Isolation of human genomic DNA for genetic neonates: a comparison occurred in the genotyping of t... more Isolation of human genomic DNA for genetic neonates: a comparison occurred in the genotyping of the UC samples. Rajatileka et al. BMC Genetics 2013, 14:105
BoxED is an outreach and widening participation initiative designed, developed and delivered by m... more BoxED is an outreach and widening participation initiative designed, developed and delivered by members of the Faculty of Health and Applied Sciences at the University of the West of England (UWE), Bristol. The scheme aims to provide inspirational and engaging workshops to secondary school pupils in the local area, particularly targeting widening participation and attainment in order to encourage a wide range of young people into higher education. The following report documents the key findings of an evaluation undertaken in conjunction with the Centre for Research in Biosciences (CRIB) and the Science Communication Unit (SCU), UWE assessing the levels of satisfaction with the service and the breadth of science capital in school children aged 11-16 years in the Bristol area. The evaluation examined BoxED activities between October 2017 and March 2018. Post-activity questionnaires and semi-structured interviews were employed to gauge opinions from a total of 376 school pupils and 2 s...
Diabetic foot ulcer (DFU) is a serious complication of diabetes mellitus, affecting roughly 25% o... more Diabetic foot ulcer (DFU) is a serious complication of diabetes mellitus, affecting roughly 25% of diabetic patients and resulting in lower limb amputation in over 70% of known cases. In addition to the devastating physiological consequences of DFU and its impact on patient quality of life, DFU has significant clinical and economic implications. Various traditional therapies are implemented to effectively treat DFU. However, emerging technologies such as bioprinting and electrospinning, present an exciting opportunity to improve current treatment strategies through the development of 3D scaffolds, by overcoming the limitations of current wound healing strategies. This review provides a summary on (i) current prevention and treatment strategies available for DFU; (ii) methods of fabrication of 3D scaffolds relevant for this condition; (iii) suitable materials and commonly used molecules for the treatment of DFU; and (iv) future directions offered by emerging technologies.
Background and aims Sleep stages begin to emerge from 20 weeks' gestational age. Typical EEG chan... more Background and aims Sleep stages begin to emerge from 20 weeks' gestational age. Typical EEG changes appear at 28-30 weeks with periods of rapid eye movement (REM) and non-REM (NREM) sleep. Sleep patterns develop into near adult like patterns by 3-5 months of age. Sleep cycles (REM/NREM) are essential for infant brain development. Caffeine is routinely used for treatment and prevention of apnea of prematurity. Its effect on sleep organisation in preterm infants has been controversial. This study aimed to find differences in sleep organisation in preterm infants before and after initiation of caffeine treatment. Methods Polysomnography was recorded in 10 preterm infants [GA 27+2-36+6 (mean 30+1) weeks, BW 790-2875 (mean 1465) g] at 34-41 (avg. 36) weeks' GA before and the day after administration of caffeine citrate loading dose (20 mg/kg). The analysis was done with visual scoring. Results Sleep quality was variable. Some infants had very interrupted sleep structure. Uninterrupted phasic REM sleep periods seemed to be more readily identified after caffeine. However, we were unable to show this by using standard indexes. There was no difference in sleep stage distribution, number of awakenings, number of sleep stage transitions or average REM period (Table 1). Conclusions We did not find any evident effect of caffeine on sleep quality in preterm infants.
The Faculty of Health and Applied Sciences at the University of the West of England, Bristol has ... more The Faculty of Health and Applied Sciences at the University of the West of England, Bristol has been piloting a schools outreach project entitled BoxED (EDucation in a Box) since 2015. School activities are inspired from the research and teaching of our academics, and the BoxED team have developed a series of hour long activities, linked to the national curriculum, which are delivered in schools across the southwest region. Alongside the somewhat obvious benefits to school pupils, this project presents numerous opportunities and benefits for our students to develop 'real-world' skills, enabling them to successfully enter the employment market on graduating from their studies. The challenge now is to ensure the success can be built upon by securing future funding to enable a greater number of students the opportunity to engage with the project and they themselves to then develop those necessary enterprise and entrepreneurial skills appropriately, alongside their chosen subject of study-ready for the 'real-world' .
Preterm delivery is associated with neurodevelopmental impairment caused by environmental and gen... more Preterm delivery is associated with neurodevelopmental impairment caused by environmental and genetic factors. Dysfunction of the excitatory amino acid transporter 2 (EAAT2) and the resultant impaired glutamate uptake can lead to neurological disorders. In this study, we investigated the role of single nucleotide polymorphisms (SNPs; g.-200C>A and g.-181A>C) in the EAAT2 promoter in susceptibility to brain injury and neurodisability in very preterm infants born at or before 32-week gestation. DNA isolated from newborns' dried blood spots were used for pyrosequencing to detect both SNPs. Association between EAAT2 genotypes and cerebral palsy, cystic periventricular leukomalacia and a low developmental score was then assessed. The two SNPs were concordant in 89.4% of infants resulting in three common genotypes all carrying two C and two A alleles in different combinations. However, in 10.6% of cases, non-concordance was found, generating six additional rare genotypes. The A ...
Archives of Disease in Childhood - Fetal and Neonatal Edition, 2014
ABSTRACT : Preterm birth is associated with adverse neurodevelopmental outcomes. The pathological... more ABSTRACT : Preterm birth is associated with adverse neurodevelopmental outcomes. The pathological mechanisms leading to adverse outcomes involve several pathways, which are not fully understood. Current methods of assessing neurological injury associated with preterm birth have limited scope and low prognostic value. Whilst structural MRI may provide detailed anatomical information about the neonatal brain, there is imperfect mapping between structure and function. A supplementary approach is the use of functional MRI (fMRI) to infer functional connectivity (FC), evaluating integration of neural activity within the brain. There is emerging evidence that children who were born preterm show long term changes in FC, and early detection of such changes offers potential to improve understanding of pathophysiology of preterm brain injury. These functional changes may be influenced by both neonatal course and underlying susceptibilities to abnormal development, including genetic risk factors. We used resting state fMRI (rs-fMRI) at 3T to examine functional brain connectivity in 20 infants born at <32 weeks of gestation, scanned at term. Infants also had genetic testing to examine polymorphisms in the EAAT2 glutamate transporter, previously associated with variation in preterm neurodevelopmental outcomes. Using a multivariate model to examine the independent contributions of demographic, genetic and clinical characteristics of the infants to FC, we identified multiple dissociable influences on functional brain networks. This is the first report of genetic variability in cerebral glutamate homeostasis influencing neonatal brain connectivity. We discuss the impact on understanding preterm brain injury, and the potential for predicting neurodevelopmental outcome by non-invasive measurement of functional brain connectivity.
The aim of this work is to test if three single nucleotide polymorphisms (SNPs) implicated in glu... more The aim of this work is to test if three single nucleotide polymorphisms (SNPs) implicated in glutamate homeostasis or signalling and cellular survival are associated with birth condition. Methods: This study is drawn from the Avon Longitudinal Study of Parents and Children. 7611 term infants were genotyped and patient outcome data retrieved from routine medical records. Exposure measures were the presence of one or more minor alleles in one of 3 SNPs (rs2284411, rs2498804, rs1835740). The primary outcome was the need for resuscitation at birth. Results: For SNP rs1835740, infants homozygous for the minor allele compared to wild-type were more likely to need resuscitation (9.2% vs. 7.0%, p=0.041) while the odds ratio for resuscitation was associated with each increasing minor allele (OR 1.17 (1.01 to 1.35)). Population attributable risk fraction was 6.5%. There was no evidence that the other two SNPs investigated were associated with birth condition. Conclusions: We have tested three candidate SNPs to measure any association with birth condition. The study revealed that the rs1835740 was associated with the need for resuscitation and Apgar scores, with a substantial population impact.
Glucose-dependent sequestration of Ca2+ into endoplasmic reticulum and its subsequent release pla... more Glucose-dependent sequestration of Ca2+ into endoplasmic reticulum and its subsequent release play an important role in the control of intracellular Ca2+ concentration, which regulates insulin secretion in pancreatic β-cells. The active uptake of cytosolic Ca2+ into endoplasmic reticulum is mediated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs). We found, using RT-PCR with isoform-specific primers, that SERCA 2 and SERCA 3 mRNAs are co-expressed in human and rat islets of Langerhans and in the RINm5F β-cell line. Immunochemical analysis also revealed the existence of two SERCA proteins with molecular masses of 110 and 115 kDa in β-cell membranes. The 115 kDa protein was identified as SERCA 2b by its reaction with an isoform-specific antibody and the 110 kDa protein most probably corresponds to SERCA 3. The presence of two functionally different SERCA isoforms raises the possibility that they are located in distinct Ca2+ stores. There is evidence that altered Ca2+ handling in...
Changes in free intracellular Ca2+ concentration regulate insulin secretion from pancreatic β-cel... more Changes in free intracellular Ca2+ concentration regulate insulin secretion from pancreatic β-cells. The existence of steep Ca2+ gradients within the β-cell requires the presence of specialized Ca2+ exclusion systems. In this study we have characterized the plasma membrane Ca2+-ATPases (PMCAs) which extrude Ca2+ from the cytoplasm. PMCA isoform- and subtype-specific mRNA expression was investigated in rodent pancreatic α- and β-cell lines, and in human and rat islets of Langerhans using reverse-transcription PCR with primers flanking the calmodulin-binding region of rat PMCA. The expression pattern of PMCA 1 and 2 was conserved in different species and islet-cell types since both rat and human islets of Langerhans and all cell lines tested contained the 1b and 2b forms. PMCA 4 isoform subtypes, however, were expressed in a cell-type-specific manner since β-cells expressed PMCA 4b only, whereas in islets of Langerhans, which contain α, β, δ and polypeptide-secreting cells, PMCA 4a an...
The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvem... more The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic beta-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative-acting globular tail domain of MyoVa decreased by approximately 50% the number of vesicles docked at the plasma membrane and by 87% the number ...
Background: Single-strand conformational polymorphism (SSCP) is still a frequently used genotypin... more Background: Single-strand conformational polymorphism (SSCP) is still a frequently used genotyping method across different fields for the detection of single nucleotide polymorphisms (SNPs) due to its simplicity, requirement for basic equipment accessible in most laboratories and low cost. This technique was previously used to detect rs4354668:A > C (g.-181A > C) SNP in the promoter of astroglial glutamate transporter (EAAT2) and the same approach was initially used here to investigate this promoter region in a cohort of newborns. Results: Unexpectedly, four distinct DNA migration patterns were identified by SSCP. Sanger sequencing revealed two additional SNPs: g.-200C > A and g.-168C > T giving a rise to a total of ten EAAT2 promoter variants. SSCP failed to distinguish these variants reliably and thus pyrosequencing assays were developed. g.-168C > T was found in heterozygous form in one infant only with minor allele frequency (MAF) of 0.0023. In contrast, g.-200C > A and-181A > C were more common (with MAF of 0.46 and 0.49, respectively) and showed string evidence of linkage disequilibrium (LD). In a systematic comparison, 16% of samples were miss-classified by SSCP with 25-31% errors in the identification of the wild-type and homozygote mutant genotypes compared to pyrosequencing or Sanger sequencing. In contrast, SSCP and pyrosequencing of an unrelated single SNP (rs1835740:C > T), showed 94% concordance. Conclusion: Our data suggest that SSCP cannot always detect reliably several closely located SNPs. Furthermore, caution is needed in the interpretation of the association studies linking only one of the co-inherited SNPs in the EAAT2 promoter to human diseases.
The brain‐spliced isoform of Myosin Va (BR‐MyoVa) plays an important role in the transport of den... more The brain‐spliced isoform of Myosin Va (BR‐MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide‐producing cells. The molecular composition of the protein complex that recruits BR‐MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG‐associated proteins granuphilin‐a/b (Gran‐a/b), BR‐MyoVa and Rab27a, a member of the Rab family of GTPases. Gran‐a/b–BR‐MyoVa interaction is direct, involves regions downstream of the Rab27‐binding domain, and the C‐terminal part of Gran‐a determines exon specificity. MyoVa and Gran‐a/b are partially colocalised on SGs and disruption of Gran‐a/b–BR‐MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient‐stimulated hormone secretion in pancreatic beta‐cells. These results indicate the existence of at least another binding partner of BR‐MyoVa that was identified as rabphilin‐3A (Rph‐3A)...
The pluripotent human embryonic carcinoma cell line NTERA2 readily differentiates into neurons wh... more The pluripotent human embryonic carcinoma cell line NTERA2 readily differentiates into neurons when exposed to retinoic acid in vitro. These neurons show characteristic morphology with long processes and they express neuronal markers TUJ-1 and NeuN. NTERA2-derived neurons can regulate Ca2+ signalling through ionotropic glutamate (iGluR) and muscarinic receptors (mAChRs). Little is known, however, about the role of metabotropic glutamate receptors (mGluRs) in these neurons. Here we show that NTERA2-derived neurons express functional mGluR5, which is involved in Ca2+ signalling. Blocking mGluR5 activity at early stages of differentiation leads to fewer dendrites and a reduction in miniature excitatory postsynaptic currents (mEPSCs). Furthermore, cells cultured in the presence of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) show reduced N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ mobilisation but increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor Ca2+ permeability. During normal neuronal development, the edited GluR2 renders AMPARs Ca2+ impermeable. The increased Ca2+ permeability of AMPARs in MPEP-treated neurons is due to the reduced expression of GluR2 subunit protein. Thus, mGluR5 activity at early stages of differentiation is likely to play a role in the development of multipotent cell-derived neurons.
Myosin- and Rab-interacting protein (MyRIP), which belongs to the protein kinase A (PKA)–anchorin... more Myosin- and Rab-interacting protein (MyRIP), which belongs to the protein kinase A (PKA)–anchoring family, is implicated in hormone secretion. However, its mechanism of action is not fully elucidated. Here we investigate the role of MyRIP in myosin Va (MyoVa)-dependent secretory granule (SG) transport and secretion in pancreatic beta cells. These cells solely express the brain isoform of MyoVa (BR-MyoVa), which is a key motor protein in SG transport. In vitro pull-down, coimmunoprecipitation, and colocalization studies revealed that MyRIP does not interact with BR-MyoVa in glucose-stimulated pancreatic beta cells, suggesting that, contrary to previous notions, MyRIP does not link this motor protein to SGs. Glucose-stimulated insulin secretion is augmented by incretin hormones, which increase cAMP levels and leads to MyRIP phosphorylation, its interaction with BR-MyoVa, and phosphorylation of the BR-MyoVa receptor rabphilin-3A (Rph-3A). Rph-3A phosphorylation on Ser-234 was inhibited...
GABA B receptors (GABA B Rs) are involved in early events during neuronal development. The presen... more GABA B receptors (GABA B Rs) are involved in early events during neuronal development. The presence of GABA B Rs in developing oligodendrocytes has not been established. Using immunofluorescent co-localization, we have identified GA-BA B R proteins in O4 marker-positive oligodendrocyte precursor cells (OPCs) in 4-day-old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABA B1abcdf and GABA B2 subunits of the GABA B R. Using semiquantitative PCR analysis with GABA B R isoform-selective primers we found that the expression level of GABA B1abd was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABA B2 isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABA B1 and GABA B2 subunits are under independent control during oligodendroglial development. Activation of GABA B Rs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABA B R activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber-based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABA B Rs in oligodendrocyte development. The presence of GABA B Rs in developing oligodendrocytes provides a new mechanism for neuronal-glial interactions during development and may offer a novel target for promoting remyelination following white matter injury.
Oligodendrocytes (OLs) are responsible for axon myelination and are the principal cells targeted ... more Oligodendrocytes (OLs) are responsible for axon myelination and are the principal cells targeted in preterm white matter injury. The cellular and molecular mechanisms involved in white matter development and immature OL injury are incompletely understood. Metabotropic glutamate receptors (mGluRs) modulate neuronal development and survival, and have recently been identified in oligodendrocyte progenitor cells (OPCs). Using the highly homogeneous CG‐4 OPC line and O4 marker‐immunoselected primary OLs, we established the differentiation stage‐specific expression profile of mGluR3 and mGluR5 mRNAs and proteins in the oligodendroglial lineage and type‐2‐astrocytes (ASTs). Our quantitative analysis indicated no changes in mGluR3, but a significant down‐regulation of mGluR5a mRNA and protein expression during differentiation of OPCs into OLs or ASTs. The down‐regulation of mGluR5a had functional consequences, with significantly fewer OLs and ASTs than OPCs responding to the group I mGluR a...
While the subcellular organisation of mitochondria is likely to influence many aspects of cell ph... more While the subcellular organisation of mitochondria is likely to influence many aspects of cell physiology, its molecular control is poorly understood. Here, we have investigated the role of the retrograde motor protein complex, dynein-dynactin, in mitochondrial localisation and morphology. Disruption of dynein function, achieved in HeLa cells either by over-expressing the dynactin subunit, dynamitin (p50), or by microinjection of an anti-dynein intermediate chain antibody, resulted in (a) the redistribution of mitochondria to the nuclear periphery, and (b) the formation of long and highly branched mitochondrial structures. Suggesting that an alteration in the balance between mitochondrial fission and fusion may be involved in both of these changes, overexpression of p50 induced the translocation of the fission factor dynamin-related protein (Drp1) from mitochondrial membranes to the cytosol and microsomes. Moreover, a dominant-negative-acting form of Drp1 mimicked the effects of p50...
Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of... more Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of insulin in response to glucose. At present, the molecular motors involved in this movement, and the mechanisms whereby they may be regulated, are undefined. To investigate the role of kinesin family members, we labelled densecore vesicles in clonal β-cells using an adenovirally expressed, vesicle-targeted green fluorescent protein(phogrin.EGFP), and employed immunoadsorption to obtain highly purified insulin-containing vesicles. Whereas several kinesin family members were expressed in this cell type, only conventional kinesin heavy chain (KHC) was detected in vesicle preparations. Expression of a dominant-negative KHC motor domain (KHCmut) blocked all vesicular movements with velocity>0.4 μm second-1, which demonstrates that kinesin activity was essential for vesicle motility in live β-cells. Moreover, expression of KHCmut strongly inhibited the sustained, but not acute,stimulation o...
Isolation of human genomic DNA for genetic neonates: a comparison occurred in the genotyping of t... more Isolation of human genomic DNA for genetic neonates: a comparison occurred in the genotyping of the UC samples. Rajatileka et al. BMC Genetics 2013, 14:105
BoxED is an outreach and widening participation initiative designed, developed and delivered by m... more BoxED is an outreach and widening participation initiative designed, developed and delivered by members of the Faculty of Health and Applied Sciences at the University of the West of England (UWE), Bristol. The scheme aims to provide inspirational and engaging workshops to secondary school pupils in the local area, particularly targeting widening participation and attainment in order to encourage a wide range of young people into higher education. The following report documents the key findings of an evaluation undertaken in conjunction with the Centre for Research in Biosciences (CRIB) and the Science Communication Unit (SCU), UWE assessing the levels of satisfaction with the service and the breadth of science capital in school children aged 11-16 years in the Bristol area. The evaluation examined BoxED activities between October 2017 and March 2018. Post-activity questionnaires and semi-structured interviews were employed to gauge opinions from a total of 376 school pupils and 2 s...
Diabetic foot ulcer (DFU) is a serious complication of diabetes mellitus, affecting roughly 25% o... more Diabetic foot ulcer (DFU) is a serious complication of diabetes mellitus, affecting roughly 25% of diabetic patients and resulting in lower limb amputation in over 70% of known cases. In addition to the devastating physiological consequences of DFU and its impact on patient quality of life, DFU has significant clinical and economic implications. Various traditional therapies are implemented to effectively treat DFU. However, emerging technologies such as bioprinting and electrospinning, present an exciting opportunity to improve current treatment strategies through the development of 3D scaffolds, by overcoming the limitations of current wound healing strategies. This review provides a summary on (i) current prevention and treatment strategies available for DFU; (ii) methods of fabrication of 3D scaffolds relevant for this condition; (iii) suitable materials and commonly used molecules for the treatment of DFU; and (iv) future directions offered by emerging technologies.
Background and aims Sleep stages begin to emerge from 20 weeks' gestational age. Typical EEG chan... more Background and aims Sleep stages begin to emerge from 20 weeks' gestational age. Typical EEG changes appear at 28-30 weeks with periods of rapid eye movement (REM) and non-REM (NREM) sleep. Sleep patterns develop into near adult like patterns by 3-5 months of age. Sleep cycles (REM/NREM) are essential for infant brain development. Caffeine is routinely used for treatment and prevention of apnea of prematurity. Its effect on sleep organisation in preterm infants has been controversial. This study aimed to find differences in sleep organisation in preterm infants before and after initiation of caffeine treatment. Methods Polysomnography was recorded in 10 preterm infants [GA 27+2-36+6 (mean 30+1) weeks, BW 790-2875 (mean 1465) g] at 34-41 (avg. 36) weeks' GA before and the day after administration of caffeine citrate loading dose (20 mg/kg). The analysis was done with visual scoring. Results Sleep quality was variable. Some infants had very interrupted sleep structure. Uninterrupted phasic REM sleep periods seemed to be more readily identified after caffeine. However, we were unable to show this by using standard indexes. There was no difference in sleep stage distribution, number of awakenings, number of sleep stage transitions or average REM period (Table 1). Conclusions We did not find any evident effect of caffeine on sleep quality in preterm infants.
The Faculty of Health and Applied Sciences at the University of the West of England, Bristol has ... more The Faculty of Health and Applied Sciences at the University of the West of England, Bristol has been piloting a schools outreach project entitled BoxED (EDucation in a Box) since 2015. School activities are inspired from the research and teaching of our academics, and the BoxED team have developed a series of hour long activities, linked to the national curriculum, which are delivered in schools across the southwest region. Alongside the somewhat obvious benefits to school pupils, this project presents numerous opportunities and benefits for our students to develop 'real-world' skills, enabling them to successfully enter the employment market on graduating from their studies. The challenge now is to ensure the success can be built upon by securing future funding to enable a greater number of students the opportunity to engage with the project and they themselves to then develop those necessary enterprise and entrepreneurial skills appropriately, alongside their chosen subject of study-ready for the 'real-world' .
Preterm delivery is associated with neurodevelopmental impairment caused by environmental and gen... more Preterm delivery is associated with neurodevelopmental impairment caused by environmental and genetic factors. Dysfunction of the excitatory amino acid transporter 2 (EAAT2) and the resultant impaired glutamate uptake can lead to neurological disorders. In this study, we investigated the role of single nucleotide polymorphisms (SNPs; g.-200C>A and g.-181A>C) in the EAAT2 promoter in susceptibility to brain injury and neurodisability in very preterm infants born at or before 32-week gestation. DNA isolated from newborns' dried blood spots were used for pyrosequencing to detect both SNPs. Association between EAAT2 genotypes and cerebral palsy, cystic periventricular leukomalacia and a low developmental score was then assessed. The two SNPs were concordant in 89.4% of infants resulting in three common genotypes all carrying two C and two A alleles in different combinations. However, in 10.6% of cases, non-concordance was found, generating six additional rare genotypes. The A ...
Archives of Disease in Childhood - Fetal and Neonatal Edition, 2014
ABSTRACT : Preterm birth is associated with adverse neurodevelopmental outcomes. The pathological... more ABSTRACT : Preterm birth is associated with adverse neurodevelopmental outcomes. The pathological mechanisms leading to adverse outcomes involve several pathways, which are not fully understood. Current methods of assessing neurological injury associated with preterm birth have limited scope and low prognostic value. Whilst structural MRI may provide detailed anatomical information about the neonatal brain, there is imperfect mapping between structure and function. A supplementary approach is the use of functional MRI (fMRI) to infer functional connectivity (FC), evaluating integration of neural activity within the brain. There is emerging evidence that children who were born preterm show long term changes in FC, and early detection of such changes offers potential to improve understanding of pathophysiology of preterm brain injury. These functional changes may be influenced by both neonatal course and underlying susceptibilities to abnormal development, including genetic risk factors. We used resting state fMRI (rs-fMRI) at 3T to examine functional brain connectivity in 20 infants born at <32 weeks of gestation, scanned at term. Infants also had genetic testing to examine polymorphisms in the EAAT2 glutamate transporter, previously associated with variation in preterm neurodevelopmental outcomes. Using a multivariate model to examine the independent contributions of demographic, genetic and clinical characteristics of the infants to FC, we identified multiple dissociable influences on functional brain networks. This is the first report of genetic variability in cerebral glutamate homeostasis influencing neonatal brain connectivity. We discuss the impact on understanding preterm brain injury, and the potential for predicting neurodevelopmental outcome by non-invasive measurement of functional brain connectivity.
The aim of this work is to test if three single nucleotide polymorphisms (SNPs) implicated in glu... more The aim of this work is to test if three single nucleotide polymorphisms (SNPs) implicated in glutamate homeostasis or signalling and cellular survival are associated with birth condition. Methods: This study is drawn from the Avon Longitudinal Study of Parents and Children. 7611 term infants were genotyped and patient outcome data retrieved from routine medical records. Exposure measures were the presence of one or more minor alleles in one of 3 SNPs (rs2284411, rs2498804, rs1835740). The primary outcome was the need for resuscitation at birth. Results: For SNP rs1835740, infants homozygous for the minor allele compared to wild-type were more likely to need resuscitation (9.2% vs. 7.0%, p=0.041) while the odds ratio for resuscitation was associated with each increasing minor allele (OR 1.17 (1.01 to 1.35)). Population attributable risk fraction was 6.5%. There was no evidence that the other two SNPs investigated were associated with birth condition. Conclusions: We have tested three candidate SNPs to measure any association with birth condition. The study revealed that the rs1835740 was associated with the need for resuscitation and Apgar scores, with a substantial population impact.
Glucose-dependent sequestration of Ca2+ into endoplasmic reticulum and its subsequent release pla... more Glucose-dependent sequestration of Ca2+ into endoplasmic reticulum and its subsequent release play an important role in the control of intracellular Ca2+ concentration, which regulates insulin secretion in pancreatic β-cells. The active uptake of cytosolic Ca2+ into endoplasmic reticulum is mediated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs). We found, using RT-PCR with isoform-specific primers, that SERCA 2 and SERCA 3 mRNAs are co-expressed in human and rat islets of Langerhans and in the RINm5F β-cell line. Immunochemical analysis also revealed the existence of two SERCA proteins with molecular masses of 110 and 115 kDa in β-cell membranes. The 115 kDa protein was identified as SERCA 2b by its reaction with an isoform-specific antibody and the 110 kDa protein most probably corresponds to SERCA 3. The presence of two functionally different SERCA isoforms raises the possibility that they are located in distinct Ca2+ stores. There is evidence that altered Ca2+ handling in...
Changes in free intracellular Ca2+ concentration regulate insulin secretion from pancreatic β-cel... more Changes in free intracellular Ca2+ concentration regulate insulin secretion from pancreatic β-cells. The existence of steep Ca2+ gradients within the β-cell requires the presence of specialized Ca2+ exclusion systems. In this study we have characterized the plasma membrane Ca2+-ATPases (PMCAs) which extrude Ca2+ from the cytoplasm. PMCA isoform- and subtype-specific mRNA expression was investigated in rodent pancreatic α- and β-cell lines, and in human and rat islets of Langerhans using reverse-transcription PCR with primers flanking the calmodulin-binding region of rat PMCA. The expression pattern of PMCA 1 and 2 was conserved in different species and islet-cell types since both rat and human islets of Langerhans and all cell lines tested contained the 1b and 2b forms. PMCA 4 isoform subtypes, however, were expressed in a cell-type-specific manner since β-cells expressed PMCA 4b only, whereas in islets of Langerhans, which contain α, β, δ and polypeptide-secreting cells, PMCA 4a an...
The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvem... more The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic beta-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative-acting globular tail domain of MyoVa decreased by approximately 50% the number of vesicles docked at the plasma membrane and by 87% the number ...
Background: Single-strand conformational polymorphism (SSCP) is still a frequently used genotypin... more Background: Single-strand conformational polymorphism (SSCP) is still a frequently used genotyping method across different fields for the detection of single nucleotide polymorphisms (SNPs) due to its simplicity, requirement for basic equipment accessible in most laboratories and low cost. This technique was previously used to detect rs4354668:A > C (g.-181A > C) SNP in the promoter of astroglial glutamate transporter (EAAT2) and the same approach was initially used here to investigate this promoter region in a cohort of newborns. Results: Unexpectedly, four distinct DNA migration patterns were identified by SSCP. Sanger sequencing revealed two additional SNPs: g.-200C > A and g.-168C > T giving a rise to a total of ten EAAT2 promoter variants. SSCP failed to distinguish these variants reliably and thus pyrosequencing assays were developed. g.-168C > T was found in heterozygous form in one infant only with minor allele frequency (MAF) of 0.0023. In contrast, g.-200C > A and-181A > C were more common (with MAF of 0.46 and 0.49, respectively) and showed string evidence of linkage disequilibrium (LD). In a systematic comparison, 16% of samples were miss-classified by SSCP with 25-31% errors in the identification of the wild-type and homozygote mutant genotypes compared to pyrosequencing or Sanger sequencing. In contrast, SSCP and pyrosequencing of an unrelated single SNP (rs1835740:C > T), showed 94% concordance. Conclusion: Our data suggest that SSCP cannot always detect reliably several closely located SNPs. Furthermore, caution is needed in the interpretation of the association studies linking only one of the co-inherited SNPs in the EAAT2 promoter to human diseases.
The brain‐spliced isoform of Myosin Va (BR‐MyoVa) plays an important role in the transport of den... more The brain‐spliced isoform of Myosin Va (BR‐MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide‐producing cells. The molecular composition of the protein complex that recruits BR‐MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG‐associated proteins granuphilin‐a/b (Gran‐a/b), BR‐MyoVa and Rab27a, a member of the Rab family of GTPases. Gran‐a/b–BR‐MyoVa interaction is direct, involves regions downstream of the Rab27‐binding domain, and the C‐terminal part of Gran‐a determines exon specificity. MyoVa and Gran‐a/b are partially colocalised on SGs and disruption of Gran‐a/b–BR‐MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient‐stimulated hormone secretion in pancreatic beta‐cells. These results indicate the existence of at least another binding partner of BR‐MyoVa that was identified as rabphilin‐3A (Rph‐3A)...
The pluripotent human embryonic carcinoma cell line NTERA2 readily differentiates into neurons wh... more The pluripotent human embryonic carcinoma cell line NTERA2 readily differentiates into neurons when exposed to retinoic acid in vitro. These neurons show characteristic morphology with long processes and they express neuronal markers TUJ-1 and NeuN. NTERA2-derived neurons can regulate Ca2+ signalling through ionotropic glutamate (iGluR) and muscarinic receptors (mAChRs). Little is known, however, about the role of metabotropic glutamate receptors (mGluRs) in these neurons. Here we show that NTERA2-derived neurons express functional mGluR5, which is involved in Ca2+ signalling. Blocking mGluR5 activity at early stages of differentiation leads to fewer dendrites and a reduction in miniature excitatory postsynaptic currents (mEPSCs). Furthermore, cells cultured in the presence of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) show reduced N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ mobilisation but increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor Ca2+ permeability. During normal neuronal development, the edited GluR2 renders AMPARs Ca2+ impermeable. The increased Ca2+ permeability of AMPARs in MPEP-treated neurons is due to the reduced expression of GluR2 subunit protein. Thus, mGluR5 activity at early stages of differentiation is likely to play a role in the development of multipotent cell-derived neurons.
Myosin- and Rab-interacting protein (MyRIP), which belongs to the protein kinase A (PKA)–anchorin... more Myosin- and Rab-interacting protein (MyRIP), which belongs to the protein kinase A (PKA)–anchoring family, is implicated in hormone secretion. However, its mechanism of action is not fully elucidated. Here we investigate the role of MyRIP in myosin Va (MyoVa)-dependent secretory granule (SG) transport and secretion in pancreatic beta cells. These cells solely express the brain isoform of MyoVa (BR-MyoVa), which is a key motor protein in SG transport. In vitro pull-down, coimmunoprecipitation, and colocalization studies revealed that MyRIP does not interact with BR-MyoVa in glucose-stimulated pancreatic beta cells, suggesting that, contrary to previous notions, MyRIP does not link this motor protein to SGs. Glucose-stimulated insulin secretion is augmented by incretin hormones, which increase cAMP levels and leads to MyRIP phosphorylation, its interaction with BR-MyoVa, and phosphorylation of the BR-MyoVa receptor rabphilin-3A (Rph-3A). Rph-3A phosphorylation on Ser-234 was inhibited...
GABA B receptors (GABA B Rs) are involved in early events during neuronal development. The presen... more GABA B receptors (GABA B Rs) are involved in early events during neuronal development. The presence of GABA B Rs in developing oligodendrocytes has not been established. Using immunofluorescent co-localization, we have identified GA-BA B R proteins in O4 marker-positive oligodendrocyte precursor cells (OPCs) in 4-day-old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABA B1abcdf and GABA B2 subunits of the GABA B R. Using semiquantitative PCR analysis with GABA B R isoform-selective primers we found that the expression level of GABA B1abd was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABA B2 isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABA B1 and GABA B2 subunits are under independent control during oligodendroglial development. Activation of GABA B Rs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABA B R activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber-based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABA B Rs in oligodendrocyte development. The presence of GABA B Rs in developing oligodendrocytes provides a new mechanism for neuronal-glial interactions during development and may offer a novel target for promoting remyelination following white matter injury.
Oligodendrocytes (OLs) are responsible for axon myelination and are the principal cells targeted ... more Oligodendrocytes (OLs) are responsible for axon myelination and are the principal cells targeted in preterm white matter injury. The cellular and molecular mechanisms involved in white matter development and immature OL injury are incompletely understood. Metabotropic glutamate receptors (mGluRs) modulate neuronal development and survival, and have recently been identified in oligodendrocyte progenitor cells (OPCs). Using the highly homogeneous CG‐4 OPC line and O4 marker‐immunoselected primary OLs, we established the differentiation stage‐specific expression profile of mGluR3 and mGluR5 mRNAs and proteins in the oligodendroglial lineage and type‐2‐astrocytes (ASTs). Our quantitative analysis indicated no changes in mGluR3, but a significant down‐regulation of mGluR5a mRNA and protein expression during differentiation of OPCs into OLs or ASTs. The down‐regulation of mGluR5a had functional consequences, with significantly fewer OLs and ASTs than OPCs responding to the group I mGluR a...
While the subcellular organisation of mitochondria is likely to influence many aspects of cell ph... more While the subcellular organisation of mitochondria is likely to influence many aspects of cell physiology, its molecular control is poorly understood. Here, we have investigated the role of the retrograde motor protein complex, dynein-dynactin, in mitochondrial localisation and morphology. Disruption of dynein function, achieved in HeLa cells either by over-expressing the dynactin subunit, dynamitin (p50), or by microinjection of an anti-dynein intermediate chain antibody, resulted in (a) the redistribution of mitochondria to the nuclear periphery, and (b) the formation of long and highly branched mitochondrial structures. Suggesting that an alteration in the balance between mitochondrial fission and fusion may be involved in both of these changes, overexpression of p50 induced the translocation of the fission factor dynamin-related protein (Drp1) from mitochondrial membranes to the cytosol and microsomes. Moreover, a dominant-negative-acting form of Drp1 mimicked the effects of p50...
Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of... more Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of insulin in response to glucose. At present, the molecular motors involved in this movement, and the mechanisms whereby they may be regulated, are undefined. To investigate the role of kinesin family members, we labelled densecore vesicles in clonal β-cells using an adenovirally expressed, vesicle-targeted green fluorescent protein(phogrin.EGFP), and employed immunoadsorption to obtain highly purified insulin-containing vesicles. Whereas several kinesin family members were expressed in this cell type, only conventional kinesin heavy chain (KHC) was detected in vesicle preparations. Expression of a dominant-negative KHC motor domain (KHCmut) blocked all vesicular movements with velocity>0.4 μm second-1, which demonstrates that kinesin activity was essential for vesicle motility in live β-cells. Moreover, expression of KHCmut strongly inhibited the sustained, but not acute,stimulation o...
Uploads
Papers by Anikó Váradi