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Excellent infection prevention and control is a vital component to the success of any practice Stephanie Carter assesses what is now required of this highly valued role in light of Covid-19.

Stephanie Carter considers the importance of targeting team members to upskill in a changing dental landscape.

Not all heroes wear capes! Dental nurse assessor and tutor Stephanie Carter considers how the joint skills of oral health education, plaque indices scoring and fluoride varnish application can add power to your position in practice

The interprofessional nature of the Primary Care Behavioral Health (PCBH) model invites potential conflicts between different ethical guidelines and principles developed by separate professional disciplines. When the foundational model of care and training on which ethical principles were developed shifts, the assumptions underlying the guidance also shifts, revealing gaps and mismatches. This article reviews the extant literature in this realm, and proposes a more unifying set of ethical guidance for interprofessional, integrated primary care practice. We discuss common ethical dilemmas unique to the PCBH model through case examples, and then apply the newly proposed ethical guideline model to these cases to illustrate how the newly proposed model can be efficient and effective navigating these dilemmas.

Brief screening tools based on the Alcohol Use Disorders Identification Test (AUDIT) consumption items (e.g., AUDIT-C and AUDIT-3) are commonly used in general medical settings to identify at-risk drinkers who may benefit from alcohol interventions. Conversely, in specialty alcohol treatment settings with a high volume of self-referrals, there may be a need for brief screening tools that can help to identify patients who are unlikely to require intensive treatments, but there has been little research on the use of AUDIT-C or AUDIT-3 in this context. The current study examined the utility of brief screeners comprised of the AUDIT consumption items for distinguishing lower-severity patients from high severity patients in a cohort of individuals self-referring to specialty alcohol treatment. Participants were adults seeking treatment for alcohol problems (N = 853) at a large public psychiatric hospital in Toronto, Canada, who completed the full 10-item AUDIT as part of an initial telephone screening with hospital staff. Results of receiver operating characteristic curve analyses showed that both the AUDIT-C and the AUDIT-3 demonstrated adequate accuracy (area under the curve; AUC 0.85) for distinguishing lower-severity patients (defined as those in AUDIT zones I, II, and III) from high-severity patients (defined as those in AUDIT zone IV). Exploratory analyses showed that the addition of AUDIT item 4 (impaired control) to the AUDIT-C and AUDIT-3 significantly improved classification accuracy (AUCs = 0.95; ps .001), and the resulting brief screeners had cut-points with good sensitivity and specificity (i.e.,80%). Results support the potential utility of brief screeners comprised of the AUDIT consumption items for distinguishing lower-severity from high severity individuals seeking specialty alcohol treatment services, which may assist with the initial screening and triage process. The addition of AUDIT item 4 improved the performance of the AUDIT-C and AUDIT-3 in this context. Future research validating these findings against external criteria, including comprehensive diagnostic information, is required.

John Playford dominated the commercial music publishing trade of mid-seventeenth-century England, encouraging musical literacy and supplying beginner books for the growing domestic amateur musical class. Playford was clearly aware of the need to attract as many customers as possible in order to succeed in a commercial business; however, very little is known about his customers. This article identifies the contemporary audiences of seventeenth-century English printed music books, building on previous scholarship including Alec Hyatt King’sSome British Collectors of Music c. 1600–1900(1963), and provides an initial record of provenance marks in surviving copies of the publications. Placing the printed book and its customer within the wider context of music-making and bookselling in seventeenth-century England develops our understanding of the social dimensions of the printed music trade, including dissemination and distribution networks.

Background Sepsis and severe focal infections represent a substantial disease burden in children admitted to hospital. We aimed to understand the burden of disease and outcomes in children with life-threatening bacterial infections in Europe. Methods The European Union Childhood Life-threatening Infectious Disease Study (EUCLIDS) was a prospective, multicentre, cohort study done in six countries in Europe. Patients aged 1 month to 18 years with sepsis (or suspected sepsis) or severe focal infections, admitted to 98 participating hospitals in the UK, Austria, Germany, Lithuania, Spain, and the Netherlands were prospectively recruited between July 1, 2012, and Dec 31, 2015. To assess disease burden and outcomes, we collected demographic and clinical data using a secured web-based platform and obtained microbiological data using locally available clinical diagnostic procedures. Findings 2844 patients were recruited and included in the analysis. 1512 (53·2%) of 2841 patients were male and median age was 39·1 months (IQR 12·4–93·9). 1229 (43·2%) patients had sepsis and 1615 (56·8%) had severe focal infections. Patients diagnosed with sepsis had a median age of 27·6 months (IQR 9·0–80·2), whereas those diagnosed with severe focal infections had a median age of 46·5 months (15·8–100·4; pInterpretation Mortality in children admitted to hospital for sepsis or severe focal infections is low in Europe. The disease burden is mainly in children younger than 5 years and is largely due to vaccine-preventable meningococcal and pneumococcal infections. Despite the availability and application of clinical procedures for microbiological diagnosis, the causative organism remained unidentified in approximately 50% of patients.

Aldosterone is a mineralocorticoid steroid hormone whose measurement in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Traditionally measurement of aldosterone has been performed by radioimmunoassay, however these assays lack specificity as they are prone to interference from structurally related steroid hormones. Herein, we have developed a novel, sensitive and specific method utilising solid phase extraction and quantitation of aldosterone from human plasma by UPLC-MS/MS. Standards, quality controls and samples (250μL) were extracted using Oasis(®) HLB 96-well plates. Extract (30μL) was injected onto a Krudcatcher UPLC In-Line Filter, 0.5μm guard column, coupled to a Kinetex PFP, 100mm×2.1mm, 2.6μm column with methanolic mobile phase gradient elution. Eluant was connected to a Waters(®) Xevo TQS tandem mass spectrometer operating in electrospray negative mode. We detected multiple reaction monitoring (MRM) transitions of m/z 359.0>189.1 for aldosterone and 366.0>194.1 for d7-aldosterone respectively, which co-eluted at 2.65min. Ion suppression was negligible. Mean recovery was 89.6%, limit of detection and lower limit of quantitation were 26pmol/L and 30pmol/L respectively. The assay was linear up to 3200pmol/L (r(2)=0.9999). Mean intra- and inter-assay imprecision and bias were all

Healthcare training environments, particularly in multidisciplinary training settings, present unique ethical dilemmas as a result of the multiple relationships faculty must balance while working with trainees. The historical and current perspectives on multiple roles in training environments will first be summarized. Evidence of a gap between the extant discipline specific guidelines and the realities of situations that occur in healthcare training will then be revealed, as illustrated in a case example. Primary care medicine training environments are highly nuanced, potentially leading to an infinite number of ambiguous situations that require a generalizable model for managing multiple roles. Rather than recommend specific modifications to existing ethical guidelines, a new model emphasizing role awareness and decision making when challenges in healthcare training settings arise is proposed. Recommendations for the case example using the model are offered. All professionals are prone to boundary transgressions; explicit training about and the maintenance of appropriate role balance will help to ensure high-functioning relationships and maximize the quality of patient care, resident education, faculty and resident satisfaction, and modeling of professional behavior to improve competencies as clinicians and educators.

The p53 tumour suppressor protein is a transcription factor that prevents oncogenic progression by activating the expression of apoptosis and cell-cycle arrest genes in stressed cells. The stability of p53 is tightly regulated by ubiquitin-dependent degradation, driven mainly by the ubiquitin ligase MDM2. In this study, we have identified USP42 as a DUB that interacts with and deubiquitinates p53. USP42 forms a direct complex with p53 and controls level of ubiquitination during the early phase of the response to a range of stress signals. Although we do not find a clear role for USP42 in controlling either the basal or fully activated levels of p53, the function of USP42 is required to allow the rapid activation of p53-dependent transcription and a p53-dependent cell-cycle arrest in response to stress. These functions of USP42 are likely to contribute to the repair and recovery of cells from mild or transient damage.

The tumor suppressor p53 is extensively regulated by post-translational modification, including modification by the small ubiquitin-related modifier SUMO. We show here that MDM2, previously shown to promote ubiquitin, Nedd8 and SUMO-1 modification of p53, can also enhance conjugation of endogenous SUMO-2/3 to p53. Sumoylation activity requires p53-MDM2 binding but does not depend on an intact RING finger. Both ARF and L11 can promote SUMO-2/3 conjugation of p53. However, unlike the previously described SUMO-1 conjugation of p53 by an MDM2-ARF complex, this activity does not depend on the ability of MDM2 to relocalize to the nucleolus. Interestingly, the SUMO consensus is not conserved in mouse p53, which is therefore not modified by SUMO-2/3. Finally, we show that conjugation of SUMO-2/3 to p53 correlates with a reduction of both activation and repression of a subset of p53-target genes.

This review represents research employing discourse analysis conducted by scholars interested in literacy issues in education across the age span—preschool to adult—during the last 10 years. Drawing from more than 300 studies, we discerned that a common theme was understanding how the literacy education of all students can be successfully accomplished. We organized the review into two complementary sections. The first section highlights discourse analytic approaches taken to investigate: Whose literacies count? Which literacies count? The second section explains the contributions the studies made organized according to five questions: What are literate identities, how are they constructed, and by whom? How are disciplinary knowledges, discourses, and identities constructed? How can schools provide students with access to school-based literacies? What are the shifting roles of literacy teachers and learners within and outside of school? How does discourse analysis research address movement within and across literacy sites and practices in a contemporary, globalized, and increasingly digitally influenced world? تلخيص البحث: تمثل هذه المراجعة الأبحاث التي يوجد فيها تحليل الخطاب الذي قام به الباحثون المهتمون بشؤون تعلم القراءة والكتابة في التعليم عبر الأعمار—من الروضة حتى بالغ سن الرشد—في السنوات العشرة الماضية. لقد حددنا موضوعا مشتركا من استجماع أكثر من 300 دراسة وهو الطريقة التي قد يتم فيها تعليم القراءة والكتابة لجميع الطلاب بالنجاح. وقد نظمنا المراجعة في قسمين متكاملين الأول يسلط الضوء على طرق الخطاب التحليلية المأخوذة للتحقيق في: لمن معارف القراءة والكتابة التي نحتاج إلى أخذها بعين الاعتبار؟ وأية معارف من هذه يجب الأخذ في الحسبان؟ والقسم الثاني يشرح مساهمات الدراسات المندرجة تحت الأصناف المبنية على خمسة أسئلة: ما هي هويات معرفة القراءة والكتابة وكيف يتم تشكيلها ومن يشكلها؟ وكيف يتم تشكيل المعارف الدراسية والخطابية والهوية؟ وكيف تستطيع المدارس أن توفر الطلاب بوسيلة للمعارف القائمة في المدرسة؟ ما هي الأدوار المتغيرة لمعلمي القراءة والكتابة وطلابهم داخل المدرسة وخارجها؟ وكيف يعالج التحليل الخطابي الحركات في مواقع القراءة والكتابة وعبرها وكذلك الممارسات التي تتم في عالم معاصر عولمي فيه يزداد التأثير الرقمي؟ 本文旨在阐述过去10年期间，学者致力于不同学龄（由学前以至成人）的读写教育问题研究时所使用的语篇分析方法。作者从300多个研究里整理出一个共通的主题，就是去理解如何使到所有学生的读写教育取得成功。本文献综述是由两个互补的章节组成。第一节重点介绍研究调查所采用的两个语篇分析的处理方法：是谁的读写文化有重要意义？哪些读写文化有重要意义？第二节根据五个问题来阐釋这些研究的贡献：1.有读写文化的人的身份认同，所指的是什么？是怎样建构而成的？由谁所建构而成的？2.各种学科知识、话语及身份认同是如何建构而成的？3.学校如何为学生提供获取校本读写文化知识的门路？4.读写教学教师与学生在校内及校外在角色上有什么转移？5.在当前全球化和日益受数码化影响的世界中，语篇分析研究如何处理流动于各种写文化网站以内及之间的文化信息及各种文化实践的问题？ Cet etat de la question presente l'analyse du discours dans les recherches des chercheurs qui s'interessent aux questions de litteratie en education— du niveau prescolaire a l'adulte — au cours des dix dernieres annees. En nous basant sur plus de 300 etudes, une preoccupation commune nous est apparue qui est de comprendre comment l'enseignement de la litteratie peut permettre a tous les eleves de reussir. Nous avons organise l'etat de la question en deux parties complementaires. La premiere partie s'interesse au discours des approches analytiques realisees pour etudier les questions suivantes: prendre en compte les litteraties de qui ? prendre en compte quelles litteraties? La seconde partie explique ce qu'ont apporte les etudes effectuees en reponse a cinq questions: que sont les identites lettrees ? comment sont-elles construites, et par qui ? Comment sont construits les avoirs disciplinaires, les discours, et les identites ? Comment les ecoles fournissent-elles aux eleves un acces aux litteraties basees sur l'ecole ? Quels sont les changements de roles des professeurs de litteratie et des apprenants dans l'ecole et hors de celle-ci ? Comment le discours de la recherche analyse prend-il en compte le mouvement au sein et au travers des sites de litteratie et des pratiques dans un monde contemporain globalise et de plus en plus influence par l'informatique. В этом обзоре представлены исследования, проведенные на протяжении последних десяти лет с помощью дискурс-анализа и посвященные проблемам развития грамотности людей разного возраста – от дошкольников до взрослых. Общая тема для более чем трехсот рассмотренных авторами источников: как добиться успеха в становлении грамотности? Обзор состоит из двух взаимодополняющих разделов. В первом описаны подходы к анализу дискурса, которые помогают понять, что принимается за образцы грамотности и кто может им соответствовать. Во втором разделе результаты исследований рассмотрены согласно пяти категориям: Как человек осознает себя в плане грамотности, на чем и кем строится эта идентичность? Как конструируется предметное или дисциплинарное знание, дискурс и идентичность? Как может школа дать учащимся доступ к школьной академической грамотности? В чем состоят и как меняются роли преподавателей грамотности и учащихся в стенах школы и за ее пределами? Как исследования дискурс-анализа отражают перемещения участников дискурса в реальном и виртуальном пространстве в современном глобализированном мире? Este repaso presenta investigacion que ha sido llevada a cabo usando analisis del discurso por estudiosos interesados en cuestiones de alfabetizacion en la educacion a todas las edades—preescolar hasta adulto—en los ultimos diez anos. Al examinar mas de 300 estudios, vimos que un tema comun es el de entender como lograr exitosamente la educacion de competencias de todos los estudiantes. Organizamos el repaso en dos secciones complementarias. Enfocamos la primera en los acercamientos analiticos del discurso que se han usado para investigar: ?Cuales competencias cuentan o valen? ?Las competencias de quienes cuentan o valen? La segunda seccion explica las contribuciones que hicieron los estudios organizadas de acuerdo a cinco temas: ?Que son identidades competentes, como se construyen, y por quienes? ?Como se construyen el conocimiento, el discurso y las identidades de las disciplinas? ?Como pueden las escuelas darles acceso a los estudiantes a las competencias basadas en la escuela? ?Cuales son los papeles cambiantes de los maestros y los estudiantes de competencias dentro y fuera de la escuela? ?Como plantea la investigacion del analisis del discurso el movimiento dentro y a traves de los sitios de competencias y practicas en este mundo contemporaneo, globalizado y digital?

Morgue is a unique multi-domain protein that contains a zinc finger motif, an F box, and a variant E2 conjugase domain. The presence of these domains suggests potentially complex and novel functions for Morgue in ubiquitination pathways. Morgue was originally identified via its gain-of-function enhancement of eye cell death phenotypes in Drosophila and ectopic expression of Morgue also influences circadian rhythms. However, there is as yet little known about Morgue’s normal developmental or physiological functions. To address this issue, we generated several morgue loss-of-function mutants via P element excision mutagenesis and analyzed the mutant phenotypes during the fly life cycle. These studies revealed that morgue null mutants are viable, though approximately 10% of the mutants exhibit defects in pupal spiracle eversion and malformations in the adult abdominal cuticle. In addition, a similar subset of morgue mutant embryos exhibited alterations in the normal number, position, or morphology of specific neurons and glia. Analysis of Morgue protein localization was addressed through generation of a transgenic fly strain that expresses a GFP::Morgue fusion protein. Use of this strain revealed Morgue protein localization in multiple cellular compartments, including nuclei, cytoplasm and membranes. Taken together, these diverse phenotypes and distribution patterns suggest pleiotropic functions for Morgue.

The p53 tumour suppressor protein is subject to numerous post-translational modifications, which coalesce in various combinations and patterns to regulate its activity. In addition to a multitude of phosphorylated serines and threonines, many of the lysine residues in p53 can be modified to regulate activity, stability and subcellular localization of the protein. This complexity is amplified by the variety of modifications that can target the same lysine residue - often with opposing effects on p53 function.

Low back pain (LBP) is a common source of disability in adults and highly prevalent in patients with painful hip pathology. Persistent LBP after hip arthroplasty is associated with lower self-reported function, however, the effect of pre-operative LBP in patients undergoing hip arthroscopy for FAI has not been evaluated. The purpose of this study was to determine whether improvements in self-reported hip function following arthroscopic surgery for femoroacetabular impingement (FAI) differed between those with and without reports of pre-operative low back pain.Cohort.Three hundred eighteen subjects undergoing primary hip arthroscopy for clinically and radiographically-confirmed FAI were recruited and consented. One hundred fifty-six of these subjects completed the International Hip Outcomes Tool (iHOT-33) and the Hip Outcome Score Activities of Daily Living Subscale (HOS-ADL) before, and six and 12 months after surgery. Subjects were grouped based on the self-reported presence or absence of LBP prior to arthroscopy. A repeated measures analysis of variance was used to determine the effects of time and low back pain on iHOT-33 and HOS-ADL scores.Seventy-five of 156 subjects (48.1%) reported LBP prior to surgery. A main effect of time was found for both outcome measures (p0.001), demonstrating improvement in self-reported outcomes over the testing period. There was a main effect of group for the iHOT-33 (LBP: 52.0 [47.9,56.0]; no LBP 57.9 [53.9,61.8]; p = 0.043) but not for the HOS-ADL (LBP: 75.2 [72.2,78.2]; no LBP 78.8 [75.9,81.7]; p = 0.088) indicating that subjects with pre-operative LBP had poorer self-reported function per the iHOT-33 compared to those without LBP.Self-reported hip function scores improved regardless of the presence of pre-operative LBP; however subjects with LBP reported poorer self-reported function per the iHOT-33 as compared to those without LBP up to 12 months post-operatively.3c.

Acute stimulation of the receptor for the hepatocyte growth factor/scatter factor Met leads to receptor monoubiquitination and down-regulation through the lysosomal degradation pathway. We have determined that the Met receptor undergoes multiple monoubiquitination as opposed to the appendage of polyubiquitin chains. Nevertheless, overexpression of ubiquitin in HEK293T cells enhances the rate of Met receptor degradation, in contrast to a point mutant of ubiquitin (K48R) that cannot form Lys48-linked polyubiquitin chains. Furthermore, an enhancement of Met degradation is also seen under conditions where the proteasome is inhibited by lactacystin. We propose that this reflects polyubiquitin-dependent sorting of Met, as the overexpression of ubiquitin but not K48R ubiquitin also restores hepatocyte growth factor-dependent phosphorylation of the endosomal coat protein Hrs from inhibition by lactacystin. Our data indicate a requirement for K48R-linked polyubiquitin for Met endosomal trafficking independent of its canonical function of targeting for proteasomal degradation.

The signal recognition particle (SRP) is required for co-translational targeting of polypeptides to the endoplasmic reticulum (ER). Once at the membrane, the precursor interacts with a complex proteinaceous machinery that mediates its translocation across the bilayer. Genetic studies in yeast have identified a number of genes whose products are involved in this complex process. These mutants offer a potentially valuable resource with which to analyze the biochemical role played by each component in the pathway. However, such analyses have been hampered by the failure to reconstitute an efficient in vitro assay for SRP-dependent translocation. We report the construction of two multicopy vectors that allow overexpression of all seven gene products required to make SRP in the yeast Saccharomyces cerevisiae. The overexpressed subunits assemble into intact and functional SRP particles, and we further demonstrate that in vitro reconstitution of co-translational translocation is greatly enhanced using cytosol from the overexpression strain. We use this assay to demonstrate that Sec63p is required for co-translational translocation in vitro and specifically identify the "J-domain" of Sec63p as crucial for this pathway.

Proteasomal activity is required for Met receptor degradation after acute stimulation with hepatocyte growth factor (HGF). Inhibition of proteasomal activity with lactacystin leads to a block in the endocytic trafficking of Met such that the receptor fails to reach late endosomes/lysosomes, where degradation by acid-dependent proteases takes place ( Hammond et al., 2001 ). In this article, we have biochemically determined Met internalization rates from the cell surface and shown that lactacystin does not inhibit the initial HGF-dependent internalization step of Met. Instead, it promotes the recycling pathway from early endosomes at the expense of sorting to late endosomes, thereby ensuring rapid return of internalized Met to the cell surface. We have used this perturbation of Met endosomal sorting by lactacystin to examine the consequences for HGF-dependent signaling outputs. In control cells HGF-dependent receptor autophosphorylation reaches a maximal level over 5–10 min but then attenuates over the ensuing 50 min. Furthermore, Met dephosphorylation can be kinetically dissociated from Met degradation. In lactacystin-treated cells, we observe a failure of Met dephosphorylation as well as Met degradation. Elements of the mitogen-activated protein kinase cascade, downstream of receptor activation, show a normal kinetic profile of phosphorylation, indicating that the mitogen-activated protein kinase pathway can attenuate in the face of sustained receptor activation. The HGF-dependent phosphorylation of a receptor substrate that is localized to clathrin-coated regions of sorting endosomes, Hrs, is dramatically reduced by lactacystin treatment. Reduction of cellular Hrs levels by short interfering RNA modestly retards Met degradation and markedly prevents the attenuation of Met phosphorylation. HGF-dependent Hrs phosphorylation and Met dephosphorylation may provide signatures for retention of the receptor in coated regions of the endosome implicated in sorting to lysosomes.

Organizations looking for new and more effective ways of handling conflict are beginning to consider the development of alternative dispute resolution and conflict management systems. This article focuses on the importance of buy-in and participation by parties and stakeholders in designing such systems. It examines the value of party buy-in and involvement and their effect on the successful application and use of the dispute resolution system. It concludes that the design of participative dispute resolution systems maximizes the opportunity for effective conflict management and the successful creation, implementation, and management of organizational change.

Background Biobank samples stored in biobanks give researchers and respiratory healthcare institutions access to datasets of analytes valuable for both diagnostic and research practices. The usefulness of these samples in clinical decision-making is highly dependent on their quality and integrity. New procedures that better preserve sample integrity and reduce degradation are being developed to meet the needs of both present and future biobanking. Hereby we present an automatic sample workflow scheme that is designed to handle high numbers of blood samples. Methods Blood fractions are aliquoted, heat sealed using novel technology, and stored in 384 tube high-density sample arrays. Results The newly developed 384 biobank rack system is especially suited for preserving identical small aliquots. We provide data on robotic processing of clinical samples at −80°C, following initial processing, analysis and shipping between laboratories throughout Europe. Subsequent to unpacking, re-sorting, and storage at these sites, the samples have been returned for analysis. Biomarker analysis of 13 common tests in the clinical chemistry unit of the hospital provides evidence of qualitative and stable logistics using the 384-sample tube system. Conclusions This technology development allows rapid access to a given sample in the frozen archive while maintaining individual sample integrity with sample tube confinement and quality management.

The p53 tumour suppressor protein is a transcription factor that prevents oncogenic progression by activating the expression of apoptosis and cell-cycle arrest genes in stressed cells. The stability of p53 is tightly regulated by ubiquitin-dependent degradation, driven mainly by the ubiquitin ligase MDM2. In this study, we have identified USP42 as a DUB that interacts with and deubiquitinates p53. USP42 forms a direct complex with p53 and controls level of ubiquitination during the early phase of the response to a range of stress signals. Although we do not find a clear role for USP42 in controlling either the basal or fully activated levels of p53, the function of USP42 is required to allow the rapid activation of p53-dependent transcription and a p53-dependent cell-cycle arrest in response to stress. These functions of USP42 are likely to contribute to the repair and recovery of cells from mild or transient damage.

The tumour suppressor p53 has been shown to be modified at its C-terminus with ubiquitin and the ubiquitin-like proteins SUMO and NEDD8. Whereas monoubiquitination of p53 is strongly associated with nuclear export, the effects of sumoylation and neddylation remain unclear. In this study we have generated p53-Ub, p53-SUMO-1 and p53-NEDD8 fusion proteins as models for the effect of these modifications on the localization and function of p53. As shown before, the ubiquitin fusion clearly drives nuclear export of p53 and we now find that this is also partially the case for a SUMO-1 fusion, which does not localise to PML bodies. In contrast a NEDD8 fusion has little effect on nuclear export, and mutating NEDD8 to more closely resemble ubiquitin did not restore nuclear export. Despite their differing subcellular localization, we find that both p53-ubiquitin and p53-NEDD8 retain similar transcriptional activity and both induce apoptosis at a similar level to unfused p53. The p53-ubiquitin fusion protein is potentially a good model for studying the role of p53 outside the nucleus. However, in our experiments we find that the export of p53 from the nucleus is not sufficient to activate its cytoplasmic apoptotic function which may depend on the ability to deubiquitinate cytoplasmic p53.

Loss of Numb could enable breast cancer cells to bypass the p53 tumor suppressor response, The cell-fate determinant Numb has recently been shown to help activate the tumor suppressor protein p53. Loss of Numb in breast cancers would result, therefore, in both the activation of the potential oncogene Notch and the diminution of tumor suppression by p53.

International audience; p53 functions to prevent malignant progression, in part by inhibiting proliferation or inducing the death of potential tumour cells. One of the most important regulators of p53 is MDM2, a RING domain E3 ligase that ubiquitinates p53, leading to both proteasomal degradation and relocation of p53 from the nucleus to the cytoplasm. Previous studies have suggested that although polyubiquitination is required for degradation, monoubiquitination of p53 is sufficient for nuclear export. Using a p53-ubiquitin fusion protein we show that ubiquitination contributes to two steps before export: exposure of a carboxy-terminal nuclear export sequence (NES), and dissociation of MDM2. Monoubiquitination can directly promote further modifications of p53 with ubiquitin-like proteins and MDM2 promotes the interaction of the SUMO E3 ligase PIASy with p53, enhancing both sumoylation and nuclear export. Our results suggest that modifications such as sumoylation can regulate the strength of the p53-MDM2 interaction and participate in driving the export of p53.

Aluminium smelter potrooms are unique in that workplace exposures to hydrogen fluoride (HF), sulfur dioxide (SO2), and particulate matter occur simultaneously for some tasks. The peak exposures to these contaminants are of increasing interest in discovering the etiology of respiratory health effects. While a variety of direct-reading instruments are available for sulfur dioxide and particulate matter, only a few exist for hydrogen fluoride. The sensors in these HF instruments have a cross-sensitivity to sulfur dioxide making it difficult to monitor HF in an environment that also contains SO2. To overcome this problem, we assessed the simultaneous use of two electrochemical instruments: one with a SO2 sensor that does not respond to HF and the second with a hydrogen fluoride sensor that responds to both HF and SO2 in a 1 ∶ 1 ratio, termed ‘total acid gas’. The difference in the response between the two instruments should indicate the HF concentration: [HF + SO2] minus SO2 equals HF. The performance characteristics of this sampling train were evaluated in the laboratory through the generation of both HF and SO2 with permeation tubes. The response and recovery times for the SO2 only instrument were acceptable (6 and 15 s, respectively), but the “total acid gas” instrument exhibited both slow response and slow recovery approaching three and six min. The association between the traditional integrated filter sampling method and the direct-reading instrument for SO2 is 0.80 (Spearman’s rho). The use of the digital filter strengthens the association between the HF direct-reading instrument and the integrated samples from 0.41 to 0.68.

The receptor for hepatocyte growth factor (HGF), Met, controls a programme of invasive growth that combines proliferation with various moto- and morphogenetic processes. This process is important for development and organ regeneration, but dysregulation in transformed tissues can contribute to cancer progression and metastasis. Acute stimulation of tissue culture cells with HGF leads to Met downregulation via degradation through an endocytic mechanism that also requires proteasome activity. Perturbation of Met trafficking on the endocytic pathway , either at the level of the internalisation step or during sorting at the early endosome, leads to altered signalling outputs. Ubiquitination of Met through the E3-ligase Cbl is required for receptor downregulation, and a mutant receptor defective in Cbl binding is able to transform cells. We discuss the hypothesis that some naturally occurring Met mutants implicated in cancer may transform cells owing to defects in their trafficking along the endosomal degradation pathway.

Urinary fluoride was evaluated as an exposure index for a prospective study of asthma in an aluminum smelter. Two studies were conducted to evaluate the relationship between airborne exposure and urinary excretion over a workweek, and to describe exposures among jobs and over time. Thirty-two subjects were evaluated on Days 1 and 3 of a 3-day workweek. On each day, spot urine samples were collected prior to the start of work and again at the end of the shift. Samples were analyzed for fluoride and expressed as milligrams fluoride per gram of creatinine. Airborne exposures to total particulate, fluoride particulate, and hydrogen fluoride (HF; using a 37-mm filter cassette containing a filter and treated back-up pad) were also evaluated on each subject. In the second study, postshift urine samples were collected from asthma study volunteers in three surveys extending over 1.5 years and analyzed for fluoride. Average airborne exposures were 15.7, 4.1, and 0.7 mg/m3 for particulates, particulate fluorides and HF, respectively, and were substantially higher among carbon setters than other workers. However, average urine fluorides among the same workers were reasonably low, 1.3 and 3.0 mg/g creatinine in pre- and postshift urine samples, respectively. Carbon setters, who routinely wore respiratory protection during high exposure periods, had urinary fluoride levels similar to those of other potroom personnel. A significant variation in dose, as expressed by postshift urinary fluoride levels, was observed between potroom and nonpotroom jobs and over three survey periods. These results suggest that postshift urinary fluorides provide a reasonable exposure index for surveillance of exposure levels for an epidemiologic study, and that a substantial variation of exposure occurs between jobs and over time. Although urinary fluorides may be used for exposure surveillance, additional details on individual exposure agents and patterns of exposure over time are required for complete assessment.

The study of cell biology via monolayer cell culture systems is not entirely adequate for the investigation of complex inter-cell and cell to extracellular matrix interactions in vitro. Three dimensional (3D) cell culture systems better mimic more complex interactions and are extremely useful in broad applications of cell biology. In human cancer biology, a 3D culture system is better able to simulate an extracellular microenvironment to study tumor cell progression and sensitivity to anticancer agents. However, variability in forming spheres has been a persistent problem. This variability has been linked to medium composition and volume, cell density, duration in culture, and most importantly, the cellular interactions with the culture dish itself. Here, we hypothesize that a hydrophilic polymer coated surface, Thermo Scientific Nunclon Sphera, inhibits cell attachment to the culture dish by blocking binding of the extracellular matrix (ECM). First we demonstrated that the adsorption of ECM proteins (e.g. Collagen I and Fibronectin) to the hydrophilic polymer coated Nunclon Sphera surface is extremely low compared to that of the standard cell culture-treated surface, implying minimal interactions between the hydrophilic polymer and the ECM. We then examined spheroid formation on the hydrophilic polymer surface in comparison to the standard cell culture treated surface. We were able to show that unlike the standard cell culture surface, the hydrophilic polymer surface was able to support spheroid development in suspension with virtually no cell attachment by several cancer cell lines including HeLa, MCF-7, HepG2, Panc-1, Saos-2, and A549. In order to show that the hydrophilic polymer coating has no deleterious effects on cell growth, we conducted spheroid culture of a cancer stem cell line, P19.CL6, in the Nunclon Sphera dishes at relatively low cell seeding densities. Over time, the spheroids of all initial seeding densities were able to grow in volume, indicating that the hydrophilic polymer coating has no adverse effect on cell survival and proliferation. Finally, we demonstrated that the performance of Nunclon Sphera surface in supporting cancer spheroid formation is comparable to that of a similar product by a different manufacturer. Overall, the data supports that the Thermo Scientific Nunclon Sphera provides a consistent and reliable culture system for non-adherent spheroid growth, aiding in three dimensional modeling of cancer cells in vitro. Citation Format: Cindy Neeley, Louise Gaarn, Tina Marwood, Robert Scott, Stephanie Carter, Joseph Granchelli. A novel cell culture surface supports effective formation of three dimensional cancer spheroids in suspension. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 208. doi:10.1158/1538-7445.AM2013-208

Background The plasma renin activity (PRA) assay measures the ability of renin to generate angiotensin I (AngI) from angiotensinogen. It is used to monitor mineralocorticoid therapy and to screen hypertensive individuals for primary aldosteronism (PA). Methods Samples were incubated in the presence of protease inhibitors for 6.5 and 24 h. The reaction was stopped by the addition of 2% ammonium hydroxide. AngI was then quantified by liquid chromatography tandem mass spectrometry using online solid-phase extraction (XLC-MS/MS). Results This method requires a sample volume of 50 μL and has an inter-assay precision 2 = 0.98), but a 37% negative bias. We also found that renin is stable in whole blood for up to 24 h at room temperature. In contrast, storage at 4°C should be avoided as prorenin cryoactivation can affect the PRA result in some patient groups. Conclusions We have developed and fully validated a semi-automated XLC-MS/MS method for the measurement of PRA. In addition, a reference range specific to this assay has been defined. We have also demonstrated that renin is stable for up to 24 h at room temperature. This will enable this assay to be extended to samples taken in primary care, potentially increasing the number of hypertensive patients who can be screened for PA.