Skin colour changes during experimentally-induced sickness

This project was supported by Swedish foundation for humanities and social sciences and a British Academy Wolfson Foundation Research Professorship grant. AH is supported by a studentship from the Biotechnology and Biological Sciences Research Council.Skin colour may be an important cue to detect sickness in humans but how skin colour changes with acute sickness is currently unknown. To determine possible colour changes, 22 healthy Caucasian participants were injected twice, once with lipopolysaccharide (LPS, at a dose of 2 ng/kg body weight) and once with placebo (saline), in a randomised cross-over design study. Skin colour across 3 arm and 3 face locations was recorded spectrophotometrically over a period of 8 hours in terms of lightness (L∗), redness (a∗) and yellowness (b∗) in a manner that is consistent with human colour perception. In addition, carotenoid status was assessed as we predicted that a decrease it skin yellowness would reflect a drop in skin carotenoids. We found an early change in skin colouration 1-3 hours post LPS injection with facial skin becoming lighter and less red whilst arm skin become darker but also less red and less yellow. The LPS injection also caused a drop in plasma carotenoids from 3 hours onwards. However, the timing of the carotenoid changes was not consistent with the skin colour changes suggesting that other mechanisms, such as a reduction of blood perfusion, oxygenation or composition. This is the first experimental study characterising skin colour associated with acute illness, and shows that changes occur early in the development of the sickness response. Colour changes may serve as a cue to health, prompting actions from others in terms of care-giving or disease avoidance. Specific mechanisms underlying these colour changes require further investigation.PostprintPeer reviewe

Lasing within live cells containing intracellular optical microresonators for barcode-type cell tagging and tracking

This work was supported by the European Union Marie Curie Career Integration Grant (PCIG12-GA-2012-334407) and the Scottish Funding Council (SUPA II). M.S. acknowledges funding by the German Science Foundation (DFG) through a Research Fellowship (SCHU 3003/1-1).We report on a laser that is fully embedded into a single live cell. By harnessing natural endocytosis of the cell we introduce a fluorescent whispering gallery mode (WGM) micro-resonator into the cell cytoplasm. On pumping with nanojoule light pulses, green laser emission is generated inside the cells. Our approach can be applied to different cell types and cells with micro-resonators remain viable for weeks under standard conditions. The characteristics of the lasing spectrum provide each cell with a barcode-type label which enables uniquely identifying and tracking of individual migrating cells. Self-sustained lasing from cells paves the way to new forms of cell tracking, intracellular sensing and adaptive imaging.PostprintPeer reviewe

An automated framework of inner segment/outer segment defect detection for retinal SD-OCT images

The integrity of inner segment/outer segment (IS/OS) has high correlation with lower visual acuity in patients suffering from blunt trauma. An automated 3D IS/OS defect detection method based on the SD-OCT images was proposed. First, 11 surfaces were automatically segmented using the multiscale 3D graph-search approach. Second, the sub-volumes between surface 7 and 8 containing IS/OS region around the fovea (diameter of mm) were extracted and flattened based on the segmented retinal pigment epithelium layer. Third, 5 kinds of texture based features were extracted for each voxel. A KNN classifier was trained and each voxel was classified as disrupted or nondisrupted and the responding defect volume was calculated. The proposed method was trained and tested on 9 eyes from 9 trauma subjects using the leave-one-out cross validation method. The preliminary results demonstrated the feasibility and efficiency of the proposed method

Tumour-associated antigenic peptides are present in the HLA class I ligandome of cancer cell line derived extracellular vesicles

Funding: Breast Cancer Now (Grant Number(s): 2018JulPR1086), Wellcome Trust (GrantNumber(s): 105621/Z/14/Z), Melville Charitable Trust.The recent success of monoclonal antibody checkpoint inhibitor therapies that enhance the ability of CD8+ T cells to detect cancer-related antigenic peptides has refocused the need to fully understand the repertoire of peptides being presented to the immune system. Whilst the peptide ligandome presented by cell surface human leucocyte antigen class I (HLA-I) molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles (EVs) remains poorly defined. Here we report the HLA-I ligandome of both the cell surface and EVs from eight breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-453, HCC 1806, HCC 1395, and HCC 1954), and additionally the melanoma cell line ESTDAB-056 and the multiple myeloma line RPMI 8226. Utilising HLA-I immunoisolation and mass spectrometry, we detected a total of 6574 peptides from the cell surface and 2461 peptides from the EVs of the cell lines studied. Within the EV HLA-I ligandome, we identified 150 peptides derived from tumour associated antigenic proteins, of which 19 peptides have been shown to elicit T cell responses in previous studies. Our data thus shows the prevalence of clinically relevant tumour-associated antigenic peptides in the HLA-I ligandome presented on EV.Publisher PDFPeer reviewe

Quantitative proteomic changes in LPS-activated monocyte-derived dendritic cells : a SWATH-MS study

We would like to thank Fiona Cooke for her help with collection of blood samples. We wish to thank the Wellcome Trust for funding the purchase of the TripleTOF 5600+ mass spectrometer (grant number 094476/Z/10/Z) and their Institutional Strategic Support Fund (grant number 097831/Z/11/Z) for funding a PhD studentship (to D.W.-M.). This work was also supported by Arthritis Research UK (grant number 21261).Dendritic cells are key immune cells that respond to pathogens and co-ordinate many innate and adaptive immune responses. Quantitative mass spectrometry using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) was performed here to determine the global alterations in monocyte-derived dendritic cells (moDCs) in response to stimulation with lipopolysaccharide (LPS). A moDC library of 4,666 proteins was generated and proteins were quantified at 0, 6 and 24 h post-LPS stimulation using SWATH-MS. At 6 h and 24 h post-LPS exposure, the relative abundance of 227 and 282 proteins was statistically significantly altered (p-value≤0.05), respectively. Functional annotation of proteins exhibiting significant changes in expression between the various time points led to the identification of clusters of proteins implicated in distinct cellular processes including interferon and interleukin signalling, endocytosis, the ER-phagosome pathway and antigen-presentation. Major histocompatibility complex (MHC) class I proteins were highly upregulated at 24 h, in SWATH-MS, whilst MHC class II proteins exhibited comparatively less change over this period. This study provides new detailed insight into the global proteomic changes that occur in moDCs during antigen processing and presentation and further demonstrates the potential of SWATH-MS for the quantitative study of proteins involved in cellular processes.Publisher PDFPeer reviewe

Salmonella exploits HLA-B27 and host unfolded protein responses to promote intracellular replication

A.N.A was funded by ARUK Fellowships Non-Clinical Career Development Fellowship Ref No: 18440. I.L. was funded by an ARUK PhD studentship Ref No: 17868. A.N.A and S.J.P were also in part funded by ARUK (grant 21261)Objective Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells. Methods Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S. enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane. Results S. enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism. Conclusions HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention.Publisher PDFPeer reviewe

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men