The Open University's repository of research publications and other research outputs Are there an... more The Open University's repository of research publications and other research outputs Are there any HOTTIPs for defining coding potential of lncRNAs, or just a lot of HOTAIR?
The OpenSTEM Labs comprises a wide range of instruments and equipment for teaching practical scie... more The OpenSTEM Labs comprises a wide range of instruments and equipment for teaching practical science at a distance. The concept of an Internet of Laboratory Things describes our approach to creating a network of experimental activities unconstrained by distance. 17000 studenthours of use in the last academic year provides insight into use at scale. Ultralow latency protocols have proved an important component for rapid-real-time control experiments. The OpenSTEM Labs ‘Internet of Laboratory Things (IoLT)’ aims to challenge the traditional university pedagogical model for lab teaching of students and teachers being co-located, during normal working hours[1]. Students are connected to state-of-the-art instrumentation and equipment for practical enquiries over the internet, where distance is no longer a barrier and where access to equipment can be available 24 hours a day. Students and teachers access the equipment via a web browser through which they can book an online session, undert...
Long noncoding RNAs (lncRNAs) have a wide range of functions in health and disease, but many rema... more Long noncoding RNAs (lncRNAs) have a wide range of functions in health and disease, but many remain uncharacterized because of their complex expression patterns and structures. The genetic loci encoding lncRNAs can be subject to accelerated evolutionary changes within the human lineage. HAR1 is a region that has a significantly altered sequence compared to other primates and is a component of two overlapping lncRNA loci, HAR1A and HAR1B. Although the functions of these lncRNAs are unknown, they have been associated with neurological disorders and cancer. Here, we explore the current state of understanding of evolution in human lncRNA genes, using the HAR1 locus as the case study.
Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammator... more Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of n...
The Open University's repository of research publications and other research outputs Are there an... more The Open University's repository of research publications and other research outputs Are there any HOTTIPs for defining coding potential of lncRNAs, or just a lot of HOTAIR?
Altered dopamine receptor labelling has been demonstrated in presymptomatic and symptomatic Hunti... more Altered dopamine receptor labelling has been demonstrated in presymptomatic and symptomatic Huntington's disease (HD) gene carriers, indicating that alterations in dopaminergic signalling are an early event in HD. We have previously described early alterations in synaptic transmission and plasticity in both the cortex and hippocampus of the R6/1 mouse model of Huntington's disease. Deficits in cortical synaptic plasticity were associated with altered dopaminergic signalling and could be reversed by D1- or D2-like dopamine receptor activation. In light of these findings we here investigated whether defects in dopamine signalling could also contribute to the marked alteration in hippocampal synaptic function. To this end we performed dopamine receptor labelling and pharmacology in the R6/1 hippocampus and report a marked, age-dependent elevation of hippocampal D1 and D2 receptor labelling in R6/1 hippocampal subfields. Yet, pharmacological inhibition or activation of D1- or D2...
ABSTRACT Huntington's disease is a fatal neurodegenerative disorder characterised by a pr... more ABSTRACT Huntington's disease is a fatal neurodegenerative disorder characterised by a progressive motor, psychiatric and cognitive decline and associated with a marked loss of neurons in the cortex and striatum of affected individuals. The disease is inherited in an autosomal dominant fashion and is caused by a trinucleotide (CAG) repeat expansion in the gene encoding the protein huntingtin. Predictive genetic testing has revealed early cognitive deficits in asymptomatic gene carriers such as altered working memory, executive function and recognition memory. The perirhinal cortex is believed to process aspects of recognition memory. Evidence from primate studies suggests that decrements in neuronal firing within this cortical region encode recognition memory and that the underlying mechanism is an activity-dependent long-term depression (LTD) of excitatory neurotransmission, the converse of long-term potentiation (LTP). We have used the R6/1 mouse model of HD to assess synaptic plasticity in the perirhinal cortex. This mouse model provides an ideal tool for investigating early and progressive changes in synaptic function in HD. We report here that LTD at perirhinal synapses is markedly reduced in R6/1 mice. We also provide evidence to suggest that a reduction in dopamine D2 receptor signalling may be implicated.
Screening of families clinically ascertained for the fragile X syndrome phenotype revealed two me... more Screening of families clinically ascertained for the fragile X syndrome phenotype revealed two mentally impaired males who were cytogenetically negative for the fragile X chromosome. In both cases, screening for the FMR1 trinucleotide expansion mutation revealed a rearrangement within the FMR1 gene. In the first case, a 660-bp deletion is present in 40% of peripheral lymphocytes. PCR and sequence analysis
A gene designated "FMR-1" has been isolated at the fragile-X locus. One exon of this ge... more A gene designated "FMR-1" has been isolated at the fragile-X locus. One exon of this gene is carried on a 5.1-kb EcoRI fragment that exhibits length variation in fragile-X patients because of amplification of or insertion into a CGG-repeat sequence. This repeat probably represents the fragile site. The EcoRI fragment also includes an HTF island that is hypermethylated in fragile-X patients showing absence of FMR-1 mRNA. In this paper, we present further evidence that the FMR-1 gene is involved in the clinical manifestation of the fragile-X syndrome and also in the expression of the cellular phenotype. A deletion including the HTF island and exons of the FMR-1 gene was detected in a fragile X-negative mentally retarded male who presented the clinical phenotype of the fragile-X syndrome. The deletion involves less than 250 kb of genomic DNA, including DXS548 and at least five exons of the FMR-1 gene. These data support the hypothesis that loss of function of the FMR-1 gene l...
Expansion of an unstable (CGG)n repeat to over 200 triplets within the promoter region of the hum... more Expansion of an unstable (CGG)n repeat to over 200 triplets within the promoter region of the human FMR1 gene leads to extensive local methylation and transcription silencing, resulting in the loss of FMRP protein and the development of the clinical features of fragile X syndrome. The causative link between (CGG)n expansion, methylation and gene silencing is unknown, although gene silencing is associated with extensive changes to local chromatin architecture. In order to determine the direct effects of increased repeat length on gene transcription in a chromatin context, we have examined the influence of FMR1 (CGG)n repeats upon transcription from the HSV thymidine kinase promoter in the Xenopus laevis oocyte. We observe a reduction in mRNA production directly associated with increasing repeat length, with a 90% reduction in mRNA production from arrays over 100 repeats in length. Using a kinetic approach, we show that this transcriptional repression is concomitant with chromatin mat...
We have recently shown that the expression of the FRAXE fragile site in Xq28 is associated with t... more We have recently shown that the expression of the FRAXE fragile site in Xq28 is associated with the expansion of a GCC trinucleotide repeat. In the families studied, FRAXE expression is also associated with mild mental handicap. Here we present data on families that previously had been diagnosed as having the fragile X syndrome but that later were found to be negative for trinucleotide repeat expansion at the FRAXA locus. In these families we demonstrate the presence of a GCC trinucleotide repeat expansion at the FRAXE locus. Studies of the FRAXE locus of normal individuals show that they have 6-25 copies of the repeat, whereas affected individuals have > 200 copies. As in the fragile X syndrome, the amplified CpG residues are methylated in affected males.
Transfection of DNA derived from a variety of tumours can induce morphological transformation of ... more Transfection of DNA derived from a variety of tumours can induce morphological transformation of certain immortalised but normally contact-inhibited cell lines. This important technique has been instrumental in the identification and subsequent molecular cloning of a number of oncogenes, including Harvey-ras. We can extend this approach for studying neoplastic potential by performing chromosome-mediated, as distinct from DNA-mediated, gene transfer. This modification offers two potentially important advantages, both of which stem from the fact that sub-chromosomal lengths of DNA are transferred. Firstly, the expression of the oncogene can be studied in its normal chromosomal milieu; potential modifying effects of linked and unlinked sequences can be evaluated. Secondly, new chromatin segments with transforming potential but too large to be transferred as naked DNA may be revealed. Our experiments illustrate some of the new insights into the molecular basis of neoplastic change which...
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, Jan 17, 2014
Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in le... more Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in leukocyte adhesion to, and subsequent migration across, brain endothelial cells (BECs), which is crucial for the development of neuroinflammatory disorders such as multiple sclerosis (MS). In contrast, microRNA-146a (miR-146a) has emerged as an anti-inflammatory molecule by inhibiting NF-κB activity in various cell types, but its effect in BECs during neuroinflammation remains to be evaluated. Here, we show that miR-146a was upregulated in microvessels of MS-active lesions and the spinal cord of mice with experimental autoimmune encephalomyelitis. In vitro, TNFα and IFNγ treatment of human cerebral microvascular endothelial cells (hCMEC/D3) led to upregulation of miR-146a. Brain endothelial overexpression of miR-146a diminished, whereas knockdown of miR-146a augmented cytokine-stimulated adhesion of T cells to hCMEC/D3 cells, nuclear translocation of NF-κB, and expression of adhesion molec...
Blood-brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple s... more Blood-brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses although their role in central nervous system (CNS) microvascular disorders is largely unknown. Here, we have identified miR-155 as a critical miRNA in neuroinflammation at the BBB. MiR-155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR-155 reduced CNS extravasation of systemic tracers both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR-155 was strongly and rapidly upregulated by inflammatory cytokines. MiR-155 upregulation mimicked cytokine-induced alterations in junctional organization and permeability whereas inhibition of endogenous miR-155 partially prevented cytokine-induced increase in permeability. Furthermore, miR-155 modulated brain endothelial barrier function by targeting not only cell-cell complex molecules such as annexin-2 and claudin-1 but also focal adhesion components such as DOCK-1 and syntenin-1. We propose brain endothelial miR-155 as a negative regulator of blood-brain barrier function that may constitute a novel therapeutic target for central nervous system neuroinflammatory disorders.
The fragile X syndrome is a common cause of mental retardation and is associated with a fragile s... more The fragile X syndrome is a common cause of mental retardation and is associated with a fragile site at Xq27.3 (FRAXA). Recently, evidence has been presented for the role of methylation and genomic imprinting in the expression of the disease. We have identified a site of methylation in patients by long range restriction mapping of the region. In this paper we present a YAC contig of this area, localise the CpG sequences which are methylated, and show by in situ hybridisation that the site of fragility lies within this region.
We describe a novel procedure, filter priming, for the generation of hybridisation probes from DN... more We describe a novel procedure, filter priming, for the generation of hybridisation probes from DNA fragments immobilised on nylon
The Open University's repository of research publications and other research outputs Are there an... more The Open University's repository of research publications and other research outputs Are there any HOTTIPs for defining coding potential of lncRNAs, or just a lot of HOTAIR?
The OpenSTEM Labs comprises a wide range of instruments and equipment for teaching practical scie... more The OpenSTEM Labs comprises a wide range of instruments and equipment for teaching practical science at a distance. The concept of an Internet of Laboratory Things describes our approach to creating a network of experimental activities unconstrained by distance. 17000 studenthours of use in the last academic year provides insight into use at scale. Ultralow latency protocols have proved an important component for rapid-real-time control experiments. The OpenSTEM Labs ‘Internet of Laboratory Things (IoLT)’ aims to challenge the traditional university pedagogical model for lab teaching of students and teachers being co-located, during normal working hours[1]. Students are connected to state-of-the-art instrumentation and equipment for practical enquiries over the internet, where distance is no longer a barrier and where access to equipment can be available 24 hours a day. Students and teachers access the equipment via a web browser through which they can book an online session, undert...
Long noncoding RNAs (lncRNAs) have a wide range of functions in health and disease, but many rema... more Long noncoding RNAs (lncRNAs) have a wide range of functions in health and disease, but many remain uncharacterized because of their complex expression patterns and structures. The genetic loci encoding lncRNAs can be subject to accelerated evolutionary changes within the human lineage. HAR1 is a region that has a significantly altered sequence compared to other primates and is a component of two overlapping lncRNA loci, HAR1A and HAR1B. Although the functions of these lncRNAs are unknown, they have been associated with neurological disorders and cancer. Here, we explore the current state of understanding of evolution in human lncRNA genes, using the HAR1 locus as the case study.
Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammator... more Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of n...
The Open University's repository of research publications and other research outputs Are there an... more The Open University's repository of research publications and other research outputs Are there any HOTTIPs for defining coding potential of lncRNAs, or just a lot of HOTAIR?
Altered dopamine receptor labelling has been demonstrated in presymptomatic and symptomatic Hunti... more Altered dopamine receptor labelling has been demonstrated in presymptomatic and symptomatic Huntington's disease (HD) gene carriers, indicating that alterations in dopaminergic signalling are an early event in HD. We have previously described early alterations in synaptic transmission and plasticity in both the cortex and hippocampus of the R6/1 mouse model of Huntington's disease. Deficits in cortical synaptic plasticity were associated with altered dopaminergic signalling and could be reversed by D1- or D2-like dopamine receptor activation. In light of these findings we here investigated whether defects in dopamine signalling could also contribute to the marked alteration in hippocampal synaptic function. To this end we performed dopamine receptor labelling and pharmacology in the R6/1 hippocampus and report a marked, age-dependent elevation of hippocampal D1 and D2 receptor labelling in R6/1 hippocampal subfields. Yet, pharmacological inhibition or activation of D1- or D2...
ABSTRACT Huntington's disease is a fatal neurodegenerative disorder characterised by a pr... more ABSTRACT Huntington's disease is a fatal neurodegenerative disorder characterised by a progressive motor, psychiatric and cognitive decline and associated with a marked loss of neurons in the cortex and striatum of affected individuals. The disease is inherited in an autosomal dominant fashion and is caused by a trinucleotide (CAG) repeat expansion in the gene encoding the protein huntingtin. Predictive genetic testing has revealed early cognitive deficits in asymptomatic gene carriers such as altered working memory, executive function and recognition memory. The perirhinal cortex is believed to process aspects of recognition memory. Evidence from primate studies suggests that decrements in neuronal firing within this cortical region encode recognition memory and that the underlying mechanism is an activity-dependent long-term depression (LTD) of excitatory neurotransmission, the converse of long-term potentiation (LTP). We have used the R6/1 mouse model of HD to assess synaptic plasticity in the perirhinal cortex. This mouse model provides an ideal tool for investigating early and progressive changes in synaptic function in HD. We report here that LTD at perirhinal synapses is markedly reduced in R6/1 mice. We also provide evidence to suggest that a reduction in dopamine D2 receptor signalling may be implicated.
Screening of families clinically ascertained for the fragile X syndrome phenotype revealed two me... more Screening of families clinically ascertained for the fragile X syndrome phenotype revealed two mentally impaired males who were cytogenetically negative for the fragile X chromosome. In both cases, screening for the FMR1 trinucleotide expansion mutation revealed a rearrangement within the FMR1 gene. In the first case, a 660-bp deletion is present in 40% of peripheral lymphocytes. PCR and sequence analysis
A gene designated "FMR-1" has been isolated at the fragile-X locus. One exon of this ge... more A gene designated "FMR-1" has been isolated at the fragile-X locus. One exon of this gene is carried on a 5.1-kb EcoRI fragment that exhibits length variation in fragile-X patients because of amplification of or insertion into a CGG-repeat sequence. This repeat probably represents the fragile site. The EcoRI fragment also includes an HTF island that is hypermethylated in fragile-X patients showing absence of FMR-1 mRNA. In this paper, we present further evidence that the FMR-1 gene is involved in the clinical manifestation of the fragile-X syndrome and also in the expression of the cellular phenotype. A deletion including the HTF island and exons of the FMR-1 gene was detected in a fragile X-negative mentally retarded male who presented the clinical phenotype of the fragile-X syndrome. The deletion involves less than 250 kb of genomic DNA, including DXS548 and at least five exons of the FMR-1 gene. These data support the hypothesis that loss of function of the FMR-1 gene l...
Expansion of an unstable (CGG)n repeat to over 200 triplets within the promoter region of the hum... more Expansion of an unstable (CGG)n repeat to over 200 triplets within the promoter region of the human FMR1 gene leads to extensive local methylation and transcription silencing, resulting in the loss of FMRP protein and the development of the clinical features of fragile X syndrome. The causative link between (CGG)n expansion, methylation and gene silencing is unknown, although gene silencing is associated with extensive changes to local chromatin architecture. In order to determine the direct effects of increased repeat length on gene transcription in a chromatin context, we have examined the influence of FMR1 (CGG)n repeats upon transcription from the HSV thymidine kinase promoter in the Xenopus laevis oocyte. We observe a reduction in mRNA production directly associated with increasing repeat length, with a 90% reduction in mRNA production from arrays over 100 repeats in length. Using a kinetic approach, we show that this transcriptional repression is concomitant with chromatin mat...
We have recently shown that the expression of the FRAXE fragile site in Xq28 is associated with t... more We have recently shown that the expression of the FRAXE fragile site in Xq28 is associated with the expansion of a GCC trinucleotide repeat. In the families studied, FRAXE expression is also associated with mild mental handicap. Here we present data on families that previously had been diagnosed as having the fragile X syndrome but that later were found to be negative for trinucleotide repeat expansion at the FRAXA locus. In these families we demonstrate the presence of a GCC trinucleotide repeat expansion at the FRAXE locus. Studies of the FRAXE locus of normal individuals show that they have 6-25 copies of the repeat, whereas affected individuals have > 200 copies. As in the fragile X syndrome, the amplified CpG residues are methylated in affected males.
Transfection of DNA derived from a variety of tumours can induce morphological transformation of ... more Transfection of DNA derived from a variety of tumours can induce morphological transformation of certain immortalised but normally contact-inhibited cell lines. This important technique has been instrumental in the identification and subsequent molecular cloning of a number of oncogenes, including Harvey-ras. We can extend this approach for studying neoplastic potential by performing chromosome-mediated, as distinct from DNA-mediated, gene transfer. This modification offers two potentially important advantages, both of which stem from the fact that sub-chromosomal lengths of DNA are transferred. Firstly, the expression of the oncogene can be studied in its normal chromosomal milieu; potential modifying effects of linked and unlinked sequences can be evaluated. Secondly, new chromatin segments with transforming potential but too large to be transferred as naked DNA may be revealed. Our experiments illustrate some of the new insights into the molecular basis of neoplastic change which...
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, Jan 17, 2014
Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in le... more Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in leukocyte adhesion to, and subsequent migration across, brain endothelial cells (BECs), which is crucial for the development of neuroinflammatory disorders such as multiple sclerosis (MS). In contrast, microRNA-146a (miR-146a) has emerged as an anti-inflammatory molecule by inhibiting NF-κB activity in various cell types, but its effect in BECs during neuroinflammation remains to be evaluated. Here, we show that miR-146a was upregulated in microvessels of MS-active lesions and the spinal cord of mice with experimental autoimmune encephalomyelitis. In vitro, TNFα and IFNγ treatment of human cerebral microvascular endothelial cells (hCMEC/D3) led to upregulation of miR-146a. Brain endothelial overexpression of miR-146a diminished, whereas knockdown of miR-146a augmented cytokine-stimulated adhesion of T cells to hCMEC/D3 cells, nuclear translocation of NF-κB, and expression of adhesion molec...
Blood-brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple s... more Blood-brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses although their role in central nervous system (CNS) microvascular disorders is largely unknown. Here, we have identified miR-155 as a critical miRNA in neuroinflammation at the BBB. MiR-155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR-155 reduced CNS extravasation of systemic tracers both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR-155 was strongly and rapidly upregulated by inflammatory cytokines. MiR-155 upregulation mimicked cytokine-induced alterations in junctional organization and permeability whereas inhibition of endogenous miR-155 partially prevented cytokine-induced increase in permeability. Furthermore, miR-155 modulated brain endothelial barrier function by targeting not only cell-cell complex molecules such as annexin-2 and claudin-1 but also focal adhesion components such as DOCK-1 and syntenin-1. We propose brain endothelial miR-155 as a negative regulator of blood-brain barrier function that may constitute a novel therapeutic target for central nervous system neuroinflammatory disorders.
The fragile X syndrome is a common cause of mental retardation and is associated with a fragile s... more The fragile X syndrome is a common cause of mental retardation and is associated with a fragile site at Xq27.3 (FRAXA). Recently, evidence has been presented for the role of methylation and genomic imprinting in the expression of the disease. We have identified a site of methylation in patients by long range restriction mapping of the region. In this paper we present a YAC contig of this area, localise the CpG sequences which are methylated, and show by in situ hybridisation that the site of fragility lies within this region.
We describe a novel procedure, filter priming, for the generation of hybridisation probes from DN... more We describe a novel procedure, filter priming, for the generation of hybridisation probes from DNA fragments immobilised on nylon
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Papers by mark hirst