development of select biomaterials and medical devices. Mosquito-inspired microneedles have been fabricated from a variety of materials, including biocompatible metals and polymers, to mimic of the mouthparts that some mosquitoes use to bite a host with minimal injury during blood collection. The salivary components that these mosquitoes use to reduce the clotting of blood extracted during the biting process provide a rich source of anticoagulants that could potentially be integrated into blood-contacting biomaterials or administered in therapeutics to reduce the risk of thrombosis. Mosquito movement, vision, and olfaction are other behaviors that also have the potential for inspiring the development of medically relevant technologies. For instance, viscoelastic proteins that facilitate mosquito movement are being investigated for use in tissue engineering and drug delivery applications. Even the non-wetting nanostructure of a mosquito eye has inspired the creation of a robust superhydrophobic surface coating that shows promise for biomaterial and drug delivery applications. Additionally, biosensors incorporating mosquito olfactory receptors have been built to detect disease-specific volatile organic compounds. Advanced technologies derived from mosquitoes, and insects in general, form a research area that is ripe for exploration and can uncover potential in further dissecting mosquito features for the continued development of novel medical innovations.

Perceived healthcare discrimination and well-being among older adults in the United States and Brazil.

Despite well-documented evidence illustrating the relationship between discrimination and health, less is known about the influence of unfair treatment when receiving medical care. Moreover, our current knowledge of cross-national and racial variations in healthcare discrimination is limited in aging populations. This article addresses these gaps using two harmonized data sets of aging populations to clarify the relationship between healthcare discrimination and health in the United States and Brazil. We use nationally representative, harmonized data from the Health and Retirement Study in the United States and the Brazilian Longitudinal Study of Aging to examine and compare perceived discrimination in the healthcare setting and its relationship to self-rated health, depression diagnosis, and depressive symptoms across national contexts. Using Poisson regression models and population attributable risk percent estimates, we found that aging adults reporting healthcare discrimination were at higher risk of poor self-rated health, diagnosed depression, and depressive symptoms. Our results also suggest that reducing perceived healthcare discrimination may contribute to improved self-rated health and mental well-being in later life across racialized societies. In two comparative settings, we highlight the differential impact of healthcare discrimination on self-rated health and depression. We describe the implications of our study's findings for national public health strategies focused on eliminating discrimination in the healthcare setting, particularly among aging countries.

Guided corona generates wettability patterns that selectively direct cell attachment inside closed microchannels.

We present a method to create plasma mediated linear protein patterns along the lengths of simple one-inlet-one-outlet straight polydimethylsiloxane microchannels by biasing the delivery of corona discharge at the capillary openings. Pattern widths ranging from 500-1,000 microm were generated in 2 mm wide microchannels with lengths of 0.5, 1.0, or 1.5 cm. Corona-treated surfaces enabled the spatial alignment of C2C12 myoblasts to the adhesive protein-coated regions, facilitating myoblast differentiation into myotubes. Although limited in precision, this protein patterning technique offers the advantages of simplicity and low cost, making it attractive for educational and research environments that lack access to extensive microfabrication facilities. The results also provide a cautionary note to those using corona discharge to increase wettability of microchannels; the surface modification may not be uniform, even within single microchannels being treated depending on settings and positioning of the corona device tips.

Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma.

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFß/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

Autophagy in the Aging and Experimental Ocular Hypertensive Mouse Model.

Purpose: To investigate autophagy in the outflow pathway and ganglion cell layer in the aging and ocular hypertensive mouse. Methods: Both 4-month-old and 18-month-old C57BL/6J and GFP-LC3 mice were subjected to unilateral injection of hypertonic saline into a limbal vein, causing sclerosis of the outflow pathway and subsequent elevation of intraocular pressure (IOP). IOP was measured on a weekly basis using a rebound tonometer. Protein expression levels of LC3B, Lamp1, and p62 were evaluated by western blot and/or immunofluorescence. Retinal ganglion cell (RGC) count was performed in whole retinal flat mounts using an anti-Brn3a antibody. Optic nerves were fixed with 4% paraformaldehyde and resin-embedded for axon counts and electron microscopy. Results: In contrast to 18-month-old mice, which developed sustained elevated IOP with a single injection, 4-month-old mice were refractory to high elevations of IOP. Interestingly, both the percentage of animals that developed elevated IOP and the mean ∆IOP were significantly higher in the transgenic mice compared to C57BL/6J. Immunofluorescence and western blot analysis showed dysregulated autophagy in the iridocorneal and retina tissues from 18-month-old mice compared to 4-month-old ones. Moreover, the LC3-II/LC3-I ratio correlated with IOP. As expected, injected hypertensive eyes displayed axonal degeneration and RGC death. RGC and axon loss were significantly exacerbated with aging, especially when combined with GFP-LC3 expression. Autophagic structures were observed in the degenerating axons. Conclusions: Our results indicate dysregulation of autophagy in the trabecular meshwork and retinal tissues with aging and suggest that such dysregulation of autophagy contributes to neurodegeneration in glaucoma.

Prevalence of Bacteremia and Bacterial Meningitis in Febrile Neonates and Infants in the Second Month of Life: A Systematic Review and Meta-analysis.

Importance: Febrile neonates (persons in the first month of life) are believed to be at higher risk for bacteremia or bacterial meningitis than infants in their second month of life. However, the true prevalence is unclear. Objective: To determine modern rates of bacteremia and bacterial meningitis in febrile neonates and infants in the second month of life presenting to an ambulatory setting. Data Sources: A comprehensive, no-limit search was conducted in PubMed using previously published search terms in February 2015 and repeated in September 2016. Study Selection: Abstracts and full texts were reviewed independently by several investigators. Studies were included if data regarding blood cultures or cerebrospinal fluid cultures from consecutive febrile infants in an ambulatory setting could be extrapolated within the age groups. To limit the analysis to the period after the availability of the Haemophilus influenzae type b vaccination, studies that collected data before 1990 were excluded. Data Extraction and Synthesis: Data were extracted in accordance with the Meta-analyses of Observational Studies in Epidemiology (MOOSE) reporting guidelines via independent abstraction by several investigators. The Newcastle-Ottawa Scale was used to assess bias. Main Outcomes and Measures: The primary outcomes were prevalence rates of bacteremia and bacterial meningitis in febrile neonates and infants in the second month of life. In neonates, prevalence rates were also estimated in the era of group B Streptococcus intrapartum antibiotic prophylaxis (after 1996). Results: In total, 7264 abstracts were screened, resulting in 188 full-text manuscripts reviewed, with 12 meeting inclusion criteria (with 15 713 culture results). For febrile neonates, the prevalence of bacteremia was 2.9% (95% CI, 2.3%-3.7%; I2 = 50%; n = 5145) and the prevalence of bacterial meningitis was 1.2% (95% CI, 0.8%-1.9%; I2 = 27%; n = 3288). In neonates in the era after group B Streptococcus prophylaxis, the prevalence of bacteremia was 3.0% (95% CI, 2.3%-3.9%; I2 = 6%; n = 2055) and the prevalence of meningitis was 1.0% (95% CI, 0.4%-2.1%; I2 = 28%; n = 1739). For febrile infants in the second month of life, the prevalence of bacteremia was 1.6% (95% CI, 0.9%-2.7%; I2 = 78%; n = 4778) and the prevalence of meningitis was 0.4% (95% CI, 0.2%-1.0%; I2 = 33%; n = 2502). Conclusions and Relevance: These findings suggest that febrile neonates have approximately twice the rate of bacteremia and meningitis as febrile infants in their second month of life.

Morphometric and computational assessments to evaluate neuron survival and maturation within compartmentalized microfluidic devices: The influence of design variation on diffusion-driven nutrient transport.

Burgeoning use of segregated microfluidic platforms that parse somas and neurites into discrete compartments is fueling unique examinations of neuronal structure and physiology in a manner impossible to achieve with non-compartmentalized systems. However, even though this line of axon-soma polarizing microfluidic devices stems from the same general design of a Campenot chamber set-up, slight deviations in device geometry appear to induce vastly different nutrient transport profiles that influence neuron survival and maturation. Here we examine the uptake of nerve growth factor (NGF) by a pheochromocytoma PC12 cell line cultured using two Campenot-like device designs, a "Standard" layout, representative of a commercial device, and a custom "Notch" layout, predicted to encourage more efficient nutrient transfer that gives rise to sustained neuron viability and extensive neurite elaboration. Exploiting in vitro culture schemes coupled with computational analyses, we identify the influence of device design geometry on the interplay between neuronal survival and maturation, gauged from morphometric assessments and the spatiotemporal distribution of NGF. Computer simulations of NGF transport within the devices revealed that the microfluidic neuron culture system is highly sensitive to change, where nutrient transport is intricately linked to device geometry and cell plating density, and premature depletion of nutrients is observed if specific design criteria are not met. This study underscores the importance of validating specific device geometries for a particular neuro-based assessment, while showcasing computational modeling as a powerful tool to achieve this goal.

Publisher Correction: Novel QUEST MRI In Vivo Measurement of Noise-induced Oxidative Stress in the Cochlea.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.