Due to strong electric fields at the surface, the absorption and scattering of electromagnetic radiation by noble metal nanoparticles are strongly enhanced. These unique properties provide the potential of designing novel optically active reagents for simultaneous molecular imaging and photothermal cancer therapy. It is desirable to use agents that are active in the near-infrared (NIR) region of the radiation spectrum to minimize the light extinction by intrinsic chromophores in native tissue. Gold nanorods with suitable aspect ratios (length divided by width) can absorb and scatter strongly in the NIR region (650-900 nm). In the present work, we provide an in vitro demonstration of gold nanorods as novel contrast agents for both molecular imaging and photothermal cancer therapy. Nanorods are synthesized and conjugated to anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies and incubated in cell cultures with a nonmalignant epithelial cell line (HaCat) and two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3). The anti-EGFR antibody-conjugated nanorods bind specifically to the surface of the malignant-type cells with a much higher affinity due to the overexpressed EGFR on the cytoplasmic membrane of the malignant cells. As a result of the strongly scattered red light from gold nanorods in dark field, observed using a laboratory microscope, the malignant cells are clearly visualized and diagnosed from the nonmalignant cells. It is found that, after exposure to continuous red laser at 800 nm, malignant cells require about half the laser energy to be photothermally destroyed than the nonmalignant cells. Thus, both efficient cancer cell diagnostics and selective photothermal therapy are realized at the same time.

This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequences to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurements of heteronuclear nuclear Overhauser effects (NOE) and spin-lattice (T1) and spin-spin (T2) 15N or 13C relaxation times with a sensitivity similar to those of many homonuclear 1H experiments. Because T1 values and heteronuclear NOEs are sensitive to high-frequency motions (10(8)-10(12) s-1) while T2 values are also a function of much slower processes, it is possible to explore dynamic events occurring over a large time scale. We have applied these techniques to investigate the backbone dynamics of the protein staphylococcal nuclease (S. Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+ and labeled uniformly with 15N. T1, T2, and NOE values were obtained for over 100 assigned backbone amide nitrogens in the protein. Values of the order parameter (S), characterizing the extent of rapid 1H-15N bond motions, have been determined. These results suggest that there is no correlation between these rapid small amplitude motions and secondary structure for S. Nase. In contrast, 15N line widths suggest a possible correlation between secondary structure and motions on the millisecond time scale. In particular, the loop region between residues 42 and 56 appears to be considerably more flexible on this slow time scale than the rest of the protein.

The phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development. Snail, a zinc-finger transcription factor, triggers this process by repressing E-cadherin expression; however, the mechanisms that regulate Snail remain elusive. Here we find that Snail is highly unstable, with a short half-life about 25 min. We show that GSK-3beta binds to and phosphorylates Snail at two consensus motifs to dually regulate the function of this protein. Phosphorylation of the first motif regulates its beta-Trcp-mediated ubiquitination, whereas phosphorylation of the second motif controls its subcellular localization. A variant of Snail (Snail-6SA), which abolishes these phosphorylations, is much more stable and resides exclusively in the nucleus to induce EMT. Furthermore, inhibition of GSK-3beta results in the upregulation of Snail and downregulation of E-cadherin in vivo. Thus, Snail and GSK-3beta together function as a molecular switch for many signalling pathways that lead to EMT.

Some statistical properties of samples of DNA sequences are studied under an infinite-site neutral model with recombination. The two quantities of interest are R, the number of recombination events in the history of a sample of sequences, and RM, the number of recombination events that can be parsimoniously inferred from a sample of sequences. Formulas are derived for the mean and variance of R. In contrast to R, RM can be determined from the sample. Since no formulas are known for the mean and variance of RM, they are estimated with Monte Carlo simulations. It is found that RM is often much less than R, therefore, the number of recombination events may be greatly under-estimated in a parsimonious reconstruction of the history of a sample. The statistic RM can be used to estimate the product of the recombination rate and the population size or, if the recombination rate is known, to estimate the population size. To illustrate this, DNA sequences from the Adh region of Drosophila melanogaster are used to estimate the effective population size of this species.

A specific assay has been developed for a blood-borne non-A, non-B hepatitis (NANBH) virus in which a polypeptide synthesized in recombinant yeast clones of the hepatitis C virus (HCV) is used to capture circulating viral antibodies. HCV antibodies were detected in six of seven human sera that were shown previously to transmit NANBH to chimpanzees. Assays of ten blood transfusions in the United States that resulted in chronic NANBH revealed that there was at least one positive blood donor in nine of these cases and that all ten recipients seroconverted during their illnesses. About 80 percent of chronic, post-transfusion NANBH (PT-NANBH) patients from Italy and Japan had circulating HCV antibody; a much lower frequency (15 percent) was observed in acute, resolving infections. In addition, 58 percent of NANBH patients from the United States with no identifiable source of parenteral exposure to the virus were also positive for HCV antibody. These data indicate that HCV is a major cause of NANBH throughout the world.

Despite the presence of mutations in APC or beta-catenin, which are believed to activate the Wnt signalling cascade constitutively, most colorectal cancers show cellular heterogeneity when beta-catenin localization is analysed, indicating a more complex regulation of Wnt signalling. We explored this heterogeneity with a Wnt reporter construct and observed that high Wnt activity functionally designates the colon cancer stem cell (CSC) population. In adenocarcinomas, high activity of the Wnt pathway is observed preferentially in tumour cells located close to stromal myofibroblasts, indicating that Wnt activity and cancer stemness may be regulated by extrinsic cues. In agreement with this notion, myofibroblast-secreted factors, specifically hepatocyte growth factor, activate beta-catenin-dependent transcription and subsequently CSC clonogenicity. More significantly, myofibroblast-secreted factors also restore the CSC phenotype in more differentiated tumour cells both in vitro and in vivo. We therefore propose that stemness of colon cancer cells is in part orchestrated by the microenvironment and is a much more dynamic quality than previously expected that can be defined by high Wnt activity.

Chromosomal microarray (CMA) is increasingly utilized for genetic testing of individuals with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). Performing CMA and G-banded karyotyping on every patient substantially increases the total cost of genetic testing. The International Standard Cytogenomic Array (ISCA) Consortium held two international workshops and conducted a literature review of 33 studies, including 21,698 patients tested by CMA. We provide an evidence-based summary of clinical cytogenetic testing comparing CMA to G-banded karyotyping with respect to technical advantages and limitations, diagnostic yield for various types of chromosomal aberrations, and issues that affect test interpretation. CMA offers a much higher diagnostic yield (15%-20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCA than a G-banded karyotype ( approximately 3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and low-level mosaicism are generally not detectable by arrays, but these are relatively infrequent causes of abnormal phenotypes in this population (<1%). Available evidence strongly supports the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with DD/ID, ASD, or MCA. G-banded karyotype analysis should be reserved for patients with obvious chromosomal syndromes (e.g., Down syndrome), a family history of chromosomal rearrangement, or a history of multiple miscarriages.

An outward current of unknown nature increases significantly when cardiac cells are treated with cyanide or subjected to hypoxia, and decreases on intracellular injection of ATP. We report here that application of the patch-clamp technique to CN-treated mammalian heart cells reveals specific K+ channels which are depressed by intracellular ATP (ATPi) at levels greater than 1 mM. For these channels, conductance in the outward direction is much larger than the inward rectifier K+ channel which is insensitive to ATP. AMP had no effect on the ATP-sensitive K+ channel, and ADP was less effective than ATP. Thus, the ATP-sensitive K+ channel seems to be important for regulation of cellular energy metabolism in the control of membrane excitability.

'New' memories are initially labile and sensitive to disruption before being consolidated into stable long-term memories. Much evidence indicates that this consolidation involves the synthesis of new proteins in neurons. The lateral and basal nuclei of the amygdala (LBA) are believed to be a site of memory storage in fear learning. Infusion of the protein synthesis inhibitor anisomycin into the LBA shortly after training prevents consolidation of fear memories. Here we show that consolidated fear memories, when reactivated during retrieval, return to a labile state in which infusion of anisomycin shortly after memory reactivation produces amnesia on later tests, regardless of whether reactivation was performed 1 or 14 days after conditioning. The same treatment with anisomycin, in the absence of memory reactivation, left memory intact. Consistent with a time-limited role for protein synthesis production in consolidation, delay of the infusion until six hours after memory reactivation produced no amnesia. Our data show that consolidated fear memories, when reactivated, return to a labile state that requires de novo protein synthesis for reconsolidation. These findings are not predicted by traditional theories of memory consolidation.

The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (tRNA). A metal ion stabilizes a kink in the mRNA that demarcates the boundary between A and P sites, which is potentially important to prevent slippage of mRNA. Metal ions also stabilize the intersubunit interface. The interactions of E-site tRNA with the 50S subunit have both similarities and differences compared to those in the archaeal ribosome. The structure also rationalizes much biochemical and genetic data on translation.

With the advent of next generation sequencing methods and progress in transcriptome analysis, it became obvious that the human genome contains much more than just protein-coding genes. In fact, up to 70% of our genome is transcribed into RNA that does not serve as templates for proteins. In this review, we focus on the emerging roles of these long non-coding RNAs (lncRNAs) in the field of tumor biology. Long ncRNAs were found to be deregulated in several human cancers and show tissue-specific expression. Functional studies revealed a broad spectrum of mechanisms applied by lncRNAs such as HOTAIR, MALAT1, ANRIL or lincRNA-p21 to fulfill their functions. Here, we link the cellular processes influenced by long ncRNAs to the hallmarks of cancer and therefore provide an ncRNA point-of-view on tumor biology. This should stimulate new research directions and therapeutic options considering long ncRNAs as novel prognostic markers and therapeutic targets.

To better understand the diversity of small silencing RNAs expressed in plants, we employed high-throughput pyrosequencing to obtain 887,000 reads corresponding to Arabidopsis thaliana small RNAs. They represented 340,000 unique sequences, a substantially greater diversity than previously obtained in any species. Most of the small RNAs had the properties of heterochromatic small interfering RNAs (siRNAs) associated with DNA silencing in that they were preferentially 24 nucleotides long and mapped to intergenic regions. Their density was greatest in the proximal and distal pericentromeric regions, with only a slightly preferential propensity to match repetitive elements. Also present were 38 newly identified microRNAs (miRNAs) and dozens of other plausible candidates. One miRNA mapped within an intron of DICER-LIKE 1 (DCL1), suggesting a second homeostatic autoregulatory mechanism for DCL1 expression; another defined the phase for siRNAs deriving from a newly identified trans-acting siRNA gene (TAS4); and two depended on DCL4 rather than DCL1 for their accumulation, indicating a second pathway for miRNA biogenesis in plants. More generally, our results revealed the existence of a layer of miRNA-based control beyond that found previously that is evolutionarily much more fluid, employing many newly emergent and diverse miRNAs, each expressed in specialized tissues or at low levels under standard growth conditions.

Point mutations frequently cause genetic diseases by disrupting the correct pattern of pre-mRNA splicing. The effect of a point mutation within a coding sequence is traditionally attributed to the deduced change in the corresponding amino acid. However, some point mutations can have much more severe effects on the structure of the encoded protein, for example when they inactivate an exonic splicing enhancer (ESE), thereby resulting in exon skipping. ESEs also appear to be especially important in exons that normally undergo alternative splicing. Different classes of ESE consensus motifs have been described, but they are not always easily identified. ESEfinder (http://exon.cshl.edu/ESE/) is a web-based resource that facilitates rapid analysis of exon sequences to identify putative ESEs responsive to the human SR proteins SF2/ASF, SC35, SRp40 and SRp55, and to predict whether exonic mutations disrupt such elements.

BACKGROUND

Although much is known about the natural history of systemic lupus erythematosus (SLE), the development of SLE autoantibodies before the diagnosis of the disease has not been extensively explored. We investigated the onset and progression of autoantibody development before the clinical diagnosis.

METHODS

The Department of Defense Serum Repository contains approximately 30 million specimens prospectively collected from more than 5 million U.S. Armed Forces personnel. We evaluated serum samples obtained from 130 persons before they received a diagnosis of SLE, along with samples from matched controls.

RESULTS

In 115 of the 130 patients with SLE (88 percent), at least one SLE autoantibody tested was present before the diagnosis (up to 9.4 years earlier; mean, 3.3 years). Antinuclear antibodies were present in 78 percent (at a dilution of 1:120 or more), anti-double-stranded DNA antibodies in 55 percent, anti-Ro antibodies in 47 percent, anti-La antibodies in 34 percent, anti-Sm antibodies in 32 percent, anti-nuclear ribonucleoprotein antibodies in 26 percent, and antiphospholipid antibodies in 18 percent. Antinuclear, antiphospholipid antibodies, anti-Ro, and anti-La antibodies were present earlier than anti-Sm and anti-nuclear ribonucleoprotein antibodies (a mean of 3.4 years before the diagnosis vs. 1.2 years, P=0.005). Anti-double-stranded DNA antibodies, with a mean onset 2.2 years before the diagnosis, were found later than antinuclear antibodies (P=0.06) and earlier than anti-nuclear ribonucleoprotein antibodies (P=0.005). For many patients, the earliest available serum sample was positive; therefore, these measures of the average time from the first positive antibody test to the diagnosis are underestimates of the time from the development of antibodies to the diagnosis. Of the 130 initial matched controls, 3.8 percent were positive for one or more autoantibodies.

CONCLUSIONS

Autoantibodies are typically present many years before the diagnosis of SLE. Furthermore, the appearance of autoantibodies in patients with SLE tends to follow a predictable course, with a progressive accumulation of specific autoantibodies before the onset of SLE, while patients are still asymptomatic.

The past decade has witnessed hundreds of reports declaring or refuting genetic association with putative Alzheimer disease susceptibility genes. This wealth of information has become increasingly difficult to follow, much less interpret. We have created a publicly available, continuously updated database that comprehensively catalogs all genetic association studies in the field of Alzheimer disease (http://www.alzgene.org). We performed systematic meta-analyses for each polymorphism with available genotype data in at least three case-control samples. In addition to identifying the epsilon4 allele of APOE and related effects, we pinpointed over a dozen potential Alzheimer disease susceptibility genes (ACE, CHRNB2, CST3, ESR1, GAPDHS, IDE, MTHFR, NCSTN, PRNP, PSEN1, TF, TFAM and TNF) with statistically significant allelic summary odds ratios (ranging from 1.11-1.38 for risk alleles and 0.92-0.67 for protective alleles). Our database provides a powerful tool for deciphering the genetics of Alzheimer disease, and it serves as a potential model for tracking the most viable gene candidates in other genetically complex diseases.

Did the end-Cretaceous mass extinction event, by eliminating non-avian dinosaurs and most of the existing fauna, trigger the evolutionary radiation of present-day mammals? Here we construct, date and analyse a species-level phylogeny of nearly all extant Mammalia to bring a new perspective to this question. Our analyses of how extant lineages accumulated through time show that net per-lineage diversification rates barely changed across the Cretaceous/Tertiary boundary. Instead, these rates spiked significantly with the origins of the currently recognized placental superorders and orders approximately 93 million years ago, before falling and remaining low until accelerating again throughout the Eocene and Oligocene epochs. Our results show that the phylogenetic 'fuses' leading to the explosion of extant placental orders are not only very much longer than suspected previously, but also challenge the hypothesis that the end-Cretaceous mass extinction event had a major, direct influence on the diversification of today's mammals.

The laboratory mouse is the most widely used mammalian model organism in biomedical research. The 2.6 × 10(9) bases of the mouse genome possess a high degree of conservation with the human genome, so a thorough annotation of the mouse genome will be of significant value to understanding the function of the human genome. So far, most of the functional sequences in the mouse genome have yet to be found, and the cis-regulatory sequences in particular are still poorly annotated. Comparative genomics has been a powerful tool for the discovery of these sequences, but on its own it cannot resolve their temporal and spatial functions. Recently, ChIP-Seq has been developed to identify cis-regulatory elements in the genomes of several organisms including humans, Drosophila melanogaster and Caenorhabditis elegans. Here we apply the same experimental approach to a diverse set of 19 tissues and cell types in the mouse to produce a map of nearly 300,000 murine cis-regulatory sequences. The annotated sequences add up to 11% of the mouse genome, and include more than 70% of conserved non-coding sequences. We define tissue-specific enhancers and identify potential transcription factors regulating gene expression in each tissue or cell type. Finally, we show that much of the mouse genome is organized into domains of coordinately regulated enhancers and promoters. Our results provide a resource for the annotation of functional elements in the mammalian genome and for the study of mechanisms regulating tissue-specific gene expression.

The endoplasmic reticulum is responsible for much of a cell's protein synthesis and folding, but it also has an important role in sensing cellular stress. Recently, it has been shown that the endoplasmic reticulum mediates a specific set of intracellular signalling pathways in response to the accumulation of unfolded or misfolded proteins, and these pathways are collectively known as the unfolded-protein response. New observations suggest that the unfolded-protein response can initiate inflammation, and the coupling of these responses in specialized cells and tissues is now thought to be fundamental in the pathogenesis of inflammatory diseases. The knowledge gained from this emerging field will aid in the development of therapies for modulating cellular stress and inflammation.

A quantitative method is described for the measurement of intralysosomal pH in living cells. Fluorescein isothiocyanate-labeled dextran (FD) is endocytized and accumulates in lysosomes where it remains without apparent degradation. The fluorescence spectrum of this compound changes with pH in the range 4-7 and is not seriously affected by FD concentration, ionic strength, or protein concentration. Living cells on coverslips are mounted in a spectrofluorometer cell and can be perfused with various media. The normal pH inside macrophage lysosomes seems to be 4.7-4.8, although it can drop transiently as low as 4.5. Exposure of the cells to various weak bases and to acidic potassium ionophores causes the pH to increase. The changes in pH are much more rapid than is the intralysosomal accumulation of the weak bases. Inhibitors of glycolysis (2-deoxyglucose) and of oxidative phosphorylation (cyanide or azide) added together, but not separately, cause the intralysosomal pH to increase. These results provide evidence for the existence of an active proton accumulation mechanism in the lysosomal membrane and support the theory of lysosomal accumulation of weak bases by proton trapping.

Much biomedical research is observational. The reporting of such research is often inadequate, which hampers the assessment of its strengths and weaknesses and of a study's generalisability. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Initiative developed recommendations on what should be included in an accurate and complete report of an observational study. We defined the scope of the recommendations to cover three main study designs: cohort, case-control, and cross-sectional studies. We convened a 2-day workshop in September 2004, with methodologists, researchers, and journal editors to draft a checklist of items. This list was subsequently revised during several meetings of the coordinating group and in e-mail discussions with the larger group of STROBE contributors, taking into account empirical evidence and methodological considerations. The workshop and the subsequent iterative process of consultation and revision resulted in a checklist of 22 items (the STROBE Statement) that relate to the title, abstract, introduction, methods, results, and discussion sections of articles. 18 items are common to all three study designs and four are specific for cohort, case-control, or cross-sectional studies. A detailed Explanation and Elaboration document is published separately and is freely available on the Web sites of PLoS Medicine, Annals of Internal Medicine, and Epidemiology. We hope that the STROBE Statement will contribute to improving the quality of reporting of observational studies.

A new measure of the extent of population subdivision as inferred from allele frequencies at microsatellite loci is proposed and tested with computer simulations. This measure, called R(ST), is analogous to Wright's F(ST) in representing the proportion of variation between populations. It differs in taking explicit account of the mutation process at microsatellite loci, for which a generalized stepwise mutation model appears appropriate. Simulations of subdivided populations were carried out to test the performance of R(ST) and F(ST). It was found that, under the generalized stepwise mutation model, R(ST) provides relatively unbiased estimates of migration rates and times of population divergence while F(ST) tends to show too much population similarity, particularly when migration rates are low or divergence times are long [corrected].

In the absence of prior knowledge about population relations, investigators frequently employ a strategy that uses the data to help them decide whether to adjust for a variable. The authors compared the performance of several such strategies for fitting multiplicative Poisson regression models to cohort data: 1) the "change-in-estimate" strategy, in which a variable is controlled if the adjusted and unadjusted estimates differ by some important amount; 2) the "significance-test-of-the-covariate" strategy, in which a variable is controlled if its coefficient is significantly different from zero at some predetermined significance level; 3) the "significance-test-of-the-difference" strategy, which tests the difference between the adjusted and unadjusted exposure coefficients; 4) the "equivalence-test-of-the-difference" strategy, which significance-tests the equivalence of the adjusted and unadjusted exposure coefficients; and 5) a hybrid strategy that takes a weighted average of adjusted and unadjusted estimates. Data were generated from 8,100 population structures at each of several sample sizes. The performance of the different strategies was evaluated by computing bias, mean squared error, and coverage rates of confidence intervals. At least one variation of each strategy that was examined performed acceptably. The change-in-estimate and equivalence-test-of-the-difference strategies performed best when the cut-point for deciding whether crude and adjusted estimates differed by an important amount was set to a low value (10%). The significance test strategies performed best when the alpha level was set to much higher than conventional levels (0.20).

Much of a cell's activity is organized as a network of interacting modules: sets of genes coregulated to respond to different conditions. We present a probabilistic method for identifying regulatory modules from gene expression data. Our procedure identifies modules of coregulated genes, their regulators and the conditions under which regulation occurs, generating testable hypotheses in the form 'regulator X regulates module Y under conditions W'. We applied the method to a Saccharomyces cerevisiae expression data set, showing its ability to identify functionally coherent modules and their correct regulators. We present microarray experiments supporting three novel predictions, suggesting regulatory roles for previously uncharacterized proteins.

Entropy, as it relates to dynamical systems, is the rate of information production. Methods for estimation of the entropy of a system represented by a time series are not, however, well suited to analysis of the short and noisy data sets encountered in cardiovascular and other biological studies. Pincus introduced approximate entropy (ApEn), a set of measures of system complexity closely related to entropy, which is easily applied to clinical cardiovascular and other time series. ApEn statistics, however, lead to inconsistent results. We have developed a new and related complexity measure, sample entropy (SampEn), and have compared ApEn and SampEn by using them to analyze sets of random numbers with known probabilistic character. We have also evaluated cross-ApEn and cross-SampEn, which use cardiovascular data sets to measure the similarity of two distinct time series. SampEn agreed with theory much more closely than ApEn over a broad range of conditions. The improved accuracy of SampEn statistics should make them useful in the study of experimental clinical cardiovascular and other biological time series.