Gender diversity in radiology: Shedding light on the 256 shades of grey.

INTRODUCTION: Despite medical school cohorts being composed of approximately 50% men and women, trainee positions in Clinical Radiology remain predominantly occupied by men. This study aims to identify reasons behind the gender gap, explore why more women are not pursuing a career in Clinical Radiology and to assess if there are gender differences in these reasons. METHODS: Prospective multi-centre study using a voluntary, anonymous questionnaire aimed at senior Medical Students and Junior Doctors. RESULTS: A total of 318 participants responded to the questionnaire - 197 women (61.9%), 114 men (35.8%), 4 non-binary (1.3%), 2 preferred not to answer gender (0.6%), 1 I/they (0.3%). The most common reasons for not considering Clinical Radiology as a specialty include 'perceived limited patient contact' (62%), 'never/rarely exposed to it' (59%) and 'too lonely' (49%). Reasons with the largest gender discrepancies were 'too technology heavy' and 'limited patient contact', both cited more frequently by women. Most respondents indicated that their opinions of Clinical Radiology may change with more exposure during medical school, more patient contact and more mentorship. CONCLUSION: This study has revealed multiple reasons why women may not pursue specialty training in Clinical Radiology. Notable differences were found in the reasonings provided by men and women. Limited Clinical Radiology exposure and mentorship in medical school result in a lack of interest in this specialty as a career option. A proposed solution is to offer more medical school lectures and more junior doctor rotations to debunk stereotypes that may be negatively impacting opinions on Clinical Radiology. More mentorship of Medical Students and Junior Doctors, particularly from women, may also help decrease the gender gap.

The PECAn image and statistical analysis pipeline identifies Minute cell competition genes and features.

Investigating organ biology often requires methodologies to induce genetically distinct clones within a living tissue. However, the 3D nature of clones makes sample image analysis challenging and slow, limiting the amount of information that can be extracted manually. Here we develop PECAn, a pipeline for image processing and statistical data analysis of complex multi-genotype 3D images. PECAn includes data handling, machine-learning-enabled segmentation, multivariant statistical analysis, and graph generation. This enables researchers to perform rigorous analyses rapidly and at scale, without requiring programming skills. We demonstrate the power of this pipeline by applying it to the study of Minute cell competition. We find an unappreciated sexual dimorphism in Minute cell growth in competing wing discs and identify, by statistical regression analysis, tissue parameters that model and correlate with competitive death. Furthermore, using PECAn, we identify several genes with a role in cell competition by conducting an RNAi-based screen.

The Gr64 cluster of gustatory receptors promotes survival and proteostasis of epithelial cells in Drosophila.

Gustatory Receptor 64 (Gr64) genes are a cluster of 6 neuronally expressed receptors involved in sweet taste sensation in Drosophila melanogaster. Gr64s modulate calcium signalling and excitatory responses to several different sugars. Here, we discover an unexpected nonneuronal function of Gr64 receptors and show that they promote proteostasis in epithelial cells affected by proteotoxic stress. Using heterozygous mutations in ribosome proteins (Rp), which have recently been shown to induce proteotoxic stress and protein aggregates in cells, we show that Rp/+ cells in Drosophila imaginal discs up-regulate expression of the entire Gr64 cluster and depend on these receptors for survival. We further show that loss of Gr64 in Rp/+ cells exacerbates stress pathway activation and proteotoxic stress by negatively affecting autophagy and proteasome function. This work identifies a noncanonical role in proteostasis maintenance for a family of gustatory receptors known for their function in neuronal sensation.

Xrp1 and Irbp18 trigger a feed-forward loop of proteotoxic stress to induce the loser status.

Cell competition induces the elimination of less-fit "loser" cells by fitter "winner" cells. In Drosophila, cells heterozygous mutant in ribosome genes, Rp/+, known as Minutes, are outcompeted by wild-type cells. Rp/+ cells display proteotoxic stress and the oxidative stress response, which drive the loser status. Minute cell competition also requires the transcription factors Irbp18 and Xrp1, but how these contribute to the loser status is partially understood. Here we provide evidence that initial proteotoxic stress in RpS3/+ cells is Xrp1-independent. However, Xrp1 is sufficient to induce proteotoxic stress in otherwise wild-type cells and is necessary for the high levels of proteotoxic stress found in RpS3/+ cells. Surprisingly, Xrp1 is also induced downstream of proteotoxic stress, and is required for the competitive elimination of cells suffering from proteotoxic stress or overexpressing Nrf2. Our data suggests that a feed-forward loop between Xrp1, proteotoxic stress, and Nrf2 drives Minute cells to become losers.

Proteotoxic stress is a driver of the loser status and cell competition.

Cell competition allows winner cells to eliminate less fit loser cells in tissues. In Minute cell competition, cells with a heterozygous mutation in ribosome genes, such as RpS3+/- cells, are eliminated by wild-type cells. How cells are primed as losers is partially understood and it has been proposed that reduced translation underpins the loser status of ribosome mutant, or Minute, cells. Here, using Drosophila, we show that reduced translation does not cause cell competition. Instead, we identify proteotoxic stress as the underlying cause of the loser status for Minute competition and competition induced by mahjong, an unrelated loser gene. RpS3+/- cells exhibit reduced autophagic and proteasomal flux, accumulate protein aggregates and can be rescued from competition by improving their proteostasis. Conversely, inducing proteotoxic stress is sufficient to turn otherwise wild-type cells into losers. Thus, we propose that tissues may preserve their health through a proteostasis-based mechanism of cell competition and cell selection.

Retromer Controls Planar Polarity Protein Levels and Asymmetric Localization at Intercellular Junctions.

The coordinated polarization of cells in the plane of a tissue, termed planar polarity, is a characteristic feature of epithelial tissues [1]. In the fly wing, trichome positioning is dependent on the core planar polarity proteins adopting asymmetric subcellular localizations at apical junctions, where they form intercellular complexes that link neighboring cells [1-3]. Specifically, the seven-pass transmembrane protein Frizzled and the cytoplasmic proteins Dishevelled and Diego localize to distal cell ends, the four-pass transmembrane protein Strabismus and the cytoplasmic protein Prickle localize proximally, and the seven-pass transmembrane spanning atypical cadherin Flamingo localizes both proximally and distally. To establish asymmetry, these core proteins are sorted from an initially uniform distribution; however, the mechanisms underlying this polarized trafficking remain poorly understood. Here, we describe the identification of retromer, a master controller of endosomal recycling [4-6], as a key component regulating core planar polarity protein localization in Drosophila. Through generation of mutants, we verify that loss of the retromer-associated Snx27 cargo adaptor, but notably not components of the Wash complex, reduces junctional levels of the core proteins Flamingo and Strabismus in the developing wing. We establish that Snx27 directly associates with Flamingo via its C-terminal PDZ binding motif, and we show that Snx27 is essential for normal Flamingo trafficking. We conclude that Wash-independent retromer function and the Snx27 cargo adaptor are important components in the endosomal recycling of Flamingo and Strabismus back to the plasma membrane and thus contribute to the establishment and maintenance of planar polarization.

Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling.

Following endocytosis into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multiprotein complex that orchestrates cargo retrieval and recycling and, importantly, is biochemically and functionally distinct from the established retromer pathway. We have called this complex 'retriever'; it is a heterotrimer composed of DSCR3, C16orf62 and VPS29, and bears striking similarity to retromer. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to CCC (CCDC93, CCDC22, COMMD) and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5ß1 integrin. Through quantitative proteomic analysis, we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, that require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major endosomal retrieval and recycling pathway.

Making, Exporting, and Modulating Wnts.

Wnt proteins activate a conserved signalling pathway that controls development and tissue homeostasis in all metazoans. The intensity of Wnt signalling must be tightly controlled to avoid diseases caused by excess or ectopic signalling. Over the years, many proteins dedicated to Wnt function have been identified, including Porcupine, which appends a palmitoleate moiety that is essential for signalling activity. This lipid inevitably affects subcellular trafficking and solubility, as well as providing a target for post-translational modulation. We review here the life history of Wnts, starting with progression through the secretory pathway, continuing with release and spread in the extracellular space, and finishing with the various proteins that dampen or inactivate Wnts in the extracellular space.

Cholera in the time of war: implications of weak surveillance in Syria for the WHO's preparedness-a comparison of two monitoring systems.

BACKGROUND: Public health breakdown from the Syrian government's targeting of healthcare systems in politically unsympathetic areas has yielded a resurgence of infectious diseases. Suspected cholera recently reappeared but conflict-related constraints impede laboratory confirmation. Given the government's previous under-reporting of infectious outbreaks and the reliance of the WHO on government reporting, we sought to assess the reliability of current surveillance systems. METHODS: We compared weekly surveillance reports of waterborne diseases from the Syrian government's (WHO-associated) Early Warning and Response System (EWARS), based in Damascus, and the independent, non-governmental Early Warning and Response Network (EWARN) headquartered in Gaziantep, Turkey. We compared raw case rates by EWARS and EWARN and assessed the quality of reporting against the WHO benchmarks. RESULTS: We identified significant under-reporting and delays in the government's surveillance. On average, EWARS reports were published 24 days (range 12-61) after the reference week compared with 11 days (5-21) for EWARN. Average completeness for EWARS was 75% (55-84%), compared with 92% for EWARN (85-99%). Average timeliness for EWARS was 79% (51-100%), compared with 88% for EWARN (70-97%). EWARS made limited use of rapid diagnostic tests, and rates of collection of stool samples for laboratory cholera testing were well below reference levels. CONCLUSIONS: In the context of the current Syrian war, the government's surveillance is inadequate due to lack of access to non-government held territory, an incentive to under-report the consequence of government attacks on health infrastructure, and an impractical insistence on laboratory confirmation. These findings should guide the WHO reform for surveillance in conflict zones.

Defining Polio: Closing the Gap in Global Surveillance.

BACKGROUND: By late 2012 the Global Polio Eradication Initiative (GPEI) had nearly eradicated this ancient infectious disease. Successful surveillance programs for acute flaccid paralysis however rely on broad governmental support for implementation. With the onset of conflict, public health breakdown has contributed to the resurgence of polio in a number of regions. The current laboratory based case definition may be a contributory factor in these regions. OBJECTIVE: We sought to compare case definition rates using strict laboratory based criteria to rates obtained using the clinical criteria in modern day Syria. We also sought to examine this distribution of cases by sub-region. METHODS: We examined the World Health Organization (WHO) reported figures for Syria from 2013-2014 using laboratory based criteria. We compared these with cases obtained when clinical criteria were applied. In addition we sought data from the opposition controlled Assistance Coordination Unit which operates in non-Government controlled areas where WHO data maybe incomplete. Cases were carefully examined for potential overlap to avoid double reporting. FINDINGS: Whilst the WHO data clearly confirmed the polio outbreak in Syria, it did so with considerable delay and with under reporting of cases, particularly from non-government controlled areas. In addition, laboratory based case definition led to a substantial underestimate of polio (36 cases) compared with those found with the clinically compatible definition (an additional 46 cases). Rates of adequate diagnostic specimens from suspected cases are well below target, no doubt reflecting the effect of conflict in these areas. CONCLUSIONS: We have identified a gap in the surveillance of polio, a global threat. The current laboratory based definition, in the setting of conflict and insecurity, leads to under diagnosis of polio with potential delays and inadequacies in coordinating effective responses to contain outbreaks and eradicate polio. Breakdown in public health measures as a contributing factor is likely to result in a resurgence of previously controlled infectious diseases. The clinical definition should be reinstituted to supplement the lab-based definition.

Notum deacylates Wnt proteins to suppress signalling activity.

Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase.

Apical deficiency triggers JNK-dependent apoptosis in the embryonic epidermis of Drosophila.

Epithelial homeostasis and the avoidance of diseases such as cancer require the elimination of defective cells by apoptosis. Here, we investigate how loss of apical determinants triggers apoptosis in the embryonic epidermis of Drosophila. Transcriptional profiling and in situ hybridisation show that JNK signalling is upregulated in mutants lacking Crumbs or other apical determinants. This leads to transcriptional activation of the pro-apoptotic gene reaper and to apoptosis. Suppression of JNK signalling by overexpression of Puckered, a feedback inhibitor of the pathway, prevents reaper upregulation and apoptosis. Moreover, removal of endogenous Puckered leads to ectopic reaper expression. Importantly, disruption of the basolateral domain in the embryonic epidermis does not trigger JNK signalling or apoptosis. We suggest that apical, not basolateral, integrity could be intrinsically required for the survival of epithelial cells. In apically deficient embryos, JNK signalling is activated throughout the epidermis. Yet, in the dorsal region, reaper expression is not activated and cells survive. One characteristic of these surviving cells is that they retain discernible adherens junctions despite the apical deficit. We suggest that junctional integrity could restrain the pro-apoptotic influence of JNK signalling.

Drosophila MCRS2 associates with RNA polymerase II complexes to regulate transcription.

Drosophila MCRS2 (dMCRS2; MCRS2/MSP58 and its splice variant MCRS1/p78 in humans) belongs to a family of forkhead-associated (FHA) domain proteins. Whereas human MCRS2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription and cell cycle progression, dMCRS2 has been largely uncharacterized. Recent data show that MCRS2 is purified as part of a complex containing the histone acetyltransferase MOF (males absent on first) in both humans and flies. MOF mediates H4K16 acetylation and regulates the expression of a large number of genes, suggesting that MCRS2 could also have a function in transcription regulation. Here, we show that dMCRS2 copurifies with RNA polymerase II (RNAP II) complexes and localizes to the 5' ends of genes. Moreover, dMCRS2 is required for optimal recruitment of RNAP II to the promoter regions of cyclin genes. In agreement with this, dMCRS2 is required for normal levels of cyclin gene expression. We propose a model whereby dMCRS2 promotes gene transcription by facilitating the recruitment of RNAP II preinitiation complexes (PICs) to the promoter regions of target genes.

The dASPP-dRASSF8 complex regulates cell-cell adhesion during Drosophila retinal morphogenesis.

BACKGROUND: Adherens junctions (AJs) provide structure to epithelial tissues by connecting adjacent cells through homophilic E-cadherin interactions and are linked to the actin cytoskeleton via the intermediate binding proteins beta-catenin and alpha-catenin. Rather than being static structures, AJs are extensively remodeled during development, allowing the cell rearrangements required for morphogenesis. Several "noncore" AJ components have been identified, which modulate AJs to promote this plasticity but are not absolutely required for cell-cell adhesion. RESULTS: We previously identified dASPP as a positive regulator of dCsk (Drosophila C-terminal Src kinase). Here we show that dRASSF8, the Drosophila RASSF8 homolog, binds to dASPP and that this interaction is required for normal dASPP levels. Our genetic and biochemical data suggest that dRASSF8 acts in concert with dASPP to promote dCsk activity. Both proteins specifically localize to AJs and are mutually required for each other's localization. Furthermore, we observed abnormal E-cadherin localization in mutant pupal retinas, correlating with aberrant cellular arrangements. Loss of dCsk or overexpression of Src elicited similar AJ defects. CONCLUSIONS: Because Src is known to regulate AJs in both Drosophila and mammals, we propose that dASPP and dRASSF8 fine tune cell-cell adhesion during development by directing dCsk and Src activity. We show that the dASPP-dRASSF8 interaction is conserved in humans, suggesting that mammalian ASPP1/2 and RASSF8, which are candidate tumor-suppressor genes, restrict the activity of the Src proto-oncogene.

C-reactive protein and interleukin-18 levels in relation to coronary heart disease: prospective cohort study from Busselton Western Australia.

BACKGROUND: Elevated levels of inflammatory markers are associated with incident coronary heart disease (CHD), but it remains controversial whether these markers provide incremental predictive value to conventional risk factors. We investigated the relationship between C-reactive protein (CRP) and interleukin-18 (IL-18) levels and risk of CHD in men and women without initial cardiovascular disease. METHODS: A prospective case-cohort design over the period 1981-2001 involving 253 incident CHD cases and a random sub-cohort of 441 subjects was used. Cox proportional hazards regression was used to estimate the relative risks (RRs) of CHD for continuous and tertiles of CRP and IL-18 after controlling for conventional risk factors. RESULTS: The multivariate-adjusted RR of CHD associated with one unit increase in log CRP in the overall population was 1.29 (1.07, 1.55; trend P=0.008). Men and women in the top compared to bottom third of CRP distribution had an adjusted RR for CHD of 1.65 (1.03-2.65; P=0.036). The multivariate RR for continuous log IL-18 was 1.34 in men, 1.63 in women and 1.36 overall, and none reached statistical significance. CONCLUSIONS: Baseline CRP but not IL-18 levels are independently predictive of future CHD. However CRP provides only modest additional predictive value over conventional risk factors and the benefit of a prevention strategy based on CRP still needs to be established.

Drosophila ASPP regulates C-terminal Src kinase activity.

Src-family kinases (SFKs) control a variety of biological processes, from cell proliferation and differentiation to cytoskeletal rearrangements. Abnormal activation of SFKs has been implicated in a wide variety of cancers and is associated with metastatic behavior (Yeatman, 2004). SFKs are maintained in an inactive state by inhibitory phosphorylation of their C-terminal region by C-terminal Src kinase (Csk). We have identified Drosophila Ankyrin-repeat, SH3-domain, and Proline-rich-region containing Protein (dASPP) as a regulator of Drosophila Csk (dCsk) activity. dASPP is the homolog of the mammalian ASPP proteins, which are known to bind to and stimulate the proapoptotic function of p53. We show that dASPP is a positive regulator of dCsk. First, dASPP loss-of-function strongly enhances the specific phenotypes of dCsk mutants in wing epithelial cells. Second, dASPP interacts physically with dCsk to potentiate the inhibitory phosphorylation of Drosophila Src (dSrc). Our results suggest a role for dASPP in maintaining epithelial integrity through dCsk regulation.