There was a substantial and significant improvement in radiologist accuracy with use of cmAssist, as demonstrated in the 7.2% increase in the area-under-the-curve (AUC) of the receiver operating characteristic (ROC) curve with two-sided p value < 0.01 for the reader group. All radiologists showed a significant improvement in their cancer detection rate (CDR) with the use of cmAssist . The readers detected between 25 and 71% (mean 51%) of the early cancers without assistance. With cmAssist, the overall reader CDR was 41 to 76% (mean 62%). The percentage increase in CDR for the reader panel was significant, ranging from 6 to 64% (mean 27%) with the use of cmAssist. There was less than 1% increase in the readers’ false-positive recalls with use of cmAssist. With the use of cmAssist TM, there was a substantial and statistically significant improvement in radiologists’ accuracy and sensitivity for detection of cancers that were originally missed. The percentage increase in CDR for the radiologists in the reader panel ranged from 6 to 64% (mean 27%) with the use of cmAssist, with negligible increase in false-positive recalls.To determine whether cmAssist™, an artificial intelligence-based computer-aided detection (AI-CAD) algorithm, can be used to improve radiologists’ sensitivity in breast cancer screening and detection. A blinded retrospective study was performed with a panel of seven radiologists using a cancer-enriched data set from 122 patients that included 90 false-negative mammograms obtained up to 5.8 years prior to diagnosis and 32 BIRADS 1 and 2 patients with a 2-year follow-up of negative diagnosis. The mammograms were performed between February 7, 2008 (earliest) and January 8, 2016 (latest), and were all originally interpreted as negative in conjunction with R2 ImageChecker CAD, version 10.0. In this study, the readers analyzed the 122 studies before and after review of cmAssist™, an AI-CAD software for mammography. The statistical significance of our findings was evaluated using Student’s The online version of this article (10.1007/s10278-019-00192-5) contains supplementary material, which is available to authorized users. Mammography is a widely accepted tool for breast cancer screening , 2. AbouDouble reading of mammograms has been shown to increase the sensitivity of screening mammography when compared to single reading by 5–15%, and this practice is still used in Europe today. In the USA, computer-aided detection (CAD) is used for more than 70% of screening exams  and inclEarly detection analysis—the study of mammograms taken more than 9 months before a cancer diagnosis—has not been as widely studied. In 2000, Warren Burhenne et al.  reportedFinancial support was provided by CureMetrix, Inc. for this study. All authors had control of the data and information submitted for publication. Because this was a retrospective study of patients’ clinical records, a waiver was obtained for Human Study IRB approval. This study has been performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments. For this type of study, formal consent is not required. All mammograms were anonymized using a Health Insurance Portability and Accountability Act (HIPAA)-compliant protocol.A set of 2D Full-Field Digital Mammograms (FFDM) was collected from a community healthcare facility in Southern California for retrospective study. The mammograms were originally interpreted by community-based radiologists using the R2 ImageChecker CAD, version 10.0 . All patients in the collected dataset were females, aged 40–90 years, who had a biopsy performed between October 2011 and March 2017. Of 1393 patients, 499 had a cancer biopsy, 973 had a benign biopsy, and 79 had both cancer and benign biopsies. None of these cases were used as training data for the cmAssist algorithm, and all the cases were quarantined for the purposes of this study.For this study, authors define a prior mammogram as one taken more than 270 days (9 months) before a tissue biopsy was performed. Of the 499 patients having biopsy-confirmed breast cancer, 317 had prior mammograms. Of the cancer patients with prior mammograms, there were 139 patients who had retrospective findings on their prior mammograms.Prior mammograms that showed retrospective findings were marked by two Mammography Quality Standards Act (MQSA)-certified “validating radiologists”  who had current mammograms and biopsy results available for reference. The markings were archived along with a written recording of location, lesion type, and tissue density. In cases of conflict, cases were reviewed again and a consensus was obtained in categorizing those lesions. These truth markings were hidden from the subsequent blinded panel of radiologists, but used for correlation of the recall results. The validating radiologists were excluded from the reader study.Actionable—the lesion that eventually was biopsied was recallable in a clinical setting,Non-actionable—the lesion that was eventually biopsied was subthreshold for recall,Excluded—ipsilateral prior lumpectomy and synthesized or three-dimensional tomography images.The validating radiologists categorized the retrospective findings on prior mammograms into one of the following categories:The cmAssist AI-CAD was considered to have correctly marked the Actionable lesion if a cmAssist marking overlapped the markings made by the validating radiologists.A total of 155 prior mammograms from 90 patients were deemed Actionable. The reader study was restricted to the oldest Actionable prior mammogram per patient, termed “Earliest Actionable.” The Earliest Actionable prior mammograms were chosen to make the data set as rigorous as possible, and to create a reasonable number of cases for a single sitting. The Earliest Actionable prior mammograms consisted of women with a mean age of 65.4 years  at diagnosis. The Earliest Actionable prior mammograms were obtained between 0.76 and 5.8 years  prior to the current mammogram.An additional 32 normal studies were included in the reader study to reduce reader bias. These patients were confirmed to have 2 or more years of subsequent normal mammograms.Time-aggregate data from 1/1/2009 through 12/31/2016 (for the institution from which mammographic studies were collected for the study presented in this paper) reveals a cancer detection rate (CDR) of 4.5 per thousand , compared to the US national average CDR of 5.1 per thousand reported by the Breast Cancer Surveillance Consortium for the period 2007–2013 [cmAssist is a recently developed AI-CAD for mammography which incorporates a popular form of artificial intelligence called deep learning. cmAssist was trained using curated mammograms from multiple institutions consisting of biopsy-proven benign and malignant lesions, as well as validated normal mammograms (BIRADS 1 and 2 studies with at least 2-year follow-up of negative diagnosis). None of the mammograms that were evaluated in this retrospective study were used in the development of cmAssist. The cmAssist algorithm is based on multiple custom deep learning-based networks that work together to achieve high sensitivity without sacrificing specificity. Furthermore, the training of the algorithms utilizes a proprietary, patent-pending data augmentation technique to enrich the different presentations of cancer and benign structures in our training set. The training set is comprised of images acquired on multiple different makes and models of mammography units. The software is vendor agnostic, runs on for presentation images (which means that raw DICOM is not needed) and no calibration is needed, which are advantages over traditional CAD.The cmAssist’s stand-alone efficacy, as measured by the traditional receiver operating characteristic (ROC) curve, is shown in Fig. A blinded reader study was performed to assess the potential clinical benefit of cmAssist for improved accuracy in cancer detection by mammographers. The enriched data set comprises of 90 false-negative Earliest Actionable prior mammograms and 32 normal exams, for a total of 122 exams.Seven MQSA-certified radiologists of various training and experience levels were recruited for the reader panel. The reader study was performed using a high-resolution viewing workstation and FDA-approved mammography viewing software  in a reading room that met MQSA standards.The cases were shown to each radiologist without any clinical information or comparison to prior studies. The readers were informed that there was a combination of normal and recallable mammograms. The cases were put into a randomly ordered worklist and displayed to each radiologist in the same order by an independent proctor. Each radiologist was asked to view each mammogram without cmAssist markings and make a clinical decision about recall. Subsequently, each radiologist was provided with the cmAssist markings and their corresponding quantitative scores  and given the opportunity to change the clinical decision, i.e., the reader has the option to add/remove areas of recall based on the review of the cmAssist results. The proctor recorded location and lesion type for recalls and whether the reader changed recommendation after cmAssist review.t test with a null hypothesis of a mean value of zero. The data was also analyzed based on lesion type  and tissue density. If the radiologist recalled the patient, but indicated the incorrect quadrant or incorrect laterality, it was considered a false-negative recall.The change in CDR and false-positive recall rate (after cmAssist compared to before cmAssist) were calculated for each radiologist. Statistical significance was calculated using the two-sided Student’s p < 0.01.The overall reader group accuracy before and after cmAssist was evaluated using ROC and AUC analysis. The statistical significance of the change in AUC was established using the bootstrapping method  with 10,n = 139) had retrospective findings and the remaining 56% of patients (n = 178) had de novo cancers, with no retrospective findings seen on their prior mammograms. Out of the patients who had retrospective findings, 90 of the 139 patients (65%) were deemed Actionable, 40 were deemed non-Actionable, and 9 were excluded , scattered , heterogeneously dense , and extremely dense .Of the 317 cancer patients with prior mammograms, 44% of the patients  and those less influenced (Group 2: Readers 4–7). Group 1 included the two less-experienced radiologists (Readers 1 and 2) and one general radiologist who had been trained over 4 decades prior to this study. These readers appeared to have high reliance on cmAssist, with reversal from no recall to recall in a significant number of the missed cancers with cmAssist assistance. One of the general radiologists (Reader 3) showed dramatic benefit (percent increase of 64% in CDR) with cmAssist assistance. Reader 1, a less-experienced general radiologist with less than 3 years in practice, had a CDR percent increase of 61% after reviewing with cmAssist assistance. As expected, the mammography fellowship-trained mammographers (Group 2) had higher CDRs than the less-experienced, general radiologists (Group 1) without cmAssist. One striking observation is that cmAssist assistance brought the sensitivity of the two least-experienced general radiologists (Group 1: Readers 1 and 2) to CDRs that exceeded the CDR of 75% of group 2 readers.The influence of the cmAssist on false-positive recalls was measured through an analysis of the 32 normal cases in the data set as shown in Table The cmAssist AI-CAD showed benefit as a decision support tool about equally for masses and calcifications, as indicated in Table p value = 0.026) and heterogeneously dense  cases. The reader CDR for fatty and extremely dense mammograms also appear to be benefited by cmAssist, but statistical significance was not demonstrated.cmAssist appears to be beneficial across all tissue densities. The increased reader CDR with the assistance of cmAssist is shown to be statistically significant in scattered (The benefit of AI-CAD in a fatty breast (tissue density = 1) is presented in Fig. An example of a missed cancer in a patient with scattered densities (tissue density = 2) is depicted in Fig. It is noted that all readers in this study appeared to ignore relatively significant number of flagged actionable lesions that would have improved their sensitivity even further. This suggests that even further improvement in reader accuracy and CDR could occur as radiologists gain experience in using cmAssist and develop more confidence in its markings and use of the neuScore . While the readers recalled an average of 3.4 additional malignant calcifications cases with benefit of cmAssist, they also disregarded an average of 6.1 flagged malignant calcifications cases. Similarly, while readers recalled an average of 6.4 additional cases of malignant masses with cmAssist assistance, they also disregarded an average of 11.4 cases of flagged malignant mass cases. Figure In addition to summarize Tables In Fig. . One can, however, compute the ROC for the readers performing as a group. The aggregate score assigned to each case is taken to be the sum of the scores by the 7 readers, and each of the 122 exams has a score between 0 and 7, inclusive. A computation of the ROC for the readers as a group is performed based on their scoring of cases before and after review of the cmAssist results. The ROCs before and after review of the cmAssist results are shown in Fig. Since each reader scores every case with a binary score (0 or 1), it is not possible to construct an ROC for each readerp < 0.01) with respect to variations in the difficulty level of the cases.First, we resampled with respect to the 122 exams, using 10,000 bootstrap samples. For each given bootstrap sample, we computed the readers-averaged ROCs before and after review of the cmAssist results. From the resulting ROCs, the change in AUC is computed for the bootstrap sample. A histogram of the percentage change in AUC, defined as 100 × (before-cmAssist AUC − after-cmAssist AUC)/before-cmAssist AUC, is shown in Fig. p < 0.01.Similarly, we perform the same analysis, also using 10,000 bootstrap samples, but we resample with respect to the readers to assess statistical significance with respect to variations in the readers’ experience level, and a histogram of the percentage change in AUC is shown in Fig. Retrospective findings may be seen on prior mammograms of breast cancer patients 52 to 75.3% of time . This raVariability in breast imaging radiologists’ performance is well recognized and has been widely reported, first by Beam and Sullivan  and, morOne of the trends seen in this study was that radiologists with the least experience and training derived the most improvement in performance with cmAssist, consistent with previous works . The felResults indicate that the sensitivity of all readers in this study appeared to be elevated due to the test setting and the enrichment of the data set with a high proportion of abnormal mammograms. This “laboratory effect” has been described, among others, by Gur et al. . These fIn this study, the radiologist panel had a slightly greater tendency to disregard flags of actionable masses over calcifications. Even the most accurate reader in the study ignored 18% of the cmAssist flagged Actionable lesions. The improvement in CDRs could have been much higher if the readers relied more on the cmAssist results for decision-making, as shown in Fig. In the short span between the completion of the reader study reported here and the time of current writing, substantial progress has been made on the stand-alone efficacy of cmAssist, as evidenced in Fig. It is again emphasized that the test cases in this study have never been provided as training cases in the development of cmAssist.There will be continued improvement in accuracy as algorithms are refined, more training data is included, and computer processing power increases. It should be noted that the maximum achievable sensitivity for cmAssist in stand-alone mode on this data set of missed cancers was 98%. Future work in false-positive reduction using AI is in progress and will lead to further improvements in accuracy for cancer detection. With the advent of 3D tomosynthesis, there is a reported further improvement in accuracy over 2D digital mammography, but with cost of higher radiation dose and expense. There has been partial clinical implementation of 3D tomosynthesis in the USA and Western Europe which is expected to grow. Most facilities still perform 2D mammography views in conjunction with 3D in part due to higher conspicuity of calcifications but also related to issues with reader confidence, archiving and comparing with prior studies. It is anticipated that the principles of AI-CAD used in development of cmAssist will translate to 3D and synthetic 2D mammograms as well.This analysis of the false-positive recall rate in this study is limited due to the relative small sample size of normal cases. However, it would not have been reasonably possible to reflect the true prevalence of malignancy in a study of this nature. Another limitation of this reader study is the lack of comparison of prior mammograms, which could have resulted in increased reader sensitivity . BecauseThis study shows how AI-based software can provide clinical benefit to radiologists in interpretation of screening mammograms. The use of AI in clinical practice may potentially expedite workflow, enhance earlier detection of cancer, and reduce false-negative mammograms. The impact of AI on medical imaging in the future is likely to be profound. To the authors’ knowledge, this is the first peer-reviewed scientific study that shows significant benefit of AI to radiologists in clinical image interpretation.ESM 1(DOCX 18 kb)


Salmonella can appear in the bloodstream within CD18 expressing phagocytes following oral ingestion in as little as 15 minutes. Here, we provide evidence that the process underlying this phenomenon is reverse transmigration. Reverse transmigration is a normal host process in which dendritic cells can reenter the bloodstream by traversing endothelium in the basal to apical direction. We have developed an in vitro reverse transmigration assay in which dendritic cells are given the opportunity to cross endothelial monolayers in the basal to apical direction grown on membranes with small pores, modeling how such cells can penetrate the bloodstream. We demonstrate that exposing dendritic cells to microbial components negatively regulates reverse transmigration. We propose that microbial components normally cause the host to toggle between positively and negatively regulating reverse transmigration, balancing the need to resolve inflammation with inhibiting the spread of microbes. We show that Salmonella in part overcomes this negative regulation of reverse transmigration with the Salmonella pathogenicity island-2 encoded type III secretion system, which increases reverse transmigration by over an order of magnitude. The SPI-2 type III secretion system does this in part, but not entirely by injecting the type III effector SpvC into infected cells. We further demonstrate that SpvC greatly promotes early extra-intestinal dissemination in mice. This result combined with the previous observation that the spv operon is conserved amongst strains of non-typhoidal Salmonella capable of causing bacteremia in humans suggests that this pathway to the bloodstream could be important for understanding human infections. Salmonella can disseminate from the gastro-intestinal (GI) tract to the blood and subsequently internal organs through three independent pathways. In what is believed to be the primary pathway, Salmonella as well as numerous other enteropathogens adhere to and invade the M cells of Peyer’s patches and are subsequently internalized by the underlying phagocytes. The infected phagocytes can then migrate to the mesenteric lymph nodes, where they can orchestrate immune responses against the microbes. As Salmonella, like many pathogens, can withstand the microbicidal activities of these cells, the bacteria are conventionally thought to passively spread throughout the host after the infected cells drain from the mesenteric lymph nodes through the thoracic duct into the bloodstream. As ingrained as this model is, there is recent evidence, which indicates that the mesenteric lymph nodes act as a firewall, largely containing oral infections, allowing for the generation of a local immune response, while shielding the host from systemic, microbial dissemination[Salmonella and Yersinia colonize these tissues in mice that completely lack Peyer’s patches with nearly identical kinetics as they do in congenic control mice[Salmonella perturbs beta-catenin-dependent signaling in gut endothelial cells, disrupting a gut vascular barrier to gain access to the bloodstream[A key component in the virulence of many successful pathogens is the ability to spread from the initial site of infection to deeper tissue. Following oral ingestion, emination, 2. In femination. Moreovetrol mice. Thus, ioodstream.Salmonella directly into the bloodstream, also bypassing the lymphatic system[Salmonella enters them however, the infected cells sometimes rapidly penetrate the bloodstream, through an as of yet, largely uncharacterized mechanism. Traversing the blood vascular endothelium in the basal to apical direction is referred to as reverse transmigration. This pathway to the bloodstream is not conventionally thought to enhance microbial virulence[Salmonella cells to the spleen, which filters the bloodstream, to engender a systemic immune response against the bacteria to combat subsequent, delayed invasion of deeper tissue through the lymphatic system[Salmonella can actively exploit reverse transmigration to bypass the lymphatic system, expediting its colonization of internal organs[In an alternative pathway, CD18 expressing phagocytes, presumably dendritic cells, can ferry ic system. These cic system. Normallvirulence, 7. Rathic system, 7. We hal organs.Salmonella manipulates reverse transmigration. Salmonella infection leads to millions of deaths world-wide every year[Salmonella enterica serovar Typhimurium (S. Typhimurium) causes gastrointestinal illness in humans, which often is not serious, but can be fatal in infants, the elderly and the immunocompromised. Also, S. Typhimurium can sometimes cause bacteremia and even septicemia in otherwise healthy individuals, which is a growing public health threat, especially in immunodeficient people, such as those infected with HIV. Salmonella enterica serovar Typhi (S. Typhi) on the other hand can cause typhoid fever, a serious systemic illness. S. Typhimurium causes a typhoid fever like disease in mice as S. Typhi does in humans. In addition to public health concerns, Salmonella is also studied because it is a convenient model pathogen.Reverse transmigration is likely relevant to numerous infectious processes including the spread of pathogenic microbes from the GI tract, lung tissue and the oral mucosa to the systemic circulation. Here we report on our studies of how very year. SalmoneSalmonella harbors two primary pathogenicity islands, termed Salmonella pathogenicity island one (SPI-1) and Salmonella pathogenicity island two (SPI-2). SPI-1 is used to invade host cells and invoke an inflammatory response and can kill host cells[Salmonella-infected cells in the bloodstream potentially through an interaction with the host protein TRIP6[ost cells–14. SPI-ost cells–17 and wost cells, 9. We sSalmonella serovars carry plasmids, which share a highly conserved locus called the spv  operon[spv genes are important for human pathogenesis as spv-carrying strains dominate among clinical isolates from patients with non-typhoidal bacteremia[spvC mutant is not defective in replication within macrophages but is attenuated in mice[Some e) operon. It has acteremia, 20. Spvacteremia, 22. An d in mice.in vitro reverse transmigration assay to model the rapid colonization of the bloodstream by Salmonella-infected cells following oral ingestion. We demonstrate that microbial components down regulate reverse transmigration and that Salmonella overcomes this by secreting SpvC and at least one additional unknown SPI-2 associated type III effector into infected dendritic cells that stimulate reverse transmigration. These results suggest that the reverse transmigration pathway to the bloodstream could be an important component of Salmonella pathogenesis.Here, we describe the development of an in vitro reverse transmigration assay adapted from Bianchi et. al.[+, presumably a mixture of macrophages and dendritic cells. Dendritic cells are the only cell type capable of reverse transmigration[We used blind wells to establish an i et. al. that modmigration. After sSalmonella in the bloodstream following oral ingestion was initially proposed to be passive on the part of the bacteria[Salmonella might deter the reverse transmigration of dendritic cells. We incubated cells with or without heat killed Salmonella for thirty minutes and then applied them to the endothelial monolayers in the blind wells. After one hour of incubation, we disassembled the blind wells and enumerated the number of dendritic cells that migrated through the endothelial monolayer into the bottom compartment with a hemocytometer. Treating the dendritic cells with dead Salmonella inhibited reverse transmigration five-fold . We chose to perform the experiments with strains lacking sipB simply to reduce the number of dendritic cells required for our assays. In subsequent mouse experiments, we did not find it necessary to use the sipB mutant background. The inclusion of gentamicin in our in vitro assay selectively killed the extracellular bacteria. We infected dendritic cells separately with the three strains and incubated them for six hours prior to performing the reverse transmigration component of the assay. While in vivo, SPI-2 associated genes can be expressed in as little as 15 minutes post-infection prior to penetrating the intestine, in vitro, in cell culture models of infection, it takes four hours for them to be induced and expression peaks at six hours post-infection[We next tested whether or not live tic cells. In agreinfection. Unlike srfH mutant, which we previously reported has about a five-fold defect in penetrating the bloodstream of mice following oral infection[srfH mutant was more efficient than wild type at triggering reverse transmigration, although the difference was not statistically significant. We observed a very large 10.5-fold defect however in our reverse transmigration assay for the mutant that could not secrete any SPI-2 effectors ..srfH mutsrfH mutant as well as the other ones we tested are resistant to the antibiotic kanamycin. In these experiments, we infected cells separately with either wild type Salmonella or a mutant, killed the extracellular bacteria with gentamicin and combined them and plated an aliquot onto Lura-Bertani (LB) agar and onto LB agar supplemented with kanamycin to determine the input ratio. We then performed the reverse transmigration assay with the remainder of the mixture and plated the output onto LB agar and LB agar augmented with kanamycin. Surprisingly, even in this assay, we could detect no effect of SrfH on reverse transmigration  or the bile duct  for extra-host dissemination. The gall bladder is an extremely beneficial niche for Salmonella as 2–6% of the time, the bacteria become asymptomatic here and can potentially be shed intermittently for the lifetime of the host[Salmonella infections are often, however, ultimately resolved by an adaptive immune response[Salmonella bypasses mesenteric lymph node confinement, accelerating its intra-host dissemination. This may increase the transmission rate by providing the bacteria with additional time to grow in their preferred sites of replication and also with more opportunities to establish a chronic, asymptomatic infection. spvC is found in non-typhoidal strains of Salmonella and is likely a virulence factor in its animal reservoir where it does cause systemic disease. The fact that it can also rapidly colonize the bloodstream of humans where it does not typically cause systemic disease may be unintentional on the part of the bacteria.It is interesting to consider why  the host–34. As S response, the bac response, 36, manSalmonella pathogenesis however as the bacteria presumably disseminate to the bloodstream from the GI tract through this pathway throughout the course of infection. Also, very few S. typhi founder cells are needed to seed the spleen and liver to cause problems as they can grow extensively there and any non-typhoidal Salmonella organisms in the bloodstream can potentially cause health problems. The fact that the spv operon is conserved among non-typhoidal Salmonella strains that cause bacteremia in humans further suggests that this pathway to the bloodstream could be important for understanding human infections.The pathway is inefficient at 30 minutes post-infection anyway with only about 1 in a million inoculated bacteria being translocated from the GI tract to the bloodstream in this time-frame. Reverse transmigration could still play an important role in It is interesting to consider potential molecular mechanisms underlying the ability of SpvC to promote reverse transmigration. SpvC deactivates, Erk1/2, p38 and JNK, 22. OnesrfH mutant, as we have shown in the past that srfH is involved in the early penetration of the bloodstream by Salmonella-infected cells following oral inoculation of mice[in vivo.It was surprising that we did not observe a defect in reverse transmigration for dendritic cells infected with a n of mice, 9. It iSalmonella in part overcomes this inhibition by secreting at least two SPI-2 effectors into them that stimulates the process by over an order of magnitude. This work provides some suggestive evidence that the CD18 expressing phagocyte pathway to the bloodstream involves reverse transmigration and that this may be an important component of the extraintestinal dissemination of Salmonella. The model described here may be useful in studying the dissemination of other pathogens and could also be used for studying metastasis and excessive inflammatory disorders.Our results demonstrate that dendritic cells down regulate reverse transmigration in the presence of microbial components and that Animals were housed, cared for, and used strictly in accordance with the USDA regulations and the NIH guide for the care and use of laboratory animals . The University of Louisville is fully accredited by the American Association for the Accreditation of Laboratory Animal Care. A full-time, specialty-trained veterinarian directs the program of animal care. The protocol was approved by the University of Louisville Institutional Animal Care and Use Committee (protocol # 12–090). All reasonable efforts were made to alleviate discomfort.Six to eight-week-old female C57BL/6J mice were obtained from Jackson labs. . Bone marrow was harvested and monocytes cryopreserved as previously described.9 bacterial cells suspended in 100μl of phosphate buffered saline (PBS). Food was withdrawn 12 hours prior to infection. Thirty minutes following infection, mice were euthanized with CO2 and blood recovered by heart puncture with a 25G needle attached to a 1mL syringe. Blood was collected in microtubes on ice containing 50 units of heparin sodium salt (Sigma) in 100μl of water to prevent coagulation. Triton X-100 was added to a final concentration of 1% to lyse host cells and the tubes incubated at 4°C with end over end rotation on a rotisserie for ten minutes. CFU were then recovered on xylose lysine deoxycholate agar plates, which are selective for Salmonella.For extra-intestinal dissemination assays, groups of 5–8, 4–6 week-old female C57BL/6J mice were orally infected by gavage with 1 X 106 cells/mL in RPMI supplemented with 10% FBS and sodium pyruvate. They were differentiated into dendritic cells by culturing them in the presence of GM-CSF (Life technologies) (20ng/mL) and IL-4 (Life technologies) (20ng/mL) for three days. The media also included 55μM ß-mercapoethanol (Life technologies). On the third day, an equal volume of media supplemented with fresh GMCSF (40ng/mL), IL-4 (40ng/mL) and 110μM ß-mercapoethanol was added. On the sixth day, TGF-ß1 (R&D systems) was added to a final concentration of 10ng/mL to induce expression of CD16, which is associated with an enhanced ability to reverse transmigrate[C166 murine endothelial-like cells were cultured in DMEM (VWR) supplemented with 10% FBS (Sigma) and sodium pyruvate (Life technologies) and passaged 1:10 every 4–5 days. Monocytes were cultured at a concentration of 1 X 10nsmigrate. Assays S. Typhimurium 14028s with an in-frame deletion of sipB described in Kidwai et. al.[srfH. We separately transduced with P22 HT int a srfH::MudJ allele[ssaK::km allele[ssaK is part of an operon of structural genes that compose the type III secretion system. This mutant cannot secrete any effectors associated with SPI-2[spvB and spvC[sipB::Km allele[spvC was PCR amplified from the virulence plasmid of S. Typhimurium 14028s and cloned into the EcoRV and SalI sites of pACYC184, under the control of the constitutive tet promoter. The construct was sequence verified.i et. al. was the dJ allele into thikm allele with estkm allele. ssaK isith SPI-2. Into pr and spvC, we tranKm allele. spvC wa5 endothelial cells were added to each well. The endothelial cells were given 5 days to form confluent monolayers. Twenty-four hours before assays began, endothelial cell media was replaced with fresh media supplemented with TNFα (Life technologies) (20ng/mL). The formation of confluent monolayers under these conditions was confirmed with diff-kwik staining.PVP-free polycarbonate membranes with 5μm pores were sterilized by autoclaving and individual membranes were submerged in 1mL of PBS (Life technologies) supplemented with 8μg of fibronectin from bovine plasma (Sigma) in 24 well plates. The membranes were coated with fibronectin overnight at 4°C. The following day, the fibronectin solution was aspirated from the wells and 5 X 106 dendritic cells were infected at an MOI of 25 in quadruplicate with the three bacterial strains. The bacteria were given one hour to invade. Under the conditions used, 1–2% of the bacteria were internalized. Next, gentamicin (Life technologies) was added to a final concentration of 100μg/mL to selectively kill the extracellular bacteria and the cells were incubated for one hour at 37°C. After the one-hour incubation the mixture was dialyzed against PBS with Slidalyzer mini dialysis devices with a 2kDa molecular weight cutoff (Thermo Fischer). Following dialysis, the mixture was incubated in media supplemented with 10μg/mL gentamicin for five hours at 37°C. After five hours, the mixture was again dialyzed against PBS and a small aliquot lysed with 1% triton X-100 for ten minutes and CFU recovered on agar plates to determine the input.The three bacterial strains were grown overnight at 37°C in LB. Approximately 1 X 10Two-hundred microliters of media containing gentamicin was added to the bottom compartment of the blind wells. Then, a membrane coated with a confluent monolayer of endothelial cells was placed upside down in the device. A second membrane was then stripped by dipping it into a solution of PBS containing 0.5% triton X-100 and 20mM ammonium hydroxide (Sigma) for 30 seconds and then rinsed in DMEM, revealing a natural extracellular matrix. This meS. Typhimurium was diluted 100-fold in cell culture media and heated to 95°C for 15 minutes. 1 X 105 dendritic cells were then resuspended in either the LB or the heat-killed Salmonella and incubated at 37°C for 30 minutes. The dendritic cells were then counted on a hemocytometer to determine the input and applied to the blind wells. They were processed as described above except instead of recovering CFU, the number of dendritic cells that traversed the endothelial monolayer was determined by concentrating the media in the bottom compartment and counting the cells with a hemocytometer.In this experiment, an equal volume of either LB or a saturated LB overnight culture of srfH, spvB, or spvC mutant. The infections were processed as described for the individual assays. After the five-hour incubation, the wild type bacteria and one of the mutants were combined and a small aliquot lysed and CFU recovered on LB-agar plates or LB-agar plates supplemented with 60μg/mL kanamycin to determine the input ratio. The reverse transmigration assay was then performed with the remainder of the mixture as described for the individual assays except after lysis of the cells in the bottom compartment, CFU were recovered on LB-agar plates or LB-agar plates supplemented with kanamycin. The number of ÇFU present on kanamycin plates was subtracted from the number present on LB plates to determine how many wild type bacteria were present. The CFU counts of the kanamycin plates revealed how many mutant bacteria were present.1 million dendritic cells were infected with either wild type bacteria or a 5 dendritic cells were infected at an MOI of 25 with the three bacterial strains and a gentamicin protection assay performed. The number of intracellular CFU at seven hours post-infection was determined by dialyzing the gentamicin and lysing the dendritic cells with triton X-100 and recovering bacteria on agar plates.In this experiment, 1.7 X 10S1 FigDendritic cells were infected separately with the three strains and a gentamicin protection assay performed. This assay was performed in triplicate on two independent occasions. There was no significant difference in the number of bacteria present with the different strains at seven hours post-infection.(TIFF)Click here for additional data file.S2 FigThe monolayers excluded greater than 99.9% of the beads.(TIFF)Click here for additional data file.


Neisseria gonorrhoeae carries one of the highest loads of RMS in its genome; between 13 to 15 of the three main types. Previous work has described their organization in the reference genome FA1090 and has inferred the associated methylated motifs. Here, we studied the structure of RMS and target methylated motifs in 25 gonococcal strains sequenced with Single Molecule Real-Time (SMRT) technology, which provides data on DNA modification. The results showed a variable picture of active RMS in different strains, with phase variation switching the activity of Type III RMS, and both the activity and specificity of a Type I RMS. Interestingly, the Dam methylase was found in place of the NgoAXI endonuclease in two of the strains, despite being previously thought to be absent in the gonococcus. We also identified the real methylation target of NgoAXII as 5′-GCAGA-3′, different from that previously described. Results from this work give further insights into the diversity and dynamics of RMS and methylation patterns in N. gonorrhoeae.Restriction-Modification systems (RMS) are one of the main mechanisms of defence against foreign DNA invasion and can have an important role in the regulation of gene expression. The obligate human pathogen  Neisseria gonorrhoeae is a sexually-transmitted pathogen that causes gonorrhoea. Antimicrobial resistance in this pathogen is of great public health concern and there have recently been an increasing number of publications aimed at analysing its transmission and the evolution of genetic determinants of antimicrobial resistance using high throughput short-read sequencing1. However, Single Molecule Real-Time (SMRT) PacBio sequencing provides deeper information about microbial genomes, including data on DNA modification, mainly methylation in the form of 6mA, 5mC or 4mC. Several studies have focused on the reference strain N. gonorrhoeae FA1090 to characterize the population of Restriction-Modification systems (RMS) in the gonococcus5 and its methylation landscape6. However, RMS and methylation status are known to vary significantly between strains of the same species, so this provides only limited knowledge about RMS and methylation in the gonococcus as a whole.N. gonorrhoeae is among the bacterial species with the highest numbers of RMS despite being naturally competent for DNA uptake7. Indeed, highly transformable bacteria have been found to contain a higher number of RMS compared with those which are less competent, acting as a defence system against the invasion of foreign DNA10. RMS are formed by a restriction endonuclease (REase) and a DNA methyltransferase (MTase) that recognize a specific pattern in the genome and act as multi-subunit complexes (Type I and III) or as independent enzymes (Type II)10. If the MTase is active, it will methylate the associated motifs and thus protect them from cleavage by the cognate REase. Bacteria can contain up to four types of RMS, although only Type I, II and III have been found in Neisseria10. Briefly, Type I RMS act as a multi-enzyme complex consisting of two MTase subunits (HsdM), two REase subunits (HsdR) and one specificity unit (HsdS) that recognizes specific asymmetric sequences, methylating both strands11. Type II RMS are generally formed by individual MTase and REase enzymes that recognize the same short palindromic motifs and perform a two-strand methylation or cleavage, respectively11. Finally, Type III RMS act as a complex of two Mod (MTase) and two Res (REase) subunits12 that recognize specific short asymmetrical motifs11. The specificity of Type I RMS is encoded by an independent gene (hsdS) that must be active for the other subunits to be functional, while Type III RMS harbour a DNA recognition domain (DRD) within the MTase10. RMS have also been associated with other roles apart from defending the genome against foreign DNA invasion, such as being involved in the epigenetic control of gene expression10. They have been described as selfish elements, tending to propagate on mobile genetic elements and promoting their own survival13. They are often associated with mobility genes, such as integrases or transposases and are sometimes flanked by repeats14. Indeed, they have been shown to have an important role in genetic flux among bacteria, as cleavage by REases provides fragments of double-stranded DNA that could be incorporated into the host genome15.10. The variation in the number of repeat copies in homopolymers or short tandem repeats, mainly due to slipped-strand mispairing during DNA replication, can cause the switch between a functional, non-functional or a different version of the gene9. It has been hypothesized that phase variation can be used by RMS to regulate genome flux8 and it has been shown to be associated with a random switching in the expression of multiple genes  in Haemophilus influenzae18, N. meningitidis19, N. gonorrhoeae2, Helicobacter pylori20, and Moraxella catarrhalis21. Apart from the Type III RMS, the Type II ‘orphan’ Dam MTase has also been shown to be involved in gene regulation in several Gram-negative bacteria9 and is even required for virulence in some pathogenic organisms such as Escherichia coli, Salmonella, Yersinia or Vibrio species22. The Dam MTase participates in the methyl-directed DNA mismatch repair system and this constrains the flexibility of the bacteria to undergo phase variation, which involves mispairing of bases during replication slippage23. It has been found in some N. lactamica and N. meningitidis strains24, however, there have been no reports of N. gonorrhoeae carrying this enzyme until now. Instead, the gonococcus and other Dam-defective Neisseria contain a dam replacing gene (drg), characterized as an endonuclease24, that has been reported to be important for adhesion and biofilm formation23.The action of some Type I and III RMS is known to be regulated by phase variation in several bacteriaN. gonorrhoeae strains using SMRT PacBio data. Results from this study give a more comprehensive insight into the link between genomics and epigenomics in the gonococcus.In this study, we characterised the population of RMS and their associated methylation specificities in 25 N. gonorrhoeae strains included in the study . An insertion of a T at position 186 of the gene creates a frameshift that causes a premature stop codon in position 62 of the protein, inactivating hsdS, and thus, the whole RMS  is found from the PacBio data (Supplementary Table Only one methylation target is currently shown for NgoAV in REBASE (5′-GCAN{8}TGC-3′). However, we also observed two other methylation targets associated with this RMS. Two strains with 1 LEAT repeat and no frameshift showed two-strand 6mA methylation in the 5′-GACN{6}TGC-3′ motif  in WHO V  contained a homologous region to NgoAXIII. Instead, genomes of N. lactamica  showed a longer version of the MTase (362 amino acids compared to 112 in N. gonorrhoeae FA1090 and 133 in the rest). This shows that N. gonorrhoeae does not have a complete NgoAXIII system, but only remnants of a MTase. Interestingly, several N. meningitidis genomes contain the VSR protein followed by a different RMS instead (NmeDI)28.RMS NgoAXIII was found as a VSR (Very Short patch Repair) protein followed by a MTase and there is no report of its target motif in REBASE. A previous study experimentally confirmed that this MTase is inactive in FA1090CGGTTNNC-3′ motif was detected by the PacBio SMRT pipeline containing single-strand m4C methylation in the underlined base. The study of the distribution of per-base IPD ratios in the WHO strains revealed that the complementary sequence was probably also methylated at 3′-TTTNGCCAANNG-5′  and a specificity unit (S). A BLASTn search of the genomic region spanning both units against the NCBI database revealed that this RMS has not been described in any other organism. A transposase immediately upstream of the RM unit indicates that this system may be mobile and acquired from an unknown source. The target motif in REBASE for this RMS is that of BcgI, 5′-GACN{5}TGA-3′, although we do not detect this motif as methylated or any other motif with 6mA methylation that could be associated with NgoAVIII , which showed double-strand 6mA 5′-GATC-3′ methylation  showed high IPDs in the underlined base . A low fraction of motifs with high IPDs in the first A of 5′-GCAGA-3′ was found in the strains with an active Type I NgoAV, and this may be due to overlapping signals between both RMS . An assembly of the PacBio reads from which the methylation data was obtained for this strain6 revealed that mod in the NgoAXII RM system in the subclone of FA1090 used for sequencing in the cited work contained 35 AGCC repeats instead of 37 in the reference genome, causing a protein truncation and thus a lack of 5′-GCAGA-3′ methylation. This difference in the number of repeats was caused by phase variation probably during subculturing, and inactivation of the mod gene has been associated with a change in gene expression17.Strangely, results from the motif analysis in FA1090 did not show signals of 5′-GCAGA-3′ methylation, although the 10 in this situation. However, methylases can be functional with a disrupted or absent endonuclease. These methylases have been associated with a regulatory rather than defensive role and have been described as ‘orphan’. In the strains under study we observed multiple examples of this in the Type I NgoAXVII  showed an orphan MTase in this element that has a 100% amino acid and nucleotide identity to the Type II M.Ngo8107ORF11P and M.Ngo5289ORFAP enzymes in REBASE  were detected in strains with a functional RMS. The change in specificity was found to be related to a combination of two factors: the number of LEAT repeats in the protein sequence of hsdS and the presence of the downstream frameshift that causes the previously-described truncation4 36 and has also been described in previous works in N. gonorrhoeae (LEAT or EATL repeats)35, but not its role in sequence specificity in combination with the downstream frameshift. Off-target methylated motifs were associated with the other Type I RMS NgoAXVII, although no associated variability was found in hsdS in this system.The analysed gonococcal genomes showed from 13 to 15 complete RMS, all of them described in the reference strain FA1090 in REBASEN. gonorrhoeae are associated with cytosine methylation. However, this type of DNA modification is difficult to detect by current PacBio sequencing, and high coverage in combination with Tet-conversion is preferable37. Only three motifs associated with 5mC or 4mC methylation were detected in a subset of the strains  recognizing the 5′-GATC-3′ motif39. Here, we observed two N. gonorrhoeae strains (NCTC10931 and NCTC12700) carrying the dam MTase gene instead of the drg gene in the NgoAXI RMS locus, followed by a truncated MTase. Thus, Dam is acting as an orphan enzyme, a form that has also been shown to regulate gene expression40. Interestingly, previous publications have suggested that strains carrying the drg gene have an advantage over those carrying dam because they have more flexibility for phase-variation, as the Dam MTase participates in DNA mismatch repair during replication39. Also, the same publications experimentally showed that strains carrying drg form more stable biofilms and have better adhesion to human cells during infection.The Dam methylase is present in some strains of N. gonorrhoeae and N. meningitidis, referred to the set of genes with an altered expression due to phase variation in a MTase17. Type I NgoAV has been considered to regulate a phasevarion as the phase variation of the G homopolymer is involved in a change in specificity16. In our work, we complement this result, as we show that the change in specificity is produced as a combination of the phase-variable poly-G and the number of LEAT repeats27. However, the best examples of phasevarion are the phase-variable Type III RMS10, in which for example modD1 has been associated with hypervirulent N. meningitidis clonal complexes19 and modA13 with enhanced biofilm formation and intracellular survival16. In a recent publication, the NgoAX Mod was experimentally inactivated in N. gonorrhoeae and a deregulation of the expression of 121 genes was observed, along with an effect on the adherence to and invasion of host epithelial cells, which are essential for infection5. In contrast, inactivation of the NgoAXII Mod enzyme showed a deregulation of 54 genes under iron-limiting conditions2. The N. gonorrhoeae population has been reported to carry a very conserved modB1 allele compared to modA13, which has led to the suggestion that NgoAX Mod is the main regulator of the epigenome, while NgoAXII regulates in specific growth conditions5. Here, we observe that indeed all the strains under study carry the modA13 and modB1 alleles, with all possible combinations of activity apparent: one of the two Mod active (methylates the target motif), both active or both inactive  avoiding overlap with the WHO panel, to further improve the representativeness of the N. gonorrhoeae genome diversity .We analysed the genomes of a total of 25 fuzznuc43. Per-base IPD ratios for the predicted motifs were extracted from the raw data and visualized in the 25 strains using R44.Genome-wide base modifications and predicted modified motifs were called using the RS_Modification_and_Motif_Analysis protocol from the PacBio SMRT Analysis software v2.3.0. Coordinates of the predicted motifs were localized in all the genomes and plasmids using the EMBOSS application 34 compared to that of unmethylated cytosines. A Mann-Whitney test was performed to evaluate statistical significance and p-values were corrected using Bonferroni45.The distribution of IPD values for each of the four bases outside any of the predicted motifs was used as the distribution of values for the four unmethylated bases. A random subsample of 10,000 unmethylated sites of each base was used. Cytosine methylation from Type II RMS was inferred by evaluating the distribution of IPD ratios in the target base in the associated motif in REBASE46 was used to detect these genes in the annotations of the genomes under study. HMMER47hmmscan was run independently on the proteome of each genome (.faa files) against Pfam(A) v3048 to complement the annotation information. R language44 was used to parse the annotation files and the results from hmmscan and extract genes with an inferred Pfam domain in the list of target REases and MTases. Genes predicted to be an REase or an MTase that were less than 10 genes distant were considered to be part of the same RMS. The rest were tagged as ‘orphan’. Results were compared to the RMS annotated for FA1090 in REBASE34. The presence or absence of every RMS in each strain was visualized using phandango49.An extended and manually curated list of Pfam domains associated with REases and MTases from a previous work34 for each MTase was compared to the list of predicted motifs obtained by the PacBio SMRT Analysis software for all the strains. In REBASE, many methylation targets are inferred across RMS that have similar amino acid sequences. However, a small change in the sequence of the DNA recognition domain of a methylase or the specificity unit among species with orthologous RMS can alter the sequence that is methylated. This means that matching the methylated motifs obtained by PacBio with the annotated methylases in REBASE is sometimes not straightforward and new methylation signals can be found that are associated with existing annotated methylases. These cases were further investigated and assigned to a RMS using the genomic data and according to the characteristics of the newly-found methylated motifs 10. Nucleotide and protein sequences of every unit of the RMS were extracted for each strain and compared using SeaView v4.6.150 to look for sources of variability within the MTases or the specificity units. These and the flanking genes were visualized using Artemis v16.0.051. Disrupted proteins were confirmed by a protein-protein BLAST against a non-redundant protein database52.The motif annotated in REBASENeisseria genus were downloaded from the Identical Protein Groups (IPG) tool of the NCBI database (accessed on 21/01/2019). These sequences were aligned using SeaView v4.6.150 and the resulting alignment trimmed using Gblocks v0.91b53 considering only positions in which at least half of the sequences do not have a gap. PhyML54 was used to build a maximum likelihood tree using the LG model and performing 100 bootstrap replicates. Final tree and metadata were visualised using iToL55.Representative protein sequences of the Dam methylase in the topGO R package56. The three ontologies were scanned . The classic and weight01 algorithms were used which do not, and do, use hierarchy information for scoring a particular GO term, respectively57. The statistical significance of the enrichment was calculated using a Fisher’s exact test between the observed and expected number of genes in each term for each algorithm. Terms with a p-value < 0.05 in both algorithms were considered as significant in the results. P-values were not corrected for multiple testing to avoid excluding significant GO terms near the cut-off. Besides, the tests are not independent when using the weight01 algorithm as it is conditioned on neighbouring terms56.A Gene Ontology (GO) enrichment analysis was performed for the genes flanking all the RMS using the Supplementary InformationSupplementary Table 3


Primary prostatic signet ring cell carcinoma is a rare form of cancer with a poor prognosis, which is generally treated with a traditional prostate adenocarcinoma therapy. This case report presents a 70-year-old diagnosed with primary prostatic signet ring cell carcinoma, treated with a combination of radiotherapy and hormone therapy and a 16 month survival without an evidence of the disease at follow up. Signet ring cell carcinoma (SRCC) is most frequently observed in the stomach and colon. It occurs less frequently in the pancreas, breast, thyroid and bladder. Primary SRCC of prostate is quite rare. Until recently, approximately 60 cases with primary prostate SRCC have been presented in the literature. The prognosis of primary prostate SRCC is poor with no clear treatment approach due to low number of cases . This reA 70-year-old patient with lower urinary tract symptoms and a PSA level of 7.26 ng/mL was presented at the hospital. The digital rectal examination did not reveal any hardness or nodules. A 12-core prostate biopsy revealed a Gleason grade 5 + 5 prostate adenocarcinoma in all quadrants and a signet ring cell component in nearly half of all quadrants A. ImmunoThe signet ring appearance in the cells occurs because the nucleus is pushed to the periphery of the cell by large intracytoplasmic vacuoles. SRCC is most commonly observed in the GI tract. Therefore, when SRCC is detected in the prostate, endoscopy, colonoscopy and abdominal CT scan are needed to exclude metastasis. This case presented here did not showed any GI tract pathological signs. Based on these findings we made the diagnosis of primary prostatic SRCC. Some studies stated that signet ring cells must constitute of at least 20–25% of the tumor to be able to have a diagnosis of primary prostatic SRCC, although other studies stated that a certain ratio of cells was not needed for diagnosis ,4,5. In In the literature, the median age for prostatic SRCC is around 68 years, which is comparable to the currently reported 70 years ,2. At thDue to the rare frequency of prostate SRCC, no standard treatment management is available. The literature shows that most primary prostatic SRCCs are treated with hormone therapy (HT), radiotherapy (RT) and radical prostatectomy (RP) or combinations of these therapies ,4,5,6,7.There is no standardized therapy for prostate SRCC due to the fact that it is a rare disease. However, application of an aggressive multi-modal therapy seems reasonable because of the poor prognosis of the disease. Radiotherapy combination applied together with hormonal therapy may be an appropriate alternative therapy for prostate SRCC treatment.


Dilatation of the cerebral ventricles is a common condition in preterm neonates with intraventricular hemorrhage. This posthemorrhagic ventricle dilatation (PHVD) can lead to lifelong neurological impairment through ischemic injury due to increased intracranial pressure, and without treatment can lead to death. Two-dimensional ultrasound (US) through the fontanelles of the patients is serially acquired to monitor the progression of PHVD. These images are used in conjunction with clinical experience and physical exams to determine when interventional therapies such as needle aspiration of the built up cerebrospinal fluid  might be indicated for a patient; however, quantitative measurements of the ventricles size are often not performed. We describe the potential utility of the quantitative three-dimensional (3-D) US measurements of ventricle volumes (VVs) in 38 preterm neonates to monitor and manage PHVD. Specifically, we determined 3-D US VV thresholds for patients who received VT in comparison to patients with PHVD who resolve without intervention. In addition, since many patients who have an initial VT will receive subsequent interventions, we determined which PHVD patients will receive additional VT after the initial one has been performed. Intraventricular hemorrhage (IVH), bleeding inside the cerebral ventricles, has been decreasing in prevalence yet remains as a common morbidity among preterm born neonates.,While the definitive treatment for PHH is the placement of a ventriculoperitoneal shunt (tubing between the ventricles and the abdomen), placement of the shunt is often delayed by several weeks to allow for the blood clot to resolve, the CSF proteins to decrease  are used to determine when neonates receive temporary interventions to removed built up CSF.–,,,,,–in vivo against the volume of CSF withdrawn during a tap as well as against MRI.Previous work with transfontanelle brain three-dimensional (3-D) US has been attempted in neonates.In this paper, we present for the first time a preliminary study used to examine whether a 3-D US-based VV and VV change measurements allow us to prognosticate, which infants with PHVD will receive a temporary intervention and who will have a spontaneous resolution of PHVD.2As part of a larger study, between April 2012 and May 2016, after an initial diagnosis of IVHInterventions were performed based on clinical judgement, with the clinician using the head circumference growth over time, the increase in ventricular dilatation on the 2-D US, as well as sometimes, but not necessarily, clinical signs and symptoms of increased ICP, such as apnea, bradycardia, and a full, tense fontanelle. Interventions were not based on 3-D US images collected during the study, as the clinical team was blinded to all 3-D US images during the course of the stay in the neonatal intensive care unit (NICU). The initial intervention at our center is a ventricular tap (VT) since it can be done at the bedside and does not require an operating room (whereas the Ommaya catheter insertion does); but if taps are thought to be needed frequently, then an external ventricle drain is usually inserted. For the purpose of this study, only patients born 2.1Images were acquired using a Philips HDI 5000 US machine with a C8-5 transducer. 3-D US imaging was performed after placing the US transducer in a handheld motorized housing that tilted the transducer about the axis at the probe tip for 8 to 9 s.During image acquisition, 2-D US images were collected on a laptop computer as the motor tilted the transducer over 9.6 s and in-house software reconstructed the 3-D US image in real-time as images were received into the computer. Technical details can be found in Ref. 2.2The lateral ventricles were manually segmented in parallel sagittal slices with 1 mm gaps between adjacent slices. A trained observer (JK) segmented all the images and a pediatric neurosurgeon verified the boundaries (SdR). Each image required between 20 and 45 min to segment. The 3-D US imaging system and ventricle segmentation have been previously validated using phantom and patient images.2.3We performed the analysis of patients who did and did not receive interventional therapies for PHVD. The VV for each scan was recorded during the first month of life for each patient as well as whether or not they eventually received a VT.2.3.1The VV for age ranges 2.3.2We calculated the rate of change of VV between two scans, and analyzed it for the first week  were generated among patients who did and did not receive interventions using 3-D US-based VVs. In the second study in patients who had previous interventions, ROC analysis was performed using 3-D US-based VVs, among patients who did not receive further interventions, and those that received at least one additional intervention. For each parameter (volume and volume change), optimal threshold for intervention was estimated using the highest product of sensitivity and specificity. In addition, the area under the ROC curve (AUC), sensitivity, and specificity were calculated. Leave-one-out thresholds were generated using the same method. Assuming a Bonferroni correction, two-sided 33.1Thirty-eight neonates were enrolled prior to the second week of life (before most patients with PHVD would receive an intervention) and were used to determine thresholds in this study. Of those, 14 patients received at least one VT. Mean age of enrolment was 9 days of life, and 12 patients were enrolled in the first week of life . Descriptions of the all study patients are shown in 3.23.2.1The patients who had interventions saw a continued increase in VV at every time interval and this can be seen in 3.2.2The rate of change in VV was highly sensitive for detecting which patients received interventions (100% sensitivity) during all age intervals monitored . The ROCy of 75% . From day of 75% . The thr studied .3.3For this study, we examined only the patients who already had a previous VT. Of the 11 patients with a previous VT, four resolved clinically without further interventions during their NICU stay. cle size . The thr of 100% .3.4For patients who did not require interventions , only 1 4In our preliminary study, we presented the potential for 3-D US-based measurements of VVs from neonatal brain images to characterize whether or not patients with PHVD will receive interventions. Specifically, neonates who go on to receive interventions have larger VVs, {mean  of life }. The paIn addition, we were able to show that our reported thresholds were able to predict that a neonate would receive an intervention 7 days before the use of the VI 97th centile +4  mm . This co3-D US-based VV acutely remains very high in the week following an intervention regardless of whether or not the patient’s PHVD is resolving (interventional patients had VV of Previous mathematical estimates of VV based on 2-D US images found that there was a significant difference between patients who received interventions and those who did not. 27 days . Brann eNeonatal patients in the NICU are also at risk of having prenatal ventriculomegaly and this finding could be indicative of abnormalities such as neural tube defects or trisomy, which require follow-up after birth. For these neonates with larger than normal ventricles, the rate of change in VV might be a better indicator of which patients have progressive VD and not just large ventricles, which are stable in volume and are likely not to contribute to signs and symptoms of increases in ICP. Therefore, while our study did not include patients with prenatal ventriculomegaly, 3-D US VV should be examined in a cohort of patients with this indication, as it could impact how these patients’ care is managed.,The relative difficulty of generating VV through manual contouring a 3-D US image might prove too difficult for translation into clinical practice and this is a limitation of the current work. In light of this and to reduce the burden and length of time of manual ventricles segmentation from 3-D US images, we have developed both a semiautomated segmentation algorithm that can be used online at the bedside and an automated segmentation algorithm that can be run offline with no user input.In segmenting the ventricles, the user did not use a purely boundary-based method as in some regions the boundaries can be difficult to detect or even not present. Since the user does not see a boundary all the time, they use prior knowledge of what shape the ventricle should be. As such, a combined approach that uses intensity information and searching for boundaries while including a shape prior has been our most successful algorithm.Additional limitations of this work include the use of VT as the initial intervention and not using quantitative US measurements to determine interventional necessity. There is a wide variation among centers in whether the intervention is performed with lumbar puncture, VTs, ventricular reservoirs, third ventriculostomy, subgaleal shunts, and/or external ventricle drains. Further confounding the use of image-based metrics, 3-D US might be easier or harder to obtain depending on the ease of access to the fontanelle after different types of treatments have been performed. Furthermore, the criteria for intervention in this study are based on clinical judgment with multiple clinicians deciding on whether or not to tap, and not on quantitative measurements, which could hinder the reproducibility depending on the degree of clinical variation in practice. These limitations and questions of the validity of 3-D US-based VV in this patient population can only be elucidated in a larger study to account for greater variation in clinical practice.As such, we have presented a potential clinical use for 3-D US within the NICU. This type of imaging appears to be promising to be able to help generate management strategies for neonates at risk of PHVD and the subsequent potential brain damage from elevated ICP. A larger, multicenter study is required to generate guidelines as this will increase the patient pool and better account for differences in clinical practice that occur in different centers.


CADASIL is a monogenic small vessel disease characterized by the accumulation of brain tissue lesions of microvascular origin leading to strokes and cognitive deficits. Both cortical and parenchymal alterations have been described using various MRI markers. However, relationships between cortical and subcortical alterations remain largely unexplored. While brain atrophy is a preponderant feature in cerebral small vessel disease, recent results in CADASIL suggest slightly larger brain volumes and increased white matter water content at early stages of the disease by comparison to controls. We hypothesized in this study that increased water content in gyral white matter balances expected brain atrophy. Direct white matter volume computation is challenging in these patients given widespread subcortical alterations. Instead, our approach was that a gyral white matter swelling would translate into a modification of the shape of cortical gyri. Our goal was then to assess the relationship between subcortical lesions and possible alteration of the cortex shape. More specifically, aims of this work were to assess 1) morphometric differences of the cortex shape between CADASIL patients and controls 2) the relationship between the cortex shape and the volume of white matter hyperintensities (WMH), a reflect of white matter alterations. Twenty-one patients at the early stage of the disease and 28 age- and sex-matched controls were included. Cortical surfaces were reconstructed from 3D-T1-weighted images. Folding power assessed from spectral analysis of gyrification and cortical morphometry using curvedness and shape index were computed as proxies of the cortex shape. Influence of segmentation errors were evaluated through the simulation of WMH in controls. As a result, patients had larger folding power and curvedness compared to controls. They also presented lower shape indices both related to sulci and gyri. In patients, the volume of WMH was associated with decreased gyral shape index. These results suggest that the cortex shape of CADASIL patients is different compared to controls and that the enlargement of gyri is related to the extent of white matter alterations. The study of the cortex shape might be another way to evaluate subcortical swelling or atrophy in various neurological disorders. NOTCH3 mutations, characterized after a normal brain development, by the accumulation of brain tissue lesions of microvascular origin as soon as the 3rd or 4th decade  is a monogenic brain disorder, due to h decade . While th decade . We alsoGiven that tissue lesions accumulate mainly in deep brain areas, while the increase of white matter water content seems to predominate in certain juxtacortical areas, we aimed to determine whether the gyral white matter was actually increased when compared to age- and sex-matched controls. Unfortunately, the gray to white matter tissue contrast may be altered in CADASIL  and the While these fine shape alterations are out of reach of advanced computational tools such as cortical thickness estimation, gyrification indices or sulcal morphometry previously used in CADASIL , 8 Figu, it can Finally, to ensure that these potential cortical alterations are actually secondary to the disease evolution and not due to innate differences with controls, we tested the relationships between cortical metrics and white matter hyperintensities (WMH), a well admitted reflect of white matter alterations in this disorder.The aims of this work were then to (1) detect subtle morphometric differences between CADASIL patients and age- and sex-matched controls using geometrical and spectral descriptors of the cortex shape (2) if present, to infer the relationship between cortex shape changes in patients and the volume of WMH.We included in our analysis other factors that may impact the cortex shape to better highlight the independent effect of WMH. Thus, volume of lacunes were computed given their potential role in cortical thinning and on macroscopic cortical abnormalities through secondary cortical neurodegeneration , 15. AlsNon-demented non-disabled  adult CADASIL patients at the early clinical stage of the disease were recruited from the CADASIL cohort followed in the National Referral Center for this disorder. Controls were drawn from a local database of healthy volunteers free of any known history of neurological disorder and without symptoms of cognitive impairment or disability as evaluated by a structured interview. Twenty-one patients and 28 controls having good image and cortical surface reconstruction qualities were included in this study.This study was carried out in accordance with the recommendations of the ethics committee of DRCD (Département de la Recherche Clinique et du Développement) of AP-HP (Assistance Publique-Hôpitaux de Paris) with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the DRCD of AP-HP.1-weighted images used for cortical surface reconstruction were obtained at 3 Tesla with a Tim-Trio MRI scanner  equipped with a 12-channel head coil, using a standard sagittal magnetization-prepared rapid acquisition gradient echo (MPRAGE) sequence . Volumes of lacunes and of WMH of presumed vascular origin were determined from 3D T1-weighted and FLAIR images obtained within 6 months on a 1.5T Signa scanner .3D TThe analysis was restricted to the left hemisphere. All subjects were right-handed.Delineation of WMH and of lacunes was performed according to the STRIVE criteria . Masks ohttp://www.brainvisa.info) was used to automatically segment the left cortical surfaces (mesh of the gray-white interface) from 3D T1-weighted images  was defined as the ratio of surface area to hull area (GGI = SA / HA) and the spherical gyrification index (SGI) was calculated as the ratio of surface area to the area of a sphere of volume equals to the brain volume (SGI = SA/4π *(3BV/4π)2/3) 2/3) . The norAFP) is a global characteristic of the hemispheric gyrification, very correlated to the sum of mean curvatures squared. This power is divided into 7 spectral bands with bandwidths of increasing (doubling) spatial frequency. The 4th, 5th, and 6th bands  gather more than 95% of total power and are associated with the variations of curvature related to the sulcal pattern  and shape index (SI) were computed vertex-wise in each left cortical surface as follows, given the two principal curvatures k1 and k2:Curvedness . The morphometric descriptors of the cortex shape were computed on lateral faces only.As segmentation errors were recurrent on the medial face of the cortical surface of patients due to periventricular WMH, we defined a lateral face by delineating a curve separating medial and lateral faces using Surfpaint Toolbox  in AnatoN = 11) were selected. The 11 patients were sex- and age-matched to 11 controls. Then, 3D T1-weighted images of patients were non-linearly co-registered to 3D T1-weighted images of matched controls using FNIRT from FSL (www.fmrib.ox.ac.uk/fsl)  . The mathttp://www.r-project.org/). Between-group comparisons were performed using t-tests or X2 tests according to variable type and linear regression models including age, gender and brain volume as covariates  and paired between one control subject and one control subject with simulated WMH  than controls. This increase in power divided up between the three sulcal bands, yet the difference between patient and control was significant only for B5 and B6. This indicates that the cortical folding of CADASIL patients required more high frequency components than the cortex of controls. Consistently, we found an increased curvedness c in patients, reflecting a more bended, sharper folding. The shape index SI brought complementary information: the shape of both sulci and gyri were different as s1 and s2 were smaller in patients compared to controls. Schematically, sulci were narrower while gyri were widened in patients  and the volume of WMH, independently of age, gender, and other imaging parameters known to alter the cortex in CADASIL, namely the volume of lacune and normalized brain volume. There were no significant associations between AFP, c or s1 and the volume of WMH. Gyral shape index descriptor s2 was found to decrease with increasing volume of WMH .To determine whether potential differences in cortex shape were actually related to disease related mechanisms, we tested via a linear model the association between each cortex morphometric descriptor , we found a significant but small effect on c and s1 . In averMH on s2 , the parIn the present study, we found that the cortex of CADASIL patients was characterized by a surface that is more sharp and more bended through both the spectral and geometrical analyses, with a difference in global shape of folds toward somehow “tightened” sulci and “expanded” gyri. Also, the level of alteration of the gyral shape descriptor was significantly associated with the extent of WMH that reflects the amount of white matter tissue changes in CADASIL. Altogether, these results support the hypothesis of a gyral white matter swelling.s2) and adjacent sulci tighten (decreased negative s1) due to lateral compression. This hypothesis could be modeled by a combination of the recent mechanical model by Tallinen et al. . Indeed in Fourier-like decomposition, higher frequencies not only contribute to periodic patterning but also to sharpness rendering. Another explanation is that the small increase in global folding power really divides-up into the three sulcal bands because it is a global effect not related to an increase in gyrification complexity nor an extension of the folding pattern.Our main hypothesis to explain these results is mechanical: the white matter swelling would be “pushing” the cortex so that its shape is slightly modified. As represented in n et al.  and the n et al.  that is s2) or much smaller  than the differences observed between groups. These results were convincing given that the simulated WMH produce much more obvious errors than our original data. Therefore, we could conclude that the observed differences were mainly driven by the disease while we could not exclude a small effect of segmentation errors on c and s1.By simulating WMH in cerebral images of controls, we have shown that segmentation errors due to uncorrected WMH near the cortex could slightly change the values of some descriptors of the cortex shape we used. However, these changes were non-significant .FD and EJ: study concept and design. EJ: acquisition of data. FD, DG, JL, CF, and EJ: analysis and interpretation of the data. FD and EJ: drafting of the manuscript. HC and EJ: study supervision. All authors: critical revision of the manuscript.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.


According to the September 2015 Institute of Medicine report, Improving Diagnosis in Health Care, each of us is likely to experience one diagnostic error in our lifetime, often with devastating consequences. Traditionally, diagnostic decision making has been the sole responsibility of an individual clinician. However, diagnosis involves an interaction among interprofessional team members with different training, skills, cultures, knowledge, and backgrounds. Moreover, diagnostic error is prevalent in the interruption-prone environment, such as the emergency department, where the loss of information may hinder a correct diagnosis.The overall purpose of this protocol is to improve team-based diagnostic decision making by focusing on data analytics and informatics tools that improve collective information management.To achieve this goal, we will identify the factors contributing to failures in team-based diagnostic decision making (aim 1), understand the barriers of using current health information technology tools for team collaboration (aim 2), and develop and evaluate a collaborative decision-making prototype that can improve team-based diagnostic decision making (aim 3).Between 2019 to 2020, we are collecting data for this study. The results are anticipated to be published between 2020 and 2021.The results from this study can shed light on improving diagnostic decision making by incorporating diagnostics rationale from team members. We believe a positive direction to move forward in solving diagnostic errors is by incorporating all team members, and using informatics.DERR1-10.2196/16047 Americans experience at least one diagnostic error in their lifetime, sometimes with devastating consequences (Institute of Medicine [IOM] report 2015). Lack of timely attention to diagnostic error can have dire implications for public health, as exemplified by the widely reported diagnostic error regarding Ebola virus infection in a Dallas hospital emergency department (ED) -6. Thus,The team-based diagnostic approach has the potential to reduce errors. Although the current diagnostic process is often the responsibility of an individual clinician, ideally the diagnostic process involves collaboration among multiple health care professionals . To manaResearch has shown that technology can positively impact provider interactions and coordination, helping group dynamics and efficiency . VariousThe informal information in a real-time workspace can help the team to communicate and interpret vital information with each other, which can improve team-based diagnostic decision making in the ED by reducing the loss of information. The objective of our study is to develop a collaborative prototype for improving team diagnostic decision making using an informatics approach.We want to focus on all types of diagnosis for adult patients who come to the ED in both the trauma and medicine units. This will ensure that we can generalize the future prototype for all ED patients. The overall methodology is described in the following 3 aims.Aim 1: identify factors contributing to failures in team-based diagnostic decision makingAim 2: understand the barriers in using health information technology (IT) tools for team collaborationAim 3: design and evaluate a collaborative decision-making prototypeThe research questions are as follows: (1) What are the specific diagnostic workflow processes that are vulnerable to failures in information gathering, integrating, interpreting, and establishing an explanation of the correct diagnosis? and (2) What specific information cues do teams share with each other to reach a diagnosis collaboratively?We will use the combination of direct observation, hierarchical task analysis (HTA), and health care failure mode and effect analysis (HFMEA) to analyze team tasks in the diagnosis process -33. HTA Step 1: observe scenarios in ED settings and transcribe the scenarios from audio recordingsStep 2: use data from the transcription to create HTA process mapsStep 3: conduct HFMEA to identify failures and improvement strategiesThe observation will start once the patient is admitted in the ED. A total of 2 research assistants will simultaneously observe the ED nurse and the attending physician. The observations will be nonintrusive, and researchers will turn on audio recorders only when the team is discussing or communicating with each other regarding the patient case -40. We wfinding diagnosis, we will merge nursing goal of finding patients home medication history as a subgoal under finding diagnosis. We will focus on the main goals associated with finding the correct diagnosis and represent the associated task steps to accomplish those goals in a hierarchical decision tree  for patient safety, information loss, misinterpretation, group conflict, and factors associated with poor communication. The team will also discuss additional failure-prone task steps found in step 2 to find potential solutions. The team will rate the severity score  for each failure-prone task step as minor (score 1), moderate, major, and catastrophic (score 4). Then, the team will also rate the probability of the occurrence of such incidents on a scale of 1 to 4 as remote (score 1: happening rarely in 2 years), uncommon (once a year), occasional (every 3-6 months), or frequent (score 4: every month). We will combine the severity and probability scores to obtain a hazard score. We will focus only on subtasks with hazard scores of 5 or greater to identify potential solutions. Finally, the team will be asked to find potential solutions, including health IT interventions, that can improve the team communication and team diagnostic decision-making process. The final results will be shown as in Each brainstorming session will be limited to 50 min, will be audio recorded and transcribed, and will occur over multiple sessions. The principal investigator will conduct a final data analysis of the transcripts to identify the high failure-prone task steps and possible solutions.We will recruit 4 ED physicians and 4 ED nurses for the observation study to increase provider diversity. For the HFMEA part of the study, we will recruit 2 ED physicians, 2 ED nurse, and 2 ED residents. A total of 14 providers will be recruited from 3 hospital sites by email and telephone, and a US $50 gift card will be provided for participation.On the basis of our pilot study sample size, we will observe 40 patient cases. We will include only adults (aged >18 years) for selecting cases. We will observe each scenario until the team reaches a consensus about the diagnosis. Previous studies have observed 32 to 50 cases for reaching data saturation ,45-47.For this aim, we will assume the ED team includes the attending physician and the attending nurse. However, we will include senior and junior-level residents, radiology physicians, other nursing staff, pharmacists, and support staff based on the makeup of that current team on that particular shift.The HTA and HFMEA methods are time consuming, specifically observation, construction of the HTA, and data analysis. However, a 3-year timeline is reasonable. In addition, there may be concern that step 2 (HTA process maps) may not generate adequate failure-prone steps. However, step 3 (HFMEA) brainstorming session by the group will also identify failure-prone steps in addition to discussing failure-prone steps found in the HTA process maps and will complement each other.What are the barriers to sharing information using current health IT tools? and (2) What are the leverage points  ion path ) for theconflict ?We will conduct a Critical Incident Technique (CIT)–based team Cognitive Task Analysis (CTA) interview -53. CTA We will ask the team members to describe a recent complex case that was challenging to solve as a team for an admitted patient. Experiences related to critical incidents in interprofessional teamwork will be evoked by asking open-ended questions: “Are there any difficulties or challenges involved in working together using the current health IT tools?” followed by “Can you describe a situation that you remember in detail when you experienced such a difficulty?” Once the situation is established with time-specific detail, follow-up questions and probes will be asked to elicit the team’s dynamic decision-making strategies to negotiate conflicts, the specific actions by each team member, and the process by which the problem was solved. We will focus on how team members prioritize and rank patient information to negotiate conflict to reach consensus.We will recruit 5 ED teams by email and telephone. Each team will consist of 4 clinicians, including 1 attending ED physician, 1 ED nurse, 1 ED resident, and 1 ED pharmacist. Inclusion criteria will be at least 1-year experience as a team member and a recent (within last 3 months) experience in working in the ED. Each clinician will receive a US $50 gift card for participation.We will use the transcripts from the audio recordings of the interviews for data analysis. All patient identifiers will be removed.The study measures are as follows: (1) cues and patterns of the team members’ preferences for using current health IT tools, (2) leverage points (cues related to shared and complementary cognition), (3) common sources of conflict and resolution strategies , and (4)A total of 2 investigators will independently code the transcripts from the team CTA interviews and merge the individual codes into subthemes and later into broader themes through a process of negotiated consensus. We will code based on a qualitative content analysis process -63. We wWe will interview 20 providers for the team CTA interviews. Previous studies used a range of 6 to 30 providers for successfully conducting similar team CTA interviews -67.CTA studies are based on memories. It can be difficult to explore past information, as key pieces of information may not be stored properly in the memory . TherefoWe will develop complex case vignettes, design the prototype, and conduct the usability study.We will design 8 complex clinical vignettes based on team-based diagnostic problems from our findings from aims 1 and 2 . We willunable to get key history becomes a major failure-prone task, then a separate tab should be created in the interface as pending information for patients). The results from aim 2 will provide specific design allocation for features such as knowledge characteristics  or expertise process requirements  and so on. For example, in this shared platform (possible diagnosis tab. Only the ED physician will be able to delete a diagnosis (shown as red strike-through in the diagnosis tab). Physicians and other team members can also assign tasks and group patients by waiting labs or completed (left side of the interface). This is an initial version only. The design will be refined based on aim 1, aim 2, and iterative design in aim 3 to ensure patient safety.The purpose of this prototype is to gather, integrate, and collect vital patient information from different team members to rank and filter information for making an informed diagnostic decision collaboratively. The results from aim 1 will inform design by allocating failure-prone task steps as the main focus in the interface  × 2 between (expertise) × 2 within (time pressure) factorial design. Each team will receive 8 vignettes presented in random order. The main effect of the presence or absence of the prototype tests the experimental question. With this design, we are also able to test for the interaction between the impact of the interface and the domain of expertise under time pressure. For example, the interface could change the interaction between time pressure and domain expertise (a 3-way interaction), eliminate the influence of time pressure overall for both ED expert and non-ED expert teams (2-way interaction), and have a main effect on quality for everyone in all conditions.To measure the effectiveness of decision making using the prototypesatisfaction level and System Usability Scale survey to understand the ease of use with the prototype. First, we will ask each provider to complete a Web-based user satisfaction survey to measure individual team members’ satisfaction for using the prototype [other successful fields when evaluating group decision support tools [We will conduct a team satisfaction survey to understand team members’ rototype . This terototype . Researcrototype . This surt tools -85. Datart tools . Finallyrt tools . Data andiagnostic accuracy, the presence of the correct diagnosis in the top 3 items of the diagnostic differential will be computed as a dichotomous (yes or no) variable. For example, if the clinical team correctly diagnoses the top 2 of the 3 diagnoses in the vignettes, it will be counted as yes. The overall team diagnostic decision quality will be an aggregate score created from the combination of the (1) correct diagnosis, (2) rating of the confidence of the final diagnosis , and (3) correctly ordered diagnostic tests. The overall score will range from 0 to 10, with correct diagnoses receiving 4 points and confidence ratings and correctly ordered tests receiving 3 points each.We will have 2 dependent variables, diagnostic accuracy and overall team diagnostic decision quality. For high time pressure as less than 3 min and low time pressure as less than 6 min.Time pressure is an independent variable because we will be assigning We will explain the procedure and ask participants to finish 4 cases under high time pressure (<3 min) and 4 cases under low time pressure (<6 min). Initially, all team members, the nurse, the resident, and the physician, will be distant and reviewing the case independently. They will use the decision-making prototype (loaded in laptops) to communicate among themselves for sharing information to establish an explanation for the diagnosis. They will have the final 1 min to discuss, as a group, the high time pressure cases and the final 2 min for low time pressure cases to reach consensus about the correct diagnosis. We will ask each team to rate their confidence in the diagnosis. We will also note the responsible team members who voice their concerns regarding each of the complex patient cases.We will use Chi-square test to evaluate association between the independent variables with the decision quality. We will use analysis of variance (ANOVA) to calculate the mean difference within and between ED expert teams’ and non-ED expert teams’ decision quality. The within- and between-group design will provide us with a sample size adequate for an ANOVA test. The proportion of decisions made with the correct diagnosis and overall decision quality will be shown as a percentage value using ANOVA. If the distribution is not normal, we will use the General Linear Model for the data analysis .The overall study measures are as follows: (1) providers’ comments about the initial design, (2) number of times assistance was required before and after demonstration, (3) scores for team decision quality, and (4) survey responses.We will recruit 12 teams with each team (6 ED experts and 6 non-ED experts) comprising a physician, a resident, and a nurse (36 providers: US $50 gift card will be provided) by emails and phone calls. The inclusion criterion for the ED team is that members should have at least 6 months’ experience working in the ED, and non-ED teams should include providers with expertise in other clinical domains.Previous studies successfully enrolled 7 to 36 providers for similar usability studies ,88-93. TReasonable efforts will be made to ensure the prototype realistically simulates a shared workspace for team collaboration. However, the assessment provides initial steps in understanding team diagnostic decision quality, serving as a foundation for future study in real-world situations.We are collecting preliminary data for this study between the period of 2019 and 2020. The results are expected to be published between 2020 and 2021.other successful fields in resolving conflict by providing a platform to coordinate team tasks.Studies have shown that uneven information can result from the exclusion of team members from messages or the failure of team members to share uniquely held information -96. StudThis protocol addresses the problem of diagnostic error through innovative approaches for reducing the loss of vital patient information and effectively sharing key information to form correct diagnosis as a team. The robustness of the methodology used in this protocol has been applied to other successful fields. Observation, HTA (aim 1), and team CTA (aim 2) methods have been applied in military, naval warfare, aviation, air traffic control, emergency services, and railway maintenance -105. ThiThe results from this study can shed light on improving diagnostic decision making by incorporating diagnostics rationale from team members. We believe a positive direction to move forward in solving diagnostic errors is by incorporating all team members, and using informatics.