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Respiratory syncytial virus (RSV) and Human metapneumovirus (hMPV), members of Pneumoviridae family are common causes of acute respiratory tract infections (ARTI) among children. Study material includes routine nasopharyngeal samples obtained during 8-year period for hMPV and one single season for RSV in children hospitalized for ARTI between 0 and 15 years at the Center Hospitalier Universitaire (CHU) Saint Pierre in Brussels. Positive samples for RSV or hMPV identified by viral culture, lateral flow chromatography test for RSV or direct fluorescent assay for hMPV were selected retrospectively. Characteristics of children hospitalized for RSV or hMPV infections were compared. Children hospitalized for RSV infection were significantly younger and requiring more respiratory support, longer hospital stay and transfers in Pediatric intensive Care Units than those hospitalized for hMPV infection. Pneumonia diagnostic and antibiotics therapies were more significantly associated with hMPV infections. In conclusion, despite their genetic similarities, RSV, and hMPV present epidemiological and clinical differences in pediatric infections. Our results should be confirmed prospectively.

Teamwork is essential in emergency medicine, but in practice it can be polluted by communication difficulties, a lack of understanding of everyone's roles and responsibilities, and a discordant definition of operating methods and objectives. Today, there is a strong awareness of the need to train medical and healthcare teams in interprofessional collaborative practice to learn how to work as a team, reduce medical errors and improve patient safety. Simulation is a recognized and effective pedagogical modality for achieving these objectives. It is now permanently established in pre- and postgraduate medical-nursing training courses in emergency medicine.Le travail en équipe est indispensable en médecine d’urgence mais, dans la pratique, il peut être pollué par des difficultés de communication, une méconnaissance des rôles et responsabilités de chacun, et une définition discordante des modes de fonctionnement et des objectifs. Aujourd’hui, il y a une forte prise de conscience de la nécessité de former les équipes médico­soignantes à la pratique collaborative interprofessionnelle pour apprendre à travailler en équipe, réduire les erreurs médicales et améliorer la sécurité des patient-e-s. La simulation est une modalité pédagogique reconnue et efficace pour atteindre ces objectifs. Elle est désormais implantée de façon pérenne dans les cursus de formation médico-soignante pré et postgraduée en médecine d’urgence.

Background Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors. Methods We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer. Results Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12V and KRASQ61H cells, respectively, 997 of which were shared (p M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS. Conclusion Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.

Since its discovery as an oncogene in 2014, mutations and amplifications in the small GTPase RIT1 (Ras-like in all tissues) have been associated with lung adenocarcinoma, myeloid malignancies, and uterine carcinosarcoma. Genome-sequencing studies in lung adenocarcinoma tumors have found that RIT1 is mutated in 2% of cases and amplified in another 14%. Several oncogenic mutations have been characterized, with the M90I variant (RIT1M90I) being most prevalent. Although targeted therapies have revolutionized the treatment of many cancers, there are no targeted therapies available for RIT1-driven diseases, and cytotoxic chemotherapy remains the only treatment option. Given its structure as a small GTPase, RIT1 itself may be difficult to drug, but identifying therapeutic vulnerabilities of RIT1-driven cancers could reveal druggable targets. Indeed, through CRISPR screening our lab recently identified the deubiquitinase USP9X as a potential regulator of RIT1 abundance and function. We hypothesize that USP9X positively regulates RIT1 protein abundance, meaning that USP9X inhibition would decrease RIT1 protein levels. It has previously been found that the protein abundance of RIT1 is important for its function, thus USP9X inhibitors could represent a novel targeted therapy for RIT1-driven diseases. There are currently two small molecule inhibitors—WP1130 and G9—that have been shown to target USP9X. We've tested these inhibitors in PC9 lung adenocarcinoma cells and found that WP1130 significantly reduced the abundance of endogenous wild-type RIT1 to 56.21% (p=0.0125, 95% CI = -71.96 to -15.61) compared to DMSO-treated cells. In a USP9X knockout (KO) PC9 cell line, treatment with WP1130 did not significantly affect RIT1 protein abundance (p=0.6001). These effects appear to be due to USP9X-targeted regulation of RIT1 given that the protein abundance of CDC20—a known target of USP9X—was reduced to 71.7% in response to WP1130 treatment (p=0.0380, 95% CI = -52.74 to -3.861), but CDC20 levels were not significantly affected in USP9X KO PC9 cells (p=0.4510). We have also tested G9 in PC9 cells and found a similar trend, but further work is ongoing to identify appropriate dosing of this inhibitor. In addition to testing the effects of WP1130 in PC9 cells, we also assessed how USP9X inhibition affects RIT1-mutant cells. For this we turned to the NCI-H2110 cell line, which is the only commercially available cell line harboring an endogenous RIT1M90I mutation. A dose response curve revealed that PC9 cells may be more sensitive to WP1130 than NCI-H2110 cells (IC50 in PC9 cells = 0.5059μM; IC50 in NCI-H2110 cells = 1.344 μM). This suggests that USP9X inhibition may be more effective at targeting wild-type RIT1, but this hypothesis must be further explored in vitro and in vivo. In summary, this work is poised to address a major unmet clinical need for the treatment of diseases characterized by RIT1 mutations and amplifications. USP9X inhibition could be a tractable means of reducing RIT1 protein abundance and abrogating the growth of RIT1-driven tumors. Citation Format: Amanda Riley, Athea Vichas, Alice Berger. Pharmacological inhibition of USP9X as a novel targeted therapy for RIT1-driven lung adenocarcinoma and other cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B028.

Respiratory syncytial virus (RSV) and Human metapneumovirus (hMPV), members of Pneumoviridae family are common causes of acute respiratory tract infections (ARTI) among children. Study material includes routine nasopharyngeal samples obtained during 8-year period for hMPV and one single season for RSV in children aged 0 to 15 years at the Centre Hospitalier Universitaire (CHU) Saint Pierre in Brussels. Positive samples for RSV or hMPV identified by viral culture, lateral flow chromatography test for RSV or direct fluorescent assay for hMPV were selected retrospectively. The medical charts of these patients were reviewed. Hospitalization rate was 37% (219/591) and 39% (187/476) for hMPV and RSV respectively. Children hospitalized for RSV infection were significantly younger and more dyspneic, requiring more respiratory support, longer hospital stay and transfers in Pediatric intensive Care Units (PICU) than those hospitalized for hMPV infection. Pneumonia diagnostic and antibiotics therapies were more significantly associated with hMPV infections.In conclusion, despite their genetic similarities, RSV and hMPV present epidemiological and clinical differences in pediatric infections.

New excavations conducted in the Circles Building (Granary) at Tel Bet Yerah, first excavated in 1946, form the basis for a revised, detailed description of the construction and use of this unique ...

International audience; Injection of recombinant tissue plasminogen activator (rt-PA) is the standard drug treatment for thrombolysis. However, rt-PA shows risk of hemorrhages and limited efficiency even at high doses. Polysaccharide-poly(isobutylcyanoacrylate) nanoparticles functionalized with fucoidan and loaded with rt-PA were designed to accumulate on the thrombus. Fucoidan has a nanomolar affinity for the P-selectin expressed by activated platelets in the thrombus. Solid spherical fluorescent nanoparticles with a hydrodynamic diameter of 136 ± 4 nm were synthesized by redox radical emulsion polymerization. The clinical rt-PA formulation was successfully loaded by adsorption on aminated nanoparticles and able to be released in vitro. We validated the in vitro fibrinolytic activity and binding under flow to both recombinant P-selectin and activated platelet aggregates. The thrombolysis efficiency was demonstrated in a mouse model of venous thrombosis by monitoring the platelet density with intravital microscopy. This study supports the hypothesis that fucoidan-nanoparticles improve the rt-PA efficiency. This work establishes the proof-of-concept of fucoidan-based carriers for targeted thrombolysis.

Treatment of lung cancer is evolving from standard cytotoxic to personalized treatment based on the molecular alterations unique to each patient’s tumor. In our recent work with TCGA and others, we identified somatic mutations in the gene encoding the RAS-related small GTPase, RIT1, in lung adenocarcinomas. In addition, somatic RIT1 mutations have also been identified in myeloid malignancies and germline RIT1 mutations are found in the developmental “RAS-opathy,” Noonan syndrome. In all these diseases, RIT1 mutations are mutually-exclusive with other RAS/MAPK pathway mutations, including mutations in EGFR, KRAS, and ALK. While our previous work demonstrated that gain-of-function RIT1 mutations induce activation of PI3K and MEK signaling, the specific effectors used by RIT1 to promote tumorigenesis are unknown. To genetically dissect the signaling pathways downstream of RIT1, KRAS, EGFR, and PIK3CA, we performed genome-wide CRISPR/Cas9 screens in four isogenic lung adenocarcinoma cell lines in which cell survival is dependent on the expression of the oncogene. Through integrative analysis of essential genes across the four cell lines, we identified synthetic lethal relationships unique and shared in each isogenic background. Among the dependencies identified was the requirement of aurora kinase pathway genes for survival of RIT1-mutant cells, raising the possibility of therapeutic aurora kinase inhibition in RIT1-positive tumors. To evaluate whether the dependence on Aurora kinases extended to small molecule inhibition, we performed a screen of 160 small molecules in clinical development or approved by the FDA. Consistent with our CRISPR/Cas9 results, we find that RIT1-mutant cells depend on Aurora kinase activity for survival. Interestingly, global phosphoproteomic profiling identified RIT1 regulation of an Aurora kinase-Haspin axis. Taken together these results identify a new arm of RIT1 signaling and nominate Aurora kinase inhibition as a potential therapeutic strategy in RIT1-mutated tumors. Citation Format: Athea Vichas, Naomi T. Nkinsi, Alice Berger. Functional multiomic profiling identifies Aurora kinase inhibition as a therapeutic strategy in RIT1-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2726.

Feline caliciviruses (FCVs) are non-enveloped RNA viruses that exhibit high genetic variation. Two reverse transcription quantitative polymerase chain reaction (RT-qPCR) FCV assays (S1 and S2) were evaluated using samples from 300 field cats. The direct detection of FCV in swabs and after propagation in cell culture, as well as the influence of storage conditions, was assessed. FCV-RNA detectability on dry swabs was similar after storage at either 4°C or -20°C, but viral burdens were maintained for a longer time period when viral transport medium was used. A total of 97 (32%) samples was considered FCV PCR-positive. Of these, 81% and 77% tested positive directly from swabs using S1 and S2, respectively; 84% and 81% tested positive after enrichment in cell culture, respectively. Combined detection by RT-PCR directly from swabs and after VI was most sensitive (up to 96%). Neither of the methods alone were able to detect all FCV-positive samples. In conclusion, clinical samples should be collected in viral transport medium, stored at ≤4°C and processed as soon as possible. The combination of cell culture with RT-qPCR or detection directly from swabs using a combination of different RT-qPCR assays is recommended to reach a high sensitivity of FCV detection.

International audience; Feline calicivirus (FCV) is a common viral pathogen in domestic cats worldwide. The variable regions of the capsid (VP1) gene of FCV have one of the highest recorded rates of molecular evolution. Understanding the genetic diversity and phylogeny of FCV is a prerequisite to exploring the epidemiology and pathogenesis of this virus and to the development of efficacious vaccine strategies. In this study, we undertook a nationwide molecular characterization of FCV using for the first time nearly complete capsid (VP1) gene sequences. Sequences from 66 FCV samples were used to investigate the correlation between viral phylogeny and several traits, including geographic origin, signalment, husbandry, FCV vaccination and co-infections. Codon-based nucleotide alignment showed that individual nucleotides and their corresponding amino acid sites were either invariant or highly variable. Using a threshold of 20 % genetic distance in variable region E, FCV samples were grouped into 52 strains, 10 of which comprised two to three samples. Significant associations between FCV phylogeny and host characteristics were found, specifically the pedigree status of the cats, and two well-supported lineages were identified in which the current FCV strain definition was confounded. No correlation between viral genetic distances and geographic distances was evident. The greater resolution of the FCV phylogeny in this study compared to previous studies can be attributed to our use of more conserved regions of the capsid (VP1) gene; nonetheless, our results were still hampered by sequence saturation. The study highlights the need for whole-genome sequences for FCV phylogeny studies.

A 23-month-old tomcat was referred to our clinic because of male behavioral problems, cryptorchidism, and an undefined intra-abdominal organ resembling a uterus. Ultrasonography and computed tomography showed 2 fluid-filled tubular structures dorsolaterally to the bladder and connected to the pelvic urethra. The cat was castrated, and the tubular structures were surgically removed. Histology identified them as Müllerian duct remnants. The testes were hypoplastic, the epididymes and deferent ducts were normal. Cytogenetic analyses revealed the presence of a mosaic 37,X/38,XY karyotype which explains the clinical findings.

Feline calicivirus (FCV) infections are associated with oral ulceration, chronic stomatitis and a limping syndrome. Epizootic outbreaks of virulent systemic disease (VSD) have been reported in the USA and Europe. Here, the molecular characterization and neutralization patterns of FCV isolates from cases of severe, non-epizootic infection associated with skin ulceration and edema are presented. Samples from eleven symptomatic cats, four in-contact cats and 27 cats with no contact with symptomatic cats were collected and tested for FCV, feline herpesvirus-1 (FHV-1), feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). Phylogenetic analyses based on the capsid (VP1) gene of FCV and virus neutralization with antisera raised against four FCV vaccine strains were performed. Nine kittens and two adult cats in two shelters and two veterinary clinics in four geographically distinct locations in Switzerland and Liechtenstein were affected. The cats showed fever, tongue and skin ulceration, head and paw edema, and occasionally jaundice, generalized edema and dyspnea. All symptomatic cats tested FCV-positive but were negative for FHV-1, FeLV and FIV, with the exception of one FIV-positive kitten. All kittens of one litter and both adult cats died. The disease did not spread to cats in the environment. Cats in the environment displayed phylogenetically distinct, but related, FCV strains. Virus neutralization patterns suggested that some cases might have been potentially prevented by vaccination with the optimal vaccine strain. In conclusion, clinicians should be aware of severe, non-epizootic forms of FCV infections with initial clinical presentations similar to VSD.

Background Cats with feline calicivirus (FCV)-related symptoms are commonly presented to veterinary practitioners. Various clinical manifestations have been attributed to FCV, i.e. upper respiratory tract disease (URTD), oral ulcerations, gingivostomatitis, limping syndrome and virulent systemic disease. Additionally, healthy cats can shed FCV. The aims of this study were 1) to investigate the frequency of FCV in cats with FCV-related symptoms and in healthy cats in Switzerland, 2) to assess risk and protective factors for infection, such as signalment, housing conditions, vaccination, and co-infection with URTD-associated pathogens, and 3) to address the association between clinical symptoms and FCV infection. Results Oropharyngeal, nasal and conjunctival swabs were collected in 24 veterinary practices from 200 FCV-suspect and 100 healthy cats originating from 19 cantons of Switzerland. The samples were tested for FCV using virus isolation and reverse-transcription real-time quantitative polymerase chain reaction (qPCR) and for feline herpesvirus-1 (FHV-1), Mycoplasma felis, Chlamydophila felis, Bordetella bronchiseptica using real-time qPCR. Within the two populations (FCV-suspect/healthy), the observed PCR prevalences were: FCV 45 %/8 %, FHV-1 20 %/9 %, C. felis 8 %/1 %, B. bronchiseptica 4 %/2 %, M. felis 47 %/31 % and any co-infections thereof 40 %/14 %. Based on multivariable regression models amongst FCV-suspect cats (odds ratio [95 % confidence interval]), co-infection with M. felis (1.75 [0.97; 3.14]), group housing (2.11 [1.02; 4.34]) and intact reproductive status (1.80 [0.99; 3.28]) were found to be risk factors for FCV infection. In healthy cats, intact reproductive status (22.2 [1.85; 266.7]) and group housing (46.4 [5.70; 377.7]) were found to be associated with FCV infection. Based on an univariable approach, FCV-suspect cats were found to be significantly less often FCV-positive when vaccinated (0.48 [0.24; 0.94]). Oral ulcerations, salivation, gingivitis and stomatitis, but not classical signs of URTD were significantly associated with FCV infection (all p

Objective. Our goals were to assist residents in acquiring skills needed to care for children at the end of life and to increase their comfort level regarding critical aspects of caring for dying children. Methods. We designed and implemented a 6-part seminar series for pediatric residents in their postgraduate year-2 at the University of Arizona Pediatric Residency Program. The series consisted of small group sessions regarding medical and legal issues facing physicians at the time of a pediatric patient’s death, symptom management in dying children, approaches to limitation of care and discussion of impending death of a child, cultural and developmental factors impacting on the care of children at the end of life, parent and sibling bereavement after the death of a child, and physician’s personal responses to the death of pediatric patients. The sessions were conducted on weekday evenings away from patient care settings and included input from community-based individuals as well as academic pediatric subspecialists. To promote active discussion, size was limited to 12 participants. Pretests and posttests were used to evaluate the success of this program. Residents’ level of comfort with important issues in end-of-life care was measured using a 5-point Likert scale. Results. Although most of the residents taking part in the seminar series had previously participated in discussions of limitation of care of a child and/or had performed advanced life support measures on a child, few had any previous training in end-of-life care. Responses of the 8 participants who completed both the pretests and posttests were evaluated for assessment of changes in comfort level in areas pertinent to end-of-life care. We found that on completion of this program, residents were significantly more comfortable with logistic issues and symptom management, discussing death and limitation of medical care with families, discussing end-of-life care with colleagues and families, handling conflicts within the health care team, working with families of varying ethnic backgrounds, guiding developmentally appropriate discussions of death, identifying and seeking out advice from a role model regarding end-of-life care, and coping with their own responses to a child’s death. Costs of this seminar series were minimal. Conclusions. An inexpensive, discussion-based seminar series successfully provided pediatric residents with basic information regarding end-of-life care and significantly increased their confidence as clinicians caring for seriously ill and dying children.

For the vast majority of cancer patients, existing knowledge of the function(s) of the mutant genes harbored by their tumor and the dependencies they induce is incomplete or non-existent since most cancer mutations are exceedingly rare. As a result, we now have long lists of candidate alleles but a paucity of targets whose biology is sufficiently well understood to guide therapeutics. Here we present an interim progress report on a pilot effort aiming to create a generalizable framework to systematically map the molecular consequences of cancer variants at scale (Target Accelerator). First, we created an efficient pipeline to generate cancer variants and generated an initial library of 1300 mutant cDNA clones corresponding to variants in lung cancer and diffuse large B-cell lymphoma as well as those nominated by “pan-cancer” computational analyses. Second, we established an industry-scale, next-generation pipeline to generate new cancer models (Cell Line Factory) directly from patient samples. We have leveraged this pipeline to process over 330 samples from 208 patients across 16 cancer types, with over 60% growing through at least 5 population doublings. We show that tumor genomics can be retained in such patient-derived models and that drug testing within 3 months is feasible. In addition, we use combinatorial molecular barcoding to rapidly generate a panel of pathway-primed human tumorigenesis models that are suitable for massively parallel multiplexed tumorigenesis assays in vivo (TumorPlex). We hypothesized that this integrated framework could be utilized to generate meaningful functional hypotheses in a high-throughput manner. To test this hypothesis, we introduced over 1000 cancer mutations into cell models and created gene expression signatures together with phenotypic data. In lung cancer, we show that the mutational impact of mutant alleles with known and unknown functions can be rapidly assessed by comparing signatures of wild-type and mutant alleles. We show that this generalizable approach, which does not require prior knowledge, can place variants of unknown significance into dominant gain-of-function and loss-of-function categories. As a complementary approach, we have used TumorPlex assays to test the tumorigenic potential of 550 mutant alleles nominated by Pan-Cancer computational analyses and discovered unexpected variants in the KRAS, AKT1, MAP2K1, ERBB2, PIK3CB, NFE2L2, FAM200A and POT1 genes as being potently tumorigenic. These proof-of-concept studies demonstrate initial feasibility of mapping cancer variant function at scale. Importantly, they demarcate a path by which mapping variant function and predicting vulnerabilities might soon be possible on a patient-by-patient basis, achieving the promise of precision functional genomics. Citation Format: Alice Berger, Eejung Kim, Angela Brooks, Yashaswi Shrestha, Yuen-Yi Tseng, Xiaoyun Wu, Nina Ilic, Lihua Zou, Atanas Kamburov, Xiaoping Yang, Cong Zhu, Paula Keskula, Sara Seepo, Andrew Hong, John Doench, Aravind Subramanian, Keith Ligon, Philip Kantoff, Katherine Janeway, Levi Garraway, David Root, Todd Golub, Matthew Meyerson, William Hahn, Gad Getz, Jesse Boehm. Towards precision functional genomics via next-generation functional mapping of cancer variants. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 957. doi:10.1158/1538-7445.AM2015-957