As part of a large-scale mutagenesis screen of the zebrafish genome, we have identified 58 mutations that affect the formation and function of the cardiovascular system. The cardiovascular system is ...particularly amenable for screening in the transparent zebrafish embryo because the heart and blood vessels are prominent and their function easily examined. We have classified the mutations affecting the heart into those that affect primarily either morphogenesis or function. Nine mutations clearly disrupt the formation of the heart. cloche deletes the endocardium. In cloche mutants, the myocardial layer forms in the absence of the endocardium but is dysmorphic and exhibits a weak contractility. Two loci, miles apart and bonnie and clyde, play a critical role in the fusion of the bilateral tubular primordia. Three mutations lead to an abnormally large heart and one to the formation of a diminutive, dysmorphic heart. We have found no mutation that deletes the myocardial cells altogether, but one, pandora, appears to eliminate the ventricle selectively. Seven mutations interfere with vascular integrity, as indicated by hemorrhage at particular sites. In terms of cardiac function, one large group exhibits a weak beat. In this group, five loci affect both chambers and seven a specific chamber (the atrium or ventricle). For example, the weak atrium mutation exhibits an atrium that becomes silent but has a normally beating ventricle. Seven mutations affect the rhythm of the heart causing, for example, a slow rate, a fibrillating pattern or an apparent block to conduction. In several other mutants, regurgitation of blood flow from ventricle to atrium is the most prominent abnormality, due either to the absence of valves or to poor coordination between the chambers with regard to the timing of contraction. The mutations identified in this screen point to discrete and critical steps in the formation and function of the heart and vasculature.
A lumbar radiculopathy model investigated pain behavioral responses after nerve root reinjury.
To gain a further understanding of central sensitization and neuroinflammation associated with chronic ...lumbar radiculopathy after repeated nerve root injury.
The pathophysiologic mechanisms associated with chronic radicular pain remain obscure. It has been hypothesized that lumbar root injury produces neuroimmunologic and neurochemical changes, sensitizing the spinal cord and causing pain responses to manifest with greater intensity and longer duration after reinjury. However, this remains untested experimentally.
Male Holtzman rats were divided into two groups: a sham group having only nerve root exposure, and a chromic group in which the nerve root was ligated loosely with chromic gut suture. Animals underwent a second procedure at 42 days. The chromic group was further divided into a reinjury group and a chromic-sham group, in which the lumbar roots were only re-exposed. Bilateral mechanical allodynia was continuously assessed throughout the study. Qualitative assessment of spinal cord glial activation and IL-beta expression was performed.
Mechanical allodynia was significantly greater on both the ipsilateral and contralateral sides after reinjury (P < 0.001), and the response did not return to baseline after reinjury, as it did with the initial injury. There were also persistent spinal astrocytic and microglial activation and interleukin-1beta expression.
The bilateral responses support central modulation of radicular pain after nerve root injury. An exaggerated and more prolonged response bilaterally after reinjury suggests central sensitization after initial injury. Neuroinflammatory activation in the spinal cord further supports the hypothesis that central neuroinflammation plays an important role in chronic radicular pain.
We have recently reported that injury to a lumbar root in a rat model of radiculopathy produces spinal glial activation associated with elevated proinflammatory cytokines. Based on our hypothesis ...that central neuroinflammatory processes may manifest clinically as radicular pain, we undertook pharmacological intervention using the immunosuppressive agent methotrexate (MTX). The L5 lumbar spinal root (central to the dorsal root ganglia) was exposed unilaterally and loosely constricted with chromic gut. In the prevention (phase I) study, MTX was administered intrathecally (1 mg/kg) and around the spinal root (1 mg/kg) at surgery and at days 2 and 4 postsurgery (group A). Saline injection was employed for the control group (group B). Sham operated animals were administered MTX to determine the potential for behavioral/neural side effects (group C). In the existing pain paradigm (phase II) study, the experiment was extended to day 14 with three additional groups. The same dose and method of delivery of MTX or saline was administered as in phase I in the first week on days 0, 2, and 4 and in the second week on days 7, 9, and 11 postsurgery. To measure the effects of MTX on existing behaviors saline was administered in the first week and MTX during the second (group D; Saline:MTX). The control group received saline during both weeks (group E; Saline:Saline). To examine the possible recurrence of radicular pain after MTX termination, MTX was given in the first week and saline in the second (group F; MTX:Saline). Gait disturbance and mechanical allodynia (using von Frey filaments) were assessed up to day 7 in the prevention study (Phase I) and day 14 in the existing pain paradigm (Phase II). The L5 spinal cord segments were harvested for assessment of immunohistochemical glial activation using the antibodies OX-42 (microglial marker) and glial fibrillary acidic protein (GFAP: astrocytic marker) and for the presence of Major Histocompatibility Complex (MHC) Class II expression. Group C (Sham+MTX) did not demonstrate any evidence of gait disturbance or mechanical allodynia after MTX administration. The rats in group B (Surgery+Saline) demonstrated mechanical allodynia from one day postsurgery to the time of euthanization. When allodynia was assessed using the 12 g von Frey filament, the MTX treated rats in group A showed significantly decreased mechanical allodynia as compared to the saline treated rats (group B) (repeated measured ANOVA, P<0.0001). In the phase II study, the rats in group D (Saline:MTX) and E (Saline:Saline) showed robust allodynia in the first week after the surgery. In the second week, mechanical allodynia significantly decreased in group D, while mechanical allodynia continued in the saline treated group (repeated measured ANOVA, P=0.0121). Allodynia was significantly attenuated in group F (MTX: Saline) as compared to the response in groups D and E at day 7 (one-way ANOVA, P<0.0001) and remained significantly lower as compared to group E up to day 11 postsurgery (one-way ANOVA, P9=0. 0013: P11=0.0048). OX-42 and GFAP expression were elevated in the gray matter of the L5 spinal section in all groups that underwent the root ligature with chromic gut (Groups A, B, D-F). There were no significant differences in glial activation between the groups. However, spinal expression of MHC II was markedly reduced in the MTX treated group as compared with the saline treated group. The exact mechanism of action of MTX in attenuating mechanical allodynia has not yet been elucidated. The present results indicate that MTX administration may offer a new treatment modality for radicular pain with or without disc herniation as well as directing new research into the development of novel immunomodulators for the treatment of chronic neuropathic and radicular pain.
Hydroxyurea, a drug widely used in therapy of several human diseases, inhibits deoxynucleotide synthesis-and, consequently, DNA synthesis-by blocking the cellular enzyme ribonucleotide reductase. ...Hydroxyurea inhibits human immunodeficiency virus-type 1 (HIV-1) DNA synthesis in activated peripheral blood lymphocytes by decreasing the amount of intracellular deoxynucleotides, thus suggesting that this drug has an antiviral effect. Hydroxyurea has now been shown to block HIV-1 replication in acutely infected primary human lymphocytes (quiescent and activated) and macrophages, as well as in blood cells infected in vivo obtained from individuals with acquired immunodeficiency syndrome (AIDS). The antiviral effect was achieved at nontoxic doses of hydroxyurea, lower than those currently used in human therapy. Combination of hydroxyurea with the nucleoside analog didanosine (2',3'-dideoxyinosine, or ddl) generated a synergistic inhibitory effect without increasing toxicity. In some instances, inhibition of HIV-1 by hydroxyurea was irreversible, even several weeks after suspension of drug treatment. The indirect inhibition of HIV-1 by hydroxyurea is not expected to generate high rates of escape mutants. Hydroxyurea therefore appears to be a possible candidate for AIDS therapy.
The Spine Patient Outcomes Research Trial (SPORT) was designed to assess the relative efficacy and cost-effectiveness of surgical and nonsurgical approaches to the treatment of common conditions ...associated with low back and leg pain.
To describe the rationale and design of the SPORT project and to discuss its strengths and limitations.
Descriptive.
First, the authors explain the rationale for embarking on SPORT, i.e., deficiencies in the existing scientific knowledge base for treatment of these conditions. Second, the authors describe the design of SPORT, including topics such as specific aims, participating sites, study population, recruitment and enrollment, study interventions, follow-up, outcomes, statistical analysis, and study governance and organization. Finally, issues that complicate the performance of randomized trials in surgery as they relate to the design and conduct of SPORT are discussed.
The SPORT project is being conducted at 11 clinical centers around the United States. It involves the simultaneous conduct of three multicenter, randomized, controlled clinical trials. The study includes patients with the three most common diagnoses for which spine surgery is performed: intervertebral disc herniation, spinal stenosis, and degenerative spondylolisthesis, and it compares the most commonly used standard surgical and nonsurgical treatments for patients with these diagnoses. By the end of enrollment the authors anticipate a total of 500 patients with intervertebral disc herniation, 370 patients with spinal stenosis, and 300 patients with degenerative spondylolisthesis in the randomized trials. Patients who meet the eligibility criteria but decline to be randomized are invited to participate in an observational cohort study. Patients are being followed for a minimum of 24 months with visits scheduled at 6 weeks and at 3, 6, 12, and 24 months.
The results of this study will provide high-quality scientific evidence to aid clinical decision-making and improve treatment outcomes for these common, costly, and, in some instances, debilitating conditions.
Background:
Topical therapy providing initial improvement and maintenance of effect after treatment of the large majority of patients with limited, mild to moderate psoriasis is not presently ...available. Previous topical retinoids have generally been either ineffective or too irritating for therapy of psoriasis.
Objective:
Our purpose was to evaluate a new topical retinoid, tazarotene, in the treatment of stable plaque psoriasis during treatment and posttreatment periods.
Methods:
In a double-blind manner, 324 patients were randomly selected to receive tazarotene 0.1% or 0.05% gel, or vehicle control, once daily for 12 weeks and were then followed up for 12 weeks after treatment.
Results:
Of the total, 318 patients could be evaluated. Tazarotene gels were superior (
p < 0.05) to vehicle, often as early as treatment week 1, in all efficacy measures: plaque elevation, scaling, and erythema; treatment response; percentage treatment success (patients with ≥50% improvement); and time to initial success. Efficacy was equivalent on target lesion sites (trunk or limbs and knees or elbows) and overall. A sustained therapeutic effect was observed for 12 weeks after treatment. Tazarotene gel was cosmetically acceptable. There was low systemic absorption, limiting toxicity to local irritation.
Conclusion:
Once-daily tazarotene was effective and safe as a topical monotherapy for plaque psoriasis, providing rapid reduction of signs and symptoms. (J Am Acad Dermatol 1997;37:85–92.)
Membrane transporters and channels (collectively the transportome) govern cellular influx and efflux of ions, nutrients, and drugs. We used oligonucleotide arrays to analyze gene expression of the ...transportome in 60 human cancer cell lines used by the National Cancer Institute for drug screening. Correlating gene expression with the potencies of 119 standard anticancer drugs identified known drug-transporter interactions and suggested novel ones. Folate, nucleoside, and amino acid transporters positively correlated with chemosensitivity to their respective drug substrates. We validated the positive correlation between SLC29A1 (nucleoside transporter ENT1) expression and potency of nucleoside analogues, azacytidine and inosine-glycodialdehyde. Application of an inhibitor of SLC29A1, nitrobenzylmercaptopurine ribonucleoside, significantly reduced the potency of these two drugs, indicating that SLC29A1 plays a role in cellular uptake. Three ABC efflux transporters (ABCB1, ABCC3, and ABCB5) showed significant negative correlations with multiple drugs, suggesting a mechanism of drug resistance. ABCB1 expression correlated negatively with potencies of 19 known ABCB1 substrates and with Baker's antifol and geldanamycin. Use of RNA interference reduced ABCB1 mRNA levels and concomitantly increased sensitivity to these two drugs, as expected for ABCB1 substrates. Similarly, specific silencing of ABCB5 by small interfering RNA increased sensitivity to several drugs in melanoma cells, implicating ABCB5 as a novel chemoresistance factor. Ion exchangers, ion channels, and subunits of proton and sodium pumps variably correlated with drug potency. This study identifies numerous potential drug-transporter relationships and supports a prominent role for membrane transport in determining chemosensitivity. Measurement of transporter gene expression may prove useful in predicting anticancer drug response.
With the advent of high-throughput sequencing technologies, much progress has been made in the identification of somatic structural rearrangements in cancer genomes. However, characterization of the ...complex alterations and their associated mechanisms remains inadequate. Here, we report a comprehensive analysis of whole-genome sequencing and DNA copy number data sets from The Cancer Genome Atlas to relate chromosomal alterations to imbalances in DNA dosage and describe the landscape of intragenic breakpoints in glioblastoma multiforme (GBM). Gene length, guanine-cytosine (GC) content, and local presence of a copy number alteration were closely associated with breakpoint susceptibility. A dense pattern of repeated focal amplifications involving the murine double minute 2 (MDM2)/cyclin-dependent kinase 4 (CDK4) oncogenes and associated with poor survival was identified in 5% of GBMs. Gene fusions and rearrangements were detected concomitant within the breakpoint-enriched region. At the gene level, we noted recurrent breakpoints in genes such as apoptosis regulator FAF1. Structural alterations of the FAF1 gene disrupted expression and led to protein depletion. Restoration of the FAF1 protein in glioma cell lines significantly increased the FAS-mediated apoptosis response. Our study uncovered a previously underappreciated genomic mechanism of gene deregulation that can confer growth advantages on tumor cells and may generate cancer-specific vulnerabilities in subsets of GBM.
We previously developed GoMiner, an application that organizes lists of 'interesting' genes (for example, under-and overexpressed genes from a microarray experiment) for biological interpretation in ...the context of the Gene Ontology. The original version of GoMiner was oriented toward visualization and interpretation of the results from a single microarray (or other high-throughput experimental platform), using a graphical user interface. Although that version can be used to examine the results from a number of microarrays one at a time, that is a rather tedious task, and original GoMiner includes no apparatus for obtaining a global picture of results from an experiment that consists of multiple microarrays. We wanted to provide a computational resource that automates the analysis of multiple microarrays and then integrates the results across all of them in useful exportable output files and visualizations.
We now introduce a new tool, High-Throughput GoMiner, that has those capabilities and a number of others: It (i) efficiently performs the computationally-intensive task of automated batch processing of an arbitrary number of microarrays, (ii) produces a human-or computer-readable report that rank-orders the multiple microarray results according to the number of significant GO categories, (iii) integrates the multiple microarray results by providing organized, global clustered image map visualizations of the relationships of significant GO categories, (iv) provides a fast form of 'false discovery rate' multiple comparisons calculation, and (v) provides annotations and visualizations for relating transcription factor binding sites to genes and GO categories.
High-Throughput GoMiner achieves the desired goal of providing a computational resource that automates the analysis of multiple microarrays and integrates results across all of the microarrays. For illustration, we show an application of this new tool to the interpretation of altered gene expression patterns in Common Variable Immune Deficiency (CVID). High-Throughput GoMiner will be useful in a wide range of applications, including the study of time-courses, evaluation of multiple drug treatments, comparison of multiple gene knock-outs or knock-downs, and screening of large numbers of chemical derivatives generated from a promising lead compound.
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