The concept of cognitive impairment intervening between normal ageing and very early dementia has been in the literature for many years. Recently, the construct of mild cognitive impairment (MCI) has been proposed to designate an early, but abnormal, state of cognitive impairment. MCI has generated a great deal of research from both clinical and research perspectives. Numerous epidemiological studies have documented the accelerated rate of progression to dementia and Alzheimer's disease (AD) in MCI subjects and certain predictor variables appear valid. However, there has been controversy regarding the precise definition of the concept and its implementation in various clinical settings. Clinical subtypes of MCI have been proposed to broaden the concept and include prodromal forms of a variety of dementias. It is suggested that the diagnosis of MCI can be made in a fashion similar to the clinical diagnoses of dementia and AD. An algorithm is presented to assist the clinician in identifying subjects and subclassifying them into the various types of MCI. By refining the criteria for MCI, clinical trials can be designed with appropriate inclusion and exclusion restrictions to allow for the investigation of therapeutics tailored for specific targets and populations.

The actin cytoskeleton mediates a variety of essential biological functions in all eukaryotic cells. In addition to providing a structural framework around which cell shape and polarity are defined, its dynamic properties provide the driving force for cells to move and to divide. Understanding the biochemical mechanisms that control the organization of actin is thus a major goal of contemporary cell biology, with implications for health and disease. Members of the Rho family of small guanosine triphosphatases have emerged as key regulators of the actin cytoskeleton, and furthermore, through their interaction with multiple target proteins, they ensure coordinated control of other cellular activities such as gene transcription and adhesion.

With a stepwise degradation and terminal labeling procedure the 3'-terminal sequence of E. coli 16S ribosomal RNA is shown to be Pyd-A-C-C-U-C-C-U-U-A(OH). It is suggested that this region of the RNA is able to interact with mRNA and that the 3'-terminal U-U-A(OH) is involved in the termination of protein synthesis through base-pairing with terminator codons. The sequence A-C-C-U-C-C could recognize a conserved sequence found in the ribosome binding sites of various coliphage mRNAs; it may thus be involved in the formation of the mRNA.30S subunit complex.

Transforming growth factor-beta (TGF-beta) proteins regulate cell function, and have key roles in development and carcinogenesis. The intracellular effectors of TGF-beta signalling, the Smad proteins, are activated by receptors and translocate into the nucleus, where they regulate transcription. Although this pathway is inherently simple, combinatorial interactions in the heteromeric receptor and Smad complexes, receptor-interacting and Smad-interacting proteins, and cooperation with sequence-specific transcription factors allow substantial versatility and diversification of TGF-beta family responses. Other signalling pathways further regulate Smad activation and function. In addition, TGF-beta receptors activate Smad-independent pathways that not only regulate Smad signalling, but also allow Smad-independent TGF-beta responses.

BACKGROUND

Next-generation sequencing technologies generate very large numbers of short reads. Even with very deep genome coverage, short read lengths cause problems in de novo assemblies. The use of paired-end libraries with a fragment size shorter than twice the read length provides an opportunity to generate much longer reads by overlapping and merging read pairs before assembling a genome.

RESULTS

We present FLASH, a fast computational tool to extend the length of short reads by overlapping paired-end reads from fragment libraries that are sufficiently short. We tested the correctness of the tool on one million simulated read pairs, and we then applied it as a pre-processor for genome assemblies of Illumina reads from the bacterium Staphylococcus aureus and human chromosome 14. FLASH correctly extended and merged reads >99% of the time on simulated reads with an error rate of <1%. With adequately set parameters, FLASH correctly merged reads over 90% of the time even when the reads contained up to 5% errors. When FLASH was used to extend reads prior to assembly, the resulting assemblies had substantially greater N50 lengths for both contigs and scaffolds.

METHODS

The FLASH system is implemented in C and is freely available as open-source code at http://www.cbcb.umd.edu/software/flash.

BACKGROUND

t.magoc@gmail.com.

MicroRNAs are endogenous approximately 23-nucleotide RNAs that can pair to sites in the messenger RNAs of protein-coding genes to downregulate the expression from these messages. MicroRNAs are known to influence the evolution and stability of many mRNAs, but their global impact on protein output had not been examined. Here we use quantitative mass spectrometry to measure the response of thousands of proteins after introducing microRNAs into cultured cells and after deleting mir-223 in mouse neutrophils. The identities of the responsive proteins indicate that targeting is primarily through seed-matched sites located within favourable predicted contexts in 3' untranslated regions. Hundreds of genes were directly repressed, albeit each to a modest degree, by individual microRNAs. Although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. The impact of microRNAs on the proteome indicated that for most interactions microRNAs act as rheostats to make fine-scale adjustments to protein output.

MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs that mediate important gene-regulatory events by pairing to the mRNAs of protein-coding genes to direct their repression. Repression of these regulatory targets leads to decreased translational efficiency and/or decreased mRNA levels, but the relative contributions of these two outcomes have been largely unknown, particularly for endogenous targets expressed at low-to-moderate levels. Here, we use ribosome profiling to measure the overall effects on protein production and compare these to simultaneously measured effects on mRNA levels. For both ectopic and endogenous miRNA regulatory interactions, lowered mRNA levels account for most >>/=84%) of the decreased protein production. These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output.

Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. Here we characterize the transcriptional landscape of the four human HOX loci at five base pair resolution in 11 anatomic sites and identify 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes and possess unique sequence motifs, and their expression demarcates broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. We identified a 2.2 kilobase ncRNA residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states.

OBJECTIVE

This report presents the revised growth charts for the United States. It summarizes the history of the 1977 National Center for Health Statistics (NCHS) growth charts, reasons for the revision, data sources and statistical procedures used, and major features of the revised charts.

METHODS

Data from five national health examination surveys collected from 1963 to 1994 and five supplementary data sources were combined to establish an analytic growth chart data set. A variety of statistical procedures were used to produce smoothed percentile curves for infants (from birth to 36 months) and older children (from 2 to 20 years), using a two-stage approach. Initial curve smoothing for selected major percentiles was accomplished with various parametric and nonparametric procedures. In the second stage, a normalization procedure was used to generate z-scores that closely match the smoothed percentile curves.

RESULTS

The 14 NCHS growth charts were revised and new body mass index-for-age (BMI-for-age) charts were created for boys and girls (http://www.cdc.gov/growthcharts). The growth percentile curves for infants and children are based primarily on national survey data. Use of national data ensures a smooth transition from the charts for infants to those for older children. These data better represent the racial/ethnic diversity and the size and growth patterns of combined breast- and formula-fed infants in the United States. New features include addition of the 3rd and 97th percentiles for all charts and extension of all charts for children and adolescents to age 20 years.

CONCLUSIONS

Created with improved data and statistical curve smoothing procedures, the United States growth charts represent an enhanced instrument to evaluate the size and growth of infants and children.

The epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib are effective treatments for lung cancers with EGFR activating mutations, but these tumors invariably develop drug resistance. Here, we describe a gefitinib-sensitive lung cancer cell line that developed resistance to gefitinib as a result of focal amplification of the MET proto-oncogene. inhibition of MET signaling in these cells restored their sensitivity to gefitinib. MET amplification was detected in 4 of 18 (22%) lung cancer specimens that had developed resistance to gefitinib or erlotinib. We find that amplification of MET causes gefitinib resistance by driving ERBB3 (HER3)-dependent activation of PI3K, a pathway thought to be specific to EGFR/ERBB family receptors. Thus, we propose that MET amplification may promote drug resistance in other ERBB-driven cancers as well.

Pfam is a widely used database of protein families and domains. This article describes a set of major updates that we have implemented in the latest release (version 24.0). The most important change is that we now use HMMER3, the latest version of the popular profile hidden Markov model package. This software is approximately 100 times faster than HMMER2 and is more sensitive due to the routine use of the forward algorithm. The move to HMMER3 has necessitated numerous changes to Pfam that are described in detail. Pfam release 24.0 contains 11,912 families, of which a large number have been significantly updated during the past two years. Pfam is available via servers in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/).

The fine detail provided by sequencing-based transcriptome surveys suggests that RNA-seq is likely to become the platform of choice for interrogating steady state RNA. In order to discover biologically important changes in expression, we show that normalization continues to be an essential step in the analysis. We outline a simple and effective method for performing normalization and show dramatically improved results for inferring differential expression in simulated and publicly available data sets.

Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.

A variety of key events in apoptosis focus on mitochondria, including the release of caspase activators (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and participation of pro- and antiapoptotic Bcl-2 family proteins. The different signals that converge on mitochondria to trigger or inhibit these events and their downstream effects delineate several major pathways in physiological cell death.

OBJECTIVE

This report provides detailed information on how the 2000 Centers for Disease Control and Prevention (CDC) growth charts for the United States were developed, expanding upon the report that accompanied the initial release of the charts in 2000.

METHODS

The growth charts were developed with data from five national health examination surveys and limited supplemental data. Smoothed percentile curves were developed in two stages. In the first stage, selected empirical percentiles were smoothed with a variety of parametric and nonparametric procedures. In the second stage, parameters were created to obtain the final curves, additional percentiles and z-scores. The revised charts were evaluated using statistical and graphical measures.

RESULTS

The 1977 National Center for Health Statistics (NCHS) growth charts were revised for infants (birth to 36 months) and older children (2 to 20 years). New body mass index-for-age (BMI-for-age) charts were created. Use of national data improved the transition from the infant charts to those for older children. The evaluation of the charts found no large or systematic differences between the smoothed percentiles and the empirical data.

CONCLUSIONS

The 2000 CDC growth charts were developed with improved data and statistical procedures. Health care providers now have an instrument for growth screening that better represents the racial-ethnic diversity and combination of breast- and formula-feeding in the United States. It is recommended that these charts replace the 1977 NCHS charts when assessing the size and growth patterns of infants, children, and adolescents.

Breast cancer is characterized by a distinct metastatic pattern involving the regional lymph nodes, bone marrow, lung and liver. Tumour cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. Here we report that the chemokine receptors CXCR4 and CCR7 are highly expressed in human breast cancer cells, malignant breast tumours and metastases. Their respective ligands CXCL12/SDF-1alpha and CCL21/6Ckine exhibit peak levels of expression in organs representing the first destinations of breast cancer metastasis. In breast cancer cells, signalling through CXCR4 or CCR7 mediates actin polymerization and pseudopodia formation, and subsequently induces chemotactic and invasive responses. In vivo, neutralizing the interactions of CXCL12/CXCR4 significantly impairs metastasis of breast cancer cells to regional lymph nodes and lung. Malignant melanoma, which has a similar metastatic pattern as breast cancer but also a high incidence of skin metastases, shows high expression levels of CCR10 in addition to CXCR4 and CCR7. Our findings indicate that chemokines and their receptors have a critical role in determining the metastatic destination of tumour cells.

This article reports the development and validation of a scale to measure global life satisfaction, the Satisfaction With Life Scale (SWLS). Among the various components of subjective well-being, the SWLS is narrowly focused to assess global life satisfaction and does not tap related constructs such as positive affect or loneliness. The SWLS is shown to have favorable psychometric properties, including high internal consistency and high temporal reliability. Scores on the SWLS correlate moderately to highly with other measures of subjective well-being, and correlate predictably with specific personality characteristics. It is noted that the SWLS is Suited for use with different age groups, and other potential uses of the scale are discussed.

There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts. However, the functional significance of these transcripts has been particularly controversial. Although there are some well-characterized examples, most (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here we report a new approach to identifying large non-coding RNAs using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified approximately 1,600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening non-coding RNAs (lincRNAs) show strong purifying selection in their genomic loci, exonic sequences and promoter regions, with greater than 95% showing clear evolutionary conservation. We also developed a functional genomics approach that assigns putative functions to each lincRNA, demonstrating a diverse range of roles for lincRNAs in processes from embryonic stem cell pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFkappaB, Sox2, Oct4 (also known as Pou5f1) and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes.

Cells of a multicellular organism are genetically homogeneous but structurally and functionally heterogeneous owing to the differential expression of genes. Many of these differences in gene expression arise during development and are subsequently retained through mitosis. Stable alterations of this kind are said to be 'epigenetic', because they are heritable in the short term but do not involve mutations of the DNA itself. Research over the past few years has focused on two molecular mechanisms that mediate epigenetic phenomena: DNA methylation and histone modifications. Here, we review advances in the understanding of the mechanism and role of DNA methylation in biological processes. Epigenetic effects by means of DNA methylation have an important role in development but can also arise stochastically as animals age. Identification of proteins that mediate these effects has provided insight into this complex process and diseases that occur when it is perturbed. External influences on epigenetic processes are seen in the effects of diet on long-term diseases such as cancer. Thus, epigenetic mechanisms seem to allow an organism to respond to the environment through changes in gene expression. The extent to which environmental effects can provoke epigenetic responses represents an exciting area of future research.

The Ribosomal Database Project (RDP) provides researchers with quality-controlled bacterial and archaeal small subunit rRNA alignments and analysis tools. An improved alignment strategy uses the Infernal secondary structure aware aligner to provide a more consistent higher quality alignment and faster processing of user sequences. Substantial new analysis features include a new Pyrosequencing Pipeline that provides tools to support analysis of ultra high-throughput rRNA sequencing data. This pipeline offers a collection of tools that automate the data processing and simplify the computationally intensive analysis of large sequencing libraries. In addition, a new Taxomatic visualization tool allows rapid visualization of taxonomic inconsistencies and suggests corrections, and a new class Assignment Generator provides instructors with a lesson plan and individualized teaching materials. Details about RDP data and analytical functions can be found at http://rdp.cme.msu.edu/.

The function of brown adipose tissue is to transfer energy from food into heat; physiologically, both the heat produced and the resulting decrease in metabolic efficiency can be of significance. Both the acute activity of the tissue, i.e., the heat production, and the recruitment process in the tissue (that results in a higher thermogenic capacity) are under the control of norepinephrine released from sympathetic nerves. In thermoregulatory thermogenesis, brown adipose tissue is essential for classical nonshivering thermogenesis (this phenomenon does not exist in the absence of functional brown adipose tissue), as well as for the cold acclimation-recruited norepinephrine-induced thermogenesis. Heat production from brown adipose tissue is activated whenever the organism is in need of extra heat, e.g., postnatally, during entry into a febrile state, and during arousal from hibernation, and the rate of thermogenesis is centrally controlled via a pathway initiated in the hypothalamus. Feeding as such also results in activation of brown adipose tissue; a series of diets, apparently all characterized by being low in protein, result in a leptin-dependent recruitment of the tissue; this metaboloregulatory thermogenesis is also under hypothalamic control. When the tissue is active, high amounts of lipids and glucose are combusted in the tissue. The development of brown adipose tissue with its characteristic protein, uncoupling protein-1 (UCP1), was probably determinative for the evolutionary success of mammals, as its thermogenesis enhances neonatal survival and allows for active life even in cold surroundings.

Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 10(10) random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.

Toll-like receptors (TLRs) are a family of innate immune-recognition receptors that recognize molecular patterns associated with microbial pathogens, and induce antimicrobial immune responses. Double-stranded RNA (dsRNA) is a molecular pattern associated with viral infection, because it is produced by most viruses at some point during their replication. Here we show that mammalian TLR3 recognizes dsRNA, and that activation of the receptor induces the activation of NF-kappaB and the production of type I interferons (IFNs). TLR3-deficient (TLR3-/-) mice showed reduced responses to polyinosine-polycytidylic acid (poly(I:C)), resistance to the lethal effect of poly(I:C) when sensitized with d-galactosamine (d-GalN), and reduced production of inflammatory cytokines. MyD88 is an adaptor protein that is shared by all the known TLRs. When activated by poly(I:C), TLR3 induces cytokine production through a signalling pathway dependent on MyD88. Moreover, poly(I:C) can induce activation of NF-kappaB and mitogen-activated protein (MAP) kinases independently of MyD88, and cause dendritic cells to mature.

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans are among the most notable prion diseases. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc. Transgenetic studies argue that PrPSc acts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein. Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrPSc. While knowledge about prions has profound implications for studies of the structural plasticity of proteins, investigations of prion diseases suggest that new strategies for the prevention and treatment of these disorders may also find application in the more common degenerative diseases.