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Hydrazone-crosslinked hydrogels are attractive protein delivery vehicles for regenerative medicine. However, each regenerative medicine application requires unique hydrogel properties to achieve an ideal outcome. The properties of a hydrogel can be impacted by numerous factors involved in its fabrication. We used design of experiments (DoE) statistical modeling to efficiently optimize the physicochemical properties of a hyaluronic acid (HA) hydrazone-crosslinked hydrogel for protein delivery for bone regeneration. We modified HA with either adipic acid dihydrazide (HA-ADH) or aldehyde (HA-Ox) functional groups and used DoE to evaluate the interactions of three input variables, the molecular weight of HA (40 or 100 kDa), the concentration of HA-ADH (1-3% w/v), and the concentration of HA-Ox (1-3% w/v), on three output responses, gelation time, compressive modulus, and hydrogel stability over time. We identified 100 kDa HA-ADH 3.00 HA-Ox 2.33 as an optimal hydrogel that met all of our design criteria, including displaying a gelation time of 3.7 minutes, compressive modulus of 62.1 Pa, and minimal mass change over 28 days. For protein delivery, we conjugated affinity proteins called affibodies that were specific to the osteogenic protein bone morphogenetic protein-2 (BMP-2) to HA hydrogels and demonstrated that our platform could control the release of BMP-2 over 28 days. Ultimately, our approach demonstrates the utility of DoE for optimizing hydrazone-crosslinked HA hydrogels for protein delivery.

Participation of Black American older adults in community-engaged research remains challenging in health sciences. The objectives of this study were to describe the specific efforts, successes, and challenges in recruiting Black American older adults in research led by the Health and Wellness in Aging Across the Lifespan core, part of the Virginia Commonwealth University Institute for Inclusion, Inquiry, and Innovation (iCubed). We conducted a cross-case analysis of 6 community-engaged research projects using the community-engaged research continuum model. Successful recruitment strategies comprised a multifaceted approach to community-based collaboration, including a wellness program with a long standing relationship with the community, engaging key stakeholders and a community advisory board, and building a community-based coalition of stakeholders. Posting flyers and modest monetary compensation remain standard recruitment strategies. The cross-case analysis offered critical lessons on the community's nature and level of engagement in research. Relationship building based on trust and respect is essential to solving complex aging issues in the community., Competing Interests: All authors declare that they have no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

The Monetary Choice Questionnaire (MCQ) is one of the most commonly used measures to assess delay discounting of reward. Reliable measurement by the MCQ is necessary for use in experimental settings or prognostic validity within clinical contexts. The present analysis expands prior work to evaluate temporal reliability and stability over an extended period, including repeated measurements, a larger and more broadly representative sample, and demonstrations of covariation with clinically significant health behaviors (e.g., cigarette use, COVID-19 vaccination, body mass index). Participants ( N = 680; 55.6% female) were recruited through crowdsourcing and completed the MCQ approximately quarterly over 2 years. Measures of reliability, stability, and correlations with clinical constructs were determined for each timepoint and pairwise comparison. Test-retest reliabilities were high across all pairwise comparisons (all r xx > .75; range = .78-.86; mean = .83). Stability was also high with within-subject effect size differences all within a less-than-small effect size range (range d z = -0.09 to 0.19; mean = 0.04). Positive associations between smoking status and delay discounting rates were observed consistent with prior clinical studies. These findings of test durability support the use of MCQ administration for repeated measurement of delay-constrained choice as a stable respondent characteristic and illustrate its association with important health behaviors over extended time periods. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Hydrazone-crosslinked hydrogels are attractive protein delivery vehicles for regenerative medicine. However, each regenerative medicine application requires unique hydrogel properties to achieve an ideal outcome. The properties of a hydrogel can be impacted by numerous factors involved in its fabrication. We used design of experiments (DoE) statistical modeling to efficiently optimize the physicochemical properties of a hyaluronic acid (HA) hydrazone-crosslinked hydrogel for protein delivery for bone regeneration. We modified HA with either adipic acid dihydrazide (HA-ADH) or aldehyde (HA-Ox) functional groups and used DoE to evaluate the interactions of three input variables, the molecular weight of HA (40 or 100 kDa), the concentration of HA-ADH (1-3% w/v), and the concentration of HA-Ox (1-3% w/v), on three output responses, gelation time, compressive modulus, and hydrogel stability over time. We identified 100 kDa HA-ADH 3.0 HA-Ox 2.33 as an optimal hydrogel that met all of our design criteria, including displaying a gelation time of 3.7 minutes, compressive modulus of 62.1 Pa, and minimal mass change over 28 days. For protein delivery, we conjugated affinity proteins called affibodies that were specific to the osteogenic protein bone morphogenetic protein-2 (BMP-2) to HA hydrogels and demonstrated that our platform could control the release of BMP-2 over 28 days. Ultimately, our approach demonstrates the utility of DoE for optimizing hydrazone-crosslinked HA hydrogels for protein delivery., Competing Interests: Conflict of Interest A.N.G., J.D., V.R.S., and M.H.H. are authors on a pending patent application encompassing some of this work (U.S. Patent Application No. 18/340,754).

Global environmental change is identified as a driver of physical transformation of coral reef islands over the past half-century, and next 100 years, posing major adaptation challenges to island nations. Here we resolve whether these recent documented changes in islands are unprecedented compared with the pre-industrial era. We utilise radiometric dating, geological, and remote sensing techniques to document the dynamics of a Maldivian reef island at millennial to decadal timescales. Results show the magnitude of island change over the past half-century (±40 m movement) is not unprecedented compared with paleo-dynamic evidence that reveals large-scale changes in island dimension, shape, beach levels, as well as positional changes of ±200 m since island formation ~1,500 years ago. Results highlight the value of a multi-temporal methodological approach to gain a deeper understanding of the dynamic trajectories of reef islands, to support development of adaptation strategies at timeframes relevant to human security., (© 2023. The Author(s).)

Background: Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited., Results: We describe a transgenic mouse line expressing CreER T2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreER T2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreER T2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreER T2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreER T2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts., Conclusion: Overall, the Alx4-CreER T2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct., (© 2021 American Association of Anatomists.)

Primary cilia are sensory and signaling hubs with a protein composition that is distinct from the rest of the cell due to the barrier function of the transition zone (TZ) at the base of the cilium. Protein transport across the TZ is mediated in part by the BBSome, and mutations disrupting TZ and BBSome proteins cause human ciliopathy syndromes. Ciliopathies have phenotypic variability even among patients with identical genetic variants, suggesting a role for modifier loci. To identify potential ciliopathy modifiers, we performed a mutagenesis screen on nphp-4 mutant Caenorhabditis elegans and uncovered a novel allele of bbs-5. Nphp-4;bbs-5 double mutant worms have phenotypes not observed in either individual mutant strain. To test whether this genetic interaction is conserved, we also analyzed zebrafish and mouse mutants. While Nphp4 mutant zebrafish appeared overtly normal, Bbs5 mutants exhibited scoliosis. When combined, Nphp4;Bbs5 double mutant zebrafish did not exhibit synergistic effects, but the lack of a phenotype in Nphp4 mutants makes interpreting these data difficult. In contrast, Nphp4;Bbs5 double mutant mice were not viable and there were fewer mice than expected carrying three mutant alleles. In addition, postnatal loss of Bbs5 in mice using a conditional allele compromised survival when combined with an Nphp4 allele. As cilia are still formed in the double mutant mice, the exacerbated phenotype is likely a consequence of disrupted ciliary signaling. Collectively, these data support an evolutionarily conserved genetic interaction between Bbs5 and Nphp4 alleles that may contribute to the variability in ciliopathy phenotypes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Atxn10 is a gene known for its role in cytokinesis and is associated with spinocerebellar ataxia (SCA10), a slowly progressing cerebellar syndrome caused by an intragenic pentanucleotide repeat expansion. Atxn10 is also implicated in the ciliopathy syndromes nephronophthisis (NPHP) and Joubert syndrome (JBTS), which are caused by the disruption of cilia function leading to nephron loss, impaired renal function, and cerebellar hypoplasia. How Atxn10 disruption contributes to these disorders remains unknown. Here, we generated Atxn10 congenital and conditional mutant mouse models. Our data indicate that while ATXN10 protein can be detected around the base of the cilium as well as in the cytosol, its loss does not cause overt changes in cilia formation or morphology. Congenital loss of Atxn10 results in embryonic lethality around E10.5 associated with pericardial effusion and loss of trabeculation. Similarly, tissue-specific loss of ATXN10 in the developing endothelium (Tie2-Cre) and myocardium (cTnT-Cre) also results in embryonic lethality with severe cardiac malformations occurring in the latter. Using an inducible Cagg-CreER to disrupt ATXN10 systemically at postnatal stages, we show that ATXN10 is also required for survival in adult mice. Loss of ATXN10 results in severe pancreatic and renal abnormalities leading to lethality within a few weeks post ATXN10 deletion in adult mice. Evaluation of these phenotypes further identified rapid epithelial-to-mesenchymal transition (EMT) in these tissues. In the pancreas, the phenotype includes signs of both acinar to ductal metaplasia and EMT with aberrant cilia formation and severe defects in glucose homeostasis related to pancreatic insufficiency or defects in feeding or nutrient intake. Collectively, this study identifies ATXN10 as an essential protein for survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bentley-Ford, Andersen, Croyle, Haycraft, Clearman, Foote, Reiter and Yoder.)

Purpose: To evaluate depth-resolved changes of corneal biomechanical properties in eyes with corneal ectasia after corneal crosslinking (CXL) using optical coherence elastography., Methods: In a prospective pilot series of eyes with corneal ectasia, a custom high-speed swept source optical coherence tomography system was used to image the cornea before and 3 months after CXL during a low-speed applanating deformation while monitoring applanation force. Cross-correlation was applied to track frame-by-frame two-dimensional optical coherence tomography speckle displacements, and the slope of force versus local axial displacement behavior during the deformation was used to produce a two-dimensional array of axial stiffness (k). These values were averaged for anterior (ka) and posterior (kp) stromal regions and expressed as a ratio (ka/kp) to assess depth-dependent differences in stiffness. CXL was performed according to the Dresden protocol with a system approved by the U.S. Food and Drug Administration., Results: Four eyes from four patients with keratoconus (n = 3) or post-LASIK ectasia (n = 1) underwent optical coherence elastography before and 3 months after CXL. The mean ka/kp was 1.03 ± 0.07 before CXL compared with 1.34 ± 0.17 after the CXL procedure. All four eyes demonstrated at least a 20% increase in the ka/kp., Conclusions: Preferential stiffening of the anterior stroma with the standard CXL protocol was demonstrated with optical coherence elastography in live human subjects., Translational Relevance: Although ex vivo studies have demonstrated anterior stiffening effects after CXL using various destructive and nondestructive methods, this report presents the first evidence of such changes in serial live human measurements.

Primary cilia are critical sensory and signaling compartments present on most mammalian cell types. These specialized structures require a unique signaling protein composition relative to the rest of the cell to carry out their functions. Defects in ciliary structure and signaling result in a broad group of disorders collectively known as ciliopathies. One ciliopathy, Bardet-Biedl syndrome (BBS; OMIM 209900), presents with diverse clinical features, many of which are attributed to defects in ciliary signaling during both embryonic development and postnatal life. For example, patients exhibit obesity, polydactyly, hypogonadism, developmental delay and skeletal abnormalities along with sensory and cognitive deficits, but for many of these phenotypes it is uncertain, which are developmental in origin. A subset of BBS proteins assembles into the core BBSome complex, which is responsible for mediating transport of membrane proteins into and out of the cilium, establishing it as a sensory and signaling hub. Here, we describe two new mouse models for BBS resulting from a targeted LacZ gene trap allele (Bbs5-/-) that is a predicted congenital null mutation and conditional (Bbs5flox/flox) allele of Bbs5. Bbs5-/- mice develop a complex phenotype consisting of increased pre-weaning lethality craniofacial and skeletal defects, ventriculomegaly, infertility and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly and pituitary abnormalities are only present when Bbs5 is disrupted prior to postnatal day 7, indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity, independent of the age of Bbs5 loss., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Large uncertainty surrounds the future physical stability of low-lying coral reef islands due to a limited understanding of the geomorphic response of islands to changing environmental conditions. Physical and numerical modelling efforts have improved understanding of the modes and styles of island change in response to increasing wave and water level conditions. However, the impact of sediment supply on island morphodynamics has not been addressed and remains poorly understood. Here we present evidence from the first physical modelling experiments to explore the effect of storm-derived sediment supply on the geomorphic response of islands to changes in sea level and energetic wave conditions. Results demonstrate that a sediment supply has a substantial influence on island adjustments in response to sea-level rise, promoting the increase of the elevation of the island while dampening island migration and subaerial volume reduction. The implications of sediment supply are significant as it improves the potential of islands to offset the impacts of future flood events, increasing the future physical persistence of reef islands. Results emphasize the urgent need to incorporate the physical response of islands to both physical and ecological processes in future flood risk models.

Background: Fetal alcohol spectrum disorder (FASD) is caused by prenatal alcohol exposure (PAE), the intake of ethanol (C 2 H 5 OH) during pregnancy. Features of FASD cover a range of structural and functional defects including congenital heart defects (CHDs). Folic acid and choline, contributors of methyl groups to one-carbon metabolism (OCM), prevent CHDs in humans. Using our avian model of FASD, we have previously reported that betaine, another methyl donor downstream of choline, prevents CHDs. The CHD preventions are substantial but incomplete. Ethanol causes oxidative stress as well as depleting methyl groups for OCM to support DNA methylation and other epigenetic alterations. To identify more compounds that can safely and effectively prevent CHDs and other effects of PAE, we tested glutathione (GSH), a compound that regulates OCM and is known as a "master antioxidant.", Methods/results: Quail embryos injected with a single dose of ethanol at gastrulation exhibited congenital defects including CHDs similar to those identified in FASD individuals. GSH injected simultaneously with ethanol not only prevented CHDs, but also improved survival and prevented other PAE-induced defects. Assays of hearts at 8 days (HH stage 34) of quail development, when the heart normally has developed 4-chambers, showed that this single dose of PAE reduced global DNA methylation. GSH supplementation concurrent with PAE normalized global DNA methylation levels. The same assays performed on quail hearts at 3 days (HH stage 19-20) of development, showed no difference in global DNA methylation between controls, ethanol-treated, GSH alone, and GSH plus ethanol-treated cohorts., Conclusions: GSH supplementation shows promise to inhibit effects of PAE by improving survival, reducing the incidence of morphological defects including CHDs, and preventing global hypomethylation of DNA in heart tissues., (© 2020 by the Research Society on Alcoholism.)

Purpose: Compare depth-resolved biomechanical properties in normal and keratoconic corneas in live human subjects using optical coherence elastography (OCE)., Methods: In a prospective series of normal and keratoconus (KC) eyes, a corneal perturbation was applied by a custom swept-source OCE system using a transparent flat lens coupled to force transducers. Cross-correlation was applied to track frame-by-frame OCT speckle displacement. Regional displacements for the anterior and posterior stroma were plotted in force versus displacement ( k ) graphs. A spatial biomechanical property ratio ( k a /k p ) was defined by dividing the maximum total displacement by the maximum force for the anterior ( k a ) and posterior cornea ( k p ) and was compared between normal and KC groups with the Mann-Whitney U test. Area under the receiver operating characteristics curve (AUROC) for differentiating normal and KC eyes was calculated for k a /k p , k max , and thinnest point of corneal thickness (TPCT)., Results: Thirty-six eyes were analyzed (21 eyes of 12 normal subjects and 15 KC eyes of 12 subjects). The k a /k p for the normal group was 1.135 ± 0.07 (mean ± standard deviation) and 1.02 ± 0.08 for the KC group ( P < 0.001), indicating a relative deficit in anterior stromal stiffness in KC eyes. AUROC was 0.91 for k a / k p , 0.95 for k max , and 1 for TPCT., Conclusions: Significant differences in depth-dependent corneal biomechanical properties were observed between normal and KC subjects., Translational Relevance: OCE was applied for the first time to human KC subjects and revealed alterations in the normal anterior-to-posterior stromal stiffness gradient, a novel and clinically accessible disease biomarker., Competing Interests: Disclosure: V.S. De Stefano, None; M.R. Ford, Cleveland Clinic (P); I. Seven, None; W.J. Dupps, Cleveland Clinic (P), Alcon (C), Zeiss (C), Glaukos (C), (Copyright 2020 The Authors.)

Purpose: There is strong evidence that abnormalities in corneal biomechanical play a causal role in corneal ectasias, such as keratoconus. Additionally, corneal crosslinking (CXL) treatment, which halts progression of keratoconus, directly appeals to corneal biomechanics. However, existing methods of corneal biomechanical assessment have various drawbacks: dependence on IOP, long acquisition times, or limited resolution. Here, we present a method that may avoid these limitations by using optical coherence tomography (OCT) to detect the endogenous random motion within the cornea, which can be associated with stromal crosslinking., Methods: Phase-decorrelation OCT (PhD-OCT), based in the theory of dynamic light scattering, is a method to spatially resolve endogenous random motion by calculating the decorrelation rate, Γ, of the temporally evolving complex-valued OCT signal. PhD-OCT images of ex vivo porcine globes were recorded during CXL and control protocols. In addition, human patients were imaged with PhD-OCT using a clinical OCT system., Results: In both the porcine cornea and the human cornea, crosslinking results in a reduction of Γ (P < 0.0001), indicating more crosslinks. This effect was repeatable in ex vivo porcine corneas (change in average Γ = -41.55 ± 9.64%, n = 5) and not seen after sham treatments (change in average Γ = 2.83 ± 12.56%, n = 5). No dependence of PhD-OCT on IOP was found, and correctable effects were caused by variations in signal-to-noise ratio, hydration, and motion., Conclusions: PhD-OCT may be a useful and readily translatable tool for investigating biomechanical properties of the cornea and for enhancing the diagnosis and treatment of patients.

Purpose: To assess depth-dependent corneal displacements in live normal subjects using optical coherence elastography (OCE)., Methods: A corneal elastography method based on swept-source optical coherence tomography (OCT) was implemented in a clinical prototype. Low amplitude corneal deformation was produced during OCT imaging with a linear actuator-driven lens coupled to force transducers. A cross-correlation algorithm was applied to track frame-by-frame speckle displacement across horizontal meridian scans. Intra-measurement force and displacement data series were plotted against each other to produce local axial stiffness approximations, k, defined by the slope of a linear fit to the force/displacement data (ignoring non-axial contributions from corneal bending). Elastographic maps displaying local k values across the cornea were generated, and the ratio of mean axial stiffness approximations for adjacent anterior and posterior stromal regions, ka/kp, was calculated. Intraclass correlation coefficients (ICC) were used to estimate repeatability., Results: Seventeen eyes (ten subjects) were included in this prospective first-in-humans translational study. The ICC was 0.84. Graphs of force vs. displacement demonstrated that, for simultaneously acquired measurements involving the same applied force, anterior stromal displacements were lower (suggesting stiffer behavior) than posterior stromal displacements. Mean ka was 0.016±0.004 g/mm and mean kp was 0.014±0.004 g/mm, giving a mean ka/kp ratio of 1.123±0.062., Conclusion: OCE is a clinically feasible, non-invasive corneal biomechanical characterization method capable of resolving depth-dependent differences in corneal deformation behavior. The anterior stroma demonstrated responses consistent with stiffer properties in compression than the posterior stroma, but to a degree that varied across normal eyes. The clinical capability to measure these differences has implications for assessing the biomechanical impact of corneal refractive surgeries and for ectasia risk screening applications., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Matthew R. Ford- Patent (Cleveland Clinic Innovations); William J. Dupps, Jr - Patent (Cleveland Clinic Innovations). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Sea-level rise and climatic change threaten the existence of atoll nations. Inundation and erosion are expected to render islands uninhabitable over the next century, forcing human migration. Here we present analysis of shoreline change in all 101 islands in the Pacific atoll nation of Tuvalu. Using remotely sensed data, change is analysed over the past four decades, a period when local sea level has risen at twice the global average (~3.90 ± 0.4 mm.yr -1 ). Results highlight a net increase in land area in Tuvalu of 73.5 ha (2.9%), despite sea-level rise, and land area increase in eight of nine atolls. Island change has lacked uniformity with 74% increasing and 27% decreasing in size. Results challenge perceptions of island loss, showing islands are dynamic features that will persist as sites for habitation over the next century, presenting alternate opportunities for adaptation that embrace the heterogeneity of island types and their dynamics.

Purpose: To evaluate corneal air-puff deformation responses and ocular geometry as predictors of Marfan syndrome., Design: Prospective observational clinical study., Methods: Sixteen investigator-derived, 4 standard Ocular Response Analyzer (ORA), and geometric variables from corneal tomography and optical biometry using Oculus Pentacam and IOL Master were assessed for discriminative value in Marfan syndrome, measuring right eyes of 24 control and 13 Marfan syndrome subjects. Area under the receiver operating characteristic (AUROC) curve was assessed in univariate and multivariate analyses., Results: Six investigator-derived ORA variables successfully discriminated Marfan syndrome. The best lone disease predictor was Concavity Min (Marfan syndrome 47.5 ± 20, control 69 ± 14, P = .003; AUROC = 0.80). Corneal hysteresis (CH) and corneal resistance factor (CRF) were decreased (Marfan syndrome CH 9.45 ± 1.62, control CH 11.24 ± 1.21, P = .01; Marfan syndrome CRF 9.77 ± 1.65, control CRF 11.03 ± 1.72, P = .01) and corneas were flatter in Marfan syndrome (Marfan syndrome Kmean 41.25 ± 2.09 diopter, control Kmean 42.70 ± 1.81 diopter, P = .046). No significant differences were observed in central corneal thickness, axial eye length, or intraocular pressure. A multivariate regression model incorporating corneal curvature and hysteresis loop area (HLA) provided the best predictive value for Marfan syndrome (AUROC = 0.85)., Conclusions: This study describes novel biodynamic features of corneal deformation responses in Marfan syndrome, including increased deformation, decreased bending resistance, and decreased energy dissipation capacity. A predictive model incorporating HLA and corneal curvature shows greatest potential for noninvasive clinical diagnosis of Marfan syndrome., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Epidemiologic studies in humans have shown associations between greater sunlight exposure, higher serum 25-hydroxycholecalciferol [25(OH)D3] concentrations, and reduced colon cancer risk. However, results from a limited number of vitamin D supplementation trials in humans have not shown a protective effect., Objective: We sought to determine whether adding to the diet increasing amounts of either 25(OH)D3, the stable metabolite measured in serum and associated with cancer risk, or cholecalciferol (vitamin D3), the compound commonly used for supplementation in humans, could reduce emergent adenomas (chemoprevention) or decrease the growth of existing adenomas (treatment) in the colons of vitamin D-sufficient rats carrying a truncation mutation of adenomatous polyposis coli (Apc), a model of early intestinal cancer., Methods: Apc(Pirc/+) rats were supplemented with either vitamin D3 over a range of 4 doses [6-1500 μg/(kg body weight · d)] or with 25(OH)D3 over a range of 6 doses [60-4500 μg/(kg body weight · d)] beginning after weaning. Rats underwent colonoscopy every other week to assess effects on adenoma number and size. At termination (140 d of age), the number of tumors in the small intestine and colon and the size of tumors in the colon were determined, and serum calcium and 25(OH)D3 measurements were obtained., Results: At lower doses (those that did not affect body weight), neither of the vitamin D compounds reduced the number of existing or emergent colonic tumors (P-trend > 0.24). By contrast, supplementation at higher doses (those that caused a suppression in body weight gain) with either 25(OH)D3 or vitamin D3 caused a dose-dependent increase in colonic tumor number in both males and females (P-trend < 0.003)., Conclusions: No evidence for protection against colon tumor development was seen with lower dose supplementation with either cholecalciferol or 25-hydroxycholecalciferol. Thus, the association between sunlight exposure and the incidence of colon cancer may involve factors other than vitamin D concentrations. Alternative hypotheses warrant investigation. Furthermore, this study provides preliminary evidence for the need for caution regarding vitamin D supplementation of humans at higher doses, especially in individuals with sufficient serum 25(OH)D3 concentrations., (© 2015 American Society for Nutrition.)

Disturbed cardiac function at an early stage of development has been shown to correlate with cellular/molecular, structural as well as functional cardiac anomalies at later stages culminating in the congenital heart defects (CHDs) that present at birth. While our knowledge of cellular and molecular steps in cardiac development is growing rapidly, our understanding of the role of cardiovascular function in the embryo is still in an early phase. One reason for the scanty information in this area is that the tools to study early cardiac function are limited. Recently developed and adapted biophotonic tools may overcome some of the challenges of studying the tiny fragile beating heart. In this chapter, we describe and discuss our experience in developing and implementing biophotonic tools to study the role of function in heart development with emphasis on optical coherence tomography (OCT). OCT can be used for detailed structural and functional studies of the tubular and looping embryo heart under physiological conditions. The same heart can be rapidly and quantitatively phenotyped at early and again at later stages using OCT. When combined with other tools such as optical mapping (OM) and optical pacing (OP), OCT has the potential to reveal in spatial and temporal detail the biophysical changes that can impact mechanotransduction pathways. This information may provide better explanations for the etiology of the CHDs when interwoven with our understanding of morphogenesis and the molecular pathways that have been described to be involved. Future directions for advances in the creation and use of biophotonic tools are discussed.

Studies suggest that standard corneal collagen crosslinking (CXL) is a safe and effective treatment to stiffen the cornea for keratoconus and other ectatic corneal disorders. The purpose of the present study was to compare the biomechanical effects of transepithelial benzalkonium chloride-EDTA (BAC-EDTA) riboflavin-UVA crosslinking to standard epithelium-off riboflavin-UVA crosslinking in a rabbit model. Corneal stiffness was quantified using optical coherence elastography at two months after treatment. The mean lateral-to-axial displacement ratio for the BAC-EDTA transepithelial CXL group was lower (greater stiffness) [0.062 ± 0.042, mean ± SD] than epithelium-off CXL (mean ± SD: 0.065 ± 0.045) or untreated control eyes (0.069 ± 0.044). Using ANOVA with Tukey correction, a statistically significant difference was found between the BAC-EDTA transepithelial CXL group and standard epithelium-off CXL (p = 0.0019) or the untreated control (p < 0.0001) groups. A graph of the probability density functions for biomechanical stiffness also showed a greater shift in stiffening in the BAC-EDTA transepithelial CXL group than the standard epithelium-off CXL or untreated control group. These results demonstrated that the biomechanical stiffening effect produced by BAC-EDTA transepithelial CXL was greater than that produced by standard epithelium-off CXL in a rabbit model., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Implantable cardioverter defibrillator (ICD) shocks terminate potentially life-threatening arrhythmias and may alter patient behavior. Patients are aware of which activities they have the ability to perform, but they may choose to avoid these behaviors after receiving a shock. This study examined ICD patient ability and avoidance of progressively exertive behaviors., Methods: ICD patients (N = 443) across the United States were surveyed using an online measure including the 12-item Duke Activity Status Index., Results: As expected, many patients reported being unable to participate in more physically exertive activities such as strenuous athletic exertion (68.8%), sex (35.4%), and running a short distance (49.0%). Avoidance rates were also relatively high, as patients who reported being able to participate in these activities also reported avoiding them (ie, strenuous athletics, 76.1%). Similarly, the majority of patients reported ability to engage in sexual activity (64.6%) but many chose to avoid sexual activity (51.0%). Multiple reasons were reported for avoidance, including fear of shock, fear of heart rate increase, doctor instruction, no desire, and an "other" option., Conclusion: Many ICD patients experience behavioral limitations because of both a perceived inability and preference to avoid exertive activities, and possibly a prescription to do so, particularly strenuous athletic exertion. Clinical and research attention to ICD patient activity levels and reasons for avoidance may improve daily functioning and help patients return to preimplant levels of activity.

Purpose: To noninvasively evaluate the effects of corneal hydration and collagen crosslinking (CXL) on the mechanical behavior of the cornea., Setting: Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, USA., Design: Experimental study., Methods: An optical coherence elastography (OCE) technique was used to measure the displacement behavior of 5 pairs of debrided human donor globes in 3 serial states as follows: edematous, normal thickness, and after riboflavin-ultraviolet-A-mediated CXL. During micromotor-controlled axial displacements with a curved goniolens at physiologic intraocular pressure (IOP), serial optical coherence tomography scans were obtained to allow high-resolution intrastromal speckle tracking and displacement measurements over the central 4.0 mm of the cornea., Results: With no imposed increase in IOP, the mean lateral to imposed axial displacement ratios were 0.035 μm/μm ± 0.037 (SD) in edematous corneas, 0.021 ± 0.02 μm/μm in normal thickness corneas, and 0.014 ± 0.009 μm/μm in post-CXL corneas. The differences were statistically significant (P<.05, analysis of variance) and indicated a 40% increase in lateral stromal resistance with deturgescence and a further 33% mean increase in relative stiffness with CXL., Conclusions: Serial perturbations of the corneal hydration state and CXL had significant effects on corneal biomechanical behavior. With an axially applied stress from a nonapplanating contact lens, displacements along the direction of the collagen lamellae were 2 orders of magnitude lower than axial deformations. These experiments show the ability of OCE to quantify clinically relevant mechanical property differences under physiologic conditions., Financial Disclosures: Proprietary or commercial disclosures are listed after the references., (Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Many studies of the response of colonic tumors to therapeutics use tumor multiplicity as the endpoint to determine the effectiveness of the agent. These studies can be greatly enhanced by accurate measurements of tumor volume. Here we present a quantitative method to easily and accurately determine colonic tumor volume. This approach uses a biocompatible alginate to create a negative mold of a tumor-bearing colon; this mold is then used to make positive casts of dental stone that replicate the shape of each original tumor. The weight of the dental stone cast correlates highly with the weight of the dissected tumors. After refinement of the technique, overall error in tumor volume was 16.9% ± 7.9% and includes error from both the alginate and dental stone procedures. Because this technique is limited to molding of tumors in the colon, we utilized the Apc(Pirc/+) rat, which has a propensity for developing colonic tumors that reflect the location of the majority of human intestinal tumors. We have successfully used the described method to determine tumor volumes ranging from 4 to 196 mm³. Alginate molding combined with dental stone casting is a facile method for determining tumor volume in vivo without costly equipment or knowledge of analytic software. This broadly accessible method creates the opportunity to objectively study colonic tumors over time in living animals in conjunction with other experiments and without transferring animals from the facility where they are maintained.

Purpose: To compare the biological effects of riboflavin-ultraviolet A (UVA) corneal cross-linking (CXL) performed with a traditional epithelium-off method to several transepithelial methods in a rabbit model. Preliminary experiments on biomechanical rigidity were also performed., Methods: Four treatment groups were included: (1) standard epithelium-off, (2) tetracaine transepithelial, (3) benzal-konium chloride-ethylenediaminetetraacetic acid (BKC-EDTA) transepithelial, and (4) femtosecond laser-assisted transepithelial riboflavin-UVA CXL. Six eyes from each treatment group and the untreated control group were analyzed at 24 hours and 2 months after treatment in wound healing studies. The TUNEL assay was performed to detect the extent of stromal cell death. Optical density was measured with a Scheimpflug analyzer. The corneal stiffening effect was quantitated in three eyes from each group using optical coherence elastography performed 2 months after treatments., Results: Twenty-four hours after CXL, stromal cell death extended full corneal thickness with both standard epithelium-off CXL and femtosecond laser-assisted CXL, but only approximately one-third stromal depth after BKC-EDTA transepithelial CXL. Negligible stromal cell death was detected with tetracaine transepithelial CXL. Cell death results were statistically different between the BKC-EDTA transepithelial CXL and standard epithelium-off CXL groups (P < .0001). Significant corneal opacity differences were noted. Standard epithelium-off CXL had the greatest density and tetracaine transepithelial CXL had the least density compared to the control group after treatment. As measured with optical coherence elastography, a trend toward greater mean stiffening was observed with BKC-EDTA transepithelial CXL than with epithelium-off CXL, femtosecond laser-assisted CXL, or tetracaine transepithelial CXL, but the result did not reach statistical significance. All of the CXL treatment groups exhibited significantly smaller variance of stiffness compared to the control group., Conclusion: In the rabbit model, BKC-EDTA transepithelial CXL produced less stromal cell death and less risk of endothelial cell damage than standard epithelium-off CXL or femtosecond laser-assisted CXL. Additional study is needed to determine whether biomechanical stiffness is significantly different between the epithelium-off CXL and transepithelial CXL groups., (Copyright 2013, SLACK Incorporated.)

The material properties of the cornea are important determinants of corneal shape and refractive power. Corneal ectatic diseases, such as keratoconus, are characterized by material property abnormalities, are associated with progressive thinning and distortion of the cornea, and represent a leading indication for corneal transplantation. We describe a corneal elastography technique based on optical coherence tomography (OCT) imaging, in which displacement of intracorneal optical features is tracked with a 2-D cross-correlation algorithm as a step toward nondestructive estimation of local and directional corneal material properties. Phantom experiments are performed to measure the effects of image noise and out-of-plane displacement on effectiveness of displacement tracking and demonstrated accuracy within the tolerance of a micromechanical translation stage. Tissue experiments demonstrate the ability to produce 2-D maps of heterogeneous intracorneal displacement with OCT. The ability of a nondestructive optical method to assess tissue under in situ mechanical conditions with physiologic-range stress levels provides a framework for in vivo quantification of 3-D corneal elastic and viscoelastic resistance, including analogs of shear deformation and Poisson's ratio that may be relevant in the early diagnosis of corneal ectatic disease.

People living without piped water and sewer can be at increased risk for diseases transmitted via the fecal-oral route. One rural Alaskan community that relies on hauling water into homes and sewage from homes was studied to determine the pathways of fecal contamination of drinking water and the human environment so that barriers can be established to protect health. Samples were tested for the fecal indicator, Escherichia coli, and the less specific indicator group, total coliforms. Shoes transported fecal contamination from outside to floor material inside buildings. Contamination in puddles on the road, in conjunction with contamination found on all-terrain vehicle (ATV) tires, supports vehicle traffic as a mechanism for transporting contamination from the dumpsite or other source areas to the rest of the community. The abundance of fecal bacteria transported around the community on shoes and ATV tires suggests that centralized measures for waste disposal as well as shoe removal in buildings could improve sanitation and health in the community.

Truly accurate dipole source localization relies on having (1) reliable, artifact-free EP/ERP (or magnetoelectroencephalogram) data to start with; (2) landmark and electrode position data obtained with a three-dimensional digitizer; (3) MRI (or CT) data from the same subject (with a means to measure the same landmarks); (4) the capability to apply different dipole and volume conductor models; and (5) the ability to coregister the functional and anatomic data to display the final source solutions. Most important is to have a thorough understanding of the fundamental principles of the localization programs and neurophysiologic processes. Localization programs are simply tools. Used incorrectly, they can yield meaningless results. Used correctly, they can provide a window into neurophysiologic functioning that is not available through any other means.

Artifact in one form or another needs to be contended with in all EEG and EP studies. Various methods have been employed to avoid, eliminate, or minimize artifact. This article has described the methods that have been available for some time and newer methods and their advantages. It is the authors' hope that through the use of these methods the accuracy of all EP and ERP measurements will be improved and promote the validity and general acceptance of EP and ERP recordings. Genuine and valuable data are contained in EPs and ERPs. The challenge is to extract relatively small voltage signals often occurring within a higher voltage background of artifact. The computing power required to perform the artifact removal/reduction procedures now is available with basic laptop and desktop computers, as are the software programs that provide the artifact removal/reduction capabilities. It may be of interest for a prudent researcher to integrate the currently available artifact rejection methods before subjecting the ERP and EP data for further analysis and subsequent publication.

Although scalp potential distributions do not uniquely determine the location and configuration of neural generators, they are important because they provide the necessary conditions that any hypothesized sources must satisfy and suggest a basis for testing alternate source hypotheses. One problem that could confound the correct interpretation of scalp potentials is the choice of reference electrode. Changing the reference may make activity patterns and waveform components appear and disappear (Pascual-Marqui et al. (1988) Int. J. Neurosci., 43: 237-249). The cortical imaging technique (CIT), a method for approximating potential fields on the cortical surface, was used to test the effects of the choice of reference electrode on these fields. Simulated and empirical evoked potential scalp-recorded referential data were mathematically analyzed for the case in which the reference (linked-ears) was arbitrarily assumed to be at zero potential, and the case in which the reference was the 'average' electrode, the arithmetic mean of all of the scalp-recorded voltages in the referential montage. The results for the two references were similar. This is encouraging because potential measurements relative to a point at infinity (zero potential) are never available and the assumption that any actual reference used for a recording is at zero potential is therefore suspect.

When evoked responses are used in clinical practice and research the measures that are most commonly considered are the latencies and amplitudes of EP components as measured at a single electrode site. Our recent studies have shown that multichannel recordings yield measures such as potential field asymmetry that may be as important as component latency and amplitude. The purpose of this short technical note is to suggest that electrode placement is critical for demonstrating interesting features of the potential field topography, specifically, bilateral, homologous generator sites. The cortical imaging technique (CIT) was used to analyze the averaged responses for a group of thirty normal young adults to a repeated tone and a random oddball tone. Recordings were obtained at 28 scalp recording sites which included 20 placements from the 10-20 system and eight additional sites. Simulated cortical maps were derived for four components, the N1 frequent response and the N2a, P3, and N3, rare minus frequent responses for three different electrode arrays. These arrangements included the full 28-channel array, a 20-channel array that excluded eight additional central sites, and a 20-channel array that included the eight additional sites and excluded peripheral sites. This study demonstrates that for these auditory paradigms, the placement of the electrodes is critically important for discriminating important features of the potential fields.

The cortical imaging technique (CIT), a mathematical method for simulating the potential fields on the surface of the brain, was used to analyze the spatio-temporal progression of the AEP P300 component (as well as the preceding and subsequent N2a and N3 components) from thirty normal adult subjects recorded in a standard "oddball" paradigm. Comparisons were made between the progressions of the endogenous event-related cognitive potentials and the exogenous stimulus-dependent potentials (N1 component). Cortical imaging results suggest that different and multiple generator sites are involved in the production of exogenous and endogenous evoked responses. We particularly note the asymmetric development of the P300 component and the apparent anterior generator sites for the N2a component. This last result is interesting because the N2a precedes the P300 component and supports an earlier frontal contribution.

Two mathematical techniques, the dipole localization method (DLM) and the cortical imaging technique (CIT), are used to analyze the resting visual response to pattern-reversal stimulation. These methods identify certain age-related features of this evoked response that are not found by using standard topographic maps. These features include the symmetry of the N1 and P1 responses. The amplitudes of the N1 and P2 responses and the latency of N2 are also significantly different between old and young groups of test subjects, findings consistent with differences seen in conventional topographical analyses. Theoretical dipole sources and simulated cortical surface maps are also constructed for the "average" normal older subject and one patient with documented progressive frontal lobe degenerative disease. Standard topographical imaging studies of this patient were unremarkable, except for the P300 auditory response. DLM and CIT analyses of the VER components were exceptional and consistent with the clinical diagnosis. These mathematical methods appear to enhance the discriminating power of traditional electrophysiological measures.

Two mathematical techniques, the dipole localization method (DLM) and the cortical imaging technique (CIT), are used to analyze the resting and P300 auditory responses in young and old normal volunteers. These methods identify certain age-related features of these evoked responses that are not found by standard topographic methods. These features include the orientation of the P200 resting response, and the laterality of the N120 response, and the eccentricity of the P300 response in the P300 stimulus condition. Theoretical dipole sources and simulated cortical surface maps are also constructed for one normal subject and one psychiatric inpatient and compared. These mathematical methods appear to enhance the discriminating power of traditional electrophysiological measures.

Self-regulation methods incorporating biofeedback are known to be useful in the treatment of psychophysiological disorders in psychologically normal patients. In this study, the effectiveness of Quieting Response (QR) training for the treatment of secondary psychophysiological complaints in psychiatric inpatients was assessed. Ten male and 27 female inpatients with a variety of secondary psychophysiological diagnoses were accepted into the study. Severely disturbed, confused, or depressed patients were excluded. All patients received QR training, which integrated EMG (electromyogram) and thermal feedback with breathing, progressive relaxation, and autogenic exercises. Daily practice of a 15-minute sequence of exercises was encouraged, and a 6-second reinforcement exercise was presented for use whenever an annoyance was encountered. No other therapeutic modalities were employed. Patients were followed for up to 2 years, and outcome was assessed with a multifactor quartile system. Rate of success at final follow-up for the inpatients (51%) was found to be equivalent to that of outpatients coming to the clinic with primary psychophysiological diagnoses (55%) and was not related to psychiatric diagnosis or nature of presenting complaint. Stepwise discriminant analysis indicated that older, less depressed individuals were more successful. Inpatients differed from nonpsychiatric patients only in the number of additional sessions received. Only one minor incident of increased psychopathology was observed. It was concluded that QR training was as effective for treating secondary psychophysiological disorders in all but the most severely disturbed inpatients as it was for the outpatient clinic population.

The differential prevalence of the histrionic and antisocial personality disorders among men and women has been attributed both to sex biases and to actual variation in disorder base rates. The present study assessed the bias and base rate explanations and examined whether sex biases are minimized by the relatively explicit diagnostic criteria in the DSM-III. Psychologists (N = 354) either diagnosed 9 DSM-III disorders from case histories that varied in the ambiguity of the antisocial and histrionic personality disorder diagnoses or rated the degree to which specific features extracted from the case histories met 10 histrionic and antisocial diagnostic criteria. The sex of the patient was either male, female, or unspecified. Sex biases were evident for the diagnoses but not for the diagnostic criteria. The results are discussed with respect to base rate effects, sex biases, and the construction of diagnostic criteria.

Although the data base describing clinical outcome following biofeedback/relaxation training is accumulating, there have been relatively few attempts to predict short-term outcome, and even fewer for long-term outcome. Significant short-term outcome predictors have been identified, and they often allude to the level of psychological distress as a major factor affecting outcome. To investigate further the role of psychological variables in outcome prediction, this project presents preliminary findings that demonstrate the relationship involving interpersonal psychological stress, interpersonal style of behavior, and outcome. With a very heterogeneous group of patients (N = 39), measures of interpersonal style of behavior were used to discriminate correctly 80% of cases by outcome at the 3-month postadmission evaluation. With a more homogeneous group having somatic disorders alone (headache, Raynaud's, etc.), the accuracy of prediction at 3 months was 90%. No measures were capable of predicting long-term outcome with accuracies even moderately greater than chance. The results indicate that style of interpersonal behavior (e.g., managerial, dependent) and, to a lesser extent, interpersonal stress is predictive of short-term outcome following biofeedback/relaxation training. Individuals who tend to like responsibility and who are more executive and independent generally benefited more than doubtful, obedient, and depressed individuals. While the resulting prediction rates were only slightly more accurate than MMPI D and Pt subscales alone, the procedures provide additional information regarding the personality characteristics of successful and unsuccessful biofeedback clients, and may also provide the clinician with information regarding treatment choice if biofeedback is provided as an adjunct to psychotherapy.

Clinical evidence for the long-term effectiveness of biofeedback related relaxation training is accumulating. The purpose of this report is to describe the population, self-regulation procedure, outcome criteria, and final outcome for patients who received Quieting Response (QR) training. Data from 340 patients who completed at least the first follow-up at 3 months is presented. Primary presenting symptoms were headaches, 72%; primary and secondary Raynaud's, 14%; hypertension, 4%; irritable colon, 4%; and miscellaneous, 6%. QR training integrated EMG and thermal feedback with deep breathing, progressive relaxation, and autogenic exercises presented on cassette tapes. Eight 1-hour weekly sessions were given, with emphasis on daily home exercises. Follow-up evaluations were at 3 months, 6 months, 1 year, and 2 years. Outcome was based on change in frequency, severity, and duration of symptoms; changes in medication; and secondary benefits. Quieting Response training was found to be most beneficial for patients with primary Raynaud's disease (18 of 23 patients, or 78% successful), classic migraines (9 of 13, or 69%), and common migraines (20 of 32, or 62%), followed by mixed headaches (79 of 131, or 60%), Raynaud's plus other symptoms (9 of 15, or 60%), and the irritable colon syndrome (7 of 13, or 54%). Less successful were patients with headaches plus other symptoms (16 of 37, or 43%), muscle contraction headaches (13 of 33, or 39%), secondary Raynaud's phenomenon (4 of 10, or 40%), and essential hypertension (5 of 15, or 33%). Speculations about the differing outcomes across symptom groups were made.

Tamoxifen has recently been suggested as treatment for endometriosis. We report a patient who developed endometriosis after starting tamoxifen therapy for benign breast disease.

Susceptibility to motion sickness is a powerful index of individual differences and can be assessed in large populations without a test of performance. Although all questionnaire techniques introduce some error, we have demonstrated that a simple set of questions can lead to a five-point rating system with adequate validity and reliability. Our observations may be useful to any investigator interested in individual differences and have been extremely effective in sorting out behavioral trends in large samples of patients.

Electroencephalogram (EEG) coherence (COH) and power measures were included in a series of stepwise discriminant analyses to determine which variables were most sensitive in the differentiation of four psychiatric inpatient groups and two major classes of psychotropic medication. Eight channels of eyes-closed, bipolar EEG activity were recorded from 74 inpatients (paranoid schizophrenics, dysthymics, major affectives receiving tricyclics, neuroleptics, or no medication, and geriatrics). Discriminant analyses were conducted for theta, alpha, and fast beta frequency bands for power variables, COH variables, and the resultant significant power and COH discriminating variables. Without exception, COH measures, usually in the alpha band, were more sensitive than power measures in differentiating the various groups. Results suggested that COH decreases with age, is greatest in paranoid schizophrenics, decreases with neuroleptic medication, and increases with tricyclic antidepressants. Group differences were interpreted in accordance with an arousal model for COH.