BACKGROUND

Mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene in lung cancers are associated with increased sensitivity of these cancers to drugs that inhibit EGFR kinase activity. However, the role of such mutations in the pathogenesis of lung cancers is unclear.

METHODS

We sequenced exons 18-21 of the EGFR TK domain from genomic DNA isolated from 617 non-small-cell lung cancers (NSCLCs) and 524 normal lung tissue samples from the same patients and 36 neuroendocrine lung tumors collected from patients in Japan, Taiwan, the United States, and Australia and from 243 other epithelial cancers. Mutation status was compared with clinicopathologic features and with the presence of mutations in KRAS, a gene in the EGFR signaling pathway that is also frequently mutated in lung cancers. All statistical tests were two sided.

RESULTS

We detected a total of 134 EGFR TK domain mutations in 130 (21%) of the 617 NSCLCs but not in any of the other carcinomas, nor in nonmalignant lung tissue from the same patients. In NSCLC patients, EGFR TK domain mutations were statistically significantly more frequent in never smokers than ever smokers (51% versus 10%), in adenocarcinomas versus cancer of other histologies (40% versus 3%), in patients of East Asian ethnicity versus other ethnicities (30% versus 8%), and in females versus males (42% versus 14%; all P < .001). EGFR TK domain mutation status was not associated with patient age at diagnosis, clinical stage, the presence of bronchioloalveolar histologic features, or overall survival. The EGFR TK domain mutations we detected were of three common types: in-frame deletions in exon 19, single missense mutations in exon 21, and in-frame duplications/insertions in exon 20. Rare missense mutations were also detected in exons 18, 20, and 21. KRAS gene mutations were present in 50 (8%) of the 617 NSCLCs but not in any tumors with an EGFR TK domain mutation.

CONCLUSIONS

Mutations in either the EGFR TK domain or the KRAS gene can lead to lung cancer pathogenesis. EGFR TK domain mutations are the first molecular change known to occur specifically in never smokers.

The non-covalent assembly of proteins that fold separately is central to many biological processes, and differs from the permanent macromolecular assembly of protein subunits in oligomeric proteins. We performed an analysis of the atomic structure of the recognition sites seen in 75 protein-protein complexes of known three-dimensional structure: 24 protease-inhibitor, 19 antibody-antigen and 32 other complexes, including nine enzyme-inhibitor and 11 that are involved in signal transduction.The size of the recognition site is related to the conformational changes that occur upon association. Of the 75 complexes, 52 have "standard-size" interfaces in which the total area buried by the components in the recognition site is 1600 (+/-400) A2. In these complexes, association involves only small changes of conformation. Twenty complexes have "large" interfaces burying 2000 to 4660 A2, and large conformational changes are seen to occur in those cases where we can compare the structure of complexed and free components. The average interface has approximately the same non-polar character as the protein surface as a whole, and carries somewhat fewer charged groups. However, some interfaces are significantly more polar and others more non-polar than the average. Of the atoms that lose accessibility upon association, half make contacts across the interface and one-third become fully inaccessible to the solvent. In the latter case, the Voronoi volume was calculated and compared with that of atoms buried inside proteins. The ratio of the two volumes was 1.01 (+/-0.03) in all but 11 complexes, which shows that atoms buried at protein-protein interfaces are close-packed like the protein interior. This conclusion could be extended to the majority of interface atoms by including solvent positions determined in high-resolution X-ray structures in the calculation of Voronoi volumes. Thus, water molecules contribute to the close-packing of atoms that insure complementarity between the two protein surfaces, as well as providing polar interactions between the two proteins.

The engagement of programmed death 1 (PD-1) to its ligands, PD-L1 and PD-L2, inhibits proliferation and cytokine production mediated by antibodies to CD3 (refs. 5,6,7). Blocking the PD-1-PD-L1 pathway in mice chronically infected with lymphocytic choriomeningitis virus restores the capacity of exhausted CD8(+) T cells to undergo proliferation, cytokine production and cytotoxic activity and, consequently, results in reduced viral load. During chronic HIV infection, HIV-specific CD8(+) T cells are functionally impaired, showing a reduced capacity to produce cytokines and effector molecules as well as an impaired capacity to proliferate. Here, we found that PD-1 was upregulated on HIV-specific CD8(+) T cells; PD-1 expression levels were significantly correlated both with viral load and with the reduced capacity for cytokine production and proliferation of HIV-specific CD8(+) T cells. Notably, cytomegalovirus (CMV)-specific CD8(+) T cells from the same donors did not upregulate PD-1 and maintained the production of high levels of cytokines. Blocking PD-1 engagement to its ligand (PD-L1) enhanced the capacity of HIV-specific CD8(+) T cells to survive and proliferate and led to an increased production of cytokines and cytotoxic molecules in response to cognate antigen. The accumulation of HIV-specific dysfunctional CD8(+) T cells in the infected host could prevent the renewal of a functionally competent HIV-specific CD8(+) repertoire.

We investigated two outbreaks of an unusual gastrointestinal illness that affected at least 47 people in Oregon and Michigan in February through March and May through June 1982. The illness was characterized by severe crampy abdominal pain, initially watery diarrhea followed by grossly bloody diarrhea, and little or no fever. It was associated with eating at restaurants belonging to the same fast-food restaurant chain in Oregon (P less than 0.005) and Michigan (P = 0.0005) and with eating any of three sandwiches containing three ingredients in common (beef patty, rehydrated onions, and pickles). Stool cultures did not yield previously recognized pathogens. However, a rare Escherichia coli serotype, O157:H7, that was not invasive or toxigenic by standard tests was isolated from 9 of 12 stools collected within four days of onset of illness in both outbreaks combined, and from a beef patty from a suspected lot of meat in Michigan. The only known previous isolation of this serotype was from a sporadic case of hemorrhagic colitis in 1975. This report describes a clinically distinctive gastrointestinal illness associated with E. coli O157:H7, apparently transmitted by undercooked meat.

Up-to-date statistics on cancer occurrence and outcome are essential for the planning and evaluation of programmes for cancer control. Since the relevant information for 2008 is not generally available as yet, we used statistical models to estimate incidence and mortality data for 25 cancers in 40 European countries (grouped and individually) in 2008. The calculations are based on published data. If not collected, national rates were estimated from national mortality data and incidence and mortality data provided by local cancer registries of the same or neighbouring country. The estimated 2008 rates were applied to the corresponding country population estimates for 2008 to obtain an estimate of the numbers of cancer cases and deaths in Europe in 2008. There were an estimated 3.2 million new cases of cancer and 1.7 million deaths from cancer in 2008. The most common cancers were colorectal cancers (436,000 cases, 13.6% of the total), breast cancer (421,000, 13.1%), lung cancer (391,000, 12.2%) and prostate cancer (382,000, 11.9%). The most common causes of death from cancer were lung cancer (342,000 deaths, 19.9% of the total), colorectal cancer (212,000 deaths, 12.3%), breast cancer (129,000, 7.5%) and stomach cancer (117,000, 6.8%).

Reverse genetics in model organisms such as Drosophila melanogaster, Arabidopsis thaliana, zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells - this broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair. Such 'genome editing' is now established in human cells and a number of model organisms, thus opening the door to a range of new experimental and therapeutic possibilities.

VPF/VEGF is a multifunctional cytokine that contributes to angiogenesis by both direct and indirect mechanisms. On the one hand, VPF/VEGF stimulates the ECs lining nearby microvessels to proliferate, to migrate, and to alter their pattern of gene expression. On the other hand, VPF/VEGF renders these same microvascular ECs hyperpermeable so that they spill plasma proteins into the extravascular space, leading to the clotting of extravasated fibrinogen with deposition of a fibrin gel. Extravascular fibrin serves as a provisional matrix that favors and supports the ingrowth of new blood vessels and other mesenchymal cells that generate mature, vascularized stroma. These same principles apply in tumors, in several examples of non-neoplastic pathology, and in physiological processes that involve angiogenesis and new stroma generation. In all of these examples, microvascular hyperpermeability and the introduction of a provisional, plasma-derived matrix precede and accompany the onset of EC division and new blood vessel formation. It would seem, therefore, that tumors have "borrowed" fundamental mechanisms that developed in multicellular organisms for purposes of tissue defense, renewal, and repair. VPF/VEGF, therefore has taught us something new about angiogenesis; namely, that vascular hyperpermeability and consequent plasma protein extravasation are important, perhaps essential, elements in its generation. However, this finding raises a paradox. While VPF/VEGF induces vascular hyperpermeability, other potent angiogenic factors apparently do not, at least in subtoxic concentrations that are more than sufficient to induce angiogenesis. Nonetheless, wherever angiogenesis has been studied, the newly generated vessels have been found to be hyperpermeable. How, therefore, do angiogenic factors other than VPF/VEGF lead to the formation of new and leaky blood vessels? We do not as yet have a complete answer to this question. One possibility is that at least some angiogenic factors mediate their effect by inducing or stimulating the expression of VPF/VEGF. In fact, there is already one clear example of this. TGF-alpha is a potent angiogenic factor but does not itself increase microvascular permeability. However, TGF-alpha strikingly upregulates VPF/VEGF expression in cultured keratinocytes and is thought to be responsible, at least in part, for the overexpression of VPF/VEGF in psoriasis. Moreover, overexpression of TGF-alpha, along with that of the EGF receptor with which it interacts, is characteristic of many malignant tumors, raising the possibility that TGF-alpha acts to stimulate VPF/VEGF expression in other types of epithelial cells and in this manner induces angiogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli in the form of death domain receptor engagement by their respective ligands under conditions where apoptotic execution is prevented. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. Here we report that necrostatin-1, a previously identified small-molecule inhibitor of necroptosis, is a selective allosteric inhibitor of the death domain receptor-associated adaptor kinase RIP1 in vitro. We show that RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. In addition, we show that two other necrostatins, necrostatin-3 and necrostatin-5, also target the RIP1 kinase step in the necroptosis pathway, but through mechanisms distinct from that of necrostatin-1. Overall, our data establish necrostatins as the first-in-class inhibitors of RIP1 kinase, the key upstream kinase involved in the activation of necroptosis.

Frontotemporal dementia (FTD) is the second most common cause of dementia in people under the age of 65 years. A large proportion of FTD patients (35-50%) have a family history of dementia, consistent with a strong genetic component to the disease. In 1998, mutations in the gene encoding the microtubule-associated protein tau (MAPT) were shown to cause familial FTD with parkinsonism linked to chromosome 17q21 (FTDP-17). The neuropathology of patients with defined MAPT mutations is characterized by cytoplasmic neurofibrillary inclusions composed of hyperphosphorylated tau. However, in multiple FTD families with significant evidence for linkage to the same region on chromosome 17q21 (D17S1787-D17S806), mutations in MAPT have not been found and the patients consistently lack tau-immunoreactive inclusion pathology. In contrast, these patients have ubiquitin (ub)-immunoreactive neuronal cytoplasmic inclusions and characteristic lentiform ub-immunoreactive neuronal intranuclear inclusions. Here we demonstrate that in these families, FTD is caused by mutations in progranulin (PGRN) that are likely to create null alleles. PGRN is located 1.7 Mb centromeric of MAPT on chromosome 17q21.31 and encodes a 68.5-kDa secreted growth factor involved in the regulation of multiple processes including development, wound repair and inflammation. PGRN has also been strongly linked to tumorigenesis. Moreover, PGRN expression is increased in activated microglia in many neurodegenerative diseases including Creutzfeldt-Jakob disease, motor neuron disease and Alzheimer's disease. Our results identify mutations in PGRN as a cause of neurodegenerative disease and indicate the importance of PGRN function for neuronal survival.

Alterations in intestinal microbiota composition are associated with several chronic conditions, including obesity and inflammatory diseases. The microbiota of older people displays greater inter-individual variation than that of younger adults. Here we show that the faecal microbiota composition from 178 elderly subjects formed groups, correlating with residence location in the community, day-hospital, rehabilitation or in long-term residential care. However, clustering of subjects by diet separated them by the same residence location and microbiota groupings. The separation of microbiota composition significantly correlated with measures of frailty, co-morbidity, nutritional status, markers of inflammation and with metabolites in faecal water. The individual microbiota of people in long-stay care was significantly less diverse than that of community dwellers. Loss of community-associated microbiota correlated with increased frailty. Collectively, the data support a relationship between diet, microbiota and health status, and indicate a role for diet-driven microbiota alterations in varying rates of health decline upon ageing.

ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/) provides a freely available archive of reports of relationships among medically important variants and phenotypes. ClinVar accessions submissions reporting human variation, interpretations of the relationship of that variation to human health and the evidence supporting each interpretation. The database is tightly coupled with dbSNP and dbVar, which maintain information about the location of variation on human assemblies. ClinVar is also based on the phenotypic descriptions maintained in MedGen (http://www.ncbi.nlm.nih.gov/medgen). Each ClinVar record represents the submitter, the variation and the phenotype, i.e. the unit that is assigned an accession of the format SCV000000000.0. The submitter can update the submission at any time, in which case a new version is assigned. To facilitate evaluation of the medical importance of each variant, ClinVar aggregates submissions with the same variation/phenotype combination, adds value from other NCBI databases, assigns a distinct accession of the format RCV000000000.0 and reports if there are conflicting clinical interpretations. Data in ClinVar are available in multiple formats, including html, download as XML, VCF or tab-delimited subsets. Data from ClinVar are provided as annotation tracks on genomic RefSeqs and are used in tools such as Variation Reporter (http://www.ncbi.nlm.nih.gov/variation/tools/reporter), which reports what is known about variation based on user-supplied locations.

In vitro studies have demonstrated antibacterial activity of essential oils (EOs) against Listeria monocytogenes, Salmonella typhimurium, Escherichia coli O157:H7, Shigella dysenteria, Bacillus cereus and Staphylococcus aureus at levels between 0.2 and 10 microl ml(-1). Gram-negative organisms are slightly less susceptible than gram-positive bacteria. A number of EO components has been identified as effective antibacterials, e.g. carvacrol, thymol, eugenol, perillaldehyde, cinnamaldehyde and cinnamic acid, having minimum inhibitory concentrations (MICs) of 0.05-5 microl ml(-1) in vitro. A higher concentration is needed to achieve the same effect in foods. Studies with fresh meat, meat products, fish, milk, dairy products, vegetables, fruit and cooked rice have shown that the concentration needed to achieve a significant antibacterial effect is around 0.5-20 microl g(-1) in foods and about 0.1-10 microl ml(-1) in solutions for washing fruit and vegetables. EOs comprise a large number of components and it is likely that their mode of action involves several targets in the bacterial cell. The hydrophobicity of EOs enables them to partition in the lipids of the cell membrane and mitochondria, rendering them permeable and leading to leakage of cell contents. Physical conditions that improve the action of EOs are low pH, low temperature and low oxygen levels. Synergism has been observed between carvacrol and its precursor p-cymene and between cinnamaldehyde and eugenol. Synergy between EO components and mild preservation methods has also been observed. Some EO components are legally registered flavourings in the EU and the USA. Undesirable organoleptic effects can be limited by careful selection of EOs according to the type of food.

Qualitative research methods could help us to improve our understanding of medicine. Rather than thinking of qualitative and quantitative strategies as incompatible, they should be seen as complementary. Although procedures for textual interpretation differ from those of statistical analysis, because of the different type of data used and questions to be answered, the underlying principles are much the same. In this article I propose relevance, validity, and reflexivity as overall standards for qualitative inquiry. I will discuss the specific challenges in relation to reflexivity, transferability, and shared assumptions of interpretation, which are met by medical researchers who do this type of research, and I will propose guidelines for qualitative inquiry.

Technologies to measure whole-genome mRNA abundances and methods to organize and display such data are emerging as valuable tools for systems-level exploration of transcriptional regulatory networks. For instance, it has been shown that mRNA data from 118 genes, measured at several time points in the developing hindbrain of mice, can be hierarchically clustered into various patterns (or 'waves') whose members tend to participate in common processes. We have previously shown that hierarchical clustering can group together genes whose cis-regulatory elements are bound by the same proteins in vivo. Hierarchical clustering has also been used to organize genes into hierarchical dendograms on the basis of their expression across multiple growth conditions. The application of Fourier analysis to synchronized yeast mRNA expression data has identified cell-cycle periodic genes, many of which have expected cis-regulatory elements. Here we apply a systematic set of statistical algorithms, based on whole-genome mRNA data, partitional clustering and motif discovery, to identify transcriptional regulatory sub-networks in yeast-without any a priori knowledge of their structure or any assumptions about their dynamics. This approach uncovered new regulons (sets of co-regulated genes) and their putative cis-regulatory elements. We used statistical characterization of known regulons and motifs to derive criteria by which we infer the biological significance of newly discovered regulons and motifs. Our approach holds promise for the rapid elucidation of genetic network architecture in sequenced organisms in which little biology is known.

Prior to the twentieth century, theories of knowledge were inherently perceptual. Since then, developments in logic, statistics, and programming languages have inspired amodal theories that rest on principles fundamentally different from those underlying perception. In addition, perceptual approaches have become widely viewed as untenable because they are assumed to implement recording systems, not conceptual systems. A perceptual theory of knowledge is developed here in the context of current cognitive science and neuroscience. During perceptual experience, association areas in the brain capture bottom-up patterns of activation in sensory-motor areas. Later, in a top-down manner, association areas partially reactivate sensory-motor areas to implement perceptual symbols. The storage and reactivation of perceptual symbols operates at the level of perceptual components--not at the level of holistic perceptual experiences. Through the use of selective attention, schematic representations of perceptual components are extracted from experience and stored in memory (e.g., individual memories of green, purr, hot). As memories of the same component become organized around a common frame, they implement a simulator that produces limitless simulations of the component (e.g., simulations of purr). Not only do such simulators develop for aspects of sensory experience, they also develop for aspects of proprioception (e.g., lift, run) and introspection (e.g., compare, memory, happy, hungry). Once established, these simulators implement a basic conceptual system that represents types, supports categorization, and produces categorical inferences. These simulators further support productivity, propositions, and abstract concepts, thereby implementing a fully functional conceptual system. Productivity results from integrating simulators combinatorially and recursively to produce complex simulations. Propositions result from binding simulators to perceived individuals to represent type-token relations. Abstract concepts are grounded in complex simulations of combined physical and introspective events. Thus, a perceptual theory of knowledge can implement a fully functional conceptual system while avoiding problems associated with amodal symbol systems. Implications for cognition, neuroscience, evolution, development, and artificial intelligence are explored.

Chronic viral infections often result in ineffective CD8 T-cell responses due to functional exhaustion or physical deletion of virus-specific T cells. However, how persisting virus impacts various CD8 T-cell effector functions and influences other aspects of CD8 T-cell dynamics, such as immunodominance and tissue distribution, remains largely unknown. Using different strains of lymphocytic choriomeningitis virus (LCMV), we compared responses to the same CD8 T-cell epitopes during acute or chronic infection. Persistent infection led to a disruption of the normal immunodominance hierarchy of CD8 T-cell responses seen following acute infection and dramatically altered the tissue distribution of LCMV-specific CD8 T cells in lymphoid and nonlymphoid tissues. Most importantly, CD8 T-cell functional impairment occurred in a hierarchical fashion in chronically infected mice. Production of interleukin 2 and the ability to lyse target cells in vitro were the first functions compromised, followed by the ability to make tumor necrosis factor alpha, while gamma interferon production was most resistant to functional exhaustion. Antigen appeared to be the driving force for this loss of function, since a strong correlation existed between the viral load and the level of exhaustion. Further, epitopes presented at higher levels in vivo resulted in physical deletion, while those presented at lower levels induced functional exhaustion. A model is proposed in which antigen levels drive the hierarchical loss of different CD8 T-cell effector functions during chronic infection, leading to distinct stages of functional impairment and eventually to physical deletion of virus-specific T cells. These results have implications for the study of human chronic infections, where similar T-cell deletion and functional dysregulation has been observed.

It is now clear that functionally specialized regulatory T (Treg) cells exist as part of the normal immune repertoire, preventing the development of pathogenic responses to both self- and intestinal antigens. Here, we report that the Treg cells that control intestinal inflammation express the same phenotype (CD25(+)CD45RB(low)CD4(+)) as those that control autoimmunity. Previous studies have failed to identify how CD25(+) Treg cells function in vivo. Our studies reveal that the immune-suppressive function of these cells in vivo is dependent on signaling via the negative regulator of T cell activation cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), as well as secretion of the immune-suppressive cytokine transforming growth factor beta. Strikingly, constitutive expression of CTLA-4 among CD4(+) cells was restricted primarily to Treg cells, suggesting that CTLA-4 expression by these cells is involved in their immune-suppressive function. These findings raise the possibility that Treg cell function contributes to the immune suppression characteristic of CTLA-4 signaling. Identification of costimulatory molecules involved in the function of Treg cells may facilitate further characterization of these cells and development of new therapeutic strategies for the treatment of inflammatory diseases.

Natural variation in gene expression is extensive in humans and other organisms, and variation in the baseline expression level of many genes has a heritable component. To localize the genetic determinants of these quantitative traits (expression phenotypes) in humans, we used microarrays to measure gene expression levels and performed genome-wide linkage analysis for expression levels of 3,554 genes in 14 large families. For approximately 1,000 expression phenotypes, there was significant evidence of linkage to specific chromosomal regions. Both cis- and trans-acting loci regulate variation in the expression levels of genes, although most act in trans. Many gene expression phenotypes are influenced by several genetic determinants. Furthermore, we found hotspots of transcriptional regulation where significant evidence of linkage for several expression phenotypes (up to 31) coincides, and expression levels of many genes that share the same regulatory region are significantly correlated. The combination of microarray techniques for phenotyping and linkage analysis for quantitative traits allows the genetic mapping of determinants that contribute to variation in human gene expression.

An acute respiratory disease, caused by a novel coronavirus (SARS-CoV-2, previously known as 2019-nCoV), the coronavirus disease 2019 (COVID-19) has spread throughout China and received worldwide attention. On 30 January 2020, World Health Organization (WHO) officially declared the COVID-19 epidemic as a public health emergency of international concern. The emergence of SARS-CoV-2, since the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, marked the third introduction of a highly pathogenic and large-scale epidemic coronavirus into the human population in the twenty-first century. As of 1 March 2020, a total of 87,137 confirmed cases globally, 79,968 confirmed in China and 7169 outside of China, with 2977 deaths (3.4%) had been reported by WHO. Meanwhile, several independent research groups have identified that SARS-CoV-2 belongs to β-coronavirus, with highly identical genome to bat coronavirus, pointing to bat as the natural host. The novel coronavirus uses the same receptor, angiotensin-converting enzyme 2 (ACE2) as that for SARS-CoV, and mainly spreads through the respiratory tract. Importantly, increasingly evidence showed sustained human-to-human transmission, along with many exported cases across the globe. The clinical symptoms of COVID-19 patients include fever, cough, fatigue and a small population of patients appeared gastrointestinal infection symptoms. The elderly and people with underlying diseases are susceptible to infection and prone to serious outcomes, which may be associated with acute respiratory distress syndrome (ARDS) and cytokine storm. Currently, there are few specific antiviral strategies, but several potent candidates of antivirals and repurposed drugs are under urgent investigation. In this review, we summarized the latest research progress of the epidemiology, pathogenesis, and clinical characteristics of COVID-19, and discussed the current treatment and scientific advancements to combat the epidemic novel coronavirus.

All fields of neuroscience that employ brain imaging need to communicate their results with reference to anatomical regions. In particular, comparative morphometry and group analysis of functional and physiological data require coregistration of brains to establish correspondences across brain structures. It is well established that linear registration of one brain to another is inadequate for aligning brain structures, so numerous algorithms have emerged to nonlinearly register brains to one another. This study is the largest evaluation of nonlinear deformation algorithms applied to brain image registration ever conducted. Fourteen algorithms from laboratories around the world are evaluated using 8 different error measures. More than 45,000 registrations between 80 manually labeled brains were performed by algorithms including: AIR, ANIMAL, ART, Diffeomorphic Demons, FNIRT, IRTK, JRD-fluid, ROMEO, SICLE, SyN, and four different SPM5 algorithms ("SPM2-type" and regular Normalization, Unified Segmentation, and the DARTEL Toolbox). All of these registrations were preceded by linear registration between the same image pairs using FLIRT. One of the most significant findings of this study is that the relative performances of the registration methods under comparison appear to be little affected by the choice of subject population, labeling protocol, and type of overlap measure. This is important because it suggests that the findings are generalizable to new subject populations that are labeled or evaluated using different labeling protocols. Furthermore, we ranked the 14 methods according to three completely independent analyses (permutation tests, one-way ANOVA tests, and indifference-zone ranking) and derived three almost identical top rankings of the methods. ART, SyN, IRTK, and SPM's DARTEL Toolbox gave the best results according to overlap and distance measures, with ART and SyN delivering the most consistently high accuracy across subjects and label sets. Updates will be published on the http://www.mindboggle.info/papers/ website.

We argue that to best comprehend many data sets, plotting judiciously selected sample statistics with associated confidence intervals can usefully supplement, or even replace, standard hypothesis-testing procedures. We note that most social science statistics textbooks limit discussion of confidence intervals to their use in between-subject designs. Our central purpose in this article is to describe how to compute an analogous confidence interval that can be used in within-subject designs. This confidence interval rests on the reasoning that because between-subject variance typically plays no role in statistical analyses of within-subject designs, it can legitimately be ignored; hence, an appropriate confidence interval can be based on the standard within-subject error term-that is, on the variability due to the subject × condition interaction. Computation of such a confidence interval is simple and is embodied in Equation 2 on p. 482 of this article. This confidence interval has two useful properties. First, it is based on the same error term as is the corresponding analysis of variance, and hence leads to comparable conclusions. Second, it is related by a known factor (√2) to a confidence interval of the difference between sample means; accordingly, it can be used to infer the faith one can put in some pattern of sample means as a reflection of the underlying pattern of population means. These two properties correspond to analogous properties of the more widely used between-subject confidence interval.

One of the main objectives in the analysis of microarray experiments is the identification of genes that are differentially expressed under two experimental conditions. This task is complicated by the noisiness of the data and the large number of genes that are examined simultaneously. Here, we present a novel technique for identifying differentially expressed genes that does not originate from a sophisticated statistical model but rather from an analysis of biological reasoning. The new technique, which is based on calculating rank products (RP) from replicate experiments, is fast and simple. At the same time, it provides a straightforward and statistically stringent way to determine the significance level for each gene and allows for the flexible control of the false-detection rate and familywise error rate in the multiple testing situation of a microarray experiment. We use the RP technique on three biological data sets and show that in each case it performs more reliably and consistently than the non-parametric t-test variant implemented in Tusher et al.'s significance analysis of microarrays (SAM). We also show that the RP results are reliable in highly noisy data. An analysis of the physiological function of the identified genes indicates that the RP approach is powerful for identifying biologically relevant expression changes. In addition, using RP can lead to a sharp reduction in the number of replicate experiments needed to obtain reproducible results.

We define a "threaded blockset," which is a novel generalization of the classic notion of a multiple alignment. A new computer program called TBA (for "threaded blockset aligner") builds a threaded blockset under the assumption that all matching segments occur in the same order and orientation in the given sequences; inversions and duplications are not addressed. TBA is designed to be appropriate for aligning many, but by no means all, megabase-sized regions of multiple mammalian genomes. The output of TBA can be projected onto any genome chosen as a reference, thus guaranteeing that different projections present consistent predictions of which genomic positions are orthologous. This capability is illustrated using a new visualization tool to view TBA-generated alignments of vertebrate Hox clusters from both the mammalian and fish perspectives. Experimental evaluation of alignment quality, using a program that simulates evolutionary change in genomic sequences, indicates that TBA is more accurate than earlier programs. To perform the dynamic-programming alignment step, TBA runs a stand-alone program called MULTIZ, which can be used to align highly rearranged or incompletely sequenced genomes. We describe our use of MULTIZ to produce the whole-genome multiple alignments at the Santa Cruz Genome Browser.

We have studied the transient expression of a cloned rabbit hemoglobin beta 1 gene after its introduction into HeLa cells. Two and one-half days after transfection using the calcium phosphate technique we extracted RNA from the entire cell population and analyzed it by the S1 nuclease hybridization assay. Transcripts were barely detectable when beta-globin gene-plasmid recombinants were used. However, 200 times more beta-globin gene transcripts were found when the beta-globin gene recombinants also contained SV40 DNA, and 90% of these transcripts (about 1000 per cell) had the same 5' end as authentic rabbit globin mRNA. In the latter case, abundant production of beta-globin protein was readily detected in a fraction of transfected cells by immunofluorescent staining. Enhancement of globin gene expression was dependent on SV40 sequences acting in cis, but independent of the viral origin of DNA replication. The enhancing activity was associated with the 72 bp repeated sequence element located at the beginning of the viral late gene region. Viral DNA fragments containing the transcriptional enhancer element could act in either orientation at many positions, including 1400 bp upstream or 3300 bp downstream from the transcription initiation site of the rabbit beta-globin gene. These studies define a class of DNA elements with a mode of action that has not been heretofore described. The activation of genes by specific enhancer elements seems to be a widespread mechanism that may be used for the regulation of gene expression.