Nonapoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of nonapoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system x(c)(-)), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the nonapoptotic destruction of certain cancer cells, whereas inhibition of this process may protect organisms from neurodegeneration.

In the early stages of apoptosis changes occur at the cell surface, which until now have remained difficult to recognize. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V is a Ca2+ dependent phospholipid-binding protein with high affinity for PS. Hence this protein can be used as a sensitive probe for PS exposure upon the cell membrane. Translocation of PS to the external cell surface is not unique to apoptosis, but occurs also during cell necrosis. The difference between these two forms of cell death is that during the initial stages of apoptosis the cell membrane remains intact, while at the very moment that necrosis occurs the cell membrane looses its integrity and becomes leaky. Therefore the measurement of Annexin V binding to the cell surface as indicative for apoptosis has to be performed in conjunction with a dye exclusion test to establish integrity of the cell membrane. This paper describes the results of such an assay, as obtained in cultured HSB-2 cells, rendered apoptotic by irradiation and in human lymphocytes, following dexamethasone treatment. Untreated and treated cells were evaluated for apoptosis by light microscopy, by measuring the amount of hypo-diploid cells using of DNA flow cytometry (FCM) and by DNA electrophoresis to establish whether or not DNA fragmentation had occurred. Annexin V binding was assessed using bivariate FCM, and cell staining was evaluated with fluorescein isothiocyanate (FITC)-labelled Annexin V (green fluorescence), simultaneously with dye exclusion of propidium iodide (PI) (negative for red fluorescence). The test described, discriminates intact cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and necrotic cells (FITC+/PI+). In comparison with existing traditional tests the Annexin V assay is sensitive and easy to perform. The Annexin V assay offers the possibility of detecting early phases of apoptosis before the loss of cell membrane integrity and permits measurements of the kinetics of apoptotic death in relation to the cell cycle. More extensive FCM will allow discrimination between different cell subpopulations, that may or may not be involved in the apoptotic process.

Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that can be isolated from many adult tissues. They can differentiate into cells of the mesodermal lineage, such as adipocytes, osteocytes and chondrocytes, as well as cells of other embryonic lineages. MSCs can interact with cells of both the innate and adaptive immune systems, leading to the modulation of several effector functions. After in vivo administration, MSCs induce peripheral tolerance and migrate to injured tissues, where they can inhibit the release of pro-inflammatory cytokines and promote the survival of damaged cells. This Review discusses the targets and mechanisms of MSC-mediated immunomodulation and the possible translation of MSCs to new therapeutic approaches.

Inositol phospholipids have long been known to have an important regulatory role in cell physiology. The repertoire of cellular processes known to be directly or indirectly controlled by this class of lipids has now dramatically expanded. Through interactions mediated by their headgroups, which can be reversibly phosphorylated to generate seven species, phosphoinositides play a fundamental part in controlling membrane-cytosol interfaces. These lipids mediate acute responses, but also act as constitutive signals that help define organelle identity. Their functions, besides classical signal transduction at the cell surface, include regulation of membrane traffic, the cytoskeleton, nuclear events and the permeability and transport functions of membranes.

Irrespective of the morphological features of end-stage cell death (that may be apoptotic, necrotic, autophagic, or mitotic), mitochondrial membrane permeabilization (MMP) is frequently the decisive event that delimits the frontier between survival and death. Thus mitochondrial membranes constitute the battleground on which opposing signals combat to seal the cell's fate. Local players that determine the propensity to MMP include the pro- and antiapoptotic members of the Bcl-2 family, proteins from the mitochondrialpermeability transition pore complex, as well as a plethora of interacting partners including mitochondrial lipids. Intermediate metabolites, redox processes, sphingolipids, ion gradients, transcription factors, as well as kinases and phosphatases link lethal and vital signals emanating from distinct subcellular compartments to mitochondria. Thus mitochondria integrate a variety of proapoptotic signals. Once MMP has been induced, it causes the release of catabolic hydrolases and activators of such enzymes (including those of caspases) from mitochondria. These catabolic enzymes as well as the cessation of the bioenergetic and redox functions of mitochondria finally lead to cell death, meaning that mitochondria coordinate the late stage of cellular demise. Pathological cell death induced by ischemia/reperfusion, intoxication with xenobiotics, neurodegenerative diseases, or viral infection also relies on MMP as a critical event. The inhibition of MMP constitutes an important strategy for the pharmaceutical prevention of unwarranted cell death. Conversely, induction of MMP in tumor cells constitutes the goal of anticancer chemotherapy.

OBJECTIVE

The worldwide prevalence estimates of attention deficit hyperactivity disorder (ADHD)/hyperkinetic disorder (HD) are highly heterogeneous. Presently, the reasons for this discrepancy remain poorly understood. The purpose of this study was to determine the possible causes of the varied worldwide estimates of the disorder and to compute its worldwide-pooled prevalence.

METHODS

The authors searched MEDLINE and PsycINFO databases from January 1978 to December 2005 and reviewed textbooks and reference lists of the studies selected. Authors of relevant articles from North America, South America, Europe, Africa, Asia, Oceania, and the Middle East and ADHD/HD experts were contacted. Surveys were included if they reported point prevalence of ADHD/HD for subjects 18 years of age or younger from the general population or schools according to DSM or ICD criteria.

RESULTS

The literature search generated 9,105 records, and 303 full-text articles were reviewed. One hundred and two studies comprising 171,756 subjects from all world regions were included. The ADHD/HD worldwide-pooled prevalence was 5.29%. This estimate was associated with significant variability. In the multivariate metaregression model, diagnostic criteria, source of information, requirement of impairment for diagnosis, and geographic origin of the studies were significantly associated with ADHD/HD prevalence rates. Geographic location was associated with significant variability only between estimates from North America and both Africa and the Middle East. No significant differences were found between Europe and North America.

CONCLUSIONS

Our findings suggest that geographic location plays a limited role in the reasons for the large variability of ADHD/HD prevalence estimates worldwide. Instead, this variability seems to be explained primarily by the methodological characteristics of studies.

Monocytes and macrophages are critical effectors and regulators of inflammation and the innate immune response, the immediate arm of the immune system. Dendritic cells initiate and regulate the highly pathogen-specific adaptive immune responses and are central to the development of immunologic memory and tolerance. Recent in vivo experimental approaches in the mouse have unveiled new aspects of the developmental and lineage relationships among these cell populations. Despite this, the origin and differentiation cues for many tissue macrophages, monocytes, and dendritic cell subsets in mice, and the corresponding cell populations in humans, remain to be elucidated.

NF-kappa B is a ubiquitous transcription factor. Nevertheless, its properties seem to be most extensively exploited in cells of the immune system. Among these properties are NF-kappa B's rapid posttranslational activation in response to many pathogenic signals, its direct participation in cytoplasmic/nuclear signaling, and its potency to activate transcription of a great variety of genes encoding immunologically relevant proteins. In vertebrates, five distinct DNA binding subunits are currently known which might extensively heterodimerize, thereby forming complexes with distinct transcriptional activity, DNA sequence specificity, and cell type- and cell stage-specific distribution. The activity of DNA binding NF-kappa B dimers is tightly controlled by accessory proteins called I kappa B subunits of which there are also five different species currently known in vertebrates. I kappa B proteins inhibit DNA binding and prevent nuclear uptake of NF-kappa B complexes. An exception is the Bcl-3 protein which in addition can function as a transcription activating subunit in th nucleus. Other I kappa B proteins are rather involved in terminating NF-kappa B's activity in the nucleus. The intracellular events that lead to the inactivation of I kappa B, i.e. the activation of NF-kappa B, are complex. They involve phosphorylation and proteolytic reactions and seem to be controlled by the cells' redox status. Interference with the activation or activity of NF-kappa B may be beneficial in suppressing toxic/septic shock, graft-vs-host reactions, acute inflammatory reactions, acute phase response, and radiation damage. The inhibition of NF-kappa B activation by antioxidants and specific protease inhibitors may provide a pharmacological basis for interfering with these acute processes.

This paper is a companion to a recent paper on fast rotation functions [Storoni et al. (2004), Acta Cryst. D60, 432-438], which showed how a Taylor-series expansion of the maximum-likelihood rotation function leads to improved likelihood-enhanced fast rotation functions. In a similar manner, it is shown here how linear and quadratic Taylor-series expansions and least-squares approximations of the maximum-likelihood translation function lead to likelihood-enhanced translation functions, which can be calculated by FFT and which are more sensitive to the correct translation than the traditional correlation-coefficient fast translation function. These likelihood-enhanced translation targets for molecular-replacement searches have been implemented in the program Phaser using the Computational Crystallography Toolbox (cctbx).

16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of 'best available' primer pairs for Bacteria and Archaea for three amplicon size classes (100-400, 400-1000, ≥ 1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.

Many networks of interest in the sciences, including social networks, computer networks, and metabolic and regulatory networks, are found to divide naturally into communities or modules. The problem of detecting and characterizing this community structure is one of the outstanding issues in the study of networked systems. One highly effective approach is the optimization of the quality function known as "modularity" over the possible divisions of a network. Here I show that the modularity can be expressed in terms of the eigenvectors of a characteristic matrix for the network, which I call the modularity matrix, and that this expression leads to a spectral algorithm for community detection that returns results of demonstrably higher quality than competing methods in shorter running times. I illustrate the method with applications to several published network data sets.

BACKGROUND

Diabetes is an increasingly important condition globally and robust estimates of its prevalence are required for allocating resources.

METHODS

Data sources from 1980 to April 2011 were sought and characterised. The Analytic Hierarchy Process (AHP) was used to select the most appropriate study or studies for each country, and estimates for countries without data were modelled. A logistic regression model was used to generate smoothed age-specific estimates which were applied to UN population estimates for 2011.

RESULTS

A total of 565 data sources were reviewed, of which 170 sources from 110 countries were selected. In 2011 there are 366 million people with diabetes, and this is expected to rise to 552 million by 2030. Most people with diabetes live in low- and middle-income countries, and these countries will also see the greatest increase over the next 19 years.

CONCLUSIONS

This paper builds on previous IDF estimates and shows that the global diabetes epidemic continues to grow. Recent studies show that previous estimates have been very conservative. The new IDF estimates use a simple and transparent approach and are consistent with recent estimates from the Global Burden of Disease study. IDF estimates will be updated annually.

Dentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts, that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue. In this study, we isolated a clonogenic, rapidly proliferative population of cells from adult human dental pulp. These DPSCs were then compared with human bone marrow stromal cells (BMSCs), known precursors of osteoblasts. Although they share a similar immunophenotype in vitro, functional studies showed that DPSCs produced only sporadic, but densely calcified nodules, and did not form adipocytes, whereas BMSCs routinely calcified throughout the adherent cell layer with clusters of lipid-laden adipocytes. When DPSCs were transplanted into immunocompromised mice, they generated a dentin-like structure lined with human odontoblast-like cells that surrounded a pulp-like interstitial tissue. In contrast, BMSCs formed lamellar bone containing osteocytes and surface-lining osteoblasts, surrounding a fibrous vascular tissue with active hematopoiesis and adipocytes. This study isolates postnatal human DPSCs that have the ability to form a dentin/pulp-like complex.

Thirty-four hundred lambda phage clones containing segments of the E. coli chromosome were isolated and used to construct a 4700 kb long integrated restriction map for eight six-base-recognizing enzymes by a rapid mass-analysis method. Our strategy was to measure the sizes of partial restriction enzyme digests by hybridization with a vector probe in a manner analogous to nucleotide sequencing. The data were sorted into groups by a computer program and the boundary clones were further correlated with each other using a mass hybridization method. These clones can be exploited for the isolation of any desired E. coli genes if their map positions are known. Also, the strategy is applicable to analyses of the genomes of other organisms.

InterProScan [E. M. Zdobnov and R. Apweiler (2001) Bioinformatics, 17, 847-848] is a tool that combines different protein signature recognition methods from the InterPro [N. J. Mulder, R. Apweiler, T. K. Attwood, A. Bairoch, A. Bateman, D. Binns, P. Bradley, P. Bork, P. Bucher, L. Cerutti et al. (2005) Nucleic Acids Res., 33, D201-D205] consortium member databases into one resource. At the time of writing there are 10 distinct publicly available databases in the application. Protein as well as DNA sequences can be analysed. A web-based version is accessible for academic and commercial organizations from the EBI (http://www.ebi.ac.uk/InterProScan/). In addition, a standalone Perl version and a SOAP Web Service [J. Snell, D. Tidwell and P. Kulchenko (2001) Programming Web Services with SOAP, 1st edn. O'Reilly Publishers, Sebastopol, CA, http://www.w3.org/TR/soap/] are also available to the users. Various output formats are supported and include text tables, XML documents, as well as various graphs to help interpret the results.

NMR chemical shifts in proteins depend strongly on local structure. The program TALOS establishes an empirical relation between 13C, 15N and 1H chemical shifts and backbone torsion angles phi and psi (Cornilescu et al. J Biomol NMR 13 289-302, 1999). Extension of the original 20-protein database to 200 proteins increased the fraction of residues for which backbone angles could be predicted from 65 to 74%, while reducing the error rate from 3 to 2.5%. Addition of a two-layer neural network filter to the database fragment selection process forms the basis for a new program, TALOS+, which further enhances the prediction rate to 88.5%, without increasing the error rate. Excluding the 2.5% of residues for which TALOS+ makes predictions that strongly differ from those observed in the crystalline state, the accuracy of predicted phi and psi angles, equals +/-13 degrees . Large discrepancies between predictions and crystal structures are primarily limited to loop regions, and for the few cases where multiple X-ray structures are available such residues are often found in different states in the different structures. The TALOS+ output includes predictions for individual residues with missing chemical shifts, and the neural network component of the program also predicts secondary structure with good accuracy.

5-azacytidine treatment of mouse C3H10T1/2 embryonic fibroblasts converts them to myoblasts at a frequency suggesting alteration of one or only a few closely linked regulatory loci. Assuming such loci to be differentially expressed as poly(A)+ RNA in proliferating myoblasts, we prepared proliferating myoblast-specific, subtracted cDNA probes to screen a myocyte cDNA library. Based on a number of criteria, three cDNAs were selected and characterized. We show that expression of one of these cDNAs transfected into C3H10T1/2 fibroblasts, where it is not normally expressed, is sufficient to convert them to stable myoblasts. Myogenesis also occurs, but to a lesser extent, when this cDNA is expressed in a number of other cell lines. The major open reading frame encoded by this cDNA contains a short protein segment similar to a sequence present in the myc protein family.

Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candidate molecular mediators of muscle atrophy, we performed transcript profiling. Although many genes were up-regulated in a single rat model of atrophy, only a small subset was universal in all atrophy models. Two of these genes encode ubiquitin ligases: Muscle RING Finger 1 (MuRF1), and a gene we designate Muscle Atrophy F-box (MAFbx), the latter being a member of the SCF family of E3 ubiquitin ligases. Overexpression of MAFbx in myotubes produced atrophy, whereas mice deficient in either MAFbx or MuRF1 were found to be resistant to atrophy. These proteins are potential drug targets for the treatment of muscle atrophy.

E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 10(9) to 10(10) transformants/micrograms with strains LE392 and DH5 alpha, and plasmids pUC18 and pBR329. The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.

Accumulating neuropsychological, electrophysiological and behavioural evidence suggests that the neural substrates of visual perception may be quite distinct from those underlying the visual control of actions. In other words, the set of object descriptions that permit identification and recognition may be computed independently of the set of descriptions that allow an observer to shape the hand appropriately to pick up an object. We propose that the ventral stream of projections from the striate cortex to the inferotemporal cortex plays the major role in the perceptual identification of objects, while the dorsal stream projecting from the striate cortex to the posterior parietal region mediates the required sensorimotor transformations for visually guided actions directed at such objects.

Although there has been considerable progress in reducing cancer incidence in the United States, the number of cancer survivors continues to increase due to the aging and growth of the population and improvements in survival rates. As a result, it is increasingly important to understand the unique medical and psychosocial needs of survivors and be aware of resources that can assist patients, caregivers, and health care providers in navigating the various phases of cancer survivorship. To highlight the challenges and opportunities to serve these survivors, the American Cancer Society and the National Cancer Institute estimated the prevalence of cancer survivors on January 1, 2012 and January 1, 2022, by cancer site. Data from Surveillance, Epidemiology, and End Results (SEER) registries were used to describe median age and stage at diagnosis and survival; data from the National Cancer Data Base and the SEER-Medicare Database were used to describe patterns of cancer treatment. An estimated 13.7 million Americans with a history of cancer were alive on January 1, 2012, and by January 1, 2022, that number will increase to nearly 18 million. The 3 most prevalent cancers among males are prostate (43%), colorectal (9%), and melanoma of the skin (7%), and those among females are breast (41%), uterine corpus (8%), and colorectal (8%). This article summarizes common cancer treatments, survival rates, and posttreatment concerns and introduces the new National Cancer Survivorship Resource Center, which has engaged more than 100 volunteer survivorship experts nationwide to develop tools for cancer survivors, caregivers, health care professionals, advocates, and policy makers.

As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 x 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 x 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10-12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10-12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-alpha for the last 24 h, where 50-70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level.

Nuclear transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the nuclear cloning approach is the derivation of 'customized' embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells-derived from mouse fibroblasts-can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.

A key step in RNA interference (RNAi) is assembly of the RISC, the protein-siRNA complex that mediates target RNA cleavage. Here, we show that the two strands of an siRNA duplex are not equally eligible for assembly into RISC. Rather, both the absolute and relative stabilities of the base pairs at the 5' ends of the two siRNA strands determine the degree to which each strand participates in the RNAi pathway. siRNA duplexes can be functionally asymmetric, with only one of the two strands able to trigger RNAi. Asymmetry is the hallmark of a related class of small, single-stranded, noncoding RNAs, microRNAs (miRNAs). We suggest that single-stranded miRNAs are initially generated as siRNA-like duplexes whose structures predestine one strand to enter the RISC and the other strand to be destroyed. Thus, the common step of RISC assembly is an unexpected source of asymmetry for both siRNA function and miRNA biogenesis.