14 research outputs found
Carolacton treatment causes delocalization of the cell division proteins PknB and DivIVa in Streptococcus mutans in vivo
No full textThe small inhibitory molecule Carolacton has been shown to cause chain formation and bulging in Streptococci, suggesting a defect in cell division, but it is not known how cell division is impaired on a molecular level. Fluorescent fusion proteins have successfully been applied to visualize protein localization and dynamics in vivo and have revolutionized our understanding of cell wall growth, cell division, chromosome replication and segregation. However, in Streptococci the required vectors are largely lacking. We constructed vectors for chromosomal integration and inducible expression of fluorescent fusion proteins based on GFP+ in S. mutans. Their applicability was verified using four proteins with known localization in the cell. We then determined the effect of Carolacton on the subcellular localization of GFP+ fusions of the cell division protein DivIVa and the serine-threonine protein kinase PknB. Carolacton caused a significant delocalization of these proteins from midcell, in accordance with a previous study demonstrating the Carolacton insensitive phenotype of a pknB deletion strain. Carolacton treated cells displayed an elongated phenotype, increased septum formation and a severe defect in daughter cell separation. GFP+ fusions of two hypothetical proteins (SMU_503 and SMU_609), that had previously been shown to be the most strongly upregulated genes after Carolacton treatment, were found to be localized at the septum in midcell, indicating their role in cell division. These findings highlight the importance of PknB as a key regulator of cell division in streptococci and indicate a profound impact of Carolacton on the coordination between peripheral and septal cell wall growth. The established vector system represents a novel tool to study essential steps of cellular metabolism
Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences
No full textThe human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates
Genetics, Cognition and Neurobiology of Schizotypal Personality: A Review of the Overlap with Schizophrenia
No full textSchizotypy refers to a set of temporally stable traits that are observed in the general population and that resemble the signs and symptoms of schizophrenia. Here, we review evidence from studies on genetics, cognition, perception, motor and oculomotor control, brain structure, brain function and psychopharmacology in schizotypy. We specifically focused on identifying areas of overlap between schizotypy and schizophrenia. Evidence was corroborated that significant overlap exists between the two, covering the behavioural, brain structural and functional as well molecular levels. In particular, several studies showed that individuals with high levels of schizotypal traits exhibit alterations in neurocognitive task performance and underlying brain function similar to the deficits seen in patients with schizophrenia. Studies of brain structure have shown both volume reductions and increases in schizotypy, pointing to schizophrenia-like deficits as well as possible protective or compensatory mechanisms. Experimental pharmacological studies have shown that high levels of schizotypy are associated with (i) enhanced dopaminergic response in striatum following administration of amphetamine and (ii) improvement of cognitive performance following administration of antipsychotic compounds. Together, this body of work suggests that schizotypy shows overlap with schizophrenia across multiple behavioural and neurobiological domains, suggesting that the study of schizotypal traits may be useful in improving our understanding of the aetiology of schizophrenia
Vestibular function and quality of life in vestibular schwannoma: does size matter?
No full textObjectives. Patients with vestibular schwannoma (VS) frequently suffer from disabling vestibular symptoms. This prospective follow-up study evaluates vestibular and auditory function and impairment of quality of life due to vertigo, dizziness and imbalance in patients with unilateral vestibular schwannoma of different sizes before/ after microsurgical or radiosurgical treatment. Methods. 38 patients with unilateral vestibular schwannoma were included. 22 received microsurgery, 16 cyberknife radiosurgery. Two follow-ups took place after a median of 50 and 186.5 days. Patients received a standardized neuro-ophthalmological examination, electronystagmography with bithermal caloric testing, and pure-tone audiometry. Quality of life was evaluated with the Dizziness Handicap Inventory (DHI). Patient data was grouped and analyzed according to the size of the VS (group 1: < 20mm vs group 2: ≥ 20mm). Results. In group 1, the median loss of vestibular function was +10.5% as calculated by Jongkees Formula (range –43;+52; group 2: median + 36%, range –56; +90). The median change of DHI scores was –9 in group 1 (range –68;30) and +2 in group 2 (–54;+20). Median loss of hearing was 4dB (-42;93) in group 1 and 12dB in group 2 (5;42).Conclusions. Loss of vestibular function in vestibular schwannoma clearly correlates with tumor size. However, loss of vestibular function was not strictly associated with a long-term deterioration of quality of life. This may be due to central compensation of vestibular deficits in long-standing large tumors. Loss of hearing before treatment was significantly influenced by the age of the patient but not by tumor size. At follow-up 1 and 2, hearing was significantly worse in those patients with a large VS and after microsurgical treatment
Counteracting protein kinase activity in the heart: The multiple roles of protein phosphatases
No full textDecades of cardiovascular research have shown that variable and flexible levels of protein phosphorylation are necessary to maintain cardiac function. A delicate balance between phosphorylated and dephosphorylated states of proteins is guaranteed by a complex interplay of protein kinases (PK) and phosphatases. Serine/threonine phosphatases, in particular members of the protein phosphatase (PP) family govern dephosphorylation of the majority of these cardiac proteins. Recent findings have however shown that PPs do not only dephosphorylate previously phosphorylated proteins as a passive control mechanism but are capable to actively control PK activity via different direct and indirect signaling pathways. These control mechanisms can take place on (epi-)genetic, (post-)transcriptional and (post-)translational levels. In addition PPs themselves are targets of a plethora of proteinaceous interaction partner regulating their endogenous activity, thus adding another level of complexity and feedback control towards this system. Finally, novel approaches are underway to achieve spatiotemporal pharmacologic control of protein phosphatases which in turn can be used to fine-tune misleaded protein kinase activity in heart disease. Taken together, this review comprehensively summarizes the major aspects of PP-mediated PK regulation and discusses the subsequent consequences of deregulated PP activity for cardiovascular diseases in depth
A dual-species co-cultivation system to study the interactions between Roseobacters and Dinoflagellates
No full textSome microalgae in nature live in symbiosis with microorganisms that can enhance or inhibit growth, thus influencing the dynamics of phytoplankton blooms. In spite of the great ecological importance of these interactions, very few defined laboratory systems are available to study them in detail. Here we present a co-cultivation system consisting of the toxic phototrophic dinoflagellate Prorocentrum minimum and the photoheterotrophic alphaproteobacterium Dinoroseobacter shibae. In a mineral medium lacking a carbon source, vitamins for the bacterium and the essential vitamin B12 for the dinoflagellate, growth dynamics reproducibly went from a mutualistic phase, where both algae and bacteria grow, to a pathogenic phase, where the algae are killed by the bacteria. The data show a Jekyll and Hyde lifestyle that had been proposed but not previously demonstrated. We used RNAseq and microarray analysis to determine which genes of D. shibae are transcribed and differentially expressed in a light dependent way at an early time-point of the co-culture when the bacterium grows very slowly. Enrichment of bacterial mRNA for transcriptome analysis was optimized, but none of the available methods proved capable of removing dinoflagellate ribosomal RNA completely. RNAseq showed that a phasin encoding gene (phaP1) which is part of the polyhydroxyalkanoate (PHA) metabolism operon represented approximately 10 % of all transcripts. Five genes for aerobic anoxygenic photosynthesis were down-regulated in the light, indicating that the photosynthesis apparatus was functional. A betaine-choline-carnitine-transporter (BCCT) that may be used for dimethylsulfoniopropionate (DMSP) uptake was the highest up-regulated gene in the light. The data suggest that at this early mutualistic phase of the symbiosis, PHA degradation might be the main carbon and energy source of D. shibae, supplemented in the light by degradation of DMSP and aerobic anoxygenic photosynthesis
Sulfate-reducing microorganisms in wetlands – fameless actors in carbon cycling and climate change
No full textFreshwater wetlands are a major source of the greenhouse gas methane but at the same time can function as carbon sink. Their response to global warming and environmental pollution is one of the largest unknowns in the upcoming decades to centuries. In this review, we highlight the role of sulfate-reducing microorganisms (SRM) in the intertwined element cycles of wetlands. Although regarded primarily as methanogenic environments, biogeochemical studies have revealed a previously hidden sulfur cycle in wetlands that can sustain rapid renewal of the small standing pools of sulfate. Thus, dissimilatory sulfate reduction, which frequently occurs at rates comparable to marine surface sediments, can contribute up to 36–50% to anaerobic carbon mineralization in these ecosystems. Since sulfate reduction is thermodynamically favored relative to fermentative processes and methanogenesis, it effectively decreases gross methane production thereby mitigating the flux of methane to the atmosphere. However, very little is known about wetland SRM. Molecular analyses using dsrAB [encoding subunit A and B of the dissimilatory (bi)sulfite reductase] as marker genes demonstrated that members of novel phylogenetic lineages, which are unrelated to recognized SRM, dominate dsrAB richness and, if tested, are also abundant among the dsrAB-containing wetland microbiota. These discoveries point towards the existence of so far unknown SRM that are an important part of the autochthonous wetland microbiota. In addition to these numerically dominant microorganisms, a recent stable isotope probing study of SRM in a German peatland indicated that rare biosphere members might be highly active in situ and have a considerable stake in wetland sulfate reduction. The hidden sulfur cycle in wetlands and the fact that wetland SRM are not well represented by described SRM species explains their so far neglected role as important actors in carbon cycling and climate change
Identification of genetic modules mediating the Jekyll and Hyde interaction of Dinoroseobacter shibae with the dinoflagellate Prorocentrum minimum
No full textThe co-cultivation of the alphaproteobacterium Dinoroseobacter shibae with the dinoflagellate Prorocentrum minimum is characterized by a mutualistic phase followed by a pathogenic phase in which the bacterium kills aging algae. Thus it resembles the Jekyll-and-Hyde interaction that has been proposed for other algae and Roseobacter. Here we identified key genetic components of this interaction. Analysis of the transcriptome of D. shibae in co-culture with P. minimum revealed growth phase dependent changes in the expression of quorum sensing (QS), the CtrA phosphorelay, and flagella biosynthesis genes. Deletion of the histidine kinase gene cckA which is part of the CtrA phosphorelay or the flagella genes fliC or flgK resulted in complete lack of growth stimulation of P. minimum in co-culture with the D. shibae mutants. By contrast, pathogenicity was entirely dependent on one of the extrachromosomal elements of D. shibae, the 191 kb plasmid. The data show that flagella and the CtrA phosphorelay are required for establishing mutualism and prove a cell density dependent killing effect of D. shibae on P. minimum which is mediated by an unknown factor encoded on the 191 kb plasmid
EMR-linked GWAS study: Investigation of variation landscape of loci for Body Mass Index in children
No full textCommon variation at the loci harboring the fat mass and obesity gene (FTO), MC4R and TMEM18 are consistently reported as being associated with obesity and body mass index especially in adult population. In order to confirm this effect in pediatric population five European ancestry cohorts from pediatric eMERGE-II network (CCHMC-BCH) were evaluated.Method:Data on 5049 samples of European ancestry were obtained from the Electronic Medical Records (EMRs) of two large academic centers in five different genotyped cohorts. For all available samples, gender, age, height and weight were collected and Body Mass Index (BMI) was calculated. To account for age and sex differences in BMI, BMI z-scores were generated using 2000 Centers of Disease Control and Prevention (CDC) growth charts. A Genome-wide association study (GWAS) was performed with BMI z-score. After removing missing data and outliers based on principal components (PC) analyses, 2860 samples were used for the GWAS study. The association between each SNP and BMI was tested using linear regression adjusting for age, gender, and PC by cohort. The effects of SNPs were modeled assuming additive, recessive and dominant effects of the minor allele. Meta-analysis was conducted using a weighted z-score approach. Results:The mean age of subjects was 9.8 years (range 2-19). The proportion of male subjects was 56%. In these cohorts, 14% of samples had a BMI≥95% and 28%≥85%. Meta analyses produced a signal at 16q12 genomic region with the best result of p=1.43x10E-07 (p (rec)=7.34E-08) for the single nucleotide polymorphism (SNP) rs8050136 at the first intron of FTO gene (z=5.26) and with no heterogeneity between cohorts (p=0.77). Imputation in this region using dense 1000-Genome and Hapmap CEU samples revealed 71 SNPs with
The struggle to find reliable results in exome sequencing data: Filtering out Mendelian errors
No full textNext Generation Sequencing studies generate a large quantity of genetic data in a relatively cost and time efficient manner and provide an unprecedented opportunity to identify candidate causative variants that lead to disease phenotypes. A challenge to these studies is the generation of sequencing artifacts by current technologies. To identify and characterize the properties that distinguish false positive variants from true variants, we sequenced a child and both parents (trio) using DNA isolated from three sources (blood, buccal cells, and saliva). The trio strategy allowed us to identify variants in the proband that could not have been inherited from the parents (Mendelian errors) and would most likely indicate sequencing artifacts. Quality control measurements were examined and three measurements were found to identify the greatest number of Mendelian errors. These included read depth, genotype quality score, and alternate allele ratio. Filtering the variants on these measurements removed ~95% of the Mendelian errors while retaining 80% of the called variants. These filters were applied independently. After filtering, the concordance between identical samples isolated from different sources was 99.99% as compared to 87% before filtering. This high concordance suggests that different sources of DNA can be used in trio studies without affecting the ability to identify causative polymorphisms. To facilitate analysis of next generation sequencing data, we developed the Cincinnati Analytical Suite for Sequencing Informatics (CASSI) to store sequencing files, metadata (e.g. relatedness information), file versioning, data filtering, variant annotation, and identify candidate causative polymorphisms that follow either de novo, rare recessive homozygous or compound heterozygous inheritance models. We conclude the data cleaning process improves the signal to noise ratio in terms of variants and facilitates the identification of candidate disease causative polymorphisms
