Dnase1-deficient mice spontaneously develop a systemic lupus erythematosus-like disease.

Systemic lupus erythematosus (SLE) is an autoimmune disease that has high morbidity and can result in multi-organ damage. SLE is characterized by dysregulated activation of T- and B-lymphocytes and the production of autoantibodies directed against nuclear components. The endonuclease deoxyribonuclease 1 (DNase1) is abundant in blood and a subset of SLE patients have mutations in DNASE1. Furthermore, a report showed that Dnase1-deficient mice develop an SLE-like disease, but these mice also carry a deletion of the gene adjacent to Dnase1, which encodes the chaperone TRAP1/HSP75. We generated a murine strain deficient in Dnase1 with an intact Trap1 gene to examine if a lack of DNase1 is responsible for the development of a spontaneous SLE-like disease. We show that the Dnase1-deficient mice do indeed develop an SLE-like phenotype with elevated autoantibody production by 9 months and kidney damage by 12 months. Notably, this model recapitulates the female bias seen in human SLE patients since female Dnase1-deficient mice produced the highest concentrations of autoantibodies and had more severe kidney damage than males. Since there is currently no cure for SLE the protective role of DNase1 as demonstrated in our study remains of great therapeutic interest.

An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis.

Identifying rare, highly penetrant risk mutations may be an important step in dissecting the molecular etiology of schizophrenia. We conducted a gene-based analysis of large (>100 kb), rare copy-number variants (CNVs) in the Wellcome Trust Case Control Consortium 2 (WTCCC2) schizophrenia sample of 1564 cases and 1748 controls all from Ireland, and further extended the analysis to include an additional 5196 UK controls. We found association with duplications at chr20p12.2 (P = 0.007) and evidence of replication in large independent European schizophrenia (P = 0.052) and UK bipolar disorder case-control cohorts (P = 0.047). A combined analysis of Irish/UK subjects including additional psychosis cases (schizophrenia and bipolar disorder) identified 22 carriers in 11 707 cases and 10 carriers in 21 204 controls [meta-analysis Cochran-Mantel-Haenszel P-value = 2 × 10(-4); odds ratio (OR) = 11.3, 95% CI = 3.7, ∞]. Nineteen of the 22 cases and 8 of the 10 controls carried duplications starting at 9.68 Mb with similar breakpoints across samples. By haplotype analysis and sequencing, we identified a tandem ~149 kb duplication overlapping the gene p21 Protein-Activated Kinase 7 (PAK7, also called PAK5) which was in linkage disequilibrium with local haplotypes (P = 2.5 × 10(-21)), indicative of a single ancestral duplication event. We confirmed the breakpoints in 8/8 carriers tested and found co-segregation of the duplication with illness in two additional family members of one of the affected probands. We demonstrate that PAK7 is developmentally co-expressed with another known psychosis risk gene (DISC1) suggesting a potential molecular mechanism involving aberrant synapse development and plasticity.

Nuclear factor κB2 p52 protein has a role in antiviral immunity through IκB kinase epsilon-dependent induction of Sp1 protein and interleukin 15.

In this study we describe a previously unreported function for NFκB2, an NFκB family transcription factor, in antiviral immunity. NFκB2 is induced in response to poly(I:C), a mimic of viral dsRNA. Poly(I:C), acting via TLR3, induces p52-dependent transactivation of a reporter gene in a manner that requires the kinase activity of IκB kinase ε (IKKε) and the transactivating potential of RelA/p65. We identify a novel NFκB2 binding site in the promoter of the transcription factor Sp1 that is required for Sp1 gene transcription activated by poly(I:C). We show that Sp1 is required for IL-15 induction by both poly(I:C) and respiratory syncytial virus, a response that also requires NFκB2 and IKKε. Our study identifies NFκB2 as a target for IKKε in antiviral immunity and describes, for the first time, a role for NFκB2 in the regulation of gene expression in response to viral infection.

Identification of mechanosensitive genes during skeletal development: alteration of genes associated with cytoskeletal rearrangement and cell signalling pathways.

BACKGROUND: Mechanical stimulation is necessary for regulating correct formation of the skeleton. Here we test the hypothesis that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in developmentally important signalling pathways in a genome wide approach. We use a mutant mouse model with altered mechanical stimulation due to the absence of limb skeletal muscle (Splotch-delayed) where muscle-less embryos show specific defects in skeletal elements including delayed ossification, changes in the size and shape of cartilage rudiments and joint fusion. We used Microarray and RNA sequencing analysis tools to identify differentially expressed genes between muscle-less and control embryonic (TS23) humerus tissue. RESULTS: We found that 680 independent genes were down-regulated and 452 genes up-regulated in humeri from muscle-less Spd embryos compared to littermate controls (at least 2-fold; corrected p-value ≤0.05). We analysed the resulting differentially expressed gene sets using Gene Ontology annotations to identify significant enrichment of genes associated with particular biological processes, showing that removal of mechanical stimuli from muscle contractions affected genes associated with development and differentiation, cytoskeletal architecture and cell signalling. Among cell signalling pathways, the most strongly disturbed was Wnt signalling, with 34 genes including 19 pathway target genes affected. Spatial gene expression analysis showed that both a Wnt ligand encoding gene (Wnt4) and a pathway antagonist (Sfrp2) are up-regulated specifically in the developing joint line, while the expression of a Wnt target gene, Cd44, is no longer detectable in muscle-less embryos. The identification of 84 genes associated with the cytoskeleton that are down-regulated in the absence of muscle indicates a number of candidate genes that are both mechanoresponsive and potentially involved in mechanotransduction, converting a mechanical stimulus into a transcriptional response. CONCLUSIONS: This work identifies key developmental regulatory genes impacted by altered mechanical stimulation, sheds light on the molecular mechanisms that interpret mechanical stimulation during skeletal development and provides valuable resources for further investigation of the mechanistic basis of mechanoregulation. In particular it highlights the Wnt signalling pathway as a potential point of integration of mechanical and molecular signalling and cytoskeletal components as mediators of the response.

Delineating the genetic heterogeneity of ALS using targeted high-throughput sequencing.

BACKGROUND: Over 100 genes have been implicated in the aetiology of amyotrophic lateral sclerosis (ALS). A detailed understanding of their independent and cumulative contributions to disease burden may help guide various clinical and research efforts. METHODS: Using targeted high-throughput sequencing, we characterised the variation of 10 Mendelian and 23 low penetrance/tentative ALS genes within a population-based cohort of 444 Irish ALS cases (50 fALS, 394 sALS) and 311 age-matched and geographically matched controls. RESULTS: Known or potential high-penetrance ALS variants were identified within 17.1% of patients (38% of fALS, 14.5% of sALS). 12.8% carried variants of Mendelian disease genes (C9orf72 8.78%; SETX 2.48%; ALS2 1.58%; FUS 0.45%; TARDBP 0.45%; OPTN 0.23%; VCP 0.23%. ANG, SOD1, VAPB 0%), 4.7% carried variants of low penetrance/tentative ALS genes and 9.7% (30% of fALS, 7.1% of sALS) carried previously described ALS variants (C9orf72 8.78%; FUS 0.45%; TARDBP 0.45%). 1.6% of patients carried multiple known/potential disease variants, including all identified carriers of an established ALS variant (p<0.01); TARDBP:c.859G>A(p.[G287S]) (n=2/2 sALS). Comparison of our results with those from studies of other European populations revealed significant differences in the spectrum of disease variation (p=1.7×10(-4)). CONCLUSIONS: Up to 17% of Irish ALS cases may carry high-penetrance variants within the investigated genes. However, the precise nature of genetic susceptibility differs significantly from that reported within other European populations. Certain variants may not cause disease in isolation and concomitant analysis of disease genes may prove highly important.

The origins of the Guinness stout yeast.

Beer is made via the fermentation of an aqueous extract predominantly composed of malted barley flavoured with hops. The transforming microorganism is typically a single strain of Saccharomyces cerevisiae, and for the majority of major beer brands the yeast strain is a unique component. The present yeast used to make Guinness stout brewed in Dublin, Ireland, can be traced back to 1903, but its origins are unknown. To that end, we used Illumina and Nanopore sequencing to generate whole-genome sequencing data for a total of 22 S. cerevisiae yeast strains: 16 from the Guinness collection and 6 other historical Irish brewing. The origins of the Guinness yeast were determined with a SNP-based analysis, demonstrating that the Guinness strains occupy a distinct group separate from other historical Irish brewing yeasts. Assessment of chromosome number, copy number variation and phenotypic evaluation of key brewing attributes established Guinness yeast-specific SNPs but no specific chromosomal amplifications. Our analysis also demonstrated the effects of yeast storage on phylogeny. Altogether, our results suggest that the Guinness yeast used today is related to the first deposited Guinness yeast; the 1903 Watling Laboratory Guinness yeast.

Global endometrial transcriptomic profiling: transient immune activation precedes tissue proliferation and repair in healthy beef cows.

BACKGROUND: All cows experience bacterial contamination and tissue injury in the uterus postpartum, instigating a local inflammatory immune response. However mechanisms that control inflammation and achieve a physiologically functioning endometrium, while avoiding disease in the postpartum cow are not succinctly defined. This study aimed to identify novel candidate genes indicative of inflammation resolution during involution in healthy beef cows. Previous histological analysis of the endometrium revealed elevated inflammation 15 days postpartum (DPP) which was significantly decreased by 30 DPP. The current study generated a genome-wide transcriptomic profile of endometrial biopsies from these cows at both time points using mRNA-Seq. The pathway analysis tool GoSeq identified KEGG pathways enriched by significantly differentially expressed genes at both time points. Novel candidate genes associated with inflammatory resolution were subsequently validated in additional postpartum animals using quantitative real-time PCR (qRT-PCR). RESULTS: mRNA-Seq revealed 1,107 significantly differentially expressed genes, 73 of which were increased 15 DPP and 1,034 were increased 30 DPP. Early postpartum, enriched immune pathways (adjusted P < 0.1) included the T cell receptor signalling pathway, graft-versus-host disease and cytokine-cytokine receptor interaction pathways. However 30 DPP, where the majority of genes were differentially expressed, the enrichment (adjusted P < 0.1) of tissue repair and proliferative activity pathways was observed. Nineteen candidate genes selected from mRNA-Seq results, were independently assessed by qRT-PCR in additional postpartum cows (5 animals) at both time points. SAA1/2, GATA2, IGF1, SHC2, and SERPINA14 genes were significantly elevated 30 DPP and are functionally associated with tissue repair and the restoration of uterine homeostasis postpartum. CONCLUSIONS: The results of this study reveal an early activation of the immune response which undergoes a temporal functional change toward tissue proliferation and regeneration during endometrial involution in healthy postpartum cows. These molecular changes mirror the activation and resolution of endometrial inflammation during involution previously classified by the degree of neutrophil infiltration. SAA1/2, GATA2, IGF1, SHC2, and SERPINA14 genes may become potential markers for resolution of endometrial inflammation in the postpartum cow.

Reducing human DNA bias in cystic fibrosis airway specimens for microbiome analysis.

Next generation sequencing (NGS) has transformed our understanding of airway microbiology, however there are methodology limitations that require consideration. The presence of high concentrations of human DNA in clinical specimens can significantly impact sequencing of the microbiome, especially in low biomass samples. Here we compared three different methods (0.025% saponin, NEBNext Microbiome DNA enrichment kit, QIAamp DNA microbiome kit) for the reduction of human DNA from six CF sputum samples and determined the impact on the microbiome detected using 16S rRNA gene sequencing. Human DNA in undepleted CF sputum accounted for 94.3% of the total DNA. Saponin, the NEBNext kit and the QIAamp kit reduced human DNA levels by an average of 38.7%, 61.8% and 94.8%, respectively. None of the depletion methods reduced total bacterial DNA concentrations. QIAamp depletion did not influence taxa richness or alpha diversity however alterations to the core genera were noted following depletion. While all methods reduced human DNA in the CF sputum samples, the QIAamp DNA microbiome kit reduced Human DNA levels significantly while leaving bacterial DNA levels unchanged. Human DNA depletion in low biomass, human DNA-dense CF sputum samples is vital for improving bacterial resolution in the CF airway microbiome.

First implication of STRA6 mutations in isolated anophthalmia, microphthalmia, and coloboma: a new dimension to the STRA6 phenotype.

Microphthalmia, anophthalmia, and coloboma (MAC) are structural congenital eye malformations that cause a significant proportion of childhood visual impairments. Several disease genes have been identified but do not account for all MAC cases, suggesting that additional risk loci exist. We used single nucleotide polymorphism (SNP) homozygosity mapping (HM) and targeted next-generation sequencing to identify the causative mutation for autosomal recessive isolated colobomatous microanophthalmia (MCOPCB) in a consanguineous Irish Traveller family. We identified a double-nucleotide polymorphism (g.1157G>A and g.1156G>A; p.G304K) in STRA6 that was homozygous in all of the MCOPCB patients. The STRA6 p.G304K mutation was subsequently detected in additional MCOPCB patients, including one individual with Matthew-Wood syndrome (MWS; MCOPS9). STRA6 encodes a transmembrane receptor involved in vitamin A uptake, a process essential to eye development and growth. We have shown that the G304K mutant STRA6 protein is mislocalized and has severely reduced vitamin A uptake activity. Furthermore, we reproduced the MCOPCB phenotype in a zebrafish disease model by inhibiting retinoic acid (RA) synthesis, suggesting that diminished RA levels account for the eye malformations in STRA6 p.G304K patients. The current study demonstrates that STRA6 mutations can cause isolated eye malformations in addition to the congenital anomalies observed in MWS.

MyD88 adaptor-like is not essential for TLR2 signaling and inhibits signaling by TLR3.

Although a clear role for the adaptor protein myeloid differentiation factor-88 (MyD88) adaptor-like (Mal, or TIRAP) in TLR4 signaling has been demonstrated, there is limited information on its role in TLR2 signaling. Here we have systematically analyzed the role of Mal in signaling by TLR2, TLR4, and as a control TLR3 in murine macrophages and dendritic cells. Mal was not required for the induction of IL-6 or NFkappaB activation at high concentrations of the TLR1/2 ligand Pam(3)Cys-Ser-(Lys)(4) or the TLR2/6 ligand macrophage-activating lipopeptide-2 and was required for these responses only at low ligand concentrations. Similarly, induction of IL-6 by Salmonella typhimurium, which is sensed by TLR2, required Mal only at low levels of bacteria. Mal was required for IL-6 induction at all concentrations of the TLR4 ligand LPS. Mal deficiency boosted IL-6 induction by the TLR3 ligand polyinosinic-polycytidylic acid. Activation of JNK, but not p38 or IkappaB degradation, was similarly potentiated in response to polyinosinic-polycytidylic acid in Mal-deficient macrophages. MyD88 was vital for all TLR2 and TLR4 responses and, similar to Mal, was also inhibitory for TLR3-dependent IL-6 and JNK induction. MyD88 interacted with the Toll/IL-1R domains of TLR1, TLR2, TLR4, and TLR6. Mal interacted with the Toll/Il-1R domains of TLR1, TLR2, and TLR4 but not with TLR6. Our study, therefore, reveals that Mal is dispensable in TLR2 signaling at high ligand concentrations in macrophages and dendritic cells, with MyD88 probably coupling to the TLR2 receptor complex at sufficient levels to allow activation. An inhibitory role for Mal in TLR3 signaling to JNK was also demonstrated.

The SNP ratio test: pathway analysis of genome-wide association datasets.

UNLABELLED: We present a tool that assesses the enrichment of significant associations from genome-wide association studies (GWAS) in a pathway context. The SNP ratio test (SRT) compares the proportion of significant to all SNPs within genes that are part of a pathway and computes an empirical P-value based on comparisons to ratios in datasets where the assignment of case/control status has been randomized. We applied the SRT to a Parkinson's disease GWAS dataset, using the KEGG database, revealing significance for Parkinson's disease and related pathways. AVAILABILITY: https://sourceforge.net/projects/snpratiotest/

The future of genomics in Ireland - focus on genomics for health.

Genomics is revolutionizing biomedical research, medicine and healthcare globally in academic, public and industry sectors alike. Concrete examples around the world show that huge benefits for patients, society and economy can be accrued through effective and responsible genomic research and clinical applications. Unfortunately, Ireland has fallen behind and needs to act now in order to catch up. Here, we identify key issues that have resulted in Ireland lagging behind, describe how genomics can benefit Ireland and its people and outline the measures needed to make genomics work for Ireland and Irish patients. There is now an urgent need for a national genomics strategy that enables an effective, collaborative, responsible, well-regulated, and patient centred environment where genome research and clinical genomics can thrive.  We present eight recommendations that could be the pillars of a national genomics health strategy.

Targeted Sequencing of 10,198 Samples Confirms Abnormalities in Neuronal Activity and Implicates Voltage-Gated Sodium Channels in Schizophrenia Pathogenesis.

BACKGROUND: Sequencing studies have pointed to the involvement in schizophrenia of rare coding variants in neuronally expressed genes, including activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-D-aspartate receptor (NMDAR) complexes; however, larger samples are required to reveal novel genes and specific biological mechanisms. METHODS: We sequenced 187 genes, selected for prior evidence of association with schizophrenia, in a new dataset of 5207 cases and 4991 controls. Included among these genes were members of ARC and NMDAR postsynaptic protein complexes, as well as voltage-gated sodium and calcium channels. We performed a rare variant meta-analysis with published sequencing data for a total of 11,319 cases, 15,854 controls, and 1136 trios. RESULTS: While no individual gene was significantly associated with schizophrenia after genome-wide correction for multiple testing, we strengthen the evidence that rare exonic variants in the ARC (p = 4.0 × 10-4) and NMDAR (p = 1.7 × 10-5) synaptic complexes are risk factors for schizophrenia. In addition, we found that loss-of-function variants and missense variants at paralog-conserved sites were enriched in voltage-gated sodium channels, particularly the alpha subunits (p = 8.6 × 10-4). CONCLUSIONS: In one of the largest sequencing studies of schizophrenia to date, we provide novel evidence that multiple voltage-gated sodium channels are involved in schizophrenia pathogenesis and confirm the involvement of ARC and NMDAR postsynaptic complexes.

Signalling adaptors used by Toll-like receptors: an update.

Research into the five Toll/IL1 receptor (TIR) adaptor proteins involved in innate immunity continues to advance. Here we outline some of the more recent findings. MyD88 has a key role in signalling by the IL1 receptor complex and TLRs. However, a MyD88-independent pathway of IL1beta signalling in neurons has been described which involves the protein kinase Akt, and which has an anti-apoptotic effect. This pathway may also be important for the mechanism whereby Alum exerts its adjuvant effect since this depends on IL1beta but is MyD88-independent. MyD88 is also involved in tumourigenesis in models of hepatocarcinoma and familial associated polyposis (FAP); negative regulation of TLR3 signalling and in PKCepsilon activation. The adaptor Mal is regulated by phosphorylation and caspase-1 cleavage. A variant form of Mal in humans termed S180L confers protection in multiple infectious diseases. TRAM is controlled by myristoylation and phosphorylation and the localisation of TRAM with TLR4 to endosomes is required for activation of IRF3 and induction of IFNbeta. Finally SARM has been shown to regulate TRIF and also appears to be involved in neuronal injury mediated by oxidative stress in mouse neurons. These advances confirm the importance for the TIR domain-containing adapters in host defence and inflammation.

Feasibility and relevance of global expression profiling of gene transcripts in serum from breast cancer patients using whole genome microarrays and quantitative RT-PCR.

BACKGROUND: Previous studies, by ourselves and others, have indicated that gene transcripts are detectable extracellularly. Advancing on this work, in order to investigate the feasibility of analysing global gene expression profiles and so the possibility in the future of identifying panels of circulating mRNA biomarkers that may be diagnostic, prognostic or predictive for cancer, here we performed the first whole genome microarray analysis of human serum. PATIENTS AND METHODS: RNA was isolated from pre-surgery serum and corresponding breast tumour and normal tissue biopsies, and from post-surgery and normal control serum. Specimens were examined using Affymetrix whole genome microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). RESULTS: Of the 54,675 mRNAs/variants analysed, approximately 8% and 45% were called Present in serum and breast tissue specimens, respectively. Differentially expressed genes were identified for each group of specimens analysed. Analysis, by qRT-PCR, of 3 selected transcripts further indicated that the nucleic acids detected were mRNA, not DNA. mRNAs are apparently present in serum and their global detection and identification can be successfully achieved using microarray technologies. CONCLUSION: The potential implication of this novel finding is that using microarrays it may be possible to identify a panel of extracellular mRNAs that are diagnostic, prognostic and/or predictive of outcome for cancer patients.

Non-random error in genotype calling procedures: implications for family-based and case-control genome-wide association studies.

The considerable data-handling requirements for genome wide association studies (GWAS) prohibit individual calling of genotypes and create a reliance on sophisticated "genotype-calling algorithms." Despite their obvious utility, the current genotyping platforms and calling-algorithms used are not without their limitations. Specifically, some genotypes are not called due to the ambiguity of the data. Any bias in the missing data could create spurious results. Using data from the Genetic Analysis Information Network (GAIN) we observed that missing genotypes are not randomly distributed throughout the homozygous and heterozygous groups. Using simulation, we examined whether the level and type of missingness observed might influence deviation from the null-hypothesis under common case-control and family-based statistical approaches. Under a case-control model, where missingness is present in a case group but not the controls, we observed bias giving rise to genome-wide significant type-I error for missingness as low as 3%. The family-based association simulations show close to nominal type-I error at 4% genotype missingness. These findings have important implications to study design, quality-control procedures and reporting of findings in GWAS.

Conduct disorder and ADHD: evaluation of conduct problems as a categorical and quantitative trait in the international multicentre ADHD genetics study.

Attention-deficit/hyperactivity disorder (ADHD) is typically characterized by inattention, excessive motor activity, impulsivity, and distractibility. Individuals with ADHD have significant impairment in family and peer relations, academic functioning, and show high co-morbidity with a wide range of psychiatric disorders including oppositional defiant disorder (ODD), conduct disorder (CD), anxiety disorder, depression, substance abuse, and pervasive developmental disorder (PDD). Family studies suggest that ADHD + CD represents a specific subtype of the ADHD disorder with familial risk factors only partly overlapping with those of ADHD alone. We performed a hypothesis-free analysis of the GAIN-ADHD sample to identify markers and genes important in the development of conduct problems in a European cohort of individuals with ADHD. Using the Family-Based Association Test (FBAT) package we examined three measures of conduct problems in 1,043,963 autosomal markers. This study is part of a series of exploratory analyses to identify candidate genes that may be important in ADHD and ADHD-related traits, such as conduct problems. We did not find genome-wide statistical significance (P < 5 x 10(-7)) for any of the tested markers and the three conduct problem traits. Fifty-four markers reached strong GWA signals (P < 10(-5)). We discuss these findings in the context of putative candidate genes and the implications of these findings in the understanding of the etiology of ADHD + CD. We aimed to achieve insight into the genetic etiology of a trait using a hypothesis-free study design and were able to identify a number of biologically interesting markers and genes for follow-up studies.

The role of neutrophil extracellular traps in rheumatic diseases.

Rheumatic diseases are a collection of disorders defined by the presence of inflammation and destruction of joints and internal organs. A common feature of these diseases is the presence of autoantibodies targeting molecules commonly expressed in neutrophils. These preformed mediators are released by neutrophils but not by other immune cells such as macrophages. Neutrophils, major players in the host innate immune response, initiate a cell death mechanism termed neutrophil extracellular trap (NET) formation as a way to ensnare pathogens. NETs are also a source of released self-molecules found in rheumatic diseases. Subsequently, research on the role of NETs in the onset, progression and resolution of inflammation in rheumatic diseases has intensified. This Review has two aims. First, it aims to highlight the mechanisms required for the generation of NETs, the research landscape of which is rapidly changing. Second, it aims to discuss the role of neutrophils and NETs in systemic lupus erythematosus, vasculitis (specifically anti-neutrophil cytoplasmic autoantibody-associated vasculitis), rheumatoid arthritis and gout. Our goal is to clarify the field of NET research in rheumatic diseases in the hope of improving the therapeutic approaches utilized for these diseases.

Diverse stimuli engage different neutrophil extracellular trap pathways.

Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways induced by five stimuli; PMA, the calcium ionophore A23187, nigericin, Candida albicans and Group B Streptococcus. We studied NET production in neutrophils from healthy donors with inhibitors of molecules crucial to PMA-induced NETs including protein kinase C, calcium, reactive oxygen species, the enzymes myeloperoxidase (MPO) and neutrophil elastase. Additionally, neutrophils from chronic granulomatous disease patients, carrying mutations in the NADPH oxidase complex or a MPO-deficient patient were examined. We show that PMA, C. albicans and GBS use a related pathway for NET induction, whereas ionophores require an alternative pathway but that NETs produced by all stimuli are proteolytically active, kill bacteria and composed mainly of chromosomal DNA. Thus, we demonstrate that NETosis occurs through several signalling mechanisms, suggesting that extrusion of NETs is important in host defence.

Itm2a silencing rescues lamin A mediated inhibition of 3T3-L1 adipocyte differentiation.

Dysregulation of adipose tissue metabolism is associated with multiple metabolic disorders. One such disease, known as Dunnigan-type familial partial lipodystrophy (FPLD2) is characterized by defective fat metabolism and storage. FPLD2 is caused by a specific subset of mutations in the LMNA gene. The mechanisms by which LMNA mutations lead to the adipose specific FPLD2 phenotype have yet to be determined in detail. We used RNA-Seq analysis to assess the effects of wild-type (WT) and mutant (R482W) lamin A on the expression profile of differentiating 3T3-L1 mouse preadipocytes and identified Itm2a as a gene that was upregulated at 36 h post differentiation induction in these cells. In this study we identify Itm2a as a novel modulator of adipogenesis and show that endogenous Itm2a expression is transiently downregulated during induction of 3T3-L1 differentiation. Itm2a overexpression was seen to moderately inhibit differentiation of 3T3-L1 preadipocytes while shRNA mediated knockdown of Itm2a significantly enhanced 3T3-L1 differentiation. Investigation of PPARγ levels indicate that this enhanced adipogenesis is mediated through the stabilization of the PPARγ protein at specific time points during differentiation. Finally, we demonstrate that Itm2a knockdown is sufficient to rescue the inhibitory effects of lamin A WT and R482W mutant overexpression on 3T3-L1 differentiation. This suggests that targeting of Itm2a or its related pathways, including autophagy, may have potential as a therapy for FPLD2.