Creating complex protocells and prototissues using simple DNA building blocks

Building synthetic protocells and prototissues hinges on the formation of biomimetic skeletal frameworks. Recreating the complexity of cytoskeletal and exoskeletal fibers, with their widely varying dimensions, cellular locations and functions, represents a major material hurdle and intellectual challenge which is compounded by the additional demand of using simple building blocks to ease fabrication and control. Here we harness simplicity to create complexity by assembling structural frameworks from subunits that can support membrane-based protocells and prototissues. We show that five oligonucleotides can anneal into nanotubes or fibers whose tunable thicknesses and lengths spans four orders of magnitude. We demonstrate that the assemblies' location inside protocells is controllable to enhance their mechanical, functional and osmolar stability. Furthermore, the macrostructures can coat the outside of protocells to mimic exoskeletons and support the formation of millimeter-scale prototissues. Our strategy could be exploited in the bottom-up design of synthetic cells and tissues, to the generation of smart material devices in medicine

Synthesis of qualitative research studies regarding the factors surrounding UK critical care trial infrastructure

© 2019 Author(s). Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJConducting clinical trials in critical care is integral to improving patient care. Unique practical and ethical considerations exist in this patient population that make patient recruitment challenging, including narrow recruitment timeframes and obtaining patient consent often in time-critical situations. Units currently vary significantly in their ability to recruit according to infrastructure and level of research activity. Aim : To identify variability in the research infrastructure of UK intensive care units (ICUs) and their ability to conduct research and recruit patients into clinical trials. Design: We evaluated factors related to intensive care patient enrolment into clinical trials in the UK. This consisted of a qualitative synthesis carried out with two datasets of in-depth interviews (distinct participants across the two datasets) conducted with 27 intensive care consultants (n=9), research nurses (n=17) and trial coordinators (n=1) from 27 units across the UK. Primary and secondary analysis of two datasets (one dataset had been analysed previously) was undertaken in the thematic analysis. Findings: The synthesis yielded an overarching core theme of Normalising Research, characterised by motivations for promoting research and fostering research-active cultures within resource constraints, with six themes under this to explain the factors influencing critical care research capacity: Organisational, Human, Study, Practical resources, Clinician, and Patient/family factors. There was a strong sense of integrating research in routine clinical practice, and recommendations are outlined. Conclusions: The central and transferable tenet of Normalising Research advocates the importance of developing a culture where research is inclusive alongside clinical practice in routine patient care and is requisite for all healthcare individuals from organisational to direct patient contact level.Peer reviewedFinal Published versio

Beneficial ex vivo immunomodulatory and clinical effects of clarithromycin in COVID-19

INTRODUCTION: Macrolide antibiotics have immunomodulatory properties which may be beneficial in viral infections. However, the precise effects of macrolides on T cell responses to COVID, differences between different macrolides, and synergistic effects with other antibiotics have not been explored. METHODS: We investigated the effect of antibiotics (amoxicillin, azithromycin, clarithromycin, and combined amoxicillin with clarithromycin) on lymphocyte intracellular cytokine levels and monocyte phagocytosis in healthy volunteer PBMCs stimulated ex vivo with SARS-CoV-2 S1+2 spike protein. A retrospective cohort study was performed on intensive care COVID-19 patients. RESULTS: Co-incubation of clarithromycin with spike protein-stimulated healthy volunteer PBMCs ex vivo resulted in an increase in CD8+ (p = 0.004) and CD4+ (p = 0.007) IL-2, with a decrease in CD8+ (p = 0.032) and CD4+ (p = 0.007) IL-10. The addition of amoxicillin to clarithromycin resulted in an increase in CD8+ IL-6 (p = 0.010), decrease in CD8+ (p = 0.014) and CD4+ (p = 0.022) TNF-alpha, and decrease in CD8+ IFN-alpha (p = 0.038). Amoxicillin alone had no effect on CD4+ or CD8+ cytokines. Co-incubation of azithromycin resulted in increased CD8+ (p = 0.007) and CD4+ (p = 0.011) IL-2. There were no effects on monocyte phagocytosis. 102 COVID-19 ICU patients received antibiotics on hospital admission; 62 (61%) received clarithromycin. Clarithromycin use was associated with reduction in mortality on univariate analysis (p = 0.023), but not following adjustment for confounders (HR = 0.540; p = 0.076). CONCLUSIONS: Clarithromycin has immunomodulatory properties over and above azithromycin. Amoxicillin in addition to clarithromycin is associated with synergistic ex vivo immunomodulatory properties. The potential benefit of clarithromycin in critically ill patients with COVID-19 and other viral pneumonitis merits further exploration

Butyrate Supplementation Exacerbates Myocardial and Immune Cell Mitochondrial Dysfunction in a Rat Model of Faecal Peritonitis

Mitochondrial dysfunction and immune cell dysfunction are commonplace in sepsis and are associated with increased mortality risk. The short chain fatty acid, butyrate, is known to have anti-inflammatory effects and promote mitochondrial biogenesis. We therefore explored the immunometabolic effects of butyrate in an animal model of sepsis. Isolated healthy human volunteer peripheral mononuclear cells were stimulated with LPS in the presence of absence of butyrate, and released cytokines measured. Male Wistar rats housed in metabolic cages received either intravenous butyrate infusion or placebo commencing 6 h following faecal peritonitis induction. At 24 h, splenocytes were isolated for high-resolution respirometry, and measurement of mitochondrial membrane potential (MMP), reactive oxygen species (mtROS), and intracellular cytokines (TNF alpha, IL-10) using flow cytometry. Isolated splenocytes from septic and septic butyrate treated rats were stimulated with LPS for 18 h and the effects of butyrate on cytokine release assessed. Ex vivo, butyrate (1.8 mM) reduced LPS-induced TNF alpha (p = 0.019) and IL-10 (p = 0.001) release by human PBMCs. In septic animals butyrate infusion reduced the respiratory exchange ratio (p < 0.001), consistent with increased fat metabolism. This was associated with a reduction in cardiac output (p = 0.001), and increased lactate (p = 0.031) compared to placebo-treated septic animals (p < 0.05). Butyrate treatment was associated with a reduction in splenocyte basal respiration (p = 0.077), proton leak (p = 0.022), and non-mitochondrial respiration (p = 0.055), and an increase in MMP (p = 0.007) and mtROS (p = 0.027) compared to untreated septic animals. Splenocyte intracellular cytokines were unaffected by butyrate, although LPS-induced IL-10 release was impaired (p = 0.039). In summary, butyrate supplementation exacerbates myocardial and immune cell mitochondrial dysfunction in a rat model of faecal peritonitis

Association Between Hypocholesterolemia and Mortality in Critically Ill Patients With Sepsis: A Systematic Review and Meta-Analysis

OBJECTIVE: To ascertain the association between cholesterol and triglyceride levels on ICU admission and mortality in patients with sepsis. DATA SOURCES: Systematic review and meta-analysis of published studies on PubMed and Embase. STUDY SELECTION: All observational studies reporting ICU admission cholesterol and triglyceride levels in critically ill patients with sepsis were included. Authors were contacted for further data. DATA EXTRACTION: Eighteen observational studies were identified, including 1,283 patients with a crude overall mortality of 33.3%. Data were assessed using Revman (Version 5.1, Cochrane Collaboration, Oxford, United Kingdom) and presented as mean difference (MD) with 95% CIs, p values, and I2 values. DATA SYNTHESIS: Admission levels of total cholesterol (17 studies, 1,204 patients; MD = 0.52 mmol/L [0.27–0.77 mmol/L]; p < 0.001; I2 = 91%), high-density lipoprotein (HDL)-cholesterol (14 studies, 991 patients; MD = 0.08 mmol/L [0.01–0.15 mmol/L]; p = 0.02; I2 = 61%), and low-density lipoprotein (LDL) cholesterol (15 studies, 1,017 patients; MD = 0.18 mmol/L [0.04–0.32 mmol/L]; p = 0.01; I2 = 71%) were significantly lower in eventual nonsurvivors compared with survivors. No association was seen between admission triglyceride levels and mortality (15 studies, 1,070 patients; MD = 0.00 mmol/L [–0.16 to 0.15 mmol/L]; p = –0.95; I2 = 79%). CONCLUSIONS: Mortality was associated with lower levels of total cholesterol, HDL-cholesterol, and LDL-cholesterol, but not triglyceride levels, in patients admitted to ICU with sepsis. The impact of cholesterol replacement on patient outcomes in sepsis, particularly in at-risk groups, merits investigation. KEYWORDS: cholesterol levels; intensive care unit; lipids; sepsis; triglyceride

Early dynamic changes to monocytes following major surgery are associated with subsequent infections

BackgroundPost-operative infections are a common cause of morbidity following major surgery. Little is understood about how major surgery perturbs immune function leading to heightened risk of subsequent infection. Through analysis of paired blood samples obtained immediately before and 24 h following surgery, we evaluated changes in circulating immune cell phenotype and function across the first 24 h, to identify early immune changes associated with subsequent infection.MethodsWe conducted a prospective observational study of adult patients undergoing major elective gastrointestinal, gynecological, or maxillofacial surgery requiring planned admission to the post-anesthetic care unit. Patients were followed up to hospital discharge or death. Outcome data collected included mortality, length of stay, unplanned intensive care unit admission, and post-operative infections (using the standardized endpoints in perioperative medicine–core outcome measures for perioperative and anesthetic care criteria). Peripheral blood mononuclear cells were isolated prior to and 24 h following surgery from which cellular immune traits including activation and functional status were assessed by multi-parameter flow cytometry and serum immune analytes compared by enzyme-linked immunosorbent assay (ELISA).ResultsForty-eight patients were recruited, 26 (54%) of whom developed a post-operative infection. We observed reduced baseline pre- and post-operative monocyte CXCR4 and CD80 expression (chemokine receptors and co-stimulation markers, respectively) in patients who subsequently developed an infection as well as a profound and selective post-operative increase in CD4+ lymphocyte IL-7 receptor expression in the infection group only. Higher post-operative monocyte count was significantly associated with the development of post-operative infection (false discovery rate &lt; 1%; adjusted p-value = 0.001) with an area under the receiver operating characteristic curve of 0.84 (p &lt; 0.0001).ConclusionLower monocyte chemotaxis markers, higher post-operative circulating monocyte counts, and reduced co-stimulatory signals are associated with subsequent post-operative infections. Identifying the underlying mechanisms and therapeutics to reverse defects in immune cell function requires further exploration

Is the inflammasome a potential therapeutic target in renal disease?

The inflammasome is a large, multiprotein complex that drives proinflammatory cytokine production in response to infection and tissue injury. Pattern recognition receptors that are either membrane bound or cytoplasmic trigger inflammasome assembly. These receptors sense danger signals including damage-associated molecular patterns and pathogen-associated molecular patterns (DAMPS and PAMPS respectively). The best-characterized inflammasome is the NLRP3 inflammasome. On assembly of the NLRP3 inflammasome, post-translational processing and secretion of pro-inflammatory cytokines IL-1β and IL-18 occurs; in addition, cell death may be mediated via caspase-1. Intrinsic renal cells express components of the inflammasome pathway. This is most prominent in tubular epithelial cells and, to a lesser degree, in glomeruli. Several primary renal diseases and systemic diseases affecting the kidney are associated with NLRP3 inflammasome/IL-1β/IL-18 axis activation. Most of the disorders studied have been acute inflammatory diseases. The disease spectrum includes ureteric obstruction, ischaemia reperfusion injury, glomerulonephritis, sepsis, hypoxia, glycerol-induced renal failure, and crystal nephropathy. In addition to mediating renal disease, the IL-1/ IL-18 axis may also be responsible for development of CKD itself and its related complications, including vascular calcification and sepsis. Experimental models using genetic deletions and/or receptor antagonists/antiserum against the NLRP3 inflammasome pathway have shown decreased severity of disease. As such, the inflammasome is an attractive potential therapeutic target in a variety of renal diseases