Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

Interfaces of Malignant and Immunologic Clonal Dynamics in Ovarian Cancer.

High-grade serous ovarian cancer (HGSC) exhibits extensive malignant clonal diversity with widespread but non-random patterns of disease dissemination. We investigated whether local immune microenvironment factors shape tumor progression properties at the interface of tumor-infiltrating lymphocytes (TILs) and cancer cells. Through multi-region study of 212 samples from 38 patients with whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T and B cell receptor sequencing, we identified three immunologic subtypes across samples and extensive within-patient diversity. Epithelial CD8+ TILs negatively associated with malignant diversity, reflecting immunological pruning of tumor clones inferred by neoantigen depletion, HLA I loss of heterozygosity, and spatial tracking between T cell and tumor clones. In addition, combinatorial prognostic effects of mutational processes and immune properties were observed, illuminating how specific genomic aberration types associate with immune response and impact survival. We conclude that within-patient spatial immune microenvironment variation shapes intraperitoneal malignant spread, provoking new evolutionary perspectives on HGSC clonal dispersion.

Epigenetic dysregulation from chromosomal transit in micronuclei.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

Clonal fitness inferred from time-series modelling of single-cell cancer genomes.

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.

Tracking the evolution of therapy-related myeloid neoplasms using chemotherapy signatures.

Patients treated with cytotoxic therapies, including autologous stem cell transplantation, are at risk for developing therapy-related myeloid neoplasms (tMN). Preleukemic clones (ie, clonal hematopoiesis [CH]) are detectable years before the development of these aggressive malignancies, although the genomic events leading to transformation and expansion are not well defined. Here, by leveraging distinctive chemotherapy-associated mutational signatures from whole-genome sequencing data and targeted sequencing of prechemotherapy samples, we reconstructed the evolutionary life-history of 39 therapy-related myeloid malignancies. A dichotomy was revealed, in which neoplasms with evidence of chemotherapy-induced mutagenesis from platinum and melphalan were hypermutated and enriched for complex structural variants (ie, chromothripsis), whereas neoplasms with nonmutagenic chemotherapy exposures were genomically similar to de novo acute myeloid leukemia. Using chemotherapy-associated mutational signatures as temporal barcodes linked to discrete clinical exposure in each patient's life, we estimated that several complex events and genomic drivers were acquired after chemotherapy was administered. For patients with prior multiple myeloma who were treated with high-dose melphalan and autologous stem cell transplantation, we demonstrate that tMN can develop from either a reinfused CH clone that escapes melphalan exposure and is selected after reinfusion, or from TP53-mutant CH that survives direct myeloablative conditioning and acquires melphalan-induced DNA damage. Overall, we revealed a novel mode of tMN progression that is not reliant on direct mutagenesis or even exposure to chemotherapy. Conversely, for tMN that evolve under the influence of chemotherapy-induced mutagenesis, distinct chemotherapies not only select preexisting CH but also promote the acquisition of recurrent genomic drivers.

Etiology and epidemiology of digital dermatitis in Australian dairy herds.

Bovine digital dermatitis (BDD) is an important cause of lameness in dairy cows worldwide. However, there is very little known about this disease in Australian herds, which are predominantly managed on pasture. The primary objectives of this cross-sectional study were to describe the presence and prevalence of BDD in Australian dairy herds and to characterize the microbiota of healthy skin and M4 lesions of BDD-affected, pasture-managed cows. Cows from 71 dairy herds were examined at milking time to identify the presence of BDD lesions. True prevalence was estimated using Bayesian methods with informative priors for sensitivity and specificity. Biopsy samples (n = 60) were collected from cows with and without BDD-lesions in 7 pasture-based herds. The microbiota in the superficial and deep strata of each tissue biopsy were characterized via sequencing of the V3-V4 region of the bacterial ribosomal RNA gene. Lesions were detected in 1,817 (11.5%) of 15,813 cows, and in 68 of 71 (95.8%) herds. The median herd-level apparent and true prevalences of BDD were 8.5 and 18.1%, respectively, but this varied considerably between farms. On farms with BDD, M4 lesions accounted for 70 to 100% of all lesions (interquartile range = 95.1 to 100%, median = 100%). M2 lesions (i.e., large ulcerative lesions) were observed at low prevalence (<2.2%) in the few herds (7/71, 9.9%) where they were found. There was a statistically significant difference in the composition of the microbiota between healthy skin and M4 lesions but not between superficial and deep tissue layers. Several gut- and effluent-associated bacterial taxa, including Lentimicrobium and Porphyromonas, which have previously been associated with BDD, were abundant in BDD lesions but not in control biopsies. Our study supports the idea that such taxa are involved in, though possibly not essential to, lesion development and persistence in pasture-managed cows in Australia. Our results also suggest that Dichelobacter may contribute to the disease process. We conclude that BDD is likely to occur in most Australian dairy farms, but that further studies are needed to identify its impact on cow welfare and productivity. Further investigation of the etiology of BDD in Australian dairy herds is also necessary to inform prevention and control strategies.

Baseline characteristics of people experiencing homelessness with a recent drug overdose in the PHOENIx pilot randomised controlled trial.

BACKGROUND: Drug-related deaths in Scotland are the highest in Europe. Half of all deaths in people experiencing homelessness are drug related, yet we know little about the unmet health needs of people experiencing homelessness with recent non-fatal overdose, limiting a tailored practice and policy response to a public health crisis. METHODS: People experiencing homelessness with at least one non-fatal street drug overdose in the previous 6 months were recruited from 20 venues in Glasgow, Scotland, and randomised into PHOENIx plus usual care, or usual care. PHOENIx is a collaborative assertive outreach intervention by independent prescriber NHS Pharmacists and third sector homelessness workers, offering repeated integrated, holistic physical, mental and addictions health and social care support including prescribing. We describe comprehensive baseline characteristics of randomised participants. RESULTS: One hundred and twenty-eight participants had a mean age of 42 years (SD 8.4); 71% male, homelessness for a median of 24 years (IQR 12-30). One hundred and eighteen (92%) lived in large, congregate city centre temporary accommodation. A quarter (25%) were not registered with a General Practitioner. Participants had overdosed a mean of 3.2 (SD 3.2) times in the preceding 6 months, using a median of 3 (IQR 2-4) non-prescription drugs concurrently: 112 (87.5%) street valium (benzodiazepine-type new psychoactive substances); 77 (60%) heroin; and 76 (59%) cocaine. Half (50%) were injecting, 50% into their groins. 90% were receiving care from Alcohol and Drug Recovery Services (ADRS), and in addition to using street drugs, 90% received opioid substitution therapy (OST), 10% diazepam for street valium use and one participant received heroin-assisted treatment. Participants had a mean of 2.2 (SD 1.3) mental health problems and 5.4 (SD 2.5) physical health problems; 50% received treatment for physical or mental health problems. Ninety-one per cent had at least one mental health problem; 66% had no specialist mental health support. Participants were frail (70%) or pre-frail (28%), with maximal levels of psychological distress, 44% received one or no daily meal, and 58% had previously attempted suicide. CONCLUSIONS: People at high risk of drug-related death continue to overdose repeatedly despite receiving OST. High levels of frailty, multimorbidity, unsuitable accommodation and unmet mental and physical health care needs require a reorientation of services informed by evidence of effectiveness and cost-effectiveness. Trial registration UK Clinical Trials Registry identifier: ISRCTN 10585019.

Probabilistic cell-type assignment of single-cell RNA-seq for tumor microenvironment profiling.

Single-cell RNA sequencing has enabled the decomposition of complex tissues into functionally distinct cell types. Often, investigators wish to assign cells to cell types through unsupervised clustering followed by manual annotation or via 'mapping' to existing data. However, manual interpretation scales poorly to large datasets, mapping approaches require purified or pre-annotated data and both are prone to batch effects. To overcome these issues, we present CellAssign, a probabilistic model that leverages prior knowledge of cell-type marker genes to annotate single-cell RNA sequencing data into predefined or de novo cell types. CellAssign automates the process of assigning cells in a highly scalable manner across large datasets while controlling for batch and sample effects. We demonstrate the advantages of CellAssign through extensive simulations and analysis of tumor microenvironment composition in high-grade serous ovarian cancer and follicular lymphoma.

Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution.

Human cancers, including breast cancers, comprise clones differing in mutation content. Clones evolve dynamically in space and time following principles of Darwinian evolution, underpinning important emergent features such as drug resistance and metastasis. Human breast cancer xenoengraftment is used as a means of capturing and studying tumour biology, and breast tumour xenografts are generally assumed to be reasonable models of the originating tumours. However, the consequences and reproducibility of engraftment and propagation on the genomic clonal architecture of tumours have not been systematically examined at single-cell resolution. Here we show, using deep-genome and single-cell sequencing methods, the clonal dynamics of initial engraftment and subsequent serial propagation of primary and metastatic human breast cancers in immunodeficient mice. In all 15 cases examined, clonal selection on engraftment was observed in both primary and metastatic breast tumours, varying in degree from extreme selective engraftment of minor (<5% of starting population) clones to moderate, polyclonal engraftment. Furthermore, ongoing clonal dynamics during serial passaging is a feature of tumours experiencing modest initial selection. Through single-cell sequencing, we show that major mutation clusters estimated from tumour population sequencing relate predictably to the most abundant clonal genotypes, even in clonally complex and rapidly evolving cases. Finally, we show that similar clonal expansion patterns can emerge in independent grafts of the same starting tumour population, indicating that genomic aberrations can be reproducible determinants of evolutionary trajectories. Our results show that measurement of genomically defined clonal population dynamics will be highly informative for functional studies using patient-derived breast cancer xenoengraftment.

Clonal genotype and population structure inference from single-cell tumor sequencing.

Single-cell DNA sequencing has great potential to reveal the clonal genotypes and population structure of human cancers. However, single-cell data suffer from missing values and biased allelic counts as well as false genotype measurements owing to the sequencing of multiple cells. We describe the Single Cell Genotyper (https://bitbucket.org/aroth85/scg), an open-source software based on a statistical model coupled with a mean-field variational inference method, which can be used to address these problems and robustly infer clonal genotypes.

Computational identification of micro-structural variations and their proteogenomic consequences in cancer.

Motivation: Rapid advancement in high throughput genome and transcriptome sequencing (HTS) and mass spectrometry (MS) technologies has enabled the acquisition of the genomic, transcriptomic and proteomic data from the same tissue sample. We introduce a computational framework, ProTIE, to integratively analyze all three types of omics data for a complete molecular profile of a tissue sample. Our framework features MiStrVar, a novel algorithmic method to identify micro structural variants (microSVs) on genomic HTS data. Coupled with deFuse, a popular gene fusion detection method we developed earlier, MiStrVar can accurately profile structurally aberrant transcripts in tumors. Given the breakpoints obtained by MiStrVar and deFuse, our framework can then identify all relevant peptides that span the breakpoint junctions and match them with unique proteomic signatures. Observing structural aberrations in all three types of omics data validates their presence in the tumor samples. Results: We have applied our framework to all The Cancer Genome Atlas (TCGA) breast cancer Whole Genome Sequencing (WGS) and/or RNA-Seq datasets, spanning all four major subtypes, for which proteomics data from Clinical Proteomic Tumor Analysis Consortium (CPTAC) have been released. A recent study on this dataset focusing on SNVs has reported many that lead to novel peptides. Complementing and significantly broadening this study, we detected 244 novel peptides from 432 candidate genomic or transcriptomic sequence aberrations. Many of the fusions and microSVs we discovered have not been reported in the literature. Interestingly, the vast majority of these translated aberrations, fusions in particular, were private, demonstrating the extensive inter-genomic heterogeneity present in breast cancer. Many of these aberrations also have matching out-of-frame downstream peptides, potentially indicating novel protein sequence and structure. Availability and implementation: MiStrVar is available for download at https://bitbucket.org/compbio/mistrvar, and ProTIE is available at https://bitbucket.org/compbio/protie. Contact: cenksahi@indiana.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Detection and Serogrouping of Dichelobacter nodosus Infection by Use of Direct PCR from Lesion Swabs To Support Outbreak-Specific Vaccination for Virulent Footrot in Sheep.

Virulent footrot is an economically significant disease in most sheep-rearing countries. The disease can be controlled with vaccine targeting the fimbriae of virulent strains of the essential causative agent, Dichelobacter nodosus However, the bacterium is immunologically heterogeneous, and 10 distinct fimbrial serogroups have been identified. Ideally, in each outbreak the infecting strains would be cultured and serogrouped so that the appropriate serogroup-specific mono- or bivalent vaccine could be administered, because multivalent vaccines lack efficacy due to antigenic competition. If clinical disease expression is suspected to be incomplete, culture-based virulence tests are required to confirm the diagnosis, because control of benign footrot is economically unjustifiable. Both diagnosis and vaccination are conducted at the flock level. The aims of this study were to develop a PCR-based procedure for detecting and serogrouping D. nodosus directly from foot swabs and to determine whether this could be done accurately from the same cultured swab. A total of 269 swabs from the active margins of foot lesions of 261 sheep in 12 Merino sheep flocks in southeastern Australia were evaluated. DNA extracts taken from putative pure cultures of D. nodosus and directly from the swabs were evaluated in PCR assays for the 16S rRNA and fimA genes of D. nodosus Pure cultures were tested also by the slide agglutination test. Direct PCR using extracts from swabs was more sensitive than culture for detecting and serogrouping D. nodosus strains. Using the most sensitive sample collection method of the use of swabs in lysis buffer, D. nodosus was more likely to be detected by PCR in active than in inactive lesions, and in lesions with low levels of fecal contamination, but lesion score was not a significant factor. PCR conducted on extracts from swabs in modified Stuart's transport medium that had already been used to inoculate culture plates had lower sensitivity. Therefore, if culture is required to enable virulence tests to be conducted, it is recommended that duplicate swabs be collected from each foot lesion, one in transport medium for culture and the other in lysis buffer for PCR.

The clonal and mutational evolution spectrum of primary triple-negative breast cancers.

Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time-to our knowledge-in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.

TITAN: inference of copy number architectures in clonal cell populations from tumor whole-genome sequence data.

The evolution of cancer genomes within a single tumor creates mixed cell populations with divergent somatic mutational landscapes. Inference of tumor subpopulations has been disproportionately focused on the assessment of somatic point mutations, whereas computational methods targeting evolutionary dynamics of copy number alterations (CNA) and loss of heterozygosity (LOH) in whole-genome sequencing data remain underdeveloped. We present a novel probabilistic model, TITAN, to infer CNA and LOH events while accounting for mixtures of cell populations, thereby estimating the proportion of cells harboring each event. We evaluate TITAN on idealized mixtures, simulating clonal populations from whole-genome sequences taken from genomically heterogeneous ovarian tumor sites collected from the same patient. In addition, we show in 23 whole genomes of breast tumors that the inference of CNA and LOH using TITAN critically informs population structure and the nature of the evolving cancer genome. Finally, we experimentally validated subclonal predictions using fluorescence in situ hybridization (FISH) and single-cell sequencing from an ovarian cancer patient sample, thereby recapitulating the key modeling assumptions of TITAN.

Evaluation of Genotypic and Phenotypic Protease Virulence Tests for Dichelobacter nodosus Infection in Sheep.

Dichelobacter nodosus is a fastidious, strictly anaerobic bacterium, an obligate parasite of the ruminant hoof, and the essential causative agent of virulent ovine footrot. The clinical disease results from a complex interplay between the pathogen, the environment, and the host. Sheep flocks diagnosed with virulent but not benign footrot in Australia may be quarantined and required to undergo a compulsory eradication program, with costs met by the farmer. Virulence of D. nodosus at least partially depends on the elaboration of a protease encoded by aprV2 and manifests as elastase activity. Laboratory virulence tests are used to assist diagnosis because clinical differentiation of virulent and benign footrot can be challenging during the early stages of disease or when the disease is not fully expressed due to unfavorable pasture conditions. Using samples collected from foot lesions from 960 sheep from 40 flocks in four different geographic regions, we evaluated the analytical characteristics of qPCR tests for the protease gene alleles aprV2 and aprB2, and compared these with results from phenotypic protease (elastase and gelatin gel) tests. There was a low level of agreement between clinical diagnosis and quantitative PCR (qPCR) test outcomes at both the flock and sample levels and poor agreement between qPCR test outcomes and the results of phenotypic virulence tests. The diagnostic specificity of the qPCR test was low at both the flock and individual swab levels (31.3% and 18.8%, respectively). By contrast, agreement between the elastase test and clinical diagnosis was high at both the flock level (diagnostic sensitivity [DSe], 100%; diagnostic specificity [DSp], 78.6%) and the isolate level (DSe, 69.5%; DSp, 80.5%).

MHC class II transactivator CIITA is a recurrent gene fusion partner in lymphoid cancers.

Chromosomal translocations are critically involved in the molecular pathogenesis of B-cell lymphomas, and highly recurrent and specific rearrangements have defined distinct molecular subtypes linked to unique clinicopathological features. In contrast, several well-characterized lymphoma entities still lack disease-defining translocation events. To identify novel fusion transcripts resulting from translocations, we investigated two Hodgkin lymphoma cell lines by whole-transcriptome paired-end sequencing (RNA-seq). Here we show a highly expressed gene fusion involving the major histocompatibility complex (MHC) class II transactivator CIITA (MHC2TA) in KM-H2 cells. In a subsequent evaluation of 263 B-cell lymphomas, we also demonstrate that genomic CIITA breaks are highly recurrent in primary mediastinal B-cell lymphoma (38%) and classical Hodgkin lymphoma (cHL) (15%). Furthermore, we find that CIITA is a promiscuous partner of various in-frame gene fusions, and we report that CIITA gene alterations impact survival in primary mediastinal B-cell lymphoma (PMBCL). As functional consequences of CIITA gene fusions, we identify downregulation of surface HLA class II expression and overexpression of ligands of the receptor molecule programmed cell death 1 (CD274/PDL1 and CD273/PDL2). These receptor-ligand interactions have been shown to impact anti-tumour immune responses in several cancers, whereas decreased MHC class II expression has been linked to reduced tumour cell immunogenicity. Thus, our findings suggest that recurrent rearrangements of CIITA may represent a novel genetic mechanism underlying tumour-microenvironment interactions across a spectrum of lymphoid cancers.

Dynamic temporal behaviour of the keyboard action on the Hammond organ and its perceptual significance.

The Hammond organ is one of earliest electronic instruments and is still used widely in contemporary popular music. One of its main sonic features is the "key-click," a transient that occurs upon note onset, caused by the mechanical bouncing of the nine electric contacts actuated during each key press. A study of the dynamic mechanical behaviour of the contact bounces is presented, showing that the velocity, the type of touch and, more in general, the temporal evolution of the key position, all affect different characteristics of the contact bounces. A second study focuses on the listener's perception of the generated sound and finds that listeners can classify sounds produced on the Hammond organ according to the type of touch and velocity used. It is concluded that the Hammond organ is a touch-responsive instrument and that the gesture used to produce a note affects the generated sound across multiple dimensions. The control available at the fingertips of the musician is therefore such that it cannot be easily reduced to a single scalar velocity parameter, as is common practice in modern digital emulations of the instrument.

Are there gender differences in locus of control specific to alcohol dependence?

AIMS AND OBJECTIVES: To investigate gender differences in locus of control in an alcohol-dependent population. BACKGROUND: Locus of control helps to explain behaviour in terms of internal (the individual is responsible) or external (outside forces, such as significant other people or chance, are responsible) elements. Past research on gender differences in locus of control in relation to alcohol dependence has shown mixed results. There is a need then to examine gender and locus of control in relation to alcohol dependence to ascertain the veracity of any locus of control differences as a function of gender. DESIGN AND METHODS: The Multidimensional Health Locus of Control form-C was administered to clients from alcohol dependence treatment centres in the West of Scotland. Independent t-tests were carried out to assess gender differences in alcohol dependence severity and internal/external aspects of locus of control. RESULTS: One hundred and eighty-eight (53% females) participants were recruited from a variety of alcohol dependence treatment centres. The majority of participants (72%) came from Alcoholics Anonymous groups. Women revealed a greater internal locus of control compared with men. Women also had a greater 'significant others' locus of control score than men. Men were more reliant on 'chance' and 'doctors' than women. All these trends were not, however, statistically significant. CONCLUSIONS: Gender differences in relation to locus of control and alcohol dependence from past studies are ambiguous. This study also found no clear statistically significant differences in locus of control orientation as a function of gender. RELEVANCE TO CLINICAL PRACTICE: This article helps nurses to contextualise health behaviours as a result of internal or external forces. It also helps nursing staff to better understand alcohol dependence treatment in relation to self-efficacy and control. Moreover, it highlights an important concept in health education theory.

Synthesis of 5'-GalNAc-Conjugated Oligonucleotides: A Comparison of Solid and Solution-Phase Conjugation Strategies.

Antisense oligonucleotides (ASOs) conjugated to triantennary N-acetyl galactosamine (GalNAc) ligands represent an emerging approach to antisense therapy. Our current generation of GalNAc-ASO conjugates link the GalNAc to the 5'-terminus of the ASO. The conjugation reaction can be accomplished using solution-phase or solid-phase techniques. Here we show a direct comparison of a solution-phase and a solid-phase conjugation strategy. The solution-phase approach, using amine-pentafluorophenyl (PFP) ester coupling, is higher yielding and gives material of slightly higher purity, but requires several additional unit operations and longer production time. The solid-phase approach, using a protected GalNAc ligand phosphoramidite, is more expedient, but results in lower yield and purity. Both strategies efficiently deliver conjugated material in excellent purity.