Multiple mechanisms are implicated in the generation of 5q35 microdeletions in Sotos syndrome

Background: Sotos syndrome (MIM 117550) is characterised by learning difficulties, overgrowth, and a typical facial appearance. Microdeletions at 5q35.3, encompassing NSD1, are responsible for ∼10% of non-Japanese cases of Sotos. In contrast, a recurrent ∼2 Mb microdeletion has been reported as responsible for ∼50% of Japanese cases of Sotos. Methods: We screened 471 cases for NSD1 mutations and deletions and identified 23 with 5q35 microdeletions. We investigated the deletion size, parent of origin, and mechanism of generation in these and a further 10 cases identified from published reports. We used "in silico" analyses to investigate whether repetitive elements that could generate microdeletions flank NSD1. Results: Three repetitive elements flanking NSD1, designated REPcen, REPmid, and REPtel, were identified. Up to 18 cases may have the same sized deletion, but at least eight unique deletion sizes were identified, ranging from 0.4 to 5 Mb. In most instances, the microdeletion arose through interchromosomal rearrangements of the paternally inherited chromosome. Conclusions: Frequency, size, and mechanism of generation of 5q35 microdeletions differ between Japanese and non-Japanese cases of Sotos. Our microdeletions were identified from a large case series with a broad range of phenotypes, suggesting that sample selection variability is unlikely as a sole explanation for these differences and that variation in genomic architecture might be a contributory factor. Non-allelic homologous recombination between REPcen and REPtel may have generated up to 18 microdeletion cases in our series. However, at least 15 cannot be mediated by these repeats, including at least seven deletions of different sizes, implicating multiple mechanisms in the generation of 5q35 microdeletions.     

Infusing Diversity and Equity Into Clinical Teaching: Training the Trainers

Clinical instructors in health care disciplines are charged with engaging students in experiential learning wherein respect and cultural sensitivity is applied. This article reports on the results of 3 diversity workshops conducted for clinical preceptors and field instructors from various disciplines. The workshops were developed in response to students’ growing concerns that their academic learning experiences were negatively affected by dissatisfying management of differences between students, faculty, and preceptors with respect to ethno‐racial group membership, socioeconomic level, and degree of privilege and power. The workshops included a didactic session that presented basic principles of social and health equity followed by small‐group reflection about various ethical and moral dilemmas that were presented in clinical education scenarios. Examples of discrimination on a variety of levels were addressed in these workshops, including race, ethnicity, immigration status, sexual orientation, religion, body size and appearance, ability, age, socioeconomic class, religious faith, and gender. The group exercises and discussion from these sessions provided valuable insight and approaches to difficult but common areas of discomfiture encountered in the clinical teaching setting. This article presents the findings from participants of these diversity workshops in order to encourage the application of equity principles into clinical teaching in midwifery and other health care education contexts.     

Multicenter evaluation of a Candida albicans peptide nucleic acid fluorescent in situ hybridization probe for characterization of yeast isolates from blood cultures

We evaluated aliquots from 244 clinical blood culture bottles that demonstrated yeasts on Gram stain using a Candida albicans peptide nucleic acid (PNA) fluorescent in situ hybridization (FISH) probe. The sensitivity, specificity, positive predictive value, and negative predictive value of the C. albicans PNA FISH test in this study were 99%, 100%, 100%, and 99.3%, respectively.     

Autofluorescence characterisation of isolated whole crypts and primary cultured human epithelial cells from normal, hyperplastic, and adenomatous colonic mucosa

BACKGROUND/AIMS: In vivo autofluorescence endoscopic imaging and spectroscopy have been used to detect and differentiate benign (hyperplastic) and preneoplastic (adenomatous) colonic lesions. This fluorescence is composed of contributions from the epithelium, lamina propria, and submucosa. Because epithelial autofluorescence in normal and diseased tissues is poorly understood, this was the focus of the present study. METHODS: Whole colonic crypts were isolated, and short term primary cultures of epithelial cells were established from biopsies of normal, hyperplastic, and adenomatous colon. Autofluorescence (488 nm excitation) was examined by confocal fluorescence microscopy. Fluorescently labelled organelle probes and transmission electron microscopy were used to identify subcellular sources of fluorescence. RESULTS: Mitochondria and lysosomes were identified as the main intracellular fluorescent components in all cell types. Normal and hyperplastic epithelial cells were weakly autofluorescent and had similar numbers of mitochondria and lysosomes, whereas adenomatous (dysplastic) epithelial cells showed much higher autofluorescence, and numerous highly autofluorescent lysosomal (lipofuscin) granules. CONCLUSIONS: Short term primary cell cultures from endoscopic biopsies provide a novel model to understand differences in colonic tissue autofluorescence at the glandular (crypt) and cellular levels. The differences between normal, hyperplastic, and adenomatous epithelial cells are attributed in part to differences in the intrinsic numbers of mitochondria and lysosomes. This suggests that the detection of colonic epithelial fluorescence alone, if possible, may be sufficient to differentiate benign (hyperplastic) from preneoplastic and neoplastic (adenomatous) colonic intramucosal lesions during in vivo fluorescence endoscopy. Furthermore, highly orange/red autofluorescent intracellular granules found only in dysplastic epithelial cells may serve as a potential biomarker.     

Use of the MGB Eclipse system and SmartCycler PCR for differentiation of Mycobacterium chelonae and M. abscessus

Although accurate in the identification of Mycobacterium species, partial 16S rRNA gene sequencing does not distinguish Mycobacterium chelonae from M. abscessus. Thus, we designed a SmartCycler PCR assay targeting the 16S-to-23S internal transcribed spacer (ITS) region with use of MGB Eclipse probes to distinguish each species. Comparison with PCR-restriction enzyme analysis of a 441-bp fragment of the hsp65 gene resulted in 100% correlation with 25 isolates of M. chelonae and 25 isolates of M. abscessus. ITS PCR performed on 90 consecutive isolates identified by partial 16S rRNA gene sequencing (26 isolates of the M. chelonae-M. abscessus complex and 64 remaining isolates, including Mycobacterium species, Nocardia species, and other aerobic actinomycetes) showed 100% specificity and sensitivity. The ITS PCR assay is accurate and specific, easy to perform, and a good supplemental test when using partial 16S rRNA gene sequencing to identify M. chelonae and M. abscessus.     

CYP2E1 inhibition enhances mallory body formation

Mallory body (MB) formation is a complex phenomenon seen in chronic liver disease. CYP2E1 may play a role in preventing MB formation since it is involved in the elimination of toxic drugs and chemicals. When mice were fed with diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks, Mallory bodies (MBs) developed in the liver at the end of this period. When DDC feeding was combined with CMZ (an efficient in vivo CYP2E1 inhibitor), more MBs formed compared to DDC feeding alone. DDC was shown to be a suicide inhibitor of CYP2E1. The level of CYP2E1 protein in the liver was further reduced by the DDC and CMZ treatment when measured by Western blot. To test whether CYP2E1 reduced MB formation, CYP2E1 knockout mice and CYP2E1 overexpressed mice were fed with DDC or DDC and CMZ for 10 weeks. MB formation increased markedly in the liver of CYP2E1 knockout mice when fed with DDC only. CYP2E1 overexpressed mice showed an increase in MB formation when the mice were fed with the combination of DDC and CMZ where the amount of CYP2E1 was reduced to levels seen in wild type mice. It was concluded that CYP2E1 inhibits MB formation by increasing the rate of elimination of DDC and/or its toxic intermediates.     

Physical and biological properties of a novel siloxane adhesive for soft tissue applications

The aim of this study was to investigate the adhesive properties of an in-house aminopropyltrimethoxysilane-methylenebisacrylamide (APTMS-MBA) siloxane system and compare them with a commercially available adhesive, n-butyl cyanoacrylate (nBCA). The ability of the material to perform as a soft tissue adhesive was established by measuring the physical (bond strength, curing time) and biological (cytotoxicity) properties of the adhesives on cartilage. Complementary physical techniques, X-ray photoelectron spectroscopy, Raman and infrared imaging, enabled the mode of action of the adhesive to the cartilage surface to be determined. Adhesion strength to cartilage was measured using a simple butt joint test after storage in phosphate-buffered saline solution at 37 degrees C for periods up to 1 month. The adhesives were also characterised using two in vitro biological techniques. A live/dead stain assay enabled a measure of the viability of chondrocytes attached to the two adhesives to be made. A water-soluble tetrazolium assay was carried out using two different cell types, human dermal fibroblasts and ovine meniscal chondrocytes, in order to measure material cytotoxicity as a function of both supernatant concentration and time. IR imaging of the surface of cartilage treated with APTMS-MBA siloxane adhesive indicated that the adhesive penetrated the tissue surface marginally compared to nBCA which showed a greater depth of penetration. The curing time and adhesion strength values for APTMS-MBA siloxane and nBCA adhesives were measured to be 60 s/0.23 MPa and 38 min/0.62 MPa, respectively. These materials were found to be significantly stronger than either commercially available fibrin (0.02 MPa) or gelatin resorcinol formaldehyde (GRF) adhesives (0.1 MPa) (P < 0.01). Cell culture experiments revealed that APTMS-MBA siloxane adhesive induced 2% cell death compared to 95% for the nBCA adhesive, which extended to a depth of approximately 100-150 microm into the cartilage surface. The WST-1 assay demonstrated that APTMS-MBA siloxane was significantly less cytotoxic than nBCA adhesive as an undiluted conditioned supernatant (P < 0.001). These results suggest that the APTMS-MBA siloxane may be a useful adhesive for medical applications.