Publications by authors named "Samuel Clokie"

26 Publications

  • Page 1 of 1

The anti-tumour activity of DNA methylation inhibitor 5-aza-2'-deoxycytidine is enhanced by the common analgesic paracetamol through induction of oxidative stress.

Cancer Lett 2021 Mar 5;501:172-186. Epub 2021 Jan 5.

School of Dentistry, Institute of Clinical Sciences, College of Medical and Dental Sciences, The University of Birmingham, Birmingham, B5 7EG, UK; Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, The University of Birmingham, Birmingham, B15 2TT, UK. Electronic address:

The DNA demethylating agent 5-aza-2'-deoxycytidine (DAC, decitabine) has anti-cancer therapeutic potential, but its clinical efficacy is hindered by DNA damage-related side effects and its use in solid tumours is debated. Here we describe how paracetamol augments the effects of DAC on cancer cell proliferation and differentiation, without enhancing DNA damage. Firstly, DAC specifically upregulates cyclooxygenase-2-prostaglandin E pathway, inadvertently providing cancer cells with survival potential, while the addition of paracetamol offsets this effect. Secondly, in the presence of paracetamol, DAC treatment leads to glutathione depletion and finally to accumulation of ROS and/or mitochondrial superoxide, both of which have the potential to restrict tumour growth. The benefits of combined treatment are demonstrated here in head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukaemia cell lines, further corroborated in a HNSCC xenograft mouse model and through mining of publicly available DAC and paracetamol responses. The sensitizing effect of paracetamol supplementation is specific to DAC but not its analogue 5-azacitidine. In summary, the addition of paracetamol could allow for DAC dose reduction, widening its clinical usability and providing a strong rationale for consideration in cancer therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.canlet.2020.12.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845757PMC
March 2021

Non-Invasive Prenatal Diagnosis of Retinoblastoma Inheritance by Combined Targeted Sequencing Strategies.

J Clin Med 2020 Oct 30;9(11). Epub 2020 Oct 30.

West Midlands Regional Genetics Service, Birmingham Women's and Children's NHS Foundation Trust, Birmingham B15 2TG, UK.

Retinoblastoma, the most common childhood eye cancer, presents in two forms: heritable or sporadic. Heritable retinoblastoma is caused by a germline mutation in the gene. Early diagnosis of children at risk of inheriting an mutation is crucial to achieve optimal clinical outcome. Currently, the majority of genetic testing is performed on newborns, which has multiple disadvantages for both families and the healthcare system. We have developed a non-invasive prenatal diagnosis (NIPD) service for retinoblastoma, available from 8 weeks' gestation, which uses a combination of massively parallel sequencing (MPS) techniques, dependent on the inheritance model. Detection of paternal or suspected de novo variants is achieved through amplicon-based MPS. NIPD of a fetus at risk of maternal inheritance is performed using capture-based targeted sequencing and relative haplotype dosage analysis. In addition, we show proof of principle of how capture-based sequencing can be used for de novo variants unsuitable for amplicon-based testing. In total, we report the NIPD of 15 pregnancies, results of which show 100% concordance with all postnatal testing performed at the time of publication ( = 12) with remaining pregnancies ongoing. NIPD of retinoblastoma therefore offers a viable alternative to newborn genetic testing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/jcm9113517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692133PMC
October 2020

Clinical Service Delivery of Noninvasive Prenatal Diagnosis by Relative Haplotype Dosage for Single-Gene Disorders.

J Mol Diagn 2020 09 15;22(9):1151-1161. Epub 2020 Jun 15.

West Midlands Regional Genetics Service, Birmingham Women's and Children's NHS Foundation Trust, Edgbaston, United Kingdom.

A relative haplotype dosage (RHDO)-based method was developed and implemented into routine clinical practice for noninvasive prenatal diagnosis (NIPD) of multiple single-gene disorders: spinal muscular atrophy, Duchenne and Becker muscular dystrophies, and cystic fibrosis. This article describes the experiences of the first 152 pregnancies to have NIPD by RHDO as part of a routine clinical service. Provision of results within a clinically useful time frame (mean, 11 calendar days) was shown to be possible, with a very low failure rate (4%), none being due to a technical failure. Where follow-up confirmatory testing was performed for audit purposes, 100% concordance was seen with the NIPD result, and no discrepancies have been reported. The robust performance of the assay, together with high sensitivity and specificity, demonstrates that NIPD by RHDO is feasible for use in a clinical setting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmoldx.2020.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471768PMC
September 2020

Resource: A multi-species multi-timepoint transcriptome database and webpage for the pineal gland and retina.

J Pineal Res 2020 Oct 8;69(3):e12673. Epub 2020 Jul 8.

Section on Neuroendocrinology, Program in Developmental Endocrinology and Genetics, Eunice Shriver Kennedy National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.

The website and database https://snengs.nichd.nih.gov provides RNA sequencing data from multi-species analysis of the pineal glands from zebrafish (Danio rerio), chicken (White Leghorn), rat (Rattus novegicus), mouse (Mus musculus), rhesus macaque (Macaca mulatta), and human (Homo sapiens); in most cases, retinal data are also included along with results of the analysis of a mixture of RNA from tissues. Studies cover day and night conditions; in addition, a time series over multiple hours, a developmental time series and pharmacological experiments on rats are included. The data have been uniformly re-processed using the latest methods and assemblies to allow for comparisons between experiments and to reduce processing differences. The website presents search functionality, graphical representations, Excel tables, and track hubs of all data for detailed visualization in the UCSC Genome Browser. As more data are collected from investigators and improved genomes become available in the future, the website will be updated. This database is in the public domain and elements can be reproduced by citing the URL and this report. This effort makes the results of 21st century transcriptome profiling widely available in a user-friendly format that is expected to broadly influence pineal research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jpi.12673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513311PMC
October 2020

De Novo Missense Variants in FBXW11 Cause Diverse Developmental Phenotypes Including Brain, Eye, and Digit Anomalies.

Am J Hum Genet 2019 09 8;105(3):640-657. Epub 2019 Aug 8.

Faculty of Health and Life Sciences, Oxford Brookes University, Oxford OX3 0BP, UK; West Midlands Regional Clinical Genetics Service and Birmingham Health Partners, Birmingham Women's and Children's National Health Service Foundation Trust, Birmingham, B15 2TG, UK. Electronic address:

The identification of genetic variants implicated in human developmental disorders has been revolutionized by second-generation sequencing combined with international pooling of cases. Here, we describe seven individuals who have diverse yet overlapping developmental anomalies, and who all have de novo missense FBXW11 variants identified by whole exome or whole genome sequencing and not reported in the gnomAD database. Their phenotypes include striking neurodevelopmental, digital, jaw, and eye anomalies, and in one individual, features resembling Noonan syndrome, a condition caused by dysregulated RAS signaling. FBXW11 encodes an F-box protein, part of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex, involved in ubiquitination and proteasomal degradation and thus fundamental to many protein regulatory processes. FBXW11 targets include β-catenin and GLI transcription factors, key mediators of Wnt and Hh signaling, respectively, critical to digital, neurological, and eye development. Structural analyses indicate affected residues cluster at the surface of the loops of the substrate-binding domain of FBXW11, and the variants are predicted to destabilize the protein and/or its interactions. In situ hybridization studies on human and zebrafish embryonic tissues demonstrate FBXW11 is expressed in the developing eye, brain, mandibular processes, and limb buds or pectoral fins. Knockdown of the zebrafish FBXW11 orthologs fbxw11a and fbxw11b resulted in embryos with smaller, misshapen, and underdeveloped eyes and abnormal jaw and pectoral fin development. Our findings support the role of FBXW11 in multiple developmental processes, including those involving the brain, eye, digits, and jaw.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2019.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731360PMC
September 2019

Non-invasive diagnosis of retinoblastoma using cell-free DNA from aqueous humour.

Br J Ophthalmol 2019 Feb 11. Epub 2019 Feb 11.

West Midlands Regional Genetics Service, Birmingham Women's and Children's NHS Foundation Trust, Birmingham, UK.

Retinoblastoma is the most common eye malignancy in childhood caused by mutations in the gene. Both alleles of the gene must be mutated for tumour development. The initial mutation may be constitutional germline or somatic (originating in one retinal cell only). Distinguishing between these alternative mechanisms is crucial, with wider implications for management of the patient and family members. Bilateral retinoblastoma is nearly always due to a constitutional mutation; however, approximately 15% of unilateral cases also carry a germline mutation, and identifying these cases is important. This can be achieved by identifying both mutation types in tumour tissue and excluding their presence in blood. Modern eye-saving chemotherapy treatment (systemic, intra-arterial and intravitreal) has resulted in fewer enucleations. As a result, tumour tissue required to identify sporadic mutation(s) is not always available. Modern intravitreal chemotherapeutic techniques for retinoblastoma involve aspiration of aqueous humour (AH), providing a novel sample source for analysis. By analysing cell-free DNA present in the AH fluid of eyes affected with retinoblastoma, we have developed a screening test capable of detecting somatic mutations that is comparable to current tests on enucleated tumour tissue. The results obtained with fluid from enucleated eyes were concordant with tumour tissue in all 10 cases analysed. In addition, AH analysis from two patients undergoing intravitreal chemotherapy successfully identified somatic variants in both cases. Our findings suggest that AH fluid is a promising source of tumour-derived DNA in retinoblastoma for analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/bjophthalmol-2018-313005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709774PMC
February 2019

mir-374-5p, mir-379-5p, and mir-503-5p Regulate Proliferation and Hypertrophic Differentiation of Growth Plate Chondrocytes in Male Rats.

Endocrinology 2018 03;159(3):1469-1478

Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland.

Growth plate chondrocytes undergo sequential differentiation to form the resting zone, the proliferative zone (PZ), and the hypertrophic zone (HZ). The important role of microRNAs (miRNAs) in the growth plate was previously revealed by cartilage-specific ablation of Dicer, an enzyme essential for biogenesis of many miRNAs. To identify specific miRNAs that regulate differentiation of PZ chondrocytes to HZ chondrocytes, we microdissected individual growth plate zones from juvenile rats and performed miRNA profiling using a solution hybridization method and miRNA sequencing. Thirty-four miRNAs were differentially expressed between the PZ and the HZ, and we hypothesized that some of the miRNAs that are preferentially expressed in the PZ may promote proliferation and inhibit hypertrophic differentiation. Consistent with this hypothesis, transfection of inhibitors for four of these miRNAs (mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p) decreased proliferation in primary epiphyseal chondrocytes. The inhibitors for three of these miRNAs (mir-374-5p, mir-379-5p, and mir-503-5p) also increased expression of multiple genes that are associated with chondrocyte hypertrophic differentiation. We next hypothesized that preferential expression of these miRNAs in the PZ is driven by the parathyroid hormone-related protein (PTHrP) concentration gradient across the growth plate. Consistent with this hypothesis, treatment of primary chondrocytes with a parathyroid hormone (PTH)/PTHrP receptor agonist, PTH1-34, increased expression of mir-374-5p, mir-379-5p, and mir-503-5p. Taken together, our findings suggest that the PTHrP concentration gradient across the growth plate induces differential expression of mir-374-5p, mir-379-5p, and mir-503-5p between the PZ and the HZ. In the PZ, the higher expression levels of these miRNAs promote proliferation and inhibit hypertrophic differentiation. In the HZ, downregulation of these miRNAs inhibits proliferation and promotes hypertrophic differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2017-00780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839730PMC
March 2018

Increased Sensitivity of Diagnostic Mutation Detection by Re-analysis Incorporating Local Reassembly of Sequence Reads.

Mol Diagn Ther 2017 12;21(6):685-692

Yorkshire Regional Genetics Service, St. James's University Hospital, 6.2 Clinical Sciences Building, Leeds, LS9 7TF, United Kingdom.

Background: Diagnostic genetic testing programmes based on next-generation DNA sequencing have resulted in the accrual of large datasets of targeted raw sequence data. Most diagnostic laboratories process these data through an automated variant-calling pipeline. Validation of the chosen analytical methods typically depends on confirming the detection of known sequence variants. Despite improvements in short-read alignment methods, current pipelines are known to be comparatively poor at detecting large insertion/deletion mutations.

Methods: We performed clinical validation of a local reassembly tool, ABRA (assembly-based realigner), through retrospective reanalysis of a cohort of more than 2000 hereditary cancer cases.

Results: ABRA enabled detection of a 96-bp deletion, 4-bp insertion mutation in PMS2 that had been initially identified using a comparative read-depth approach. We applied an updated pipeline incorporating ABRA to the entire cohort of 2000 cases and identified one previously undetected pathogenic variant, a 23-bp duplication in PTEN. We demonstrate the effect of read length on the ability to detect insertion/deletion variants by comparing HiSeq2500 (2 × 101-bp) and NextSeq500 (2 × 151-bp) sequence data for a range of variants and thereby show that the limitations of shorter read lengths can be mitigated using appropriate informatics tools.

Conclusions: This work highlights the need for ongoing development of diagnostic pipelines to maximize test sensitivity. We also draw attention to the large differences in computational infrastructure required to perform day-to-day versus large-scale reprocessing tasks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s40291-017-0304-xDOI Listing
December 2017

Dominant Mutations in GRM1 Cause Spinocerebellar Ataxia Type 44.

Am J Hum Genet 2017 Sep;101(3):451-458

Nuffield Department of Clinical Neurosciences, University of Oxford, 6th Floor West Wing, John Radcliffe Hospital, Oxford OX3 9DU, UK; Oxford Centre for Genomic Medicine, Oxford University Hospitals NHS Trust, Oxford OX3 7HE, UK. Electronic address:

The metabotropic glutamate receptor 1 (mGluR1) is abundantly expressed in the mammalian central nervous system, where it regulates intracellular calcium homeostasis in response to excitatory signaling. Here, we describe heterozygous dominant mutations in GRM1, which encodes mGluR1, that are associated with distinct disease phenotypes: gain-of-function missense mutations, linked in two different families to adult-onset cerebellar ataxia, and a de novo truncation mutation resulting in a dominant-negative effect that is associated with juvenile-onset ataxia and intellectual disability. Crucially, the gain-of-function mutations could be pharmacologically modulated in vitro using an existing FDA-approved drug, Nitazoxanide, suggesting a possible avenue for treatment, which is currently unavailable for ataxias.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2017.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591020PMC
September 2017

Non-invasive prenatal diagnosis of spinal muscular atrophy by relative haplotype dosage.

Eur J Hum Genet 2017 04 25;25(4):416-422. Epub 2017 Jan 25.

West Midlands Regional Genetics Laboratory, Birmingham Women's NHS Foundation Trust, Birmingham, UK.

Although technically possible, few clinical laboratories across the world have implemented non-invasive prenatal diagnosis (NIPD) for selected single-gene disorders, mostly owing to the elevated costs incurred. Having previously proven that NIPD for X-linked disorders can be feasibly implemented in clinical practice, we have now developed a test for the NIPD of an autosomal-recessive disorder, spinal muscular atrophy (SMA). Cell-free DNA was extracted from maternal blood and prepared for massively parallel sequencing on an Illumina MiSeq by targeted capture enrichment of single-nucleotide polymorphisms across a 6 Mb genomic window on chromosome 5 containing the SMN1 gene. Maternal, paternal and proband DNA samples were also tested for haplotyping purposes. Sequencing data was analysed by relative haplotype dosage (RHDO). Six pregnant SMA carriers and 10 healthy pregnant donors were recruited through the NIPSIGEN study. Inheritance of the maternally and paternally derived alleles of the affected SMN1 gene was determined in the foetus by RHDO analysis for autosomal-recessive disorders. DNA from the proband (for SMA carriers) or an invasively obtained foetal sample (for healthy pregnant donors) was used to identify the maternal and paternal reference haplotypes associated with the affected SMN1 gene. Results for all patients correlated with known outcomes and showed a testing specificity and sensitivity of 100%. On top of showing high accuracy and reliability throughout the stages of validation, our novel test for NIPD of SMA is also affordable and viable for implementation into clinical service.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ejhg.2016.195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386415PMC
April 2017

Non-invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies by relative haplotype dosage.

Prenat Diagn 2016 Apr 23;36(4):312-20. Epub 2016 Feb 23.

West Midlands Regional Genetics Laboratory, Birmingham Women's NHS Foundation Trust, Birmingham, UK.

Objective: Development of an accurate and affordable test for the non-invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies (DMD/BMD) to implement in clinical practice.

Method: Cell-free DNA was extracted from maternal blood and prepared for massively parallel sequencing on an Illumina MiSeq by targeted capture enrichment of single nucleotide polymorphisms (SNPs) across the dystrophin gene on chromosome X. Sequencing data were analysed by relative haplotype dosage.

Results: Seven healthy pregnant donors and two pregnant DMD carriers all bearing a male fetus were recruited through the non-invasive prenatal diagnosis for single gene disorders study. Non-invasive prenatal diagnosis testing was conducted by relative haplotype dosage analysis for X-linked disorders where the genomic DNA from the chorionic villus sampling (for healthy pregnant donors) or from the proband (for pregnant DMD carriers) was used to identify the reference haplotype. Results for all patients showed a test accuracy of 100%, when the calculated fetal fraction was >4% and correlated with known outcomes. A recombination event was also detected in a DMD patient.

Conclusion: Our new test for NIPD of DMD/BMD has been shown to be accurate and reliable during initial stages of validation. It is also feasible for implementation into clinical service.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pd.4781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864947PMC
April 2016

Chronic FLT3-ITD Signaling in Acute Myeloid Leukemia Is Connected to a Specific Chromatin Signature.

Cell Rep 2015 Aug 23;12(5):821-36. Epub 2015 Jul 23.

School of Immunity and Infection, College of Medicine and Dentistry, University of Birmingham, Birmingham B15 2TT, UK. Electronic address:

Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect the epigenetic regulatory machinery and signaling molecules, leading to a block in hematopoietic differentiation. Constitutive signaling from mutated growth factor receptors is a major driver of leukemic growth, but how aberrant signaling affects the epigenome in AML is less understood. Furthermore, AML cells undergo extensive clonal evolution, and the mutations in signaling genes are often secondary events. To elucidate how chronic growth factor signaling alters the transcriptional network in AML, we performed a system-wide multi-omics study of primary cells from patients suffering from AML with internal tandem duplications in the FLT3 transmembrane domain (FLT3-ITD). This strategy revealed cooperation between the MAP kinase (MAPK) inducible transcription factor AP-1 and RUNX1 as a major driver of a common, FLT3-ITD-specific gene expression and chromatin signature, demonstrating a major impact of MAPK signaling pathways in shaping the epigenome of FLT3-ITD AML.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2015.06.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726916PMC
August 2015

Evidence That Up-Regulation of MicroRNA-29 Contributes to Postnatal Body Growth Deceleration.

Mol Endocrinol 2015 Jun 13;29(6):921-32. Epub 2015 Apr 13.

Section on Growth and Development (F.K., A.A.N., G.R., J.B., J.C.L.) and Section on Neuroendocrinology (S.J.C.), Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Center for Molecular Medicine and Pediatric Endocrinology Unit (A.C.A., O.N.), Department of Women's and Children's Health, Karolinska Institutet and Karolinska University Hospital, SE-171 76 Stockholm, Sweden.

Body growth is rapid in infancy but subsequently slows and eventually ceases due to a progressive decline in cell proliferation that occurs simultaneously in multiple organs. We previously showed that this decline in proliferation is driven in part by postnatal down-regulation of a large set of growth-promoting genes in multiple organs. We hypothesized that this growth-limiting genetic program is orchestrated by microRNAs (miRNAs). Bioinformatic analysis identified target sequences of the miR-29 family of miRNAs to be overrepresented in age-down-regulated genes. Concomitantly, expression microarray analysis in mouse kidney and lung showed that all members of the miR-29 family, miR-29a, -b, and -c, were strongly up-regulated from 1 to 6 weeks of age. Real-time PCR confirmed that miR-29a, -b, and -c were up-regulated with age in liver, kidney, lung, and heart, and their expression levels were higher in hepatocytes isolated from 5-week-old mice than in hepatocytes from embryonic mouse liver at embryonic day 16.5. We next focused on 3 predicted miR-29 target genes (Igf1, Imp1, and Mest), all of which are growth-promoting. A 3'-untranslated region containing the predicted target sequences from each gene was placed individually in a luciferase reporter construct. Transfection of miR-29 mimics suppressed luciferase gene activity for all 3 genes, and this suppression was diminished by mutating the target sequences, suggesting that these genes are indeed regulated by miR-29. Taken together, the findings suggest that up-regulation of miR-29 during juvenile life drives the down-regulation of multiple growth-promoting genes, thus contributing to physiological slowing and eventual cessation of body growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2015-1047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447640PMC
June 2015

Molecular signatures of mouse TRPV1-lineage neurons revealed by RNA-Seq transcriptome analysis.

J Pain 2014 Dec 2;15(12):1338-1359. Epub 2014 Oct 2.

Anesthesia Section, Department of Perioperative Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland. Electronic address:

Unlabelled: Disorders of pain neural systems are frequently chronic and, when recalcitrant to treatment, can severely degrade the quality of life. The pain pathway begins with sensory neurons in dorsal root or trigeminal ganglia, and the neuronal subpopulations that express the transient receptor potential cation channel, subfamily V, member 1 (TRPV1) ion channel transduce sensations of painful heat and inflammation and play a fundamental role in clinical pain arising from cancer and arthritis. In the present study, we elucidate the complete transcriptomes of neurons from the TRPV1 lineage and a non-TRPV1 neuroglial population in sensory ganglia through the combined application of next-gen deep RNA-Seq, genetic neuronal labeling with fluorescence-activated cell sorting, or neuron-selective chemoablation. RNA-Seq accurately quantitates gene expression, a difficult parameter to determine with most other methods, especially for very low and very high expressed genes. Differentially expressed genes are present at every level of cellular function from the nucleus to the plasma membrane. We identified many ligand receptor pairs in the TRPV1 population, suggesting that autonomous presynaptic regulation may be a major regulatory mechanism in nociceptive neurons. The data define, in a quantitative, cell population-specific fashion, the molecular signature of a distinct and clinically important group of pain-sensing neurons and provide an overall framework for understanding the transcriptome of TRPV1 nociceptive neurons.

Perspective: Next-gen RNA-Seq, combined with molecular genetics, provides a comprehensive and quantitative measurement of transcripts in TRPV1 lineage neurons and a contrasting transcriptome from non-TRPV1 neurons and cells. The transcriptome highlights previously unrecognized protein families, identifies multiple molecular circuits for excitatory or inhibitory autocrine and paracrine signaling, and suggests new combinatorial approaches to pain control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jpain.2014.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469214PMC
December 2014

The Lhx9 homeobox gene controls pineal gland development and prevents postnatal hydrocephalus.

Brain Struct Funct 2015 20;220(3):1497-509. Epub 2014 Mar 20.

Section on Neuroendocrinology, Program in Developmental Endocrinology and Genetics, The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 49 Convent Drive, Room 6A82, Bethesda, MD, 20892-4510, USA.

Lhx9 is a member of the LIM homeobox gene family. It is expressed during mammalian embryogenesis in the brain including the pineal gland. Deletion of Lhx9 results in sterility due to failure of gonadal development. The current study was initiated to investigate Lhx9 biology in the pineal gland. Lhx9 is highly expressed in the developing pineal gland of the rat with transcript abundance peaking early in development; transcript levels decrease postnatally to nearly undetectable levels in the adult, a temporal pattern that is generally similar to that reported for Lhx9 expression in other brain regions. Studies with C57BL/6J Lhx9(-/-) mutant mice revealed marked alterations in brain and pineal development. Specifically, the superficial pineal gland is hypoplastic, being reduced to a small cluster of pinealocytes surrounded by meningeal and vascular tissue. The deep pineal gland and the pineal stalk are also reduced in size. Although the brains of neonatal Lhx9(-/-) mutant mice appear normal, severe hydrocephalus develops in about 70% of the Lhx9(-/-) mice at 5-8 weeks of age; these observations are the first to document that deletion of Lhx9 results in hydrocephalus and as such indicate that Lhx9 contributes to the maintenance of normal brain structure. Whereas hydrocephalus is absent in neonatal Lhx9(-/-)mutant mice, the neonatal pineal gland in these animals is hypoplastic. Accordingly, it appears that Lhx9 is essential for early development of the mammalian pineal gland and that this effect is not secondary to hydrocephalus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00429-014-0740-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197116PMC
January 2016

pY RNA1-s2: a highly retina-enriched small RNA that selectively binds to Matrin 3 (Matr3).

PLoS One 2014 18;9(2):e88217. Epub 2014 Feb 18.

Section on Neuroendocrinology, Program in Developmental Endocrinology and Genetics, The Eunice Shriver Kennedy National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.

The purpose of this study was to expand our knowledge of small RNAs, which are known to function within protein complexes to modulate the transcriptional output of the cell. Here we describe two previously unrecognized, small RNAs, termed pY RNA1-s1 and pY RNA1-s2 (processed Y RNA1-stem -1 and -2), thereby expanding the list of known small RNAs. pY RNA1-s1 and pY RNA1-s2 were discovered by RNA sequencing and found to be 20-fold more abundant in the retina than in 14 other rat tissues. Retinal expression of pY RNAs is highly conserved, including expression in the human retina, and occurs in all retinal cell layers. Mass spectrometric analysis of pY RNA1-S2 binding proteins in retina indicates that pY RNA1-s2 selectively binds the nuclear matrix protein Matrin 3 (Matr3) and to a lesser degree to hnrpul1 (heterogeneous nuclear ribonucleoprotein U-like protein). In contrast, pY RNA1-s1 does not bind these proteins. Accordingly, the molecular mechanism of action of pY RNA1-s2 is likely be through an action involving Matr3; this 95 kDa protein has two RNA recognition motifs (RRMs) and is implicated in transcription and RNA-editing. The high affinity binding of pY RNA1-s2 to Matr3 is strongly dependent on the sequence of the RNA and both RRMs of Matr3. Related studies also indicate that elements outside of the RRM region contribute to binding specificity and that phosphorylation enhances pY RNA-s2/Matr3 binding. These observations are of significance because they reveal that a previously unrecognized small RNA, pY RNA1-s2, binds selectively to Matr3. Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism. The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0088217PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928194PMC
January 2015

Female-specific induction of rat pituitary dentin matrix protein-1 by GnRH.

Mol Endocrinol 2013 Nov 1;27(11):1840-55. Epub 2013 Oct 1.

Eunice Kennedy Shriver National Institute of Child Health and Human Development, Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. or

Hypothalamic GnRH is the primary regulator of reproduction in vertebrates, acting via the G protein-coupled GnRH receptor (GnRHR) in pituitary gonadotrophs to control synthesis and release of gonadotropins. To identify elements of the GnRHR-coupled gene network, GnRH was applied in a pulsatile manner for 6 hours to a mixed population of perifused pituitary cells from cycling females, mRNA was extracted, and RNA sequencing analysis was performed. This revealed 83 candidate-regulated genes, including a large number coding for secreted proteins. Most notably, GnRH induces a greater than 600-fold increase in expression of dentin matrix protein-1 (Dmp1), one of five members of the small integrin-binding ligand N-linked glycoprotein gene family. The Dmp1 response is mediated by the GnRHR, not elicited by other hypothalamic releasing factors, and is approximately 20-fold smaller in adult male pituitary cells. The sex-dependent Dmp1 response is established during the peripubertal period and independent of the developmental pattern of Gnrhr expression. In vitro, GnRH-induced expression of this gene is coupled with release of DMP1 in extracellular medium through the regulated secretory pathway. In vivo, pituitary Dmp1 expression in identified gonadotrophs is elevated after ovulation. Cell signaling studies revealed that the GnRH induction of Dmp1 is mediated by the protein kinase C signaling pathway and reflects opposing roles of ERK1/2 and p38 MAPK; in addition, the response is facilitated by progesterone. These results establish that DMP1 is a novel secretory protein of female rat gonadotrophs, the synthesis and release of which are controlled by the hypothalamus through the GnRHR signaling pathway. This advance raises intriguing questions about the intrapituitary and downstream effects of this new player in GnRH signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2013-1068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3805844PMC
November 2013

MicroRNAs in the pineal gland: miR-483 regulates melatonin synthesis by targeting arylalkylamine N-acetyltransferase.

J Biol Chem 2012 Jul;287(30):25312-24

Section on Neuroendocrinology, Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.

MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ~75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3"-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408182PMC
http://dx.doi.org/10.1074/jbc.M112.356733DOI Listing
July 2012

Circadian changes in long noncoding RNAs in the pineal gland.

Proc Natl Acad Sci U S A 2012 Aug 3;109(33):13319-24. Epub 2012 Aug 3.

Section on Neuroendocrinology, Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

Long noncoding RNAs (lncRNAs) play a broad range of biological roles, including regulation of expression of genes and chromosomes. Here, we present evidence that lncRNAs are involved in vertebrate circadian biology. Differential night/day expression of 112 lncRNAs (0.3 to >50 kb) occurs in the rat pineal gland, which is the source of melatonin, the hormone of the night. Approximately one-half of these changes reflect nocturnal increases. Studies of eight lncRNAs with 2- to >100-fold daily rhythms indicate that, in most cases, the change results from neural stimulation from the central circadian oscillator in the suprachiasmatic nucleus (doubling time = 0.5-1.3 h). Light exposure at night rapidly reverses (halving time = 9-32 min) levels of some of these lncRNAs. Organ culture studies indicate that expression of these lncRNAs is regulated by norepinephrine acting through cAMP. These findings point to a dynamic role of lncRNAs in the circadian system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1207748109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3421215PMC
August 2012

Crx broadly modulates the pineal transcriptome.

J Neurochem 2011 Oct 2;119(2):262-74. Epub 2011 Sep 2.

Department of Neuroscience and Pharmacology, Panum Institute, University of Copenhagen, Copenhagen, Denmark.

Cone-rod homeobox (Crx) encodes Crx, a transcription factor expressed selectively in retinal photoreceptors and pinealocytes, the major cell type of the pineal gland. In this study, the influence of Crx on the mammalian pineal gland was studied by light and electron microscopy and by use of microarray and qRTPCR technology, thereby extending previous studies on selected genes (Furukawa et al. 1999). Deletion of Crx was not found to alter pineal morphology, but was found to broadly modulate the mouse pineal transcriptome, characterized by a>2-fold down-regulation of 543 genes and a>2-fold up-regulation of 745 genes (p<0.05). Of these, one of the most highly up-regulated (18-fold) was Hoxc4, a member of the Hox gene family, members of which are known to control gene expression cascades. During a 24-h period, a set of 51 genes exhibited differential day/night expression in pineal glands of wild-type animals; only eight of these were also day/night expressed in the Crx⁻/⁻ pineal gland. However, in the Crx⁻/⁻ pineal gland 41 genes exhibited differential night/day expression that was not seen in wild-type animals. These findings indicate that Crx broadly modulates the pineal transcriptome and also influences differential night/day gene expression in this tissue. Some effects of Crx deletion on the pineal transcriptome might be mediated by Hoxc4 up-regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1471-4159.2011.07405.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422619PMC
October 2011

The interaction between casein kinase Ialpha and 14-3-3 is phosphorylation dependent.

FEBS J 2009 Dec 27;276(23):6971-84. Epub 2009 Oct 27.

Institute of Structural Biology, Edinburgh University, UK.

We have previously shown that casein kinase (CK) Ialpha from mammalian brain phosphorylates 14-3-3 zeta and tau isoforms on residue 233. In the present study, we show that CKIalpha associates with 14-3-3 both in vitro and in vivo. The interaction between CKIalpha and 14-3-3 is dependent on CKIalpha phosphorylation, unlike centaurin-alpha1 (also known as ADAP1), which binds to unphosphorylated CKIalpha on the same region. CKIalpha preferentially interacts with mammalian eta and gamma 14-3-3 isoforms, and peptides that bind to the 14-3-3 binding pocket prevent this interaction. The region containing Ser218 in this CKIalpha binding site was mutated and the interaction between CKIalpha and 14-3-3 was reduced. We subsequently identified a second phosphorylation-dependent 14-3-3 binding site within CKIalpha containing Ser242 that may be the principal site of interaction. We also show that both fission and budding yeast CKI kinase homologues phosphorylate mammalian and budding yeast (BMH1 and BMH2) 14-3-3 at the equivalent site.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1742-4658.2009.07405.xDOI Listing
December 2009

Pigment composition and adaptation in free-living and symbiotic strains of Acaryochloris marina.

FEMS Microbiol Ecol 2007 Jul 27;61(1):65-73. Epub 2007 Apr 27.

MOAC Doctoral Training Centre, University of Warwick, Coventry, UK.

Acaryochloris marina strains have been isolated from several varied locations and habitats worldwide demonstrating a diverse and dynamic ecology. In this study, the whole cell photophysiologies of strain MBIC11017, originally isolated from a colonial ascidian, and the free-living epilithic strain CCMEE5410 are analyzed by absorbance and fluorescence spectroscopy, laser scanning confocal microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent protein analysis. We demonstrate pigment adaptation in MBIC11017 and CCMEE5410 under different light regimes. We show that the higher the incident growth light intensity for both strains, the greater the decrease in their chlorophyll d content. However, the strain MBIC11017 loses its phycobiliproteins relative to its chlorophyll d content when grown at light intensities of 40 microE m(-2) s(-1) without shaking and 100 microE m(-2) s(-1) with shaking. We also conclude that phycobiliproteins are absent in the free-living strain CCMEE5410.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1574-6941.2007.00320.xDOI Listing
July 2007

BCR kinase phosphorylates 14-3-3 Tau on residue 233.

FEBS J 2005 Aug;272(15):3767-76

School of Biomedical and Clinical Laboratory Sciences, University of Edinburgh, UK.

The breakpoint cluster region protein, BCR, has protein kinase activity that can auto- and trans-phosphorylate serine, threonine and tyrosine residues. BCR has been implicated in chronic myelogenous leukaemia as well as important signalling pathways, and as such its interaction with 14-3-3 is of major interest. 14-3-3tau and zeta isoforms have been shown previously to be phosphorylated in vitro and in vivo by BCR kinase on serine and threonine residue(s) but site(s) were not determined. Phosphorylation of 14-3-3 isoforms at distinct sites is an important mode of regulation that negatively affects interaction with Raf kinase and Bax, and potentially influences the dimerization of 14-3-3. In this study we have further characterized the BCR-14-3-3 interaction and have identified the site phosphorylated by BCR. We show here that BCR interacts with at least five isoforms of 14-3-3 in vivo and phosphorylates 14-3-3tau on Ser233 and to a lesser extent 14-3-3zeta on Thr233. We have previously shown that these two isoforms are also phosphorylated at this site by casein kinase 1, which, in contrast to BCR, preferentially phosphorylates 14-3-3zeta.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1742-4658.2005.04765.xDOI Listing
August 2005