Hearing molecules: contributions from genetic deafness.

Considerable progress has been made over the past decade identifying many genes associated with deafness. With the identification of these hereditary deafness genes and the proteins they encode, molecular elements of basic hearing mechanisms emerge. As functional studies of these molecular elements become available, we can put together the pieces of the puzzle and begin to reach an understanding of the molecular mechanisms of hearing. The goal of this review is to discuss studies over the past decade that address the function of the proteins implicated in genetic deafness and to place them in the context of basic molecular mechanisms in hearing. The first part of this review highlights structural and functional features of the cochlea and auditory nerve. This background will provide a context for the second part, which addresses the molecular mechanisms underlying cochlear function as elucidated by genetic causes of deafness.

Use of sedation for routine diagnostic upper gastrointestinal endoscopy: a European Society of Gastrointestinal Endoscopy Survey of National Endoscopy Society Members.

BACKGROUND/AIMS: Sedation rates may vary among countries, depending on patients' and endoscopists' preferences. The aim of this survey was to investigate the rate of using premedication for routine diagnostic upper gastrointestinal (UGI) endoscopy in endoscopy societies, members of the European Society of Gastrointestinal Endoscopy (ESGE). METHODS: We evaluated a multiple-choice questionnaire which was e-mailed to representatives of national endoscopy societies, which are members of the ESGE. The questionnaire had 14 items referring to endoscopy practices in each country and the representatives' endoscopy units. RESULTS: The response rate was 76% (34/45). In 47% of the countries, less than 25% of patients undergo routine diagnostic UGI endoscopy with conscious sedation. In 62% of the responders' endoscopy units, patients are not asked their preference for sedation and do not sign a consent form (59%). Common sedatives in use are midazolam (82%), diazepam (38%) or propofol (47%). Monitoring equipment is not available 'in most of the endoscopy units' in 46% (13/28) of the countries. Though they were available in 91% of the national representatives' endoscopy units, they are rarely (21%) used to monitor unsedated routine diagnostic UGI endoscopy. CONCLUSIONS: In about 50% of ESGE-related countries, less than 25% of patients are sedated for routine diagnostic UGI endoscopy. Major issues to improve include availability of monitoring equipment and the use of a consent form.

Toenail onychomycosis in Estonia.

The aim of our study was to evaluate the clinical features, predisposing factors and pathogens of toenail onychomycosis in Estonia. During study period we interviewed and examined 436 dermatological patients with clinical signs of toenail onychomycosis in all counties of Estonia. In 69% of cases, the clinical diagnosis of onychomycosis was confirmed by the mycological analysis. The most common clinical symptoms of onychomycosis both in mycologically proven and non-proven cases were discolorization of nail plate, hyperkeratosis and brittle nails. The number of infected toenails positively correlated with patients' age. On average, patient had 5.4 infected nails. In 78% of culture-positive cases, a dermatophyte was isolated as the causative agent, in 10% yeasts and in 7% moulds. In 6% of culture-positive cases we reported mixed infections. Trichophyton rubrum was the most common pathogen. The high occurrence of mixed infections, clinical symptoms characteristic to long lasting or chronic infection and high number of infected toenails indicate that Estonian patients have more advanced stage of toenail onychomycosis compared with other western and central European countries.

Phylogenetic motif detection by expectation-maximization on evolutionary mixtures.

The preferential conservation of transcription factor binding sites implies that non-coding sequence data from related species will prove a powerful asset to motif discovery. We present a unified probabilistic framework for motif discovery that incorporates evolutionary information. We treat aligned DNA sequence as a mixture of evolutionary models, for motif and background, and, following the example of the MEME program, provide an algorithm to estimate the parameters by Expectation-Maximization. We examine a variety of evolutionary models and show that our approach can take advantage of phylogenic information to avoid false positives and discover motifs upstream of groups of characterized target genes. We compare our method to traditional motif finding on only conserved regions. An implementation will be made available at http://rana.lbl.gov.

Chick cochlear hair cell exocytosis mediated by dihydropyridine-sensitive calcium channels.

1. A semi-intact preparation of the chick basilar papilla was developed to study calcium-dependent neurotransmitter release by tall hair cells (avian equivalent of cochlear inner hair cells). 2. Tall hair cell depolarization resulted in changes in cell membrane capacitance (DeltaC(m)) that reflected cell surface area increases following synaptic vesicle exocytosis and provided a surrogate measure of neurotransmitter release. Both calcium current (I(Ca)) and DeltaC(m) were reversibly blocked by cobalt, and exhibited a similar bell-shaped dependency on voltage with a peak response around -10 mV. 3. Pharmacological agents selective for L-type calcium channels were employed to assess the role of this channel type in neurotransmitter exocytosis. Nimodipine, a dihydropyridine (DHP) antagonist, suppressed I(Ca) and blocked DeltaC(m). Conversely, the DHP agonist Bay K 8644 increased both I(Ca) and DeltaC(m) amplitude nearly 3-fold. These findings suggest that chick tall hair cell neurotransmitter release is mediated by calcium influx through L-type calcium channels.

Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.

The purpose of this study was to classify breast carcinomas based on variations in gene expression patterns derived from cDNA microarrays and to correlate tumor characteristics to clinical outcome. A total of 85 cDNA microarray experiments representing 78 cancers, three fibroadenomas, and four normal breast tissues were analyzed by hierarchical clustering. As reported previously, the cancers could be classified into a basal epithelial-like group, an ERBB2-overexpressing group and a normal breast-like group based on variations in gene expression. A novel finding was that the previously characterized luminal epithelial/estrogen receptor-positive group could be divided into at least two subgroups, each with a distinctive expression profile. These subtypes proved to be reasonably robust by clustering using two different gene sets: first, a set of 456 cDNA clones previously selected to reflect intrinsic properties of the tumors and, second, a gene set that highly correlated with patient outcome. Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.

Visualizing associations between genome sequences and gene expression data using genome-mean expression profiles.

The combination of genome-wide expression patterns and full genome sequences offers a great opportunity to further our understanding of the mechanisms and logic of transcriptional regulation. Many methods have been described that identify sequence motifs enriched in transcription control regions of genes that share similar gene expression patterns. Here we present an alternative approach that evaluates the transcriptional information contained by specific sequence motifs by computing for each motif the mean expression profile of all genes that contain the motif in their transcription control regions. These genome-mean expression profiles (GMEP's) are valuable for visualizing the relationship between genome sequences and gene expression data, and for characterizing the transcriptional importance of specific sequence motifs. Analysis of GMEP's calculated from a dataset of 519 whole-genome microarray experiments in Saccharomyces cerevisiae show a significant correlation between GMEP's of motifs that are reverse complements, a result that supports the relationship between GMEP's and transcriptional regulation. Hierarchical clustering of GMEP's identifies clusters of motifs that correspond to binding sites of well-characterized transcription factors. The GMEP's of these clustered motifs have patterns of variation across conditions that reflect the known activities of these transcription factors. Software that computed GMEP's from sequence and gene expression data is available under the terms of the Gnu Public License from http://rana.lbl.gov/.

Patients with allergic and irritant contact dermatitis are characterized by striking change of iron and oxidized glutathione status in nonlesional area of the skin.

To assess the consequences of oxidative stress in allergic and irritant contact dermatitis, we compared the iron level, unsaturated iron-binding capacity, total iron binding capacity, the percentage saturation of iron-binding capacity, the amount of diene conjugates as well as the amounts of total glutathione, reduced glutathione, oxidized glutathione, and the oxidized glutathione/reduced glutathione ratio in skin homogenate from lesional and nonlesional skin. Lesional skin samples were obtained from positive patch test sites to 5% NiSO4 in five subjects, and from chronic contact dermatitis lesions on the hands, which had exacerbated over 3--9 wk in six subjects. Contact dermatitis caused at least a 4-fold increase in the iron level in the lesional skin area compared with the nonlesional skin area (p < 0.02). The increase in the iron level depended on the duration of contact dermatitis and was accompanied by high unsaturated iron-binding capacity and total iron-binding capacity values in the positive patch test sites (p < 0.05), and by a high percentage saturation value in the chronic contact dermatitis lesions (p < 0.05). We found high indices for iron, total iron-binding capacity and diene conjugates in the apparently healthy skin of the patients with persistent contact dermatitis that significantly (p < 0.05) exceeded the corresponding values in the patients with only patch test reactions. In summary, we have succeeded in providing evidence that generalized oxidative damage of the skin occurs as a consequence of contact dermatitis in a restricted area.

Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays.

We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

The Stanford Microarray Database.

The Stanford Microarray Database (SMD) stores raw and normalized data from microarray experiments, and provides web interfaces for researchers to retrieve, analyze and visualize their data. The two immediate goals for SMD are to serve as a storage site for microarray data from ongoing research at Stanford University, and to facilitate the public dissemination of that data once published, or released by the researcher. Of paramount importance is the connection of microarray data with the biological data that pertains to the DNA deposited on the microarray (genes, clones etc.). SMD makes use of many public resources to connect expression information to the relevant biology, including SGD [Ball,C.A., Dolinski,K., Dwight,S.S., Harris,M.A., Issel-Tarver,L., Kasarskis,A., Scafe,C.R., Sherlock,G., Binkley,G., Jin,H. et al. (2000) Nucleic Acids Res., 28, 77-80], YPD and WormPD [Costanzo,M.C., Hogan,J.D., Cusick,M.E., Davis,B.P., Fancher,A.M., Hodges,P.E., Kondu,P., Lengieza,C., Lew-Smith,J.E., Lingner,C. et al. (2000) Nucleic Acids Res., 28, 73-76], Unigene [Wheeler,D.L., Chappey,C., Lash,A.E., Leipe,D.D., Madden,T.L., Schuler,G.D., Tatusova,T.A. and Rapp,B.A. (2000) Nucleic Acids Res., 28, 10-14], dbEST [Boguski,M.S., Lowe,T.M. and Tolstoshev,C.M. (1993) Nature Genet., 4, 332-333] and SWISS-PROT [Bairoch,A. and Apweiler,R. (2000) Nucleic Acids Res., 28, 45-48] and can be accessed at http://genome-www.stanford.edu/microarray.

Peer-based professional development viewed through the lens of transformative learning.

The goal of professional development is improved practice through change-changes in ways of doing or thinking about one's work. Traditional approaches, including group instruction and individualized coaching, emphasize the unidirectional flow of information from expert to novice. This article foregrounds a nontraditional peer-based modality, the peer learning partnership, which promotes joint reflection and reciprocal learning between professionals. A qualitative case study of a peer-based community college faculty development initiative reveals participants' perceptions of the role peer partnering played in their learning. Findings are discussed relative to selected theories, specifically transformative learning. Recommendations are offered on using peer-based approaches for professional development and transformation.

Genomic expression programs in the response of yeast cells to environmental changes.

We explored genomic expression patterns in the yeast Saccharomyces cerevisiae responding to diverse environmental transitions. DNA microarrays were used to measure changes in transcript levels over time for almost every yeast gene, as cells responded to temperature shocks, hydrogen peroxide, the superoxide-generating drug menadione, the sulfhydryl-oxidizing agent diamide, the disulfide-reducing agent dithiothreitol, hyper- and hypo-osmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. A large set of genes (approximately 900) showed a similar drastic response to almost all of these environmental changes. Additional features of the genomic responses were specialized for specific conditions. Promoter analysis and subsequent characterization of the responses of mutant strains implicated the transcription factors Yap1p, as well as Msn2p and Msn4p, in mediating specific features of the transcriptional response, while the identification of novel sequence elements provided clues to novel regulators. Physiological themes in the genomic responses to specific environmental stresses provided insights into the effects of those stresses on the cell.

Morbidity after midline mandibulotomy and radiation therapy.

PURPOSE: To assess the morbidity of mandibulotomy in patients treated for neoplasms of the oropharynx and oral cavity, and to determine if postoperative radiation therapy to the mandibulotomy site carries an increased risk of complications. PATIENTS AND METHODS: The medical charts of 30 patients treated between 1992 and 1996 undergoing midline mandibulotomy for tumors of the oral cavity (7 patients) and oropharynx (23 patients) were retrospectively reviewed. Three patients presented with recurrent disease, 1 of whom was previously irradiated. Twenty-five patients received postoperative radiation after mandibulotomy to a median dose of 60 Gy to the primary tumor bed, whereas 5 patients were treated with surgery alone. The patients were separated into those whose mandibulotomy site was within the radiation treatment field (n = 9), and those whose site was shielded (n = 10). Median follow-up was 27.8 months (range 5-81 months). End points included significant pain involving the mandibulotomy site, trismus, malocclusion, wound infection, osteoradionecrosis, and time to oral intake. RESULTS: There were no postoperative deaths. Minor wound infection or breakdown occurred in 4/30 patients (13%). All of these resolved with local care and parenteral antibiotics. More serious complications involving the mandibulotomy occurred in 2 patients (7%). One patient had chronic wound drainage at the mandibular osteotomy site, which healed after plate removal. Another patient developed osteoradionecrosis. No patient developed trismus or malocclusion. With a median follow-up of 27.8 months, 4 patients have recurred locally. The complication rate was 11% for patients whose mandibulotomy site was irradiated, and 30% for those whose site was shielded. CONCLUSION: Mandibulotomy can be safely performed in patients who are likely to require postoperative external radiation.

Molecular portraits of human breast tumours.

Human breast tumours are diverse in their natural history and in their responsiveness to treatments. Variation in transcriptional programs accounts for much of the biological diversity of human cells and tumours. In each cell, signal transduction and regulatory systems transduce information from the cell's identity to its environmental status, thereby controlling the level of expression of every gene in the genome. Here we have characterized variation in gene expression patterns in a set of 65 surgical specimens of human breast tumours from 42 different individuals, using complementary DNA microarrays representing 8,102 human genes. These patterns provided a distinctive molecular portrait of each tumour. Twenty of the tumours were sampled twice, before and after a 16-week course of doxorubicin chemotherapy, and two tumours were paired with a lymph node metastasis from the same patient. Gene expression patterns in two tumour samples from the same individual were almost always more similar to each other than either was to any other sample. Sets of co-expressed genes were identified for which variation in messenger RNA levels could be related to specific features of physiological variation. The tumours could be classified into subtypes distinguished by pervasive differences in their gene expression patterns.

The yeast mitochondrial transport proteins: new sequences and consensus residues, lack of direct relation between consensus residues and transmembrane helices, expression patterns of the transport protein genes, and protein-protein interactions with other proteins.

Mitochondrial transport proteins (MTP) typically are homodimeric with a 30-kDa subunit with six transmembrane helices. The subunit possesses a sequence motif highly similar to Pro X Asp/Glu X X Lys/Arg X Arg within each of its three similar 10-kDa segments. Four (YNL083W, YFR045W, YPR021C, YDR470C) of the 35 yeast (S. cerevisiae) MTP genes were resequenced since the masses of their proteins deviate significantly from the typical 30 kDa. We now find these four proteins to have 545, 285, 902, and 502 residues, respectively. Together with only four other MTPs, the sequences of YPR021C and YDR470C show substitutions of some of the five residues that are absolutely conserved among the 12 MTPs with identified transport function and 17 other MTPs. We do now find these five consensus residues also in the new sequences of YNL083W and YFR045W. Additional analyses of the 35 yeast MTPs show that the location of transmembrane helix sequences do not correlate with the general consensus residues of the MTP family; protein segments connecting the six transmembrane helices and facing the intermembrane space are not uniformly short (about 20 residues) or long (about 40 residues) when facing the matrix; most MTPs have at least one transmembrane helix for which the sum of the negative hydropathy values of all residues yields a very small negative value, suggesting a membrane location bordering polar faces of other transmembrane helices or a non-transmembrane location. The extra residues of the three large MTPs are hydrophilic and at the N-terminal. The 200-residue N-terminal segment of YNL083W has four putative Ca2+-binding sites. The 500-residue N-terminal segment of YPR021C shows sequence similarity to enzymes of nucleic acid metabolism. cDNA microarray data show that YNL083W is expressed solely during sporulation, while the expressions of YFR045W, YPR021C, and YDR470C are induced by various stress situations. These results also show that the 35 MTP genes are expressed under a rather diverse set of metabolic conditions that may help identify the function of the proteins. Interestingly, yeast two-hybrid screens, that will also be useful in identifying the function of MTPs, indicate that MIR1, AAC3, YOR100C, and YPR011C do interact with non-MTPs.

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas.

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (V(H)) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

Preoperative imaging to predict orbital invasion by tumor.

BACKGROUND: Our purpose was to examine the accuracy of preoperative imaging in assessing tumor invasion of the orbit and nasolacrimal system. METHODS: Nineteen preoperative CT and 17 preoperative MR images from patients at risk for orbital invasion were retrospectively reviewed. Invasion was corroborated by pathologic and intraoperative assessment. RESULTS: Tumor adjacent to the periorbita was the most sensitive predictor of orbital invasion (90%) for both CT and MRI. Extraocular muscle involvement on MRI (100%) and orbital fat obliteration (80% MRI, 86% CT) had the highest positive predictive values of the criteria evaluated. Extraocular muscle displacement and enhancement were less accurate (<65%) predictors. No one criterion was >79% accurate in predicting orbital invasion. Six or more positive criteria predicted invasion with 67% sensitivity and 80% specificity (accuracy, 72%). CT was more accurate than MRI in seven of nine criteria. Invasion of the nasolacrimal system was predicted accurately (89%). CONCLUSIONS: Although preoperative imaging can aid in surgical planning, it should not replace intraoperative assessment in ambiguous cases of orbital invasion.

Large-scale identification of secreted and membrane-associated gene products using DNA microarrays.

Membrane-associated and secreted proteins are an important class of proteins and include receptors, transporters, adhesion molecules, hormones and cytokines. Although algorithms have been developed to recognize potential amino-terminal membrane-targeting signals or transmembrane domains in protein sequences, their accuracy is limited and they require knowledge of the entire coding sequence, including the N terminus, which is not currently available for most of the genes in most organisms, including human. Several experimental approaches for identifying secreted and membrane proteins have been described, but none have taken a comprehensive genomic approach. Furthermore, none of these methods allow easy classification of clones from arrayed cDNA libraries, for which large-scale gene-expression data are now becoming available through the use of DNA microarrays. We describe here a rapid and efficient method for identifying genes that encode secreted or membrane proteins. mRNA species bound to membrane-associated polysomes were separated from other mRNAs by sedimentation equilibrium or sedimentation velocity. The distribution of individual transcripts in the 'membrane-bound' and 'cytosolic' fractions was quantitated for thousands of genes by hybridization to DNA microarrays. Transcripts known to encode secreted or membrane proteins were enriched in the membrane-bound fractions, whereas those known to encode cytoplasmic proteins were enriched in the fractions containing mRNAs associated with free and cytoplasmic ribosomes. On this basis, we identified over 275 human genes and 285 yeast genes that are likely to encode previously unrecognized secreted or membrane proteins.