Large, living biological specimens present challenges to existing optical imaging techniques because of their absorptive and scattering properties. We developed selective plane illumination microscopy (SPIM) to generate multidimensional images of samples up to a few millimeters in size. The system combines two-dimensional illumination with orthogonal camera-based detection to achieve high-resolution, optically sectioned imaging throughout the sample, with minimal photodamage and at speeds capable of capturing transient biological phenomena. We used SPIM to visualize all muscles in vivo in the transgenic Medaka line Arnie, which expresses green fluorescent protein in muscle tissue. We also demonstrate that SPIM can be applied to visualize the embryogenesis of the relatively opaque Drosophila melanogaster in vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections that are becoming increasingly difficult to combat because of emerging resistance to all current antibiotic classes. The evolutionary origins of MRSA are poorly understood, no rational nomenclature exists, and there is no consensus on the number of major MRSA clones or the relatedness of clones described from different countries. We resolve all of these issues and provide a more thorough and precise analysis of the evolution of MRSA clones than has previously been possible. Using multilocus sequence typing and an algorithm, BURST, we analyzed an international collection of 912 MRSA and methicillin-susceptible S. aureus (MSSA) isolates. We identified 11 major MRSA clones within five groups of related genotypes. The putative ancestral genotype of each group and the most parsimonious patterns of descent of isolates from each ancestor were inferred by using BURST, which, together with analysis of the methicillin resistance genes, established the likely evolutionary origins of each major MRSA clone, the genotype of the original MRSA clone and its MSSA progenitor, and the extent of acquisition and horizontal movement of the methicillin resistance genes. Major MRSA clones have arisen repeatedly from successful epidemic MSSA strains, and isolates with decreased susceptibility to vancomycin, the antibiotic of last resort, are arising from some of these major MRSA clones, highlighting a depressing progression of increasing drug resistance within a small number of ecologically successful S. aureus genotypes.

With the availability of a dense genome-wide map of single nucleotide polymorphisms (SNPs), a central issue in human genetics is whether it is now possible to use linkage disequilibrium (LD) to map genes that cause disease. LD refers to correlations among neighbouring alleles, reflecting 'haplotypes' descended from single, ancestral chromosomes. The size of LD blocks has been the subject of considerable debate. Computer simulations and empirical data have suggested that LD extends only a few kilobases (kb) around common SNPs, whereas other data have suggested that it can extend much further, in some cases greater than 100 kb. It has been difficult to obtain a systematic picture of LD because past studies have been based on only a few (1-3) loci and different populations. Here, we report a large-scale experiment using a uniform protocol to examine 19 randomly selected genomic regions. LD in a United States population of north-European descent typically extends 60 kb from common alleles, implying that LD mapping is likely to be practical in this population. By contrast, LD in a Nigerian population extends markedly less far. The results illuminate human history, suggesting that LD in northern Europeans is shaped by a marked demographic event about 27,000-53,000 years ago.

Soil salinity is a major abiotic stress in plant agriculture worldwide. This has led to research into salt tolerance with the aim of improving crop plants. However, salt tolerance might have much wider implications because transgenic salt-tolerant plants often also tolerate other stresses including chilling, freezing, heat and drought. Unfortunately, suitable genetic model systems have been hard to find. A recently discovered halophytic plant species, Thellungiella halophila, now promises to help in the detection of new tolerance determinants and operating pathways in a model system that is not limited to Arabidopsis traits or ecotype variations.

The ligands for programmed cell death 1 (PD-1), an immunoinhibitory receptor belonging to CD28/cytotoxic T lymphocyte antigen 4 family, are PD-1 ligand 1 and 2 (PD-Ls). Recent reports suggest that the aberrant expression of PD-Ls on tumor cells impairs antitumor immunity, resulting in the immune evasion of the tumor cells. Although an inverse correlation between the expression level of PD-Ls and patients' prognosis has been reported for several malignant tumors, the follow-up period was limited because of the lack of the antibody (Ab) applicable to paraffin-embedded specimens. Here we generated a new Ab against PD-1 ligand 1 (PD-L1) and analyzed the expression level of PD-Ls in human ovarian cancer using paraffin-embedded specimens. Patients with higher expression of PD-L1 had a significantly poorer prognosis than patients with lower expression. Although patients with higher expression of PD-1 ligand 2 also had a poorer prognosis, the difference was not statistically significant. A significant inverse correlation was observed between PD-L1 expression and the intraepithelial CD8(+) T lymphocyte count, suggesting that PD-L1 on tumor cells directly suppresses antitumor CD8(+) T cells. Multivariate analysis showed the expression of PD-L1 on tumor cells and intraepithelial CD8(+) T lymphocyte count are independent prognostic factors. The PD-1/PD-L pathway can be a good target for restoring antitumor immunity in ovarian cancer.

SMN1 and SMN2 (survival motor neuron) encode identical proteins. A critical question is why only the homozygous loss of SMN1, and not SMN2, results in spinal muscular atrophy (SMA). Analysis of transcripts from SMN1/SMN2 hybrid genes and a new SMN1 mutation showed a direct relationship between presence of disease and exon 7 skipping. We have reported previously that the exon-skipped product SMNDelta7 is partially defective for self-association and SMN self-oligomerization correlated with clinical severity. To evaluate systematically which of the five nucleotides that differ between SMN1 and SMN2 effect alternative splicing of exon 7, a series of SMN minigenes was engineered and transfected into cultured cells, and their transcripts were characterized. Of these nucleotide differences, the exon 7 C-to-T transition at codon 280, a translationally silent variance, was necessary and sufficient to dictate exon 7 alternative splicing. Thus, the failure of SMN2 to fully compensate for SMN1 and protect from SMA is due to a nucleotide exchange (C/T) that attenuates activity of an exonic enhancer. These findings demonstrate the molecular genetic basis for the nature and pathogenesis of SMA and illustrate a novel disease mechanism. Because individuals with SMA retain the SMN2 allele, therapy targeted at preventing exon 7 skipping could modify clinical outcome.

To identify selective steps in metastasis, those that eliminate nonmetastatic tumor cells more efficiently than metastatic cells, we have evaluated the sequential dissemination of tumor cells from a mammary fatpad, using both metastatic (4T1 and 66cl4) and nonmetastatic (67NR, 168FARN, and 4TO7) subpopulations of a single mouse mammary tumor. Each of these variant subpopulations is resistant to one or more selective drugs so they could be quantitatively identified by colony formation in selective media. We found that the 2 metastatic cell lines metastasized by different routes and that the nonmetastatic tumor cell lines failed at different points in dissemination. Line 67NR did not leave the primary site; clonogenic tumor cells were not detected in the nodes, blood, or lungs during the experiment (7 weeks). Tumor line 168FARN disseminated from the primary tumor because clonogenic cells were cultured from the draining lymph nodes throughout the experiment. However, dissemination essentially stopped in the node as cells were rarely isolated from blood, lungs, or lives. Whether 168FARN cells failed to reach these tissues or were killed very rapidly after traversing the lymph node is unknown. Line 4TO7 cells disseminated via the blood and were consistently recovered from lungs by day 19 but failed to proliferate. This panel of 5 subpopulations thus identifies different points of selective failure in tumor cell dissemination and should be valuable in the assessment of antimetastatic therapies.

3-Methyladenine (5 mM) inhibits endogenous protein degradation in isolated rat hepatocytes by about 60%, while having no adverse effect on the degradation of an exogenous protein (asialofetuin), on protein synthesis, or on intracellular ATP levels. 3-Methyladenine appears to act specifically upon the autophagic/lysosomal pathway of degradation, as judged from its lack of effect in the presence of amino acids or a lysosomotropic amine (propylamine). The effect of the purine is not mediated by amino acids because the inhibition of protein degradation is accompanied by a significant depression of intracellular amino acid levels. The ability of 3-methyladenine to suppress the formation of electron microscopically visible autophagosomes suggests that it may be regarded as a specific inhibitor of autophagy.

World wheat grain yields increased substantially in the 1960s and 1970s because farmers rapidly adopted the new varieties and cultivation methods of the so-called 'green revolution'. The new varieties are shorter, increase grain yield at the expense of straw biomass, and are more resistant to damage by wind and rain. These wheats are short because they respond abnormally to the plant growth hormone gibberellin. This reduced response to gibberellin is conferred by mutant dwarfing alleles at one of two Reduced height-1 (Rht-B1 and Rht-D1) loci. Here we show that Rht-B1/Rht-D1 and maize dwarf-8 (d8) are orthologues of the Arabidopsis Gibberellin Insensitive (GAI) gene. These genes encode proteins that resemble nuclear transcription factors and contain an SH2-like domain, indicating that phosphotyrosine may participate in gibberellin signalling. Six different orthologous dwarfing mutant alleles encode proteins that are altered in a conserved amino-terminal gibberellin signalling domain. Transgenic rice plants containing a mutant GAI allele give reduced responses to gibberellin and are dwarfed, indicating that mutant GAI orthologues could be used to increase yield in a wide range of crop species.

Eye movements, eye blinks, cardiac signals, muscle noise, and line noise present serious problems for electroencephalographic (EEG) interpretation and analysis when rejecting contaminated EEG segments results in an unacceptable data loss. Many methods have been proposed to remove artifacts from EEG recordings, especially those arising from eye movements and blinks. Often regression in the time or frequency domain is performed on parallel EEG and electrooculographic (EOG) recordings to derive parameters characterizing the appearance and spread of EOG artifacts in the EEG channels. Because EEG and ocular activity mix bidirectionally, regressing out eye artifacts inevitably involves subtracting relevant EEG signals from each record as well. Regression methods become even more problematic when a good regressing channel is not available for each artifact source, as in the case of muscle artifacts. Use of principal component analysis (PCA) has been proposed to remove eye artifacts from multichannel EEG. However, PCA cannot completely separate eye artifacts from brain signals, especially when they have comparable amplitudes. Here, we propose a new and generally applicable method for removing a wide variety of artifacts from EEG records based on blind source separation by independent component analysis (ICA). Our results on EEG data collected from normal and autistic subjects show that ICA can effectively detect, separate, and remove contamination from a wide variety of artifactual sources in EEG records with results comparing favorably with those obtained using regression and PCA methods. ICA can also be used to analyze blink-related brain activity.

The precise role played by HIV-specific cytotoxic T lymphocytes (CTL) in HIV infection remains controversial. Despite strong CTL responses being generated during the asymptomatic phase, the virus persists and AIDS ultimately develops. It has been argued that the virus is so variable, and the virus turnover so great that escape from CTL recognition would occur continually, but so far there is limited evidence for CTL escape. The opposing argument is that evidence for CTL escape is present but hard to find because multiple anti-HIV immune responses are acting simultaneously during the asymptomatic phase of infection. We describe six donors who make a strong CTL response to an immunodominant HLA-B27-restricted epitope. In the two donors who progressed to AIDS, CTL escape to fixation by the same mutation was observed, but only after 9-12 years of epitope stability. CTL escape may play an important role in the pathogenesis of HIV infection.

Eosinophilic esophagitis (EoE) is a clinicopathologic condition of increasing recognition and prevalence. In 2007, a consensus recommendation provided clinical and histopathologic guidance for the diagnosis and treatment of EoE; however, only a minority of physicians use the 2007 guidelines, which require fulfillment of both histologic and clinical features. Since 2007, the number of EoE publications has doubled, providing new disease insight. Accordingly, a panel of 33 physicians with expertise in pediatric and adult allergy/immunology, gastroenterology, and pathology conducted a systematic review of the EoE literature (since September 2006) using electronic databases. Based on the literature review and expertise of the panel, information and recommendations were provided in each of the following areas of EoE: diagnostics, genetics, allergy testing, therapeutics, and disease complications. Because accumulating animal and human data have provided evidence that EoE appears to be an antigen-driven immunologic process that involves multiple pathogenic pathways, a new conceptual definition is proposed highlighting that EoE represents a chronic, immune/antigen-mediated disease characterized clinically by symptoms related to esophageal dysfunction and histologically by eosinophil-predominant inflammation. The diagnostic guidelines continue to define EoE as an isolated chronic disorder of the esophagus diagnosed by the need of both clinical and pathologic features. Patients commonly have high rates of concurrent allergic diatheses, especially food sensitization, compared with the general population. Proved therapeutic options include chronic dietary elimination, topical corticosteroids, and esophageal dilation. Important additions since 2007 include genetic underpinnings that implicate EoE susceptibility caused by polymorphisms in the thymic stromal lymphopoietin protein gene and the description of a new potential disease phenotype, proton pump inhibitor-responsive esophageal eosinophila. Further advances and controversies regarding diagnostic methods, surrogate disease markers, allergy testing, and treatment approaches are discussed.

BACKGROUND

Acute aortic dissection is a life-threatening medical emergency associated with high rates of morbidity and mortality. Data are limited regarding the effect of recent imaging and therapeutic advances on patient care and outcomes in this setting.

OBJECTIVE

To assess the presentation, management, and outcomes of acute aortic dissection.

METHODS

Case series with patients enrolled between January 1996 and December 1998. Data were collected at presentation and by physician review of hospital records.

METHODS

The International Registry of Acute Aortic Dissection, consisting of 12 international referral centers.

METHODS

A total of 464 patients (mean age, 63 years; 65.3% male), 62.3% of whom had type A dissection.

METHODS

Presenting history, physical findings, management, and mortality, as assessed by history and physician review of hospital records.

RESULTS

While sudden onset of severe sharp pain was the single most common presenting complaint, the clinical presentation was diverse. Classic physical findings such as aortic regurgitation and pulse deficit were noted in only 31.6% and 15.1% of patients, respectively, and initial chest radiograph and electrocardiogram were frequently not helpful (no abnormalities were noted in 12.4% and 31.3% of patients, respectively). Computed tomography was the initial imaging modality used in 61.1%. Overall in-hospital mortality was 27.4%. Mortality of patients with type A dissection managed surgically was 26%; among those not receiving surgery (typically because of advanced age and comorbidity), mortality was 58%. Mortality of patients with type B dissection treated medically was 10.7%. Surgery was performed in 20% of patients with type B dissection; mortality in this group was 31.4%.

CONCLUSIONS

Acute aortic dissection presents with a wide range of manifestations, and classic findings are often absent. A high clinical index of suspicion is necessary. Despite recent advances, in-hospital mortality rates remain high. Our data support the need for continued improvement in prevention, diagnosis, and management of acute aortic dissection.

Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors. However, such iPS cells contain a large number of viral vector integrations, any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming, complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia. Two transient transfection-reprogramming methods have been published to address this issue. However, the efficiency of both approaches is extremely low, and neither has been applied successfully to human cells so far. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. When the single-vector reprogramming system was combined with a piggyBac transposon, we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models.

BACKGROUND

The causes of intellectual disability remain largely unknown because of extensive clinical and genetic heterogeneity.

METHODS

We evaluated patients with intellectual disability to exclude known causes of the disorder. We then sequenced the coding regions of more than 21,000 genes obtained from 100 patients with an IQ below 50 and their unaffected parents. A data-analysis procedure was developed to identify and classify de novo, autosomal recessive, and X-linked mutations. In addition, we used high-throughput resequencing to confirm new candidate genes in 765 persons with intellectual disability (a confirmation series). All mutations were evaluated by molecular geneticists and clinicians in the context of the patients' clinical presentation.

RESULTS

We identified 79 de novo mutations in 53 of 100 patients. A total of 10 de novo mutations and 3 X-linked (maternally inherited) mutations that had been previously predicted to compromise the function of known intellectual-disability genes were found in 13 patients. Potentially causative de novo mutations in novel candidate genes were detected in 22 patients. Additional de novo mutations in 3 of these candidate genes were identified in patients with similar phenotypes in the confirmation series, providing support for mutations in these genes as the cause of intellectual disability. We detected no causative autosomal recessive inherited mutations in the discovery series. Thus, the total diagnostic yield was 16%, mostly involving de novo mutations.

CONCLUSIONS

De novo mutations represent an important cause of intellectual disability; exome sequencing was used as an effective diagnostic strategy for their detection. (Funded by the European Union and others.).

We reviewed evidence of the clinical implications and burden of malaria in pregnancy. Most studies come from sub-Saharan Africa, where approximately 25 million pregnant women are at risk of Plasmodium falciparum infection every year, and one in four women have evidence of placental infection at the time of delivery. P falciparum infections during pregnancy in Africa rarely result in fever and therefore remain undetected and untreated. Meta-analyses of intervention trials suggest that successful prevention of these infections reduces the risk of severe maternal anaemia by 38%, low birthweight by 43%, and perinatal mortality by 27% among paucigravidae. Low birthweight associated with malaria in pregnancy is estimated to result in 100,000 infant deaths in Africa each year. Although paucigravidae are most affected by malaria, the consequences for infants born to multigravid women in Africa may be greater than previously appreciated. This is because HIV increases the risk of malaria and its adverse effects, particularly in multigravidae, and recent observational studies show that placental infection almost doubles the risk of malaria infection and morbidity in infants born to multigravidae. Outside Africa, malaria infection rates in pregnant women are much lower but are more likely to cause severe disease, preterm births, and fetal loss. Plasmodium vivax is common in Asia and the Americas and, unlike P falciparum, does not cytoadhere in the placenta, yet, is associated with maternal anaemia and low birthweight. The effect of infection in the first trimester, and the longer term effects of malaria beyond infancy, are largely unknown and may be substantial. Better estimates are also needed of the effects of malaria in pregnancy outside Africa, and on maternal morbidity and mortality in Africa. Global risk maps will allow better estimation of potential impact of successful control of malaria in pregnancy.

Glioblastomas are lethal cancers characterized by florid angiogenesis promoted in part by glioma stem cells (GSCs). Because hypoxia regulates angiogenesis, we examined hypoxic responses in GSCs. We now demonstrate that hypoxia-inducible factor HIF2alpha and multiple HIF-regulated genes are preferentially expressed in GSCs in comparison to non-stem tumor cells and normal neural progenitors. In tumor specimens, HIF2alpha colocalizes with cancer stem cell markers. Targeting HIFs in GSCs inhibits self-renewal, proliferation, and survival in vitro, and attenuates tumor initiation potential of GSCs in vivo. Analysis of a molecular database reveals that HIF2A expression correlates with poor glioma patient survival. Our results demonstrate that GSCs differentially respond to hypoxia with distinct HIF induction patterns, and HIF2alpha might represent a promising target for antiglioblastoma therapies.

Synchronous rhythms represent a core mechanism for sculpting temporal coordination of neural activity in the brain-wide network. This review focuses on oscillations in the cerebral cortex that occur during cognition, in alert behaving conditions. Over the last two decades, experimental and modeling work has made great strides in elucidating the detailed cellular and circuit basis of these rhythms, particularly gamma and theta rhythms. The underlying physiological mechanisms are diverse (ranging from resonance and pacemaker properties of single cells to multiple scenarios for population synchronization and wave propagation), but also exhibit unifying principles. A major conceptual advance was the realization that synaptic inhibition plays a fundamental role in rhythmogenesis, either in an interneuronal network or in a reciprocal excitatory-inhibitory loop. Computational functions of synchronous oscillations in cognition are still a matter of debate among systems neuroscientists, in part because the notion of regular oscillation seems to contradict the common observation that spiking discharges of individual neurons in the cortex are highly stochastic and far from being clocklike. However, recent findings have led to a framework that goes beyond the conventional theory of coupled oscillators and reconciles the apparent dichotomy between irregular single neuron activity and field potential oscillations. From this perspective, a plethora of studies will be reviewed on the involvement of long-distance neuronal coherence in cognitive functions such as multisensory integration, working memory, and selective attention. Finally, implications of abnormal neural synchronization are discussed as they relate to mental disorders like schizophrenia and autism.

BACKGROUND

Cognitive function in older adults is related to independent living and need for care. However, few studies have addressed whether improving cognitive functions might have short- or long-term effects on activities related to living independently.

OBJECTIVE

To evaluate whether 3 cognitive training interventions improve mental abilities and daily functioning in older, independent-living adults.

METHODS

Randomized, controlled, single-blind trial with recruitment conducted from March 1998 to October 1999 and 2-year follow-up through December 2001.

METHODS

Volunteer sample of 2832 persons aged 65 to 94 years recruited from senior housing, community centers, and hospital/clinics in 6 metropolitan areas in the United States.

METHODS

Participants were randomly assigned to 1 of 4 groups: 10-session group training for memory (verbal episodic memory; n = 711), or reasoning (ability to solve problems that follow a serial pattern; n = 705), or speed of processing (visual search and identification; n = 712); or a no-contact control group (n = 704). For the 3 treatment groups, 4-session booster training was offered to a 60% random sample 11 months later.

METHODS

Cognitive function and cognitively demanding everyday functioning.

RESULTS

Thirty participants were incorrectly randomized and were excluded from the analysis. Each intervention improved the targeted cognitive ability compared with baseline, durable to 2 years (P<.001 for all). Eighty-seven percent of speed-, 74% of reasoning-, and 26% of memory-trained participants demonstrated reliable cognitive improvement immediately after the intervention period. Booster training enhanced training gains in speed (P<.001) and reasoning (P<.001) interventions (speed booster, 92%; no booster, 68%; reasoning booster, 72%; no booster, 49%), which were maintained at 2-year follow-up (P<.001 for both). No training effects on everyday functioning were detected at 2 years.

CONCLUSIONS

Results support the effectiveness and durability of the cognitive training interventions in improving targeted cognitive abilities. Training effects were of a magnitude equivalent to the amount of decline expected in elderly persons without dementia over 7- to 14-year intervals. Because of minimal functional decline across all groups, longer follow-up is likely required to observe training effects on everyday function.

BACKGROUND

Randomized trials of surgery for epilepsy have not been conducted, because of the difficulties involved in designing and implementing feasible studies. The lack of data supporting the therapeutic usefulness of surgery precludes making strong recommendations for patients with epilepsy. We conducted a randomized, controlled trial to assess the efficacy and safety of surgery for temporal-lobe epilepsy.

METHODS

Eighty patients with temporal-lobe epilepsy were randomly assigned to surgery (40 patients) or treatment with antiepileptic drugs for one year (40 patients). Optimal medical therapy and primary outcomes were assessed by epileptologists who were unaware of the patients' treatment assignments. The primary outcome was freedom from seizures that impair awareness of self and surroundings. Secondary outcomes were the frequency and severity of seizures, the quality of life, disability, and death.

RESULTS

At one year, the cumulative proportion of patients who were free of seizures impairing awareness was 58 percent in the surgical group and 8 percent in the medical group (P<0.001). The patients in the surgical group had fewer seizures impairing awareness and a significantly better quality of life (P<0.001 for both comparisons) than the patients in the medical group. Four patients (10 percent) had adverse effects of surgery. One patient in the medical group died.

CONCLUSIONS

In temporal-lobe epilepsy, surgery is superior to prolonged medical therapy. Randomized trials of surgery for epilepsy are feasible and appear to yield precise estimates of treatment effects.

BACKGROUND

General population survey data are presented on the lifetime prevalence of suicide attempts as well as transition probabilities to onset of ideation, plans among ideators, and attempts among ideators either with or without a plan. Risk factors for these transitions are also studied.

METHODS

Data are from part II of the National Comorbidity Survey, a nationally representative survey carried out from 1990 to 1992 in a sample of 5877 respondents aged 15 to 54 years to study prevalences and correlates of DSM-III-R disorders. Transitions are estimated using life-table analysis. Risk factors are examined using survival analysis.

RESULTS

Of the respondents, 13.5% reported lifetime ideation, 3.9% a plan, and 4.6% an attempt. Cumulative probabilities were 34% for the transition from ideation to a plan, 72% from a plan to an attempt, and 26% from ideation to an unplanned attempt. About 90% of unplanned and 60% of planned first attempts occurred within 1 year of the onset of ideation. All significant risk factors (female, previously married, age less than 25 years, in a recent cohort, poorly educated, and having 1 or more of the DSM-III-R disorders assessed in the survey) were more strongly related to ideation than to progression from ideation to a plan or an attempt.

CONCLUSIONS

Prevention efforts should focus on planned attempts because of the rapid onset and unpredictability of unplanned attempts. More research is needed on the determinants of unplanned attempts.

Until around 1990, most multigene families were thought to be subject to concerted evolution, in which all member genes of a family evolve as a unit in concert. However, phylogenetic analysis of MHC and other immune system genes showed a quite different evolutionary pattern, and a new model called birth-and-death evolution was proposed. In this model, new genes are created by gene duplication and some duplicate genes stay in the genome for a long time, whereas others are inactivated or deleted from the genome. Later investigations have shown that most non-rRNA genes including highly conserved histone or ubiquitin genes are subject to this type of evolution. However, the controversy over the two models is still continuing because the distinction between the two models becomes difficult when sequence differences are small. Unlike concerted evolution, the model of birth-and-death evolution can give some insights into the origins of new genetic systems or new phenotypic characters.

Decision makers in health care are increasingly interested in using high-quality scientific evidence to support clinical and health policy choices; however, the quality of available scientific evidence is often found to be inadequate. Reliable evidence is essential to improve health care quality and to support efficient use of limited resources. The widespread gaps in evidence-based knowledge suggest that systematic flaws exist in the production of scientific evidence, in part because there is no consistent effort to conduct clinical trials designed to meet the needs of decision makers. Clinical trials for which the hypothesis and study design are developed specifically to answer the questions faced by decision makers are called pragmatic or practical clinical trials (PCTs). The characteristic features of PCTs are that they (1) select clinically relevant alternative interventions to compare, (2) include a diverse population of study participants, (3) recruit participants from heterogeneous practice settings, and (4) collect data on a broad range of health outcomes. The supply of PCTs is limited primarily because the major funders of clinical research, the National Institutes of Health and the medical products industry, do not focus on supporting such trials. Increasing the supply of PCTs will depend on the development of a mechanism to establish priorities for these studies, significant expansion of an infrastructure to conduct clinical research within the health care delivery system, more reliance on high-quality evidence by health care decision makers, and a substantial increase in public and private funding for these studies. For these changes to occur, clinical and health policy decision makers will need to become more involved in all aspects of clinical research, including priority setting, infrastructure development, and funding.

This review is focused on purinergic neurotransmission, i.e., ATP released from nerves as a transmitter or cotransmitter to act as an extracellular signaling molecule on both pre- and postjunctional membranes at neuroeffector junctions and synapses, as well as acting as a trophic factor during development and regeneration. Emphasis is placed on the physiology and pathophysiology of ATP, but extracellular roles of its breakdown product, adenosine, are also considered because of their intimate interactions. The early history of the involvement of ATP in autonomic and skeletal neuromuscular transmission and in activities in the central nervous system and ganglia is reviewed. Brief background information is given about the identification of receptor subtypes for purines and pyrimidines and about ATP storage, release, and ectoenzymatic breakdown. Evidence that ATP is a cotransmitter in most, if not all, peripheral and central neurons is presented, as well as full accounts of neurotransmission and neuromodulation in autonomic and sensory ganglia and in the brain and spinal cord. There is coverage of neuron-glia interactions and of purinergic neuroeffector transmission to nonmuscular cells. To establish the primitive and widespread nature of purinergic neurotransmission, both the ontogeny and phylogeny of purinergic signaling are considered. Finally, the pathophysiology of purinergic neurotransmission in both peripheral and central nervous systems is reviewed, and speculations are made about future developments.