The interplay between the biochemical and biomechanical properties to the establishment of perineuronal nets

Perineuronal nets (PNNs) are an important pericellular matrix structure that form around the soma and dendrites of sub-populations of neurons in the mature central nervous system (CNS). They have been implicated in regulating synaptic plasticity, increasing synaptic stability, neuroprotection and memory formation. They are composed of hyaluronan (HA), chondroitin sulfate proteoglycans (CSPGs), Haplns and tenascin-R, that can assemble in many different potential configurations to form morphologically distinct PNNs. Currently there is a proposed model for the assembly of this extracellular matrix structure, but the detailed mechanism for the stabilisation of the morphologies of PNNs is not yet known. The aim of this thesis project is to characterise the biochemical heterogeneity of PNNs and understand how PNN molecules assemble to form a pericellular coat with distinct morphologies, while ascertaining the biomechanical contribution of each molecule to the pericellular coat. Using Western blotting, regional variations in the molecular composition of PNN-associated molecules were observed that may present an opportunity to modulate PNNs region specifically in the future. Different splice variants and levels of glycosylation of CSPGs were profiled in different CNS regions. To analyse the assembly of PNNs in real-time and measure the biomechanical properties of the film, an in vitro methodology was established using quartz crystal microbalance with dissipation monitoring (QCM-D). The PNN film was modelled using a tethered film of HA, to which HA and proteoglycan link protein 1 (Hapln1) and aggrecan (Acan) were sequentially added. Hapln1 bound to HA films stably, while binding of Acan was reversible, though could be stabilised by sequentially adding Acan to Hapln1-presenting HA films. While the density of HA films influenced the interaction of Acan, increasing the density of pre-bound Hapln1 did not. Furthermore, Hapln1 addition caused a rigidification of HA films, while Acan addition caused a softening effect, highlighting the important role of Hapln1 in stabilising the PNN matrix and a potential mechanism for regulating the biomechanical properties of PNNs. As cells in the CNS can mechanosense, understanding these mechanisms may offer new therapeutic avenues for targeting neurological diseases correlated with changes to PNNs. In addition, a new methodology for sizing surface-anchored GAG chains was established using QCM-D by utilising the relationship between the ratio of ∆D/-∆f and GAG size. This methodology highlights the effect of HA size on the biomechanical properties of the PNN film and presents a potential future methodology for the characterisation of PNN HA size. Mechanisms for biomechanically manipulating PNN films by increasing HA size and varying the density of Hapln1 and HA suggest PNNs may partially function via a biomechanical signalling mechanism in addition to already established mechanisms

A gene expression resource generated by genome-wide lacZ profiling in the mouse

Knowledge of the expression profile of a gene is a critical piece of information required to build an understanding of the normal and essential functions of that gene, and any role it may play in the development or progression of disease. High throughput, large scale efforts are on-going internationally to characterise reporter tagged knockout mouse lines. As part of that effort, we report an open access adult mouse expression resource in which the expression profile of 424 genes has been assessed in up to 47 different organs, tissues and sub-structures using a lacZ reporter gene. Many specific and informative expression patterns were noted. Expression was most commonly observed in the testis and brain, and was most restricted in white adipose tissue and mammary gland. Over half of the genes assessed presented with an absent or localised expression pattern (categorised as 0-10 positive structures). A link between complexity of expression profile and viability of homozygous null animals was observed; inactivation of genes expressed in ≥21 structures was more likely to result in reduced viability by postnatal day 14 compared with more restricted expression profiles. For validation purposes, this mouse expression resource was compared with Bgee, a federated composite of RNA based expression datasets. Strong agreement was observed indicating a high degree of specificity in our data. Furthermore, there were 1,207 observations of expression of a particular gene in an anatomical structure where Bgee had no data, indicating a large amount of novelty in our dataset. Examples of expression data corroborating and extending genotype-phenotype associations and supporting disease gene candidacy are presented to demonstrate the potentia

A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces

International audienceHyaluronan (HA) is a major component of peri-and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift (ΔD) and the negative frequency shift (− Δf) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from ~ 2 kDa tetrasaccharides to ~ 1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the ΔD/ − Δf ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coating

A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces

AbstractHyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift (ΔD) and the negative frequency shift (− Δf) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from ~ 2 kDa tetrasaccharides to ~ 1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the ΔD/ − Δf ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.</jats:p

A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces

AbstractHyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift (ΔD) and the negative frequency shift (-Δf) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from ∼2 kDa tetrasaccharides to ∼1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the ΔD/-Δf ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.</jats:p

A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis

A gene expression resource generated by genome-wide lacZ profiling in the mouse

Knowledge of the expression profile of a gene is a critical piece of information required to build an understanding of the normal and essential functions of that gene and any role it may play in the development or progression of disease. High-throughput, large-scale efforts are on-going internationally to characterise reporter-tagged knockout mouse lines. As part of that effort, we report an open access adult mouse expression resource, in which the expression profile of 424 genes has been assessed in up to 47 different organs, tissues and sub-structures using a lacZ reporter gene. Many specific and informative expression patterns were noted. Expression was most commonly observed in the testis and brain and was most restricted in white adipose tissue and mammary gland. Over half of the assessed genes presented with an absent or localised expression pattern (categorised as 0-10 positive structures). A link between complexity of expression profile and viability of homozygous null animals was observed; inactivation of genes expressed in ≥21 structures was more likely to result in reduced viability by postnatal day 14 compared with more restricted expression profiles. For validation purposes, this mouse expression resource was compared with Bgee, a federated composite of RNA-based expression data sets. Strong agreement was observed, indicating a high degree of specificity in our data. Furthermore, there were 1207 observations of expression of a particular gene in an anatomical structure where Bgee had no data, indicating a large amount of novelty in our data set. Examples of expression data corroborating and extending genotype-phenotype associations and supporting disease gene candidacy are presented to demonstrate the potential of this powerful resource

Animal-assisted psychotherapy for young people with behavioural problems in residential care

The aim of this study was to evaluate the impact of an animal-assisted psychotherapy (AAP) programme on clinical symptoms, personal adjustment and adaptive skills in a group of adolescents in residential care who had experienced childhood trauma and who presented mental health problems and difficulties adapting to the care home environment. The 87 participants (Mage = 15.17, SD = 1.53) were divided into two groups: a treatment group (25 girls and 27 boys; Mage = 15.00, SD = 1.55) and a control group (9 girls and 26 boys; Mage = 15.42, SD = 1.50). The programme consisted of 34 sessions involving both group (23 sessions) and individual (11 sessions) AAP. The Behaviour Assessment System for Children (BASC) was used to evaluate clinical and adaptive dimensions of behaviour and personality. The results indicated that, in comparison with controls, the young people who took part in the AAP programme reported a significant improvement on two measures of internalising symptoms, namely depression and sense of inadequacy. Although no significant differences were observed in relation to externalising symptoms, the adolescents who received the AAP programme showed improved social skills in terms of their ability to interact satisfactorily with peers and adults in the care home environment, as well as a more positive attitude towards teachers at school. These results suggest that AAP may be a promising treatment for young people who have experienced childhood trauma and who subsequently find it difficult to adapt to the residential care settingThis work was supported by the Basque Government [grant number IT892-16

Capacity of the Golgi Apparatus for Cargo Transport Prior to Complete Assembly

In yeast, particular emphasis has been given to endoplasmic reticulum (ER)-derived, cisternal maturation models of Golgi assembly while in mammalian cells more emphasis has been given to golgins as a potentially stable assembly framework. In the case of de novo Golgi formation from the ER after brefeldin A/H89 washout in HeLa cells, we found that scattered, golgin-enriched, structures formed early and contained golgins including giantin, ranging across the entire cis to trans spectrum of the Golgi apparatus. These structures were incompetent in VSV-G cargo transport. Second, we compared Golgi competence in cargo transport to the kinetics of addition of various glycosyltransferases and glycosidases into nascent, golgin-enriched structures after drug washout. Enzyme accumulation was sequential with trans and then medial glycosyltransferases/glycosidases found in the scattered, nascent Golgi. Involvement in cargo transport preceded full accumulation of enzymes or GPP130 into nascent Golgi. Third, during mitosis, we found that the formation of a golgin-positive acceptor compartment in early telophase preceded the accumulation of a Golgi glycosyltransferase in nascent Golgi structures. We conclude that during mammalian Golgi assembly components fit into a dynamic, first-formed, multigolgin-enriched framework that is initially cargo transport incompetent. Resumption of cargo transport precedes full Golgi assembly