Prespacer processing and specific integration in a Type I-A CRISPR system

This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (REF: BB/M021017/1 to MFW).The CRISPR–Cas system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. Adaptation is dependent on the Cas1 and Cas2 proteins along with varying accessory proteins. Here we analyse the process in Sulfolobus solfataricus, showing that while Cas1 and Cas2 catalyze spacer integration in vitro, host factors are required for specificity. Specific integration also requires at least 400 bp of the leader sequence, and is dependent on the presence of hydrolysable ATP, suggestive of an active process that may involve DNA remodelling. Specific spacer integration is associated with processing of prespacer 3′ ends in a PAM-dependent manner. This is reflected in PAM-dependent processing of prespacer 3′ ends in vitro in the presence of cell lysate or the Cas4 nuclease, in a reaction consistent with PAM-directed binding and protection of prespacer DNA. These results highlight the diverse interplay between CRISPR–Cas elements and host proteins across CRISPR types.Publisher PDFPeer reviewe

Infrared Surface Brightness Fluctuations of the Coma Elliptical NGC 4874 and the Value of the Hubble Constant

We have used the Keck I Telescope to measure K-band surface brightness fluctuations (SBFs) of NGC 4874, the dominant elliptical galaxy in the Coma cluster. We use deep HST WFPC2 optical imaging to account for the contamination due to faint globular clusters and improved analysis techniques to derive measurements of the SBF apparent magnitude. Using a new SBF calibration which accounts for the dependence of K-band SBFs on the integrated color of the stellar population, we measure a distance modulus of 34.99+/-0.21 mag (100+/-10 Mpc) for the Coma cluster. The resulting value of the Hubble constant is 71+/-8 km/s/Mpc, not including any systematic error in the HST Cepheid distance scale.Comment: ApJ Letters, in press. Uses emulateapj5.st

Impacts of the Primary School Free Breakfast Initiative on socio-economic inequalities in breakfast consumption among 9–11-year-old schoolchildren in Wales

Objectives - Universal interventions may widen or narrow inequalities if disproportionately effective among higher or lower socio-economic groups. The present paper examines impacts of the Primary School Free Breakfast Initiative in Wales on inequalities in children's dietary behaviours and cognitive functioning.<p></p> Design Cluster - randomised controlled trial. Responses were linked to free school meal (FSM) entitlement via the Secure Anonymised Information Linkage databank. Impacts on inequalities were evaluated using weighted school-level regression models with interaction terms for intervention × whole-school percentage FSM entitlement and intervention × aggregated individual FSM entitlement. Individual-level regression models included interaction terms for intervention × individual FSM entitlement.<p></p> Setting - Fifty-five intervention and fifty-six wait-list control primary schools.<p></p> Subjects - Approximately 4500 children completed measures of dietary behaviours and cognitive tests at baseline and 12-month follow-up.<p></p> Results School-level models indicated that children in intervention schools ate a greater number of healthy items for breakfast than children in control schools (b = 0·25; 95 % CI 0·07, 0·44), with larger increases observed in more deprived schools (interaction term b = 1·76; 95 % CI 0·36, 3·16). An interaction between intervention and household-level deprivation was not significant. Despite no main effects on breakfast skipping, a significant interaction was observed, indicating declines in breakfast skipping in more deprived schools (interaction term b = −0·07; 95 % CI −0·15, −0·00) and households (OR = 0·67; 95 % CI 0·46, 0·98). No significant influence on inequality was observed for the remaining outcomes.<p></p> Conclusions - Universal breakfast provision may reduce socio-economic inequalities in consumption of healthy breakfast items and breakfast skipping. There was no evidence of intervention-generated inequalities in any outcomes

Probabilistic Inductive Classes of Graphs

Models of complex networks are generally defined as graph stochastic processes in which edges and vertices are added or deleted over time to simulate the evolution of networks. Here, we define a unifying framework - probabilistic inductive classes of graphs - for formalizing and studying evolution of complex networks. Our definition of probabilistic inductive class of graphs (PICG) extends the standard notion of inductive class of graphs (ICG) by imposing a probability space. A PICG is given by: (1) class B of initial graphs, the basis of PICG, (2) class R of generating rules, each with distinguished left element to which the rule is applied to obtain the right element, (3) probability distribution specifying how the initial graph is chosen from class B, (4) probability distribution specifying how the rules from class R are applied, and, finally, (5) probability distribution specifying how the left elements for every rule in class R are chosen. We point out that many of the existing models of growing networks can be cast as PICGs. We present how the well known model of growing networks - the preferential attachment model - can be studied as PICG. As an illustration we present results regarding the size, order, and degree sequence for PICG models of connected and 2-connected graphs.Comment: 15 pages, 6 figure

Evidence of neutral transcriptome evolution in plants

The transcriptome of an organism is its set of gene transcripts (mRNAs) at a defined spatial and temporal locus. Because gene expression is affected markedly by environmental and developmental perturbations, it is widely assumed that transcriptome divergence among taxa represents adaptive phenotypic selection. This assumption has been challenged by neutral theories which propose that stochastic processes drive transcriptome evolution. To test for evidence of neutral transcriptome evolution in plants, we quantified 18 494 gene transcripts in nonsenescent leaves of 14 taxa of Brassicaceae using robust cross-species transcriptomics which includes a two-step physical and in silicobased normalization procedure based on DNA similarity among taxa. Transcriptome divergence correlates positively with evolutionary distance between taxa and with variation in gene expression among samples. Results are similar for pseudogenes and chloroplast genes evolving at different rates. Remarkably, variation in transcript abundance among root-cell samples correlates positively with transcriptome divergence among root tissues and among taxa. Because neutral processes affect transcriptome evolution in plants, many differences in gene expression among or within taxa may be nonfunctional, reflecting ancestral plasticity and founder effects. Appropriate null models are required when comparing transcriptomes in space and time

Duration of shedding of respiratory syncytial virus in a community study of Kenyan children

Background: Our understanding of the transmission dynamics of respiratory syncytial virus (RSV) infection will be better informed with improved data on the patterns of shedding in cases not limited only to hospital admissions. Methods: In a household study, children testing RSV positive by direct immunofluorescent antibody test (DFA) were enrolled. Nasal washings were scheduled right away, then every three days until day 14, every 7 days until day 28 and every 2 weeks until a maximum of 16 weeks, or until the first DFA negative RSV specimen. The relationship between host factors, illness severity and viral shedding was investigated using Cox regression methods. Results: From 151 families a total of 193 children were enrolled with a median age of 21 months (range 1-164 months), 10% infants and 46% male. The rate of recovery from infection was 0.22/person/day (95% CI 0.19-0.25) equivalent to a mean duration of shedding of 4.5 days (95%CI 4.0-5.3), with a median duration of shedding of 4 days (IQR 2-6, range 1-14). Children with a history of RSV infection had a 40% increased rate of recovery i.e. shorter duration of viral shedding (hazard ratio 1.4, 95% CI 1.01-1.86). The rate of cessation of shedding did not differ significantly between males and females, by severity of infection or by age. Conclusion: We provide evidence of a relationship between the duration of shedding and history of infection, which may have a bearing on the relative role of primary versus re-infections in RSV transmission in the community

Control of cyclic oligoadenylate synthesis in a type III CRISPR system

This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (REF: BB/M000400 /1 to MFW), and a Royal Society Challenge Grant (REF: CH160014 to MFW).The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3' end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The 'RNA shredding' activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence.Publisher PDFPeer reviewe

Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

Royal Society Challenge Grant [REF: CH160014 to M.F.W.]; Biotechnology and Biological Sciences Research Council [REF: BB/S000313/1 to M.F.W.]. Funding for open access charge: Institutional Block Grant.The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.Publisher PDFPeer reviewe

Toll-like receptor 4, but not neutrophil extracellular Traps, Promote IFN Type I expression to enhance Th2 responses to Nippostrongylus brasiliensis

The induction of Th2 responses is thought to be multifactorial, and emerge from specific pathways distinct from those associated with antagonistic antibacterial or antiviral Th1 responses. Here, we show that the recognition of non-viable Nippostrongylus brasiliensis (Nb) in the skin induces a strong recruitment of monocytes and neutrophils and the release of neutrophil extracellular traps (NETs). Nb also activates toll-like receptor 4 (TLR4) signaling with expression of Ifnb transcripts in the skin and the development of an IFN type I signature on helminth antigen-bearing dendritic cells in draining lymph nodes. Co-injection of Nb together with about 10,000 Gram-negative bacteria amplified this TLR4-dependent but NET-independent IFN type I response and enhanced the development of Th2 responses. Thus, a limited activation of antibacterial signaling pathways is able to boost antihelminthic responses, suggesting a role for bacterial sensing in the optimal induction of Th2 immunity

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea. A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOSTM clones with insert sizes ~20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs. Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter b-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue. This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae