Gene Ontology annotations and resources.

The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bioinformatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources.

The integrated stress response contributes to tRNA synthetase-associated peripheral neuropathy.

Dominant mutations in ubiquitously expressed transfer RNA (tRNA) synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth (CMT) disease. Genetic evidence in mouse and Drosophila models suggests a gain-of-function mechanism. In this study, we used in vivo, cell type­specific transcriptional and translational profiling to show that mutant tRNA synthetases activate the integrated stress response (ISR) through the sensor kinase GCN2 (general control nonderepressible 2). The chronic activation of the ISR contributed to the pathophysiology, and genetic deletion or pharmacological inhibition of Gcn2 alleviated the peripheral neuropathy. The activation of GCN2 suggests that the aberrant activity of the mutant tRNA synthetases is still related to translation and that inhibiting GCN2 or the ISR may represent a therapeutic strategy in CMT.

Earthquakes, active faults, and geothermal areas in the imperial valley, california.

A dense seismograph network in the Imperial Valley recorded a series of earthquake swarms along the Imperial and Brawley faults and a diffuse pattern of earthquakes along the San Jacinto fault. Two known geothermal areas are closely associated with these earthquake swarms. This seismicity pattern demonstrates that seismic slip is occurring along both the Imperial-Brawley and San Jacinto fault systems.

Point mugu, california, earthquake of 21 february 1973 and its aftershocks.

Seismological investigations show that the Point Mugu earthquake involved north-south crustal shortening deep within the complex fault zone that marks the southern front of the Transverse Ranges province. This earthquake sequence results from the same stress system responsible for the deformation in this province in the Pliocene through Holocene and draws attention to the significant earthquake hazard that the southern frontal fault system poses to the Los Angeles metropolitan area.

Seismicity remotely triggered by the magnitude 7.3 landers, california, earthquake.

The magnitude 7.3 Landers earthquake of 28 June 1992 triggered a remarkably sudden and widespread increase in earthquake activity across much of the western United States. The triggered earthquakes, which occurred at distances up to 1250 kilometers (17 source dimensions) from the Landers mainshock, were confined to areas of persistent seismicity and strike-slip to normal faulting. Many of the triggered areas also are sites of geothermal and recent volcanic activity. Static stress changes calculated for elastic models of the earthquake appear to be too small to have caused the triggering. The most promising explanations involve nonlinear interactions between large dynamic strains accompanying seismic waves from the mainshock and crustal fluids (perhaps including crustal magma).

The Mouse Gene Expression Database (GXD).

The Gene Expression Database (GXD) is a community resource of gene expression information for the laboratory mouse. By combining the different types of expression data, GXD aims to provide increasingly complete information about the expression profiles of genes in different mouse strains and mutants, thus enabling valuable insights into the molecular networks that underlie normal development and disease. GXD is integrated with the Mouse Genome Database (MGD). Extensive interconnections with sequence databases and with databases from other species, and the development and use of shared controlled vocabularies extend GXD's utility for the analysis of gene expression information. GXD is accessible through the Mouse Genome Informatics web site at http://www.informatics.jax.org/ or directly at http://www.informatics.jax.org/menus/expression_menu. shtml.

The Gene Ontology (GO) database and informatics resource.

The Gene Ontology (GO) project (http://www. geneontology.org/) provides structured, controlled vocabularies and classifications that cover several domains of molecular and cellular biology and are freely available for community use in the annotation of genes, gene products and sequences. Many model organism databases and genome annotation groups use the GO and contribute their annotation sets to the GO resource. The GO database integrates the vocabularies and contributed annotations and provides full access to this information in several formats. Members of the GO Consortium continually work collectively, involving outside experts as needed, to expand and update the GO vocabularies. The GO Web resource also provides access to extensive documentation about the GO project and links to applications that use GO data for functional analyses.

A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice.

We have used a gene-trap vector and mouse embryonic stem (ES) cells to screen for insertional mutations in genes developmentally regulated at 8.5 days of embryogenesis (dpc). From 38,730 cell lines with vector insertions, 393 clonal integrations had disrupted active transcription units, as assayed by beta-galactosidase reporter gene expression. From these lines, 290 clones were recovered and injected into blastocysts to assay for reporter gene expression in 8.5-dpc chimeric mouse embryos. Of these, 279 clones provided a sufficient number of chimeric embryos for analysis. Thirty-six (13%) showed restricted patterns of reporter-gene expression, 88 (32%) showed widespread expression and 155 (55%) failed to show detectable levels of expression. Further analysis showed that approximately one-third of the clones that did not express detectable levels of the reporter gene at 8.5 dpc displayed reporter gene activity at 12.5 dpc. Thus, a large proportion of the genes that are expressed in ES cells are either temporally or spatially regulated during embryogenesis. These results indicate that gene-trap mutageneses in embryonic stem cells provide an effective approach for isolating mutations in a large number of developmentally regulated genes.

The myeloid zinc finger gene (MZF-1) delays retinoic acid-induced apoptosis and differentiation in myeloid leukemia cells.

The myeloid zinc finger gene, MZF-1, is a hematopoietic transcription factor expressed in developing myeloid cells. To characterize further the role of MZF-1 in myelopoiesis, we used retroviral gene transduction to overexpress MZF-1 in HL-60 cells to produce HL-60-MZF-1 cells. HL-60 cells respond to retinoic acid (RA) with growth inhibition, granulocytic differentiation and apoptosis. However, HL-60-MZF-1 cells exposed to RA continue to proliferate in response to RA as evidenced by a higher percentage of cells in S phase, higher peak cell counts, and later peak cell counts. Morphologic differentiation of the RA-induced HL-60-MZF-1 cells is delayed with half as many of the HL-60-MZF-1 cells compared to the wild-type HL-60 cells that are differentiated after 3 days of RA, although both cells types responded with 80-95% mature granulocytes after 6 days of RA. Apoptosis was delayed in the MZF-1 transduced cells as measured by internucleosomal DNA fragmentation patterns, the terminal transferase end labeling reaction (TUNEL), and quantitation of fragmented DNA by the diphenylamine reaction. Several markers of differentiation were identical in both HL-60 and HL-60-MZF-1 cells including CD11b, CD33, CD34, CD13, CD16 and CD14. However, following 6 days of RA, only half as many HL-60-MZF-1 cells expressed CD18 compared to the wild-type HL-60 cells. Expression of the bcl-2 proto-oncogene transcript and protein was higher in the HL-60-MZF-1 cells compared to wild-type HL-60s and expression persisted for 5 days following RA in the HL-60-MZF-1 cells compared to only 3 days in the parental HL-60 cells suggesting that bcl-2 may contribute to the inhibition of apoptosis. Overexpression of MZF-1 had no effect on PMA-induced monocyte/macrophage differentiation of HL-60 cells. Together these findings indicate that MZF-1 can stimulate cell proliferation and delay RA-induced differentiation and apoptosis in HL-60 cells. MZF-1 may function in a similar role in myelopoiesis allowing myeloid precursors to expand their numbers before going on to terminally differentiate.

A controlled trial of the cost benefit of computerized bayesian aminoglycoside administration.

We studied the effect of a bayesian pharmacokinetic dosing program on the outcome of aminoglycoside therapy in patients with clinical infections. Patients were randomized to a control (dosing based on physician choice; n = 75) or experimental group (dosing based on the bayesian program; n = 72). Both groups used serum aminoglycoside concentration data when making dosing decisions. Improved response rates were seen in the experimental (60%; 42/68) compared with the control group (48%; 36/68). A higher, but not statistically significant, incidence of toxicity was found in the control (7/75; 9.7%) versus the experimental group (4/72; 5.1%). Mean length of total hospital stay was significantly longer for patients in the control group (20.3 days) compared with the experimental group (16.0 days) (p = 0.028). The variables from multivariate analysis with a significant impact on length of stay were patient group and length of aminoglycoside therapy. On the basis of a reduced length of stay, a potential cost savings of $1311 per patient can be achieved.

Lymphatic invasion in pigmented nevi.

All 124 pigmented nevi registered at the Canadian Tumour Reference Centre between July 1958 and May 1969 were reviewed. Nevus cells invading endothelial lined spaces were observed in serial sections from five cases. The significance of this finding is discussed in relation to published reports of the presence of nevus cells in lymph nodes.

Wound healing trajectories as predictors of effectiveness of therapeutic agents.

BACKGROUND: One goal of wound healing research is to discover agents to accelerate healing. Regulatory agencies have suggested stringent criteria to determine efficacy, that of 100% wound closure. Data analysis at a single point such as 100% closure does not provide detailed information about agent effectiveness over the entire span of healing. HYPOTHESIS: Wound healing trajectories can provide such information and can be used to demonstrate utility as alternative end points for wound healing trials. DESIGN: Data from 160 patients in 11 clinical trials of diabetic foot ulcers conducted at 2 centers were evaluated. Wound healing trajectories were constructed for patients whose wounds healed (100% closure) and those whose did not (<100% closure) over a 20-week period. The percentage of patients achieving total healing vs time of treatment was plotted and divided into patients receiving a test agent or placebo. RESULTS: The healing trajectories were almost identical for patients achieving complete healing at the 2 centers, as were the trajectories for patients with less than 100% closure. However, the trajectories of patients achieving total healing were significantly different from those not achieving 100% closure. Fifty-two percent of all patients achieved 100% healing by 20 weeks; 61% of patients receiving an experimental agent had total healing compared with 39% of placebo-treated patients. Linear regression suggested that all patients would achieve total healing by 37 weeks. CONCLUSIONS: Since wound healing trajectories for diabetic foot ulcers treated at 2 centers so closely mimic one another, trajectories might be useful efficacy end points, and used to compare significant points along a continuum rather than a single static end point. Shifting of the wound healing trajectory from an impaired to a more ideal course may be considered when determining efficacy of new wound treatments.

Characterization of the cholinergic neuronal differentiation of the human neuroblastoma cell line LA-N-5 after treatment with retinoic acid.

Analysis of the molecular factors that control cellular differentiation in mammalian embryos is difficult due to the small amount of material available from embryos and their inaccessibility during gestation. One way to circumvent these limitations is to use model systems that allow the study of differentiation in vitro. In this study we have characterized the response of a human neuroblastoma cell line, LA-N-5, to the differentiation-inducing agent, all-trans retinoic acid (RA) using 23 markers that are characteristic of neural crest cells and some of their derivatives. Following induction with RA, the neural crest-like LA-N-5 cells undergo differentiation into cholinergic neurons with increased expression of a variety of neural-specific markers including neurofilaments, growth associated protein-43, tetanus toxin binding sites, receptors for neurotrophic factors, neuropeptides, choline acetyl transferase, vesicular acetylcholine transporter, and acetylcholinesterase with a concomitant decrease in the expression of non-neuronal markers. These results provide the basis for the use of retinoic acid-induced differentiation of LA-N-5 cells as a model system to study molecular events associated with the differentiation of cholinergic neurons.

Differentiation of LA-N-5 neuroblastoma cells into cholinergic neurons: methods for differentiation, immunohistochemistry and reporter gene introduction.

The use of model systems derived from cell lines has been a valuable tool in understanding the molecules and cellular processes that govern differentiation processes (T.R. Breitman, S.E. Selonick, S.J. Collins, Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid, Proc. Natl. Acad. Sci. USA 77 (1980) 2936-2940 [2]; N. Gomez, S. Traverse, P. Cohen, Identification of a MAP kinase in phaeochromocytoma (PC12) cells, FEBS Lett. 314 (1992) 461-465 [4]). The use of such systems provides an inexpensive, quick and simple way to identify and test molecules that can be further studied in more complex in vivo experiments. Some cell lines such as embryonic stem cells can be induced to differentiate in vitro, however, the differentiation is difficult to control and most often leads to the generation of a wide variety of cell types. Cell lines derived from sources committed to a restricted cell fate provide an opportunity to examine cell growth and differentiation within a specific cell type (G.M. Keller, In vitro differentiation of embryonic stem cells, Curr. Opin. Cell Biol. 7 (1995) 862-869 [10]). In this article we describe a simple system for the differentiation of the human neuroblastoma cell line LA-N-5 into cholinergic neurons using all-trans retinoic acid (G. Han, B. Chang, M.J. Connor, N. Sidell, Enhanced potency of 9-cis versus all-trans retinoic acid to induce the differentiation of human neuroblastoma cells, Differentiation, 59 (1995) 61-69 [5]; D.P. Hill, K.R. Robertson, Characterization of the cholinergic neuronal differentiation of the human neuroblastoma cell line LA-N-5 after treatment with retinoic acid, Dev. Brain Res. 102 (1997) 53-67 [6]; J.A. Robson, N. Sidell, Ultrastructural features of a human neuroblastoma cell line treated with retinoic acid, Neuroscience 14 (1985) 1149-1162 [12]; N. Sidell, C.A. Lucas, G.W. Kreutzberg, Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line, Exp. Cell Res. 155 (1984) 305-309 [14]). These cells provide a setting for the study of cholinergic neuronal differentiation and of the factors that influence that process. We also discuss procedures that can be used to study gene expression in LA-N-5 cells by immunohistochemistry and reporter gene analysis.

Initial healing rates of venous ulcers: are they useful as predictors of healing?

Clinical trials that follow venous ulcers to complete healing can be costly because of the prolonged healing time involved. Initial healing rates in venous ulcers are calculated by 2 methods, which are based on a metric using wound area and perimeter. It has been proposed that these rates allow the prediction of complete healing and that they may be useful as surrogate end points for clinical trials. The objective of this study was to compare the 2 proposed methods for calculating initial healing rates and determine their usefulness in predicting the healing of venous ulcers. Venous leg ulcers from patients enrolled in a randomized, double-blind, placebo-controlled study were measured weekly for up to 12 weeks. Their healing status was determined for up to 24 weeks. Initial healing rates were calculated using the 2 proposed methods. The ability of these rates to predict time to complete healing was assessed. Information from 17 patients was available. The initial healing rates, calculated by either method, were quite similar; both methods produced the same median value of 0.046 cm/week in our patients. Five of the patients had negative initial healing rates, which do not allow any prediction of a healing time. Three of 7 patients predicted to heal within 24 weeks failed to do so. One of the 5 patients was predicted to heal at some time after 24 weeks but actually healed within 24 weeks. None of the 5 patients with negative initial healing rates healed within 24 weeks. Initial healing rates, as calculated by either method, have limited utility in describing healing curves and predicting a healing time. This poor predictive ability argues against using these initial healing rates as surrogate end points for clinical trials. The great variability observed in venous ulcer healing curves may limit the development of useful predictive models in this patient population.

Long-term outcome study of growth factor-treated pressure ulcers.

BACKGROUND: Exogenous application of growth factors have been reported in an attempt to accelerate healing of chronic wounds. Most of the trials were of brief duration with short to no follow-up periods. Long-term outcome studies are sparse for pressure ulcer therapies with success rates around 30% for both operative and nonoperative treatments. METHODS: Follow-up evaluations were performed serially up to 12 months for patients completing a 35 day blinded, placebo-controlled cytokine clinical trial of pressure ulcers. RESULTS: Fifty-four of 61 patients completed the follow-up period with 68.5% of the patients (37 of 54) being healed after 1 year. Of patients healing > or =85% during the active treatment phase, 84.6% were healed after 1 year compared with 61% of those that healed <85% during treatment (P <0.05). CONCLUSION: Long-term outcome was better in this growth factor trial than with surgical or standard nonoperative treatment of pressure ulcers. Since only patients receiving exogenously applied cytokines achieved >85% closure during the treatment phase of the trial, the excellent long-term outcome appears attributable to the cytokine therapy.

Autologous fat injection for vocal cord medialization in the canine larynx.

This study examined the use of autologous fat as an alternative to Teflon and collagen as the implantable material in vocal cord medialization. Five animals underwent left recurrent laryngeal nerve sections with subsequent fat harvest and implantation into the left true vocal cords. Three animals were killed after 48 hours and 2 after 3 weeks; their larynges were examined with light microscopy. The results of the 48-hour samples show mode-rate acute inflammation and few areas of focal necrosis. The 3-week samples show no necrotic foci, minimal foreign-body reaction, and maintenance of structure and volume of the injected fat. Autologous fat may prove to be a valuable alternative to nonautologous injectable material in vocal cord augmentation.

Necrotizing vasculitis in gynecological surgery.

Extensive clinical investigation of asymptomatic vasculitis found in routine histopathological studies following gynecological surgery may often be unnecessary. In 11 patients necrotizing angiitis was an incidental finding in surgically removed uterine and uterine adnexal tissues. Following irradiation for urinary bladder carcinoma, one patient died during radical surgery: vasculitis was found in surgically resected pelvic organs, colon and kidneys. In other patients, vasculitis may have been attributable to drugs, e.g. penicillin, or to local arthus-like reaction following cone biopsy of cervix. Available from 6 of these cases, follow-up data presented no evidence of progressive systemic vascular disease, supporting the view that incidentally encountered necrotizing angiitis of uterus and uterine adnexa, like that of the appendix, may be innocuous.

The efficacy of 5% Sulfamylon solution for the treatment of contaminated explanted human meshed skin grafts.

Large TBSA burns have a deficiency of skin graft donor sites necessitating meshed skin autografts, cultured epithelial autografts or biosynthetic skin substitutes. Because these do not effect immediate complete biological closure of the wound, the burn victim remains at risk for life-threatening infection. Topical antimicrobials can protect colonization of these grafts from becoming invasive sepsis. However, many of these agents are cytotoxic to new partially keratinized epithelial cells. This study using a model of epithelialization kinetics of human meshed skin grafts explanted to athymic 'nude' rats evaluated: (1) the effect of bacterial colonization on the rate of closure of meshed graft interstices; (2) the efficacy of 5% Sulfamylon solution for bacterial control and (3) the effect on interstitial closure rates caused by control of bacterial proliferation. Results showed the rate of interstitial closure was progressive over 7 days in noncontaminated grafts treated with moistened saline dressings. Areas of total closure of a 1:1.5 meshed graft were seen as early as 5 days. When grafts were inoculated with 10(2) or 10(3) Pseudomonas aeruginosa organisms and treated with saline moistened dressings, the resultant bacterial load rose to 10(6) organisms, less than 3% of the interstices closed and grafts were destroyed. With the same organism level of contamination, bacterial levels were eradicated with topical 5% Sulfamylon solution, interstitial closure rates returned to normal and areas of total meshed graft closure were seen by day 4. These data demonstrate the efficacy of 5% Sulfamylon solution on epithelialization kinetics of contaminated meshed skin grafts.