The structural investigation of biological macromolecules is indispensable in understanding the molecular mechanisms underlying diseases. Several structural biology techniques have been introduced to unravel the structural facets of biomolecules. Among these, the electron cryomicroscopy (cryo-EM) method microcrystal electron diffraction (MicroED) has produced atomic resolution structures of important biological and small molecules. Since its inception in 2013, MicroED established a demonstrated ability for solving structures of difficult samples using vanishingly small crystals. However, membrane proteins remain the next big frontier for MicroED. The intrinsic properties of membrane proteins necessitate improved sample handling and imaging techniques to be developed and optimized for MicroED. Here, we summarize the milestones of electron crystallography of two-dimensional crystals leading to MicroED of three-dimensional crystals. Then, we focus on four different membrane protein families and discuss representatives from each family solved by MicroED. Microcrystal electron diffraction (MicroED) is a structural determination technique that integrates the two methodological pillars of structural biology — crystallography and electron microscopy (EM) ,2. As of− Å−2) of electron beam while the crystal is continuously rotated. As the crystal is rotated, MicroED data are collected as a movie using a fast camera as a continuous series of frames that finely sample the reciprocal space. Because continuous rotation MicroED is analogous to the rotation method in X-ray crystallography [For MicroED, microcrystals are prepared by standard crystallization procedures . Once crlography  data prolography ,5,6  have been extremely successful. With the bicelle method, aqueous lipid-detergent assemblies form bilayered disks ,30,34 thSome membrane proteins that are abundantly expressed in cells naturally form ordered arrays within the plasma membrane. Classic examples of naturally occurring arrays are the hexagonal lattice of bacteriorhodopsin (bR)  and the When naturally occurring 2D arrays are not available, membrane proteins can be crystallized in membranes by slow dialysis. Membrane protein 2D crystals are produced from detergent-solubilized protein dialyzed into a lipidic environment. Slow removal of detergent molecules in the presence of detergent-solubilized lipids allows the formation of vesicles, tubules and/or sheets into which the membrane proteins insert and densely pack into 2D arrays . PerfectBecause many membrane protein 2D crystals tend to stack and naturally pack into thin 3D crystals it became important to develop a robust method capable of extracting the necessary information for structure solution from such thin crystals. In 2013, the method MicroED was developed for exactly this purpose. Lysozyme, which is an excellent soluble protein test sample was the first protein structure solved by electron diffraction of multi-layered (about 10 layers) 3D crystals  and with2+-ATPase and the non-selective ion channel NaK [2+-ATPase and the sodium-conducting channel NaK.Soon after Shi et al.  publishennel NaK  which de2+-ATPase is a P-type ATPase capable of pumping Ca2+ into the sarcoendoplasmic reticulum lumen. The detailed mechanism on the transport cycle of Ca2+-ATPase has been reported through biochemical and structural studies. In 2000, the first atomic resolution structure of Ca2+-ATPase has been solved by X-ray crystallography at 2.6 Å [2+-ATPase has eluded structural determination because it can only form tubular crystals in the absence of Ca2+ and 3D microcrystals in the presence of millimolar Ca2+ [2+-ATPase was determined and charge properties described. MicroED coulomb potential maps revealed pronounced details of the two Ca2+ binding sites, including charged states of acidic residues Asp800 and Glu908 [+ was bound at the entrance of the selectivity filter shedding light to how Na+ enters the channel  receptor [Recently, two new studies reported the use of MicroED in LCP- and bicelle-crystallized membrane proteins ,55. As mreceptor . This alreceptor . This apFIB milling has been more successful in thinning out membrane protein crystals, and removing thick lipid deposits around crystals before MicroED analyses. 2A receptor (A2AAR) was successfully determined by MicroED directly from nanocrystals grown in LCP. By converting the LCP matrix into the sponge phase and cryo-FIB milling the crystals into 200-nm thick lamellae, the structure of A2AAR was solved at 2.8 Å from a single nanocrystal [More than 800 different GPCRs exist in humans and these proteins are the largest pharmaceutical targets ,63. Tradocrystal  (Figure ocrystal ,67. ThroMicroED unlocks new strategies for membrane protein structural biology. Since 2013, four membrane protein structures have been reported by the method in which, regardless of the crystallization technique, high-resolution structures can be attained. MicroED's ability to obtain structures from tiny and single crystals abolishes limitations imposed by the intrinsic properties of membrane proteins especially those that crystallize poorly and purify in low quantities. Through Coulomb potential maps, MicroED provides unambiguous assignment of charges of ions and residues found within the protein that can provide meaningful detail into mechanisms of transporters and channels. Moreover, the combination of LCP and MicroED allows for the investigation of lipid interactions vital for membrane protein structures, carried out through the application of FIB milling. MicroED, in combination with LCP crystallography and Coulomb potential maps, prove to be a powerful technique that delivers high-resolution structures and details underlying molecular mechanisms of membrane proteins. Together with other cryo-EM modalities, MicroED promises to transform the field of structural biology.MicroED can deliver atomic resolution structures from vanishingly small crystals. This cryo-EM method is gaining momentum and becoming an important player in determining structures of membrane proteins embedded in a lipid matrix.By using MicroED structures of important material can be determined and charge properties probed in a way which is not possible by X-ray crystallography.Now that methods for studying membrane proteins in lipids have been developed and demonstrated the path is clear for examining samples that were thus far beyond reach.


Accumulation of lipid droplets (LDs) was recently observed in pyometra-affected uteri. As data about their nature and function are missing we intended to compare the localization, quality and quantity of LDs in canine healthy and pyometra-affected tissues and in an in vitro model.We characterized LDs in healthy and pyometra uterine tissue samples as well as in canine endometrial epithelial cells (CEECs) in vitro by means of histochemistry, immunohistochemistry, transmission electron microscopy, western blot, and RT-qPCR. Oil Red O (ORO) staining and quantification as well as p-phenylenediamine staining showed a higher number of LDs in epithelial cells of pyometra samples. Immunohistochemistry revealed that the amount of LDs coated by perilipin2 (PLIN2) protein was also higher in pyometra samples. Transmission electron microscopy showed an increase of LD size in surface and glandular epithelial cells of pyometra samples. In cell culture experiments with CEECs, supplementation with oleic acid alone or in combination with cholesterol lead to an increased LD accumulation. The expression of PLIN2 at protein and mRNA level was also higher upon oleic acid supplementation. Most LDs were double positive for ORO and PLIN2. However, ORO positive LDs lacking PLIN2 coating or LDs positive for PLIN2 but containing a lipid class not detectable by ORO staining were identified.We found differences in the healthy and pyometra-affected endometrium with respect to LDs size. Moreover, several kinds of LDs seem to be present in the canine endometrium. In vitro studies with CEECs could show their responsiveness to external lipids. Since epithelial cells reacted only to oleic acid stimulation, we assume that the cyclic lipid accumulation in the canine endometrium is based mainly on triglycerides and might serve as energy provision for the developing early embryo. Further studies are necessary to verify the complex role of lipids in the healthy and pyometra-affected canine endometrium.The online version contains supplementary material available at 10.1186/s12917-022-03321-5. The canine endometrium undergoes massive characteristic morphological changes during each sexual cycle. The remodeling includes the connective tissue of the endometrial stroma and also the surface, crypt, and glandular epithelium. One astonishing phenomenon is the accumulation of lipid droplets (LDs) in the surface and crypt epithelium of the canine endometrium during the luteal phase of the sexual cycle. This aggregation of LDs in the canine endometrium has been reported before –3; howevIn the pyometra affected uterus, a characteristic lipid storage pattern has been observed also in the surface and crypt epithelium . PyometrLipid droplet accumulation in epithelial cells has formerly often been interpreted as a sign of degeneration. Meanwhile, it is clear that LDs are not only storage compartments for different classes of lipids in a cell, but they have also been identified as important dynamic cellular organelles and signal exchange platforms with key functions in lipid and energy homeostasis . TherefoOur objective was to investigate the localization, quality and quantity of LDs in healthy and pyometra tissue samples to better understand their role the pathophysiology of pyometra in the dog. Futhermore, we assessed the expression of PLIN proteins  on the protein and mRNA level to analyze their association with LDs. As we hypothesize that steroid hormones might be involved in the formation and accumulation of lipids in the canine endometrium, we conducted an experimental in vitro approach to study the potential of endometrial epithelial cells to response to external lipids along with steroid hormone stimulation to mimic sexual cycle conditions. This knowledge might help to elucidate the nature and function of LDs in the canine endometrium, which could be involved in both, physiological and pathophysiological processes.In brief, LDs were detected in uterine tissue samples from healthy and pyometra-affected bitches by means of histochemical methods (Oil Red O and/or p-phenylenediamine staining) and additionally characterized by the immunohistochemical detection of PLIN1, PLIN2, and PLIN3 as well as size distribution analysis using transmission electron microscopy. Canine endometrial epithelial cells in vitro models were used to further study the LDs biology under external lipid and hormone supplementation, focusing on LDs amount and interaction with lipid droplet coating proteins PLIN1–3.n = 21; age from 9mo to 9y, documented body weight from 4.7 kg to 32,3 kg) and pyometra-affected bitches  of different breeds were used. Pyometra cases  were determined as hyperplastic with severe inflammation, many/large cysts, increased endometrium/myometrium ratio and fibroblastic proliferation [All tissue samples were withdrawn from the archive of the VetBiobank/VetCore Facility for Research of the VetMedUni Vienna, Austria . Archiveferation . Samplesferation .Oil Red O (ORO) staining was applied on cryosections of healthy and pyometra affected endometrial cases for general lipid demonstration. Oil Red O stock solution was made by dissolving 0.5 g of ORO substance  in 100 ml of 2-propanol . Working solution was prepared by diluting the stock solution with distilled water in a ratio of 6:4, 24 hours before the staining procedure. Cryosections were formaldehyde fixed , rinsed with distilled water, incubated in 60% 2-propanol (5 min at room temperature) followed by staining in ORO working solution (10 min at room temperature). Afterwards, samples were briefly rinsed in 60% 2-propanol  and submerged into distilled water for 3 min. Cells were counterstained with hematoxylin solution for 3 min  followed by the blueing step in tap water. Finally, coverslips were mounted using Aquatex solution .For frozen tissue sections a semi-quantitative estimation score  was applied to evaluate the amount of lipid within the surface and glandular epithelium.n = 3) and pyometra affected canine endometria (n = 3) were fixed in 3% buffered glutaraldehyde . After washing in 0.1 M phosphate buffer  samples were postfixed in 1% osmium tetroxide  for 2 hours at room temperature. Afterwards, samples were embedded in epoxy-resin (Serva). Semi-thin sections were cut at 0.8 μm, stained with toluidine blue (Merck) for general histological evaluation. Ultrathin sections were prepared (70 nm) and contrasted with uranyl acetate  and lead citrate (Merck), and evaluated by a transmission electron microscope . Images from surface and glandular epithelium were made to assess the amount of LDs (LDs/nuclei) and to measure LDs diameter .Samples of healthy (Semi-thin tissue sections (0.8 μm) of osmicated, resin-embedded tissue - as prepared for transmission electron microscopy (see above) - were used for lipid assessment with p-phenylenediamine (PPD). PPD staining is optimally applicable to identify lipids by light microscopy on semi-thin sections . This stKi67 immunostaining was used to confirm the luteal stage of the endometrial paraffin sections of healthy endometria. Immunostaining for PLINs  was applied for LD analysis in both, healthy and pyometra-affected canine endometrial FFPE samples.2O2 for 60 min, followed by a washing step in distilled water. Antigen retrieval was done by heating sections in 0.01 M citrate buffer, pH 6. Afterwards, sections were incubated in 10% normal goat serum (Sigma Aldrich) for 60 min to minimize unspecific binding, followed by the incubation with the primary antibody .Western blots were applied to verify the specificity of the antibodies used for canine tissue. Frozen tissue samples were cut into small pieces, further disrupted and homogenised using a dounce homogenizer in RIPA lysis buffer , 1% Nonidet P-40 , 0.1% sodium dodecyl sulfate (Serva) supplemented with 1% (v/v) protease and phosphatase inhibitors . Lysates were then incubated on ice for 30 min and vortexed occasionally. To shred the DNA, lysates were pushed through a 20-G needle several times followed by centrifugation for 15 min at 4 °C using 10,621 x g. The soluble supernatant fraction was stored at − 80 °C until further analysis. Protein concentration was measured using DC™ Protein Assay  according to the manufacturer’s recommendations. Protein extracts (20 μg protein/lane) were separated on 10% polyacrylamide minigels for SDS-PAGE and analyzed as described previously . The pri2 at 37 °C. Cells were regularly passaged twice a week using trypsin solution  with a usual split ratio 1:4.Canine endometrial epithelial cells (cEECs)  were gro4 cells per well of a 24-well plate  with or without glass coverslips . Alternatively, the same amount of cells was seeded per well of the 4-well glass chamberslide . The next day, treatment with oleic acid , cholesterol , or a combination thereof and solvent control ethanol  was performed for 24 hours. For hormone treatment experiments, cells were cultured in the presence of progesterone , 17β-estradiol , oleic acid  or a combination thereof for 24 hours. Afterwards, cells were washed in PBS and fixed with 4% neutral buffered formaldehyde (10 min at room temperature). After subsequent washing with water, cells grown on a glass coverslips were either air dried and stored for further histological analyses or subjected immediately to ORO staining. Cells grown in wells without coverslips were used for ORO staining followed by spectrophotometric quantification immediately. For quantitative analyses, data obtained from four independent experiments are shown.For lipid and/or steroid hormone treatment, cells were seeded at a concentration of 4 × 10To stain cultured cells in wells, fixed cells were incubated with 60% 2-propanol solution (250 μl/well for 5 min) followed by replacement with 250 μl of ORO (Serva) working solution for 10 min. After staining, wells were briefly washed first with 500 μl of 60% 2-propanol solution followed by water washing step. Staining of cells grown on glass coverslips was done as described above, doing all staining and washing steps in a glass cuvette. After staining, coverslips were mounted to the glass slides upside down using Aquatex (Merck).Image analysis for quantitative assessment of LDs in cultured canine endometrial cells after ORO staining was done with the open source software FIJI  and a seFurther quantification of the ORO staining signal in canine endometrial epithelial cells was performed as described previously . BrieflyCanine endometrial epithelial cells were grown on either glass coverslips or chamber slides as described above, fixed in 4% neutral buffered formaldehyde for 10 min at room temperature, washed in distilled water and air-dried. For permeabilization, cells on coverslips/chamber slides were incubated in 0.2% TritonX-100 (Merck) in PBS for 15 min at 4 °C, followed by a washing step in PBS and incubation with the primary antibody overnight. Signal of the secondary HRP labelled antibody was detected using Invitrogen™ Alexa Fluor™ 488 Tyramide-Reagent  according to manufacturer’s guidelines, nuclei were counterstained using DAPI  and mounted with coverslip and Aqua Poly MountTM .Image analysis for quantitative assessment of PLIN2 immunostained LDs in cultured canine endometrial cells after ORO staining were done as described above (ORO staining) with the open source software FIJI and a self-designed macro plugin.As ORO is known to have a red fluorescence emission  double lFASN), synaptosome-associated protein 23 (SNAP23), acetyl-CoA carboxylase 1 (ACACA), fatty acid binding protein 4 (FABP4) as well as genes for PLIN1, PLIN2, and PLIN3. Canine endometrial epithelial cells were lysed in TRI Reagent  and stored at − 80 °C until further processing. RNA extraction and DNase I treatment were done with the Direct-zol RNA Miniprep Kit (Zymo Research) according to the manufacturer’s instructions. Reverse transcription (RT) was performed with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher). No-RT controls (without RT enzyme) were included for each sample to monitor the amplification of contaminating DNA. Primer for qPCR (Table OAZ1), ribosomal protein L27 (RPL27), ribosomal protein L32 (RPL32) and ribosomal protein L8 (RPL8) were included for normalization. The RG expression stability was assessed with the RefFinder tool [RPL27 and RPL32 were selected for normalization. Target gene Cq values were normalized to the mean of the selected RGs and relative fold changes were calculated with the comparative 2-ΔΔCT method [We analyzed the mRNA expression levels of genes involved in the fat metabolism encoding for fatty acid synthase  were conducted with GraphPad Prism 8.4.3 . A The sexual cycle stage of healthy uterine tissue sections was determined by Ki67 immunostaining pattern. As characteristic for luteal stage, proliferative activity was generally low; Ki67 immunohistochemistry resulted in a characteristic staining pattern with only scattered positive cells in the surface epithelium, superficial glands, and stroma. Ki67 immunostaining was absent in basal glands. All pyometra samples had a high proliferative activity within the stroma (inflammatory cells) and scattered positive cells in the surface and crypt epithelium (not shown). To visualize the general amount of LDs in the healthy and diseased endometrium, we applied ORO histochemistry on frozen tissue sections. Scoring of ORO staining in tissue sections of healthy canine endometrium on average revealed a moderate amount of LDs in the surface epithelium whereas the glandular epithelium was almost free of LDs Fig. . PyometrImmunohistochemistry for different members of the PLIN family  intended to specify the LD coating and structure in the canine endometrium. Although all PLIN proteins are somehow involved in LD formation and/or LD coating, their distribution in the canine endometrium varied greatly. PLIN1 was negative in all endometrial structures, but stained lipid vacuoles in unilocular fat cells that were present occasionally in the perimetrium (not shown). Therefore, quantification as initially intended for the epithelial endometrial cells was not possible for PLIN1.PLIN2 immunostaining clearly marked the membrane of LDs. Amount and distribution of PLIN2-positive LDs in the surface epithelium was comparable with ORO staining. In contrast, a high number of PLIN2 positive LDs was also observed in the endometrial glandular epithelium, whereas ORO staining was absent in most endometrial glands. In pyometra specimens, the amount of PLIN2-positive LDs was generally higher compared to healthy samples Fig. . PLIN3 wWestern blot detection of PLIN2 and PLIN3 protein was performed in whole tissue extracts prepared from healthy and diseased canine uteri. Specific bands for PLIN2 (50 kDa) and PLIN3 (40 kDa) were observed with different intensities among the tested samples as well as in corresponding positive controls.PPD staining of resin semi-thin sections showed distinguishable LDs as marked by different dyeing. Large, light brown, homogenous LDs were observed in the surface epithelium of healthy samples, whereas only scattered tiny, dark stained LDs were seen in the glands. LDs in pyometra samples were generally larger and stained darker compared to LDs in healthy samples Fig. . TransmiORO staining was used to qualitatively and quantitatively assess LDs in canine endometrial epithelial cells grown in culture medium with and without lipid supplementation to determine processing of external lipids by these cells. Cells grown without lipid supplementation (not treated and ethanol diluent supplemented groups) revealed only a limited and comparable amount of LDs of relatively homogeneous size, distributed irregularly within the cell. However, a significant rise of LDs was observed in cells treated with oleic acid alone or in combination with cholesterol Fig. A. Cell cTo quantify the intensity of the ORO staining signal, image analysis as well as spectrophotometric quantification was performed. Using image analysis, quantitative data represent the percentage of area of ORO signal calculated out of the whole area covered with cells. A significant rise in positive signals was observed when comparing not treated groups or ethanol diluent supplemented groups to cells supplemented with oleic acid. Supplementation with a combination of both oleic acid and cholesterol did not yield a further increase in signal, whereas supplementation with cholesterol alone had no effect Fig. B. SimilaDue to the partly differential PLIN2 and ORO staining patterns in tissue sections and cell cultures, we applied a double labelling method combining immunofluorescence for PLIN2 and histochemical ORO staining to elucidate if both markers are concomitantly present on/in LDs in vitro. Multiplex-labelled cell cultures of canine endometrial epithelial cells revealed the presence of ORO and PLIN2 single positive droplet-like structures as well as co-labelled LDs independent of treatment Fig. .Fig. 5MuTransmission electron microscopy of cultured canine endometrial epithelial cells revealed a moderate number of LDs in untreated cells, after supplementation with oleic acid 56.5 μg/ml) or/and cholesterol (50 μg/ml) the number of LD increased significantly. LDs were frequently framed by glycogen , providing fatty acids for prostaglandins, protection against lipotoxicity, facilitating coordination and communication between intracellular organelles, and many others . LDs inThe incidence of lipids in the endometrium – especially in conjunction with the development of a corpus luteum - has early been observed . Their pThe aim of the present study was to characterize the LDs in the epithelial cells of the canine endometrium by classical histochemical lipid staining (ORO) but also by means of immunohistochemistry, identifying LD coating proteins (PLINs), as these have been found to be predominantly associated with LDs in certain cell types (reviewed in ). ORO stIn the present study members of the mammalian PLIN family were assessed by immunohistochemistry in healthy and pyometra affected tissue sections. Ki67 immunostaining patterns suggestl that in canine pyometra affected endometrium the characteristic cyclic patterns are lost, as has been described before ; this haTo enable the investigation of the responsiveness of canine endometrial cells to external lipid sources and thereby the dynamics of LD formation, we made an experimental in vitro approach by using primary canine endometrial cells . In lineAs LD accumulation peaks in luteal phase, we assessed a potential hormonal regulation of the lipid processing. Other authors , 39, 49 FASN) mRNA was previously detected in the human endometrium [FASN mRNA concentrations were higher in biopsies removed during the luteal phase than the follicular phase of the menstrual cycle [FASN mRNA levels were observed in white adipose tissue after treatment with trilactic glyceride [FASN mRNA levels in canine endometrial epithelial cells. The synaptosomal-associated protein 23 (SNAP23) is involved in LD formation and fusion as mentioned above [SNAP23 mRNA levels in treated cells. Surprisingly, similar as for FASN, no significant changes for SNAP23 were observed among the tested samples. The fatty acid binding protein 4 (FABP4) was found to be expressed by epithelial cells of the proliferative endometrium, as well as epithelial and stromal cells of secretory endometrium [FABP4 has an important role in the establishment and maintenance of pregnancy, with decreased expression of the protein in the endometrium possibly linked to pregnancy loss [FABP4 levels were remarkably increased under high ω-3 PUFA exposure [ACACA) in uterine tissue or cells have been presented up to the publication date of the current study. However, treatment of primary hepatocytes with cholesterol increased the mRNA level of ACACA and FASN [FASN, SNAP23, ACACA, FABP4) were not changed after external lipid stimulation of canine endometrial epithelial cells.Next to the detection of LD associated proteins we investigated mRNA levels of several genes involved in LD formation and lipid metabolism. Fatty acid synthase (ometrium . Its mRNal cycle . Enhancelyceride . In our ed above –54 as weed above . Our datometrium . FABP4 hncy loss . FABP4 lexposure , howeverand FASN . This isIn the frame of this investigation we intended to promote the analysis of LDs in the canine endometrium as they have not gained much attention until now. However, due to their vast presence in the uterus, specific functions during the reproductive process are likely. We found differences in the healthy and pyometra-affected endometrium and could show the responsiveness of endometrial epithelial cells to external lipids. Moreover, several kinds of LDs seem to be present in the canine endometrium. In conclusion, LDs were found in endometrial surface and glandular epithelium in both healthy and pyometra-affected bitches. We assume that LDs in the canine uterus mainly contains triglycerides and therefore in general provide energy for the developing embryo. A clear increase in LD size was observed in samples from pyometra-affected bitches, in both evaluated epithelial regions. Therefore, we speculate that LDs might be involved in the pathophysiological processes of pyometra. Limiting factors of our study are the relative low number of cases and the restriction of one region of the uterus which was due to availability of fresh frozen material. Nevertheless, we hope that with this study we created a focal point that encourages further groups to join the lipid research in reproduction.Additional file 1.


The IO Score is a 27-gene immuno-oncology (IO) classifier that has previously predicted benefit to immune checkpoint inhibitor (ICI) therapy in triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). It generates both a continuous score and a binary result using a defined threshold that is conserved between breast and lung. Herein, we aimed to evaluate the IO Score’s binary threshold in ICI-naïve TCGA bladder cancer patients (TCGA-BLCA) and assess its clinical utility in metastatic urothelial cancer (mUC) using the IMvigor210 clinical trial treated with the ICI, atezolizumab.We identified a list of tumor immune microenvironment (TIME) related genes expressed across the TCGA breast, lung squamous and lung adenocarcinoma cohorts  and then examined the expression of these 939 genes in TCGA-BLCA, to identify patients as having high inflammatory gene expression. Using this as a test of classification, we assessed the previously established threshold of IO Score. We then evaluated the IO Score with this threshold in the IMvigor210 cohort for its association with overall survival (OS).In TCGA-BLCA, IO Score positive patients had a strong concordance with high inflammatory gene expression (p < 0.0001). Given this concordance, we applied the IO Score to the ICI treated IMvigor210 patients. IO Score positive patients (40%) had a significant Cox proportional hazard ratio (HR) of 0.59 (95% CI 0.45–0.78 p < 0.001) for OS and improved median OS (15.6 versus 7.5 months) compared to IO Score negative patients. The IO Score remained significant in bivariate models combined with all other clinical factors and biomarkers, including PD-L1 protein expression and tumor mutational burden.The IMvigor210 results demonstrate the potential for the IO Score as a clinically useful biomarker in mUC. As this is the third tumor type assessed using the same algorithm and threshold, the IO Score may be a promising candidate as a tissue agnostic marker of ICI clinical benefit. The concordance between IO Score and inflammatory gene expression suggests that the classifier is capturing common features of the TIME across cancer types.The online version contains supplementary material available at 10.1186/s12967-022-03563-9. Immune checkpoint inhibitors targeting programmed cell death 1 or its ligand, programmed cell death 1 ligand 1,  are active in many different tumor types and may modulate the immune response by impacting a common immune regulatory pathway. To date, observed benefit across tumor types has been modest despite co-development of candidate biomarkers for identifying responders to ICI therapy, including immunohistochemistry (IHC) for PD-L1 and tumor mutation burden (TMB). These biomarkers are limited in their value as predictive markers due to inconsistent scoring methods and differing thresholds for positivity, often specifically tailored to tumor types or even clinical studies . AdditioAtezolizumab was the first FDA approved ICI in mUC based on the initial results of IMvigor210, a non-randomized Phase II clinical trial involving two different cohorts of mUC. Cohort I NCT02951767) was comprised of 119 patients who were ineligible to receive cisplatin 67 was co. Based oThe IO Score has been validated as a biomarker that identifies patients likely to benefit from ICIs in TNBC and NSCLC –15. ThesWhile the IO Score was developed in TNBC, its three components are not unique to TNBC but collectively measure ubiquitous features across tumors of epithelial origin. Given that urothelial carcinoma is likewise of epithelial origin, we sought to test the clinical utility of the IO Score to identify patients who benefit from the administration of ICIs in bladder cancer. We first evaluated the predefined IO Score threshold using the IM, MSL, and M subtypes to classify ICI naïve samples from TCGA-BLCA. Using the predefined threshold for IO Score we then tested its association with treatment outcome in patients from the IMvigor210 study. Finally, we compared these results to the previously published extensive exploratory analysis of candidate biomarkers, biomarker signatures, and clinical factors . T. T36]. TThe IMvigor210 combined platinum resistant and platinum contraindicated patients for a total of 429 subjects of which 348 patients were successfully RNA sequenced [Given the failure of the IMvigor211 to reach the primary endpoint of improved OS in IC2/3 patients, we performed an exploratory analysis to determine if IC2/3 positive patients who were also IO Score positive has better OS Fig. C. These In order to test whether IO Score was quantitatively related to objective response, the continuous IO Score was plotted against the four objective response categories  . This wiThe demonstrated clinical utility of the IO Score may be due to the unsupervised clustering performed to first identify robust classifiers before proceeding to building the algorithm. This approach to biomarker development is similar to the approach taken by Perou and colleagues who demonstrated the use of hierarchical clustering of gene expression data to identify likely responders to endocrine therapy within hormone positive breast cancer. Their work led to the development of a targeted biomarker panel, PAM50 , now an Given our prior approach and published data in TNBC and NSCLC, we believe that by assessing the IO Score as a classifier of the TIME in ICI naïve patients, we can better inform clinical decision making for mUC patients due to the IO Score’s strong association with response to ICI therapy in multiple tumor types. Future studies in both bladder cancers and additional tumor types using randomized clinical trial samples are warranted.Additional file 1: Figure S1. Average score for the continuous IO Score by response The IO Score was significant by trend , complete response, objective response, and disease control rate . CR = complete response, PR = partial response, SD = stable disease, and PD = progressive disease. Objective Response is CR or PR versus SD or PD and Disease Control is CR, PR, or SD versus PD. **p≤0.01, ***p≤0.001.Additional file 2: Figure S2A.. IO Score Independence with Additional Clinical Factors and Genomic Biomarkers Demonstrating IO Score independence with various genomic signatures in a series of bivariate Cox Proportional Hazards. In all cases the median of the signature was used as a threshold for positive or negative. A more complete description of each of these signatures can be found in the work of Mariathasan and colleagues [lleagues .Additional file 3. Figure S2B. IO Score Independence with Additional Clinical Factors and Genomic Biomarkers Demonstrating IO Score independence with various genomic signatures in a series of bivariate Cox Proportional Hazards. In all cases the median of the signature was used as a threshold for positive or negative. A more complete description of each of these signatures can be found in the work of Mariathasan and colleagues [lleagues .Additional file 4. Figure S2C. IO Score Independence with Additional Clinical Factors and Genomic Biomarkers Demonstrating IO Score independence with various genomic signatures in a series of bivariate Cox Proportional Hazards. In all cases the median of the signature was used as a threshold for positive or negative. A more complete description of each of these signatures can be found in the work of Mariathasan and colleagues [lleagues .Additional file 5: Figure S3A. Identifying Additional Eligible Patients by Combining IO Score and TMB (A) Kaplan Meier curves showing individual OS results for TMB-high. (B) Kaplan Meier curves showing the combined results considering patients that were either IO Score+ or TMB -high versus those that were negative for both. (C) Percentage of patients positive for TMB-high/IO Score, and a combined population of either TMB-high or IO Score+. (D) Median and 2-year OS rates for each individual marker and combined population of either IO Score+ or TMB-high.


MUTYH mutations cause predisposition to colorectal adenomas and carcinoma. However, the mechanistic progression from germline MUTYH mutations to MUTYH-Associated Polyposis (MAP) is incompletely understood. Here, we sequence normal tissue DNAs from 10 individuals with MAP. Somatic base substitution mutation rates in intestinal epithelial cells were elevated 2 to 4-fold in all individuals, except for one showing a 31-fold increase, and were also increased in other tissues. The increased mutation burdens were of multiple mutational signatures characterised by C > A changes. Different mutation rates and signatures between individuals are likely due to different MUTYH mutations or additional inherited mutations in other BER pathway genes. The elevated base substitution rate in normal cells likely accounts for the predisposition to neoplasia in MAP. Despite ubiquitously elevated mutation rates, individuals with MAP do not display overt evidence of premature ageing. Thus, accumulation of somatic mutations may not be sufficient to cause the global organismal functional decline of ageing.Cellular DNA damage caused by reactive oxygen species is repaired by the base excision repair (BER) pathway which includes the DNA glycosylase MUTYH. Inherited biallelic  MUTYH have been shown to predispose patients to colorectal cancers. Here, the authors show that MUTYH mutations lead to an increased somatic base substitution mutation rate in normal intestinal epithelial cells, which is the likely cause for the increased cancer risk.Inherited mutations in  A ubiquitous source of potential mutations is DNA damage caused by reactive oxygen species (ROS) which are formed as by-products of aerobic metabolism9. ROS cause a variety of DNA lesions, the most common being 8-oxoguanine (8-OG)10. As a consequence of mispairing with adenine during DNA replication, 8-OG can cause G:C > T:A (referred to as C > A for brevity) transversion mutations11. Under normal circumstances, 8-OG and its consequences are efficiently mitigated by the Base Excision Repair (BER) pathway effected by DNA glycosylases; oxoguanine DNA glycosylase (OGG1) removes 8-OG12 and MutY DNA glycosylase (MUTYH) removes adenines misincorporated opposite 8-OG13.The genomes of all normal human cells are thought to acquire mutations during the course of life. However, the mutation rates of normal cells and the processes of DNA damage, repair and replication that underlie them are incompletely understoodMUTYH engineered in experimental systems can impair its glycosylase activity, reducing its ability to excise mispaired bases and leading to an increased rate of predominantly C > A mutations18. MUTYH mutations inherited in the germline in humans cause an autosomal recessive syndrome  characterised by intestinal adenomatous polyposis and an elevated risk of early onset colorectal and duodenal cancer22. The age of onset and the burden of intestinal polyps are highly variable between individuals, ranging from 10 s to 100 s leading to a substantially increased incidence of colorectal cancer27. Risks of other cancer types are also thought to be increased28.Mutations in 33. However, whether there is an increased mutation rate in normal cells from individuals with biallelic germline MUTYH mutations is unknown. If present in normal cells, understanding the magnitude of the increase in mutation rate, the tissues and cell types in which it occurs, the proportion of cells which show it, the mutational processes responsible and the effects of early neoplastic change would provide insight into the genesis of the elevated cancer risk observed in these individuals.Colorectal adenomas and carcinomas from individuals with MAP show a predominance of C > A mutations consistent with the presence of an elevated mutation rate attributed to defective MUTYH functionIn this study we perform whole-genome sequencing of normal cells from individuals with MAP. Using whole-genome sequencing we characterise the mutation rates and mutational processes in healthy tissues at a near-single-cell resolution. This study identifies the mutational processes that are associated with neoplastic transformation and are likely to underpin the increased cancer risk observed in this population of high-risk individuals.MUTYH mutations were studied. These included five missense mutation homozygotes , three compound heterozygotes for the same pair of missense mutations (MUTYHY179C+/− G396D+/−), and two siblings homozygous for a nonsense mutation (MUTYHY104*+/+). These MUTYH germline mutations have all been previously recognised as predisposing to MAP23. All 10 individuals had colorectal polyposis, with between 16 and >100 colonic adenomas, six were known to have duodenal polyps, five had colorectal cancer and one developed jejunal and pancreatic neuroendocrine cancer  were isolated from the 10 individuals with germline MUTYH mutations  mutation burdens of individual crypts ranged from a median for each individual of 2294 to 33,350, equating to mutation rates of 92-1446 SBS/year, 2-31-fold higher than normal crypts from wild-type individuals (~46 SBS/year) Fig. , Methods3 was observed in PD44890, a 16 year old male with MUTYHY179C+/− G396D+/− who had an aggressive clinical phenotype with a very large number of adenomas and two different primary cancers at an early age. By contrast, the nine other individuals showed only 2- to 4-fold increases in mutation rate compared to wild type. The reason for this substantial difference is not clear. However, in addition to the MUTYH mutations, PD44890 carried two heterozygous germline missense variants in OGG1  than individuals with MUTYHY179C+/+ , MUTYHY104*+/+  or MUTYHG286E+/+  . The results, therefore, indicate that different MUTYH genotypes confer differentially elevated mutation rates and that the extent of the mutation rate increase can be modified by other factors.There was also evidence of differences in mutation rates between the various ied Fig. . Excludi43.SBS mutation rates in coding exons in normal intestinal crypts from MAP individuals were also elevated compared to wild-type individuals  Fig.  than norP = < 10−4), which is higher than in wild-type controls 42. The cause of this modestly elevated ID rate is not clear. In two MAP individuals additional mutational processes could explain the higher burdens observed in these cases. In PD44890 the high ID mutation rate (ID rate 6/yr) was, at least partially, explained by the presence of an additional ID generating mutational process associated with exposure to the mutagen colibactin produced by a strain of E.Coli47 present in the colonic microbiome of some people (see below). In PD50747 (ID rate 6/yr), a previously undescribed sporadic ID signature IDA was identified which was not present in other MAP individuals (described below). Structural rearrangements and copy number changes were only observed in a small number of normal intestinal crypts, at similar frequencies to those in wild-type controls .Small insertion and deletion (ID) mutations accumulated at a rate of 2.1 ID/yr  1.2–3.0, OGG1 germline variants) . Three de novo extracted signatures, N1-N3, accounted for the majority of mutations, all of which were mainly characterised by C > A mutations Fig. . Two of 50. SBS18, thought to result from DNA damage due to reactive oxygen species, has previously been reported in normal colorectal cells3 and many types of cancer33 and is characterised by C > A mutations predominantly at ACA, CCA, GCA and TCT trinucleotide contexts (mutated base underlined)  than in individuals with the MUTYHY179C+/+, MUTYHY104*+/+ and MUTYHG286E+/+ genotypes , resembled reference signature SBS18 (https://cancer.sanger.ac.uk/cosmic/signatures) but showed differences, notably with over representation of C > A mutations at GCA and, to a lesser extent, CCA and ACA trinucleotides (mutated base underlined)  in PD44890 (see above) is superimposed on the mutational signature produced by the MUTYH germline mutations to generate N2 .The de novo extracted mutational signature N2, which primarily contributes to the mutational spectra of crypts from PD44890  were nonsense mutations, mirroring the broader exome-wide increase in nonsense mutations  and is responsible for the elevated ID rate in this individual 50 that allows mutation calling from single DNA molecules and thus accurately discovers somatic mutations in tissues in which multiple clonal lineages are intimately mixed.To investigate whether the elevated mutation rates and mutational signatures observed in intestinal epithelium caused by defective MUTYH are present in other cell types, peripheral blood and tissue lymphocyte DNAs from individuals with biallelic MUTYH mutations were higher than wild-type controls . The relative increases in blood mutation rates were lower than in intestinal crypts from each individual . The signatures associated with defective MUTYH, SBS18 and SBS36, contributed the excess mutations in all samples Fig.  who exhibited a substantially higher mutation rate than others of this genotype, and showed a much accelerated rate of colorectal adenoma development  although the increases appear lesser in extent than in intestinal cells. The propensity to generate SBS18 mutations appears greater in wild type intestinal cells than in other cell types49 and this may also be contributory. Extending this study to investigate somatic mutagenesis in a greater number of tissues with varying cancer incidence and in larger cohorts of individuals may offer further insight into the role mutation rates and mutational signatures play in tissue specific cancer risk.As for many other cancer predisposition syndromes, it is unclear why In summary, we report elevated somatic base substitution rates characterised by distinctive mutational signatures in normal tissues from individuals with MAP. These findings underscore previous observations that elevated somatic base substitution rates are largely tolerated by cells and do not overtly accelerate the process of ageing. It is likely, however, that increased mutation rates in normal intestinal cells throughout life lead to increased rates of accumulation of driver mutations and, hence, the procession of neoplastic clones culminating in cancer.This research complies with all relevant ethical regulations. MAP patients were recruited as part of Wales Research Ethics Committee (REC) 12-WA0071 and 15-WA0075 and samples collected were approved for use in this project by REC 18/ES/0133. Normal healthy controls were recruited as part of the following UK Research Ethics Committee (REC) studies; 15/WA/0131, 15/EE/0152, 18/ES/0133 and 08/h0304/85 + 5.Informed consent was obtained from all participants and no monetary compensation was offered for their participation. Consent was obtained for publication of demographics including age, sex, phenotypic features and other potentially identifiable data. A complete list of study participants and tissue samples is summarised in Supplementary Data Frozen whole blood underwent DNA extraction using the Gentra Puregene Blood Kit (Qiagen). Briefly, 1–2 ml of frozen blood were thawed, lysed in RBC lysis solution and centrifuged. Cell pellet was resuspended in cell lysis solution and incubated at 37 °C for 2 h. RNA and protein were degraded using RNase A solution and protein precipitation solution. DNA was precipitated with isopropanol.Tissues were embedded in Optimal Cutting Temperature (OCT) compound, frozen histological sections were cut at 25–30 µm and mounted on polyethylene naphthalate (PEN) slides and fixed in 70% ethanol for 5 minutes followed by two washes with phosphate buffered saline for 1 min each. Slides were manually stained in haematoxylin and eosin using a conventional staining protocol. A subset of samples were fixed in RNAlater (Sigma Aldrich) according to manufacturer’s instructions. Fixed tissue samples were embedded in paraffin using a Tissue-Tek tissue processing machine (Sakura). No formalin was used in the preparation, storage, fixation or processing of samples. Processed tissue blocks were embedded in paraffin wax, sectioned to 10 µm thickness and mounted onto PEN slides (Leica). Tissue slides were stained using a standard haematoxylin and eosin (H&E) protocol. Slides were temporarily cover-slipped and scanned on a NanoZoomer S60 Slide Scanner (Hamamatsu), images were viewed with NDP.View2 software (Hamamatsu).All tissues studied were carefully reviewed using the following approach: (1) tissue blocks were reviewed by a pathologist / clinician at the time of sampling and classified based on their macroscopic appearance as being normal or adenoma. (2) Following histological sectioning and high-resolution scanning, tissue sections were categorised as being normal, adenoma or cancer. Slides that were indeterminate were referred to a gastrointestinal pathologist for review. (3) Prior to laser-capture microdissection, each crypt was carefully inspected using the 40x digital scan and classified as being normal or dysplastic / adenoma. Initial review, in stages 2 and 3, was principally undertaken by an experienced clinician with a special interest in gastrointestinal pathology. Glands were classified as adenomatous if they bore histological features indicating them to be dysplastic or likely dysplastic and harboured a cancer “driver” mutation.Laser capture microdissection was undertaken using a LMD7000 microscope (Leica) into a skirted 96-well PCR plate. Cell lysis was undertaken using 20 µl proteinase-K PicoPureⓇ DNA Extraction kit (ArcturusⓇ). Samples were incubated at 65 °C for 3 h followed by proteinase denaturation at 75 °C for 30 min. Thereafter samples were stored at −20 °C prior to DNA library preparation.37. This method was employed as it allows for high quality DNA library preparation from a very low starting quantity of material (from 100-500 cells). In brief, gDNA was purified from cell lysates using bead purification. Enzymatic fragmentation, end-repair and dA-tailing was performed using NEBNext Ultra II FS DNA Library Prep Kit (New England BioLabs). Indexing and PCR amplification was subsequently performed (12 cycles). DNA library concentration was assessed after library preparation and used to guide choice of samples to take forward to DNA sequencing. The minimum library concentration was 5 ng/µL and libraries with >15 ng/µL were preferentially chosen. 150 bp paired-end Illumina reads were prepared with Unique Dual Index barcodes (Illumina). DNA sequencing was undertaken on a NovaSeq 6000 platform using an XP kit (Illumina). Samples were multiplexed in pools of 6-24 samples. Pools were sequenced to achieve a coverage of ~30x per sample.DNA library preparation of micro-dissected tissue samples was undertaken using a bespoke low-input enzymatic-fragmentation-based library preparation method55. Mutations were called using an unmatched normal synthetic bam file to retain early embryonic and somatic mutations. Post-processing filters were applied to remove low-input library preparation specific artefacts and germline mutations using a previously described method56. Filters applied were: (1) common single nucleotide polymorphisms were removed by filtering against a panel of 75 unmatched normal samples57 (2) to remove mapping artefacts, mutations were required to have a minimum median read alignment score of mutant reads (ASMD ≥ 140) and fewer than half of the reads supporting the mutation should be clipped (CLPM = 0) (3) a filter to remove overlapping reads that result from the relatively short insert size, which could lead to double counting of variant reads; and (4) a filter to remove cruciform DNA structures that can arise during the low-input library preparation method.Sequencing reads were aligned to NCBI human genome GRCh37 using the Burrow-Wheeler Alignment (BWA-MEM). Single Base Substitutions (SBS) were called using the ‘Cancer Variants through Expectation Maximization’ algorithm (CaVEMan)https://github.com/TimCoorens/Unmatched_NormSeq. Mutations were aggregated per patient and a read pile-up was performed using an in-house algorithm (cgpVAF) to tabulate the read count of mutant and reference reads per sample for each mutation locus. Germline mutations were filtered out using an exact binomial test which distinguishes germline from somatic variants using the aggregate read counts across all samples from the same patient56. In brief, the read depth across all samples from each individual was calculated (median in this study 496-fold). This high coverage yields a very precise estimate of the true VAF of each mutation. While the VAF estimates of the earliest embryonic SBS and germline variants from samples sequenced at 30x might overlap, the VAFs from the much higher coverage achieved by aggregating all samples from each individual, are distinguishable using statistical testing. To achieve this, the beta-binomial test was applied. The over dispersion parameter (rho) threshold for genuine variants of rho > 0.1 was used.Next, we applied multiple filters to remove germline variants and potential artefacts whilst retaining bona fide embryonic and somatic variants. This approach has been detailed in previous publications and the code for these filters can be found at Phylogenetic trees were created using MPBoot (version 1.1.0 bootstrapped – 1000 times) and mutations were mapped to branches using maximum likelihood assignment.58 using the same synthetic unmatched normal sample employed in SBS mutation calling. ID calls were filtered to remove calls with a quality score of <300  and a read depth of less than 15. Thereafter, ID filtering was performed in a similar manner as SBS to remove germline variants and library preparation / sequencing artefacts.Indels (ID) were called using Pindel59 as part of the in the ascatNGS package60 (https://github.com/Crick-CancerGenomics/ascat). Bulk blood samples or phylogenetically unrelated normal samples were used as matched normals. ASCAT was run with default parameters. A bespoke filtering algorithm - ascatPCA - was used to reduce the number of false-positive calls that can arise when analysing genome sequences from normal tissue (https://github.com/hj6-sanger/ascatPCA). ascatPCA extracts a noise profile by aggregating the LogR ratio from across a panel of normal unrelated samples and subtracts this signature from that observed in the sample being analysed using principal component analysis.Somatic copy-number variants (CNVs) were called using the Allele‐Specific Copy number Analysis of Tumours (ASCAT) algorithmWhole-genome sequences were analysed for somatic structural variants (SVs) using the Genomic Rearrangement Identification Software Suite (GRIDSS). In preparation for this analysis, genomes were remapped to Human Genome Version 38 and GRIDSS was run using the same matched normal as used for CNV analysis. Coordinates for SV calls were subsequently converted back to GRCh37. SV calls in L1 transposon donor regions and fragile sites were excluded from the final SV analysis.https://github.com/nicolaroberts/hdp), based on the hierarchical Dirichlet process61, was used to extract mutational signatures. Analysis of mutational signatures using this package has been applied to normal tissues previously4. In brief, this nonparametric Bayesian method models categorical count data using the hierarchical Dirichlet process. A hierarchical structure is established using patients as the first tier (parent nodes) and individual samples as the second tier (dependent nodes). Uniform Dirichlet priors were applied across all samples. The algorithm creates a mutation catalogue for each sample and infers the distribution of signatures in any one sample using a Gibbs sampler. We performed mutational signatures analysis per-branch, counting each branch of the phylogenetic tree as a distinct sample to avoid double counting of mutations. Since the MCMC process scales linearly with the number of counts, we randomly subsampled each branch to a maximum of 2500 total substitutions. Branches with fewer than 100 mutations were excluded from the mutational signature extraction. No reference signatures were included as priors.The R package, HDP (https://github.com/kgori/sigfit)62. To avoid overfitting, a limited subset of reference mutational signatures were included for each patient corresponding to the HDP signatures that were identified in that individual.Next, to estimate the contribution of each mutational process, mutational signatures were refitted to all mutation counts using the R package sigfit  the inherent challenges of accurately estimating mutation burden in highly mutated samples and (2) the appreciable contamination of reference signatures with SBS1 and SBS5. To partially address this, we used the extracted HDP component corresponding to SBS36 in the refitting stage which has lower SBS1 and SBS5 contamination than the COSMIC reference SBS36 signature. Nevertheless, in individual PD44890 where SBS18 and SBS36 exposures are many tens of times greater than the normal mutation rate, the estimates of SBS1 and SBS5 are substantially lower than would be expected.Ageing signatures SBS1 and SBS5 are present in all normal intestinal cryptsSettings / parameters used for mutational signature extraction:SBS Signature Extraction - Hierarchical Dirichlet Process (HDP)Chains: 20 MCMC chainsIterations: 40,000Burn-in: 20,000Samples: 100 / chainSignature components identified: 9Component Names: HDP N0-HDP N8SBS Signature Extraction – Sigprofilerinput_type: vcfstartProcess: 1endProcess: 15totalIterations: 1000cpu: −1hierarchy: Truerefgen: GRCh37genome_build: GRCh37mtype: [‘default’]init: randomSignature extraction was performed using two independent methods; the Hierarchical Dirichlet Process (HDP) and SigProfiler. De novo extraction was performed to extract / identify mutational signatures. HDP signature extraction yielded 9 signature components (N0-N8) . To prevent overfitting, a limited subset of reference signatures was used corresponding to HDP components identified in that patient. Furthermore, any signatures occurring at less than 10% exposure were excluded to prevent over-fitting. Therefore 7 mutational signatures were refitted; SBS1, SBS5, SBS17b, SBS18, SBS36, SBS38 and SBS88.Signatures were re-fitted to the mutation counts for each branch of the phylogenetic tree to establish the absolute contributions of each mutational signature using the R package SigFit  based algorithm SigProfiler. Using the same input data, SigProfiler generated 5 signature components  mapped to branches of the phylogenetic trees were analysed. dNdScv was run using the following parameters; max_coding_muts_per_sample = 5000 and max_muts_per_gene_per_sample = 20. Genes with a qval of <0.05 were considered to be under positive selection.Cancer driver mutations were identified using two methods aiming to identify genes and mutations in this cohort that are subject to positive selection. Firstly, to identify mutations in cancer genes under positive selection in an unbiased manner, we ran a modified dNdS method2. Analysis was restricted to somatic mutations (SBS and ID) in protein coding regions and mutations were filtered using lists of known cancer genes; mutations in samples from intestinal epithelium were filtered using a list of 90 genes associated with colorectal cancer which includes genes that are common in small bowel adenocarcinoma3. Samples from all other tissues, including blood, were filtered using a pan-cancer list of 369 driver genes63. Genes were then characterised according to their predominant molecular behaviour: dominant, recessive or intermediate (those demonstrating aspects of both types of behaviour), using the COSMIC Cancer Gene Census64. Potential hotspot mutations were annotated using the cBioportal MutationMapper database . Mutations meeting the following criteria were considered to be driver mutations: truncating mutations  in recessively acting genes, known activating hotspot mutations in dominant (and recessive) genes. Lastly, mutations that were in neither of the above categories but were characterised by the MutationMapper database as being ‘likely oncogenic’ were also included in the final driver mutation catalogue. We then compared the frequency of driver mutations in histologically normal crypts with MUTYH mutations to a cohort of n = 445 normal intestinal crypts3 from wild-type individuals that were analysed using the same method.A second phase of cancer driver mutation analysis was undertaken, identifying mutations in this cohort that are codified in cancer mutation databases and exhibit characteristic traits of driver mutations, an approach that has been previously employed in the study of normal tissuesMUTYH mutation. The wild-type cohort was generated as part of a previously published study3 and comprises n = 445 normal intestinal crypts that were processed using the same laboratory methods employed in this paper. While most of the bioinformatic analysis in the original paper followed the same pipeline employed in this study, there were some small differences in the methods used for filtering. Therefore, we re-filtered the mutations in the wild-type cohort using the same parameters employed in the MUTYH cohort. Mutation burden estimates were corrected for sensitivity in the same manner as the MUTYH cohort (described below). This was particularly important as the wild-type cohort was sequenced at a lower median coverage than the MUTYH cohort (~16-fold vs ~28-fold). In the original study, Lee-Six et al report a mutation burden of 43.6 SBS/yr in normal crypts. Here, using data that was re-filtered, we obtain an estimate of the mutation rate that is highly concordant (46 SBS/yr).Data generated in this study was compared to a cohort of healthy individuals with no germline 47. First, we sampled a range of sequence coverage values from a Poisson distribution centred around the mean coverage for the sample being analysed. Next, using these values we simulated the number of sequencing reads at each site using a truncated binomial distribution based on the median VAF of each sample. The sensitivity was then calculated as the fraction of simulated mutation calls with 4 or more reads, which is the minimum number of reads that the SBS mutation calling algorithm, CaVEMan, requires to call a mutation. The genome-wide mutation SBS burden was then corrected by dividing it by the estimated sensitivity to give the corrected SBS mutation count.The sensitivity of calling mutations in a genome sequence is strongly influenced by the depth of sequencing coverage and clonality of the sample. Natural variation in sequencing coverage and the clonality of samples may, therefore, influence the sensitivity to call mutations and hence the genome-wide mutation burden estimate. To account for these differences, we calculated the sensitivity of mutation calling from its two principal determinants, sequencing coverage and clonality using a previously validated methodMUTYH germline genotypes and the re-filtered wild-type control data. Fold-changes in the SBS mutation rate were calculated by dividing the modelled mutation rates for the five different MUTYH germline mutation groupings by the modelled mutation rate for the wild-type group.Statistical modelling was performed to assess the mutation rate associated with each germline genotype. A linear mixed-effects model was used to assess the SBS mutation rate for each of the main 66. To assess whether telomere shortening is altered in normal tissues from individuals with MAP compared with wild-type controls, we used a bioinformatic method to assess the telomere content of DNA sequencing files called TelomereHunter. TelomereHunter has previously been applied to the study of telomere biology in cancer samples and normal tissues68. We applied TelomereHunter to estimate the telomere content in normal intestinal crypts with MUTYH mutations (n = 144) and in wild-type controls (n = 445). Next, we used linear mixed-effects modelling to assess the rate of telomere attrition in the MUTYH and wild-type cohorts. No significant difference was observed, thus implying that telomere maintenance is not overtly dysregulated in individuals with germline MUTYH mutations.Telomere attrition is a hallmark of ageing observed in normal cellsMUTYH mutations was extracted as outlined above. Samples from normal healthy control was obtained and processed using the following method. Whole blood was diluted with PBS and mononuclear cells (MNC) were isolated using lymphoprepTM (STEMCELL Technologies) density gradient centrifugation. The red blood cell and granulocyte fraction of the blood was then removed. The MNC fraction was depleted of red blood cells by lysis steps involving 3 incubations at room temperature for 20 mins/10 mins/10 mins respectively with RBC lysis buffer (BioLegend). Tissue lymphocytes were isolated from Peyer’s patches in intestinal mucosa using laser capture microdissection and subjected to protein lysis as outlined above. Cell lysates were processed and whole genome sequenced using the NanoSeq protocol.DNA from bulk blood samples from individuals with germline 50.Our modified duplex sequencing method, called NanoSeq, relies on blunt-end restriction enzymes to fragment the genome in order to avoid errors associated to the filling of 5′ overhangs and the extension of internal nicks during end repair after sonication. Our modified method has error rates < 5e-9Given the uneven frequencies of trinucleotides in the digested genome, the strong filtering of common SNPs sites , and the strong dependence of mutation rates on trinucleotide contexts, our estimates of mutation burdens are normalised and projected onto genomic trinucleotide frequencies.t denote the count of a given trinucleotide of type i = 1…32. The frequency of each trinucleotide is calculated separately for the genome j = 1…6 classes of substitution where the mutated base is a pyrimidine. Let sij denote the count of substitution j in trinucleotide context i, giving a total of 96 substitution classes. Each substitution count is corrected as follows (Formula 3):https://github.com/cancerit.Mutation calling algorithms are available at https://github.com/TimCoorens/Unmatched_NormSeq.Code for filtering mutation calls is available at https://github.com/nicolaroberts/hdp and https://github.com/kgori/sigfit and https://github.com/AlexandrovLab.Software for mutational signature analysis is available at https://github.com/cancerit/NanoSeq.Software for analysis of duplex / NanoSeq data is provided at Parameters used for these various pieces of software have been included in the manuscript methods section and supplementary information.Let Further information on research design is available in the Supplementary InformationSupplementary Data Table 1Supplementary Data Table 2Supplementary Data Table 3Reporting Summary


Most prognostic signatures for colorectal cancer (CRC) are developed to predict overall survival (OS). Gene signatures predicting recurrence-free survival (RFS) are rarely reported, and postoperative recurrence results in a poor outcome. Thus, we aim to construct a robust, individualized gene signature that can predict both OS and RFS of CRC patients.via univariate Cox regression analysis and Venn diagram. These genes were then submitted to least absolute shrinkage and selection operator (LASSO) regression analysis and followed by multivariate Cox regression analysis to obtain an optimal gene signature. Kaplan–Meier (K–M), calibration curves and receiver operating characteristic (ROC) curves were used to evaluate the predictive performance of this signature. A nomogram integrating prognostic factors was constructed to predict 1-, 3-, and 5-year survival probabilities. Function annotation and pathway enrichment analyses were used to elucidate the biological implications of this model.Prognostic genes that were significantly associated with both OS and RFS in GSE39582 and TCGA cohorts were screened A total of 186 genes significantly associated with both OS and RFS were identified. Based on these genes, LASSO and multivariate Cox regression analyses determined an 8-gene signature that contained ATOH1, CACNB1, CEBPA, EPPHB2, HIST1H2BJ, INHBB, LYPD6, and ZBED3. Signature high-risk cases had worse OS in the GSE39582 training cohort  and the TCGA validation cohort  and worse RFS in both cohorts . The area under the curves (AUCs) of this model in the training and validation cohorts were all around 0.7, which were higher or no less than several previous models, suggesting that this signature could improve OS and RFS prediction of CRC patients. The risk score was related to multiple oncological pathways. CACNB1, HIST1H2BJ, and INHBB were significantly upregulated in CRC tissues.A credible OS and RFS prediction signature with multi-cohort and cross-platform compatibility was constructed in CRC. This signature might facilitate personalized treatment and improve the survival of CRC patients. Colorectal cancer (CRC) is the third most common cancer in the world and the second leading cause of cancer-related death . In the As CRC treatment has entered the era of precision medicine, many studies have endeavored to accurately evaluate patient survival in various ways. Traditional clinicopathological features, such as C-reactive protein , tumor sAs far as we know, only three gene signatures have previously predicted both OS and RFS, and the accuracy remains to be improved –17. In thttps://www.ncbi.nlm.nih.gov/geo/) database and used as the training set. The TCGA cohort (N = 368) was downloaded from the TCGA hub at UCSC Xena (https://tcga.xenahubs.net) and used for external validation.Two public CRC cohorts with clinical and gene expression data were used for survival analyses in this study. Among them, the GSE39582 cohort (N = 536) was retrieved from the Gene Expression Omnibus  in the GSE39582 and TCGA cohorts. Then, a Venn diagram  was emplPatients in each cohort were then assigned matched risk scores and they were divided into low- and high-risk groups based on the medium value of these risk scores. Kaplan–Meier (K–M) survival curves, univariate Cox analyses, and calibration curves were adopted to evaluate the prognostic performance of this signature. Time-dependent area under the curve (AUC) values were employed to compare the predictive accuracy of clinical predictors and the risk signature. Moreover, receiver operating characteristic (ROC) curves were used to compare the predictive ability of our signature with nine recently published prognostic signatures for CRC patients –28.The nomogram is a widely used method to quantitatively predict patient survival. To facilitate the clinical application of this signature, we established the nomogram based on prognostic variables derived from univariate Cox regression analysis in the GSE39582 training cohort to predict 1-, 3-, and 5-year survival probabilities. The predictive performance of the Nomogram was validated in the TCGA cohort through ROC curves.To preliminarily clarify the underlying mechanism of the high risk score-resulted unfavorable prognosis, genes significantly correlated with risk scores (p <0.05) were identified by the Pearson correlation analysis in the GSE39582 and TCGA cohorts, respectively. A Venn diagram was applied to determine common correlated genes. These genes were then submitted to Gene Ontology-Biological Process (GO-BP) analysis and The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the DAVID website , 30, resIHC assays were performed as previously reported . To quanThe statistical analyses and graphic study were conducted using R software (version 3.5.2). K–M survival curves with log-rank tests were executed by the ‘survival’ package. ROC analyses were plotted by the ‘survivalROC’ package. Time-dependent AUC values were generated using the ‘timeROC’ package. In Cox regression analyses, we estimated the hazard ratios (HRs) of CRC subgroups with standard clinicopathological variables: age at diagnosis (≥65 vs <65), gender, and tumor size (≥T2 vs <T2), lymph node invasion (≥N1 vs <N1), metastatic spread (M1 vs M0), disease stage (≥II vs <II), chemotherapy and resection margin (>R0 vs R0). Continuous risk scores were classified into low- and high-risk groups according to the medium value of their risk scores. Parameters in univariate and multivariate Cox analyses were generated from the ‘survival’ package and were visualized using the ‘forestplot’ package. LASSO regression analysis was conducted by the ‘glmnet’ package. The nomogram and calibration curves were produced by the ‘rms’ R package. Boxplots depicting the distribution of gene expression and risk scores were derived from the ‘ggpubr’ package. P <0.05 was considered significant.K–M survival analysis demonstrated that patients in the high-risk group had a significantly decreased OS . The results of the univariate Cox regression analyses confirmed that the 8-gene signature and several clinical indicators were risk factors for OS  in human CRC tissues and matched adjacent normal tissues were detected through IHC analyses. The results showed that CACNB1, HIST1H2BJ, and INHBB were significantly overexpressed in CRC tissues , the Natural Science Fund of Shanghai (20ZR1457200), and the Shanghai Sailing Program (21YF1459300).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The clinical significance of hyperoxia in critically ill patients remains unclear –3. Oxida2) results from the interplay between multiple factors: fraction of oxygen delivered by the ventilator (FiO2), fraction of oxygen in sweep gas delivered to the membrane (FbO2), ventilation: perfusion (V/Q) matching in the lung and the balance of ECMO flow and native cardiac output.Patients requiring Venoarterial extracorporeal membrane oxygenation (VA-ECMO), may be at increased risk from hyperoxia due to pre-existing oxidative stress from ischemia reperfusion injury and a high prevalence of extreme hyperoxia , 7. As sSeveral observational studies have examined the association between hyperoxia during VA-ECMO and patient outcomes and the findings are inconsistent , 9–12. TInteractions between ECMO and patient physiology High PaO2, particularly measured at the right radial artery, may reflect poor native cardiac output. In these cases, the mixing point is proximal to the innominate artery with high PO2 levels reflecting hyperoxic ECMO blood flow in right radial blood samples. Few studies have meaningfully considered the interplay between VA-ECMO physiology and disease course  vs. 2.85 [95% CI = 1.12–7.37] respectively). Questions remain around specific PaO2 thresholds at which hyperoxia becomes clinically significant and the importance of hyperoxia duration. These questions are of particular relevance as Moussa et al., like others, confirm a dose-dependent relationship between hyperoxia and morality  of 92–96%) compared the liberal group (in whom the FbO2 remained at 1.0 while the FiO2 was adjusted to SpO2 of 97–100%). Twice daily post oxygenator blood gases are being collected for seven days. Patients are randomised within 6 h of ECMO initiation, to limit hyperoxia exposure in the conservative arm and achieve meaningful group separation. Disability at 6 months is a key secondary outcome, particularly because hyperoxia has been associated with poor neurological outcome [Ultimately randomised controlled trials are needed to establish the causal link between oxygen exposure and clinical outcomes in VA-ECMO patients. The BLENDER trial (NCT03841084) has recruited 214-patients to September 2022. It examines the number of ICU free days in patients exposed to a conservative oxygen strategy (combined manipulation of FbO outcome . AdditioThe management of oxygenation in critically ill patients remains a complex question. We commend the efforts of Moussa et al., and others that are helping untangle the narrative of hyperoxia in VA-ECMO; we eagerly await further evidence to guide clinical practice .


XIST system to silence one chromosome 21 in vitro. T21 caused the dysregulation of Notch signaling in iPSCs, potentially affecting cell-type programming. Further analyses identified dysregulation of pathways important for two cell types: neurogenesis and angiogenesis. Angiogenesis is essential to many bodily systems, yet is understudied in DS; therefore, we focused next on whether T21 affects endothelial cells. An in vitro assay for microvasculature formation revealed a cellular pathology involving delayed tube formation in response to angiogenic signals. Parallel transcriptomic analysis of endothelia further showed deficits in angiogenesis regulators. Results indicate a direct cell-autonomous impact of T21 on endothelial function, highlighting the importance of angiogenesis, with wide-reaching implications for development and disease progression.Despite the prevalence of Down syndrome (DS), little is known regarding the specific cell pathologies that underlie this multi-system disorder. To understand which cell types and pathways are more directly affected by trisomy 21 (T21), we used an inducible-  Moon and Lawrence examine the immediate effects of trisomy 21 silencing and find angiogenesis and neurogenesis pathways, including Notch signaling, affected as early as pluripotency. In endothelial cells, functional analyses show that trisomy delays the angiogenic response for microvessel formation and transcriptomics show a parallel impact on angiogenic regulators and signal-response and cytoskeleton processes. Down syndrome (DS), caused by trisomy of chromosome 21 (chr21) occurs in 1 in every 700 live births in the United States, and conceptions have a high rate of spontaneous loss. Despite the clinical and societal effects of DS, the cellular pathologies that underlie this important syndrome remain poorly understood. Trisomy 21 (T21) consistently leads to mild to moderate intellectual disability in children, which can progress in severity in adults. T21 sharply increases risks for other medical conditions, particularly congenital heart defects, early-onset Alzheimer disease (AD) and acute megakaryoblastic leukemia . DespiteXIST RNA, which coats one female X chromosome in cis (XIST transgene into one chr21 in DS patient-derived induced pluripotent stem cells (iPSCs), and demonstrated robust gene silencing across one chr21 in cis  analysis in iPSCs to determine how correcting T21 expression levels affect genome-wide pathways while maintaining pluripotency. The PSC state—in some sense, a theoretical tabula rasa—allows the analysis of any effects of chr21 dosage on the gene expression landscape and developmental programming from the onset. Pluripotent cells express an unusually large fraction of the genome, including many genes expressed later in specific cell types , thus poPathway analysis revealed changes in specific genes relevant to current or new hypotheses of DS research, leading us to investigate endothelial cells (ECs) and angiogenesis. While the results shown here are relevant to neurodevelopment, we focused more on angiogenesis because this was unanticipated and less studied, and any effect on angiogenesis could affect multiple systems during development and adulthood. For example, vascular deficits could contribute to pulmonary hypertension, early-onset AD, low incidence of solid tumors, neurodevelopment, or even the higher risk of autism in DS . Limitedin vitro cellular phenotype, demonstrating for the first time that T21 expression affects ECs and angiogenesis.In summary, the results of the initial transcriptomic analysis in a panel of DS iPSCs identified gene pathways affected by chr21 overexpression, which then led us to examine angiogenesis in DS iPSC-derived ECs. Results reveal a previously unreported XIST and identify the more direct transcriptome-wide changes caused by chr21 dosage, distinct from changes in the developmental cell state. XIST could silence one chr 21 in iPSC cultures after 20 days of induction -induced nduction . The micin situ hybridization (FISH), we quantified transcription foci for the chr21 gene, APP, which we recently found is one of the last genes silenced  induced the expression of XIST, but by day 6, XIST+ cells overwhelmingly show that the APP allele was silenced  rather than between different isogenic cell lines. This avoids epigenetic and potentially genetic variability between lines, as evidenced by PC2 in TET3G transgene but lacking the XIST transgene . While some studies have suggested not all chr21 genes are overexpressed due to potential feedback regulation, these results indicate that all or most expressed mRNAs approximate the theoretical one-third reduction.MAP2, a non-chr21 gene commonly used as a neuronal marker  expression changes in the 4 clones just after completion of chr21 silencing. As shown in l marker , which cl marker , we notestaining . A subsestaining . That stprograms .XIST−/+ comparisons, and gene expression across each chromosome did not strongly correlate with the Letourneau et al. dataset. While not examined extensively, we also did not detect GEDDs comparing disomic and trisomic isogenic lines, consistent with other results  catalog for biological processes. When visualizing the GO terms of upregulated genes in hierarchical order, four major categories—angiogenesis, neurogenesis, cytoskeleton organization, and response to stimulus—were apparent  and 3B. TTYH1, a non-chr21 gene recently reported to upregulate Notch via gamma-secretase  along with the binding receptors of semaphorin—NRP2, PLXNB1, PLXND1—were differentially expressed in our dataset; these genes are known to promote angiogenesis, endothelial proliferation, and survival, as well as functions related to cell extension and migrational guidance in ECs , kinase insert domain receptor (KDR), CD31, and vascular endothelial (VE)-cadherin by IF , we subsequently found that selecting for puromycin resistance (inserted with TET3G transgene) ensured that dox induced XIST in 100% of cells (and maintained expression through differentiation)  dox did not induce ion) see . Figuresion) see . Silenciion) see ; notablyion) see . We noteion) see  showing ion) see  (Goncharion) see . In addiion) see .RAC2, PARVB, FMOD, some collagen genes). We note that we did not see any gene sets enriched for apoptosis or cell death, consistent with similar vitality of trisomic cultures . Corresponding to results here and elsewhere, DS patients have been found to have elevated levels of ANGPT1 and ANGPT2, which are protective against tumor metastasis, providing insight into the less solid tumors in DS  are more responsive to angiogenic cues and more readily form tubes within the first hour  as the predominant effect, and connected this to reduced terminal differentiation of neurons . The effect on Notch signaling was notable, even in undifferentiated iPSCs. A recent study using this system also found increased Notch signaling , but statistical corrections are imperfect and could introduce some error.This study used a panel of all-isogenic sublines of iPS cells and relied mostly on direct comparison of identical cell populations (different culture dishes), with and without induced chr21 silencing. While this approach is designed to minimize variables between people (or even isogenic lines), comparison of all-isogenic cells limits findings to the genetic background of one individual. Using this “reductionist” approach allowed us to show a direct, cell-autonomous effect to Chr21 expression on certain pathways and angiogenic function; however, clinical studies in diverse individuals are a key complementary approach, required to draw conclusions about how generalizable findings here are to the broader DS population. Angiogenesis is understudied in DS; however, we have cited several clinical studies that suggest that vascular changes are more broadly evident. Finally, we incorporated controls to correct for the effects of dox on individual genes (independent of Detailed methods are provided in the online version of this paper and include the following:Jeanne.Lawrence@umassmed.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jeanne B. Lawrence (This study did not generate new unique reagents.RNA sequencing datasets generated by this study have been deposited at GEO (GSE166849).This paper does not report original code.Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.TET3G, at the AAVS1 safe harbor locus. Each transgene line with the exception of C5A was generated from an independent XIST transgene integration event into an intron of the DYRK1A locus. XIST is expressed when treated with dox, which was described by TET3G but not the XIST transgene were included as dox controls (“Tri” and “Dis”). All iPSCs were cultured with Essential 8 medium (ThermoFisher) in a feeder-free condition on vitronectin-coated plates. Cells were maintained at 37°C, 20% O2, and 5% CO2, and passaged every 3–5 days (~80% confluency) using 500 uM EDTA in PBS. Expression of XIST was induced by adding doxycycline at a final concentration of 500 ng/mL while maintaining cultures in the pluripotent stage or directly upon differentiation. Experiments for dox treatment in iPSC cultures were performed twice.The endothelial cell differentiation of DS hiPSC lines were adapted from in situ hybridization (FISH) staining were adapted from previously published protocols (XIST RNA. The APP probe was generated using a BAC from BACPAC Resources (RP11-910G8) and labeled via nick translation with Digoxigenin-dUTP (Roche). To dual stain for protein and RNA, RNAsin (Promega) was added to the primary and secondary antibody stains as described by Coverslips prepared for RNA fluorescence rotocols . In brieFiji (IF and RNA FISH images used a Zeiss AxioObserver 7 with a Flash 4.0 LT CMOS camera (Hamamatsu). Brightness and contrast were corrected in Fiji , 2015 toRNA samples were extracted using Trizol® Reagent (ThermoFisher) adapted from the manufacturer’s instructions, using 100% ethanol in place of isopropanol. Precipitated RNA was resuspended in RNase- and DNase-free water and DNA digestion was performed using DNase I (Roche) with 2U/ul of RNasin® Plus (Promega) for 1 h at room temperature. RNeasy® Mini Kit was used for RNA purification and DNase I removal following the RNA Cleanup protocol from the manufacturer’s instructions. RNA quality and purity was assessed via Fragment Analyzer . All samples received an RQN score of >8.0.The NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina®, NEBNext® Poly (A) mRNA Magnetic Isolation Module, and NEBNext® Multiplex Oligos for Illumina® were used to prepare sequencing libraries following the manufacturer’s instructions . All libraries were prepared with 1 ug of starting RNA. Sequencing was performed by the University of Massachusetts Medical School Deep Sequencing Core with 7–10 million reads per sample.Cells from monolayer endothelial cell differentiation were dissociated using TrypLE Express (ThermoFisher) according to manufacturer’s instructions, dissociated in a wash buffer . Dissociated cells were run through a 30 uM MACS SmartStrainer (Miltenyi Biotec). One million cells were resuspended in the wash buffer. Conjugated antibodies were added to each sample and incubated for 1 h. Cells were washed and resuspended with 300 ul of flow cytometry running buffer . The Flow Cytometry Core Facility detected the staining on BD LSR II Flow Cytometer and analysis was completed using FlowJo software .Angiogenesis Analyzer  were used as dox controls. Genes differentially expressed in the transgenic and control lines after treatment of dox  were excluded from pathway analysis. We used goana for GO enrichment analysis from the R package limma, correcting for transcript length bias (AmiGO web application (Libraries were aligned to genome build GRch38 using HISAT2  and mapp package . Multi-mwithin R . Gene depheatmap . For the package  and incogth bias , and vislication .Graphs were generated using GraphPad Prism 9 and R. Specific details regarding statistical tests, value of n, and other graph features are detailed in the figure legends. RNA sequencing and analysis methods are detailed above.1Table S2Table S3


Background: Face masks are a major tool for reducing the spread of COVID-19 has been the use of face masks because (1) they protect the wearer from aerosol laden virus in the environment and (2) they reduce aerosol emissions to the environment from infected individuals. Methods that quantify the fitted mask filtration efficiency for protection of the wearer are well established . In contrast, current methods for assessing face-mask containment efficiency are generally semiquantitative and rely on measurement of a very low concentration of aerosols emitted from a healthy or infected human, or the use of mannequins in which a high concentration of surrogate aerosols can be introduced inside the mask. Methods: Expanding on our standard methods used for fitted face-mask filtration efficiency, we designed a small-volume, low-ventilation chamber to accommodate a seated study participant. The study participant wore a ported face mask to allow introduction of a stream of 0.05 μm NaCl particles at a constant concentration (TSI 8026 particle generator) into the mask space. The ambient chamber concentration was continuously measured by a TSI 3775 condensation particle counter sampling 2 feet (~2 m) in front of the participant’s head over a series of three 3-minute periods: (1) resting, (2) reading out loud, and (3) repeated forceful coughing (2 × 10 coughs) (~450 L/min peak flows). Figure Results: Table Conclusions: Our preliminary data on fitted containment efficiency of masks under different conditions suggest that the fitted containment efficiency closely mimics their performance for personal protection. This information that may aid in providing optimum source control in indoor environments.Funding: NoneDisclosures: None