An investigation into cholinergic interactions in the rat pineal gland

The mammalian pineal gland is mainly innervated by the sympathetic nervous system which modulates the activity of indole pathway enzymes and the secretion of pineal hormones. Recently researchers have demonstrated and characterized the presence of muscarinic cholinergic receptors in the pineal gland. However the role of these receptors remains unclear. In an attempt to investigate the role of cholinergic receptors in the pineal gland, a number of studies were carried out on the various steps in the indole metabolic pathway, using various agents which act on the cholinergic system. Investigations using pineal organ cultures showed that stimulation of these muscarinic cholinergic receptor sites with a parasympathomimetic agent, a rise in levels of aHT occurred without a concomitant increase in aMT levels. Further organ culture experiments using the cholinergic agonist acetylcholine and anticholinesterase agent physostigmine, produced a similar rise in aHT without altering aMT levels. This acetylcholine-induced rise in aHT levels were not altered by the ganglion blocking agent hexamethonium whilst the antimuscarinic agent atropine prevented the acetylcholine-induced rise in aHT levels. Thesefindings suggest that cholinergic agents may play a role in regulating indoleamine synthesis in the pineal gland. Cyclic-AMP assay studies showed that acetylcholine increases pineal cAMP levels significantly and does not influence the isoproterenol-induced cAMP rise in the pineal gland. The cAMP regulator cAMP-phosphodiesterase (cAMP-PDE) was found to increase significantly in the presence of the anticholinesterase agent physostigmine. NAT enzyme studies revealed that physostigmine does not affect NAT enzyme levels significantly and HIOMT studies showed that this agent does not inhibit HIOMT activity. The mechanism by which acetylcholine and physostigmine are able to cause a increase in aHT and not aMT levels needs to be researched further. Acetylcholinesterase enzyme assay studies revealed that the AChE enzyme undergoes a diurnal rhythm in the pineal gland with activity being higher during the day and lower at night. Investigations using the drug reserpine showed that this rhythm is not under the control of the sympathetic nervous system. Further research needs to be done however, in determining whether or not this enzyme is present in the pineal gland to regulate the levels of acetylcholine interacting with muscarinic receptors in the gland, or for some other reason. Choline acetyltransferase studies demonstrate the presence of the enzyme in the rat brain cerebral cortex as well as showing that melatonin increases ChAT enzyme activity in this tissue. This suggests that melatonin plays a role in cholinergic transmission there. ChAT activity could not be measured in the pineal gland however. Muscarinic receptor binding studies also carried out on rat brain cerebral cortex show that melatonin enhances cholinergic receptor affinity and receptor number in this tissue. In summary, data presented herein concur with proposals that: i) the cholinergic system affects the indole metabolic pathway by causing a rise in aRT but not aMT levels. ii) cholinergic agonist acetylcholine causes cAMP levels to rise with a concomitant increase in cAMP-PDE levels. iii) the enzyme acetylcholinesterase undergoes a diurnal rhythm in the pineal gland which is not under the control of the sympathetic nervous system. iv) the activity of the enzyme choline acetyltransferase is increased by melatonin in the rat brain cerebral cortex suggesting that melatonin facilitates cholinergic transmission in this tissue. v) melatonin enhances cholinergic receptor affinity and receptor number in the cerebral cortex of rat brain

Comparative Analysis of Root Microbiomes of Rice Cultivars with High and Low Methane Emissions Reveals Differences in Abundance of Methanogenic Archaea and Putative Upstream Fermenters.

Rice cultivation worldwide accounts for ∼7 to 17% of global methane emissions. Methane cycling in rice paddies is a microbial process not only involving methane producers (methanogens) and methane metabolizers (methanotrophs) but also other microbial taxa that affect upstream processes related to methane metabolism. Rice cultivars vary in their rates of methane emissions, but the influence of rice genotypes on methane cycling microbiota has been poorly characterized. Here, we profiled the rhizosphere, rhizoplane, and endosphere microbiomes of a high-methane-emitting cultivar (Sabine) and a low-methane-emitting cultivar (CLXL745) throughout the growing season to identify variations in the archaeal and bacterial communities relating to methane emissions. The rhizosphere of the high-emitting cultivar was enriched in methanogens compared to that in the low emitter, whereas the relative abundances of methanotrophs between the cultivars were not significantly different. Further analysis of cultivar-sensitive taxa identified families enriched in the high emitter that are associated with methanogenesis-related processes. The high emitter had greater relative abundances of sulfate-reducing and iron-reducing taxa which peak earlier in the season than methanogens and are necessary to lower soil oxidation reduction potential before methanogenesis can occur. The high emitter also had a greater abundance of fermentative taxa which produce methanogenesis precursors (acetate, CO2, and H2). Furthermore, the high emitter was enriched in taxa related to acetogenesis which compete with methanogens for CO2 and H2 These taxa were enriched in a spatio-specific manner and reveal a complex network of microbial interactions on which plant genotype-dependent factors can act to affect methanogenesis and methane emissions.IMPORTANCE Rice cultivation is a major source of anthropogenic emissions of methane, a greenhouse gas with a potentially severe impact on climate change. Emission variation between rice cultivars suggests the feasibility of breeding low-emission rice, but there is a limited understanding of how genotypes affect the microbiota involved in methane cycling. Here, we show that the root microbiome of the high-emitting cultivar is enriched both in methanogens and in taxa associated with fermentation, iron, and sulfate reduction and acetogenesis, processes that support methanogenesis. Understanding how cultivars affect microbes with methanogenesis-related functions is vital for understanding the genetic basis for methane emission in rice and can aid in the development of breeding programs that reduce the environmental impact of rice cultivation

Finding Diagnostically Useful Patterns in Quantitative Phenotypic Data.

Trio-based whole-exome sequence (WES) data have established confident genetic diagnoses in ∼40% of previously undiagnosed individuals recruited to the Deciphering Developmental Disorders (DDD) study. Here we aim to use the breadth of phenotypic information recorded in DDD to augment diagnosis and disease variant discovery in probands. Median Euclidean distances (mEuD) were employed as a simple measure of similarity of quantitative phenotypic data within sets of ≥10 individuals with plausibly causative de novo mutations (DNM) in 28 different developmental disorder genes. 13/28 (46.4%) showed significant similarity for growth or developmental milestone metrics, 10/28 (35.7%) showed similarity in HPO term usage, and 12/28 (43%) showed no phenotypic similarity. Pairwise comparisons of individuals with high-impact inherited variants to the 32 individuals with causative DNM in ANKRD11 using only growth z-scores highlighted 5 likely causative inherited variants and two unrecognized DNM resulting in an 18% diagnostic uplift for this gene. Using an independent approach, naive Bayes classification of growth and developmental data produced reasonably discriminative models for the 24 DNM genes with sufficiently complete data. An unsupervised naive Bayes classification of 6,993 probands with WES data and sufficient phenotypic information defined 23 in silico syndromes (ISSs) and was used to test a "phenotype first" approach to the discovery of causative genotypes using WES variants strictly filtered on allele frequency, mutation consequence, and evidence of constraint in humans. This highlighted heterozygous de novo nonsynonymous variants in SPTBN2 as causative in three DDD probands

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year

Genome-Wide Association Study in BRCA1 Mutation Carriers Identifies Novel Loci Associated with Breast and Ovarian Cancer Risk

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7×10-8, HR = 1.14, 95% CI: 1.09-1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4×10-8, HR = 1.27, 95% CI: 1.17-1.38) and 4q32.3 (rs4691139, P = 3.4×10-8, HR = 1.20, 95% CI: 1.17-1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific associat

The contribution of X-linked coding variation to severe developmental disorders

Abstract: Over 130 X-linked genes have been robustly associated with developmental disorders, and X-linked causes have been hypothesised to underlie the higher developmental disorder rates in males. Here, we evaluate the burden of X-linked coding variation in 11,044 developmental disorder patients, and find a similar rate of X-linked causes in males and females (6.0% and 6.9%, respectively), indicating that such variants do not account for the 1.4-fold male bias. We develop an improved strategy to detect X-linked developmental disorders and identify 23 significant genes, all of which were previously known, consistent with our inference that the vast majority of the X-linked burden is in known developmental disorder-associated genes. Importantly, we estimate that, in male probands, only 13% of inherited rare missense variants in known developmental disorder-associated genes are likely to be pathogenic. Our results demonstrate that statistical analysis of large datasets can refine our understanding of modes of inheritance for individual X-linked disorders

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Comparative Analysis of Root Microbiomes of Rice Cultivars with High and Low Methane Emissions Reveals Differences in Abundance of Methanogenic Archaea and Putative Upstream Fermenters.

Rice cultivation worldwide accounts for ∼7 to 17% of global methane emissions. Methane cycling in rice paddies is a microbial process not only involving methane producers (methanogens) and methane metabolizers (methanotrophs) but also other microbial taxa that affect upstream processes related to methane metabolism. Rice cultivars vary in their rates of methane emissions, but the influence of rice genotypes on methane cycling microbiota has been poorly characterized. Here, we profiled the rhizosphere, rhizoplane, and endosphere microbiomes of a high-methane-emitting cultivar (Sabine) and a low-methane-emitting cultivar (CLXL745) throughout the growing season to identify variations in the archaeal and bacterial communities relating to methane emissions. The rhizosphere of the high-emitting cultivar was enriched in methanogens compared to that in the low emitter, whereas the relative abundances of methanotrophs between the cultivars were not significantly different. Further analysis of cultivar-sensitive taxa identified families enriched in the high emitter that are associated with methanogenesis-related processes. The high emitter had greater relative abundances of sulfate-reducing and iron-reducing taxa which peak earlier in the season than methanogens and are necessary to lower soil oxidation reduction potential before methanogenesis can occur. The high emitter also had a greater abundance of fermentative taxa which produce methanogenesis precursors (acetate, CO2, and H2). Furthermore, the high emitter was enriched in taxa related to acetogenesis which compete with methanogens for CO2 and H2 These taxa were enriched in a spatio-specific manner and reveal a complex network of microbial interactions on which plant genotype-dependent factors can act to affect methanogenesis and methane emissions.IMPORTANCE Rice cultivation is a major source of anthropogenic emissions of methane, a greenhouse gas with a potentially severe impact on climate change. Emission variation between rice cultivars suggests the feasibility of breeding low-emission rice, but there is a limited understanding of how genotypes affect the microbiota involved in methane cycling. Here, we show that the root microbiome of the high-emitting cultivar is enriched both in methanogens and in taxa associated with fermentation, iron, and sulfate reduction and acetogenesis, processes that support methanogenesis. Understanding how cultivars affect microbes with methanogenesis-related functions is vital for understanding the genetic basis for methane emission in rice and can aid in the development of breeding programs that reduce the environmental impact of rice cultivation

Data from: Compositional shifts in root-associated bacterial and archaeal microbiota track the plant life-cycle in field-grown rice

Bacterial communities associated with roots impact the health and nutrition of the host plant. The dynamics of these microbial assemblies over the plant life cycle is however, not well understood. Here, we use dense temporal sampling of 1588 samples from root spatial compartments to characterize the bacterial and archaeal components of the root-associated microbiota of field grown rice (Oryza sativa) over the course of three consecutive growing seasons, as well as two sites in diverse geographic regions. The root microbiota was found to be highly dynamic during the vegetative phase of plant growth, then stabilized compositionally for the remainder of the life cycle. Bacterial and archaeal taxa conserved between field sites were defined as predictive features of rice plant age by modeling using a random forests approach. The age-prediction models revealed that drought stressed plants have developmentally immature microbiota compared to unstressed plants. Further, by using genotypes with varying developmental rates, we show that shifts in the microbiome are correlated with rates of developmental transitions rather than age alone, such that different microbiota compositions reflect juvenile and adult life stages. These results suggest a model for successional dynamics of the root-associated microbiota over the plant life cycle