De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology

Application of Endophytic Bacillus subtilis and Salicylic Acid to Improve Wheat Growth and Tolerance under Combined Drought and Fusarium Root Rot Stresses

In nature, plants are constantly exposed to a varied abiotic and biotic stresses or their combinations, limiting the productivity of major crops, including wheat. Combinations of drought and soil-borne Fusarium-instigated diseases are the most common combinations of stresses, significantly reducing wheat yield around the world. Here, were analyzed the potential of application of endophytic bacteria Bacillus subtilis (strain 10–4) together with the natural signal molecule salicylic acid (SA) to improve growth and tolerance of Triticum aestivum L. (wheat) plants under combined drought and Fusarium culmorum-instigated root rot (FRR) stresses. It was revealed that pre-sowing treatment with B. subtilis 10–4, SA, and B. subtilis 10–4 + SA, both under normal and combined drought conditions, notably reduced (by 50–80% or more) the incidence of FRR development in wheat plants, with the most notable effect for B. subtilis 10–4 + SA (wherein disease symptoms were almost absent). Moreover, B. subtilis 10–4, SA, and especially B. subtilis 10–4 + SA increased plant growth (root and shoot length, fresh and dry biomass) under normal (up to 20–50%), drought (up to 15–40%), FRR (up to 15–30%), and combined drought + FRR stresses (up to 20%), with the maximum effect for B. subtilis 10–4 + SA. Additionally, B. subtilis 10–4, SA, and B. subtilis 10–4 + SA decreased stress (drought, FRR, and combined drought + FRR)-instigated lipid peroxidation and osmotic damages of plant cells. The findings indicate that endophytic bacteria B. subtilis 10–4 alone and in a mixture with SA may be used as an effective eco-friendly agent to improve wheat growth and tolerance under the influence of drought, FRR, and combinations of these stresses

A Screening-Based Method for Identifying Patients with REM Sleep Behaviour Disorder in a Danish Community Setting

Isolated REM sleep behaviour disorder (iRBD) is a predictive marker of prodromal Lewy body disease. iRBD prevalence in the general population is around 1%, but it remains under-diagnosed, even though symptoms are alleviated by medication. We developed a population screening strategy and identified 16 iRBD patients by conducting telephone interviews and polysomnography examinations. We compared our population-screened cohort with sleep-center referred patients and found higher MoCA scores and lower MDS-UPDRS-III scores in our patients. In conclusion, screening can be used to identify iRBD patients in a cost-effective manner with the benefit of identifying patients at a very early disease stage

Objective intestinal function in patients with idiopathic REM sleep behavior disorder

Background: Parkinson's disease is characterized by pathological alpha-synuclein accumulation and cell death, which has been hypothesized to originate in peripheral nerve terminals and subsequently spread via autonomic nerves. Supporting this, most Parkinson's disease patients experience autonomic non-motor symptoms such as constipation, often years prior to diagnosis. Objective: We aimed to study gastrointestinal transit time, colonic volume, and peristaltic movements in idiopathic REM Sleep Behavior Disorder patients, a prodromal marker of Parkinson's disease or Dementia with Lewy bodies. Methods: Twenty-two patients were included and compared to previously published data from Parkinson's disease patients and controls. Gastrointestinal transit time, computed tomography-based volume estimation, and colonic motility were performed as markers of gastrointestinal function and autonomic involvement. Subjective constipation symptoms were evaluated with two different questionnaires. Results: Gastrointestinal transit time was increased in 33% (p = 0.039) and colonic volume in 48% (p = 0.0049) of patients. Colonic transit time measured by the 3D-Transit system was increased in 70% (p = 0.0326) and the number of fast peristaltic colonic movements was reduced (p = 0.015). Mean small intestinal transit time was comparable to Parkinson's disease patients, although not significantly different compared to controls (p = 0.18). Subjective constipation symptoms were present in 18 or 419/0 , depending on type of questionnaire. Conclusions: Total gastrointestinal transit time, colonic volume, and 3D-Transit colonic transit time were significantly increased compared to controls, although not to the extent seen in medicated Parkinson's patients. Limited correlation was seen between subjective constipation and objective markers. The findings support that marked GI dysfunction is present in the early prodromal PD phase

Imaging progressive peripheral and central dysfunction in isolated REM sleep behaviour disorder after 3 years of follow-up

Introduction: Most patients with isolated rapid eye movement sleep behaviour disorder (iRBD) convert to Parkinson's disease (PD), dementia with Lewy bodies, or multiple system atrophy within 15 years of diagnosis. Furthermore, iRBD patients develop non-motor symptoms similar to those of manifest PD patients and display dysfunction of the sympathetic and parasympathetic nervous system, comparable to that seen in PD. However, progression rates of autonomic dysfunction in iRBD have not been studied with objective measures in detail, which is the aim of this study. Methods: Twenty-two iRBD patients were included at baseline and 14 participated in follow-up after 3 years. Colonic transit time (CTT) was examined using radio opaque markers, colonic volume was defined on abdominal computed tomography (CT) scans, Iodine-123-metaiodobenzylguanidine ([123I]MIBG) scintigraphy was performed to assess cardiac sympathetic innervation, and 3,4-dihydroxy-6-(18F) fluoro-L-phenylalanine ([18F]FDOPA) positron emission tomography (PET) scan determined nigrostriatal dopamine storage capacity. All examinations were performed at baseline and after 3 years. Results: iRBD patients displayed increased CTT (p = 0.001) and colonic volume (p = 0.01) at follow-up compared to baseline. Furthermore, [123I]MIBG uptake and [18F]FDOPA uptake showed progressive reductions at follow-up (p = 0.02 and p = 0.002, respectively). No correlations were seen between changes in intestinal or cardiac measurements and dopaminergic function. Conclusion: Using objective markers, the present study documented that intestinal dysfunction and cardiac sympathetic degeneration worsen in the majority of iRBD patients over a 3-year period. The absent correlation between these markers and nigrostriatal dopaminergic dysfunction suggests that progressive gastrointestinal and cardiac dysfunction in iRBD is caused mainly by non-dopaminergic mechanisms

In-vivo staging of pathology in REM sleep behaviour disorder: a multimodality imaging case-control study

Background Accumulating evidence suggests that a-synuclein aggregates-a defining pathology of Parkinson's disease-display cell-to-cell transmission. a-synuclein aggregation is hypothesised to start in autonomic nerve terminals years before the appearance of motor symptoms, and subsequently spread via autonomic nerves to the spinal cord and brainstem. To assess this hypothesis, we investigated sympathetic, parasympathetic, noradrenergic, and dopaminergic innervation in patients with idiopathic rapid eye movement (REM) sleep behaviour disorder, a prodromal phenotype of Parkinson's disease. Methods In this prospective, case-control study, we recruited patients with idiopathic REM sleep behaviour disorder, confirmed by polysomnography, without clinical signs of parkinsonism or dementia, via advertisement and through sleep clinics in Denmark. We used C-11-donepezil PET and CT to assess cholinergic (parasympathetic) gut innervation, I-123-metaiodobenzylguanidine (MIBG) scintigraphy to measure cardiac sympathetic innervation, neuromelanin-sensitive MRI to measure integrity of pigmented neurons of the locus coeruleus, C-11-methylreboxetine (MeNER) PET to assess noradrenergic nerve terminals originating in the locus coeruleus, and F-18-dihydroxyphenylalanine (DOPA) PET to assess nigrostriatal dopamine storage capacity. For each imaging modality, we compared patients with idiopathic REM sleep behaviour disorder with previously published reference data of controls without neurological disorders or cognitive impairment and with symptomatic patients with Parkinson's disease. We assessed imaging data using one-way ANOVA corrected for multiple comparisons. Findings Between June 3, 2016, and Dec 19, 2017, we recruited 22 consecutive patients with idiopathic REM sleep behaviour disorder to the study. Compared with controls, patients with idiopathic REM sleep behaviour disorder had decreased colonic C-11-donepezil uptake (-0.322, 95% CI-0.112 to -0.531; p=0.0020), I-123-MIBG heart: mediastinum ratio (-0.508, -0.353 to -0.664; p0.99), neuromelanin-sensitive MRI (p=0.96), and C-11-MeNER (p=0.56). By contrast, 15 (71%) of 21 patients with idiopathic REM sleep behaviour disorder had (1)8F-DOPA Ki values within normal limits, whereas all patients with Parkinson's disease had significantly decreased F-18-DOPA Ki values when compared with patients with idiopathic REM sleep behaviour disorder (p<0.0001). Interpretation Patients with idiopathic REM sleep behaviour disorder had fully developed pathology in the peripheral autonomic nervous system and the locus coeruleus, equal to that in diagnosed Parkinson's disease. These patients also showed noradrenergic thalamic denervation, but most had normal putaminal dopaminergic storage capacity. This caudorostral gradient of dysfunction supports the hypothesis that a-synuclein pathology in Parkinson's disease initially targets peripheral autonomic nerves and then spreads rostrally to the brainstem. Copyright (c) 2018 Elsevier Ltd. All rights reserved