Bedform migration in a mixed sand and cohesive clay intertidal environment and implications for bed material transport predictions

Many coastal and estuarine environments are dominated by mixtures of non-cohesive sand and cohesive mud. The migration rate of bedforms, such as ripples and dunes, in these environments is important in determining bed material transport rates to inform and assess numerical models of sediment transport and geomorphology. However, these models tend to ignore parameters describing the physical and biological cohesion (resulting from clay and extracellular polymeric substances, EPS) in natural mixed sediment, largely because of a scarcity of relevant laboratory and field data. To address this gap in knowledge, data were collected on intertidal flats over a spring-neap cycle to determine the bed material transport rates of bedforms in biologically-active mixed sand-mud. Bed cohesive composition changed from below 2 vol% up to 5.4 vol% cohesive clay, as the tide progressed from spring towards neap. The amount of EPS in the bed sediment was found to vary linearly with the clay content. Using multiple linear regression, the transport rate was found to depend on the Shields stress parameter and the bed cohesive clay content. The transport rates decreased with increasing cohesive clay and EPS content, when these contents were below 2.8 vol% and 0.05 wt%, respectively. Above these limits, bedform migration and bed material transport was not detectable by the instruments in the study area. These limits are consistent with recently conducted sand-clay and sand-EPS laboratory experiments on bedform development. This work has important implications for the circumstances under which existing sand-only bedform migration transport formulae may be applied in a mixed sand-clay environment, particularly as 2.8 vol% cohesive clay is well within the commonly adopted definition of “clean sand”

Assessing the potential for gene therapy in a mouse model of SYNGAP1 haploinsufficiency-associated disorder

SYNGAP1 (Synaptic Ras GTPase activating protein 1) haploinsufficiency-associated disorder or intellectual developmental disorder, autosomal dominant 5 (MRD5) is caused by autosomal dominant loss-of-function mutations in the SYNGAP1 gene. De novo SYNGAP1 mutations represent one of the most common causes of non-syndromic intellectual disability (NSID) (OMIM #603384). The most common symptoms include moderate-to-severe intellectual disability (ID), epilepsy and autism spectrum phenotypes. SYNGAP1 protein is a component of the postsynaptic density complex where it acts as one of the key effectors of N-methyl-D-aspartate (NMDA) receptor downstream pathways. It is involved in regulatory pathways such as the trafficking of the α-amino-3-hydroxy-5-mehyl-4-isoxazolepropionic acid (AMPA) receptors, regulation of AMPA receptor translation and the regulation of several forms of synaptic plasticity. With the aim to study SYNGAP1 protein function and its involvement in the pathophysiology of the disease, several rodent models have been created and extensively characterised. These studies revealed phenotypes which partially resemble the symptomatology observed in humans such as seizure susceptibility, hyperactivity and impulsivity. It has been shown that early restoration of normal SYNGAP1 protein levels prevents the onset of haploinsufficiency-associated phenotypes in the mouse. This, and the monogenic origin of the disorder, makes SYNGAP1 a candidate target for gene replacement therapeutic approaches. I hypothesised that restoration of SYNGAP1 levels via Adeno Associated Virus (AAV)-mediated gene transfer during early postnatal development, will prevent the appearance or ameliorate the severity of behavioural phenotypes observed in the mouse model of SYNGAP1 haploinsufficiency. Therefore, the aim of this project was to evaluate molecular therapies to test this hypothesis. To this end, I first defined a robust battery of behavioural tests in order to confirm and identify deficits to use as endpoints to evaluate the efficacy of gene therapy products. Well-established and novel phenotypes were evaluated in wild-type and Syngap1+/- mice at different postnatal developmental time points. Syngap1+/- mice showed increased locomotor activity, anxiety-like phenotype and a risk-taking like behaviour. No genotype effect was observed in tests assessing compulsive behaviour, working memory, motor function and general well-being and depth perception. For the first generation of candidate therapeutic constructs two different promoters were used, the minimal murine Mecp2 promoter (MeP) 229 and the synthetic JeT promoter. MeP229 regulated constructs were expressing three different Myc-tagged human SYNGAP1 isoforms, A2, A1 and B2 while the JeT regulated construct the Myc-tagged human SYNGAP1 isoform A1. In vitro evaluation of protein expression revealed low levels of plasmid-derived SYNGAP1 produced from the MeP229 regulated constructs while a higher amount of Myc-tagged SYNGAP1 protein was detectable from the JeT regulated cassette via both fluorescence immunocytochemistry and immunoblotting. Protein expression from the vectorised ssAAV9/JeT-hSYNGAP1_A1-Myc was confirmed in the brain in vivo at 5 weeks post intracerebroventricular injection. A therapeutic dose escalation efficacy study assessing ssAAV9/JeT-hSYNGAP1_A1-Myc was then conducted in Syngap1+/- mice (1E10, 5E10 and 1E11 vg/mouse). Overall, these data revealed no amelioration of hyperactivity or anxiety phenotypes, but a modest trend toward the amelioration of the risk-taking behaviour was observed in a platform departure test. Post-hoc immunoblot analysis confirmed a dose-dependent expression of the vector-derived SYNGAP1. However absolute levels of SYNGAP1 protein measured in different brain areas were below physiological levels of the native protein. Subcellular localisation analysis in whole hippocampal synaptosomal preparation showed that the vector-derived protein was correctly translocated to the synaptic compartment, mimicking endogenous patterns of protein localisation

Bi-allelic TARS mutations are associated with brittle hair phenotype

Brittle and “tiger-tail” hair is the diagnostic hallmark of trichothiodystrophy (TTD), a rare recessive disease associated with a wide spectrum of clinical features including ichthyosis, intellectual disability, decreased fertility, and short stature. As a result of premature abrogation of terminal differentiation, the hair is brittle and fragile and contains reduced cysteine content. Hypersensitivity to UV light is found in about half of individuals with TTD; all of these individuals harbor bi-allelic mutations in components of the basal transcription factor TFIIH, and these mutations lead to impaired nucleotide excision repair and basal transcription. Different genes have been found to be associated with non-photosensitive TTD (NPS-TTD); these include MPLKIP (also called TTDN1), GTF2E2 (also called TFIIEβ), and RNF113A. However, a relatively large group of these individuals with NPS-TTD have remained genetically uncharacterized. Here we present the identification of an NPS-TTD-associated gene, threonyl-tRNA synthetase (TARS), found by next-generation sequencing of a group of uncharacterized individuals with NPS-TTD. One individual has compound heterozygous TARS variants, c.826A>G (p.Lys276Glu) and c.1912C>T (p.Arg638∗), whereas a second individual is homozygous for the TARS variant: c.680T>C (p.Leu227Pro). We showed that these variants have a profound effect on TARS protein stability and enzymatic function. Our results expand the spectrum of genes involved in TTD to include genes implicated in amino acid charging of tRNA, which is required for the last step in gene expression, namely protein translation. We previously proposed that some of the TTD-specific features derive from subtle transcription defects as a consequence of unstable transcription factors. We now extend the definition of TTD from a transcription syndrome to a “gene-expression” syndrome.</p

Protein instability associated with AARS1 and MARS1 mutations causes trichothiodystrophy

Trichothiodystrophy (TTD) is a rare hereditary neurodevelopmental disorder defined by sulfur-deficient brittle hair and nails and scaly skin, but with otherwise remarkably variable clinical features. The photosensitive TTD (PS-TTD) forms exhibits in addition to progressive neuropathy and other features of segmental accelerated aging and is associated with impaired genome maintenance and transcription. New factors involved in various steps of gene expression have been identified for the different non-photosensitive forms of TTD (NPS-TTD), which do not appear to show features of premature aging. Here, we identify alanyl-tRNA synthetase 1 and methionyl-tRNA synthetase 1 variants as new gene defects that cause NPS-TTD. These variants result in the instability of the respective gene products alanyl- and methionyl-tRNA synthetase. These findings extend our previous observations that TTD mutations affect the stability of the corresponding proteins and emphasize this phenomenon as a common feature of TTD. Functional studies in skin fibroblasts from affected individuals demonstrate that these new variants also impact on the rate of tRNA charging, which is the first step in protein translation. The extension of reduced abundance of TTD factors to translation as well as transcription redefines TTD as a syndrome in which proteins involved in gene expression are unstable

Bi-allelic TARS Mutations Are Associated with Brittle Hair Phenotype

Brittle and “tiger-tail” hair is the diagnostic hallmark of trichothiodystrophy (TTD), a rare recessive disease associated with a wide spectrum of clinical features including ichthyosis, intellectual disability, decreased fertility, and short stature. As a result of premature abrogation of terminal differentiation, the hair is brittle and fragile and contains reduced cysteine content. Hypersensitivity to UV light is found in about half of individuals with TTD; all of these individuals harbor bi-allelic mutations in components of the basal transcription factor TFIIH, and these mutations lead to impaired nucleotide excision repair and basal transcription. Different genes have been found to be associated with non-photosensitive TTD (NPS-TTD); these include MPLKIP (also called TTDN1), GTF2E2 (also called TFIIEβ), and RNF113A. However, a relatively large group of these individuals with NPS-TTD have remained genetically uncharacterized. Here we present the identification of an NPS-TTD-associated gene, threonyl-tRNA synthetase (TARS), found by next-generation sequencing of a group of uncharacterized individuals with NPS-TTD. One individual has compound heterozygous TARS variants, c.826A>G (p.Lys276Glu) and c.1912C>T (p.Arg638∗), whereas a second individual is homozygous for the TARS variant: c.680T>C (p.Leu227Pro). We showed that these variants have a profound effect on TARS protein stability and enzymatic function. Our results expand the spectrum of genes involved in TTD to include genes implicated in amino acid charging of tRNA, which is required for the last step in gene expression, namely protein translation. We previously proposed that some of the TTD-specific features derive from subtle transcription defects as a consequence of unstable transcription factors. We now extend the definition of TTD from a transcription syndrome to a “gene-expression” syndrome