Generation, Characterization, and Treatment of Functional Zebrafish Models for Musculoskeletal Disorders

Musculoskeletal disorders in humans present with a broad range of phenotypes that vary in severity, onset, and genetic cause. The musculoskeletal system is comprised of complex and diverse tissues that make up the majority of total body mass. Thus, mutations in many different genes with differing structure and function can produce phenotypically similar developmental and morphological defects in the muscle and skeleton. In addition, the muscular and skeletal systems develop non-autonomously, wherein development and function of each will affect development of the other. Thus, mutations in genes responsible for development of this complex system have the potential to influence morphogenesis outside of their tissue of expression. Many different gene defects have the potential to cause musculoskeletal disorders and to properly treat afflicted patients it is necessary to understand the underlying causes of pathogenesis. One such example of pathogenic morphogenesis is Distal Arthrogryposis (DA), a group of ten congenital disorders characterized by contractures of the joints of the distal limbs that presents at birth. The most severe form of DA, Freeman-Sheldon syndrome (DA2A), is most commonly associated with autosomal dominant missense mutations in the motor domain of MYH3, a sarcomeric embryonic myosin heavy chain gene expressed in developing skeletal muscle. While this gene is expressed only transiently in skeletal muscle tissue, symptoms present in the structure of skeletal joints and persist beyond birth. To examine the disease mechanism responsible for developing skeletal muscle and joint contracture and to explore treatment options for afflicted patients, we created the most commonly observed human DA2A pathogenic variant (MYH3R672H) in the analogous gene in zebrafish (smyhc1R673H). The heterozygous zebrafish had contractures of the spine that modeled the phenotype of DA2A patient’s muscle contractures. We observed shortened muscle fibers and sarcomeres, disorganized skeletal muscle tissue, and disrupted motor activity. The phenotype was rescued with an actin-myosin inhibitor, para-aminoblebbistatin, suggesting that contractures are caused by constitutive muscle contraction. While we were able to characterize the phenotype of this pathogenic variant, many variants of unknown significance have been recorded in the motor domain of MYH3. Given the labor and time required to generate missense mutants using traditional methods, a higher throughput means of mutant generation is necessary to examine the function of dozens of reported variants. To develop a higher throughput method of analyzing single variants, we created zebrafish lines that would be expected to enable high-throughput screening. A GFP reporter tag at the end of smyhc1 is used as a marker for successful homology directed repair. A deletion is induced where reported variants cluster in the human analog that also serves to put GFP out of frame. In-frame repair and integration of a variant of interest via a repair template returns GFP into frame, allowing for efficient screening of F0 embryos. Finally, we analyzed a fibronectin domain containing 1 (fndc1) zebrafish knockout to better understand its association with human scoliosis. fndc1 zebrafish mutants displayed a higher bone mineral density and slightly larger vertebral centra, though scoliosis was not present. Using zebrafish to model musculoskeletal disease, we were able to elucidate disease mechanisms of distal arthrogryposis and scoliosis, as well as develop more efficient methods for variant analysis in zebrafish

Models of distal arthrogryposis and lethal congenital contracture syndrome

Distal arthrogryposis and lethal congenital contracture syndromes describe a broad group of disorders that share congenital limb contractures in common. While skeletal muscle sarcomeric genes comprise many of the first genes identified for Distal Arthrogyposis, other mechanisms of disease have been demonstrated, including key effects on peripheral nerve function. While Distal Arthrogryposis and Lethal Congenital Contracture Syndromes display superficial similarities in phenotype, the underlying mechanisms for these conditions are diverse but overlapping. In this review, we discuss the important insights gained into these human genetic diseases resulting from in vitro molecular studies and in vivo models in fruit fly, zebrafish, and mice

Universal Credit and In-Work Conditionality – a productive turn?

This report explores employer perspectives of Universal Credit (the new working age benefit for those who are unemployed or on a low income), and the likely impact of ‘in-work conditionality’ on firms’ behaviour and productivity. Under the policy, working social security claimants may be expected to increase their pay through progressing and/or taking on additional hours of work (UC replaces Working Tax Credits). Employers are key to outcomes arising from active labour market policies and their response to new expectations placed on low-income workers will be pivotal to the policy’s effects. However, policy and research has neglected to consider employer perspectives of UC, or likely impact on firms’ behaviour and productivity

MYH3-associated distal arthrogryposis zebrafish model is normalized with para-aminoblebbistatin

Distal arthrogryposis (DA) is group of syndromes characterized by congenital joint contractures. Treatment development is hindered by the lack of vertebrate models. Here, we describe a zebrafish model in which a common MYH3 missense mutation (R672H) was introduced into the orthologous zebrafish gene smyhc1 (slow myosin heavy chain 1) (R673H). We simultaneously created a smyhc1 null allele (smyhc

Re-structuring of marine communities exposed to environmental change

Species richness is the most commonly used but controversial biodiversity metric in studies on aspects of community stability such as structural composition or productivity. The apparent ambiguity of theoretical and experimental findings may in part be due to experimental shortcomings and/or heterogeneity of scales and methods in earlier studies. This has led to an urgent call for improved and more realistic experiments. In a series of experiments replicated at a global scale we translocated several hundred marine hard bottom communities to new environments simulating a rapid but moderate environmental change. Subsequently, we measured their rate of compositional change (re-structuring) which in the great majority of cases represented a compositional convergence towards local communities. Re-structuring is driven by mortality of community components (original species) and establishment of new species in the changed environmental context. The rate of this re-structuring was then related to various system properties. We show that availability of free substratum relates negatively while taxon richness relates positively to structural persistence (i.e., no or slow re-structuring). Thus, when faced with environmental change, taxon-rich communities retain their original composition longer than taxon-poor communities. The effect of taxon richness, however, interacts with another aspect of diversity, functional richness. Indeed, taxon richness relates positively to persistence in functionally depauperate communities, but not in functionally diverse communities. The interaction between taxonomic and functional diversity with regard to the behaviour of communities exposed to environmental stress may help understand some of the seemingly contrasting findings of past research

Household-level predictors of the presence of servants in Northern Orkney, Scotland, 1851–1901

Servants were an important part of the northwestern European household economy in the preindustrial past. This study examines household-level characteristics that are predictive of the presence of rural servants using data from Orkney, Scotland. The number of servants present in a household is related to household composition, landholding size, and the marital status of the household head. In addition, the sex of the particular servant hired reveals that the labor of male and female servants is not fungible. The sex of the servant hired is related to the ratio of male and female household members of working age, the occupation of the head, household composition, and the size of the household\u27s landholding

Maternal proviral load and vertical transmission of Human T cell Lymphotropic Virus type 1 in Guinea-Bissau

The relative importance of routes of transmission of human T cell lymphotropic virus type 1 (HTLV-1) in Guinea-Bissau is largely unknown; vertical transmission is thought to be important, but there are very few existing data. We aimed to examine factors associated with transmission in mothers and children in Guinea-Bissau, where HTLV-1 is endemic (prevalence of 5% in the adult population). A cross-sectional survey was performed among mothers and their children (aged <15 years) in a rural community in Guinea-Bissau. A questionnaire to identify risk factors for infection and a blood sample were obtained. HTLV-1 proviral load in peripheral blood was determined and PCR was performed to compare long terminal repeat (LTR) sequences in mother-child pairs. Fourteen out of 55 children (25%) of 31 HTLV-1-infected mothers were infected versus none of 70 children of 30 uninfected mothers. The only factor significantly associated with HTLV-1 infection in the child was the proviral load of the mother; the risk of infection increased significantly with the log(10) proviral load in the mother's peripheral blood (OR 5.5, 95% CI 2.1-14.6, per quartile), adjusted for weaning age and maternal income. HTLV-1 sequences of the LTR region obtained from mother-child pairs were identical within pairs but differed between the pairs. Vertical transmission plays an important role in HTLV-1 transmission in this community in Guinea-Bissau. The risk of transmission increases with the mother's proviral load in the peripheral blood. Identical sequences in mother-child pairs give additional support to the maternal source of the children's infectio

Interactions among mitochondrial proteins altered in glioblastoma

Mitochondrial dysfunction is putatively central to glioblastoma (GBM) pathophysiology but there has been no systematic analysis in GBM of the proteins which are integral to mitochondrial function. Alterations in proteins in mitochondrial enriched fractions from patients with GBM were defined with label-free liquid chromatography mass spectrometry. 256 mitochondrially-associated proteins were identified in mitochondrial enriched fractions and 117 of these mitochondrial proteins were markedly (fold-change &#8805;2) and significantly altered in GBM (p &#8804; 0.05). Proteins associated with oxidative damage (including catalase, superoxide dismutase 2, peroxiredoxin 1 and peroxiredoxin 4) were increased in GBM. Protein–protein interaction analysis highlighted a reduction in multiple proteins coupled to energy metabolism (in particular respiratory chain proteins, including 23 complex-I proteins). Qualitative ultrastructural analysis in GBM with electron microscopy showed a notably higher prevalence of mitochondria with cristolysis in GBM. This study highlights the complex mitochondrial proteomic adjustments which occur in GBM pathophysiology

Narratives for Lengyel funerary practice

This paper, dedicated to the memory of István Zalai-Gaál, presents formally modelled date estimates for the sequence of Lengyel funerary pottery in western Hungary, eastern Austria and south-west Slovakia. It is an extension of the dating and modelling already carried out by the project, The Times of Their Lives (ToTL), on the major Lengyel aggregation, including burials, at Alsónyék-Bátaszék in south-east Transdanubia. Key aspects of the Lengyel culture and its research history are discussed, including the challenges of establishing its sequence with greater precision and of affirming its significance in wider Neolithic narratives. The many contributions of István Zalai-Gaál are noted. Results from Alsónyék are summarised. The present study concentrates on furnished Lengyel graves, using the analysis of Lengyel funerary pottery from western Hungary, eastern Austria and south-west Slovakia. A full catalogue of the 121 pot types identified by István Zalai-Gaál is presented, and correspondence analysis of the occurrence of 113 of these types in 247 graves suggests a seriation for these grave-assemblages. The new radiocarbon dating programme for the sequence of Lengyel funerary pottery was designed within the framework of Bayesian chronological modelling. We aimed to provide formal date estimates for the use of different pot types and for their combinations in different phases of the seriation. We also aimed to estimate the period during which furnished Lengyel burial occurred, the pace of its introduction and demise, the date and duration of use of individual cemeteries, and the intensity of Lengyel funerary practice through time. In addition to the 159 radiocarbon measurements from 141 graves at Alsónyék (29 of which appear in the correspondence analyses presented here), 91 radiocarbon measurements are reported from 57 graves across the Lengyel distribution under discussion, all but 16 achieved by the ToTL project. These provide three chronological models for the sequence of Lengyel furnished burials: one based on the chronology of individual cemeteries and other contexts; another combining the seriation based on István Zalai-Gaál’s typology with the radiocarbon dates; and the third combining the seriation previously suggested by Diaconescu (2014a) with the radiocarbon dates. Details of the models, and of dietary analysis to investigate the possibility of offsets, are described. Lengyel furnished burial appears to have emerged rapidly, probably during the course of the 49th century cal BC, across the whole area in question (although the bulk of the earliest burials may have been in the north). Furnished burial increased in popularity in south-east Transdanubia during the 48th century cal BC, although there was a decline in the practice further north at this time. Furnished Lengyel burial appears to have become less frequent after c. 4600 cal BC, although the practice finally ended in the second half of the 46th century cal BC. Implications for the character of Lengyel communities and wider narratives of Lengyel development as a whole are discussed