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Molecular technologies have revolutionized the field of wildlife disease ecology, allowing the detection of outbreaks, novel pathogens, and invasive strains. In particular, metabarcoding approaches, defined here as tools used to amplify and sequence universal barcodes from a single sample (e.g., 16S rRNA for bacteria, ITS for fungi, 18S rRNA for eukaryotes), are expanding our traditional view of host–pathogen dynamics by integrating microbial interactions that modulate disease outcome. Here, I provide an analysis from the perspective of the field of amphibian disease ecology, where the emergence of multi-host pathogens has caused global declines and species extinctions. I reanalyzed an experimental mesocosm dataset to infer the functional profiles of the skin microbiomes of coqui frogs (Eleutherodactylus coqui), an amphibian species that is consistently found infected with the fungal pathogen Batrachochytrium dendrobatidis and has high turnover of skin bacteria driven by seasonal shifts. I found that the metabolic activities of microbiomes operate at different capacities depending on the season. Global enrichment of predicted functions was more prominent during the warm-wet season, indicating that microbiomes during the cool-dry season were either depauperate, resistant to new bacterial colonization, or that their functional space was more saturated. These findings suggest important avenues to investigate how microbes regulate population growth and contribute to host physiological processes. Overall, this study highlights the current challenges and future opportunities in the application of metabarcoding to investigate the causes and consequences of disease in wild systems.

Symbiotic interactions can determine the evolutionary trajectories of host species, influencing genetic variation through selection and changes in demography. In the context of strong selective pressures such as those imposed by infectious diseases, symbionts providing defences could contribute to increase host fitness upon pathogen emergence. Here, we generated genome-wide data of an amphibian species to find evidence of evolutionary pressures driven by two skin symbionts: a batrachochytrid fungal pathogen and an antifungal bacterium. Using demographic modelling, we found evidence of decreased effective population size, probably due to pathogen infections. Additionally, we investigated host genetic associations with infection status, antifungal bacterium abundance and overall microbiome diversity using structural equation models. We uncovered relatively lower nucleotide diversity in infected frogs and potential heterozygote advantage to recruit the candidate beneficial symbiont and fight infections. Our models indicate that environmental conditions have indirect effects on symbiont abundance through both host body traits and microbiome diversity. Likewise, we uncovered a potential offsetting effect among host heterozygosity-fitness correlations, plausibly pointing to different ecological and evolutionary processes among the three species due to dynamic interactions. Our findings revealed that evolutionary pressures not only arise from the pathogen but also from the candidate beneficial symbiont, and both interactions shape the genetics of the host. Our results advance knowledge about multipartite symbiotic relationships and provide a framework to model ecological and evolutionary dynamics in wild populations. Finally, our study approach can be applied to inform conservation actions such as bioaugmentation strategies for other imperilled amphibians affected by infectious diseases.

As globalization lowers geographic barriers to movement, coinfection with novel and enzootic pathogens is increasingly likely. Novel and enzootic pathogens can interact synergistically or antagonistically, leading to increased or decreased disease severity. Here we examine host immune responses to coinfection with two closely related fungal pathogens: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Both pathogens have had detrimental effects on amphibian populations, with Bd now largely enzootic, while Bsal is currently spreading and causing epizootics. Recent experimental work revealed that newts coinfected with Bd and Bsal had significantly higher mortality than those infected with either pathogen alone. Here we characterize host immunogenomic responses to chytrid coinfection relative to single infection. Across several classes of immune genes including pattern recognition receptors, cytokines, and MHC, coinfected host gene expression was weakly upregulated or comparable to that seen in single Bd infection, but significantly decreased when compared to Bsal infection. Combined with strong complement pathway downregulation and keratin upregulation, these results indicate that coinfection with Bd and Bsal compromises immune responses active against Bsal alone. As Bsal continues to invade naïve habitats where Bd is enzootic, coinfection will be increasingly common. If other Bd-susceptible species in the region have similar responses, interactions between the two pathogens could cause severe population and community-level declines.

The objective of this study is to find a contrast-enhanced CT-radiomic signature to predict clinical incomplete response in patients affected by hepatocellular carcinoma who underwent locoregional treatments.190 patients affected by hepatocellular carcinoma treated using focal therapies (radiofrequency or microwave ablation) from September 2018 to October 2020 were retrospectively enrolled. Treatment response was evaluated on a per-target-nodule basis on the 6-months follow-up contrast-enhanced CT or MR imaging using the mRECIST criteria. Radiomics analysis was performed using an in-house developed open-source R library. Wilcoxon-Mann-Whitney test was applied for univariate analysis; features with a p-value lower than 0.05 were selected. Pearson correlation was applied to discard highly correlated features (cut-off=0.9). The remaining features were included in a logistic regression model and receiver operating characteristic curves; sensitivity, specificity, positive and negative predictive value were also computed. The model was validated performing 2000 bootstrap resampling.56 treated lesions from 42 patients were selected. Treatment responses were: complete response for 26 lesions (46.4%), 18 partial responses (32.1%), 10 stable diseases (17.9%), 2 progression diseases (3.6%). Area-Under-Curve value was 0.667 (95% CI: 0.527-0.806); accuracy, sensitivity, specificity, positive and negative predictive values were respectively 0.66, 0.85, 0.50, 0.59 and 0.79.This contrast-enhanced CT-based model can be helpful to early identify poor responder's hepatocellular carcinoma patients and personalize treatments.

While some pathogens are limited to single species, others can colonize many hosts, likely contributing to the emergence of novel disease outbreaks. Despite this biodiversity threat, traits associated with host niche expansions are not well understood in multihost pathogens. Here, we aimed to uncover functional machinery driving multihost invasion by focusing on Batrachochytrium dendrobatidis (Bd), a pathogen that infects the skin of hundreds of amphibians worldwide. We performed a meta-analysis of Bd gene expression using data from published infection experiments and newly generated profiles. We analysed Bd transcriptomic landscapes across the skin of 14 host species, reconstructed Bd isolates phylogenetic relationships, and inferred the origin and evolutionary history of differentially expressed genes under a phylogenetic framework comprising other 12 zoosporic fungi. Bd displayed plastic infection strategies when challenged by hosts with different disease susceptibility. Our analyses identified sets of differentially expressed genes under host environments with similar infection outcome. We stressed nutritional immunity and gene silencing as important processes required to overcome challenging skin environments in less susceptible hosts. Overall, Bd genes expressed during amphibian skin exploitation have arisen mainly via gene duplications with great family expansions, increasing the gene copy events previously described for this fungal species. Finally, we provide a comprehensive gene data set that can be used to further examine eco-evolutionary hypotheses for this host-pathogen system. Our study supports the idea that host environments exert contrasting selective pressures, such that gene expression plasticity could be one of the evolutionary keys leading to the success of multihost pathogens.

A persistent two-month long outbreak of Ranavirus in a natural community of amphibians contributed to a mass die-off of gopher frog tadpoles (Lithobates capito) and severe disease in striped newts (Notophthalmus perstriatus) in Florida. Ongoing mortality in L. capito and signs in N. perstriatus continued for five weeks after the first observation. Hemorrhagic disease and necrosis were diagnosed from pathological examination of L. capito tadpoles. We confirmed detection of a Frog Virus 3 (FV3)-like Ranavirus via quantitative PCR in all species. Our findings highlight the susceptibility of these species to Rv and the need for long-term disease surveillance during epizootics.

The human paraoxonases family (PONs) includes three calcium-dependent esterases: PON1, PON2, and PON3. The presence of PONs mRNA in human lungs is known, however, their enzymatic activity and subcellular localization have not been sufficiently explored.In this work, the presence of PONs in human lung tissues, at both mRNA and protein levels, was confirmed by Real-Time RT-PCR and Western blot analysis. Moreover, the activities of PONs were determined in cytosol and microsomes of 30 subjects and in mitochondria of 8 representative lung tissues using selective and non-selective substrates. Besides, to exclude the possible contribution of other esterases on PON1 organophosphate activity, the effect of bis-p-nitrophenyl phosphate (BNPP) and phenylmethylsulfonyl fluoride (PMSF), esterase inhibitors, and ethylenediaminetetraacetic acid (EDTA), a general paraoxonase inhibitor, was tested. Finally, the presence and activities of PONs in the A549 pulmonary cell line were also evaluated in order to be used as a model for studies on paraoxonases' metabolism.Our results demonstrated high interindividual variability in both PONs mRNA/protein levels and enzymatic activities and pointed out the presence of all PONs in human lungs and their subcellular distribution in the cytosol, microsomes, and mitochondria.These findings add further information to our knowledge of pulmonary metabolism and, given that PON1 can metabolize some drugs used for respiratory diseases, the presence of PON1 activity in the lung tissue should no longer be ignored in the development of treatment plans and the design of new drugs.

Study question Do stage and grade of breast cancer impact the number of retrieved mature oocytes during controlled ovarian stimulation for fertility preservation? Summary answer Stage and grade of breast cancer do not impact the number of retrieved mature oocytes. Higher grade breast cancer requires higher gonadotropin doses during stimulation. What is known already Cancer can impair ovarian response by unknown mechanisms. Some authors suggest that it could be detrimental on fertility because it elicits a catabolic state, increasing stress hormone levels. Some studies have also shown that ovarian response to controlled ovarian stimulation (COS) is, in some way, compromised in oncological patients. Little is known about the impact of different types of cancer on ovarian reserve, and specifically whether higher stage and grade could compromise egg retrieval during fertility preservation (FP) techniques. Study design, size, duration: Retrospective cohort study evaluating data of FP treatment cycles among women with breast cancer at the Oncofertility Unit of San Raffaele Hospital, Milan in the period from 2011 to 2019. Participants/materials, setting, methods Inclusion criteria were: breast cancer diagnosis; age 22–41; oocyte cryopreservation after stimulation with a random start GnRH-antagonist protocol. Patients receiving chemotherapy before FP were excluded. We compared outcomes between low-stage (stage I) and high-stage (stage II-III) patients and low-grade (G1-G2) and high-grade (G3) patients. Main study outcome was the total number of retrieved mature oocytes. Univariate analysis was performed by Mann-Whitney test, Kruskal-Wallis test and Fisher’s exact test. Multivariate analysis was performed by logistic regression. Main results and the role of chance 101 stimulation cycles were included. High-stage disease patients were significantly younger than low-stage. Median antral follicle count (AFC) was 12 in low-stage and 10 in high-stage (age-adjusted p = 0.92) and median anti-mullerian hormone (AMH) levels were 1.9 ug/L in low-stage and 1.8 ug/L in high-stage (age-adjusted p = 0.22). No significant difference in stimulation protocols and follicle-stimulating hormone (FSH) start and total dose could be detected between the 2 groups. Median number of vitrified oocytes was 7 in both groups (p = 0.75). No significant difference could be observed in median AFC (13 vs 10, p = 0.14) and AMH levels (2.1 vs 1.5, p = 0.88) between low-grade and high-grade disease patients. When adjusting for age, AFC was found to be significantly lower in high-grade disease patients (p = 0.03). Patients with high-grade tumors were stimulated with higher doses of FSH (age-adjusted p-value=0.03). Median number of vitrified oocytes was 6 in low-grade patients and 7 in high-grade (p = 0.35). In a multivariate model including age, cancer stage, cancer grade and molecular classification, the only significant factor found to be inversely associated with AFC was cancer grade (OR 3.6; 95% CI 0.7 – 6.5, p = 0.01), while only age was significantly associated with oocyte retrieval (OR 0.4; 95% CI 0.01 – 0.9, p = 0.04). Limitations, reasons for caution The main limitations of our study are its retrospective design and the small sample size. Wider implications of the findings: Fertility preservation counselling and ovarian stimulation protocols of breast cancer patients could be implemented with cancer grade. Trial registration number Not applicable

Invasive fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) are causing mortality events and population declines in amphibians around the world. B. salamandrivorans has not been found in the Americas but is predicted to emerge there given the presence of competent hosts, high volume import pathways, and lack of effective biosecurity measures. Eastern newts (Notophthalmus viridescens) are the most abundant and widespread salamanders in United States with known susceptibility to both fungi. However, little is known about how their current Bd infections will interact with novel Bsal infections. Here, we ran a series of experiments in which we exposed newts to each fungal pathogen, combinations of both fungi, and three Bd isolates to: (1) determine if natural exposure to Bd provides protection against Bsal, (2) quantify the effect of co-infections on newt survival, and (3) examine if resistance responses are general to multiple lineages. We found that hosts exposed under simultaneous co-infections experienced 78% mortality over a period of 18 weeks, which was driven by the persistence of Bsal because newts cleared Bd infection within a month. In a subsequent experiment, resistant newts also resolved infections of three Bd genotypes, suggesting that the defense mechanism is effective across all lineages. However, re-exposing these individuals with a tenfold higher dose (106Bd zoospores) increased their susceptibility to Bsal, and 93% of them sustained Bsal infections. Our findings will improve the estimates of decline risk for a species that could serve as a potential superspreader of Bsal in North America.

Animal-associated microbiomes are integral to host health, yet key biotic and abiotic factors that shape host-associated microbial communities at the global scale remain poorly understood. We investigated global patterns in amphibian skin bacterial communities, incorporating samples from 2,349 individuals representing 205 amphibian species across a broad biogeographic range. We analysed how biotic and abiotic factors correlate with skin microbial communities using multiple statistical approaches. Global amphibian skin bacterial richness was consistently correlated with temperature-associated factors. We found more diverse skin microbiomes in environments with colder winters and less stable thermal conditions compared with environments with warm winters and less annual temperature variation. We used bioinformatically predicted bacterial growth rates, dormancy genes and antibiotic synthesis genes, as well as inferred bacterial thermal growth optima to propose mechanistic hypotheses that may explain the observed patterns. We conclude that temporal and spatial characteristics of the host’s macro-environment mediate microbial diversity. National Science Foundation/[DEB-1146284]/NSF/Estados Unidos National Science Foundation/[IOS-1121758]/NSF/Estados Unidos National Science Foundation/[DEB-1310036]/NSF/Estados Unidos John Templeton Foundation/[]/JTF/Estados Unidos Deutsche Forschungsgemeinschaft/[]/DFG/Alemania Deutsche Forschungsgemeinschaft/[VE247/9-1]/DFG/Alemania Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/[]/CAPES/Brasil Fundação de Amparo à Pesquisa do Estado de São Paulo/[#2013/50741-7]/FAPESP/Brasil Conselho Nacional de Desenvolvimento Científico e Tecnológico/[]/CNPq/Brasil Simons Foundation/[429440, WTW]//Estados Unidos Deutscher Akademischer Austauschdienst/[]/DAAD/Alemania Universidad de Costa Rica/[801-B2-029]/UCR/Costa Rica Ministerio de Ciencia, Tecnología y Telecomunicaciones/[849-PINN-2015]/MICITT/Costa Rica National Research Foundation of Korea/[2015R1D1A1A01057282]/NRF/Corea del Sur UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Estructuras Microscópicas (CIEMIC)

A persistent 2-month long outbreak of Ranavirus in a natural community of amphibians contributed to a mass die-off of gopher frog tadpoles (Lithobates capito) and severe disease in striped newts (Notophthalmus perstriatus) in Florida. Ongoing mortality in L. capito and disease signs in N. perstriatus continued for 5 weeks after the first observation. Hemorrhagic disease and necrosis were diagnosed from pathological examination of L. capito tadpoles. We confirmed detection of a frog virus 3 (FV3)-like Ranavirus via quantitative PCR in all species. Our findings highlight the susceptibility of these species to Rv and the need for long-term disease surveillance during epizootics.

BackgroundSince the COVID-19 vaccination campaign was launched all over Europe, there has been general agreement on how benefits of SARS-CoV2 vaccines outweigh the risks in patients with rare connective tissue diseases (rCTDs). Yet, there is still limited evidence regarding safety and efficacy of such vaccines in these patients, especially in the long-term. For this reason, in the framework of ERN-ReCONNET, an observational long-term study (VACCINATE) was designed in order to explore the long-term outcome of COVID-19 vaccination in rCTDs patients. The consent form was developed thanks to the involvement of the ERN ReCONNET ePAG Advocates (European Patients Advocacy Group).ObjectivesTo evaluate the safety profile of COVID-19 vaccination in rCTDs patients and the potential impact on disease activity. Primary endpoints were the prevalence of adverse events (AEs) and of disease exacerbations post-vaccination. Secondary endpoints were the proportion of serious adverse events (SAEs) and adverse events of special interest for COVID-19 (adapted from https://brightoncollaboration.us/wp-content/uploads/2021/01/SO2_D2.1.2_V1.2_COVID-19_AESI-update-23Dec2020-review_final.pdf)MethodsThe first ad-interim analysis of the VACCINATE study involved 9 ERN-ReCONNET Network centres. Patients over 18 years of age with a known rCTD and who received vaccine against COVID-19 were eligible for recruitment. Demographic data and diagnoses were collected at the time of enrolment, while the appearance of AEs and potential disease exacerbations were monitored after one week from each vaccination dose, and then after 4, 12 and 24 weeks from the second dose. A disease exacerbation was defined as at least one of the following: new manifestations attributable to disease activity, hospitalization, increase in PGA from previous evaluation, addition of corticosteroids or immunosuppressants.ResultsA cohort of 300 patients (261 females, mean age 52, range 18-85) was recruited. Systemic lupus erythematosus (44%) and systemic sclerosis (16%) were the most frequent diagnoses, followed by Sjogren’s syndrome (SS,12%), idiopathic inflammatory myositis (IMM,10%), undifferentiated connective tissue disease (UCTD,8%), mixed connective tissue disease (MCTD,4%), Ehlers-Danlos’s syndrome (EDS,4%), antiphospholipid syndrome (APS,2%). AEs appearing 7 days after the first and second doses were reported in 93 (31%) and 96 (32%) patients respectively, mainly represented by fatigue, injection site reaction, headache, fever and myalgia. Otitis, urticaria, Herpes Simplex-related rash, stomatitis, migraine with aura, vertigo, tinnitus and sleepiness were reported with very low frequency. Less than 2% of patients experienced AEs within 24 weeks from the second dose. No SAEs or AEs of special interest were observed in the study period. There were 25 disease exacerbations (8%), 7 of which severe. The highest number of exacerbations was observed after 4 weeks from the second dose (12 within week 4, 6 within week 12 and 7 within week 24). Disease exacerbation was most frequent in patients with EDS (33%) and MCTD (25%).ConclusionThis preliminary analysis shows that COVID-19 vaccination is safe in rCTDs patients. AEs appear most often early after vaccination and are usually mild. Disease exacerbations are not frequent, but can be potentially severe and tend to occur most frequently within the first month after vaccination. Exacerbations can also occur 3-6 months after vaccination, although a causal relationship with the vaccination remains to be established. Our present data underline the importance of long-term observational studies.Table 1.AEs and disease exacerbations per diseaseDiagnosisPatients enrolled (%) (n=300)EAs after 1st and 2nd dose (%)Exacerbations (%)APS25714EDS45033IIM10527MCTD44225SS12598SLE44698SSC16492UCTD850-AcknowledgementsVACCINATE is a study promoted by the European Reference Network on rare and complex connective tissue diseases, ERN ReCONNET. This publication was funded by the European Union’s Health Programme (2014-2020)Disclosure of InterestsNone declared