Evaluation of an automated high-level disinfection technology for ultrasound transducers

SummaryBackgroundUltrasound transducer reprocessing is required to prevent the transmission of infections between patients. In some regions, reprocessing practices are not sufficient to achieve high-level disinfection (HLD), which can result in contaminated probes. Furthermore, current manual HLD methods use toxic chemicals and are prone to operator error/variability. The development of automated, non-toxic HLD disinfection devices may reduce the risk of transmission and reduce safety risks for operators and patients. This study investigated the disinfection efficacy of a hydrogen peroxide-based, automated HLD device, the Trophon® EPR, against a range of international standards.MethodsDisinfection efficacy was assessed in carrier and simulated use tests against 21 different species of bacteria, fungi and viruses. Carrier tests were performed by placing carriers throughout the disinfection chamber and measuring the log reduction in viable organisms following disinfection. These tests were performed according to Association of Analytical Communities International Official Methods and European and ASTM International Standards for bactericidal, fungicidal, mycobactericidal, sporicidal and virucidal disinfection. Simulated use tests involving the disinfection of six widely used ultrasound probe models were conducted according to ASTM-E1837-96 using Mycobacterium terrae as a test organism.ResultsThe device satisfied criteria for HLD and sporicidal disinfection efficacy under all standards tested.ConclusionsAutomated, hydrogen peroxide-based disinfection devices offer an alternative to manual ultrasound probe disinfection technologies. Such devices reduce the risks of operator error and can improve patient and operator safety by preventing exposure to toxic chemicals. The adoption of next-generation disinfection devices may help to decrease infection risk and improve patient safety

Differential regulation of the alpha-globin locus by Kruppel-like factor 3 in erythroid and non-erythroid cells

Background: Krüppel-like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor with diverse biological roles. During erythropoiesis, KLF3 acts as a feedback repressor of a set of genes that are activated by Krüppel-like Factor 1 (KLF1). Noting that KLF1 binds α-globin gene regulatory sequences during erythroid maturation, we sought to determine whether KLF3 also interacts with the α-globin locus to regulate transcription. Results: We found that expression of a human transgenic α-globin reporter gene is markedly up-regulated in fetal and adult erythroid cells of Klf3−/− mice. Inspection of the mouse and human α-globin promoters revealed a number of canonical KLF-binding sites, and indeed, KLF3 was shown to bind to these regions both in vitro and in vivo. Despite these observations, we did not detect an increase in endogenous murine α-globin expression in Klf3−/− erythroid tissue. However, examination of murine embryonic fibroblasts lacking KLF3 revealed significant de-repression of α-globin gene expression. This suggests that KLF3 may contribute to the silencing of the α-globin locus in non-erythroid tissue. Moreover, ChIP-Seq analysis of murine fibroblasts demonstrated that across the locus, KLF3 does not occupy the promoter regions of the α-globin genes in these cells, but rather, binds to upstream, DNase hypersensitive regulatory regions. Conclusions: These findings reveal that the occupancy profile of KLF3 at the α-globin locus differs in erythroid and non-erythroid cells. In erythroid cells, KLF3 primarily binds to the promoters of the adult α-globin genes, but appears dispensable for normal transcriptional regulation. In non-erythroid cells, KLF3 distinctly binds to the HS-12 and HS-26 elements and plays a non-redundant, albeit modest, role in the silencing of α-globin expression. </p

Extracellular vesicle associated long non-coding RNAs functionally enhance cell viability

Cells communicate with one another to create microenvironments and share resources. One avenue by which cells communicate is through the action of exosomes. Exosomes are extracellular vesicles that are released by one cell and taken up by neighbouring cells. But how exosomes instigate communication between cells has remained largely unknown. We present evidence here that particular long non-coding RNA molecules are preferentially packaged into exosomes. We also find that a specific class of these exosome associated non-coding RNAs functionally modulate cell viability by direct interactions with l-lactate dehydrogenase B (LDHB), high-mobility group protein 17 (HMG-17), and CSF2RB, proteins involved in metabolism, nucleosomal architecture and cell signalling respectively. Knowledge of this endogenous cell to cell pathway, those proteins interacting with exosome associated non-coding transcripts and their interacting domains, could lead to a better understanding of not only cell to cell interactions but also the development of exosome targeted approaches in patient specific cell-based therapies. Keywords: Non-coding RNA, Extracellular RNA, Exosomes, Retroelement, Pseudogen

RNA Directed Modulation of Phenotypic Plasticity in Human Cells

<div><p>Natural selective processes have been known to drive phenotypic plasticity, which is the emergence of different phenotypes from one genome following environmental stimulation. Long non-coding RNAs (lncRNAs) have been observed to modulate transcriptional and epigenetic states of genes in human cells. We surmised that lncRNAs are governors of phenotypic plasticity and drive natural selective processes through epigenetic modulation of gene expression. Using heat shocked human cells as a model we find several differentially expressed transcripts with the top candidates being lncRNAs derived from retro-elements. One particular retro-element derived transcripts, Retro-EIF2S2, was found to be abundantly over-expressed in heat shocked cells. Over-expression of Retro-EIF2S2 significantly enhanced cell viability and modulated a predisposition for an adherent cellular phenotype upon heat shock. Mechanistically, we find that this retro-element derived transcript interacts directly with a network of proteins including 40S ribosomal protein S30 (FAU), Eukaryotic translation initiation factor 5A (EIF5A), and Ubiquitin-60S ribosomal protein L40 (UBA52) to affect protein modulated cell adhesion pathways. We find one motif in Retro-EIF2S2 that exhibits binding to FAU and modulates phenotypic cell transitions from adherent to suspension states. The observations presented here suggest that retroviral derived transcripts actively modulate phenotypic plasticity in human cells in response to environmental selective pressures and suggest that natural selection may play out through the action of retro-elements in human cells.</p></div

The effects of heat shock on gene expression and the top candidate lncRNAs differentially modulated.

<p>(<b>A</b>) The summary of differentially expressed genes at 30 minutes post heat shock (45°C) in HEK293 cells was determined by transcriptome deep sequencing. (<b>B</b>) The top candidate heat-shock induced transcripts containing retroviral derived retrogenes that were experimentally validated further. (<b>C</b>) The top-candidate retrogenes are also increased in expression 30 minutes following heat shock at 50°C. (<b>D</b>) The top-candidate retrogene expression is significantly reduced 24hrs post-heat shock. (<b>E</b>) Retro-Cox6A, RPS20 and RPL32a are increased in expression while Retro-EIF2S2 is not affected by heat shock at 42°C. For C-E the averages of triplicate treated cultures are shown with the standard deviations and * indicates significant differences (p<0.05) as determined by a paired two-sided T-test.</p

The effects of Retrogene RNA expression on phenotypic plasticity in heat shocked HEK293 cells.

<p>(<b>A</b>) Differentially transfected HEK293 cells were characterized for the effects of lncRNA expression on cell viability at various times post-transfection. The averages of triplicate treated cells are shown with the standard error of the mean and p values shown from a paired two-sided T-test. (<b>B</b>) An image of control and heat shocked adherent and suspension cells immediately after and 24hrs post-heat shock. (<b>C</b>) The experimental plan for determining the effects of overexpression of Retro-EIF2S2 and Retro-Cox6A on cellular states during heat shock. (<b>D-E</b>) The over-expression of Retro-EIF2S2 and Retro-Cox6A predisposes heat shocked cells at 96hrs post-heat shock towards an adherent state relative to untransfected controls at both 48°C and 50°C. (<b>E</b>) The effects of truncated variants of Retro-EIF2S2 to modulate suspension cells relative to the Retro-EIF2S2 in heat shocked cells. (<b>F</b>) The truncated variant EIF3 and a focus on the conserved hairpin stem-loop structure relative to full-length Retro-EIF2S2. Transfected and heat shock treated cultures were assessed 96hrs post-head shock (45°C for 30 min). For (A and D-F) the averages of triplicate treated cells are shown with the standard error of the mean and significance p<0.10 shown (*).</p

Mechanism of Retro-EIF2S2 modulation of adherent cellular phenotypes.

<p>(<b>A</b>) Characterization of Retro-EIF2S2 associated proteins. Biotin-labeled Retro-EIF2S2 was immunoprecipated and bound proteins determined relative GFP-Biotin controls. Triplicate transfected cultures were pooled and IP-Mass Spec performed. The top hits are shown with their % coverage of the protein. (<b>B</b>) Full-length Retro-EIF2S2 immunoprecipitates with FAU following heat shock. (<b>C-D</b>) siRNA repression of (<b>C</b>) FAU, EIF5A and UBA (35-50nM siRNA, Dharmacon siRNAs) followed by heat shock results in (<b>D</b>) a reduction in viable suspension relative to adherent cell numbers 72hrs post-transfection. (<b>E</b>) The over-expression of full-length Retro-EIF1 and truncated EIF3 significantly increase the number of viable suspension cells relative to the control (reverse Retro-EIF2S2). (<b>F</b>) The truncated EIF3 binds FAU as determined by RIP relative to the EIF reverse control. (<b>G</b>) A model for Retro-EIF2S2 regulation of phenotypic states. Heat shock induced activation of Retro-EIF2S2 can result in either adherent cell phenotypes, if bound to FAU, or if Retro-EIF2S2 is inhibited and unable to bind FAU then a suspension cellular state. (<b>H</b>) Schematic summary of Retro-EIF2S2 directed plasticity in HEK293 cells. Adherent vs. suspension cells is contrasted in transfected cells over 120hrs. For B-F triplicate treated cultures are shown with the standard error of the means and p values from a paired T-test.</p

<i>MYCNOS</i> functions as an antisense RNA regulating <i>MYCN</i>

<div><p>Amplification or overexpression of neuronal MYC (MYCN) is associated with poor prognosis of human neuroblastoma. Three isoforms of the MYCN protein have been described as well as a protein encoded by an antisense transcript (<i>MYCNOS</i>) that originates from the opposite strand at the MYCN locus. Recent findings suggest that some antisense long non-coding RNAs (lncRNAs) can play a role in epigenetically regulating gene expression. Here we report that <i>MYCNOS</i> transcripts function as a modulator of the <i>MYCN</i> locus, affecting <i>MYCN</i> promoter usage and recruiting various proteins, including the Ras GTPase-activating protein-binding protein G3BP1, to the upstream <i>MYCN</i> promoter. Overexpression of <i>MYCNOS</i> results in a reduction of upstream <i>MYCN</i> promoter usage and increased MYCN expression, suggesting that the protein-coding <i>MYCNOS</i> also functions as a regulator of <i>MYCN</i> ultimately controlling <i>MYCN</i> transcriptional variants. The observations presented here demonstrate that protein-coding transcripts can regulate gene transcription and can tether regulatory proteins to target loci.</p></div