Zebrafish earns its stripes for in vivo ASC speck dynamics

Assembly of the ASC speck is critical for signaling by the inflammasome. In this issue, Kuri et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201703103) use live microscopy to track fluorescently tagged endogenous ASC in the zebrafish, describing the molecular domains driving ASC speck assembly and identifying a key role for macrophages in ASC speck removal in vivo

Dimerization and auto-processing induce caspase-11 protease activation within the non-canonical inflammasome

Caspase-11 is a cytosolic sensor and protease that drives innate immune responses to the bacterial cell wall component, LPS. Caspase-11 provides defence against cytosolic Gram-negative bacteria; however, excessive caspase-11 responses contribute to murine endotoxic shock. Upon sensing LPS, caspase-11 assembles a higher order structure called the non-canonical inflammasome that enables the activation of caspase-11 protease function, leading to gasdermin D cleavage and cell death. The mechanism by which caspase-11 acquires protease function is, however, poorly defined. Here, we show that caspase-11 dimerization is necessary and sufficient for eliciting basal caspase-11 protease function, such as the ability to auto-cleave. We further show that during non-canonical inflammasome signalling, caspase-11 self-cleaves at site (D285) within the linker connecting the large and small enzymatic subunits. Self-cleavage at the D285 site is required to generate the fully active caspase-11 protease (proposed here to be p32/p10) that mediates gasdermin D cleavage, macrophage death, and NLRP3-dependent IL-1β production. This study provides a detailed molecular mechanism by which LPS induces caspase-11-driven inflammation and cell death to provide host defence against cytosolic bacterial infection

Doubling the number of health graduates in Zambia: estimating feasibility and costs

<p>Abstract</p> <p>Background</p> <p>The Ministry of Health (MoH) in Zambia is operating with fewer than half of the human resources for health (HRH) necessary to meet basic population health needs. Responding urgently to address this HRH crisis, the MoH plans to double the annual number of health training graduates in the next five years to increase the supply of health workers. The feasibility and costs of achieving this initiative, however, are unclear.</p> <p>Methods</p> <p>We determined the feasibility and costs of doubling training institution output through an individual school assessment framework. Assessment teams, comprised of four staff from the MoH and Clinton Health Access Initiative, visited all of Zambia's 39 public and private health training institutions from 17 April to 19 June 2008. Teams consulted with faculty and managers at each training institution to determine if student enrollment could double within five years; an operational planning exercise carried out with school staff determined the investments and additional operating costs necessary to achieve expansion. Cost assumptions were developed using historical cost data.</p> <p>Results</p> <p>The individual school assessments affirmed the MoH's ability to double the graduate output of Zambia's public health training institutions. Lack of infrastructure was determined as a key bottleneck in achieving this increase while meeting national training quality standards. A total investment of US$58.8 million is required to meet expansion infrastructure needs, with US$ 35.0 million (59.5%) allocated to expanding student accommodation and US$23.8 million (40.5$ 58.0 million over the five-year scale-up period. Total cost of expansion is estimated at US$ 116.8 million over five years.</p> <p>Conclusions</p> <p>Historic underinvestment in training institutions has crippled Zambia's ability to meet expansion ambitions. There must be significant investments in infrastructure and faculty to meet quality standards while expanding training enrollment. Bottom-up planning can be used to translate national targets into costed implementation plans for expansion at each school.</p

Data-driven normalization strategies for high-throughput quantitative RT-PCR

Background: High-throughput real-time quantitative reverse transcriptase polymerase chain reaction (qPCR) is a widely used technique in experiments where expression patterns of genes are to be profiled. Current stage technology allows the acquisition of profiles for a moderate number of genes (50 to a few thousand), and this number continues to grow. The use of appropriate normalization algorithms for qPCR-based data is therefore a highly important aspect of the data preprocessing pipeline

Phosphoinositide 3-kinase δ regulates membrane fission of Golgi carriers for selective cytokine secretion

The PI3K isoform p110δ is required for TNF trafficking and secretion

Expression analysis of G Protein-coupled receptors in mouse macrophages

Background. Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Results. Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate- elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. Conclusion. The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery

Update of the FANTOM web resource: from mammalian transcriptional landscape to its dynamic regulation

The international Functional Annotation Of the Mammalian Genomes 4 (FANTOM4) research collaboration set out to better understand the transcriptional network that regulates macrophage differentiation and to uncover novel components of the transcriptome employing a series of high-throughput experiments. The primary and unique technique is cap analysis of gene expression (CAGE), sequencing mRNA 5′-ends with a second-generation sequencer to quantify promoter activities even in the absence of gene annotation. Additional genome-wide experiments complement the setup including short RNA sequencing, microarray gene expression profiling on large-scale perturbation experiments and ChIP-chip for epigenetic marks and transcription factors. All the experiments are performed in a differentiation time course of the THP-1 human leukemic cell line. Furthermore, we performed a large-scale mammalian two-hybrid (M2H) assay between transcription factors and monitored their expression profile across human and mouse tissues with qRT-PCR to address combinatorial effects of regulation by transcription factors. These interdependent data have been analyzed individually and in combination with each other and are published in related but distinct papers. We provide all data together with systematic annotation in an integrated view as resource for the scientific community (http://fantom.gsc.riken.jp/4/). Additionally, we assembled a rich set of derived analysis results including published predicted and validated regulatory interactions. Here we introduce the resource and its update after the initial releas