Hippocampal Engrams Generate Variable Behavioral Responses and Brain-Wide Network States.

Freezing is a defensive behavior commonly examined during hippocampal-mediated fear engram reactivation. How these cellular populations engage the brain and modulate freezing across varying environmental demands is unclear. To address this, we optogenetically reactivated a fear engram in the dentate gyrus subregion of the hippocampus across three distinct contexts in male mice. We found that there were differential amounts of light-induced freezing depending on the size of the context in which reactivation occurred: mice demonstrated robust light-induced freezing in the most spatially restricted of the three contexts but not in the largest. We then utilized graph theoretical analyses to identify brain-wide alterations in cFos expression during engram reactivation across the smallest and largest contexts. Our manipulations induced positive interregional cFos correlations that were not observed in control conditions. Additionally, regions spanning putative "fear" and "defense" systems were recruited as hub regions in engram reactivation networks. Lastly, we compared the network generated from engram reactivation in the small context with a natural fear memory retrieval network. Here, we found shared characteristics such as modular composition and hub regions. By identifying and manipulating the circuits supporting memory function, as well as their corresponding brain-wide activity patterns, it is thereby possible to resolve systems-level biological mechanisms mediating memory's capacity to modulate behavioral states.

Basolateral Amygdala Astrocytes Are Engaged by the Acquisition and Expression of a Contextual Fear Memory.

Astrocytes are key cellular regulators within the brain. The basolateral amygdala (BLA) is implicated in fear memory processing, yet most research has entirely focused on neuronal mechanisms, despite a significant body of work implicating astrocytes in learning and memory. In the present study, we used in vivo fiber photometry in C57BL/6J male mice to record from amygdalar astrocytes across fear learning, recall, and three separate periods of extinction. We found that BLA astrocytes robustly responded to foot shock during acquisition, their activity remained remarkably elevated across days in comparison to unshocked control animals, and their increased activity persisted throughout extinction. Further, we found that astrocytes responded to the initiation and termination of freezing bouts during contextual fear conditioning and recall, and this behavior-locked pattern of activity did not persist throughout the extinction sessions. Importantly, astrocytes do not display these changes while exploring a novel context, suggesting that these observations are specific to the original fear-associated environment. Chemogenetic inhibition of fear ensembles in the BLA did not affect freezing behavior or astrocytic calcium dynamics. Overall, our work presents a real-time role for amygdalar astrocytes in fear processing and provides new insight into the emerging role of these cells in cognition and behavior.SIGNIFICANCE STATEMENT We show that basolateral amygdala astrocytes are robustly responsive to negative experiences, like shock, and display changed calcium activity patterns through fear learning and memory. Additionally, astrocytic calcium responses become time locked to the initiation and termination of freezing behavior during fear learning and recall. We find that astrocytes display calcium dynamics unique to a fear-conditioned context, and chemogenetic inhibition of BLA fear ensembles does not have an impact on freezing behavior or calcium dynamics. These findings show that astrocytes play a key real-time role in fear learning and memory.

Evaluation of a psychoneurological symptom cluster in patients with breast or digestive cancer: a longitudinal observational study.

BACKGROUND: A psychoneurological symptom cluster composed of cancer-related fatigue, emotional distress, sleep difficulties, and pain is very common among patients with cancer. Cognitive difficulties are also frequently associated with this cluster. Network analyses allow for an in-depth understanding of the relationships between symptoms in a cluster. This paper details the study protocol of a longitudinal assessment of the psychoneurological symptom cluster in two distinct cohorts: breast cancer and digestive cancer survivors, using network analyses. METHODS: Over two years, the symptoms involved in the psychoneurological symptom cluster, along with other common symptoms (e.g., digestive symptoms, financial difficulties) and variables (i.e., self-compassion, coping strategies) will be assessed in two cohorts: breast cancer survivors (N = 240) and digestive cancer survivors (N = 240). Online questionnaires will be completed at baseline, then 6, 12 and 24 months later. Network analyses will be used to assess the configuration of the symptom cluster at each measurement time and in each cohort. Comparison of networks between two measurement times or between the two cohorts will also be done with network comparison tests. DISCUSSION: This study will enable a better understanding of the relationships between common symptoms endured by patients with cancer. The results will be employed to develop more cost-effective interventions which, ultimately, will significantly improve the quality of life of patients with breast or digestive cancer. TRIAL REGISTRATION: ClinicalTrials.gov (NCT05867966). Registered on the 27th of April 2023. url: https://classic. CLINICALTRIALS: gov/ct2/show/NCT05867966 .

The m6A RNA demethylase FTO is a HIF-independent synthetic lethal partner with the VHL tumor suppressor.

Loss of the von Hippel-Lindau (VHL) tumor suppressor is a hallmark feature of renal clear cell carcinoma. VHL inactivation results in the constitutive activation of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 and their downstream targets, including the proangiogenic factors VEGF and PDGF. However, antiangiogenic agents and HIF-2 inhibitors have limited efficacy in cancer therapy due to the development of resistance. Here we employed an innovative computational platform, Mining of Synthetic Lethals (MiSL), to identify synthetic lethal interactions with the loss of VHL through analysis of primary tumor genomic and transcriptomic data. Using this approach, we identified a synthetic lethal interaction between VHL and the m6A RNA demethylase FTO in renal cell carcinoma. MiSL identified FTO as a synthetic lethal partner of VHL because deletions of FTO are mutually exclusive with VHL loss in pan cancer datasets. Moreover, FTO expression is increased in VHL-deficient ccRCC tumors compared to normal adjacent tissue. Genetic inactivation of FTO using multiple orthogonal approaches revealed that FTO inhibition selectively reduces the growth and survival of VHL-deficient cells in vitro and in vivo. Notably, FTO inhibition reduced the survival of both HIF wild type and HIF-deficient tumors, identifying FTO as an HIF-independent vulnerability of VHL-deficient cancers. Integrated analysis of transcriptome-wide m6A-seq and mRNA-seq analysis identified the glutamine transporter SLC1A5 as an FTO target that promotes metabolic reprogramming and survival of VHL-deficient ccRCC cells. These findings identify FTO as a potential HIF-independent therapeutic target for the treatment of VHL-deficient renal cell carcinoma.

Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5.

Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite's protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including 'avirulent' ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.

Clinical prediction models for diagnosis of COVID-19 among adult patients: a validation and agreement study.

BACKGROUND: Since the beginning of the pandemic, hospitals have been constantly overcrowded, with several observed waves of infected cases and hospitalisations. To avoid as much as possible this situation, efficient tools to facilitate the diagnosis of COVID-19 are needed. OBJECTIVE: To evaluate and compare prediction models to diagnose COVID-19 identified in a systematic review published recently using performance indicators such as discrimination and calibration measures. METHODS: A total of 1618 adult patients present at two Emergency Department triage centers and for whom qRT-PCR tests had been performed were included in this study. Six previously published models were reconstructed and assessed using diagnostic tests as sensitivity (Se) and negative predictive value (NPV), discrimination (Area Under the Roc Curve (AUROC)) and calibration measures. Agreement was also measured between them using Kappa's coefficient and IntraClass Correlation Coefficient (ICC). A sensitivity analysis has been conducted by waves of patients. RESULTS: Among the 6 selected models, those based only on symptoms and/or risk exposure were found to be less efficient than those based on biological parameters and/or radiological examination with smallest AUROC values (< 0.80). However, all models showed good calibration and values above > 0.75 for Se and NPV but poor agreement (Kappa and ICC < 0.5) between them. The results of the first wave were similar to those of the second wave. CONCLUSION: Although quite acceptable and similar results were found between all models, the importance of radiological examination was also emphasized, making it difficult to find an appropriate triage system to classify patients at risk for COVID-19.

Patients' self-triage for unscheduled urgent care: a preliminary study on the accuracy and factors affecting the performance of a Belgian self-triage platform.

BACKGROUND: Management of unscheduled urgent care is a complex concern for many healthcare providers. Facing the challenge of appropriately dispatching unscheduled care, primary and emergency physicians have collaboratively implemented innovative strategies such as telephone triage. Currently, new original solutions tend to emerge with the development of new technologies. We created an interactive patient self-triage platform, ODISSEE, and aimed to explore its accuracy and potential factors affecting its performance using clinical case scenarios. METHODS: The ODISSEE platform was developed based on previously validated triage protocols for out-of-hours primary care. ODISSEE is composed of 18 icons leading to algorithmic questions that finally provide an advised orientation (emergency or primary care services). To investigate ODISSEE performance, we used 100 clinical case scenarios, each associated with a preestablished orientation determined by a group of experts. Fifteen volunteers were asked to self-triage with 50 randomly selected scenarios using ODISSEE on a digital tablet. Their triage results were compared with the experts' references. RESULTS: The 15 participants performed a total of 750 self-triages, which matched the experts references regarding the level of care in 85.6% of the cases. The orientation was incorrect in 14.4%, with an undertriage rate of 1.9% and an overtriage rate of 12.5%. The tool's specificity and sensitivity to advise participants on the appropriate level of care were 69% (95% CI: 64-74) and 97% (95% CI: 95-98) respectively. When combined with advice on the level of urgency, the tool only found the correct orientation in 68.4% with 9.2% of undertriages and 22.4% of overtriages. Some participant characteristics and the types of medical conditions demonstrated a significant association with the tool performance. CONCLUSION: Self-triage apps, such as the ODISSEE platform, could represent an innovative method to allow patients to self-triage to the most appropriate level of care. This study based on clinical vignettes highlights some positive arguments regarding ODISSEE safety, but further research is needed to assess the generalizability of such tools to the population without equity issues.

Defining the impact of mutation accumulation on replicative lifespan in yeast using cancer-associated mutator phenotypes.

Mutations accumulate within somatic cells and have been proposed to contribute to aging. It is unclear what level of mutation burden may be required to consistently reduce cellular lifespan. Human cancers driven by a mutator phenotype represent an intriguing model to test this hypothesis, since they carry the highest mutation burdens of any human cell. However, it remains technically challenging to measure the replicative lifespan of individual mammalian cells. Here, we modeled the consequences of cancer-related mutator phenotypes on lifespan using yeast defective for mismatch repair (MMR) and/or leading strand (Polε) or lagging strand (Polδ) DNA polymerase proofreading. Only haploid mutator cells with significant lifetime mutation accumulation (MA) exhibited shorter lifespans. Diploid strains, derived by mating haploids of various genotypes, carried variable numbers of fixed mutations and a range of mutator phenotypes. Some diploid strains with fewer than two mutations per megabase displayed a 25% decrease in lifespan, suggesting that moderate numbers of random heterozygous mutations can increase mortality rate. As mutation rates and burdens climbed, lifespan steadily eroded. Strong diploid mutator phenotypes produced a form of genetic anticipation with regard to aging, where the longer a lineage persisted, the shorter lived cells became. Using MA lines, we established a relationship between mutation burden and lifespan, as well as population doubling time. Our observations define a threshold of random mutation burden that consistently decreases cellular longevity in diploid yeast cells. Many human cancers carry comparable mutation burdens, suggesting that while cancers appear immortal, individual cancer cells may suffer diminished lifespan due to accrued mutation burden.

Training of radiotherapy professionals: status, content, satisfaction and improvement suggestions in the Greater Region.

BACKGROUND: The initial training of Radiation Oncology professionals can vary widely across Europe. The aim of this study was to assess the status and content of the initial training programs currently implemented in the Greater Region: Lorraine (Nancy, France), Saarland (Homburg, Germany), Luxembourg, and Liège (Wallonia, Belgium). METHODS: A survey was developed to investigate (1) the overall satisfaction, learning objectives, and teaching methods used during initial training programs and (2) the perceptions of the importance of key professional competencies as described by the CanMEDS (a framework that identifies and describes the abilities physicians require to effectively meet the health care needs of the people they serve). In addition, open-ended questions were used to elicit opinions on room for improvement. Participants (N = 38) were physicians (radiation oncologists (RO) seniors and residents) and radiation therapists (RTTs). RESULTS: Only 21.1% of the respondents declared having acquired all the competencies required for their professional practice during their initial training. Heterogeneity in teaching methods was noted within professional programs but there is no difference between those from RO and RTT in the teaching of technical and relational skills. Relational skills were not addressed in a range of 39.5-57.9% of respondent's curricula. More practical lessons were deemed necessary to improve radiotherapy (RT) training programs. CONCLUSIONS: Radiation oncology professionals expressed the need for more practical teaching, especially in the training of non-technical skills. Regarding the perceived importance of professional aptitudes, radiation oncology professionals highlighted medical and relational skills as the most important competencies.

Validation of a French-language version of TeamSTEPPS® T-TPQ and T-TAQ questionnaires.

Teamwork training and evaluation are essential to enhance safety and quality of care. The lack of the psychometric testing of the TeamSTEPPS® Teamwork Attitudes Questionnaire (T-TAQ) and Teamwork Perceptions Questionnaire (T-TPQ) across different language and cultural settings has questioned their widespread use because such attitudes and perceptions are highly subjective and context-bound. The present study aims to translate the T-TAQ and T-TPQ into the French language and validate the psychometric properties of the two questionnaires in a public health context. A forward-backward translation process, panel reviewing, and pilot testing in two rounds were followed to develop the French versions. Confirmatory factor analysis (CFA) and Cronbach's alpha were used to examine the factor structure and internal consistency, whereas two-way mixed Intraclass Correlation Coefficient (ICC) was performed to assess test-retest reliability. A total of 235 healthcare professionals in the French-speaking community of Belgium completed the T-TAQ and T-TPQ. After two to four weeks, 102 participants took part in the second round. Despite good fit indices as revealed by the CFA and Cronbach's alpha from 0.53 to 0.75 for the five dimensions of the T-TAQ and 0.76 to 0.79 for the T-TPQ, the squared correlations among the constructs were higher than the average variance extracted. Two-way mixed ICCs indicated fair to good test-retest reliability for all the five constructs of the two questionnaires, except the leadership scale of the T-TAQ. The French-language versions of the T-TAQ and T-TPQ were semantically equivalent and culturally relevant with adequate test-retest reliability as compared to the English versions. These two instruments might be used to capture the overall attitude toward teamwork and perceptions of team skills and behaviors. Yet, further research is advisable to refine the scales to establish the discriminant validity of the different dimensions and discriminative power of the instruments.

Direct regulation of GAS6/AXL signaling by HIF promotes renal metastasis through SRC and MET.

Dysregulation of the von Hippel-Lindau/hypoxia-inducible transcription factor (HIF) signaling pathway promotes clear cell renal cell carcinoma (ccRCC) progression and metastasis. The protein kinase GAS6/AXL signaling pathway has recently been implicated as an essential mediator of metastasis and receptor tyrosine kinase crosstalk in cancer. Here we establish a molecular link between HIF stabilization and induction of AXL receptor expression in metastatic ccRCC. We found that HIF-1 and HIF-2 directly activate the expression of AXL by binding to the hypoxia-response element in the AXL proximal promoter. Importantly, genetic and therapeutic inactivation of AXL signaling in metastatic ccRCC cells reversed the invasive and metastatic phenotype in vivo. Furthermore, we define a pathway by which GAS6/AXL signaling uses lateral activation of the met proto-oncogene (MET) through SRC proto-oncogene nonreceptor tyrosine kinase to maximize cellular invasion. Clinically, AXL expression in primary tumors of ccRCC patients correlates with aggressive tumor behavior and patient lethality. These findings provide an alternative model for SRC and MET activation by growth arrest-specific 6 in ccRCC and identify AXL as a therapeutic target driving the aggressive phenotype in renal clear cell carcinoma.

Effect of animal-source food supplement prior to and during pregnancy on birthweight and prematurity in rural Vietnam: a brief study description.

BACKGROUND: Maternal nutritional status is an important predictor of infant birthweight. Most previous attempts to improve birthweight through multiple micronutrient supplementation have been initiated after women are pregnant. Interventions to improve maternal nutritional status prior to conception may be more effective in preventing low birthweight and improving other infant health outcomes. OBJECTIVE: To compare the effects of maternal supplementation with animal-source food from preconception to term or from mid-gestation to term with routine prenatal care on birthweight, the prevalence of preterm births, intrauterine growth restriction, and infant growth during the first 12 months of life and on maternal nutrient status and the incidence of maternal and infant infections. METHODS: Young women from 29 rural communes in northwestern Vietnam were recruited when they registered to marry and were randomized to one of three interventions: animal-source food supplement 5 days per week from marriage to term (approximately 13 months), animal-source food supplement 5 days per week from 16 weeks of gestation to term (approximately 5 months), or routine prenatal care without supplementalfeeding. Data on infant birthweight and gestational age, maternal and infant anthropometry, micronutrient status, and infections in the infant and mother were collected at various time points. RESULTS: In a preliminary study of women of reproductive age in this area of Vietnam, 40% of the women were underweight (body mass index < 18.5) and anemic. About 50% had infections. Rice was the dietary staple, and nutrient-rich, animal-source foods were rarely consumed by women. Iron, zinc, vitamin A, folate, and vitamin B12 intakes were inadequate in about 40% of the women. The study is still ongoing, and further data are not yet available. CONCLUSIONS: The results of this study will provide important data regarding whether improved intake of micronutrient-rich animal-source foods that are locally available and affordable before and during pregnancy improves maternal and infant health and development. This food-based approach may have global implications regarding how and when to initiate sustainable nutritional interventions to improve maternal and infant health.

Engram reactivation mimics cellular signatures of fear.

Engrams, or the physical substrate of memory, recruit heterogeneous cell types. Targeted reactivation of neurons processing discrete memories drives the behavioral expression of memory, though the underlying landscape of recruited cells and their real-time responses remain elusive. To understand how artificial stimulation of fear affects intra-hippocampal neuron-astrocyte dynamics as well as their behavioral consequences, we express channelrhodopsin-2 in an activity-dependent manner within dentate gyrus neurons while recording both cell types with fiber photometry in hippocampal ventral CA1 across learning and memory. Both cell types exhibit shock responsiveness, with astrocytic calcium events uniquely modulated by fear conditioning. Optogenetic stimulation of a hippocampus-mediated engram recapitulates coordinated calcium signatures time locked to freezing, mirroring those observed during natural fear memory recall. Our findings reveal cell-type-specific dynamics in the hippocampus during freezing behavior, emphasizing neuronal-astrocytic coupling as a shared mechanism enabling both natural and artificially induced memory retrieval and the behavioral expression of fear.

Host tracheal and intestinal microbiomes inhibit Coccidioides growth in vitro.

Coccidioidomycosis, also known as Valley fever, is a disease caused by the fungal pathogen Coccidioides. Unfortunately, patients are often misdiagnosed with bacterial pneumonia, leading to inappropriate antibiotic treatment. The soil Bacillus subtilis-like species exhibits antagonistic properties against Coccidioides in vitro; however, the antagonistic capabilities of host microbiota against Coccidioides are unexplored. We sought to examine the potential of the tracheal and intestinal microbiomes to inhibit the growth of Coccidioides in vitro. We hypothesized that an uninterrupted lawn of microbiota obtained from antibiotic-free mice would inhibit the growth of Coccidioides, while partial in vitro depletion through antibiotic disk diffusion assays would allow a niche for fungal growth. We observed that the microbiota grown on 2×GYE (GYE) and Columbia colistin and nalidixic acid with 5% sheep's blood agar inhibited the growth of Coccidioides, but microbiota grown on chocolate agar did not. Partial depletion of the microbiota through antibiotic disk diffusion revealed diminished inhibition and comparable growth of Coccidioides to controls. To characterize the bacteria grown and identify potential candidates contributing to the inhibition of Coccidioides, 16S rRNA sequencing was performed on tracheal and intestinal agar cultures and murine lung extracts. We found that the host bacteria likely responsible for this inhibition primarily included Lactobacillus and Staphylococcus. The results of this study demonstrate the potential of the host microbiota to inhibit the growth of Coccidioides in vitro and suggest that an altered microbiome through antibiotic treatment could negatively impact effective fungal clearance and allow a niche for fungal growth in vivo. IMPORTANCE: Coccidioidomycosis is caused by a fungal pathogen that invades the host lungs, causing respiratory distress. In 2019, 20,003 cases of Valley fever were reported to the CDC. However, this number likely vastly underrepresents the true number of Valley fever cases, as many go undetected due to poor testing strategies and a lack of diagnostic models. Valley fever is also often misdiagnosed as bacterial pneumonia, resulting in 60%-80% of patients being treated with antibiotics prior to an accurate diagnosis. Misdiagnosis contributes to a growing problem of antibiotic resistance and antibiotic-induced microbiome dysbiosis; the implications for disease outcomes are currently unknown. About 5%-10% of symptomatic Valley fever patients develop chronic pulmonary disease. Valley fever causes a significant financial burden and a reduced quality of life. Little is known regarding what factors contribute to the development of chronic infections and treatments for the disease are limited.

Comparison between the WHO-CFICPS and the PRISMA classification of safety-related events in a radiation oncology department.

INTRODUCTION: Describing Safety-Related Events (SREs) in a radiotherapy (RT) department and comparing WHO-CFICPS (World Health Organization's Conceptual Framework For The International Classification For Patient Safety) and PRISMA (Prevention and Recovery Information System for Monitoring and Analysis) methods for classifying SREs. METHODS: From February 2017 to October 2020, two Quality Managers (QMs) randomly classified 1173 SREs using 13 incident types of WHO-CFICPS. The same two QMs, reclassified the same SREs according to 20 PRISMA incident codes. Statistical analysis was performed to assess the association between the 13 incident types of WHO-CFICPS and the 20 PRISMA codes. The chi-squared and post-hoc tests using adjusted standardized residuals were applied to detect the association between the two systems. RESULTS: There was a significant association between WHO-CFICPS incident types and PRISMA codes (P < 0.001). Ninety-two percent of all SREs were categorized using 4 of 13 WHO-CFICPS incident types including Clinical Process/Procedure (n = 448, 38.2%), Clinical Administration (n = 248, 21.1%), Documentation (n = 226, 19.2%) and Resources/Organizational Management (n = 15,613.3%). According to PRISMA classification, 14 of the 20 codes were used to describe the same SREs. PRISMA captured 41 Humans Skill Slips from 226 not better defined WHO-CFICPS Documentation Incidents, 38 Human Rule-based behaviour Qualification from not better defined 447 Clinical Process/Procedure and 40 Organization Management priority events from 156 not better defined WHO-CFICPS Resources/Organizational Management events (P < 0.001). CONCLUSION: Although there was a significant association between WHO-CFICPS and PRISMA, The PRISMA method provides a more detailed insight into SREs compared to WHO-CFICPS in a RT department.

Host tracheal and intestinal microbiomes inhibit Coccidioides growth in vitro.

Coccidioidomycosis, also known as Valley fever, is a disease caused by the fungal pathogen Coccidioides. Unfortunately, patients are often misdiagnosed with bacterial pneumonia leading to inappropriate antibiotic treatment. Soil bacteria B. subtilis-like species exhibits antagonistic properties against Coccidioides in vitro; however, the antagonistic capabilities of host microbiota against Coccidioides are unexplored. We sought to examine the potential of the tracheal and intestinal microbiomes to inhibit the growth of Coccidioides in vitro. We hypothesized that an uninterrupted lawn of microbiota obtained from antibiotic-free mice would inhibit the growth of Coccidioides while partial in vitro depletion through antibiotic disk diffusion assays would allow a niche for fungal growth. We observed that the microbiota grown on 2xGYE (GYE) and CNA w/ 5% sheep's blood agar (5%SB-CNA) inhibited the growth of Coccidioides, but that grown on chocolate agar does not. Partial depletion of the microbiota through antibiotic disk diffusion revealed that microbiota depletion leads to diminished inhibition and comparable growth of Coccidioides growth to controls. To characterize the bacteria grown and narrow down potential candidates contributing to the inhibition of Coccidioides, 16s rRNA sequencing of tracheal and intestinal agar cultures and murine lung extracts was performed. The identity of host bacteria that may be responsible for this inhibition was revealed. The results of this study demonstrate the potential of the host microbiota to inhibit the growth of Coccidioides in vitro and suggest that an altered microbiome through antibiotic treatment could negatively impact effective fungal clearance and allow a niche for fungal growth in vivo.

Correction: Civil war and death in Yemen: Analysis of SMART survey and ACLED data, 2012-2019.

[This corrects the article DOI: 10.1371/journal.pgph.0000581.].

Novel Frizzled-specific antibody-based Wnt mimetics and Wnt superagonists selectively activate WNT/ß-catenin signaling in target tissues.

WNTs are essential factors for stem cell biology, embryonic development, and for maintaining homeostasis and tissue repair in adults. Difficulties in purifying WNTs and their lack of receptor selectivity have hampered research and regenerative medicine development. While breakthroughs in WNT mimetic development have overcome some of these difficulties, the tools developed so far are incomplete and mimetics alone are often not sufficient. Here, we developed a complete set of WNT mimetic molecules that cover all WNT/ß-catenin-activating Frizzleds (FZDs). We show that FZD1,2,7 stimulate salivary gland expansion in vivo and salivary gland organoid expansion. We further describe the discovery of a novel WNT-modulating platform that combines WNT and RSPO mimetics' effects into one molecule. This set of molecules supports better organoid expansion in various tissues. These WNT-activating platforms can be broadly applied to organoids, pluripotent stem cells, and in vivo research, and serve as bases for future therapeutic development.

Assessment of rare BRCA1 and BRCA2 variants of unknown significance using hierarchical modeling.

Current evidence suggests that the genetic risk of breast cancer may be caused primarily by rare variants. However, while classification of protein-truncating mutations as deleterious is relatively straightforward, distinguishing as deleterious or neutral the large number of rare missense variants is a difficult on-going task. In this article, we present one approach to this problem, hierarchical statistical modeling of data observed in a case-control study of contralateral breast cancer (CBC) in which all the participants were genotyped for variants in BRCA1 and BRCA2. Hierarchical modeling permits leverage of information from observed correlations of characteristics of groups of variants with case-control status to infer with greater precision the risks of individual rare variants. A total of 181 distinct rare missense variants were identified among the 705 cases with CBC and the 1,398 controls with unilateral breast cancer. The model identified three bioinformatic hierarchical covariates, align-GV, align-GD, and SIFT scores, each of which was modestly associated with risk. Collectively, the 11 variants that were classified as adverse on the basis of all the three bioinformatic predictors demonstrated a stronger risk signal. This group included five of six missense variants that were classified as deleterious at the outset by conventional criteria. The remaining six variants can be considered as plausibly deleterious, and deserving of further investigation (BRCA1 R866C; BRCA2 G1529R, D2665G, W2626C, E2663V, and R3052W). Hierarchical modeling is a strategy that has promise for interpreting the evidence from future association studies that involve sequencing of known or suspected cancer genes.

Consensus on the Objectives of an Educational Intervention for Patients with Oropharyngeal Dysphagia and Their Informal Caregivers: A Delphi Study.

Purpose: In the absence of literature allowing for an evidence-based approach to therapeutic patient education (TPE) in Oropharyngeal Dysphagia (OD), this study aims to reach a consensus of experts on the content of a competency framework of an educational program for OD patients and their informal caregivers (ICGs). Methods: We used the Delphi consensus-building method. Four categories of experts were recruited: 12 patients, 17 ICGs, 46 healthcare professionals (HCP) (experienced in OD, not necessarily certified in TPE), and 19 experts in TPE (trained individuals to set up and run TPE programs not necessarily HCPs). The content of the questionnaire of the first round (R) was established according to the result of a scoping review and the opinion of an expert committee. We carried out three rounds. In R1 and R2, we collected the opinions on the relevance (7-point Likert-type scale) and on comprehensiveness (YES/No question and asking participants to propose additional content). Participants were also invited to leave comments on each objective. In R3, we asked the participants to give their opinion about the relevance of the objectives again and asked them to rank the themes from highest to lowest priority. Results: Objectives were considered relevant for all participants if they reached consensus when the interquartile (IQR) ≤ 1, and if the median indicated agreement (Mdn ≥ 6) (6= appropriate, 7 = totally appropriate). Following three rounds, the final content of the educational program is composed of 23 educational objectives organized in 13 themes with an agreement about relevance amongst all participants (Mdn ≥ 6; IQR ≤ 1). The comprehensiveness criterion received also a consensus (IQR ≤ 1). The participants ranked the theme "normal swallowing vs difficulty swallowing" as the highest priority. Conclusion: This Delphi study resulted in a consensus, on the content of a competency framework of an educational program for OD patients and their ICGs. Further steps are needed to construct learning activities based on these objectives before testing their feasibility and efficacy.