The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor.

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids

The expansion of available bioparts and the development of CRISPR/Cas9 for genetic engineering of Parageobacillus thermoglucosidasius

Growing public concern over the environmental and health impacts caused by the use of fossil fuels, coupled with diminishing reserves, is now compelling the pursuit for an alternative source. The search for alternative methods to manufacture products conventionally derived from fossil fuels, has led to research into microbial conversion of lignocellulosic biomass into analogous derivatives. Though many well established renewable energy methods currently exist, research into the production of petroleum-based products, such as transportation fuel and platform chemicals, is currently insufficient. Second generation biofuels have successfully overcome numerous problems associated with original biofuel production; however, many challenges are prevalent for established mesophilic organisms such as Escherichia coli or Saccharomyces cerevisiae. Thermophilic species have been presented as a potential platform strain to circumvent these problems. Typically non-fastidious and catabolically flexible organisms, thermophiles can ferment both hexose and pentose sugars, facilitate lower overall fermentation costs and are generally more robust organisms, a trait valuable for industrial processes. Unfortunately, current genetic tools are not well established to achieve the necessary yields to replace both the fossil fuel industry and the currently used organisms. This study details the identification and subsequent testing of additional genetic tools, to expand the ability for genetic engineering in the thermophile Parageobacillus thermoglucosidasius. The capacity to express exogenous DNA is often predicated on the availability of necessary gene expression elements for complete optimisation of synthetic systems. The identification and expression of the first two thermostable fluorescent proteins in P. thermoglucosidasius allowed the characterisation of several important gene expression elements, which included a collection of promoters, ribosomal binding sites and transcriptional terminators. Subsequently, these tools can now be utilised for precise expression of both endogenous and exogenous DNA in P. thermoglucosidasius. The initial subset of libraries were further successfully utilised to enable the first expression of a CRISPR/Cas9 complex, isolated from Streptococcus thermophilus, in P. thermoglucosidasius. This bacterial adaptive immunity system was then repurposed to facilitate successful genome editing in P. thermoglucosidasius, verified by the removal of the endogenous acetate kinase gene. The efficacy of CRISPR/Cas9 for genome engineering was further demonstrated by its ability to integrate exogenous DNA into both the chromosome and the extrachromosomal megaplasmid present in P. thermoglucosidasius. Cas9 was also validated as an effective method of curing plasmids, through the removal of the megaplasmids, pNCI001 and pNCI002, present in P. thermoglucosidasius. The curing displayed no essential function of either megaplasmid under the conditions tested, but implied the presence of a carbon utilisation pathway, for degradation of aromatic hydrocarbons. The curing of the megaplasmids indicated no beneficial effects and therefore displayed their potential as a vehicle for heterologous expression, due to their stability and increased copy number. Validation of the Cas9-based technology was further achieved through the deletion of the native R-M systems, which revealed an increase in transformation efficiency. This increase can be co-opted to offset the reduction in transformation efficiency observed through the introduction of Cas9 and furthermore, facilitate more efficient allele exchange. The ability to increase succinate production was a further demonstration of the applicability of these developed tools for genome engineering of P. thermoglucosidasius. The production of this important platform chemical, typically produced from petroleum, was achieved through the deletion of several genes from within the central carbon utilisation pathways, using the Cas9 technology exploited in this study. Deletion of succinate dehydrogenase resulted in a 3.5-fold increase compared to WT and produced a base-line strain for further deletions, which will enable the diversion of carbon flux towards succinate production. Both the tools and strains presented in this study can be built upon to synthetically produce an industrially useful strain of P. thermoglucosidasius. This demonstrates significant potential for the future production of biofuels and platform chemicals from lignocellulosic biomass

The effectiveness, acceptability and cost-effectiveness of psychosocial interventions for maltreated children and adolescents: an evidence synthesis.

BACKGROUND: Child maltreatment is a substantial social problem that affects large numbers of children and young people in the UK, resulting in a range of significant short- and long-term psychosocial problems. OBJECTIVES: To synthesise evidence of the effectiveness, cost-effectiveness and acceptability of interventions addressing the adverse consequences of child maltreatment. STUDY DESIGN: For effectiveness, we included any controlled study. Other study designs were considered for economic decision modelling. For acceptability, we included any study that asked participants for their views. PARTICIPANTS: Children and young people up to 24 years 11 months, who had experienced maltreatment before the age of 17 years 11 months. INTERVENTIONS: Any psychosocial intervention provided in any setting aiming to address the consequences of maltreatment. MAIN OUTCOME MEASURES: Psychological distress [particularly post-traumatic stress disorder (PTSD), depression and anxiety, and self-harm], behaviour, social functioning, quality of life and acceptability. METHODS: Young Persons and Professional Advisory Groups guided the project, which was conducted in accordance with Cochrane Collaboration and NHS Centre for Reviews and Dissemination guidance. Departures from the published protocol were recorded and explained. Meta-analyses and cost-effectiveness analyses of available data were undertaken where possible. RESULTS: We identified 198 effectiveness studies (including 62 randomised trials); six economic evaluations (five using trial data and one decision-analytic model); and 73 studies investigating treatment acceptability. Pooled data on cognitive-behavioural therapy (CBT) for sexual abuse suggested post-treatment reductions in PTSD [standardised mean difference (SMD) -0.44 (95% CI -4.43 to -1.53)], depression [mean difference -2.83 (95% CI -4.53 to -1.13)] and anxiety [SMD -0.23 (95% CI -0.03 to -0.42)]. No differences were observed for post-treatment sexualised behaviour, externalising behaviour, behaviour management skills of parents, or parental support to the child. Findings from attachment-focused interventions suggested improvements in secure attachment [odds ratio 0.14 (95% CI 0.03 to 0.70)] and reductions in disorganised behaviour [SMD 0.23 (95% CI 0.13 to 0.42)], but no differences in avoidant attachment or externalising behaviour. Few studies addressed the role of caregivers, or the impact of the therapist-child relationship. Economic evaluations suffered methodological limitations and provided conflicting results. As a result, decision-analytic modelling was not possible, but cost-effectiveness analysis using effectiveness data from meta-analyses was undertaken for the most promising intervention: CBT for sexual abuse. Analyses of the cost-effectiveness of CBT were limited by the lack of cost data beyond the cost of CBT itself. CONCLUSIONS: It is not possible to draw firm conclusions about which interventions are effective for children with different maltreatment profiles, which are of no benefit or are harmful, and which factors encourage people to seek therapy, accept the offer of therapy and actively engage with therapy. Little is known about the cost-effectiveness of alternative interventions. LIMITATIONS: Studies were largely conducted outside the UK. The heterogeneity of outcomes and measures seriously impacted on the ability to conduct meta-analyses. FUTURE WORK: Studies are needed that assess the effectiveness of interventions within a UK context, which address the wider effects of maltreatment, as well as specific clinical outcomes. STUDY REGISTRATION: This study is registered as PROSPERO CRD42013003889. FUNDING: The National Institute for Health Research Health Technology Assessment programme

Coparenting after marital dissolution and children's mental health: a systematic review

Research has shown that coparenting is a vital family mechanism in predicting mental health in children and adolescents. Considering the increasing prevalence of marital dissolution in Western societies, the objective of this systematic review was to summarize the key results of empirical studies that tested the association between mental health of children and coparenting after marital dissolution.info:eu-repo/semantics/publishedVersio

The Coiled Coils of Cohesin Are Conserved in Animals, but Not In Yeast

The SMC proteins are involved in DNA repair, chromosome condensation, and sister chromatid cohesion throughout Eukaryota. Long, anti-parallel coiled coils are a prominent feature of SMC proteins, and are thought to serve as spacer rods to provide an elongated structure and to separate domains. We reported recently that the coiled coils of mammalian condensin (SMC2/4) showed moderate sequence divergence (approximately 10-15%) consistent with their functioning as spacer rods. The coiled coils of mammalian cohesins (SMC1/3), however, were very highly constrained, with amino acid sequence divergence typically <0.5%. These coiled coils are among the most highly conserved mammalian proteins, suggesting that they make extensive contacts over their entire surface.Here, we broaden our initial analysis of condensin and cohesin to include additional vertebrate and invertebrate organisms and multiple species of yeast. We found that the coiled coils of SMC1/3 are highly constrained in Drosophila and other insects, and more generally across all animal species. However, in yeast they are no more constrained than the coils of SMC2/4 and Ndc80/Nuf2p, suggesting that they are serving primarily as spacer rods.SMC1/3 functions for sister chromatid cohesion in all species. Since its coiled coils apparently serve only as spacer rods in yeast, it is likely that this is sufficient for sister chromatid cohesion in all species. This suggests an additional function in animals that constrains the sequence of the coiled coils. Several recent studies have demonstrated that cohesin has a role in gene expression in post-mitotic neurons of Drosophila, and other animal cells. Some variants of human Cornelia de Lange Syndrome involve mutations in human SMC1/3. We suggest that the role of cohesin in gene expression may involve intimate contact of the coiled coils of SMC1/3, and impose the constraint on sequence divergence

Modes of Aβ toxicity in Alzheimer’s disease

Alzheimer’s disease (AD) is reaching epidemic proportions, yet a cure is not yet available. While the genetic causes of the rare familial inherited forms of AD are understood, the causes of the sporadic forms of the disease are not. Histopathologically, these two forms of AD are indistinguishable: they are characterized by amyloid-β (Aβ) peptide-containing amyloid plaques and tau-containing neurofibrillary tangles. In this review we compare AD to frontotemporal dementia (FTD), a subset of which is characterized by tau deposition in the absence of overt plaques. A host of transgenic animal AD models have been established through the expression of human proteins with pathogenic mutations previously identified in familial AD and FTD. Determining how these mutant proteins cause disease in vivo should contribute to an understanding of the causes of the more frequent sporadic forms. We discuss the insight transgenic animal models have provided into Aβ and tau toxicity, also with regards to mitochondrial function and the crucial role tau plays in mediating Aβ toxicity. We also discuss the role of miRNAs in mediating the toxic effects of the Aβ peptide

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The expansion of available bioparts and the development of CRISPR/Cas9 for genetic engineering of Parageobacillus thermoglucosidasius

Growing public concern over the environmental and health impacts caused by the use of fossil fuels, coupled with diminishing reserves, is now compelling the pursuit for an alternative source. The search for alternative methods to manufacture products conventionally derived from fossil fuels, has led to research into microbial conversion of lignocellulosic biomass into analogous derivatives. Though many well established renewable energy methods currently exist, research into the production of petroleum-based products, such as transportation fuel and platform chemicals, is currently insufficient. Second generation biofuels have successfully overcome numerous problems associated with original biofuel production; however, many challenges are prevalent for established mesophilic organisms such as Escherichia coli or Saccharomyces cerevisiae. Thermophilic species have been presented as a potential platform strain to circumvent these problems. Typically non-fastidious and catabolically flexible organisms, thermophiles can ferment both hexose and pentose sugars, facilitate lower overall fermentation costs and are generally more robust organisms, a trait valuable for industrial processes. Unfortunately, current genetic tools are not well established to achieve the necessary yields to replace both the fossil fuel industry and the currently used organisms. This study details the identification and subsequent testing of additional genetic tools, to expand the ability for genetic engineering in the thermophile Parageobacillus thermoglucosidasius. The capacity to express exogenous DNA is often predicated on the availability of necessary gene expression elements for complete optimisation of synthetic systems. The identification and expression of the first two thermostable fluorescent proteins in P. thermoglucosidasius allowed the characterisation of several important gene expression elements, which included a collection of promoters, ribosomal binding sites and transcriptional terminators. Subsequently, these tools can now be utilised for precise expression of both endogenous and exogenous DNA in P. thermoglucosidasius. The initial subset of libraries were further successfully utilised to enable the first expression of a CRISPR/Cas9 complex, isolated from Streptococcus thermophilus, in P. thermoglucosidasius. This bacterial adaptive immunity system was then repurposed to facilitate successful genome editing in P. thermoglucosidasius, verified by the removal of the endogenous acetate kinase gene. The efficacy of CRISPR/Cas9 for genome engineering was further demonstrated by its ability to integrate exogenous DNA into both the chromosome and the extrachromosomal megaplasmid present in P. thermoglucosidasius. Cas9 was also validated as an effective method of curing plasmids, through the removal of the megaplasmids, pNCI001 and pNCI002, present in P. thermoglucosidasius. The curing displayed no essential function of either megaplasmid under the conditions tested, but implied the presence of a carbon utilisation pathway, for degradation of aromatic hydrocarbons. The curing of the megaplasmids indicated no beneficial effects and therefore displayed their potential as a vehicle for heterologous expression, due to their stability and increased copy number. Validation of the Cas9-based technology was further achieved through the deletion of the native R-M systems, which revealed an increase in transformation efficiency. This increase can be co-opted to offset the reduction in transformation efficiency observed through the introduction of Cas9 and furthermore, facilitate more efficient allele exchange. The ability to increase succinate production was a further demonstration of the applicability of these developed tools for genome engineering of P. thermoglucosidasius. The production of this important platform chemical, typically produced from petroleum, was achieved through the deletion of several genes from within the central carbon utilisation pathways, using the Cas9 technology exploited in this study. Deletion of succinate dehydrogenase resulted in a 3.5-fold increase compared to WT and produced a base-line strain for further deletions, which will enable the diversion of carbon flux towards succinate production. Both the tools and strains presented in this study can be built upon to synthetically produce an industrially useful strain of P. thermoglucosidasius. This demonstrates significant potential for the future production of biofuels and platform chemicals from lignocellulosic biomass

Spacing and the transition from calculation to retrieval

Many arithmetic problems can be solved in two ways: by a calculation involving several steps, and by direct retrieval of the answer. With practice on particular problems, memory retrieval tends to supplant calculation—an important aspect of skill learning. We asked how the distribution of practice on particular problems affects this kind of learning. In two experiments, subjects repeatedly worked through sets of multi-digit multiplication problems. The size of the trained problem set was varied. The smaller set size (with shorter average time between problem repetitions) showed faster responses and an earlier transition to retrieval during training. However, in a test session presented days later, the pattern reversed, with faster responses and more retrieval for the large set size. Evidently, maximizing the occurrence of direct retrieval within training is not the best way to promote learning to retrieve the answer. Practical implications are discussed