Gene discovery and mechanism of disease in the myopathies

Congenital myopathy and muscular dystrophy are two groups of inherited muscle diseases characterised by muscle weakness, and sub-classified by hallmark pathological features within a skeletal muscle biopsy. In order to understand the pathogenesis of inherited muscle disorders, and develop or apply therapies based on mechanistic insight, one must elucidate deep knowledge of the associated gene, genetic variant and the function of the encoded protein. This thesis focuses on three aspects of gene discovery in the inherited myopathies: (1) Identification of a novel variant and phenotype for a known disease gene; (2) understanding the functional role of a recently identified disease gene in skeletal muscle biology and disease; and (3) discovering a novel disease gene for congenital myopathy. We identified the first recessive variant within ACTA1 (encoding α-skeletal actin) as the genetic cause of congenital muscular dystrophy with rigid spine. This case uniquely describes recessive ACTA1 variants where α-skeletal actin protein is expressed. The unique clinical and histological presentation expands the spectrum of ACTA1 disease, and will help guide clinical care and future genetic diagnoses. Our team identified LMOD3 (leiomodin-3) as a novel disease gene for severe nemaline myopathy (NM). KLHL40 (encoding kelch-like family member 40) is another disease gene for severe NM. A recent study suggests mouse Klhl40 protects mouse Lmod3 protein from proteasome-mediated degradation, with the mechanistic basis of KLHL40-NM resulting from secondary loss of LMOD3. We investigated the regulation of human LMODs by human KLHL40, and unexpectedly found evidence that disputes the central paradigm that KLHL40 protects LMOD3 from proteasome-mediated degradation. We identified PYROXD1 as a new genetic cause of early-onset congenital myopathy. We provide the first characterisation of PYROXD1 as a nuclear-cytoplasmic oxidoreductase and our discovery highlights oxidative distress as a core mechanistic pathway in the myopathies. We derived a mouse model of Pyroxd1 deficiency, determining that global loss of mouse Pyroxd1 is embryonic lethal. We subsequently developed a mouse model with skeletal muscle knock-out of Pyroxd1 – as a means to elucidate the role of PYROXD1 in biology and disease

Building a Better Description

Universally practiced across the disciplines, description is also consistently devalued or overlooked. In this introduction to the special issue "Description Across Disciplines," Sharon Marcus, Heather Love, and Stephen Best propose that description is a critical practice more complex (and less contradictory) than its detractors have taken it to be. They argue that turning critical attention toward description’s nuances gives us access to the ways that scholars conventionally assign and withhold value and prestige. The authors set forth a number of principles (using their contributors’ essays as a guide) toward the end of "building a better description.

Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding

The polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology. In combination with High Throughput Sequencing (HTS), PCR is widely used to quantify transcript abundance for RNA-seq, and in the context of analysis of T and B cell receptor repertoires. In this study, we combine DNA barcoding with HTS to quantify PCR output from individual target molecules. We develop computational tools that simulate both the PCR branching process itself, and the subsequent subsampling which typically occurs during HTS sequencing. We explore the influence of different types of heterogeneity on sequencing output, and compare them to experimental results where the efficiency of amplification is measured by barcodes uniquely identifying each molecule of starting template. Our results demonstrate that the PCR process introduces substantial amplification heterogeneity, independent of primer sequence and bulk experimental conditions. This heterogeneity can be attributed both to inherited differences between different template DNA molecules, and the inherent stochasticity of the PCR process. The results demonstrate that PCR heterogeneity arises even when reaction and substrate conditions are kept as constant as possible, and therefore single molecule barcoding is essential in order to derive reproducible quantitative results from any protocol combining PCR with HTS

Dynamic Perturbations of the T-Cell Receptor Repertoire in Chronic HIV Infection and following Antiretroviral Therapy

HIV infection profoundly affects many parameters of the immune system and ultimately leads to AIDS, yet which factors are most important for determining resistance, pathology, and response to antiretroviral treatment - and how best to monitor them - remain unclear. We develop a quantitative high-throughput sequencing pipeline to characterize the TCR repertoires of HIV-infected individuals before and after antiretroviral therapy, working from small, unfractionated samples of peripheral blood. This reveals the TCR repertoires of HIV(+) individuals to be highly perturbed, with considerably reduced diversity as a small proportion of sequences are highly overrepresented. HIV also causes specific qualitative changes to the repertoire including an altered distribution of V gene usage, depletion of public TCR sequences, and disruption of TCR networks. Short-term antiretroviral therapy has little impact on most of the global damage to repertoire structure, but is accompanied by rapid changes in the abundance of many individual TCR sequences, decreases in abundance of the most common sequences, and decreases in the majority of HIV-associated CDR3 sequences. Thus, high-throughput repertoire sequencing of small blood samples that are easy to take, store, and process can shed light on various aspects of the T-cell immune compartment and stands to offer insights into patient stratification and immune reconstitution

Feature selection using a one dimensional naïve Bayes' classifier increases the accuracy of support vector machine classification of CDR3 repertoires.

MOTIVATION: Somatic DNA recombination, the hallmark of vertebrate adaptive immunity, has the potential to generate a vast diversity of antigen receptor sequences. How this diversity captures antigen specificity remains incompletely understood. In this study we use high throughput sequencing to compare the global changes in T cell receptor β chain complementarity determining region 3 (CDR3β) sequences following immunization with ovalbumin administered with complete Freund's adjuvant (CFA) or CFA alone. RESULTS: The CDR3β sequences were deconstructed into short stretches of overlapping contiguous amino acids. The motifs were ranked according to a one-dimensional Bayesian classifier score comparing their frequency in the repertoires of the two immunization classes. The top ranking motifs were selected and used to create feature vectors which were used to train a support vector machine. The support vector machine achieved high classification scores in a leave-one-out validation test reaching  : >90% in some cases. SUMMARY: The study describes a novel two-stage classification strategy combining a one-dimensional Bayesian classifier with a support vector machine. Using this approach we demonstrate that the frequency of a small number of linear motifs three amino acids in length can accurately identify a CD4 T cell response to ovalbumin against a background response to the complex mixture of antigens which characterize Complete Freund's Adjuvant. AVAILABILITY AND IMPLEMENTATION: The sequence data is available at www.ncbi.nlm.nih.gov/sra/?term¼SRP075893 The Decombinator package is available at github.com/innate2adaptive/Decombinator The R package e1071 is available at the CRAN repository https://cran.r-project.org/web/packages/e1071/index.html CONTACT: [email protected] information: Supplementary data are available at Bioinformatics online

Brown Dwarfs in Young Moving Groups from Pan-STARRS1. I. AB Doradus

Substellar members of young ($\lesssim$150 Myr) moving groups are valuable benchmarks to empirically define brown dwarf evolution with age and to study the low-mass end of the initial mass function. We have combined Pan-STARRS1 (PS1) proper motions with optical$-$IR photometry from PS1, 2MASS and $\textit{WISE}$ to search for substellar members of the AB Dor Moving Group within $\approx$50 pc and with spectral types of late-M to early-L, corresponding to masses down to $\approx$30 M$_{Jup}$ at the age of the group ($\approx$125 Myr). Including both photometry and proper motions allows us to better select candidates by excluding field dwarfs whose colors are similar to young AB~Dor Moving Group members. Our near-IR spectroscopy has identified six ultracool dwarfs (M6$-$L4; $\approx$30$-$100 M$_{Jup}$) with intermediate surface gravities (INT-G) as candidate members of the AB Dor Moving Group. We find another two candidate members with spectra showing hints of youth but consistent with field gravities. We also find four field brown dwarfs unassociated with the AB Dor Moving Group, three of which have INT-G gravity classification. While signatures of youth are present in the spectra of our $\approx$125 Myr objects, neither their $J-K$ nor $W1-W2$ colors are significantly redder than field dwarfs with the same spectral types, unlike younger ultracool dwarfs. We also determined PS1 parallaxes for eight of our candidates and one previously identified AB Dor Moving Group candidate. Although radial velocities (and parallaxes, for some) are still needed to fully assess membership, these new objects provide valuable insight into the spectral characteristics and evolution of young brown dwarfs.Comment: ApJ, accepte

Specificity, Privacy, and Degeneracy in the CD4 T Cell Receptor Repertoire Following Immunization.

T cells recognize antigen using a large and diverse set of antigen-specific receptors created by a complex process of imprecise somatic cell gene rearrangements. In response to antigen-/receptor-binding-specific T cells then divide to form memory and effector populations. We apply high-throughput sequencing to investigate the global changes in T cell receptor sequences following immunization with ovalbumin (OVA) and adjuvant, to understand how adaptive immunity achieves specificity. Each immunized mouse contained a predominantly private but related set of expanded CDR3β sequences. We used machine learning to identify common patterns which distinguished repertoires from mice immunized with adjuvant with and without OVA. The CDR3β sequences were deconstructed into sets of overlapping contiguous amino acid triplets. The frequencies of these motifs were used to train the linear programming boosting (LPBoost) algorithm LPBoost to classify between TCR repertoires. LPBoost could distinguish between the two classes of repertoire with accuracies above 80%, using a small subset of triplet sequences present at defined positions along the CDR3. The results suggest a model in which such motifs confer degenerate antigen specificity in the context of a highly diverse and largely private set of T cell receptors

How U.S. Ocean Policy and Market Power Can Reform the Coral Reef Wildlife Trade

As the world’s largest importer of marine ornamental species for the aquaria, curio, home décor, and jewelry industries, the United States has an opportunity to leverage its considerable market power to promote more sustainable trade and reduce the effects of ornamental trade stress on coral reefs worldwide. Evidence indicates that collection of some coral reef animals for these trades has caused virtual elimination of local populations, major changes in age structure, and promotion of collection practices that destroy reef habitats. Management and enforcement of collection activities in major source countries such as Indonesia and the Philippines remain weak. Strengthening US trade laws and enforcement capabilities combined with increasing consumer and industry demand for responsible conservation can create strong incentives for improving management in source countries. This is particularly important in light of the March 2010 failure of the parties to the Convention on International Trade in Endangered Species (CITES) to take action on key groups of corals

Relation of Trihalomethane Concentrations in Public Water Supplies to Stillbirth and Birth Weight in Three Water Regions in England

We investigated the association between total trihalomethanes (TTHMs) and risk of stillbirth and low and very low birth weight in three water regions in England, 1992–1998; associations with individual trihalomethanes (THMs) were also examined. Modeled estimates of quarterly TTHM concentrations in water zones, categorized as low (< 30 μg/L), medium (30–59 μg/L), or high (≥60 μg/L), were linked to approximately 1 million routine birth and stillbirth records using maternal residence at time of birth. In one region, where there was a positive socioeconomic deprivation gradient across exposure categories, there was also a positive, significant association of TTHM with risk of stillbirth and low and very low birth weight. Overall summary estimates across the three regions using a random-effects model to allow for between-region heterogeneity in exposure effects showed small excess risks in areas with high TTHM concentrations for stillbirths [odds ratio (OR) = 1.11; 95% confidence interval (CI), 1.00–1.23), low birth weight (OR = 1.09; 95% CI, 0.93–1.27), and very low birth weight (OR = 1.05; 95% CI, 0.82–1.34). Among the individual THMs, chloroform showed a similar pattern of risk as TTHM, but no association was found with concentrations of bromodichloromethane or total brominated THMs. Our findings overall suggest a significant association of stillbirths with maternal residence in areas with high TTHM exposure. Further work is needed looking at cause-specific stillbirths and effects of other disinfection by-products and to help differentiate between alternative (noncausal) explanations and those that may derive from the water supply

Causation events of stud laceration injuries in rugby union

Laceration injuries in rugby union account for approximately 6% of all injuries sustained during match play. Commentators often cite the design of studded footwear as a causal factor in laceration injuries. In order to assess the laceration injury risk of different stud designs, there is a need to develop a testing protocol that is able to replicate the laceration injury event. This study used a questionnaire to identify the play scenarios that result in laceration injuries. The questionnaire was answered by 191 rugby players, of which 72% had experienced one or more stud injuries during their career which hindered them playing rugby. Half of the laceration injuries described by the respondents came from the ruck, and 27% from a tackle. When analysing free-text responses, a deliberate stamp was described in 35% of the responses and a tackle from behind was described in 14% of responses. These injury scenarios are considered to be the dominant cause of laceration injuries. In future work the identified injury scenarios will be replicated in simulated play and kinetic and kinematic measurements will be recorded. This will inform test parameters for future assessment of laceration injury risk of stud designs