Pharmacokinetics and Safety of Micafungin in Infants Supported With Extracorporeal Membrane Oxygenation

Candida is a leading cause of infection in infants on extracorporeal membrane oxygenation (ECMO). Optimal micafungin dosing is unknown in this population because ECMO can alter drug pharmacokinetics (PK)

MAGE-A cancer/testis antigens inhibit MDM2 ubiquitylation function and promote increased levels of MDM4

Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically

An animal model to evaluate the function and regulation of the adaptively evolving stress protein SEP53 in oesophageal bile damage responses

Squamous epithelium in mammals has evolved an atypical stress response involving down-regulation of the classic HSP70 protein and induction of sets of proteins including one named SEP53. This atypical stress response might be due to the unusual environmental pressures placed on squamous tissue. In fact, SEP53 plays a role as an anti-apoptotic factor in response to DNA damage induced by deoxycholic acid stresses implicated in oesophageal reflux disease. SEP53 also has a genetic signature characteristic of an adaptively and rapidly evolving gene, and this observation has been used to imply a role for SEP53 in immunity. Physiological models of squamous tissue are required to further define the regulation and function of SEP53. We examined whether porcine squamous epithelium would be a good model to study SEP53, since this animal suffers from a bile-reflux disease in squamous oesophageal tissue. We have (1) cloned and sequenced the porcine SEP53 locus from porcine bacterial artificial chromosome genomic DNA, (2) confirmed the strikingly divergent nature of the C-terminal portion of the SEP53 gene amongst mammals, (3) discovered that a function of the conserved N-terminal domain of the gene is to maintain cytoplasmic localisation, and (4) examined SEP53 expression in normal and diseased porcine pars oesophagea. SEP53 expression in porcine tissue was relatively confined to gastric squamous epithelium, consistent with its expression in normal human squamous epithelium. Immunohistochemical staining for SEP53 protein in normal and damaged pars oesophagea demonstrated significant stabilisation of SEP53 protein in the injured tissue. These results suggest that porcine squamous epithelium would be a robust physiological model to examine the evolution and function of the SEP53 stress pathway in modulating stress-induced responses in squamous tissue

Energy applications of ionic liquids

Ionic liquids offer a unique suite of properties that make them important candidates for a number of energy related applications. Cation–anion combinations that exhibit low volatility coupled with high electrochemical and thermal stability, as well as ionic conductivity, create the possibility of designing ideal electrolytes for batteries, super-capacitors, actuators, dye sensitised solar cells and thermoelectrochemical cells. In the field of water splitting to produce hydrogen they have been used to synthesize some of the best performing water oxidation catalysts and some members of the protic ionic liquid family co-catalyse an unusual, very high energy efficiency water oxidation process. As fuel cell electrolytes, the high proton conductivity of some of the protic ionic liquid family offers the potential of fuel cells operating in the optimum temperature region above 100 °C. Beyond electrochemical applications, the low vapour pressure of these liquids, along with their ability to offer tuneable functionality, also makes them ideal as CO2 absorbents for post-combustion CO2 capture. Similarly, the tuneable phase properties of the many members of this large family of salts are also allowing the creation of phase-change thermal energy storage materials having melting points tuned to the application. This perspective article provides an overview of these developing energy related applications of ionic liquids and offers some thoughts on the emerging challenges and opportunities

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Model depicting the effect of MAGE-A on MDM2 and MDM4.

<p>MDM2 and MDM4 interact through their respective RING finger domains (located at their C-termini). This interaction allows both the activation of MDM2 ubiquitylation function and the ubiquitylation of MDM4 itself leading to MDM4 destruction. MAGE-A interacts preferentially with MDM2 via the N-terminal hydrophobic pocket and the RING finger. The model predicts that this will compete with MDM4 for binding to MDM2, leading to elevated levels of MDM4.</p

Immunohistochemical analysis of MAGE-A and MDM4 in a cohort of 225 human primary breast cancer specimens.

<p>^aThe P value was achieved using Fisher's exact test</p><p>^bThe Odds ratio determines the likelihood of elevated MDM4 staining if the tumor is MAGE-A positive as opposed to MAGE-A negative.</p><p>Immunohistochemical analysis of MAGE-A and MDM4 in a cohort of 225 human primary breast cancer specimens.</p

MAGE-A2 interacts with the p53-binding hydrophobic cleft in the N-terminus of MDM2.

<p>(A) The structure of the N-terminus (p53-binding domain) of MDM2 (1YCR) showing the positions of MAGE-A2 binding peptides 51–65 (red) and 91–105 (magenta). The backbone is shown in green. The locations of the amino acids at the flanks of each of these peptides are shown. (B) Schematic showing the structure of the GST-MDM2 mini-protein, MP1, used in this experiment. A version in which GST was substituted by GFP and was used for cultured cell expression and immunoprecipitation analysis. (C) Co-immunoprecipitation analysis was carried out following expression of GFP-MP1 (or GFP alone as control) in H1299 cells together with MAGE-A2 or p53 (as positive control). (D) GST pull-down assays were performed in which ³⁵S-radiolabelled MAGE-A2, or ³⁵S-radiolabelled p53 as control, were captured on glutathione sepharose 4B beads using GST linked to the MDM2 mini-protein, MP1, which encompasses amino acids 1–110 including the hydrophobic p53-binding cleft. The association of MAGE-A2 or p53 was measured in the presence of increasing concentrations of Nutlin-3a. The co-precipitating MAGE-A2 and p53 proteins were detected by SDS-PAGE followed by fluorography. Note that <i>in vitro</i> transcription/translation of p53 gives rise to two polypeptides. (E) Quantification of the pull-down experiment following densitometry. In all cases the data are representative of at least three independent experiments.</p