Intergenic disease-associated regions are abundant in novel transcripts.

BACKGROUND: Genotyping of large populations through genome-wide association studies (GWAS) has successfully identified many genomic variants associated with traits or disease risk. Unexpectedly, a large proportion of GWAS single nucleotide polymorphisms (SNPs) and associated haplotype blocks are in intronic and intergenic regions, hindering their functional evaluation. While some of these risk-susceptibility regions encompass cis-regulatory sites, their transcriptional potential has never been systematically explored. RESULTS: To detect rare tissue-specific expression, we employed the transcript-enrichment method CaptureSeq on 21 human tissues to identify 1775 multi-exonic transcripts from 561 intronic and intergenic haploblocks associated with 392 traits and diseases, covering 73.9 Mb (2.2%) of the human genome. We show that a large proportion (85%) of disease-associated haploblocks express novel multi-exonic non-coding transcripts that are tissue-specific and enriched for GWAS SNPs as well as epigenetic markers of active transcription and enhancer activity. Similarly, we captured transcriptomes from 13 melanomas, targeting nine melanoma-associated haploblocks, and characterized 31 novel melanoma-specific transcripts that include fusion proteins, novel exons and non-coding RNAs, one-third of which showed allelically imbalanced expression. CONCLUSIONS: This resource of previously unreported transcripts in disease-associated regions ( http://gwas-captureseq.dingerlab.org ) should provide an important starting point for the translational community in search of novel biomarkers, disease mechanisms, and drug targets.

Cardiac catheterization: beyond stenoses.

Coronary angiography remains the gold standard for the clinical assessment of atherosclerotic heart disease. Findings other than percent stenosis can often enhance mortality risk determinations.

A partnership that matters: collaborative interdisciplinary ministry among parish nurses and faith group leaders.

We describe our experience of developing two university nursing courses and Clinical Pastoral Education (CPE) programs for parish nurses. We note the differences between nursing prepared and theologically prepared professionals. A vignette demonstrates our joint work and adaptation of the McGill Model of Nursing.

A phase I study of the antimetabolite (E)-2'-fluoromethylene-2'-deoxycytidine (MDL 101,731) administered as a twice-weekly infusion.

PURPOSE: (E)-2'-fluoromethylene-2'-deoxycytidine is a novel antimetabolite. Preclinical tests have shown antiproliferative activity in various human tumor xenograft models and have also indicated that efficacy is greatest with frequent dosing schedules. We conducted a phase I trial of MDL 101,731 infusion in humans with advanced cancer to determine the maximum tolerated dose and the dose-limiting toxicities of this drug when administered on a twice-weekly schedule. PATIENTS AND METHODS: The drug was administered on a twice-weekly schedule for 3 weeks, followed by a 2-week rest. The initial dose was 16 mg/m2. This was reduced to 12 mg/m2 if persistent neutropenia occurred. All toxicity in the first six patients resolved by the end of the first rest week. The schedule was changed to 3 weeks of therapy followed by 1 rest week for the subsequent four patients. RESULTS: Dose escalation beyond 16 mg/m2 was not feasible because of dose-limiting toxicities, principally hematologic. No irreversible or life-threatening toxicity was seen. Grade 2 noninfectious fever, mucositis, and anorexia were also seen. In patients with stable disease, there was a heavily pretreated patient with rectal cancer in whom a 38% reduction in indicator lesions of 7 months' duration occurred. DISCUSSION: (E)-2'-fluoromethylene-2'-deoxycytidine is a novel antimetabolite with evidence of anticancer activity in heavily pretreated patients. The maximum tolerated dose when the agent is given twice weekly is 16 mg/m2.

A phase I study of paclitaxel for mobilization of peripheral blood progenitor cells.

We conducted a phase I trial to determine the dose and schedule of paclitaxel, when given together with filgrastim, which would optimally promote mobilization of stem cells with tolerable toxicity. Dose escalation began at 275 mg/m2 3 h infusion. Dose-limiting neuropathy was observed at the 300 mg/m2 dose level. A second dose escalation was conducted utilizing 24 h infusion schedules, beginning at 225 mg/m2. Dose escalation was continued by 25 mg/m2 increments to 300 mg/m2, at which dose neuropathy was again dose-limiting. The recommended dose and schedule of paclitaxel for the purpose of mobilization of stem cells, when given together with filgrastim, are 275 mg/m2 as a 24 h infusion. The median stem cell yield after this dose of paclitaxel was 6.6 x 10(6) CD34+ cells/kg/apheresis (range 3.6 x 10(6)-7.7 x 10(6)).

Educating parish nurses.

During the early 1980s, as government health care reimbursement systems changed in the United States, an interest developed in strengthening healing alliances between health care facilities and faith communities. At the same time, Reverend Granger Westberg recognized the untapped resources of professional nurses that could be made available to parishioners. This recognition led to Westberg's pioneering of parish nursing as a way for faith communities to reclaim their healing mission. The value of reconnecting nurses to their traditional roots in faith communities spread quickly across the United States, and there are now over 3,000 registered nurses practising within American faith communities.

Consistency between US dietary fat intake and serum total cholesterol concentrations: the National Health and Nutrition Examination Surveys.

The National Health and Nutrition Examination Surveys (NHANESs) are conducted periodically to assess the health and nutritional status of the US population by means of standardized interviews and physical examinations. Since the early 1970s there have been three national cross-sectional surveys: NHANES I, 1971-1974; NHANES II, 1976-1980; and NHANES III, phase 1, 1988-1991. During the 18 y between the midpoint of NHANES I (1972) and the midpoint of phase 1 of NHANES III (1990), the age-adjusted mean percentage of energy from fat declined from 36.4% to 34.1% for adults aged 20-74 y. Trend data are shown for dietary fat and cholesterol as well as for serum cholesterol from NHANES I (1971-1975) to NHANES III (1988-1991) by age, sex, and race-ethnicity. The results document a decline in dietary fat, saturated fat, dietary cholesterol, and serum cholesterol. The observed changes reflect those that are predicted by the classic Keys and Hegsted formulas. Changes in reported intake are matched by similar shifts in the food supply for sources of these nutrients. These changes suggest that the Healthy People 2000 goal of reducing the mean serum cholesterol concentration of US adults to < or = 200 mg/dL (5.17 mmol/L) is attainable. The changes in diet are promising, yet we are challenged to achieve greater reductions in the intake of total fat and saturated fatty acids.

Cardiovascular health risks related to overweight.

Cross-sectional surveys of the civilian noninstitutionalized population of the United States, including in-home interviews and clinical examinations, were employed to examine trends in consumption of energy and fat, prevalence of overweight in the population, the association of overweight with levels of blood pressure and blood cholesterol, and the prevalence of high blood pressure and high blood cholesterol among the overweight compared with the nonoverweight. Data from participants 20 years of age and older are reported. Study results suggest that total mean energy intake, although generally accepted to be underreported in dietary surveys, may have increased. Total fat and saturated fat intake as a percent of energy decreased, but remained above recommended levels. Overweight has increased in the population, despite decreases in the prevalence of high blood pressure and high blood cholesterol levels. Increased levels of overweight, reported as body mass index, are associated with increased cardiovascular risk factors of high blood pressure and high blood cholesterol. These data suggest the need for health care practitioners to emphasize the requirement for energy balance (or weight loss if overweight, ie, not at a "healthy weight"). A focus on fat intake alone without emphasis on energy balance is inadequate for achieving and maintaining recommended weight.

A promotion ladder for teachers at Harvard Medical School: experience and challenges.

The authors describe the development, implementation, and institutionalization at Harvard Medical School of a promotion ladder that recognizes the teaching and scholarly contributions of full-time clinical faculty. They also discuss the challenges that arose during this process, for example, how to make the new track creditable and attractive to both the appointed faculty and the faculty at large. The criteria developed for promotion focus on a candidate's skills and accomplishments in teaching, scholarship, clinical work, and departmental service. The authors present the elements of these criteria for the three professional levels of the ladder and outline the appointment process, including the steps for consideration of a given promotion. The development of this teacher-clinician ladder has had a positive influence on faculty who are committed to teaching by allowing recognition of their contributions in a track held to be the equal of the other full-time tracks in a medical faculty traditionally committed to research and patient care. Data are given for the 70 faculty who were promoted over the five years from 1989 to 1994. The true success of this promotion ladder will be measured only over time through its impact on the educational enterprise within the medical school and its hospitals, and its capacity to both successfully advance the careers of qualified medical educators and further the development of the field of medical education.

Brain spectrin: of mice and men.

This article reviews our current knowledge of the structure of alpha spectrins and beta spectrins in the brain, as well as their location and expression within neural tissue. We discuss the known protein interactions of brain spectrin isoforms, and then describe results that suggest an important role for spectrin (alpha SpII sigma 1/beta SpII sigma 1) in the Ca(2+)-regulated release of neurotransmitters. Evidence that supports a role for spectrin in the docking of synaptic vesicles to the presynaptic plasma membrane and as a Ca2+ sensor protein that unclamps the fusion machinery is described, along with the Casting the Line model, which summarizes the information. We finish with a discussion of the value of spectrin and ankyrin-deficient mouse models in deciphering spectrin function in neural tissue.

Brain alpha erythroid spectrin: identification, compartmentalization, and beta spectrin associations.

Using isoform and subunit specific antibodies we have determined the presence, localization, and beta spectrin associations of alpha erythroid spectrin, alpha SpI sigma*, as well as alpha non-erythroid spectrin, alpha SpII sigma 1, in mouse brain. Peptide specific antibodies against unique sequences within the beta SpII sigma 1, non-erythroid beta spectrin isoform, and within beta SpI sigma 1, erythrocyte beta spectrin isoform were used to compare the immunolocalization of beta spectrin subunit isoforms with that of alpha spectrin subunit isoforms and to immunoprecipitate spectrin tetramers in order to identify the subunit components by immunoblot analysis. The specificity and sensitivity of antibodies for isoform specific alpha and beta subunits was determined by immunodot and immunoblot methods. Immunohistochemical analyses indicated that beta SpI sigma 2 is located in neuronal somata and dendrites in mouse cerebellum. beta SpII sigma 1 is located in the medullary layer, chiefly composed of axonal tracts. Parallel immunohistochemical analysis with antibodies for the alpha and beta spectrin isoforms revealed that antibodies specific for the alpha subunit of erythrocyte spectrin (alpha SpI sigma 1) localized antigen to the somata and dendrites of cerebellar granule cell neurons, a pattern similar to that for the localization of the erythroid beta subunit (beta SpI sigma 2). In contrast antibodies specific for the non-erythroid alpha subunit (alpha SpII sigma 1) localized antigen to axons in the cerebellum corresponding to the pattern for the non-erythroid beta subunit (beta SpII sigma 1). The distinct localization of antigens by antisera which recognize either the alpha subunit of red blood cell spectrin or the alpha subunit of non-erythroid brain spectrin, together with the correspondence of their localization with appropriate beta subunits, clearly indicate that brain contains at least two species of spectrin each with distinct alpha and beta subunits. Immunoprecipitation experiments of cerebellar extracts using beta spectrin peptide specific antibodies followed by immunoblotting analysis confirmed the association of an erythroid alpha subunit isoform with a beta erythroid subunit isoform, as well as the association of non-erythroid alpha and beta subunits. In addition the immunoblot analysis of the immunoprecipitated material suggested there are minor populations of various hybrid tetramers in brain consisting of mixed erythroid and non-erythroid subunits. In summary these data collectively demonstrate that in mouse brain there are at least two alpha spectrin subunits, one erythroid alpha SpI sigma* and one non-erythroid alpha SpII sigma 1; these associate with an erythroid beta SpI sigma 1, and a non-erythroid beta SpII sigma 1 in the cerebellum of mouse.(ABSTRACT TRUNCATED AT 400 WORDS)

Correlation between lipid-associated sialic acid and tumor burden in melanoma.

Recent studies have suggested that lipid-associated sialic acid (LSA) may be a useful tumor marker for monitoring patients with melanoma, but the relationship between LSA and tumor burden has not been previously studied. We therefore examined LSA levels in 240 patients of whom 169 had no clinical evidence of disease (NED) and 71 had metastatic disease. There was a statistically significant difference in LSA levels in patients with NED compared with metastatic disease as well as those with high tumor burden compared with low or intermediate tumor burden. There was no difference between the groups with low and intermediate tumor burden. An LSA level of 25 mg/dl provided a positive predictive value of 70% and a negative value of approximately 80%. Our data show that LSA levels correlate with tumor burden in patients with melanoma.

Neuron-specific enolase as a tumor marker in metastatic melanoma.

Neuron-specific enolase (NSE) has been shown by some investigators to be a useful tumor marker for melanoma, but the relationship between NSE and tumor burden has not been extensively studied. We therefore examined NSE levels in 240 patients of whom 169 had no clinical evidence of disease (NED) and 71 had metastatic disease. There was no statistically significant difference in NSE levels in patients with NED compared with metastatic disease as well as those with high tumor burden compared with low or intermediate tumor burden. In addition, the mean absolute values of NSE, despite a slight elevation with tumor burden, were within the normal range (< 20 ng/ml). Our data suggest that NSE levels measured by the method used in our study are of no benefit in melanoma.

Influence of contextual features on the activation of ambiguous word meanings.

Three studies examined whether initial meaning activation is sensitive to context. Experiment 1 demonstrated that contextually appropriate targets were activated more than inappropriate targets. Experiment 2 evaluated activation across intervals of 0, 300, and 600 ms. Constraining sentences activated contextually appropriate meanings over inappropriate meanings. This was maintained across the intervals for highly salient targets. Less-salient targets, although initially activated, were no longer activated 300 ms following the homograph. Experiment 3 converged on context-sensitive activation following a 50-ms exposure of the sentence-final homograph. Conclusions are (a) initial meaning activation can be sensitive to context, (b) when a homograph is instantiated, it is congruent with a broad scope of targets, and (c) less-salient targets receive less activation over the time course.

In vitro demyelination by serum antibody from patients with Guillain-Barré syndrome requires terminal complement complexes.

Serum from 7 patients who had acute-phase Guillain-Barré syndrome with high anti-peripheral nerve myelin antibody activity (54 to 210 units/ml) was compared with serum from 3 patients in the recovery phase (0 to 17 units/ml) and serum from 7 disease control subjects (0 to 24 units/ml) and 7 normal control subjects (0 to 7 units/ml) for its ability to demyelinate rodent dorsal root ganglion cultures. The demyelinating capacity of each serum was quantitated by counting the percent of damaged internodal segments in each of four cultures. All sera from patients in the acute phase GBS caused 50 to 78% demyelination, in contrast with 6 to 19% by the sera from all 3 patients in the recovery phase and all other control subjects. The degree of demyelination correlated with anti-peripheral nerve myelin antibody activity of the sera and demyelination was complement-dependent. Further, cultures were treated with an immunoglobulin M (IgM) fraction of an acute-phase Guillain-Barré syndrome plasma plus normal human serum depleted of complement component C7. Only those cultures treated with IgM and C7-depleted human serum reconstituted with purified C7 resulted in 50.8% demyelination, which was significantly greater than the 14.2 to 16.2% demyelination observed in the presence of heat-inactivated, C7-depleted human serum plus purified C7 or in the absence of C7 or antibody. In summary, our work suggests that anti-peripheral nerve myelin antibody in Guillain-Barré syndrome mediated complement dependent-demyelination of rodent dorsal root ganglion cultures. Further, this in vitro demyelination required generation of activation complexes of the terminal complement cascade.

Schwann cell plasminogen activator is regulated by neurons.

The synthesis and release of plasminogen activators (PAs) in co-cultures of embryonic rat dorsal root ganglion nerve cells (NCs) and Schwann cells (SCs) were examined by metabolic labeling, immunoprecipitation, immunodepletion, SDS-PAGE, zymography, and a two-step esterolytic assay. Metabolic labeling of SC cultures followed by immunoprecipitation of the conditioned medium (CM) demonstrated that cultured SCs synthesized and released tissue type PA (tPA). Failure of amiloride to inhibit PA activity in SCCM indicated that urokinase PA (uPA) was unlikely to contribute significantly to PA activity in SCCM. Experimental manipulation of the NCs and SCs suggested that NCs regulated SC derived PA. Total PA activity increased in SCCM 10-14-fold by 6 days after removal of NCs. Multiple molecular weight forms of PAs were detected by SDS-PAGE followed by zymography. A PA approximately 95 kDa was absent in co-cultures of SCs + NCs but prominent by 4 days postdenervation; PA approximately 50-70 kDa increased through 8 days postdenervation and PA approximately 25 kDa, present in SC + NC cultures, was absent 8 days after removal of NCs. Upon reintroduction of NCs to denervated cultures (SCs), the pattern of PAs detected in culture medium was transitional between innervated and denervated cultures. Immunodepletion experiments using conditioned medium from denervated SC cultures indicated that various molecular weight forms of PA detected in SCCM by zymography were immunologically related to tPA. These studies demonstrate that SCs synthesized and released tPA in a tissue culture model of peripheral nerve and that one mechanism for regulation of PA released by SCs was by association with NCs. This regulation occurred in cultures of both myelinating and nonmyelinating Schwann cells and thus was not dependent on the state of myelination.

Quantitative static secondary ion mass spectrometry of molecular ions from 1-beta-3,4-dihydroxyphenylalanine (L-dopa) and indolic derivatives.

Traditionally, static secondary ion mass spectrometry (SIMS) is believed to yield qualitative information and very little quantitative information. A method to obtain quantitative molecular ion data from organic static SIMS analysis of L-DOPA and related compounds is presented. Linear calibration curves have been constructed by integrating the protonated molecular ion to silver ion peak area ratios over a known ion dosage and plotting versus the original sample concentration.