Nitric Oxide on Extracorporeal Membrane Oxygenation in Neonates and Children (NECTAR Trial): Protocol for a Randomized Controlled Trial

Background Extracorporeal membrane oxygenation (ECMO) provides support for the pulmonary or cardiovascular function of children in whom the predicted mortality risk remains very high. The inevitable host inflammatory response and activation of the coagulation cascade due to the extracorporeal circuit contribute to additional morbidity and mortality in these patients. Mixing nitric oxide (NO) into the sweep gas of ECMO circuits may reduce the inflammatory and coagulation cascade activation during ECMO support. Objective The purpose of this study is to test the feasibility and safety of mixing NO into the sweep gas of ECMO systems and assess its effect on inflammation and coagulation system activation through a pilot randomized controlled trial. Methods The Nitric Oxide on Extracorporeal Membrane Oxygenation in Neonates and Children (NECTAR) trial is an open-label, parallel-group, pilot randomized controlled trial to be conducted at a single center. Fifty patients who require ECMO support will be randomly assigned to receive either NO mixed into the sweep gas of the ECMO system at 20 ppm for the duration of ECMO or standard care (no NO) in a 1:1 ratio, with stratification by support type (veno-venous vs veno-arterial ECMO). Results Outcome measures will focus on feasibility (recruitment rate and consent rate, and successful inflammatory marker measurements), the safety of the intervention (oxygenation and carbon dioxide control within defined parameters and methemoglobin levels), and proxy markers of efficacy (assessment of cytokines, chemokines, and coagulation factors to assess the impact of NO on host inflammation and coagulation cascade activation, clotting of ECMO components, including computer tomography scanning of oxygenators for clot assessments), bleeding complications, as well as total blood product use. Survival without ECMO and the length of stay in the pediatric intensive care unit (PICU) are clinically relevant efficacy outcomes. Long-term outcomes include neurodevelopmental assessments (Ages and Stages Questionnaire, Strength and Difficulties Questionnaire, and others) and quality of life (Pediatric Quality of Life Inventory and others) measured at 6 and 12 months post ECMO cannulation. Analyses will be conducted on an intention-to-treat basis. Conclusions The NECTAR study investigates the safety and feasibility of NO as a drug intervention during extracorporeal life support and explores its efficacy. The study will investigate whether morbidity and mortality in patients treated with ECMO can be improved with NO. The intervention targets adverse outcomes in patients who are supported by ECMO and who have high expected mortality and morbidity. The study will be one of the largest randomized controlled trials performed among pediatric patients supported by ECMO. Trial Registration Australian New Zealand Clinical Trials Registry ACTRN12619001518156; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=376869 International Registered Report Identifier (IRRID) DERR1-10.2196/4376

Study protocol: NITric oxide during cardiopulmonary bypass to improve Recovery in Infants with Congenital heart defects (NITRIC trial): a randomised controlled trial

Introduction Congenital heart disease (CHD) is a major cause of infant mortality. Many infants with CHD require corrective surgery with most operations requiring cardiopulmonary bypass (CPB). CPB triggers a systemic inflammatory response which is associated with low cardiac output syndrome (LCOS), postoperative morbidity and mortality. Delivery of nitric oxide (NO) into CPB circuits can provide myocardial protection and reduce bypass-induced inflammation, leading to less LCOS and improved recovery. We hypothesised that using NO during CPB increases ventilator-free days (VFD) (the number of days patients spend alive and free from invasive mechanical ventilation up until day 28) compared with standard care. Here, we describe the NITRIC trial protocol. Methods and analysis The NITRIC trial is a randomised, double-blind, controlled, parallel-group, two-sided superiority trial to be conducted in six paediatric cardiac surgical centres. One thousand three-hundred and twenty infants <2 years of age undergoing cardiac surgery with CPB will be randomly assigned to NO at 20 ppm administered into the CPB oxygenator for the duration of CPB or standard care (no NO) in a 1:1 ratio with stratification by age (<6 and ≥6 weeks), single ventricle physiology (Y/N) and study centre. The primary outcome will be VFD to day 28. Secondary outcomes include a composite of LCOS, need for extracorporeal membrane oxygenation or death within 28 days of surgery; length of stay in intensive care and in hospital; and, healthcare costs. Analyses will be conducted on an intention-to-treat basis. Preplanned secondary analyses will investigate the impact of NO on host inflammatory profiles postsurgery. Ethics and dissemination The study has ethical approval (HREC/17/QRCH/43, dated 26 April 2017), is registered in the Australian New Zealand Clinical Trials Registry (ACTRN12617000821392) and commenced recruitment in July 2017. The primary manuscript will be submitted for publication in a peer-reviewed journal. Trial registration number ACTRN12617000821392.</p

Cavin-1/PTRF alters prostate cancer cell-derived extracellular vesicle content and internalization to attenuate extracellular vesicle-mediated osteoclastogenesis and osteoblast proliferation

Background: Tumour-derived extracellular vesicles (EVs) play a role in tumour progression; however, the spectrum of molecular mechanisms regulating EV secretion and cargo selection remain to be fully elucidated. We have reported that cavin-1 expression in prostate cancer PC3 cells reduced the abundance of a subset of EV proteins, concomitant with reduced xenograft tumour growth and metastasis. Methods: We examined the functional outcomes and mechanisms of cavin-1 expression on PC3-derived EVs (PC3-EVs). Results: PC3-EVs were internalized by osteoclast precursor RAW264.7 cells and primary human osteoblasts (hOBs) in vitro, stimulating osteoclastogenesis 37-fold and hOB proliferation 1.5-fold, respectively. Strikingly, EVs derived from cavin-1-expressing PC3 cells (cavin-1-PC3-EVs) failed to induce multinucleate osteoblasts or hOB proliferation. Cavin-1 was not detected in EVs, indicating an indirect mechanism of action. EV morphology, size and quantity were also not affected by cavin-1 expression, suggesting that cavin-1 modulated EV cargo recruitment rather than release. While cavin-1-EVs had no osteoclastogenic function, they were internalized by RAW264.7 cells but at a reduced efficiency compared to control EVs. EV surface proteins are required for internalization of PC3-EVs by RAW264.7 cells, as proteinase K treatment abolished uptake of both control and cavin-1-PC3-EVs. Removal of sialic acid modifications by neuraminidase treatment increased the amount of control PC3-EVs internalized by RAW264.7 cells, without affecting cavin-1-PC3-EVs. This suggests that cavin-1 expression altered the glycosylation modifications on PC3-EV surface. Finally, cavin-1 expression did not affect EV in vivo tissue targeting as both control and cavin-1-PC3-EVs were predominantly retained in the lung and bone 24 hours after injection into mice. Discussion: Taken together, our results reveal a novel pathway for EV cargo sorting, and highlight the potential of utilizing cavin-1-mediated pathways to attenuate metastatic prostate cancer

Dengue virus NS1 protein activates immune cells via TLR4 but not TLR2 or TLR6

The secreted hexameric form of the dengue virus (DENV) non-structural protein 1 (NS1) has recently been shown to elicit inflammatory cytokine release and disrupt endothelial cell monolayer integrity. This suggests that circulating NS1 contributes to the vascular leak that plays a major role in the pathology of dengue haemorrhagic fever and shock. Pathways activated by NS1 are thus of great interest as potential therapeutic targets. Recent works have separately implicated both toll-like receptor 4 (TLR4) and the TLR2/6 heterodimer in immune cell activation by NS1. Here we have used mouse gene knockout macrophages and antibodies blocking TLR function in human peripheral blood mononuclear cells to show that recombinant NS1, expressed and purified from eukaryotic cells, induces cytokine production via TLR4 but not TLR2/6. Furthermore, the commercial Escherichia coli-derived recombinant NS1 preparation used in other work to implicate TLR2/6 in the response is not correctly folded and appears to be contaminated by several microbial TLR ligands. Thus TLR4 remains a therapeutic target for DENV infections, with TLR4 antagonists holding promise for the treatment of dengue disease

A transient disruption of fibroblastic transcriptional regulatory network facilitates trans-differentiation

Transcriptional Regulatory Networks (TRNs) coordinate multiple transcription factors (TFs) in concert to maintain tissue homeostasis and cellular function. The re-establishment of target cell TRNs has been previously implicated in direct trans-differentiation studies where the newly introduced TFs switch on a set of key regulatory factors to induce de novo expression and function. However, the extent to which TRNs in starting cell types, such as dermal fibroblasts, protect cells from undergoing cellular reprogramming remains largely unexplored. In order to identify TFs specific to maintaining the fibroblast state, we performed systematic knockdown of 18 fibroblast-enriched TFs and analyzed differential mRNA expression against the same 18 genes, building a Matrix-RNAi. The resulting expression matrix revealed seven highly interconnected TFs. Interestingly, suppressing four out of seven TFs generated lipid droplets and induced PPARG and CEBPA expression in the presence of adipocyte-inducing medium only, while negative control knockdown cells maintained fibroblastic character in the same induction regime. Global gene expression analyses further revealed that the knockdown-induced adipocytes expressed genes associated with lipid metabolism and significantly suppressed fibroblast genes. Overall, this study reveals the critical role of the TRN in protecting cells against aberrant reprogramming, and demonstrates the vulnerability of donor cell's TRNs, offering a novel strategy to induce transgene-free trans-differentiations

Evaluating the Sensitivity of Mycobacterium tuberculosis to Biotin Deprivation Using Regulated Gene Expression

In the search for new drug targets, we evaluated the biotin synthetic pathway of Mycobacterium tuberculosis (Mtb) and constructed an Mtb mutant lacking the biotin biosynthetic enzyme 7,8-diaminopelargonic acid synthase, BioA. In biotin-free synthetic media, ΔbioA did not produce wild-type levels of biotinylated proteins, and therefore did not grow and lost viability. ΔbioA was also unable to establish infection in mice. Conditionally-regulated knockdown strains of Mtb similarly exhibited impaired bacterial growth and viability in vitro and in mice, irrespective of the timing of transcriptional silencing. Biochemical studies further showed that BioA activity has to be reduced by approximately 99% to prevent growth. These studies thus establish that de novo biotin synthesis is essential for Mtb to establish and maintain a chronic infection in a murine model of TB. Moreover, these studies provide an experimental strategy to systematically rank the in vivo value of potential drug targets in Mtb and other pathogens

Simultaneous Analysis of Multiple Mycobacterium tuberculosis Knockdown Mutants In Vitro and In Vivo

Mycobacterium tuberculosis (Mtb) represents one of the most persistent bacterial threats to human health and new drugs are needed to limit its impact. Conditional knockdown mutants can help validate new drug targets, but the analysis of individual mutants is laborious and time consuming. Here, we describe quantitative DNA tags (qTags) and their use to simultaneously analyze conditional Mtb knockdown mutants that allowed silencing the glyoxylate and methylcitrate cycles (via depletion of isocitrate lyase, ICL), the serine protease Rv3671c, and the core subunits of the mycobacterial proteasome, PrcB and PrcA. The impact of gene silencing in multi-strain cultures was determined by measuring the relative abundance of mutant-specific qTags with real-time PCR. This achieved accurate quantification over a broad range of qTag abundances and depletion of ICL, Rv3671c, or PrcBA resulted in the expected impairment of growth of Mtb with butyrate as the primary carbon source, survival during oxidative stress, acid stress and starvation. The impact of depleting ICL, Rv3671c, or PrcBA in multi-strain mouse infections was analyzed with two approaches. We first measured the relative abundance of mutant-specific qTags in total chromosomal DNA isolated from bacteria that were recovered from infected lungs on agar plates. We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue. Both strategies confirmed that inactivation of Rv3671c and PrcBA severely reduced persistence of Mtb in mice. The multi-strain infections furthermore suggested that silencing ICL not only prevented growth of Mtb during acute infections but also prevented survival of Mtb during chronic infections. Analyses of the ICL knockdown mutant in single-strain infections confirmed this and demonstrated that silencing of ICL during chronic infections impaired persistence of Mtb to the extent that the pathogen was cleared from the lungs of most mice

Septic Shock: A Genomewide Association Study and Polygenic Risk Score Analysis.

Previous genetic association studies have failed to identify loci robustly associated with sepsis, and there have been no published genetic association studies or polygenic risk score analyses of patients with septic shock, despite evidence suggesting genetic factors may be involved. We systematically collected genotype and clinical outcome data in the context of a randomized controlled trial from patients with septic shock to enrich the presence of disease-associated genetic variants. We performed genomewide association studies of susceptibility and mortality in septic shock using 493 patients with septic shock and 2442 population controls, and polygenic risk score analysis to assess genetic overlap between septic shock risk/mortality with clinically relevant traits. One variant, rs9489328, located in AL589740.1 noncoding RNA, was significantly associated with septic shock (p = 1.05 × 10-10); however, it is likely a false-positive. We were unable to replicate variants previously reported to be associated (p < 1.00 × 10-6 in previous scans) with susceptibility to and mortality from sepsis. Polygenic risk scores for hematocrit and granulocyte count were negatively associated with 28-day mortality (p = 3.04 × 10-3; p = 2.29 × 10-3), and scores for C-reactive protein levels were positively associated with susceptibility to septic shock (p = 1.44 × 10-3). Results suggest that common variants of large effect do not influence septic shock susceptibility, mortality and resolution; however, genetic predispositions to clinically relevant traits are significantly associated with increased susceptibility and mortality in septic individuals

Deletion of Wntless in myeloid cells exacerbates liver fibrosis and the ductular reaction in chronic liver injury

Background: Macrophages play critical roles in liver regeneration, fibrosis development and resolution. They are among the first responders to liver injury and are implicated in orchestrating the fibrogenic response via multiple mechanisms. Macrophages are also intimately associated with the activated hepatic progenitor cell (HPC) niche or ductular reaction that develops in parallel with fibrosis. Among the many macrophage-derived mediators implicated in liver disease progression, a key role for macrophage-derived Wnt proteins in driving pro-regenerative HPC activation towards a hepatocellular fate has been suggested. Wnt proteins, in general, however, have been associated with both pro-and anti-fibrogenic activities in the liver and other organs. We investigated the role of macrophage-derived Wnt proteins in fibrogenesis and HPC activation in murine models of chronic liver disease by conditionally deleting Wntless expression, which encodes a chaperone essential for Wnt protein secretion, in LysM-Cre-expressing myeloid cells (LysM-Wls mice)

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution