In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells

The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl₂, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.     

NMR <Superscript>1</Superscript>H,<Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone and <Superscript>13</Superscript>C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here ¹H^{N, 15}N, and ¹³C resonance assignments for the 19.3 kDa (aa 1–169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS^G12C-GDP), using heteronuclear, multidimensional NMR spectroscopy. Backbone ¹H–¹⁵N correlations have been assigned for all non-proline residues, except for the first methionine residue.     

Multi-centre randomised controlled trial of a smart phone-based event recorder alongside standard care versus standard care for patients presenting to the Emergency Department with palpitations and pre-syncope - the IPED (Investigation of Palpitations in the ED) study: study protocol for a randomised controlled trial

Background

Palpitations and pre-syncope are together responsible for 300,000 annual Emergency Department (ED) attendances in the United Kingdom (UK). Diagnosis of the underlying rhythm is difficult as many patients are fully recovered on ED arrival; and examination and presenting electrocardiogram (ECG) are commonly normal. The only way to establish the underlying heart rhythm is to capture an ECG during symptoms. Recent technology advances have led to several novel ECG monitoring devices appearing on the market. This trial aims to compare the symptomatic rhythm detection rate at 90?days of one such smart phone-based event recorder (AliveCor Heart Monitor and AliveECG) with standard care for participants presenting to the ED with palpitations and pre-syncope and no obvious cause in the ED.

Methods/Design

This is a multi-centre hospital ED / Acute Medical Unit (AMU) open label, randomised controlled trial. Participants will be recruited in 10 tertiary and district general hospitals in the UK. Participants aged ≥?16?years presenting with an episode of palpitations or pre-syncope with no obvious cause and whose underlying ECG rhythm during these episodes remains undiagnosed after clinical assessment will be included. Participants will be randomised to either: (1) the intervention arm, standard care plus the use of a smart phone-based event recorder; or (2) the control arm, standard care. Primary endpoint will be symptomatic rhythm detection rate at 90?days. A number of secondary clinical, process and cost-effectiveness endpoints will be collected and analysed. Analysis will be on an intention-to-treat basis.

Discussion

The Investigation of Palpitations in the ED (IPED) study aims to recruit 242 participants across 10 hospital sites. It will be the first study to investigate the ability of a smart phone-based event recorder to detect symptomatic cardiac rhythms compared to standard care for ED patients with palpitations and pre-syncope with no obvious cause in the ED. This smart phone event recorder will allow ED patients who have presented with palpitations or pre-syncope to record their ECG tracing if they have a further episode and may increase the rate of underlying rhythm diagnosis.

Trial registration

ClinicalTrials.gov, NCT02783898. Registered on 26 May 2016.

     

Modelling antisepsis using defined populations of facultative and anaerobic wound pathogens grown in a basally perfused biofilm model

An in vitro model was developed to assess the effects of topical antimicrobials on taxonomically defined wound biofilms. Biofilms were exposed over seven days to povidone-iodine, silver acetate or polyhexamethylene biguanide (PHMB) at concentrations used in wound dressings. The rank order of tolerance in multi-species biofilms, based on an analysis of the average bacterial counts over time was P. aeruginosa > methicillin-resistant Staphylococcus aureus (MRSA) > B. fragilis > S. pyogenes. The rank order of effectiveness for the antimicrobials in the biofilm model was povidone-iodine > PHMB > silver acetate. None of the test compounds eradicated P. aeruginosa or MRSA from the biofilms although all compounds except silver acetate eliminated S. pyogenes. Antimicrobial effectiveness against bacteria grown in multi-species biofilms did not correlate with planktonic susceptibility. Defined biofilm populations of mixed-species wound pathogens could be maintained in the basal perfusion model, facilitating the efficacy testing of treatments regimens and potential dressings against multi-species biofilms composed of wound isolates.     

“Animals Like Us”: Identifying with Nonhuman Animals and Support for Nonhuman Animal Rights

While anthropomorphizing nonhuman animals has been shown to increase identification with them and, by extension, concern for their wellbeing, little research has directly tested whether identifying with nonhuman animals is similarly associated with concern for their wellbeing. We tested hypotheses related to this premise across three cross-sectional studies. In study 1 (n = 224), we tested the hypothesis that therians—a group of people who self-identify with nonhuman animals, show greater concern for nonhuman animal rights than non-therian furries—people with a fan-like interest in media featuring anthropomorphized animal characters. In study 2 (n = 206), we further tested this hypothesis using implicit and explicit measures of identification with nonhuman animals to predict behavioral intentions to support nonhuman animal rights. In study 3 (n = 182), we tested the generalizability of our findings in a sample of undergraduate students. Taken together, the studies show that explicit, but not implicit, identification with nonhuman animals predicts greater support for their rights. The implications of these findings for research on anthropomorphism and animal rights activism are discussed, as well as the limitations of these findings and possible avenues for future research.     

SNX18 regulates ATG9A trafficking from recycling endosomes by recruiting Dynamin‐2

Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18‐induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin‐2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1‐ and WIPI2‐positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin‐2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.