Automated, computerized, feature-based phenotype analysis of slit lamp images of the mouse lens

Longitudinal studies of a variety of transgenic mouse models for lens development can create substantial challenges in database management and analysis. We report a novel, automated, feature-based informatics approach to screening lens phenotypes in a large database of slit lamp images. Digital slit lamp images of normal and abnormal lenses in eyes of wild type (wt), SC1 null and SPARC null transgenic mice were recorded for quantitative evaluation of their structural phenotype. The images were processed to improve the contrast of structural features that corresponded to rings of opacity and fluctuations in scattering intensity in the lenses. Measurable attributes were assigned to the features in the lens images and given as an output vector of 46 dimensions. Characteristic patterns were correlated with the structural phenotype of each mutant and wt lens and a statistical fit for each phenotype was defined. The genotype was identified correctly in nearly 85% of the slit lamp images on the basis of an automated computer analysis of the lens structural phenotype. The automated computer algorithm has the potential to evaluate a large database of slit lamp images and distinguish mouse genotypes on the basis of lens phenotypes objectively using a neural network analysis of the structural features observed in the slit lamp images. The neural network approach is a promising technology for objective evaluation of genotype/phenotype relationships based on structural features and light scattering in lenses. Further improvements in the automated method can be expected to simplify and increase the accuracy and efficiency of the feature based analysis of structural phenotypes linked to genetic variation.     

An extracellular matrix infrastructure provides support for murine secondary palatal shelf remodelling

A cricial part of secondary palate morphogenesis is the movement of the palatal shelves from an initial vertical position on either side of the tongue to a final horizontal one above it to achieve palate closure. The immunocytochemical localization of extracellular matrix (ECM) molecules in the palatal shelf during this remodelling and reorientation revealed the existence of an ECM infrastructure within the mesenchyme. The major components of this infrastructure were collagen III, fibronectin, and hyaluronate (HA). With remodelling, HA's domain within the mesenchyme was expanded, whereas those of fibronectin and collagen III became more circumscribed. The expansion of an HA-rich matrix within the mesenchyme is thought to be crucial for palatal reorientation. The results of this study suggest that, as this expansion occurs, it is modulated by collagen and fibronectin components of the ECM infrastructure. Prior to shelf remodelling, this infrastructure may be anchored by a specialized region of the midoral epithelial-mesenchymal interface and the subjacent mesenchyme which is characterized by the unique distribution of collagen III, fibronectin, and tenascin. The midoral palatal epithelium also may play a role in directing shelf expansion. This epithelial region undergoes changes in cell packing and epithelial cell layering that correlate with shelf remodelling. These changes occur concomitantly with changes in the expression of collagen III, collagen IV, and laminin within the underlying basement membrane. The localization and patterning of tenascin within the developing palate suggests that it not only contributes to the postulated anchoring structure of the midoral epithelial-mesenchymal region, but also plays a role in the determining the fate of the medial edge epithelial cells during the final stage of palate closure.© Willey-Liss, Inc.     

Inability to assess breath sounds during air medical transport by helicopter

This study assessed the capabilities of a traditional and an amplified stethoscope used by flight nurses to assess breath sound during air medical transport in an MBB BO-105 helicopter. We developed a normal breath sound model using a prerecorded tape of breath sounds interspersed with segments without breath sounds; the recorder had been placed in the chest wall of a resuscitation training manikin. Flight nurses completed control listening sessions in a quiet environment and experimental sessions during flight using a traditional stethoscope for half of the sessions and an amplified stethoscope for the remaining half. In the quiet environment, flight nurses accurately reported the presence or absence of breath sounds in 110 (92%) of 120 trials. During helicopter flight, none of the flight nurses heard breath sounds during any of the recorded segments with either the traditional stethoscope or the amplified stethoscope. We conclude that flight nurses are unable to hear normal breath sounds using a traditional or amplified stethoscope during flight in a medically configured MBB BO-105 helicopter. Improved stethoscopes, innovative methods of listening, and reduction of aircraft noise are potential solutions to the problems of breath sound assessment during air medical transport.     

Morphological and biochemical responses of cultured thyroid cells to thyrotropin

In the thyroid gland, TSH stimulates cAMP formation, exocytosis of precursor (noniodinated) thyroglobulin, endocytosis of thyroglobulin, and proteolytic processing of the thyroglobulin to form thyroid hormones. In this report we describe TSH effects on cAMP levels, microtubules, microfilaments, myosin fibrils, and the morphology of cultured thyroid follicle cells. The cells were normally cultured in the presence of 10 mU/ml TSH, and fresh TSH produced no stimulation when assayed for cAMP production in a 15-min assay. When such cells were cultured for up to 72 h in the absence of TSH and then assayed for cAMP production, the basal levels were much reduced, but fresh TSH stimulated cAMP levels half-maximally at 1 mU/ml and up to 50-fold at 20 mU/ml. Microtubules, myosin fibers, and microfilaments were demonstrated by indirect immunofluorescent staining. Fluorescent staining of fibers was observed in cells fixed before lysis and in cells lysed before fixation. In control cells grown without hormone, microtubules originated near the nucleus and extended to the cell periphery. Myosin-containing fibrils traversed the cell or radiated from foci. Microfilaments spanned the cell in a stress fiber pattern. After incubation with 20 mU/ml TSH and 4 mM isobutylmethylxanthine (IBMX) for 10-20 min, the microtubules in up to half of the cells appeared altered and more granular, and the cell periphery was scalloped. After 15-30 min with TSH and IBMX, normal myosin fibers were replaced with a fine lattice-work, peripheral staining disappeared, and the proportion of nonfibrous myosin increased. Stress fibers demonstrated with antibody to actin also disappeared, and the peripheral structures observed in normal cells became fragmented. Incubation with forskolin or TSH and IBMX for 2-3 h resulted in arborization of 30-60% of the cells that contained bundles of microtubules, myosin fibers, or microfilaments into dendrite-like processes and increased staining near the nucleus. At 5 h, more than 80% of the cells were arborized. These morphological changes were less pronounced with IBMX alone and minimal with TSH alone. The time course of cAMP levels observed basally or after TSH, forskolin, or TSH and IBMX was consistent with the relative effects of these agents on arborization. These studies are consistent with effects of cAMP on microtubules, myosin-containing fibrils, and microfilaments and may provide a basis for the morphological response to TSH.     

The effectiveness of esophageal stethoscopy in a simulated in-flight setting

Purpose: Previous research has confirmed the inability of flight nurses in an airborne BO-105 helicopter to hear breath sounds using normal or amplified transthoracic stethoscopy. The purpose of this study was to determine whether esophageal stethoscopy enabled effective auscultation of breath sounds in a simulated in-flight environment.

Methods: The cabin-sound environment of an in-flight BO-105 was recorded and recreated in an audiology laboratory, where five flight nurses were evaluated listening to taped breath sounds via an esophageal stethoscope. This audiotape model, validated in a previously published study, used a tape consisting of 24 20-second segments. Each segment, the beginning of which was marked with a beep signal, consisted of 20 seconds of silence or breath sounds. The distal (esophageal) end of the esophageal stethoscope was attached to the tape recorder; the intensity level of breath sounds heard at the stethoscope earpiece was calibrated to equate the sound level of actual esophageal breath sounds recorded on a volunteer.

Results: All nurses correctly identified the 24 taped segments as silent or including breath sounds 100% of the time.

Conclusion: In the simulated environment tested, esophageal stethoscopy enabled 100% accuracy in identification of breath sounds, as compared with previously reported 0% efficacy for standard transthoracic auscultation. Study in the actual patient-care environment is indicated to confirm the usefulness of esophageal stethoscopy in the in-flight setting.     

Unreported alcohol use was common but did not impact hepatitis C cure in HIV‐infected persons who use drugs

We investigated the prevalence and impact of heavy alcohol use on the hepatitis C virus (HCV) care continuum amongst HIV/HCV co‐infected persons who use drugs. In the CHAMPS study, 144 HIV/HCV co‐infected persons were randomized to contingent cash incentives, peer mentors and usual care to evaluate the impact on HCV care. Alcohol use was ascertained using the 10‐item AUDIT (hazardous: male ≥8, female ≥4) and phosphatidylethanol (PEth) (heavy: ≥50 ng/mL), an alcohol biomarker. Log binomial regression was used to evaluate the association between heavy alcohol use and failure to initiate treatment and to achieve sustained virologic response (SVR). Of the 135 participants with PEth data, median age was 55 years, 59% were male, 92% were Black, 91% reported a history of drug use, and 97% were on antiretroviral therapy. Hazardous drinking was reported on AUDIT by 28% of participants, and 35% had heavy alcohol use by PEth. Of the 47 individuals with a PEth ≥50 ng/mL, 23 (49%) reported no or minimal alcohol use by AUDIT. HCV treatment was initiated in 103 of 135 participants, and SVR was achieved in 92%. PEth ≥50 ng/mL (Relative Risk [RR] 0.72, 95% CI 0.35‐1.48) was not significantly associated with failure to initiate HCV treatment or failure to achieve SVR (RR 0.85, 95% CI 0.46‐1.57).In conclusion, alcohol use was common and frequently not detected by self‐report. However, heavy alcohol use, even when measured objectively, was not associated with failure to initiate HCV treatment or to achieve cure.     

An inducible mouse model for skin cancer reveals distinct roles for gain- and loss-of-function p53 mutations

Mutations in ras and p53 are the most prevalent mutations found in human nonmelanoma skin cancers. Although some p53 mutations cause a loss of function, most result in expression of altered forms of p53, which may exhibit gain-of-function properties. Therefore, understanding the consequences of acquiring p53 gain-of-function versus loss-of-function mutations is critical for the generation of effective therapies for tumors harboring p53 mutations. Here we describe an inducible mouse model in which skin tumor formation is initiated by activation of an endogenous K-ras^G12D allele. Using this model we compared the consequences of activating the p53 gain-of-function mutation p53^R172H and of deleting the p53 gene. Activation of the p53^R172H allele resulted in increased skin tumor formation, accelerated tumor progression, and induction of metastasis compared with deletion of p53. Consistent with these observations, the p53^R172H tumors exhibited aneuploidy associated with centrosome amplification, which may underlie the mechanism by which p53^R172H exerts its oncogenic properties. These results clearly demonstrate that p53 gain-of-function mutations confer poorer prognosis than loss of p53 during skin carcinogenesis and have important implications for the future design of therapies for tumors that exhibit p53 gain-of-function mutations.     

Effect of oral purine load and allopurinol on the crystallization of calcium salts in urine of patients with hyperuricosuric calcium urolithiasis

The pathogenetic importance of hyperuricosuria in the formation of calcium-containing renal stones was sought in 11 patients with hyperuricosuric calcium urolithiasis. The renal excretion of uric acid was altered over a wide range, from low normal values with a low purine diet and/or allopurinol therapy, to high values with an oral purine load. These fluctuations in urinary uric acid were compared with measures of urinary “crystallization” determined concurrently. As the renal excretion of urlc acid increased, urine specimens became more supersaturated with respect to monosodium urate. These changes were associated with a decline in the formation product ratio of calcium oxalate, a finding indicating that the spontaneous nucleation of calcium oxalate was facilitated. In separate experiments, “colloidal” or bound urate fraction, obtained by ultrafiltration, was found to be significantly increased in samples which were supersaturated with respect to monosodium urate than in undersaturated samples. Although unproved, monosodium urate so formed may facilitate nucleation of calcium oxalate, directly via heterogeneous nucleation or indirectly by removal of inhibitors of calcium oxalate nucleation. The results supported the following scheme: hyperuricosuria → supersaturation with respect to monosodium urate → formation of monosodium urate → nucleation of calcium oxalate.Conversely, treatment with allopurinol was shown to lower the urinary saturation with respect to monosodhim urate, commensurate with a decline in the renal excretion of uric acid. These changes were accompanied by a retardation of nucleation and of crystal growth of calcium oxalate. These studies provide the rationale, albeit partial, for the use of allopurinol in hyperuricosuric calcium urolithiasis.