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Objective Challenges providing cleft/craniofacial care in rural communities are often reported, leading to disparities in resources available to clinicians. The purpose of this study was to identify the impact of rurality on caseloads and practice patterns of speech-language pathologists (SLPs) regarding speech and velopharyngeal function for children with cleft lip and/or palate (CL/P).Design A national, survey of US-based SLPs (N = 359 respondents) investigated resources, comfort level, caseloads, and practice patterns for children with CL/P. Sub-county classifications that delineated levels of rurality were utilized. Descriptive statistics and chi-square analyses were conducted to determine the impact of population density on assessment and referral decisions.Results Nearly 83% of SLPs reported providing care for a child with CL/P and 41.4% of these SLPs reported five or more children with CL/P on caseload throughout their career. There were no significant differences in rurality of practice setting and the likelihood of treating a child with CL/P. Significant differences were present between rural, town, suburban, and metropolitan-based SLPs regarding available resources (p= 0.035). SLPs in rural settings reported feeling uncomfortable treating children with CL/P compared to those in metropolitan settings (p= 0.02). Distance to the cleft/craniofacial team and comfort levels impacted referral decisions.Conclusions Most SLPs report having children with CL/P on caseload regardless of practice location. Rurality impacted assessment and referral decisions, especially surrounding access to resources and comfort levels engaging in team care. Findings have implications for developing support systems and reducing barriers for rural SLPs working with children born with CL/P.

Objective The use of magnetic resonance imaging (MRI) in the assessment process for children with cleft/craniofacial conditions remains uncommon, particularly for velopharyngeal assessments. The purpose of this study was to analyze the perceived clinical utility of MRI for cleft/craniofacial providers and identify barriers that exist for clinical translation of this imaging modality to this population of patients.Methods A 38-item survey was disseminated to craniofacial team providers. Workplace context and demographics, MRI as a research and clinical tool, access and barriers for use of MRI, and needs for successfully establishing MRI protocols at clinical sites were investigated. Descriptive statistics were used to identify differences in the clinical use of MRI across disciplines. Chi-square analyses were conducted to determine how different specialties perceived potential barriers.Results Respondents reported that MRI is likely to be beneficial for clinical assessments (93.5%) and that this imaging modality is available for use (83.8%). However, only 11.8% of providers indicated the use of MRI in their clinical assessments. This discrepancy highlights a potential disconnect between perceived use and implementation of this imaging methodology on cleft and craniofacial teams. A number of barriers were identified by providers. Challenges and opportunities for clinical translation of MRI protocols were highlighted.Conclusion Results may guide the development for improved clinical feasibility and implementation of MRI for clinical planning in this population of patients. Reported barriers highlight additional areas for translational research and the potential for the development of clinical tools related to MRI assessment and protocol implementation.

Background/Context: Most students with disabilities receive the majority of their instruction in general education classrooms. Yet, general education teachers persistently describe feeling unprepared to academically support students with disabilities in those spaces. Because disabled students are typically excluded from mathematics education research, and because special education researchers typically describe mathematics teaching and learning in ways that are incongruent with ambitious mathematics instruction, there is arguably a lack of guidance for these teachers. In the absence of clear guidance, teachers may turn to the well-established mathematical ability hierarchy, which positions disabled students (among others) as less capable.Purpose/Objective/Research Question/Focus of Study: The purpose of this study was to uncover teachers’ talk about the mathematical capabilities of students with (and without) disabilities. Existing coding schemes (perhaps inadvertently) treat teachers’ views as uniform across students despite evidence that teachers hold different views of different students, in part because of the multiple and varied identities that students bring to the classroom. By using an adapted interview protocol, which yielded more (and more nuanced) analytic categories, I foregrounded students’ disability status as a factor that could relate to differences in teachers’ conceptions of who they view as mathematically capable.Research Design: I interviewed general education mathematics teachers (N= 20) about their students (n= 407) using an adapted version of Jackson et al.’s (2017) semi-structured protocol that focused on uncovering teachers’ usages of diagnostic and prognostic frames. I used open and concept coding to develop an expanded version of Jackson et al.’s coding scheme and then applied the new coding framework to the entire data set. I used student demographic data to compare within-group percentages, noticing to what degree students with disabilities were represented within particular qualitative categories in relation to their representation within the entire data set. I also used transformed data to estimate two multinomial logistic regressions: one that used diagnostic frames as the outcome variable, and one that used prognostic frames as the outcome variable. Both models used students’ disability status and teacher dummy codes as predictor variables.Conclusions/Recommendations: The majority of teachers in this sample explained mathematical struggle in unproductiveterms and said they would aim instructional adjustment at unproductiveoutcomes for students with and without disabilities. However, students with disabilities were overrepresented in unproductive categories and underrepresented in productive categories in relation to both diagnostic and prognostic frames. Regression analyses indicated that a student was statistically less likely to get a productive diagnostic or prognostic frame if they had a disability label. Findings from this study highlight the necessity of including teachers’ views of their students’ mathematical capabilities in instructional improvement efforts. Second, they indicate that student-level factors, such as disability status, relate to qualitatively and quantitatively meaningful differences in teachers’ views of their students, suggesting the importance of attending to broader narratives around constructs that may be associated with teachers’ views, and the subsequent enactment of those views, in mathematics instruction.

Objective Sphincter pharyngoplasty is a surgical method to treat velopharyngeal dysfunction. However, surgical failure is often noted and postoperative assessment frequently reveals low-set pharyngoplasties. Past studies have not quantified pharyngoplasty tissue changes that occur postoperatively and gaps remain related to the patient-specific variables that influence postoperative change. The purpose of this study was to utilize advanced three-dimensional imaging and volumetric magnetic resonance imaging (MRI) data to visualize and quantify pharyngoplasty insertion site and postsurgical tissue changes over time.Design A prospective, repeated measures design was used for the assessment of craniometric and velopharyngeal variables postsurgically. Imaging was completed across two postoperative time points. Tissue migration, pharyngoplasty dimensions, and predictors of change were analyzed across imaging time points.Results Significant differences were present between the initial location of pharyngoplasty tissue and the pharyngoplasty location 2 to 4 months postoperatively. The average postoperative inferior movement of pharyngoplasty tissue was 6.82 mm, although notable variability was present across participants. The pharyngoplasty volume decreased by 30%, on average.Conclusions Inferior migration of the pharyngoplasty tissue was present in all patients. Gravity, scar contracture, and patient-specific variables likely interact, impacting final postoperative pharyngoplasty location. The use of advanced imaging modalities, such as 3D MRI, allows for the quantification and visualization of tissue change. There is a need for continued identification of patient-specific factors that may impact the amount of inferior tissue migration and scar contracture postoperatively.

Many students who experience difficulty with mathematics may also require behavioral support. We suggest educators create an appropriate scope and sequence for mathematics intervention by focusing on the critical mathematics content. Educators can then design the intervention with a common session structure. Finally, educators can embed and intensify supports for a student by increasing the dosage, focusing on the alignment of the intervention, and attending to transfer.

Stem cell transplantation therapies are currently under investigation for central nervous system disorders. Although preclinical models show benefit, clinical translation is somewhat limited by the absence of reliable noninvasive methods to confirm targeting and monitor transplanted cells in vivo. Here, we assess a novel magnetic resonance imaging (MRI) contrast agent derived from magnetotactic bacteria, magneto‐endosymbionts (MEs), as a translatable methodology for in vivo tracking of stem cells after intracranial transplantation. We show that ME labeling provides robust MRI contrast without impairment of cell viability or other important therapeutic features. Labeled cells were visualized immediately post‐transplantation and over time by serial MRI in nonhuman primate and mouse brain. Postmortem tissue analysis confirmed on‐target grft location, and linear correlations were observed between MRI signal, cell engraftment, and tissue ME levels, suggesting that MEs may be useful for determining graft survival or rejection. Overall, these findings indicate that MEs are an effective tool for in vivo tracking and monitoring of cell transplantation therapies with potential relevance to many cellular therapy applications. Translation of cell therapies for central nervous system disorders is often limited by an inability to noninvasively identify and track transplanted cells in vivo. McGinley et al report a novel MRI contrast agent based on magnetotactic bacteria that enables visualization and long‐term tracking of cell grafts in nonhuman primate and murine brain.

PurposeDental and occlusal anomalies are common in individuals with cleft lip and palate, placing them at risk for speech sound distortions. Speech-language pathologists and dental practitioners frequently interact when presented with clinical problems secondary to dental and occlusal anomalies. This is especially true when treating a child with a cleft lip and palate. The speech sound error types must be identified and their etiologies evaluated to develop appropriate management plans. The purpose of this review article is to describe the types of dental and occlusal anomalies that are common in individuals with cleft lip and/or palate and discuss the impact these anomalies have on articulation.MethodA review of the literature was completed with a focus on prevalence, definitions, and descriptions of dental and occlusal anomalies in individuals with cleft lip and palate. The impact of dental/occlusal anomalies on speech production is described.Results and ConclusionsThere is sufficient evidence that dental and occlusal anomalies have an impact on articulation, but the relationship of dental or occlusal status is not always a direct one. The phonemes most affected by aberrant oral conditions are sibilants, though other phonemes may be impacted as well. These speech errors are considered to be obligatory oral distortions in that they are made in response to an oral structural defect. These speech errors are not typically amenable to speech therapy, but rather require orthodontic and/or surgical correction. Such treatment can be effective in improving these errors, often spontaneously, however, speech therapy may be necessary (and most effective) following correction of the dental and/or occlusal anomalies if errors persist.

Background/PurposeWhen working with a specialized population, it is necessary to have the appropriate clinical and academic training. However, many speech-language pathologists report being ill-prepared regarding best practice when evaluating and treating patients with low-incidence disorders, particularly cleft palate/craniofacial anomalies and fluency disorders. The purpose of this study was to compare differences in graduate speech-language pathology coursework in the United States across two low-incidence disorder areas: cleft/craniofacial anomalies and fluency disorders.MethodA review of the accredited graduate curricula offerings within these domains was completed. Information whether coursework in these areas was offered, if the course was taught as a full course or embedded within a related course, or whether the content was required or an elective, and if the course was taught by an expert was obtained.ResultsSignificant differences were present in the amount, quality, and type of course content offered for cleft/craniofacial anomalies compared to fluency disorders. Only 72.83% of graduate speech-language pathologist programs offered content in cleft/craniofacial anomalies. Approximately one out of every four programs (27.17%) did not provide this content within the graduate curriculum. In contrast, content in fluency disorders was taught in 99.28% of graduate programs.ConclusionResults demonstrate that graduate training in topics related to cleft/craniofacial anomalies is significantly limited, particularly in comparison to another low-incidence communication disorder.

This clinical report describes the implementation of magnetic resonance imaging (MRI) to evaluate a patient with long-standing velopharyngeal dysfunction. She was referred to the craniofacial clinic at age 10 with no prior surgical history and subsequently completed a Furlow palatoplasty due to a suspected submucous cleft palate. However, results were unfavorable with minimal improvement in speech or resonance. The clinical presentation, treatment, outcomes, and contributions from MRI for secondary surgical planning are described. Addition of MRI into the clinical workflow provided insights into the anatomy and physiology of the velopharyngeal mechanism that were unable to be obtained from nasendoscopy and speech evaluation alone.

We construct a demographically informed model of age specific healthcare consumption for 36 countries of widely varying income and wealth, in the National Transfer Accounts project. We project healthcare consumption to 2060 using a modified Lee-Carter technique. In our modification, GDP per capita plays the role of time in explaining changes in the age-pattern of health consumption as countries become wealthier. We find that rising wealth mainly affects health consumption at older-ages.

Objective: Palpation is often used to identify C1, an intraoperative landmark, for placement of the pharyngoplasty. However, little is known about the relationship between the palatal plane (PP) and this cervical spine landmark across select variables. This study seeks to analyze variations in the height of velopharyngeal closure relative to C1 across differing cleft types and age groups.Design: Retrospective, cross-sectional analysis.Setting: Large, multidisciplinary center for craniofacial disorders.Methods: Clinical lateral cephalograms were analyzed in nonsyndromic patients who underwent primary palatoplasty. Regression analysis and analysis of covariance were completed to determine how age and cleft type impact underlying cervical and velopharyngeal measures.Results: Age (P< .001) and cleft type (P= .036) were significant predictors of the distance between the height of velopharyngeal closure and C1. Those with greater severity of clefting demonstrated larger distances between the height of velopharyngeal closure and C1. Compared to normative data, children with cleft palate have significantly larger distances between the PP and C1. The height of velopharyngeal closure above C1 was observed to range from 3.6 to 12.6 mm across cleft populations.Conclusions: This study demonstrates the variability in C1 as a landmark across variables including cleft type and age. Because of differences in the height of velopharyngeal closure across cleft types relative to C1, it is necessary to preoperatively quantify the vertical distance between the PP and palpable intraoperative landmark, C1, to determine the appropriate height of pharyngoplasty insertion.

Pediatric acute myeloid leukemia (pAML) is typified by high relapse rates and a relative paucity of somatic DNA mutations. Although seminal studies show that splicing factor mutations and mis-splicing fuel therapy-resistant leukemia stem cell (LSC) generation in adults, splicing deregulation has not been extensively studied in pAML. Herein, we describe single-cell proteogenomics analyses, transcriptome-wide analyses of FACS-purified hematopoietic stem and progenitor cells followed by differential splicing analyses, dual-fluorescence lentiviral splicing reporter assays, and the potential of a selective splicing modulator, Rebecsinib, in pAML. Using these methods, we discover transcriptomic splicing deregulation typified by differential exon usage. In addition, we discover downregulation of splicing regulator RBFOX2 and CD47splice isoform upregulation. Importantly, splicing deregulation in pAML induces a therapeutic vulnerability to Rebecsinib in survival, self-renewal, and lentiviral splicing reporter assays. Taken together, the detection and targeting of splicing deregulation represent a potentially clinically tractable strategy for pAML therapy.

Adenosine deaminase acting on RNA1 (ADAR1) preserves genomic integrity by preventing retroviral integration and retrotransposition during stress responses. However, inflammatory-microenvironment-induced ADAR1p110 to p150 splice isoform switching drives cancer stem cell (CSC) generation and therapeutic resistance in 20 malignancies. Previously, predicting and preventing ADAR1p150-mediated malignant RNA editing represented a significant challenge. Thus, we developed lentiviral ADAR1 and splicing reporters for non-invasive detection of splicing-mediated ADAR1 adenosine-to-inosine (A-to-I) RNA editing activation; a quantitative ADAR1p150 intracellular flow cytometric assay; a selective small-molecule inhibitor of splicing-mediated ADAR1 activation, Rebecsinib, which inhibits leukemia stem cell (LSC) self-renewal and prolongs humanized LSC mouse model survival at doses that spare normal hematopoietic stem and progenitor cells (HSPCs); and pre-IND studies showing favorable Rebecsinib toxicokinetic and pharmacodynamic (TK/PD) properties. Together, these results lay the foundation for developing Rebecsinib as a clinical ADAR1p150 antagonist aimed at obviating malignant microenvironment-driven LSC generation.

3D printing of biological tissues has been of increasing interest to the biomaterials community in part because of its potential to produce spatially heterogeneous constructs. Such technology is particularly promising for orthopedic applications, which require the generation of complex geometries to match patient anatomy and complex microstructures to produce spatial heterogeneity and anisotropy. Prior research has demonstrated the capacity to create precisely shaped 3D printed constructs using biocompatible alginate hydrogels. However, alginate is extremely compliant and brittle, and high-density collagen hydrogels could be a preferable option for load-bearing applications. This research focused on developing and evaluating a method of printing soft tissue implants with high-density collagen hydrogels using a commercially available 3D printer, modified for tissue-engineering purposes. The tissue constructs, seeded with primary meniscal fibrochondrocytes, were evaluated using measures of geometric fidelity, cell viability, mechanical properties, and fiber microstructure. The constructs were found to be mechanically stable and were able to support and maintain cell growth. Furthermore, heterogeneous 3D-printed constructs were generated, consisting of discrete domains with distinct mechanical properties.

Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1E912Amutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28Bupregulation. A small-molecule tool compound antagonizes ADAR1’s effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling.

ABSTRACTAccumulating evidence suggests a connection between asthma development and colonization with nontypeable Haemophilus influenzae(NTHi). Specifically, nasopharyngeal colonization of human infants with NTHi within 4 weeks of birth is associated with an increased risk of asthma development later in childhood. Monocytes derived from these infants have aberrant inflammatory responses to common upper respiratory bacterial antigens compared to those of cells derived from infants who were not colonized and do not go on to develop asthma symptoms in childhood. In this study, we hypothesized that early-life colonization with NTHi promotes immune system reprogramming and the development of atypical inflammatory responses. To address this hypothesis in a highly controlled model, we tested whether colonization of mice with NTHi on day of life 3 induced or exacerbated juvenile airway disease using an ovalbumin (OVA) allergy model of asthma. We found that animals that were colonized on day of life 3 and subjected to induction of allergy had exacerbated airway disease as juveniles, in which exacerbated airway disease was defined as increased cellular infiltration into the lung, increased amounts of inflammatory cytokines interleukin-5 (IL-5) and IL-13 in lung lavage fluid, decreased regulatory T cell-associated FOXP3gene expression, and increased mucus production. We also found that colonization with NTHi amplified airway resistance in response to increasing doses of a bronchoconstrictor following OVA immunization and challenge. Together, the murine model provides evidence for early-life immune programming that precedes the development of juvenile airway disease and corroborates observations that have been made in human children.

Arterial hemodynamic shear stress and blood vessel stiffening both significantly influence the arterial endothelial cell (EC) phenotype and atherosclerosis progression, and both have been shown to signal through cell-matrix adhesions. However, the cooperative effects of fluid shear stress and matrix stiffness on ECs remain unknown. To investigate these cooperative effects, we cultured bovine aortic ECs on hydrogels matching the elasticity of the intima of compliant, young, or stiff, aging arteries. The cells were then exposed to laminar fluid shear stress of 12 dyn/cm2. Cells grown on more compliant matrices displayed increased elongation and tighter EC-cell junctions. Notably, cells cultured on more compliant substrates also showed decreased RhoA activation under laminar shear stress. Additionally, endothelial nitric oxide synthase and extracellular signal-regulated kinase phosphorylation in response to fluid shear stress occurred more rapidly in ECs cultured on more compliant substrates, and nitric oxide production was enhanced. Together, our results demonstrate that a signaling cross talk between stiffness and fluid shear stress exists within the vascular microenvironment, and, importantly, matrices mimicking young and healthy blood vessels can promote and augment the atheroprotective signals induced by fluid shear stress. These data suggest that targeting intimal stiffening and/or the EC response to intima stiffening clinically may improve vascular health.

In this report we describe a preparation of silver wires (SWs) on gold mirrors and its application to surface enhanced fluorescence (SEF) using a new methodology. Silica protected gold mirrors were drop-coated with a solution of silver triangular nanoprisms. The triangular nanoprisms were slowly air-dried to get silver wires that self-assembled on the gold mirrors. Fluorescence enhancement was studied using methyl azadioxatriangulenium chloride (Me-ADOTA·Cl) dye in PVA spin-coated on a clean glass coverslip. New Plasmonic Platforms (PPs) were assembled by placing a mirror with SWs in contact with a glass coverslip spin-coated with a uniform Me-ADOTA·Cl film. It was shown that surface enhanced fluorescence is a real phenomenon, not just an enhancement of the fluorescence signal due to an accumulation of the fluorophore on rough nanostructure surfaces. The average fluorescence enhancement was found to be about 15-fold. The lifetime of Me-ADOTA·Cl dye was significantly reduced (∼4 times) in the presence of SWs. Moreover, fluorescence enhancement and lifetime did not show any dependence on the excitation light polarization.

Background:We previously showed that intra-amniotic lipopolysaccharide (LPS) amplifies alveolar hypoplasia induced by postnatal hyperoxia. We determined whether the priming effect of intra-amniotic LPS amplifies hyperoxia-induced airway hyperreactivity (AHR).Methods:LPS or normal saline was injected into the amniotic cavities of pregnant rats at the 20th day of gestation. After birth, rat pups were exposed to 60% O2or air for 14 d. On postnatal day 14, rat pups underwent forced oscillometry, which included a challenge with nebulized methacholine, and the lungs were harvested for morphological studies.Results:Hyperoxia significantly increased airway reactivity and decreased compliance. Intra-amniotic LPS further increased hyperoxia-induced AHR but did not further impair respiratory system compliance. Hyperoxia-induced changes in lung parenchymal and small airway morphology were not further altered by intra-amniotic LPS. However, combined exposure to intra-amniotic LPS and hyperoxia increased the proportion of degranulating mast cells in the hilar airways.Conclusion:Intra-amniotic LPS amplified postnatal hyperoxia-induced AHR. This was associated with increased airway mast cell degranulation, which has previously been linked with hyperoxia-induced AHR. There were no morphologic changes of parenchyma or airways that would account for the LPS augmentation of hyperoxia-induced AHR.

The mechanical properties of the extracellular matrix play an important role in maintaining cellular function and overall tissue homeostasis. Recently, a number of hydrogel systems have been developed to investigate the role of matrix mechanics in mediating cell behavior within three-dimensional environments. However, many of the techniques used to modify the stiffness of the matrix also alter properties that are important to cellular function including matrix density, porosity and binding site frequency, or rely on amorphous synthetic materials. In a recent publication, we described the fabrication, characterization and utilization of collagen gels that have been non-enzymatically glycated in their unpolymerized form to produce matrices of varying stiffness. Using these scaffolds, we showed that the mechanical properties of the resulting collagen gels could be increased 3-fold without significantly altering the collagen fiber architecture. Using these matrices, we found that endothelial cell spreading and outgrowth from multi-cellular spheroids changes as a function of the stiffness of the matrix. Our results demonstrate that non-enzymatic collagen glycation is a tractable technique that can be used to study the role of 3D stiffness in mediating cellular function. This commentary will review some of the current methods that are being used to modulate matrix mechanics and discuss how our recent work using non-enzymatic collagen glycation can contribute to this field.

Objectives: To document levels of cardiovascular disease (CVD), diagnosed and undiagnosed risk factors and clinical management of CVD risk in rural Māori.

To document levels of cardiovascular disease (CVD), diagnosed and undiagnosed risk factors and clinical management of CVD risk in rural Māori.

We assessed the ability of a gamma-secretase inhibitor to promote the in vitrodifferentiation of induced embryonic pancreatic precursor cell aggregates into functional islet-like clusters when encapsulated within a three-dimensional hydrogel. Undifferentiated pancreatic precursor cells were isolated from E.15 rat embryos, dissociated into single cells, and aggregated in suspension-rotation culture. Aggregates were photoencapsulated into poly(ethylene glycol) hydrogels with entrapped collagen type 1 and cultured for 14 days with or without a gamma-secretase inhibitor. Gene expression, proinsulin content, and C-peptide release were measured to determine differentiation and maturation of encapsulated precursor cell aggregates. In the control medium, scattered breakthrough beta cell differentiation was observed; however, cells remained largely insulin negative. Upon addition of a gamma-secretase inhibitor the majority of cells in clusters became insulin positive, and insulin per DNA and glucose-stimulated insulin release measurements for these cultures were comparable with those for adult rat islets. Cluster counts after culture day 14 were 88% of those initially encapsulated, demonstrating excellent cluster survival in hydrogel culture. These results indicate that concerted differentiation of pancreatic precursor cell aggregates into functionally mature islet-like clusters can be achieved in poly(ethylene glycol)-based hydrogel cultures by blocking cell contact-mediated Notch signaling with a gamma-secretase inhibitor.

Premature infants are at increased risk of developing airway hyper-reactivity (AHR) after oxidative stress and inflammation. Mast cells contribute to AHR partly by mediator release, so we sought to determine whether blocking mast cell degranulation or recruitment prevents hyperoxia-induced AHR, mast cell accumulation, and airway smooth muscle (ASM) changes. Rats were exposed at birth to air or 60% O2for 14 d, inducing significantly increased AHR in the latter group, induced by nebulized methacholine challenge and measured by forced oscillometry. Daily treatment (postnatal d 1–14) with intraperitoneal cromolyn prevented hyperoxia-induced AHR, as did treatment with imatinib on postnatal d 5–14, compared with vehicle treated controls. Cromolyn prevented mast cell degranulation in the trachea but not hilar airways and blocked mast cell accumulation in the hilar airways. Imatinib treatment completely blocked mast cell accumulation in tracheal/hilar airway tissues. Hyperoxia-induced AHR in neonatal rats is mediated, at least in part, via the mast cell.

Premature infants are at increased risk of developing airway hyper-reactivity (AHR) after oxidative stress and inflammation. Mast cells contribute to AHR partly by mediator release, so we sought to determine whether blocking mast cell degranulation or recruitment prevents hyperoxia-induced AHR, mast cell accumulation, and airway smooth muscle (ASM) changes. Rats were exposed at birth to air or 60% O2for 14 d, inducing significantly increased AHR in the latter group, induced by nebulized methacholine challenge and measured by forced oscillometry. Daily treatment (postnatal d 1–14) with intraperitoneal cromolyn prevented hyperoxia-induced AHR, as did treatment with imatinib on postnatal d 5–14, compared with vehicle treated controls. Cromolyn prevented mast cell degranulation in the trachea but not hilar airways and blocked mast cell accumulation in the hilar airways. Imatinib treatment completely blocked mast cell accumulation in tracheal/hilar airway tissues. Hyperoxia-induced AHR in neonatal rats is mediated, at least in part, via the mast cell.

Development of an alternative source of functional, transplantable β-cells to replace or supplement cadaveric tissue is critical to the future success of islet cell transplantation therapy. Embryonic pancreatic precursor cells are desirable as a renewable source of β-cells as they are both proliferative and inherently capable of pancreatic cell differentiation. We have previously shown that precursor cells undergo selective β-cell differentiation when dissociated and photoencapsulated in a polyethylene glycol (PEG) hydrogel network; however, these cells remained immature and were not glucose responsive. Collagen type 1 supports mature cell viability and function in many cell types and we hypothesized that incorporating it within our gels may support differentiating β-cells and facilitate β-cell maturation. For these studies, collagen-1 was entrapped with dissociated pancreatic precursor cells in a PEG hydrogel matrix (PEGCol) with the following key findings: (1) mature, glucose-responsive, islet-like structures differentiated from spontaneously forming precursor cell clusters in PEGCol, but not unmodified PEG, hydrogels; (2) a balance existed between providing sufficient collagen-1 signaling to support precursor cell development and providing an overabundance of adhesive sites allowing contaminating mesenchymal cells to thrive’ and (3) mechanical stability provided by the PEG hydrogel platform is important for successful precursor cell culture, as PEGCol hydrogels encourage glucose responsiveness and high-insulin gene expression, while pure collagen gel cultures, with the same collagen concentration, have negligible insulin gene expression. These results indicate that PEGCol hydrogels are a useful culture platform to promote differentiation of a glucose-responsive β-cell population from dissociated precursor cells.

Continuing advances in islet cell transplantation have been promising; however, several limitations, including severe shortage of transplantable islets, hinder the widespread use of this therapy. Pancreatic precursor cells are one alternative to cadaveric donor islets. These cells found in the developing pancreatic buds are capable of self-renewal and also have the innate ability to become insulin-producing β-cells. For this work, bioinert polyethylene glycol (PEG) hydrogels were chosen as the supportive three-dimensional matrix for encapsulation of dissociated pancreatic precursor cells obtained from the dorsal pancreatic bud of day-15 rat embryos. This culture system was selected in order to eliminate cell–extracellular matrix and cell–cell signal heterogeneity present when intact pancreatic buds are embedded in protein-based gels, the typical in vitroculture conditions used to study this cell population. In this study it was found that (1) dissociated precursor cells maintain a robust viability for 7 days in PEG hydrogel culture, (2) encapsulated cells selectively differentiate into insulin-expressing β-cells, and (3) differentiated β-cells have releasable insulin stores, but are not achieving a mature, glucose responsive phenotype. These findings suggest that encapsulating dissociated pancreatic precursor cells in an environment designed to minimize the heterogeneous signaling cues present during development or in standard culture conditions generates a population highly enriched in pancreatic β-cells; however, future efforts must focus on achieving glucose responsiveness in this cell population. Further, these results indicate that differentiation down a β-cell lineage may be the default pathway in pancreatic development.

Desvenlafaxine succinate (DVS) is a recently introduced antagonist of the human norepinephrine and serotonin transporters (hNET and hSERT, respectively), currently in clinical development for use in the treatment of major depressive disorder and vasomotor symptoms associated with menopause. Initial evaluation of the pharmacological properties of DVS (J Pharmacol Exp Ther318:657–665, 2006) revealed significantly reduced potency for the hNET expressed in membranes compared with whole cells when competing for [3H]nisoxetine (NIS) binding. Using hNET in transfected human embryonic kidney-293 cells, this difference in potency for DVS at sites labeled by [3H]NIS was found to distinguish DVS, the DVS analog rac-(1-[1-(3-chloro-phenyl)-2-(4-methylpiperazin-1-yl)-ethyl]cyclohexanol (WY-46824), methylphenidate, and the cocaine analog 3β-(4-iodophenyl)tropane-2β-carboxylic acid methyl ester (RTI-55) from other hNET antagonists, such as NIS, mazindol, tricyclic antidepressants, and cocaine. These differences seem not to arise from preparation-specific perturbations of ligand intrinsic affinity or antagonist-specific surface trafficking but rather from protein conformational alterations that perturb the relationships between distinct hNET binding sites. In an initial search for molecular features that differentially define antagonist binding determinants, we document that Val148 in hNET transmembrane domain 3 selectively disrupts NIS binding but not that of DVS.

Desvenlafaxine succinate (DVS) is a recently introduced antagonist of the human norepinephrine and serotonin transporters (hNET and hSERT, respectively), currently in clinical development for use in the treatment of major depressive disorder and vasomotor symptoms associated with menopause. Initial evaluation of the pharmacological properties of DVS (J Pharmacol Exp Ther 318:657-665, 2006) revealed significantly reduced potency for the hNET expressed in membranes compared with whole cells when competing for [(3)H]nisoxetine (NIS) binding. Using hNET in transfected human embryonic kidney-293 cells, this difference in potency for DVS at sites labeled by [(3)H]NIS was found to distinguish DVS, the DVS analog rac-(1-[1-(3-chloro-phenyl)-2-(4-methylpiperazin-1-yl)-ethyl]cyclohexanol (WY-46824), methylphenidate, and the cocaine analog 3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester (RTI-55) from other hNET antagonists, such as NIS, mazindol, tricyclic antidepressants, and cocaine. These differences seem not to arise from preparation-specific perturbations of ligand intrinsic affinity or antagonist-specific surface trafficking but rather from protein conformational alterations that perturb the relationships between distinct hNET binding sites. In an initial search for molecular features that differentially define antagonist binding determinants, we document that Val148 in hNET transmembrane domain 3 selectively disrupts NIS binding but not that of DVS.

The electrochemical (platinum and glassy carbon electrodes, cyclic and rotating disk electrode voltammetry, bulk electrolysis, voltammetric simulation) and chemical oxidation (NO+) of the organoamidoplatinum(ii) complex, [Pt{((p-HC₆F₄)NCH₂)₂}(py)₂], has been studied in acetonitrile and acetone. The initial process is a complicated function of concentration, temperature, and method of oxidation. The products formed have been probed by in situ spectroelectrochemical techniques (UV/Visible and EPR spectroscopy), and ex situ by 1H, 19F and 195Pt NMR spectroscopy and electrospray mass spectrometry. Under conditions of cyclic voltammetry, the initial oxidation process (at high concentration) is an overall irreversible one-electron process, complicated by crossover of current on the reverse scan. This and many (but not all) features are simulated by a sequence of electron transfer steps and coupled chemical reactions which requires the formation of two structurally different dinuclear intermediates. Regeneration of [Pt{((p-HC₆F₄)NCH₂)₂}(py)₂] by reduction is possible under short timescale conditions, but not long timescale conditions, implying that the finally observed product from long timescale experiments may be oligomeric. The observation of moderately stable diamagnetic diplatinum(iii) compounds is attributed to the formation of bridged complexes containing Pt–Pt bonds. Oxidation of all platinum(iii) intermediates gives rise to the same or very closely related platinum(iv) complexes. Features of the organoamide ligand that enable the formation of moderately stable platinum(iii) complexes are considered.

The aim of this study was to use pharmacological inhibition of protein kinase A and mutation of potential protein kinase A phosphorylation sites to determine the role of protein kinase A-catalyzed phosphorylation of the dopamine D1receptor in agonist-stimulated desensitization and internalization of the receptor. To facilitate purification and imaging of the D1receptor, we attached a polyhistidine tag to the amino terminus and enhanced green fluorescent protein to the carboxyl terminus of the receptor (D1-EGFP). D1-EGFP was similar to the untagged D1receptor in terms of affinity for agonist and antagonist ligands, coupling to G proteins, and stimulation of cyclic AMP accumulation. D1-EGFP and two mutants in which either Thr268 or Ser380 was replaced with Ala were stably expressed in NS20Y neuroblastoma cells. Pretreatment with the protein kinase A inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) or substitution of Ala for Thr268 reduced agonist-stimulated phosphorylation of the receptor and resulted in diminished trafficking of the receptor to the perinuclear region of the cell. Substitution of Ala for Thr268 had no effect, however, on agonist-induced receptor sequestration or desensitization of cyclic AMP accumulation. Substitution of Ala for Ser380 had no effect on D1receptor phosphorylation, sequestration, desensitization, or trafficking to the perinuclear region. We conclude that protein kinase A-dependent phosphorylation of the D1receptor on Thr268 regulates a late step in the sorting of the receptor to the perinuclear region of the cell, but that phosphorylation of Thr268 is not required for receptor sequestration or maximal desensitization of cyclic AMP accumulation.

This paper describes the development and application of scaling laws relating bulk-degradation behavior of poly(lactic acid)−poly(ethylene glycol)−poly(lactic acid) (PLA-b-PEG-b-PLA) copolymer hydrogels to the ever-changing transport properties during network degradation. Specifically, the relationships developed were used to extend previous work and to predict the effects of cross-linking density and hydrolytic degradation rate on the controlled release of high molecular weight proteins from these hydrogels. Theoretical drug release profiles, obtained using scaling laws, agree qualitatively with experimental observations and show a decrease in the protein release rate with an increase in protein size, extent of macromer functionalization, and macromer concentration. Quantitative predictions of release behavior were possible under certain experimental conditions.

Background: Ethanol withdrawal seizure‐prone (WSP) and ‐resistant (WSR) mice have been genetically selected for differences in handling‐induced convulsion severity during withdrawal from chronic ethanol administration. Importantly, drug‐naïve mice from these selected lines also differ in handling‐induced convulsion severity. Different N‐methyl‐D‐aspartate (NMDA) receptor subunit and splice variant associations confer varying sensitivities to ethanol, and may play a role in the different behavioral responses of the WSP and WSR mice.

Abstract:To determine if catechol-O-methyltransferase (COMT) metabolizes catecholamines within cell lines used for heterologous expression of plasmalemmal transporters and alters the measured characteristics of 3H-substrate transport, the uptake of monoamine transporter substrates was assessed in three cell lines (C6 glioma, L-M fibroblast, and HEK293 cells) that had been transfected with the recombinant human transporters. Uptake and cellular retention of 3H-catecholamines was increased by up to fourfold by two COMT inhibitors, tropolone and Ro 41-0960, with potencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT, [3H]serotonin and [3H]MPP+, was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 mM) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [3H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 mM) decreased the apparent Kmof transport from 0.61 µMto 0.34 µMwithout significantly altering the maximal velocity of transport. These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter deposition and thus the apparent kinetic characteristics of transport.

MRL-lpr/lpr mice spontaneously develop various manifestations of autoimmunity including an inflammatory arthropathy and immune complex glomerulonephritis. This study examines the role of nitric oxide, a molecule with proinflammatory actions, in the pathogenesis of MRL-lpr/lpr autoimmune disease. MRL-lpr/lpr mice excreted more urinary nitrite/nitrate (an in vivo marker of nitric oxide production) than did mice of normal strains and MRL-(+/+) and B6-lpr/lpr congenic strains. In addition, MRL-lpr/lpr peritoneal macrophages had an enhanced capacity to produce nitric oxide in vitro as well as increased nitric oxide synthase activity, and certain tissues from MRL-lpr/lpr mice had increased expression of inducible nitric oxide synthase (NOS) mRNA and increased amounts of material immunoreactive for inducible NOS. Oral administration of NG-monomethyl-L-arginine, a nitric oxide synthase inhibitor, prevented the development of glomerulonephritis and reduced the intensity of inflammatory arthritis in MRL-lpr/lpr mice. By using interspecific backcross mice, the gene for inducible NOS (Nosi) was mapped to mouse chromosome 11. This chromosomal localization was different from those loci that we have previously demonstrated to be linked to enhanced susceptibility to renal disease in an MRL-lpr/lpr cross. However, the chromosomal location of the NOS gene was consistent with an insulin-dependent diabetes locus identified in an analysis of nonobese diabetic (NOD) mice. These results suggest that elevated nitric oxide production could be important in the pathogenesis of autoimmunity, and that treatments to block the production of nitric oxide or block its effects might be valuable therapeutically.

The clinical application of computerized continuous-infusion pressure-monitored sialography using an aqueous contrast medium during the investigation of 296 salivary glands is described. A comparison between the pressure changes and the sialographic features revealed a significant (P less than 0.001) correlation between raised filling pressure (greater than 60 mmHg) and the presence of an obstructive lesion (stricture, mucous plug, sialolith). Occasionally a raised filling pressure was observed although the associated sialography films were normal. The sensitivity of the technique was 86.4%. It is concluded that the monitoring of pressure changes occurring during sialography can alert the clinician to the presence of high filling pressure and may also provide additional evidence of obstructive changes within salivary glands.

The microbial degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 was studied, and two major products were isolated and identified as 7 alpha, 12 beta-dihydroxyandrosta-1,4-diene-3,17-dione and 7 alpha, 12 alpha-dihydroxy-3-oxopregna-1,4-diene-20-carboxylic acid. Four minor products were isolated and evidence is given for the following structures: 7 alpha, 12 alpha-dihydroxyandrosta-1,4-diene-3,17-dione, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 7 alpha, 12 beta, 17 beta-trihydroxyandrosta-1,4-dien-3-one and 7 alpha, 12 alpha-dihydroxy-3-oxopregn-4-ene-20-carboxylic acid. The significance of the production of the steroid products is discussed, along with the possible enzymic mechanisms responsible for their production.

CONSCIOUSNESS is a word with many meanings. Schiller1 remarked that it may connote attending, sensing, being alive, being responsive to stimuli, having insight, being able to communicate, and the psychoanalysts give it another interpretation. It can also be described as a state of passive awareness independent of speech or movement. Such a definition applies to crude as distinct from discriminative consciousness and will be the definition used here.Le Gros Clarke2 has drawn attention to the function of the thalamic and brain-stem reticular system as the possible structural basis of what may be called a general background of consciousness. Interest in the reticular system as the activator of consciousness originates largely from animal experimental work of Magoun3 and others. Clinical evidence in support of this hypothesis is rare, and it is partly for this reason that a case admitted to the Royal Edinburgh Hospital for Mental and

1) An attempt is made to clarify and standardize the technique and modification of electro-convulsive therapy.2) The literature is briefly reviewed.3) A technique suitable for outpatient, hospital or private practice use is suggested and discussed. Kindly and proper handling of the patient, the time element, nursing requirements, and the training of medical staff were among the factors considered in the description of this technique.4) A total of 1863 treatments were given to 246 female patients by the author over an arbitrary period and under exactly similar conditions. The limitation of the trial to females is coincidental.5) For the first time 8 different muscle relaxants are compared and assessed in the same paper. Six of these relaxants were given to 25 of the patients in a partially unsuccessful attempt to give each of these patients a trial with each relaxant on at least one occasion.6) It is concluded that physical disease or age do not contra-indicate the use of E.C.T. provided this is modified.7) It is further concluded that suxethonium bromide is at least as satisfactory, and in most cases more satisfactory, than the other relaxant drugs used for the modification of electro-shock therapy. The drugs were assessed for this purpose in terms of occurrence of fracture, degree of apnoea produced, contra-indications, antidotes, effect upon blood pressure and pulse, production of histamine-like effects, excessive secretions, cyanosis, subjective ill effects, extent of muscle fasciculations, degree of relaxation, modification of the convulsion, time required, and finally—ease of handling.