We describe here a general Amber force field (GAFF) for organic molecules. GAFF is designed to be compatible with existing Amber force fields for proteins and nucleic acids, and has parameters for most organic and pharmaceutical molecules that are composed of H, C, N, O, S, P, and halogens. It uses a simple functional form and a limited number of atom types, but incorporates both empirical and heuristic models to estimate force constants and partial atomic charges. The performance of GAFF in test cases is encouraging. In test I, 74 crystallographic structures were compared to GAFF minimized structures, with a root-mean-square displacement of 0.26 A, which is comparable to that of the Tripos 5.2 force field (0.25 A) and better than those of MMFF 94 and CHARMm (0.47 and 0.44 A, respectively). In test II, gas phase minimizations were performed on 22 nucleic acid base pairs, and the minimized structures and intermolecular energies were compared to MP2/6-31G* results. The RMS of displacements and relative energies were 0.25 A and 1.2 kcal/mol, respectively. These data are comparable to results from Parm99/RESP (0.16 A and 1.18 kcal/mol, respectively), which were parameterized to these base pairs. Test III looked at the relative energies of 71 conformational pairs that were used in development of the Parm99 force field. The RMS error in relative energies (compared to experiment) is about 0.5 kcal/mol. GAFF can be applied to wide range of molecules in an automatic fashion, making it suitable for rational drug design and database searching.

The development of a 10-item self-report scale (EPDS) to screen for Postnatal Depression in the community is described. After extensive pilot interviews a validation study was carried out on 84 mothers using the Research Diagnostic Criteria for depressive illness obtained from Goldberg's Standardised Psychiatric Interview. The EPDS was found to have satisfactory sensitivity and specificity, and was also sensitive to change in the severity of depression over time. The scale can be completed in about 5 minutes and has a simple method of scoring. The use of the EPDS in the secondary prevention of Postnatal Depression is discussed.

The ability to identify active compounds (³hits²) from large chemical libraries accurately and rapidly has been the ultimate goal in developing high-throughput screening (HTS) assays. The ability to identify hits from a particular HTS assay depends largely on the suitability or quality of the assay used in the screening. The criteria or parameters for evaluating the ³suitability² of an HTS assay for hit identification are not well defined and hence it still remains difficult to compare the quality of assays directly. In this report, a screening window coefficient, called ³Z-factor,² is defined. This coefficient is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements, and therefore is suitable for assay quality assessment. The Z-factor is a dimensionless, simple statistical characteristic for each HTS assay. The Z-factor provides a useful tool for comparison and evaluation of the quality of assays, and can be utilized in assay optimization and validation.

The evolutionary history of a set of taxa is usually represented by a phylogenetic tree, and this model has greatly facilitated the discussion and testing of hypotheses. However, it is well known that more complex evolutionary scenarios are poorly described by such models. Further, even when evolution proceeds in a tree-like manner, analysis of the data may not be best served by using methods that enforce a tree structure but rather by a richer visualization of the data to evaluate its properties, at least as an essential first step. Thus, phylogenetic networks should be employed when reticulate events such as hybridization, horizontal gene transfer, recombination, or gene duplication and loss are believed to be involved, and, even in the absence of such events, phylogenetic networks have a useful role to play. This article reviews the terminology used for phylogenetic networks and covers both split networks and reticulate networks, how they are defined, and how they can be interpreted. Additionally, the article outlines the beginnings of a comprehensive statistical framework for applying split network methods. We show how split networks can represent confidence sets of trees and introduce a conservative statistical test for whether the conflicting signal in a network is treelike. Finally, this article describes a new program, SplitsTree4, an interactive and comprehensive tool for inferring different types of phylogenetic networks from sequences, distances, and trees.

This paper describes DARTEL, which is an algorithm for diffeomorphic image registration. It is implemented for both 2D and 3D image registration and has been formulated to include an option for estimating inverse consistent deformations. Nonlinear registration is considered as a local optimisation problem, which is solved using a Levenberg-Marquardt strategy. The necessary matrix solutions are obtained in reasonable time using a multigrid method. A constant Eulerian velocity framework is used, which allows a rapid scaling and squaring method to be used in the computations. DARTEL has been applied to intersubject registration of 471 whole brain images, and the resulting deformations were evaluated in terms of how well they encode the shape information necessary to separate male and female subjects and to predict the ages of the subjects.

Plasmid expression vectors have been constructed that direct the synthesis of foreign polypeptides in Escherichia coli as fusions with the C terminus of Sj26, a 26-kDa glutathione S-transferase (GST; EC 2.5.1.18) encoded by the parasitic helminth Schistosoma japonicum. In the majority of cases, fusion proteins are soluble in aqueous solutions and can be purified from crude bacterial lysates under non-denaturing conditions by affinity chromatography on immobilised glutathione. Using batch wash procedures several fusion proteins can be purified in parallel in under 2 h with yields of up to 15 micrograms protein/ml of culture. The vectors have been engineered so that the GST carrier can be cleaved from fusion proteins by digestion with site-specific proteases such as thrombin or blood coagulation factor Xa, following which, the carrier and any uncleaved fusion protein can be removed by absorption on glutathione-agarose. This system has been used successfully for the expression and purification of more than 30 different eukaryotic polypeptides.

The Huntington's disease (HD) gene has been mapped in 4p16.3 but has eluded identification. We have used haplotype analysis of linkage disequilibrium to spotlight a small segment of 4p16.3 as the likely location of the defect. A new gene, IT15, isolated using cloned trapped exons from the target area contains a polymorphic trinucleotide repeat that is expanded and unstable on HD chromosomes. A (CAG)n repeat longer than the normal range was observed on HD chromosomes from all 75 disease families examined, comprising a variety of ethnic backgrounds and 4p16.3 haplotypes. The (CAG)n repeat appears to be located within the coding sequence of a predicted approximately 348 kd protein that is widely expressed but unrelated to any known gene. Thus, the HD mutation involves an unstable DNA segment, similar to those described in fragile X syndrome, spino-bulbar muscular atrophy, and myotonic dystrophy, acting in the context of a novel 4p16.3 gene to produce a dominant phenotype.

There has been much recent interest in using magnetic resonance diffusion imaging to provide information about anatomical connectivity in the brain, by measuring the anisotropic diffusion of water in white matter tracts. One of the measures most commonly derived from diffusion data is fractional anisotropy (FA), which quantifies how strongly directional the local tract structure is. Many imaging studies are starting to use FA images in voxelwise statistical analyses, in order to localise brain changes related to development, degeneration and disease. However, optimal analysis is compromised by the use of standard registration algorithms; there has not to date been a satisfactory solution to the question of how to align FA images from multiple subjects in a way that allows for valid conclusions to be drawn from the subsequent voxelwise analysis. Furthermore, the arbitrariness of the choice of spatial smoothing extent has not yet been resolved. In this paper, we present a new method that aims to solve these issues via (a) carefully tuned non-linear registration, followed by (b) projection onto an alignment-invariant tract representation (the "mean FA skeleton"). We refer to this new approach as Tract-Based Spatial Statistics (TBSS). TBSS aims to improve the sensitivity, objectivity and interpretability of analysis of multi-subject diffusion imaging studies. We describe TBSS in detail and present example TBSS results from several diffusion imaging studies.

The field of neuroscience has, after a long period of looking the other way, again embraced emotion as an important research area. Much of the progress has come from studies of fear, and especially fear conditioning. This work has pinpointed the amygdala as an important component of the system involved in the acquisition, storage, and expression of fear memory and has elucidated in detail how stimuli enter, travel through, and exit the amygdala. Some progress has also been made in understanding the cellular and molecular mechanisms that underlie fear conditioning, and recent studies have also shown that the findings from experimental animals apply to the human brain. It is important to remember why this work on emotion succeeded where past efforts failed. It focused on a psychologically well-defined aspect of emotion, avoided vague and poorly defined concepts such as "affect," "hedonic tone," or "emotional feelings," and used a simple and straightforward experimental approach. With so much research being done in this area today, it is important that the mistakes of the past not be made again. It is also time to expand from this foundation into broader aspects of mind and behavior.

High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.

CHARMM (Chemistry at HARvard Molecular Mechanics) is a highly versatile and widely used molecular simulation program. It has been developed over the last three decades with a primary focus on molecules of biological interest, including proteins, peptides, lipids, nucleic acids, carbohydrates, and small molecule ligands, as they occur in solution, crystals, and membrane environments. For the study of such systems, the program provides a large suite of computational tools that include numerous conformational and path sampling methods, free energy estimators, molecular minimization, dynamics, and analysis techniques, and model-building capabilities. The CHARMM program is applicable to problems involving a much broader class of many-particle systems. Calculations with CHARMM can be performed using a number of different energy functions and models, from mixed quantum mechanical-molecular mechanical force fields, to all-atom classical potential energy functions with explicit solvent and various boundary conditions, to implicit solvent and membrane models. The program has been ported to numerous platforms in both serial and parallel architectures. This article provides an overview of the program as it exists today with an emphasis on developments since the publication of the original CHARMM article in 1983.

CONCLUSIONS

The variant call format (VCF) is a generic format for storing DNA polymorphism data such as SNPs, insertions, deletions and structural variants, together with rich annotations. VCF is usually stored in a compressed manner and can be indexed for fast data retrieval of variants from a range of positions on the reference genome. The format was developed for the 1000 Genomes Project, and has also been adopted by other projects such as UK10K, dbSNP and the NHLBI Exome Project. VCFtools is a software suite that implements various utilities for processing VCF files, including validation, merging, comparing and also provides a general Perl API.

BACKGROUND

http://vcftools.sourceforge.net

Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to saturation in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).

Interleukin-10 (IL-10), first recognized for its ability to inhibit activation and effector function of T cells, monocytes, and macrophages, is a multifunctional cytokine with diverse effects on most hemopoietic cell types. The principal routine function of IL-10 appears to be to limit and ultimately terminate inflammatory responses. In addition to these activities, IL-10 regulates growth and/or differentiation of B cells, NK cells, cytotoxic and helper T cells, mast cells, granulocytes, dendritic cells, keratinocytes, and endothelial cells. IL-10 plays a key role in differentiation and function of a newly appreciated type of T cell, the T regulatory cell, which may figure prominently in control of immune responses and tolerance in vivo. Uniquely among hemopoietic cytokines, IL-10 has closely related homologs in several virus genomes, which testify to its crucial role in regulating immune and inflammatory responses. This review highlights findings that have advanced our understanding of IL-10 and its receptor, as well as its in vivo function in health and disease.

Pfam is a widely used database of protein families, currently containing more than 13,000 manually curated protein families as of release 26.0. 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/). Here, we report on changes that have occurred since our 2010 NAR paper (release 24.0). Over the last 2 years, we have generated 1840 new families and increased coverage of the UniProt Knowledgebase (UniProtKB) to nearly 80%. Notably, we have taken the step of opening up the annotation of our families to the Wikipedia community, by linking Pfam families to relevant Wikipedia pages and encouraging the Pfam and Wikipedia communities to improve and expand those pages. We continue to improve the Pfam website and add new visualizations, such as the 'sunburst' representation of taxonomic distribution of families. In this work we additionally address two topics that will be of particular interest to the Pfam community. First, we explain the definition and use of family-specific, manually curated gathering thresholds. Second, we discuss some of the features of domains of unknown function (also known as DUFs), which constitute a rapidly growing class of families within Pfam.

Molecular structures and sequences are generally more revealing of evolutionary relationships than are classical phenotypes (particularly so among microorganisms). Consequently, the basis for the definition of taxa has progressively shifted from the organismal to the cellular to the molecular level. Molecular comparisons show that life on this planet divides into three primary groupings, commonly known as the eubacteria, the archaebacteria, and the eukaryotes. The three are very dissimilar, the differences that separate them being of a more profound nature than the differences that separate typical kingdoms, such as animals and plants. Unfortunately, neither of the conventionally accepted views of the natural relationships among living systems--i.e., the five-kingdom taxonomy or the eukaryote-prokaryote dichotomy--reflects this primary tripartite division of the living world. To remedy this situation we propose that a formal system of organisms be established in which above the level of kingdom there exists a new taxon called a "domain." Life on this planet would then be seen as comprising three domains, the Bacteria, the Archaea, and the Eucarya, each containing two or more kingdoms. (The Eucarya, for example, contain Animalia, Plantae, Fungi, and a number of others yet to be defined). Although taxonomic structure within the Bacteria and Eucarya is not treated herein, Archaea is formally subdivided into the two kingdoms Euryarchaeota (encompassing the methanogens and their phenotypically diverse relatives) and Crenarchaeota (comprising the relatively tight clustering of extremely thermophilic archaebacteria, whose general phenotype appears to resemble most the ancestral phenotype of the Archaea.

The European Working Group on Sarcopenia in Older People (EWGSOP) developed a practical clinical definition and consensus diagnostic criteria for age-related sarcopenia. EWGSOP included representatives from four participant organisations, i.e. the European Geriatric Medicine Society, the European Society for Clinical Nutrition and Metabolism, the International Association of Gerontology and Geriatrics-European Region and the International Association of Nutrition and Aging. These organisations endorsed the findings in the final document. The group met and addressed the following questions, using the medical literature to build evidence-based answers: (i) What is sarcopenia? (ii) What parameters define sarcopenia? (iii) What variables reflect these parameters, and what measurement tools and cut-off points can be used? (iv) How does sarcopenia relate to cachexia, frailty and sarcopenic obesity? For the diagnosis of sarcopenia, EWGSOP recommends using the presence of both low muscle mass + low muscle function (strength or performance). EWGSOP variously applies these characteristics to further define conceptual stages as 'presarcopenia', 'sarcopenia' and 'severe sarcopenia'. EWGSOP reviewed a wide range of tools that can be used to measure the specific variables of muscle mass, muscle strength and physical performance. Our paper summarises currently available data defining sarcopenia cut-off points by age and gender; suggests an algorithm for sarcopenia case finding in older individuals based on measurements of gait speed, grip strength and muscle mass; and presents a list of suggested primary and secondary outcome domains for research. Once an operational definition of sarcopenia is adopted and included in the mainstream of comprehensive geriatric assessment, the next steps are to define the natural course of sarcopenia and to develop and define effective treatment.

Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7. Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF. The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding. A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients.

BACKGROUND

Trastuzumab, a monoclonal antibody against human epidermal growth factor receptor 2 (HER2; also known as ERBB2), was investigated in combination with chemotherapy for first-line treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer.

METHODS

ToGA (Trastuzumab for Gastric Cancer) was an open-label, international, phase 3, randomised controlled trial undertaken in 122 centres in 24 countries. Patients with gastric or gastro-oesophageal junction cancer were eligible for inclusion if their tumours showed overexpression of HER2 protein by immunohistochemistry or gene amplification by fluorescence in-situ hybridisation. Participants were randomly assigned in a 1:1 ratio to receive a chemotherapy regimen consisting of capecitabine plus cisplatin or fluorouracil plus cisplatin given every 3 weeks for six cycles or chemotherapy in combination with intravenous trastuzumab. Allocation was by block randomisation stratified by Eastern Cooperative Oncology Group performance status, chemotherapy regimen, extent of disease, primary cancer site, and measurability of disease, implemented with a central interactive voice recognition system. The primary endpoint was overall survival in all randomised patients who received study medication at least once. This trial is registered with ClinicalTrials.gov, number NCT01041404.

RESULTS

594 patients were randomly assigned to study treatment (trastuzumab plus chemotherapy, n=298; chemotherapy alone, n=296), of whom 584 were included in the primary analysis (n=294; n=290). Median follow-up was 18.6 months (IQR 11-25) in the trastuzumab plus chemotherapy group and 17.1 months (9-25) in the chemotherapy alone group. Median overall survival was 13.8 months (95% CI 12-16) in those assigned to trastuzumab plus chemotherapy compared with 11.1 months (10-13) in those assigned to chemotherapy alone (hazard ratio 0.74; 95% CI 0.60-0.91; p=0.0046). The most common adverse events in both groups were nausea (trastuzumab plus chemotherapy, 197 [67%] vs chemotherapy alone, 184 [63%]), vomiting (147 [50%] vs 134 [46%]), and neutropenia (157 [53%] vs 165 [57%]). Rates of overall grade 3 or 4 adverse events (201 [68%] vs 198 [68%]) and cardiac adverse events (17 [6%] vs 18 [6%]) did not differ between groups.

CONCLUSIONS

Trastuzumab in combination with chemotherapy can be considered as a new standard option for patients with HER2-positive advanced gastric or gastro-oesophageal junction cancer.

BACKGROUND

F Hoffmann-La Roche.

Amplified marker-gene sequences can be used to understand microbial community structure, but they suffer from a high level of sequencing and amplification artifacts. The UPARSE pipeline reports operational taxonomic unit (OTU) sequences with ≤1% incorrect bases in artificial microbial community tests, compared with >3% incorrect bases commonly reported by other methods. The improved accuracy results in far fewer OTUs, consistently closer to the expected number of species in a community.

<AbstractText>In late Decem<em>be</em>r, 2019, patients presenting with viral pneumonia due to an unidentified microbial agent were reported in Wuhan, China. A novel coronavirus was subsequently identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV). As of Jan 26, 2020, more than 2000 cases of 2019-nCoV infection have <em>be</em>en confirmed, most of which involved people living in or visiting Wuhan, and human-to-human transmission has <em>be</em>en confirmed.</AbstractText><AbstractText>We did next-generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions determined by rapid amplification of cDNA ends. Phylogenetic analysis of these 2019-nCoV genomes and those of other coronaviruses was used to determine the evolutionary history of the virus and help infer its likely origin. Homology modelling was done to explore the likely receptor-binding properties of the virus.</AbstractText><AbstractText>The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Phylogenetic analysis revealed that 2019-nCoV fell within the subgenus Sar<em>be</em>covirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV. Notably, homology modelling revealed that 2019-nCoV had a similar receptor-binding domain structure to that of SARS-CoV, despite amino acid variation at some key residues.</AbstractText><AbstractText>2019-nCoV is sufficiently divergent from SARS-CoV to <em>be</em> considered a new human-infecting <em>be</em>tacoronavirus. Although our phylogenetic analysis suggests that bats might <em>be</em> the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans. Importantly, structural analysis suggests that 2019-nCoV might <em>be</em> able to bind to the angiotensin-converting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation.</AbstractText><AbstractText>National Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, Chinese Academy of Sciences, Shandong First Medical University.</AbstractText>

The role of the adaptive immune response in controlling the growth and recurrence of human tumors has been controversial. We characterized the tumor-infiltrating immune cells in large cohorts of human colorectal cancers by gene expression profiling and in situ immunohistochemical staining. Collectively, the immunological data (the type, density, and location of immune cells within the tumor samples) were found to be a better predictor of patient survival than the histopathological methods currently used to stage colorectal cancer. The results were validated in two additional patient populations. These data support the hypothesis that the adaptive immune response influences the behavior of human tumors. In situ analysis of tumor-infiltrating immune cells may therefore be a valuable prognostic tool in the treatment of colorectal cancer and possibly other malignancies.

Large phylogenomics data sets require fast tree inference methods, especially for maximum-likelihood (ML) phylogenies. Fast programs exist, but due to inherent heuristics to find optimal trees, it is not clear whether the best tree is found. Thus, there is need for additional approaches that employ different search strategies to find ML trees and that are at the same time as fast as currently available ML programs. We show that a combination of hill-climbing approaches and a stochastic perturbation method can be time-efficiently implemented. If we allow the same CPU time as RAxML and PhyML, then our software IQ-TREE found higher likelihoods between 62.2% and 87.1% of the studied alignments, thus efficiently exploring the tree-space. If we use the IQ-TREE stopping rule, RAxML and PhyML are faster in 75.7% and 47.1% of the DNA alignments and 42.2% and 100% of the protein alignments, respectively. However, the range of obtaining higher likelihoods with IQ-TREE improves to 73.3-97.1%. IQ-TREE is freely available at http://www.cibiv.at/software/iqtree.

The National Institute on Aging and the Alzheimer's Association charged a workgroup with the task of developing criteria for the symptomatic predementia phase of Alzheimer's disease (AD), referred to in this article as mild cognitive impairment due to AD. The workgroup developed the following two sets of criteria: (1) core clinical criteria that could be used by healthcare providers without access to advanced imaging techniques or cerebrospinal fluid analysis, and (2) research criteria that could be used in clinical research settings, including clinical trials. The second set of criteria incorporate the use of biomarkers based on imaging and cerebrospinal fluid measures. The final set of criteria for mild cognitive impairment due to AD has four levels of certainty, depending on the presence and nature of the biomarker findings. Considerable work is needed to validate the criteria that use biomarkers and to standardize biomarker analysis for use in community settings.