Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

CCR5 is the major HIV-1 co-receptor, and individuals homozygous for a 32-bp deletion in CCR5 are resistant to infection by CCR5-tropic HIV-1. Using engineered zinc-finger nucleases (ZFNs), we disrupted CCR5 in human CD34(+) hematopoietic stem/progenitor cells (HSPCs) at a mean frequency of 17% of the total alleles in a population. This procedure produces both mono- and bi-allelically disrupted cells. ZFN-treated HSPCs retained the ability to engraft NOD/SCID/IL2rgamma(null) mice and gave rise to polyclonal multi-lineage progeny in which CCR5 was permanently disrupted. Control mice receiving untreated HSPCs and challenged with CCR5-tropic HIV-1 showed profound CD4(+) T-cell loss. In contrast, mice transplanted with ZFN-modified HSPCs underwent rapid selection for CCR5(-/-) cells, had significantly lower HIV-1 levels and preserved human cells throughout their tissues. The demonstration that a minority of CCR5(-/-) HSPCs can populate an infected animal with HIV-1-resistant, CCR5(-/-) progeny supports the use of ZFN-modified autologous hematopoietic stem cells as a clinical approach to treating HIV-1.

1. A method is described for the trace iodination of immunoglobulins and other serum proteins by a system consisting of lactoperoxidase, hydrogen peroxide and iodide. 2. gammaG immunoglobulin that had been labelled to a specific radioactivity of 5muc/mug. by use of carrier-free [(125)I]iodide gave no evidence of denaturation when analysed by electrophoresis and density-gradient ultracentrifugation. 3. Tryptic hydrolysis and peptide ;mapping' of a completely characterized peptide radioiodinated by this method showed that the [(125)I]iodide was bound to tyrosyl residues. 4. Proteins differ in their susceptibility to iodination by this method. Human gammaG immunoglobulin, for example, is iodinated more than ten times as readily as is human alpha(2)-macroglobulin under the same conditions. 5. Lactoperoxidase catalyses the iodination of proteins much more readily than does horseradish peroxidase.

A new tetrazolium analog of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was evaluated as a substitute for MTT in the microculture screening assay for in vitro cell growth. This new tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), in the presence of phenazine methosulfate (PMS), gave a water-soluble formazan product that had an absorbance maximum at 490-500 nm in phosphate-buffered saline. The amount of colored product formed was proportional to the number of cells and the time of incubation of the cells with MTS/PMS. MTS/PMS was reactive in all the cell lines tested which included mouse leukemia L1210 cells, mouse Ehrlich tumor cells, mouse 3T3 fibroblasts, and human colon tumor cells (HT-29). HT-29 and 3T3 fibroblasts reduced MTS/PMS more efficiently than they reduced MTT. Comparable to the amount of product formed from MTT, MTS/PMS gave excellent product formation. The IC50 value for pyrazoloimidazole obtained using MTS/PMS was 200 microM; for 5-fluoro-2'-deoxyuridine, the IC50 value was 0.9 nM. These values compared very favorably with the IC50 values obtained by direct cell counts. Further, the same IC50 values were obtained when the absorbances of the formazan product in the 96-well plates were determined after different times of incubation.

By introduction of recombinant plasmids into monkey CV1 cells, we have unambiguously demonstrated that sequences entirely within the 72 bp repeat, which is located upstream of the SV40 early region, are crucial for T-antigen expression in vivo. We have also shown that a DNA fragment containing the 72 bp repeat, inserted directly before chicken conalbumin or adenovirus-2 major late promoter sequences in chimeric plasmids where these promoters replace that of the SV40 early genes, caused a dramatic increase in the expression of T-antigen in vivo. This effect was independent of the orientation of the 72 bp repeat, but was sensitive to its location within the plasmid, when the 72 bp repeat was separated from the promoter sequences, T-antigen expression was reduced. Insertion of the 72 bp repeat into equivalent plasmids containing no known eukaryotic promoter sequences (plasmids which were not detectably expressed in vivo) gave rise to a measurable, but smaller level of expression. The stimulation of expression by the 72 bp repeat is cis-acting : it required covalent linkage to the recombinant. We discuss the possibility that the 72 bp repeat region in SV40 may act as a bi-directional entry site for RNA polymerase B such that promoter sequences linked to the repeat are more efficiently utilised.

BACKGROUND

The spectrum of disorders of the brain is large, covering hundreds of disorders that are listed in either the mental or neurological disorder chapters of the established international diagnostic classification systems. These disorders have a high prevalence as well as short- and long-term impairments and disabilities. Therefore they are an emotional, financial and social burden to the patients, their families and their social network. In a 2005 landmark study, we estimated for the first time the annual cost of 12 major groups of disorders of the brain in Europe and gave a conservative estimate of €386 billion for the year 2004. This estimate was limited in scope and conservative due to the lack of sufficiently comprehensive epidemiological and/or economic data on several important diagnostic groups. We are now in a position to substantially improve and revise the 2004 estimates. In the present report we cover 19 major groups of disorders, 7 more than previously, of an increased range of age groups and more cost items. We therefore present much improved cost estimates. Our revised estimates also now include the new EU member states, and hence a population of 514 million people.

OBJECTIVE

To estimate the number of persons with defined disorders of the brain in Europe in 2010, the total cost per person related to each disease in terms of direct and indirect costs, and an estimate of the total cost per disorder and country.

METHODS

The best available estimates of the prevalence and cost per person for 19 groups of disorders of the brain (covering well over 100 specific disorders) were identified via a systematic review of the published literature. Together with the twelve disorders included in 2004, the following range of mental and neurologic groups of disorders is covered: addictive disorders, affective disorders, anxiety disorders, brain tumor, childhood and adolescent disorders (developmental disorders), dementia, eating disorders, epilepsy, mental retardation, migraine, multiple sclerosis, neuromuscular disorders, Parkinson's disease, personality disorders, psychotic disorders, sleep disorders, somatoform disorders, stroke, and traumatic brain injury. Epidemiologic panels were charged to complete the literature review for each disorder in order to estimate the 12-month prevalence, and health economic panels were charged to estimate best cost-estimates. A cost model was developed to combine the epidemiologic and economic data and estimate the total cost of each disorder in each of 30 European countries (EU27+Iceland, Norway and Switzerland). The cost model was populated with national statistics from Eurostat to adjust all costs to 2010 values, converting all local currencies to Euro, imputing costs for countries where no data were available, and aggregating country estimates to purchasing power parity adjusted estimates for the total cost of disorders of the brain in Europe 2010.

RESULTS

The total cost of disorders of the brain was estimated at €798 billion in 2010. Direct costs constitute the majority of costs (37% direct healthcare costs and 23% direct non-medical costs) whereas the remaining 40% were indirect costs associated with patients' production losses. On average, the estimated cost per person with a disorder of the brain in Europe ranged between €285 for headache and €30,000 for neuromuscular disorders. The European per capita cost of disorders of the brain was €1550 on average but varied by country. The cost (in billion €PPP 2010) of the disorders of the brain included in this study was as follows: addiction: €65.7; anxiety disorders: €74.4; brain tumor: €5.2; child/adolescent disorders: €21.3; dementia: €105.2; eating disorders: €0.8; epilepsy: €13.8; headache: €43.5; mental retardation: €43.3; mood disorders: €113.4; multiple sclerosis: €14.6; neuromuscular disorders: €7.7; Parkinson's disease: €13.9; personality disorders: €27.3; psychotic disorders: €93.9; sleep disorders: €35.4; somatoform disorder: €21.2; stroke: €64.1; traumatic brain injury: €33.0. It should be noted that the revised estimate of those disorders included in the previous 2004 report constituted €477 billion, by and large confirming our previous study results after considering the inflation and population increase since 2004. Further, our results were consistent with administrative data on the health care expenditure in Europe, and comparable to previous studies on the cost of specific disorders in Europe. Our estimates were lower than comparable estimates from the US.

CONCLUSIONS

This study was based on the best currently available data in Europe and our model enabled extrapolation to countries where no data could be found. Still, the scarcity of data is an important source of uncertainty in our estimates and may imply over- or underestimations in some disorders and countries. Even though this review included many disorders, diagnoses, age groups and cost items that were omitted in 2004, there are still remaining disorders that could not be included due to limitations in the available data. We therefore consider our estimate of the total cost of the disorders of the brain in Europe to be conservative. In terms of the health economic burden outlined in this report, disorders of the brain likely constitute the number one economic challenge for European health care, now and in the future. Data presented in this report should be considered by all stakeholder groups, including policy makers, industry and patient advocacy groups, to reconsider the current science, research and public health agenda and define a coordinated plan of action of various levels to address the associated challenges.

CONCLUSIONS

Political action is required in light of the present high cost of disorders of the brain. Funding of brain research must be increased; care for patients with brain disorders as well as teaching at medical schools and other health related educations must be quantitatively and qualitatively improved, including psychological treatments. The current move of the pharmaceutical industry away from brain related indications must be halted and reversed. Continued research into the cost of the many disorders not included in the present study is warranted. It is essential that not only the EU but also the national governments forcefully support these initiatives.

A monoclonal antibody was produced to human peripheral blood T cells. This hybridoma antibody, termed OKT4, was reactive by indirect immunofluorescence with only 55-60% of the peripheral blood T cell population (OKT4+) and unreactive with normal B cells, null cells, and macrophages. The OKT4- T cell population contained the previously described TH2+ subset that has been shown to contain cytotoxic/suppressor cells. With cell-sorter separation of OKT4+ and OKT4- cells, it was shown that these T cell subsets were functionally discrete. Both gave proliferative responses with concanavalin A, alloantigens, and phytohemagglutinin although OKT4+ cells were much more responsive to the latter. OKT4+ cells alone responded to soluble antigens whereas OKT4- cells alone were cytotoxic after alloantigenic sensitization of unfractionated T cells. However, both OKT4+ and OKT4- cells were required during sensitization for optimal development of cytotoxicity. These data suggest that the OKT4+ subset represents a helper population and that the OKT4- subset contains the cytotoxic effector population. OKT4 could be a valuable reagent for determining alterations of these functional subsets in human diseases.

Abscisic acid (ABA), a plant hormone, is involved in responses to environmental stresses such as drought and high salinity, and is required for stress tolerance. ABA is synthesized de novo in response to dehydration. 9-cis-epoxycarotenoid dioxygenase (NCED) is thought to be a key enzyme in ABA biosynthesis. Here we demonstrate that the expression of an NCED gene of Arabidopsis, AtNCED3, is induced by drought stress and controls the level of endogenous ABA under drought-stressed conditions. Overexpression of AtNCED3 in transgenic Arabidopsis caused an increase in endogenous ABA level, and promoted transcription of drought- and ABA-inducible genes. Plants overexpressing AtNCED3 showed a reduction in transpiration rate from leaves and an improvement in drought tolerance. By contrast, antisense suppression and disruption of AtNCED3 gave a drought-sensitive phenotype. These results indicate that the expression of AtNCED3 plays a key role in ABA biosynthesis under drought-stressed conditions in Arabidopsis. We improved drought tolerance by gene manipulation of AtNCED3 causing the accumulation of endogenous ABA.

A myeloma line has been developed which produces no globulin chains of its own, has a duplication of 8.7 h, fuses effectively with B-lymphoblasts and produces stable hybrids. An enhancing effect of macrophages on hybridoma yields has been observed. Among the fusing agents tested, PEG of mol.wt. 4000 gave the best results, 20 degrees C being the optimum working temperature. The maintenance medium of choice has been found to be Iscove's with 10% FCS. Direct exposure of fusion cultures to a selective medium with hypoxanthine, aminopterine and thymidine reduced the labor involved and increased the yield. A mechanical device for changing the medium has been designed. The replacement of standard trays by microtrays resulted in a higher frequency of surviving hybrids. By using a feeder layer, the spleen cell input can be reduced 50-fold. At such low multiplicities the positive cultures arise predominantly from single hybrids, eliminating the need for subsequent cloning. The hybrids can be labelled and will yield in serum-free medium. Since at least a third of them inherit the fast growth rate of their myeloma parent and keep producing over 2000 antibody molecules per second, readaptation to ascitic growth is also superfluous. A simplified technique of producing monoclonal antibodies is given in detail, together with the experimental evidence prompting modifications of the classical method of Köhler and Milstein (1975, 1976).

What are the neural substrates of food reward? Are reward and pleasure identical? Can taste pleasure be assessed in animals? Is reward necessarily conscious? These questions have re-emerged in recent years, and there is now sufficient evidence to prompt re-examination of many preconceptions concerning reward and its relation to brain systems. This paper reviews evidence from many sources regarding both the psychological structure of food reward and the neural systems that mediate it. Special attention is paid to recent evidence from "tasty reactivity" studies of affective reactions to food. I argue that this evidence suggests the following surprising possibilities regarding the functional components and brain substrates of food reward. (1) Reward contains distinguishable psychological or functional components--"liking" (pleasure/palatability) and "wanting" (appetite/incentive motivation). These can be manipulated and measured separately. (2) Liking and wanting have separable neural substrates. Mediation of liking related to food reward involves neurotransmitter systems such as opioid and GABA/benzodiazepine systems, and anatomical structures such as ventral pallidum and brainstem primary gustatory relays. Mediation of wanting related to food reward involves mesotelencephalic dopamine systems, and divisions of nucleus accumbens and amygdala. Both liking and wanting arise from vastly distributed neural systems, but the two systems are separable. (3) Neural processing of food reward is not confined to the limbic forebrain. Aspects of food reward begin to be processed in the brainstem. A neural manipulation can enhance reward or produce aversion but no single lesion or transection is likely abolish all properties of food reward. (4) Both wanting and liking can exist without subjective awareness. Conscious experience can distort or blur the underlying reward process that gave rise to it. Subjective reports may contain false assessments of underlying processes, or even fail at all to register important reward processes. The core processes of liking and wanting that constitute reward are distinct from the subjective report or conscious awareness of those processes.

Estimates of body surface area were made based on measurement of 81 subjects, ranging from premature infants to adults. SA was calculated geometrically for each subject from 34 body measurements, and the values obtained compared with those based on previously published formulas and graphs. The most widely used formula, that of Du Bois and Du Bois, increasingly underestimated SA as values fell below 0.7 m2; the disparity was greatest in the newborn infant (7.96%). Closer agreement was obtained with the equations and nomograms of Body, Brody, Faber and Melcher, and Sendroy and Cecchini, although minor deviations were noted in some age ranges. The formula SA (m2) = weight (kg)0.5378 X height (cm)0.3964 X 0.024265, derived from the measured data by multiple regression analysis, gave a good fit for all values of SA from less than 0.2 m2 to greater than 2.0 m2 (r = 0.998). This formula was used to construct nomograms for estimation of SA in infants, children, and adults from height (length) and weight.

We are an intensely social species--it has been argued that our social nature defines what makes us human, what makes us conscious or what gave us our large brains. As a new field, the social brain sciences are probing the neural underpinnings of social behaviour and have produced a banquet of data that are both tantalizing and deeply puzzling. We are finding new links between emotion and reason, between action and perception, and between representations of other people and ourselves. No less important are the links that are also being established across disciplines to understand social behaviour, as neuroscientists, social psychologists, anthropologists, ethologists and philosophers forge new collaborations.

The AAA+ ATPases are enzymes containing a P-loop NTPase domain, and function as molecular chaperones, ATPase subunits of proteases, helicases or nucleic-acid-stimulated ATPases. All available sequences and structures of AAA+ protein domains were compared with the aim of identifying the definitive sequence and structure features of these domains and inferring the principal events in their evolution. An evolutionary classification of the AAA+ class was developed using standard phylogenetic methods, analysis of shared sequence and structural signatures, and similarity-based clustering. This analysis resulted in the identification of 26 major families within the AAA+ ATPase class. We also describe the position of the AAA+ ATPases with respect to the RecA/F1, helicase superfamilies I/II, PilT, and ABC classes of P-loop NTPases. The AAA+ class appears to have undergone an early radiation into the clamp-loader, DnaA/Orc/Cdc6, classic AAA, and "pre-sensor 1 beta-hairpin" (PS1BH) clades. Within the PS1BH clade, chelatases, MoxR, YifB, McrB, Dynein-midasin, NtrC, and MCMs form a monophyletic assembly defined by a distinct insert in helix-2 of the conserved ATPase core, and additional helical segment between the core ATPase domain and the C-terminal alpha-helical bundle. At least 6 distinct AAA+ proteins, which represent the different major clades, are traceable to the last universal common ancestor (LUCA) of extant cellular life. Additionally, superfamily III helicases, which belong to the PS1BH assemblage, were probably present at this stage in virus-like "selfish" replicons. The next major radiation, at the base of the two prokaryotic kingdoms, bacteria and archaea, gave rise to several distinct chaperones, ATPase subunits of proteases, DNA helicases, and transcription factors. The third major radiation, at the outset of eukaryotic evolution, contributed to the origin of several eukaryote-specific adaptations related to nuclear and cytoskeletal functions. The new relationships and previously undetected domains reported here might provide new leads for investigating the biology of AAA+ ATPases.

Hybrid myeloma cell lines secreting monoclonal antibodies to tubulin have been prepared using rat myelomas and spleen cells from rats immunized with yeast tubulin. A comparison between the results obtained with the rat myeloma Y3-Ag 1.2.3., which secretes a light chain, and a new line, YB2/O, which does not, shows that they are both excellent parental lines and that the second produces hybrids with no myeloma chain components. The antitubulin antibodies in the serum of rats bearing two of the hybrid myeloma tumors gave titers of up to 1:10(6) from which large amounts of monoclonal antibodies could be easily purified. They recognized tubulin from yeast as well as from birds and mammals. The two antibodies gave clear immunofluorescent staining of yeast mitotic spindles as well as the interphase microtubule network of tissue culture cells. Some difference in the pattern of immunofluorescence staining of yeast cells and nuclei was observed between the two antibodies. The purified antibodies could be conjugated to colloidal gold particles and used for direct labeling of yeast microtubules for electron microscopy.

Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of P(tet) promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter. To remove these limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox sensitivity. Five new rtTAs were identified, of which two have greatly improved properties. The most promising new transactivator, rtTA2(S)-M2, functions at a 10-fold lower Dox concentration than rtTA, is more stable in eukaryotic cells, and causes no background expression in the absence of Dox. The coding sequences of the new reverse TetR mutants fused to minimal activation domains were optimized for expression in human cells and synthesized. The resulting transactivators allow stringent regulation of target genes over a range of 4 to 5 orders of magnitude in stably transfected HeLa cells. These rtTA versions combine tightness of expression control with a broad regulatory range, as previously shown for the widely applied tTA.

BACKGROUND

Neurosurgical resection is the standard treatment for subependymal giant-cell astrocytomas in patients with the tuberous sclerosis complex. An alternative may be the use of everolimus, which inhibits the mammalian target of rapamycin, a protein regulated by gene products involved in the tuberous sclerosis complex.

METHODS

Patients 3 years of age or older with serial growth of subependymal giant-cell astrocytomas were eligible for this open-label study. The primary efficacy end point was the change in volume of subependymal giant-cell astrocytomas between baseline and 6 months. We gave everolimus orally, at a dose of 3.0 mg per square meter of body-surface area, to achieve a trough concentration of 5 to 15 ng per milliliter.

RESULTS

We enrolled 28 patients. Everolimus therapy was associated with a clinically meaningful reduction in volume of the primary subependymal giant-cell astrocytoma, as assessed on independent central review (P<0.001 for baseline vs. 6 months), with a reduction of at least 30% in 21 patients (75%) and at least 50% in 9 patients (32%). Marked reductions were seen within 3 months and were sustained. There were no new lesions, worsening hydrocephalus, evidence of increased intracranial pressure, or necessity for surgical resection or other therapy for subependymal giant-cell astrocytoma. Of the 16 patients for whom 24-hour video electroencephalography data were available, seizure frequency for the 6-month study period (vs. the previous 6-month period) decreased in 9, did not change in 6, and increased in 1 (median change, -1 seizure; P=0.02). The mean (±SD) score on the validated Quality-of-Life in Childhood Epilepsy questionnaire (on which scores can range from 0 to 100, with higher scores indicating a better quality of life) was improved at 3 months (63.4±12.4) and 6 months (62.1±14.2) over the baseline score (57.8±14.0). Single cases of grade 3 treatment-related sinusitis, pneumonia, viral bronchitis, tooth infection, stomatitis, and leukopenia were reported.

CONCLUSIONS

Everolimus therapy was associated with marked reduction in the volume of subependymal giant-cell astrocytomas and seizure frequency and may be a potential alternative to neurosurgical resection in some cases, though long-term studies are needed. (Funded by Novartis; ClinicalTrials.gov number, NCT00411619.).

The duplication of entire genomes has long been recognized as having great potential for evolutionary novelties, but the mechanisms underlying their resolution through gene loss are poorly understood. Here we show that in the unicellular eukaryote Paramecium tetraurelia, a ciliate, most of the nearly 40,000 genes arose through at least three successive whole-genome duplications. Phylogenetic analysis indicates that the most recent duplication coincides with an explosion of speciation events that gave rise to the P. aurelia complex of 15 sibling species. We observed that gene loss occurs over a long timescale, not as an initial massive event. Genes from the same metabolic pathway or protein complex have common patterns of gene loss, and highly expressed genes are over-retained after all duplications. The conclusion of this analysis is that many genes are maintained after whole-genome duplication not because of functional innovation but because of gene dosage constraints.

To test the predictive values of and independent contributions to cardiovascular and all-cause mortality of various arterial parameters exploring characteristics of the arterial wall at different sites, we studied prospectively 110 stable end-stage renal disease patients on hemodialysis. These parameters involved carotid diameter, carotid intima-media thickness, carotid compliance, carotid distensibility, carotid incremental elastic modulus, aortic diameter, aortic pulse wave velocity, and the presence of arterial calcifications measured at the sites of the carotid artery, abdominal aorta, iliofemoral axis, and legs. The presence of calcifications was analyzed semiquantitatively as a score (0 to 4) according to the number of arterial sites with calcifications. During a follow-up of 53+/-21 months (mean+/-SD), 25 cardiovascular and 14 noncardiovascular deaths occurred. In univariate analysis, the carotid incremental elastic modulus was the most closely related to prognosis. Risk of death increased with the number of vascular sites involved by calcifications. Moreover, information (in terms of prediction) given by carotid elastic incremental modulus was additive to the presence and extent of vascular calcification-related prediction value. Adjusted hazard ratios of all-cause and cardiovascular mortality for an increase of 1 unit in calcification score were 1.9 (95% confidence interval [CI], 1.4 to 2.6) and 2.6 (95% CI, 1.5 to 4.4), respectively (P<0.001 for both). Adjusted hazard ratios of all-cause and cardiovascular mortality for a 1-SD increase in carotid incremental elastic modulus were 1.6 (95% CI, 1.2 to 2.2) and 1.7 (95% CI, 1.2 to 2.4), respectively (P<0.01 for both). The results of this study showed that the presence and extent of vascular calcifications were strong predictors of cardiovascular and all-cause mortality. Carotid incremental elastic modulus gave additional predictive value.

OBJECTIVE

To evaluate how well quality of life is being measured in the medical literature and to offer a new approach to the measurement.

METHODS

Original English-language articles having the term "quality of life" in their titles were identified from a recent Quality-of-Life Bibliography and from two MEDLINE searches. Articles were eligible for review only if they described or used one or more "quality-of-life" instruments.

METHODS

Twenty-five articles were randomly selected from each of the three data sources.

METHODS

Each article was reviewed for its compliance with two sets of criteria having several components, which are cited under "Data Synthesis."

RESULTS

(1) Investigators conceptually defined quality of life in only 11 (15%) of the 75 articles; identified the targeted domains in only 35 (47%); gave reasons for selecting the chosen quality-of-life instruments in only 27 (36%); and aggregated their results into a composite quality-of-life score in only 27 (38%) of 71 eligible articles. (2) No article distinguished "overall" quality of life from health-related quality of life; patients were invited to give their own separate rating for quality of life in only 13 articles (17%); and among 71 eligible articles, patients were asked to supplement the stipulated items with personal responses in only nine (13%) and to rate the importance of individual items in only six (8.5%).

CONCLUSIONS

Because quality of life is a uniquely personal perception, denoting the way that individual patients feel about their health status and/or nonmedical aspects of their lives, most measurements of quality of life in the medical literature seem to aim at the wrong target. Quality of life can be suitably measured only by determining the opinions of patients and by supplementing (or replacing) the instruments developed by "experts."

Ribosome synthesis is a highly complex and coordinated process that occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells. Based on the protein composition of several ribosomal subunit precursors recently characterized in yeast, a total of more than 170 factors are predicted to participate in ribosome biogenesis and the list is still growing. So far the majority of ribosomal factors have been implicated in RNA maturation (nucleotide modification and processing). Recent advances gave insight into the process of ribosome export and assembly. Proteomic approaches have provided the first indications for a ribosome assembly pathway in eukaryotes and confirmed the dynamic character of the whole process.

An automated refinement procedure (ARP) for protein models is proposed, and its convergence properties discussed. It is comparable to the iterative least-squares minimization/difference Fourier synthesis approach for small molecules. ARP has been successfully applied to three proteins, and for two of them resulted in models very similar to those obtained by conventional least-squares refinement and rebuilding with FRODO. In real time ARP is about ten times faster than conventional refinement. In its present form ARP requires high (2.0 A or better) resolution data, which should be of high quality and a starting protein model having about 75% of the atoms in roughly the correct position. For the third protein at 2.4 A resolution, ARP was significantly less powerful but nevertheless gave definite improvement, in the density map at least.

The consequences of cardiac excitation-contraction coupling by calcium-induced calcium release were studied theoretically, using a series of idealized models solved by analytic and numerical methods. "Common-pool" models, those in which the trigger calcium and released calcium pass through a common cytosolic pool, gave nearly all-or-none regenerative calcium releases (in disagreement with experiment), unless their loop gain was made sufficiently low that it provided little amplification of the calcium entering through the sarcolemma. In the linear (small trigger) limit, it was proven rigorously that no common-pool model can give graded high amplification unless it is operated on the verge of spontaneous oscillation. To circumvent this problem, we considered two types of "local-control" models. In the first type, the local calcium from a sarcolemmal L-type calcium channel directly stimulates a single, immediately opposed SR calcium release channel. This permits high amplification without regeneration, but requires high conductance of the SR channel. This problem is avoided in the second type of local control model, in which one L-type channel triggers a regenerative cluster of several SR channels. Statistical recruitment of clusters results in graded response with high amplification. In either type of local-control model, the voltage dependence of SR calcium release is not exactly the same as that of the macroscopic sarcolemmal calcium current, even though calcium is the only trigger for SR release. This results from the existence of correlations between the stochastic openings of individual sarcolemmal and SR channels. Propagation of regenerative calcium-release waves (under conditions of calcium overload) was analyzed using analytically soluble models in which SR calcium release was treated phenomenalogically. The range of wave velocities observed experimentally is easily explained; however, the observed degree of refractoriness to wave propagation requires either a strong dependence of SR calcium release on the rate of rise of cytosolic calcium or localization of SR release sites to one point in the sarcomere. We conclude that the macroscopic behavior of calcium-induced calcium release depends critically on the spatial relationships among sarcolemmal and SR calcium channels, as well as on their kinetics.

As is the case for embryo-derived stem cells, application of reprogrammed human induced pluripotent stem cells is limited by our understanding of lineage specification. Here we demonstrate the ability to generate progenitors and mature cells of the haematopoietic fate directly from human dermal fibroblasts without establishing pluripotency. Ectopic expression of OCT4 (also called POU5F1)-activated haematopoietic transcription factors, together with specific cytokine treatment, allowed generation of cells expressing the pan-leukocyte marker CD45. These unique fibroblast-derived cells gave rise to granulocytic, monocytic, megakaryocytic and erythroid lineages, and demonstrated in vivo engraftment capacity. We note that adult haematopoietic programs are activated, consistent with bypassing the pluripotent state to generate blood fate: this is distinct from haematopoiesis involving pluripotent stem cells, where embryonic programs are activated. These findings demonstrate restoration of multipotency from human fibroblasts, and suggest an alternative approach to cellular reprogramming for autologous cell-replacement therapies that avoids complications associated with the use of human pluripotent stem cells.

Cardiac macrophages are crucial for tissue repair after cardiac injury but are not well characterized. Here we identify four populations of cardiac macrophages. At steady state, resident macrophages were primarily maintained through local proliferation. However, after macrophage depletion or during cardiac inflammation, Ly6c(hi) monocytes contributed to all four macrophage populations, whereas resident macrophages also expanded numerically through proliferation. Genetic fate mapping revealed that yolk-sac and fetal monocyte progenitors gave rise to the majority of cardiac macrophages, and the heart was among a minority of organs in which substantial numbers of yolk-sac macrophages persisted in adulthood. CCR2 expression and dependence distinguished cardiac macrophages of adult monocyte versus embryonic origin. Transcriptional and functional data revealed that monocyte-derived macrophages coordinate cardiac inflammation, while playing redundant but lesser roles in antigen sampling and efferocytosis. These data highlight the presence of multiple cardiac macrophage subsets, with different functions, origins, and strategies to regulate compartment size.