Fluorescently Labeled DNA Interacts with Competence and Recombination Proteins and Is Integrated and Expressed Following Natural Transformation of Bacillus subtilis

During competence, Bacillus subtilis is able to take up DNA from its environment through the process of transformation. We investigated the ability of B. subtilis to take up fluorescently labeled DNA and found that it is able to take up fluorescein-dUTP-, DyLight 550-dUTP-, and DyLight 650-dUTP-labeled DNA. Transformation with labeled DNA containing an antibiotic cassette resulted in uptake of the labeled DNA and also generated antibiotic-resistant colonies. DNA is primarily taken up at the pole, as it can be seen to colocalize with ComFC, which is a component of the competence machinery. The DNA is taken up rapidly and can be seen to localize with (the actively searching form of) RecA. Colocalization with a homologous locus on the chromosome increases over time. Using microfluidics, we observed replacement of the homologous locus and subsequent expression of the integrated labeled and unlabeled DNA, although whether the integrated DNA contains labeled nucleotides needs to be determined conclusively. Integrated DNA in cells with a doubling time of 60 min is expressed on average 6 h 45 min after the addition of DNA and 4 h 45 min after the addition of fresh medium. We also found that the expression of the incoming DNA under these conditions can occur before cell division and, thus, before complete exit from the competence state. Because the competence machinery is conserved among naturally competent bacteria, this method of labeling is also suitable for studying transformation of other naturally competent bacteria.IMPORTANCE We used DNA that was covalently labeled with fluorescent nucleotides to investigate the transformation process of Bacillus subtilis at the molecular level. We show that the labeled DNA colocalizes with components of the competence machinery, the chromosome, and the recombination protein RecA. Using time-lapse microscopy and microfluidics, we visualized, in real-time, the uptake of fluorescently labeled DNA. We found that under these conditions, cell division is not required for the expression of integrated DNA. Because the competence machinery is conserved in naturally competent bacteria, this method can also be used to investigate the transformation process in many other bacterial species

Near Horizon Geometry of Rotating Black Holes in Five Dimensions

We interpret the general rotating black holes in five dimensions as rotating black strings in six dimensions. In the near horizon limit the geometry is locally AdS_3 x S_3, as in the nonrotating case. However, the global structure couples the AdS_3 and the S_3, giving angular velocity to the S_3. The asymptotic geometry is exploited to count the microstates and recover the precise value of the Bekenstein- Hawking entropy, with rotation taken properly into account. We discuss the perturbation spectrum of the rotating black hole, and its relation to the underlying conformal field theory.Comment: 18 pages, late

Analyses of competent and non-competent subpopulations of Bacillus subtilis reveal yhfW, yhxC and ncRNAs as novel players in competence

Upon competence-inducing nutrient-limited conditions, only part of the Bacillus subtilis population becomes competent. Here, we separated the two subpopulations by fluorescence-assisted cell sorting (FACS). Using RNA-seq, we confirmed the previously described ComK regulon. We also found for the first time significantly downregulated genes in the competent subpopulation. The downregulated genes are not under direct control by ComK but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell division and replication are halted. By investigating the proteome during competence, we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post-transcriptional level. In the competent subpopulation, yhfW was newly identified as being highly upregulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK-binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ΔyhfW mutant. RNA-seq analysis of ΔyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC

DNGR1-mediated deletion of A20/Tnfaip3 in dendritic cells alters T and B-cell homeostasis and promotes autoimmune liver pathology

Dendritic cells (DCs) are central regulators of tolerance versus immunity. The outcome depends amongst others on DC subset and activation status. Whereas CD11b+ type 2 conventional DCs (cDC2s) initiate proinflammatory helper T (Th)-cell responses, CD103+ cDC1s are crucial for regulatory T-cell (Treg) induction and CD8+ T-cell activation. DC activation is controlled by the transcription factor NF-κB. Ablation of A20/Tnfaip3, a critical regulator of NF-κB activation, in DCs leads to constitutive DC activation and development of systemic autoimmunity. We hypothesized that the activation status of cDCs controls the development of autoimmunity. To target cDCs, DNGR1(Clec9a)-cre-mediated excision of A20/Tnfaip3 was used through generation of Tnfaip3fl/flxClec9a+/cre (Tnfaip3DNGR1−KO) mice. Immune cell activation was evaluated at 31-weeks of age. We found that DNGR1-cre-mediated deletion of A20/Tnfaip3 resulted in liver pathology characterized by inflammatory infiltrates adjacent to the portal triads. Both cDC subsets as well as monocyte-derived DCs (moDCs) in Tnfaip3DNGR1−KO livers harbored an activated phenotype. Specifically, the costimulatory molecule CD40 in liver cDCs and moDCs was regulated by A20/Tnfaip3 expression. Livers from Tnfaip3DNGR1−KO mice had augmented prop

Self-stigma and cognitive insight in individuals at ultra-high risk for psychosis

BackgroundImpaired cognitive insight and increased self-stigma have been consistently reported in individuals diagnosed with schizophrenia spectrum disorders, but little is known about its presence in individuals at ultra-high risk of developing a psychosis, although self-stigma is associated with transition.to psychosis. The current study examined whether self-stigma is already present in individuals at ultra-high risk of psychosis, and whether this is associated with impaired cognitive insight.Methods184 participants were recruited divided over three groups, namely individuals diagnosed with a schizophrenia spectrum disorder (SSD; n = 92, 34% females), individuals at ultra-high risk for psychosis (UHR; n = 43, 59% females) and general population controls (GPC; n = 49, 27% females). All participants completed assessments on demographic information (gender, age, education), and cognitive insight. In addition, participants with SSD and individuals at UHR completed a questionnaire on self-stigma.ResultsThe level of self-stigma did not differ between individuals at UHR and individuals diagnosed with SSD. Cognitive insight also did not differ significantly between the three groups, but the subscale self-reflection differed between the three groups [F(2,184) = 4.20, p = 0.02], with the UHR and SSD groups showing more self-reflection. Pearson’s correlation analyses showed that in individuals at UHR total cognitive insight and its self-reflection subscale were significantly associated with the alienation subscale of self-stigma, and in individuals with SSD self-certainty subscale of cognitive insight was significantly associated with stereotype endorsement.ConclusionFindings show that self-stigma was already present in the UHR phase, to a similar degree as in individuals with a diagnosis of a SSD, and is thus not dependent of previous experience of having a label of SSD. Cognitive insight in individuals at UHR of psychosis appears to be intact, but individuals at UHR showed more self-reflectiveness, and individuals at risk with high cognitive insight also experience high levels of self-stigma. Overall findings from our study suggest that pre-emptive interventions targeting self-stigma, while considering cognitive insight, are needed early on in manifestation of psychotic illness, preferably already in the UHR phase

Comparison of a home-based (multi) systemic intervention to promoting Medication AdheRence and Self-management among kidney transplant recipients with care-as-usual: the MARS randomized controlled trial protocol

BACKGROUND: After kidney transplantation non-adherence and inadequate self-management undermine clinical outcomes and quality of life. Both have been demonstrated to be substantial in all age groups. However, interventions promoting adherence and self-management among kidney transplant recipients that have proven to be effective are scarce. In this study we aim to develop and test an intervention to optimize adherence and self-management. In this article we describe the background and design of the trial entitled 'promoting Medication AdheRence and Self-management among kidney transplant recipients' (MARS-trial)'. METHODS/DESIGN: This is a single-center, parallel arm randomized controlled trial. Nonadherent kidney transplant recipients aged 12 years or older are eligible for inclusion. Patients will be randomly assigned to either the experimental or a control group. The control group will receive care-as-usual. The experimental group will receive care-as-usual plus the MARS-intervention. The MARS-intervention is an outreaching intervention, based on the principles of (multi) systemic therapy which means involving the social network. A standardized intervention protocol is used for consistency but we will tailor the behavior change techniques used to the specific needs and determinants

Bacillus subtilis: sporulation, competence and the ability to take up fluorescently labelled DNA

In dit proefschrift zijn competentie, transformatie en sporulatie van Bacillus subtilis bestudeerd. We hebben gevonden dat de master regulator van sporulatie SPo0A direct bindt aan de origin of replication. Het effect van Spo0A op replicatie is het sterkst wanneer sporulatie geïnduceerd wordt tijdens exponentiële groei. De competente staat van B. subtilis is onderzocht met RNA-seq en differentiële expressie van non-coding RNAs is aangetoond. Er zijn down-regulated genen gevonden in de competente sub-populatie en met uitzondering van een, hebben al deze genen een up-regulated anti-sense RNA. Een aantal belangrijke eiwitten werd gevonden die gereguleerd worden op eiwit post-transcriptioneel niveau. Een nieuw hoog differentieel tot expressie komend gen, yhfW, is gevonden en afwezigheid van dit gen heeft een negatief effect op competentie. Analyse van het metaboloom toonde aan dat afwezigheid van yhfW resulteert in lagere niveaus van TCA-cyclus metabolieten tijdens competentie. Tijdens sporulatie leidt afwezigheid van yhfW tot hogere niveaus van Spo0A. We laten voor de eerste keer zien dat Bacillus subtilis getransformeerd kan worden met fluorescent DNA. Het fluorescente DNA gaat interactie aan met het competentie complex, het recombinatie eiwit RecA en het chromosoom. Door gebruik van microfluidics tonen we aan dat het fluorescente DNA tot expressie komt na integratie, en dat celdeling niet noodzakelijk is voor expressie van geïntegreerd DNA

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