MUC5B (rs35705950) is the strongest risk factor for sporadic and familiar IPF, as well as a predictor of outcome. However, there are no data on the effect of MUC5B rs35705950 genotype on the prognosis of IPF patients on antifibrotic treatment. The aim of this study is to determine, in a phenotypically well-characterized population of patients with IPF treated with antifibrotics, the impact of MUC5B rs35705950 genotype on disease progression and survival.A common variant located in the promoter region of MUC5B rs35705950 by PCR amplification and Sanger sequencing.88 IPF patients on antifibrotic treatment were followed-up from 2014 until transplantation, death or end of follow-up (December 2019). Disease progression was defined as a forced vital capacity (FVC) loss ≥ 5% per year. All patients were genotyped for MUC5B rs35705950 T allele was not associated with a faster decline in FVC. Conversely, at the end of the follow-up, overall survival in carriers of the TT/TG genotype was longer compared to that of the GG genotype carriers. FVC (L) at baseline and time to respiratory failure at rest were independent predictors of worse prognosis.Out of 88 patients, 61 (69%) carried the mutant T allele (TT or TG) and 27 (31%) did not (GG). Carriage of the MUC5B rs35705950 T allele is associated with longer survival, highlighting the usefulness of MUC5B genetic data in clinical decision making.In IPF patients on antifibrotic treatment, carriage of the The online version contains supplementary material available at 10.1186/s12931-021-01694-z. Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrosing interstitial lung disease of unknown origin, characterized by relentless respiratory failure leading to death within 3–5 years from diagnosis . IPF is MUC5B gene transcription start site on 11p15 (rs35705950) was present in 38% of subjects with sporadic IPF and in 34% of subjects with familial interstitial pneumonia [MUC5B rs35705950 with IPF has been replicated in several independent cohorts [In 2011, Seibold and colleagues, using a genome-wide linkage analysis, demonstrated that the minor allele (T) of a single nucleotide polymorphism (SNP) located 3 kb upstream of the MUC5B encodes a mucin 5B precursor protein that contributes to airway mucus production and homeostasis [MUC5B dysregulation contributes to IPF development are currently unknown, MUC5B overexpression may cause mucociliary dysfunction, retention of particles and disruption of the normal reparative mechanisms in the distal lung, leading to chronic fibroproliferation and regenerative process that results in honeycomb cyst formation [eostasis . Althougormation –14.MUC5B rs35705950 T allele not only predisposes to IPF but has also been associated with improved survival, although this latter association remains debated and somehow controversial.MUC5B rs35705950 genotype on disease behavior and survival of IPF patients on antifibrotic treatment. To the best of our knowledge, this has never been investigated before.With this background, the aim of our study was to evaluate the influence of In this longitudinal retrospective study, we analyzed a consecutively collected cohort of well-characterized Caucasian adult patients with sporadic IPF referred to our center between April 2014 and September 2018. Patients were followed-up until transplantation, death or end of follow-up (December 2019), and those who permanently discontinued treatment were excluded from the study. Eighty-eight patients were included in the study  or stable (< 5%pred.), as previously reported , 16. ImpThe progression-free survival (PFS) was calculated from the time of treatment initiation until functional progression, which was defined as absolute FVC% pred. loss ≥ 5% compared to the basal FVC% pred.Based on the level of oxygen in the blood (PaO2), we defined respiratory failure when this value was < 60 mmHg (8.0 kPa).The time to development of respiratory failure (RF) on exercise and at rest was defined as the time from treatment initiation and development of RF.The occurrence of acute exacerbation of IPF, defined as an acute worsening of dyspnea with bilateral ground glass opacities superimposed on the UIP pattern not fully explained by fluid overload , has beeMUC5B rs35705950 genotyping. Based on their MUC5B genotype, patients were then divided in two groups (TT/TG or GG genotype).Blood sample was taken for each patient included in the study for DNA extraction and The study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the University Hospital of Padova (4280/AO/17). Informed consent was obtained for all study participants.Sample processing were described in the Additional file Categorical variables are described as absolute (n) and relative values (%) whereas continuous variables are described as median and interquartile range. To compare demographic data and baseline clinical characteristics between TT/TG and GG genotypes, Chi square test and Fisher’s exact test for categorical variables and Mann–Whitney U test for continuous variables were used, as appropriate. Due to the low number of events, in survival analysis, death and death/lung transplantation were combined. Survival was estimated using the Kaplan–Meier method and the p-value of the log-rank test was reported. Analysis on progression was conducted using the Cumulative Incidence Functions (CIF) to account for competing risks.Clinical characteristics were evaluated to determine their relationship with survival in a univariate analysis of Cox proportional hazards regression testing. The time dependency was evaluated via visual examination of Schoenfeld residuals plot. Variables with a statistically significant association with overall survival on univariate analysis were included in a multivariate Cox proportional hazard regression test to find factors independently associated with disease progression.All data were analyzed using SPSS Software version 25.0  and R software. P-values < 0.05 were considered statistically significant.Clinical and functional characteristics at baseline for the entire study population are shown in Table MUC5B rs35705950 T was 42% (74/176), while the frequency of the wild type G allele was 58% (102/176). The MUC5B rs35705950 genotype frequencies met the Hardy–Weinberg equilibrium  either in homozygosity or in heterozygosity, the population was categorized in two groups: patients with TT/TG genotype n = 61, 69%) and with GG genotype     and lower FVC (L) at treatment initiation  were significantly associated with mortality   and FVC at treatment start (2.6L), a significantly higher percentage of GG genotype carriers had a FVC lower than the median value , whereas no differences were observed with regard to development of RF at rest , lower lung function decline and mortality [The finding of our study is in line with previous work by Peljto et al. , who desortality .CO. One bias that makes it difficult to compare these studies was the T allele frequency of 20%, consistently lower to that reported in previous studies [MUC5B polymorphisms depends on the individual’s ethnic background, with a lower prevalence reported among Asians compared to white non-Hispanics [Conversely, Jiang and colleagues showed that T allele was associated to increased mortality in a Chinese population ; specifi studies , 6, 7  rarely determine significant clinical or biological effects, except for conferring increased disease susceptibility. Conversely, rare variants  tend to be highly penetrant with substantial phenotypic effect. The wild type (G) and mutant (T) allele may interact with distinct environmental factor to determine opposite effect on disease susceptibility and prognosis, but this needs to be explored further. Intuitively, carriers of the T allele may have a better survival than noncarriers as a result of a slower disease progression, but this does not seem to be the case. Indeed, evidence of an association of less severe pathological changes and MUC5B polymorphism is reported, but it is not clear how these changes were defined [The genetic peculiarity of the  defined . MoreoveIPF population in our cohort had a relatively stable disease under antifibrotics, with FVC decline of approximately 50 mL/year, similar in TT/TG and GG carriers. Moreover, the survival rate was very high, up to 70% at 5 years with only 5 cases of acute exacerbations leading the patient to death, confirming the efficacy of antifibrotic treatment in reducing mortality and also acute exacerbations. This rate is higher to that reported in literature; indeed, a recent study described survival rate of the INSIGHT-IPF registry  of nearlMUC5B genotype [Similarly to FVC decline, no between-group difference was observed in progression-free survival, that was nearly two years, supporting the beneficial effect of antifibrotic treatment in IPF, irrespective of genotype –30.MUC5B genotype was associated with survival independently from FVC.However, at treatment initiation the two groups differed in terms of FVC, which was an independent predictor of mortality. Functional differences between TT and GG genotypes were described also by Peljto and coworkers , but autGiven the prognostic role of FVC in IPF, it is not surprising that patients with a lower FVC at baseline had a worse survival, and that patients with more preserved lung function at diagnosis live longer , 32. WhaMUC5B rs35705950 T allele is associated with overproduction and accumulation of mucin in distal airspaces and this could lead to an impaired mucociliary activity, that may trigger cough [MUC5B has an important role in airway immunity, similar to other mucins, by capturing and removing infectious agents through mucociliary clearance . MUC5B rer cough . TherefoMUC5B gene polymorphism and honeycomb cysts, one of the pathologic hallmarks of IPF. In subjects with IPF, regions of dense accumulation of MUC5B were observed in areas of microscopic honeycombing and involved patchy staining of the metaplastic epithelia lining the honeycomb cysts [Another potential consequence of mucociliary dysfunction is the retention of inhaled substances  and endogenous inflammatory debris that over time may result in temporally and spatially distinct areas of microscopic scaring and progressive fibroproliferation in the lung. In this regard, Seibold  reportedmb cysts .MUC5B polymorphism is associated with a more typical subpleural distribution of fibrosis and with a greater proportion of confident radiological diagnosis (probable UIP and UIP) [. In our cohort, the presence of T allele MUC5B polymorphism was associated with a lower percentage of radiological diagnosis, which implies that carriers of the T allele did not have a CT pattern of UIP and required a histological diagnostic confirmation. In the study by Chung and coworkers [These pathological changes are reflected in the radiological abnormalities, characteristic of IPF. Indeed, and UIP) . In our oworkers , no infoOur study has some limitation. Firstly, the study population is relatively small and there is no independent validation cohort. Secondly, the retrospective nature of the study might have introduced unintentional biases. However, the study population was carefully characterized and enrolled consecutively, which may have mitigated the selection bias. Finally, although we selected only sporadic cases, three patients were younger than 50 years, which makes one wonder about familiar disease. To the best of our knowledge they are all sporadic cases, although telomere gene mutations screening and monitoring extended to their family members would be needed to detect family aggregation.In conclusion, we have shown for the first time that MUC5B rs35705950 genotype does not seem to affect response to antifibrotic treatment in patients with IPF. In addition, carriage of the mutant T allele is associated with longer survival in IPF patients on antifibrotic treatment. Larger studies and genotyping of additional genes involved in disease pathogenesis are needed to assess the role of genotype stratification in clinical trial design and in clinical decision making.Additional file 1. Sample processing.Additional file 2: Table S1. MUC5B rs35705950 genotype frequency.Additional file 3: Table S2. Predictive factors of progression in the entire population of IPF patients treated with antifibrotics.Additional file 4: Table S3. Occurrence of respiratory failure (RF) at rest and FVC (L) at treatment initiation according to MUC5B genotype (TT/TG vs. GG patients).


Apc-mutated Pirc rats, and in age-paired wt F344 rats. Principal Coordinates Analysis indicated that samples’ distribution was driven by age, with samples of young rats  separated from older ones . Diversity analysis showed significant differences between FM and MAM in older Pirc rats, and between MAM of both Pirc and wt rats and the tumour microbiota, enriched in Enterococcus, Escherichia/Shigella, Proteus and Bifidobacteriaceae. In young animals, Pirc FM was enriched in the genus Delftia, while wt FM was enriched in Lactobacillus and Streptococcus. Some CRC biomarkers and faecal short chain fatty acids (SCFAs) were also measured. Colon proliferation and DClK1 expression, a pro-survival mucosal marker, were higher in Pirc than in wt rats, while the mucin MUC2, was lower in Pirc rats. Branched SCFAs were higher in Pirc than in wt animals. By Spearman analysis CRC biomarkers correlated with FM (in both young and old rats) and with MAM (in young rats), suggesting a specific relationship between the gut microbiota profile and these functional mucosal parameters deserving further investigation.Faecal (FM) and colon mucosal associated microbiota (MAM) were studied in a model of colorectal cancer (CRC), the  Moreover, not only the faecal microbiota, but also the bacterial community associated with the colon mucosa may be an important determinant of CRC risk6. In fact, it has been shown that bacterial biofilms associated with the surface of colon epithelium are increased in a subset of sporadic CRC7 and in the apparently normal colon mucosa of patients with familial adenomatous polyposis (FAP), a genetic syndrome causing high risk of developing CRC5. These biofilms contain species subtypes of Escherichia coli and Bacteroides fragilis, encoding genes for oncotoxins (colibactin) and B. fragilis toxin (bft).Epidemiological and experimental studies show that variation in the composition of the intestinal microbiota affects colorectal cancer (CRC) risk, accordingly, a number of studies indicates that the faecal microbiota of colon cancer patients differs from that of healthy subjectsAPC gene, responsible for the inherited predisposition to intestinal carcinogenesis in FAP patients and present in the majority of sporadic CRC , are considered early, necessary events in the development of the disease8. Accordingly, rodent models mutated in Apc gene have been widely used to study the mechanisms of colon carcinogenesis, but also the effect of drugs or environmental factors, such as diet. Pirc rats (F344/NTac-Apcam1137) carry a heterozygous germinal mutation in Apc leading to the spontaneous development of tumours along the intestine, notably in the colon10, thus representing a relevant experimental model to study the process of colon tumorigenesis including the influence of external factors or individual host factors, such as the intestinal microbiota11.In humans, mutations in the Apc-mutated rodents gave conflicting results. Son et al. reported that the microbiota associated to the intestinal mucosa of Apc-mutated Min mice was enriched with Bacteroidetes spp. compared with wt animals6. Similarly, we recently documented that the bacterial community associated with the mucosa of Pirc rats presents some differences when compared with that of wt rats12, thus suggesting variations in animals at higher CRC risk. On the contrary, Ericsson et al. documented that the faecal microbiota of Pirc rats and wt rats are similar13.Previous studies on the intestinal bacterial community in 10, and in older rats (11-month-old), when colon tumours are present. In addition, since microbiota may affect cancer risk with various mechanisms such as the production of metabolites in the intestinal lumen, in the same rats we also studied faecal short chain fatty acids (SCFAs), deriving from gut microbial fermentation of indigestible fibres and other substrates. SCFAs contribute to the homeostasis of colonocytes14 and were not studied before in Pirc rats. Moreover, we also determined some mucosal parameters that have been associated with CRC risk and used as cancer biomarkers. MUC2 is the main mucin secreted by colonocytes, whose alterations have been associated with an increased CRC risk16; CD68, is a pan-macrophage marker used as an index of inflammation, a phenomenon associated with high CRC risk. Since Apc mutations have been reported to affect colon proliferation and cell survival8, we also studied PCNA expression, as a marker of proliferation, together with DClK1 expression, a microtubule-associated protein kinase regulating pro-survival signalling18 .Given these contrasting results and the importance to characterize Pirc rats as a relevant model of CRC, we though it of interest to study the composition of their intestinal bacterial community in both the faeces and the colon mucosa. For this purpose, we compared the microbiota profiles of Pirc and wt rats at two different ages: 1 month-old, when the colon mucosa is morphologically normal with no macroscopic lesionsFinally, to have a glimpse on the composite relations between the faecal and mucosal microbiota, SCFAs as its produced metabolites, and the cancer biomarkers measured in the mucosa, the data obtained were analysed with a multivariate approach  to look for a correlation between all these different parameters.9. Twenty-six rats (13 wt and 13 Pirc) were maintained in polyethylene cages with their respective mothers up to 4 weeks of age , then separated and fed with Teklad Global Rodents Diet® (ENVIGO) until 11 months of age. Rats were euthanized at one month (T1: 7 Pirc rats and 8 wt rats) and eleven months (T11: 6 Pirc rats and 5 wt rats) of age and the entire colon and small intestine opened to check for the presence of macroscopic tumours, which were counted and the diameter measured (Supplementary Table Pirc rats (F344/NTac-Apcam1137) and wild type (wt) Fisher F344 rats were originally obtained by the National Institutes of Health (NIH), Rat Resource and Research Center (RRRC)  and bred in CESAL . The colony is maintained by mating heterozygous Pirc rats with wt and pups genotyped at 3 weeks of ageThe ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the Italian Ministry of Health (in accordance with EU Directive 2010/63/EU). Formal approval to conduct the experiments was obtained from the Animal Subjects Review Board of the University of Florence. Experiments have been reported according to ARRIVE guidelines. All experiments were in accordance with the Commission for Animal Experimentation of the Italian Ministry of Health and with EU Directive 2010/63/EU for animal experiments.12. Briefly, faecal pellets, colon mucosa and tumour samples were collected in RNAlater  and stored at − 80 °C until DNA extraction with DNeasy PowerLyzer PowerSoil Kit . Library preparation and sequencing of the hypervariable region V3–V4 of the 16S rRNA gene were performed by using an Illumina MiSeq platform with a 300-bp paired-end reads protocol. The obtained reads were pre-processed with CUTADAPT19 to remove primers and Illumina adapters, while SICKLE was used to remove low quality portions of the reads20. OTUs/ASVs identification was performed in MICCA (ver. 1.7.2)21 with miccaotu command, using the UNOISE3 algorithm22, while taxonomy was assigned using the RDP classifier (ver 2.11)23 against the RDP database.Bacterial DNA extraction, sequencing, and sequencing data analysis were performed according to a recent work from our grouphttps://www.ebi.ac.uk/ena/browser/view/PRJEB40657.The datasets generated during and/or analysed during the current study are available in the ENA repository, under accession number PRJEB40657 at 10. CD-68 and DClK1 expressions were also evaluated in NM and tumours with immunohistochemistry experiments as reported25. Apoptosis was evaluated in histological longitudinal sections (4 µm thick) of the NM and tumours stained with hematoxylin–eosin, as reported10.Proliferative activity in the normal colon mucosa (NM) and in tumours was evaluated measuring Proliferating Cell Nuclear Antigen (PCNA) immunoreactivity with a mouse monoclonal antibody , as describedg for 15 min at 4 °C. Protein content of supernatants was measured accordingly to DC Protein Assay kit instructions . Equal volumes of supernatants containing 30 μg of proteins were allowed to dry on nitrocellulose membrane (Invitrogen) for 30 min and then washed under gentle agitation at RT for 1 h with 0.05% Tween-PBS solution (T-PBS) containing 5% dry milk . Membranes were incubated for 1 h with 1:1000 dilution of anti-MUC2 antibody (sc-15334 Santa Cruz Biotech.), then washed with T-PBS and incubated for another 1 h with anti-rabbit IgG HRP-conjugated antibody . Finally, protein spots were visualized using the enhanced chemiluminescence procedure with Immobilon Horseradish Peroxidase Substrate (Millipore) and quantified by densitometric analysis using the Quantity-One software (Bio-Rad).MUC2 protein levels were measured by dot blot assay. Proteins were extracted from scraped NM samples harvested at sacrifice from the proximal colon and kept at − 80 °C. Samples were homogenized in RIPA-buffer containing 1% protease cocktail inhibitors and 1% phosphatase inhibitors , sonicated for 15 s and centrifuged at 1.000×Faeces were freshly collected and stored at − 80 °C. At use, each sample was thawed and weighted (weight range 500–800 mg); then added sodium bicarbonate 10 mM (1:1 w/v) in a 1.5 mL centrifuge tube. The obtained suspension was then mixed with the aid of a sterile wooden stick and briefly shaken in a vortex apparatus, extracted in ultrasonic bath (15 min) and then centrifuged at 4 °C at 13.000 rpm for 90 min. The supernatant was collected, transferred in 1.5 mL centrifuge tube and stored at − 20 °C until use.For the analysis, these supernatant samples were thawed, briefly centrifuged at 5000 rpm and resuspended for 5 min in ultrasonic bath. The SCFAs were then extracted as follows: an aliquot of 100 μL of sample solution (corresponding to 0.1 mg of stool sample) was added with 10 μL of internal standard (ISTD) mixture, 1 mL of tert-butyl-methyl ether and 50 μL of 1.0 M HCl solution in 1.5 mL centrifuge tube. Afterwards, each tube was shaken in a vortex apparatus for 2 min, centrifuged at 10,000 rpm for 5 min; the solvent layer was finally transferred in auto-sampler vial and analysed by Gas-Chromatography–Mass Spectrometry (GC–MS) method, using an Agilent GC–MS system composed with 5971 single quadrupole mass spectrometer, 5890 gas-chromatograph and 7673 autosampler. Details of the method are described in 26. Sequencing count data were parsed into R with phyloseq27, and prior to further analysis, reads counts were transformed with CSS  followed by logarithm transformation as implemented in metagenomeSeq package28. Alpha diversity in bacterial communities was explored using three indices: Species Richness , Evenness index , and Shannon index . Differences in alpha diversity indices between samples categories, were assessed with T-test. OTUs distribution in the mucosal and tumour adherent microbiota community was further explored with rank-abundance curves, obtained with the package BiodiversityR29. Beta diversity of bacterial communities was explored and visualized using Principal Coordinate Analysis (PCoA) ordinations based on the Bray–Curtis dissimilarity, with the phyloseq package. Associations of bacterial community diversity with experimental factors  were assessed with permutational analysis of variance  in the vegan package30, and with Hierarchical Clustering on Principal Components (HCPC) analysis in the FactoMineR package31. In HCPC, dimensionality of microbiota data is first reduced with the aid of principal component analysis, and then hierarchical clustering is applied. LEfSe  analysis32 on the CSS transformed abundances, was performed using an online Galaxy implementation  to identify plausible bacterial biomarker(s) able to separate different groups . Significance threshold for Kruskal–Wallis rank-sum test was 0.05, and threshold for LDA  was 2. Data from the SCFAs analysis, and from the determination of mucosal biomarkers  were presented as means ± SE; differences between Pirc and wt rats and between samples at T1 and T11 were analysed with two–way ANOVA considering the effect of genotype and age. All analyses were carried out using GraphPad Prism 5.0 and STATGRAPHICS Centurion XVI.II. p values of ≤ 0.05 were considered statistically significant. Mantel test was used to assess relations between the structure of the bacterial community and the measured variables  as previously described34.Statistical analysis of the microbiota data was performed using R Software as describedEscherichia/Shigella, Streptococcus, and Bacteroides genera, in decreasing order of dominance and relative abundance, respectively , by alpha diversity estimation . Furthermore, on the second ordination axis . Moreover, PERMANOVA performed on T1 or T11 samples separately , PCoA based on Bray–Curtis distances was performed Fig. . PCoA orBacterial community composition Fig. A shows tBacteroides and Clostridium XIVa were the most abundant genera in almost all samples, with highest relative abundances in faecal samples at T1. On the contrary, Oscillibacter was less abundant at T1, and increased at T11 in all samples, with exception of tumours. In general, as an effect of temporal variation (from T1 to T11), the relative abundances of Ruminococcus, Oscillibacter, Streptococcus, and Lachnospiraceae increase with rats’ aging, while those of Bacteroides, Clostridium XIVa, and Parabacteroides decrease .Heatmap analysis Fig. D on thesles Fig. D. At theDelftia was enriched in the faecal bacterial community of Pirc rats, while Lactobacillus and Streptococcus in the faecal bacterial community of wt animals  from faecal samples in young rats, 105 OTUs were shared between Pirc and wt core microbiota, while a comparable number of exclusive “core OTUs” were found for each genotype , comparing faecal samples of Pirc and wt rats, a higher number of differences was found Fig. B. GammapT11 Fig. , 352 OTUCandidatus, Saccharibacteria, Roseburia, Clostridium XI, and Turicibacter were enriched in Pirc rats. Interestingly, in Pirc rats, when adherent microbiota of tumours was compared with adherent microbiota of their normal mucosa  compared to wt (N = 4), while only 16 OTUs were shared Fig. . By compAnaeroplasma, Bacteroides, Saccharibacteria, Roseburia, and Clostridium XI in 11-month-old Pirc rats, while Oscillibacter and Clostridium IV are enriched in 11 months old wt rats.Based on the LEfSe and on the relative abundance analyses “ we can iSCFAs were measured in the faeces of Pirc and wt rats at the two different ages  and the Euclidean distance matrices of: (i) concentration of individual SCFAs ; and (ii) values of functional parameters  on the same set of samples. The test was performed on different set of samples on the basis of age and sample type , is of particular interest, faithfully reproducing many traits of human CRC biology, in particular the development of tumours in the colon, the very tract of the intestine more frequently affected by cancer in humans11. Notwithstanding its relevance as a model to study CRC, insight into the Pirc gut microbiota is still scarce.Accumulating evidences, driven by the metagenomics breakthrough during the last decade, are linking CRC to specific signatures of what could be considered an “oncogenic” microbiotaIn the present work we aimed at defining the structure of both the faecal and mucosal-associated microbiota in Pirc rats, along with its changes during the colorectal tumorigenesis process and its association with host functional parameters and with microbial metabolic markers.13 reporting that the faecal microbial community is shaped by age (1 month and 4 months), rather than by genotype (Pirc vs wt). On the other hand, considering separately only old rats (T11), diversity analysis showed differences in bacterial community clearly related to the genotype . Moreover, beta diversity analysis, revealed that the main factor shaping faecal and mucosal bacterial communities was the rat age with a low contribution from rat genotype. This result is in agreement with a previous study in Pirc rats. MoreoveBacteroides, Escherichia/Shigella, and Streptococcus genera, a result, that at least for, Escherichia/Shigella was also confirmed by the LEfSe analysis which identified this genus as a marker for the tumour microbiota compared to the normal mucosa of Pirc rats. Members of the Escherichia/Shigella genus such as pathogenic E. coli, can acquire the ability to produce genotoxins, which could be involved in the carcinogenesis process, as observed in colon cancer-associated E. coli strain37 in Min mice and by several reports indicating that E. coli frequently colonizes cancer lesions and neighbouring epithelium39.Intriguingly, alpha diversity analysis highlighted a strong difference in the Evenness and Shannon’s indices of the microbiota adherent to the tumours with respect to all other samples, together with unaltered Richness compared with the normal mucosa. Three OTUs dominated the tumour microbiota which was otherwise similar to the normal mucosal microbiota from the same subject. This evidence, together with the observed similarity in beta diversity between normal mucosa and tumour adherent microbiota, suggests that the latter is dominated by a subset of the same OTUs that normally colonizes the mucosa, which, by some mechanism, expands and dominates the community in the tumour tissue. These 3 dominant OTUs were identified as belonging to the Bacteroides fragilis and E. coli were reported as associated to the biofilm of both the normal mucosa and the polyps in FAP patients. Besides reporting the presence of such bacterial strain, Dejea et al.5 demonstrated their carcinogenic effect when inoculated in pathogen-free wt mice, especially when a co-inoculation of both strains was performed. The evaluation of mucosal biofilm was beyond the scope of this work, nevertheless the observed dominance of OTUs from the Bacteroides and Escherichia/Shigella on Pirc tumours respect to normal mucosa, suggests that in Pirc rats, a similar phenomenon could be present.Moreover, specific endotoxin producing strains of Anaeroplasma, Bacteroides, Saccharibacteria, Roseburia, and Clostridium XI, while Oscillibacter and Clostridium IV were enriched in 11 months old wt rats.LEfSe analysis indicated that the microbiota adherent to the normal mucosa of old Pirc rats (T11) was enriched in 12, with Pirc rat colon mucosa enriched in Clostridium cluster XI , while the wt colon mucosa was enriched in Clostridium clusters IV, a bacterial group including several anti-inflammatory and butyrate-producing species40.These finding are in agreement with recently reported significant differences between the two genotypesDelftia and Corynebacterium genera were identified as markers of Pirc genotype in faeces and mucosa, respectively this specific finding could imply a causal relationship with tumour development since. LPS from Delftia tsuruhatensis can regulate cell proliferation and differentiation in murine colonic crypt-derived organoids41. Regarding the genus Corynebacterium, Zorron et al.42 found a higher relative abundance in colorectal invasive cancers suggesting a role of this bacterial genus in cancer progression. We also found that Lactobacillus and Streptococcus are enriched in the faecal bacterial community of wt, while in mucosal-associated bacterial community in wt rats was enriched in Burkholderiales.Regarding differential abundance analysis in young rats (when tumours are not present), as reported above differences between Pirc and wt rats were almost negligible with diversity analysis. However, we identified a set of possible markers of the Pirc microbiota in faeces and in mucosa which are interesting given that the absence of tumours in these animals (T1) reduces possible interferences on the microbiota composition from the peculiar tumour’s environment. Accordingly, in these young rats, 44. The relative abundance of proteolytic bacteria such as Enterococcaceae as found in tumour samples may influence BSCFAs production45, leading to an increase in the production of detrimental putrefactive products such as ammonia, hydrogen sulphide and indoles44. We did not find other significant differences in SCFAs level in the faeces. Regarding functional mucosal parameters associated with cancer risk, we confirm a higher level of proliferation in the colon mucosa of Pirc rats, as previously documented by our group10. On the other hand, we did not find any difference between the two strains of rats in the expression of the pan-macrophage marker CD-68, although the expression of this marker was higher in older rats (irrespectively from strain). Although it is possible that indeed inflammation in the colon is not different between the two strains of rats, it is also possible that the macrophage accumulation in older rats overcomes any possible difference in CD-68 staining between the two strains of animals. Moreover, we should stress that although CD-68 is a commonly used marker of macrophages, including those associated with CRC, it is not able to discriminate between the different subtypes of macrophages , a this fact constitutes a limitation of this study. Indeed, the phenotypic recognition of the different type of macrophages would have required the use of a double staining with additional markers, a characterization that could be deepen in future studies46. In addition, we showed that the expression of MUC2, the most abundant mucin secreted in the colon, was lower in Pirc rats compared with wt animals, and that this expression was significantly higher in older rats than in young ones, irrespectively from the strain of the rats. Several studies have found that MUC2 plays a crucial role in protecting the gut and maintaining intestinal homeostasis15. In particular, the lack of MUC2 has been related to colon cancer development in Muc2−/− deficient mice15. The lower expression of MUC2 in Pirc rats could be linked with their mutation in Apc, a genetic alteration leading to up-regulation of Wnt-signalling pathway which negatively regulates MUC2 expression47. Moreover, based on evidences showing that the mucus layer attenuates the cytotoxic activity of the E. coli phylotype B2 in vitro48, it is possible to speculate that the lower level of MUC2 in Pirc rats may expose their epithelium to potentially cytotoxic bacterial strains48, an hypothesis that should be verified in vivo, labelling with probes recognizing those specific bacteria the colon mucosa of both wt and Pirc rats5, as well of clinical specimens from patients at various CRC risk. Finally, in this work, we showed for the first time that the expression of DClK1, a microtubule-associated protein kinase identified in the intestine as a marker of tuft cells and regulating pro-survival signalling, was significantly higher in the crypts of Pirc rats compared with those of wt rats, in agreement with higher expression found in Apc-mutated min mice17.Stepping from microbiota analysis to metabolite and mucosal functional parameters, we found that BSCFAs, mainly isovalerate, isobutyrate, and 2‐methylvalerate, were higher in Pirc genotype. BSCFAs are the bacterial fermentation products of undigested proteins and peptides reaching the colonTo acquire a glimpse on the possible relations between the microbiota composition, the SCFAs as its produced metabolites, and the functional parameters of the mucosa as a consequence, we further analysed the whole data of microbiota composition, the whole set of SCFAs measured, and the whole set of functional parameters with a multivariate approach (i.e. Mantel Test on Spearman correlation). A significant Spearman correlation in Mantel test implies a similar structure in the two distance matrices compared, hence, that some kind of relation exists between the measured set of variables. Both Pirc and wt rats were included with all the data measured on the same set of animals, thus representing different aspects of their gut biology. Interestingly, in young rats, the microbial community composition in the mucosa was significantly correlated to the functional parameters measured at the same time, while the microbial communities in faeces resulted correlated with borderline significance. Thus, even if the diversity analysis alone failed to highlight a difference between Pirc and wt microbial communities’ composition in young rats, the evidence of a correlation with functional parameters, most of which presents significant differences between Pirc and wt rats , suggests that some differences are present.Apc and in age-paired wt rats. PCoA on the samples collected indicates that the distribution of both Pirc and wt rats was mainly driven by the age of the rats and, partially by sample type  with significant effect of the different genotype on older animals.We performed a meta-taxonomic analysis of faecal and colon mucosal adherent microbiota in Pirc rats, mutated in the CRC key gene Differential abundance analysis identified a set of possible markers for the Pirc genotype microbiota in faeces and in colon mucosa, in particular at 11 months of age, as well as markers of microbiota adherent to tumours respect to that adherent to the normal mucosa.We also showed that BSCFAs were higher in Pirc rats and that some functional parameters representing CRC biomarkers  were significantly different in Pirc rats as compared to wt rats.Finally, we found a significant correlation between the microbiota and these CRC biomarkers. Since the differences in these biomarkers are significantly different between the two strains, this result suggests a specific relationship between the microbiota profile and the colon mucosa which should be deepen studying in both pre-clinical and clinical specimens, not only the faecal microbiota but also that associated with the colon mucosa.Supplementary Tables.Supplementary Information.


Oryza sativa L.) lines to produce hybrids can obtain great heterosis. However, PTSGMS rice lines exhibit low stigma vitality when high-temperature (HT) stress happens during anthesis. Jasmonates (JAs) are novel phytohormones and play vital roles in mediating biotic and abiotic stresses. Little is known, however, if and how JAs could alleviate the harm of HT stress during anthesis to the stigma vitality of PTSGMS lines. This study investigated the question. Two PTSGMS lines and one restorer line of rice were pot-grown and subjected to normal temperature and HT stress during anthesis. The stigma exertion rate, sigma fresh weight, stigma area, contents of JAs, hydrogen peroxide (H2O2), and ascorbic acid (AsA), activity of catalase in stigmas, and the number of pollens germinated on the stigma of PTSGMS lines were determined. The results showed that a rice line with higher JAs content in the stigma under HT stress showed lower H2O2 content, higher AsA content and catalase activity in stigmas, larger stigma area, heavier stigma fresh weight, more pollens germinated on the stigma, and higher fertilization and seed-setting and rates. Applying methyl JAs during anthesis to rice panicles decreased the accumulation of reactive oxygen species and enhanced stigma vitality, thereby increasing fertilization and seed-setting rates of the hybrids of PTSGMS rice lines under HT stress. The results demonstrate that JAs attenuate the injury of HT stress to the stigma vitality of PTSGMS rice lines through enhancing antioxidant ability.Using photothermosensitive genic male sterile (PTSGMS) rice ( Oryza sativa L.), namely, a hybrid produced by a photothermosensitive genic male sterile (PTSGMS) line crossed with a restorer line, could produce great heterosis  activity of PTSGMS rice lines , and reduce antioxidant ability, such as decreases in catalase activity and ascorbic acid (AsA) content, in plant organs , such as hydrogen peroxide  lines currently used in China because of their greater heterosis than other PTSGMS rice lines  during the rice-growing season (May–October) in 2017, 2018, and 2019. Two PTSGMS rice  for both normal temperature (NT) and HT treatments with three rice lines in each phytotron. Treatment details were described by Chen et al. , 2021. B2O2, and AsA; and catalase activity in stigmas. Each physiological measurement had three biological replications.Four hundred panicles showing spikelet opening were tagged from 30 pots of each rice line and each temperature treatment. The spikelets that opened at 9:00 h on the first day during treatment were sampled at 12:00 and 14:00 h on the first day during treatment, 14:00 h on the second day during treatment, and 14:00 h on the first day after treatment (the third day from the treatment), respectively, for the measurements of stigma area; stigma fresh weight; stigma exertion rate; the contents of JA, MeJA, HThe opened spikelets with an angle ≥10° between the palea and lemma showing the exertion of single stigma and/or dual stigmas on the panicle that headed out from the sheath of flag leaf were marked and counted at every 30 min from 07:00 to 15:00 until the spikelets on each tagged panicle were fully opened. The total number of opened spikelets and spikelets with single exerted stigma and/or with dual exerted stigmas from 18 tagged panicles were counted and recorded from the first day of temperature treatment to the finish of the whole heading period, with six panicles as one replication. The percentage of single stigma exertion, dual stigma exertion, and total stigma exertion on a panicle were calculated using the following formulas:For each PTSGMS line, 30 tagged spikelets that opened on the top of panicle at 9:00 h on the second day during treatment were fully fertilized with the pollens from the restorer line and then were sampled from each treatment. After 30 min of fertilization, 10 pollinated stigmas from each treatment were quickly taken out using tweezers and put into the precooled formaldehyde–acetic acid–ethanol fixative solution for 24 h, and then the pretreated samples were reserved in 70% (vol/vol) alcohol. Before the observation of pollen germination, the samples were put in 50% (vol/vol), 30% (vol/vol), and 10% (vol/vol) alcohol and distilled water, respectively, and rehydrated for 10 min each time. Ten M NaOH solution at 56°C was used to soften the samples after rehydration for about 10 min, and 0.1% (vol/vol) aniline blue dyeing solution was also used to stain the samples for 12–24 h. The stained samples were washed with distilled water twice and then put on a glass slide. After adding a drop of glycerin and covering a cover slip, the samples on the slide were used to observe the number of pollens germinated on the stigmas under a fluorescent microscope and used the image editing software  for calculation. The stigma area was also observed using a fluorescent microscope with the help of image editing software ZEN 2012.2O2 in the stigma was determined using the method of Avramova et al.  from 10 pots for each treatment were harvested at maturity for the determination of grain yield and yield components as described previously  was sprayed to the panicles that headed out from the sheaths of flag leaves, with 4 mL per panicle. The MeJA concentration of 100 μM was chosen because such a concentration was observed to be an optimal dose for enhancing the tolerance of rice to HT stress  accumulation in the stigmas and stigma receptivity were qualitatively analyzed as described by diaminobenzidine (DAB) stain  ethanol for 3 times at every 30 min, after the samples were incubated at 25°C in the dark for 2 and 24 h, respectively. Accumulation of ROS in samples was then observed under a microscope. The Image-iT™ LIVE Green Reactive Oxygen Species Detection Kit (I36007) was used for quantifying the ROS accumulation in live cells and stigma receptivity according to Prasad and Djanaguiraman  than for Peiai 64S . In contrast, the catalase activity in stigmas was significantly increased at T1, but decreased at T2, T3, and T4 under HT, relative to that under NT  with variation among the 3 lines . On averr = 0.976** to 0.992** (r = 0.848* to 0.980**), whereas they were negatively correlated with H2O2 content in stigmas with r = −0.853* to −0.890*  and Control-2 (spraying water under HT), spraying MeJA under NT (NT+MeJA) and spraying MeJA under HT (HT+MeJA) significantly increased JA and MeJA contents in stigmas at 12:00 h (T1) and 14:00 h (T2) on the first day during the temperature treatment and 14:00 h on the second day during the temperature treatment (T3) for all the test rice lines . ContentSimilar to JA and MeJA contents, catalase activity and AsA contents in stigmas were significantly increased for both PTSGMS lines under NT+MeJA and HT+MeJA, especially under HT+MeJA when compared with those under Conrol-1 and Control-2, . Applyin2O2 in stigmas was decreased by applying MeJA, and the decrease was significant under HT, relative to the control  JAs contents in stigmas varied with PTSGMS rice lines, and a PTSGMS rice line with higher contents of JAs in stigmas exhibited stronger thermotolerance; (2) applying MeJA could alleviate stigma impairment caused by HT stress during anthesis of PTSGMS lines via maintaining the redox homeostasis. These findings would have great significance in the production of “two-line method” hybrid rice. First, a rice cultivar or a PTSGMS rice line with strong thermotolerance could be bred by enhancing JAs biosynthesis through classical breeding methods and/or genetic engineering technology. Second, MeJA could be used as a chemical regulator to alleviate HT stress on the stigmas of PTSGMS rice lines by inhibiting ROS and enhancing AOS. Moreover, there is a report showing that a cytoplasmic male sterile rice line treated with MeJA under NT during anthesis substantially increased the percentage of filled spikelets and the seed yield when the line was crossed with a restorer line  showing normal male fertility significantly decreased fertilization rate and seed-setting rate under both NT and HT during anthesis . A similIt is suggested that MeJA could trigger cleistogamy sometimes in cereals, which can reduce sterility under heat stress conditions (Honda et al., 2O2 content in the stigmas, heavier fresh weight of stigmas, larger single stigma area, greater exertion rate of stigmas, and more number of pollens geminated on the stigma, leading to higher seed yield when the line was crossed with a restorer rice line. JAs could alleviate the injury to stigmas caused by HT stress during anthesis through strengthening AOS and suppressing ROS production in the stigmas of PTSGMS rice lines. A higher level of JAs in the stigmas under HT stress could be used as a criterion or a physiological trait for breeding and selecting a PTSGMS line with thermotolerance, and MeJA could be used as a chemical regulator to minimize the adverse effect of HT stress on the stigma vitality of PTSGMS rice lines. Further work is needed to verify the roles of JAs in responding to HT stress under different conditions including different genetic backgrounds and to understand the molecular mechanism by which JAs assuage the harm of HT stress to stigma vitality of rice.A PTSGMS line with higher JA and MeJA contents in stigmas under HT stress had better traits reflecting stigma vitality including higher AsA content and catalase activity and lower HThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JC and JY designed the research. JC, WM, KF, HS, YZ, YS, CL, and JH performed the research. JC, KF, KZ, and ZW analyzed the data. JC and JY wrote the paper. All authors reviewed 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.


Togaviridae. Disturbances in the natural transmission cycle of this virus result in outbreaks in equines and humans, leading to high case fatality in the former and acute febrile illness or neurological disease in the latter. Although a considerable amount of knowledge exists on the eco-epidemiology of North American EEEV strains, little is known about MADV. In Brazil, the most recent isolations of MADV occurred in 2009 in the States of Paraíba and Ceará, northeast Brazil. Because of that, health authorities have recommended vaccination of animals in these regions. However, in 2019 an equine encephalitis outbreak was reported in a municipality in Ceará. Here, we present the isolation of MADV from two horses that died in this outbreak. The full-length genome of these viruses was sequenced, and phylogenetic analyses performed. Pathological findings from postmortem examination are also discussed. We conclude that MADV is actively circulating in northeast Brazil despite vaccination programs, and call attention to this arbovirus that likely represents an emerging pathogen in Latin America.Madariaga virus (MADV) is a member of the eastern equine encephalitis virus (EEEV) complex that circulates in Central and South America. It is a zoonotic, mosquito-borne pathogen, belonging to the family  Alphavirus, family Togaviridae, and have a positive-sense, single-stranded, nonsegmented RNA genome of approximately 11.7 Kb [Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV), and Venezuelan equine encephalitis virus (VEEV) are neurotropic, mosquito-borne alphaviruses of zoonotic and public health importance circulating in the Americas . They be 11.7 Kb ,3. Initi 11.7 Kb . Further 11.7 Kb .Culiseta melanura mosquitoes [The primary transmission cycle of EEEV leading to endemicity is enzootic; however, infections of animal species that are not the main reservoirs and of humans occur when factors disturbing the natural transmission cycles result in spillover events. Despite the important fact that EEEV infection can result in severe disease or death in equines and humans, these are considered dead-end hosts and, thus, do not contribute to EEEV transmission. In its natural cycle, NA EEEV is transmitted among passerine birds by squitoes ,7; howevOutbreaks of encephalitis and death in equines caused by SA EEEV strains have been reported in Central and South American countries in the past decades ,9,10,11 In Brazil, impactful equine epizootics caused by EEEV have been reported in all five Brazilian regions, with the first EEEV isolations having occurred in the 1940s, followed by several reports in the next decades ,21,22,23Circulation of SA EEEV in Brazil has also been evidenced by serosurveys, which have been mostly performed during equine epizootics or in areas where equine encephalitis cases had been previously reported. An early serosurvey in 1940–41 in the State of Minas Gerais (southeast (SE) region) performed during an epizootic with high case-fatality rates showed a seropositivity of 44.4% among alive equines . In a reVirus isolation and serosurveys in other animals have also been carried out in Brazil to infer their susceptibility to EEEV infection and/or potential role as reservoirs in the virus’s transmission cycle. EEEV has been isolated from avian species ,16, terrIn humans, SA EEEV was isolated once from a person in the 1950s in NE Brazil , and detHere, we present the isolation and full-length genome sequencing of SE EEEV (MADV) isolated in 2019 from two horses that died with encephalitis in the State of Ceará, NE Brazil. We then discuss their phylogeny, pathological findings, and the relevance of MADV active circulation in Brazil.Diffuse hyperemia was the only central nervous system (CNS) gross lesion observed in the two horses upon necropsy. One cerebrum section of animal 164 was examined by histopathology and no lesions were observed. Examination of two cerebrum sections of horse 165 revealed the following histopathologic findings: (1) moderate lymphoplasmacytic encephalitis was present mainly in the cerebral cortex (gray matter) and with a lesser extent in the white matter; (2) some vessels had swollen endothelium surrounded by one or three layers of lymphocytes and plasma cells; (3) nonsuppurative perivascular inflammation was seen in multifocal areas of the leptomeninges; (4) glial reaction was characterized by multiple areas with microglial proliferation in the neuroparenchyma. Microscopic changes as described above for horse 165 were indicative of viral infection.8 and 1 × 108 PFU/mL, respectively. Plaques were similar in both samples and measured approximately 2–3 mm in diameter (Titers of 164 and 165 P5 were 1.5 × 10diameter .Samples 164 and 165 were negative in the genus-specific flavivirus RT-PCR (no fragments were amplified). In the genus-specific alphavirus RT-PCR, a strong 434-bp fragment (expected band size) was observed in both samples. In the specific equine encephalitis virus PCRs, both samples tested negative for WEEV, positive for VEEV , and positive for EEEV . The controls worked properly in all PCRs. Amplified fragments in the equine encephalitis virus PCRs are shown in Except for the positive control (HSV-1), no sample yielded visible bands in the universal herpesvirus PCR.The first sequenced fragments (143–600 Kb) from 164 and 165 led to the identification of EEEV/MADV through BLASTn. The full-length genome was successfully sequenced through the Sanger method after several rounds of RT-PCRs. The genomes have 11,605 nucleotides and contain two ORFs. The first ORF has 7416 nucleotides and encodes the nonstructural proteins (nsP1–nsP4), while the second ORF is 3729 nucleotides long and encodes the structural polyprotein . A conserved “leaky” stop codon was identified at position 5614. There was 99.9% of nucleotide identity between the genomes of 164 and 165, with only 9 mismatches. The sequences of samples 164 and 165 were submitted to GenBank .The maximum likelihood phylogenetic tree  supportsThe equine encephalitis outbreak reported here leading to the isolation of MADV occurred in 2019 in the State of Ceará, where an outbreak had been reported in 2009 . In addiEndemicity of MADV in Brazil is truly relevant for animal and human health considering that infection with this pathogen can result in high morbidity and mortality in equines ,29 and hThe clinical signs observed in the two horses  that ultimately died, appear to be common in cases of eastern equine encephalitis ,29,38. OHere, lesions in horse 165 were more pronounced in the cerebral cortex; however, other regions of the CNS were not sampled for histopathology and more conclusions could not be reached. Importantly, the microscopic lesions in this animal, including the nonsuppurative encephalitis, were indicative of viral infection. Microglial proliferation and nonsuppurative inflammation, especially in the gray matter, can occur in a more advanced stage of this type of viral infection . NeutropAs the samples tested negative for rabies and because histology observations were indicative of viral infection, virus isolation and molecular diagnostic tests were carried out and led to the final diagnosis. We call attention to the fact that the viruses had to be amplified in tissue culture and the published flavivirus and alphavirus genus-specific RT-PCRs protocols had to be optimized to work properly (based on the positive controls). This is important as the low amount of virus in the original samples and the lack of optimizations in molecular assays can lead to false negative results. Issues concerning sample collection/processing and molecular assays must be considered as these are directly associated with the success of surveillance and control programs of zoonotic diseases.The phylogenetic analyses showed that MADV#164 MADV#165 clustered within lineage III. Although strains from lineages II and IV  have been detected in Brazil, the most recent studies have identified lineage III strains ,22,23, sAedes taeniorhynchus [Aedes spp. [Culex spp., especially from Cx. (Melanoconion) spp. indicate that species of this genus may be playing an important role in transmission. Interestingly, Cx. (Melanoconion) spp. from a region in Brazil where several MADV epizootics have occurred  exhibited the most eclectic feeding behavior compared to other mosquito species [According to the evolutionary profile of MADV and to comparisons with NA EEEV and the closely related VEEV, it has been hypothesized that although some bird species are susceptible to MADV infection, the main amplifying reservoirs may be ground-dwelling mammals. In Brazil, studies with SA EEEV/MADV in the N region have shown that several bird species are susceptible to infection, given that some species exhibited (low) seropositivity. However, in these studies the seropositivity rates and the number of avian species potentially susceptible to MADV were considerably lower compared to Saint-Louis encephalitis virus (SLEV) and WEEV, which are arboviruses that have avian hosts as the main amplifying reservoirs . In addirhynchus  and othedes spp.  in Brazi species  and some species —excellenMADV is a potential emerging pathogen in Latin America and the several knowledge gaps regarding its transmission cycle, pathogenicity in different hosts, and attack rates, among others, warrant further research at many levels. Surveillance programs should start considering MADV as an endemic zoonotic agent in Brazil and, according to reports on MADV detection in acute human febrile illnesses in Venezuela , Haiti , and BraOn 30 May 2019, cases of acute equine neurological disease were notified to the local health authorities in Irauçu, State of Ceará, NE Brazil. One out of twelve, and six out of twelve horses from two farms (farm 1 and farm 2) became sick and died within five days from the onset of clinical signs. One horse from each farm was necropsied for postmortem diagnosis. Both horses were 5-year-old crossbreeds of unknown gender that exhibited signs like ataxia, paresis, circling, and knuckling, that progressed to recumbency and death. Upon necropsy, CNS fragments were collected and sent to the designated laboratory. Horses were the only affected species on the farms. Previous vaccination history was unknown, except that the herd had not been vaccinated for rabies. Farm 1 is located at 03°46′8.7″ latitude and 39°47′51.1″ longitude, and farm 2 at 03°46′31″ latitude and 39°46′12″ longitude. The farms are 3.2 km apart from each other.Fresh CNS samples received by the Animal Health Laboratory at the Animal Defense Agency in the State of Minas Gerais  were processed and tested for rabies by the direct fluorescent antibody and mouse inoculation tests, for which they were negative. For histopathology analysis, one or two sections of the cerebral hemispheres were fixed in 10% formalin, processed by routine histologic methods, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin/eosin (HE), according to Luna .Samples were then sent to the Laboratory of Animal Virology Research at the Veterinary School  of Universidade Federal de Minas Gerais and the Virology Department at Instituto Aggeu Magalhães/Fundação Oswaldo Cruz for the experiments describe hereafter.g for 10 min at 4 °C. The supernatant was then collected and filtered through a 0.22 μm filter. Virus isolation was performed according to the protocol described by Pinheiro et al. [2 incubator. The inoculum was removed, and medium was added into the flasks, which were incubated for approximately 3–5 days until most cells exhibited a cytopathic effect. The flask content was then harvested, centrifuged at 1000× g for 5 min, and the supernatant stored at −80 °C. The negative control consisted of cells inoculated with PBS. Four more rounds of virus amplification were performed the same way but instead of using homogenized CNS tissue, an aliquot of the previous passage was used for cell inoculation.Frozen CNS fragments from the two horses  were homogenized using mortar and pestle and diluted 1:20 (w/v) in phosphate buffered saline (PBS) containing antibiotics and antifungal. The suspensions were vortexed for 15 sec, kept at 4 °C overnight, and centrifuged at 4000× o et al. , with mo−1 to 10−6 dilutions after medium removal). After a 1 h adsorption at 37 °C/5% CO2 the inoculum was removed, and semi-solid medium (1.5% carboxymethyl cellulose in 2× tissue culture medium) was added to the wells. Cells were incubated at 37 °C/5% CO2 for 6 days and then fixed in 4% formalin after medium removal. Plaques were stained with 1% crystal violet , washed and counted visually for titer calculation, which was expressed as plaque forming units per mL (PFU/mL).Plaque characterization and virus titer of 164 and 165 passage 5 (P5) were determined through plaque assays in Vero cells . Briefly2O. Samples were stored at −80 °C until further analysis. In addition, RNA of Yellow Fever virus (YFV) strain 17DD and Chikungunya virus (CHIKV) strain PB302 that had been cultivated in Vero cells was extracted using the same protocol.A 250 uL aliquot of 164 and 165 P5 frozen stocks was used for RNA extraction using TRIzol Reagent , according to the manufacturer’s instructions. After the 75% ethanol wash, the precipitate was air-dried and dissolved in 30 uL of Ultra-Pure H2O and CHIKV PB302 RNA.A genus-specific nested RT-PCR targeting the NS5 flavivirus gene was optimized based on the method described by Sanchez-Seco et al. . In the 2O and YFV 17DD RNA.A genus-specific alphavirus RT-PCR was optimized based on the strategy reported by Pfeffer et al. . In the 2O and CHIKV PB302; positive controls were not available.The product from the RT reaction with the genus-specific alphavirus reverse primer (described above) was used in a modified nested, primer-specific PCR for WEEV, VEEV, and EEEV, following a previously described strategy . For tha2O. Herpesvirus PCR was then performed according to VanDevanter [2O.A nested-PCR to detect herpesviruses was performed with samples 164 and 165. For that, DNA was extracted using 500 µL of 164 and 165 frozen stocks using the phenol:chloroform extraction protocol described by ThermoFisher  , except Devanter . In the http://blast.ncbi.nlm.nih.gov, accessed on 26 November 2020) showed high similarity of the amplified material with MADV sequences deposited in the database. Next, whole genome sequencing of both samples was achieved through Sanger sequencing. Initially, primers were designed based on a MADV sequence retrieved from GenBank (accession number KR132531.1) and then sequences obtained from the first-round sequencing were used for further primer design to be used in RT and PCR. RT reactions were first performed with the reverse primers as described above (except that 4 µL of RNA were used and RT reaction occurred for 1 h), and PCR was then performed as described above for the genus-specific alphavirus PCR, except that the annealing temperature varied from 52 to 65 °C and the extension time was adapted to the size of each fragment to be amplified (1 min/Kb). Primers were designed with the “A plasmid Editor-ApE” program. The sequences of the primers designed to obtain the full-length genome sequencing of the samples are shown in The amplicons of samples 164 and 165 from the genus-specific alphavirus PCR and the equine encephalitis virus PCRs were sequenced by the chain termination (Sanger) method with the same primers used in the respective PCRs. Nucleotide sequence similarity searching using the Basic Local Alignment Search Tool (BLASTn) (Sequences were aligned with reference sequences obtained from GenBank  using MA


Allium cepa L.) to be applied as packaging is attractive, due to its chemical properties and biodegradable characteristics. Thus, we tested the hypothesis that edible onion film can positively influence the sensory properties, quality and increasing shelf life of beef burgers patties. The experiment was designed in a 4 × 2 factorial scheme, with two treatments (beef burgers patties with or without edible onion film) at an interval of four storage times  at 4 °C. The uncoated burger patties (control) suffered the most intense color modifications during the storage (p < 0.05). The luminosity index was higher (p < 0.05) in the control at all storage times, except at day 6, and redness, yellowness and chrome were higher (p < 0.05) in the edible onion film patties at all storage times. The pH of the beef burger patties was lower (p < 0.05) at all storage times when the edible onion film was applied. For the texture profile, only the chewiness was affected, as the inclusion of the edible onion film improved the chewing of the beef burgers patties over the storage time (p < 0.05). Additionally, there was an inhibition of the microbial growth of mesophiles and psychrophiles with the application of the edible onion film in beef burgers patties. The use of edible onion film improved the perception of panelists for the variables texture, color, flavor, odor and overall appearance, and increased the preference of panelists. The edible onion film is recommended for preserving beef burgers patties, as it delays the proliferation of unwanted microorganisms, stabilizes and improves the color parameters and sensory attributes, and increases the overall acceptance of the consumer.The production of edible film from onion ( Beef is a food with high nutritional value and is one of the most consumed protein sources in the world and therefore deserves special attention in terms of its preservation and consumption . HoweverThus, beef becomes an excellent culture medium for microbial growth, whether due to favorable intrinsic factors, such as: chemical composition, high water activity, and pH decline rate; or by extrinsic factors, such as: humidity, temperature and chemical composition of the atmosphere . These fAnother important factor to be considered is the processing of meat into meat products, such as the beef burger patties, which does not significantly modify the original nutritional qualities, but attributes organoleptic characteristics such as color, flavor and aroma, characteristic of each process . In addiIn this aspect of preservation, this is one of the main functions of food packaging systems, as it separates food from the surrounding environment, reducing the interaction with spoilage factors  and preventing losses of desirable compounds , thus extending the shelf life of foods . In addiThe application of new technologies to improve quality foods and longer commercial time, or shelf life, has been increasing remarkably, in view of the high potential offered by alternative techniques, often combined with traditional methods in terms of preservation. The application of new technologies to prolong the shelf life and improve food sensory quality has been increasing remarkably. In the meat industry, alternative techniques often combined with traditional preservation methods are able to reduce undesirable biochemical effects, such as reducing oxidative processes and microorganism contamination .In the new global economy, petroleum-derived polymers used in packaging applications have become a central issue as their value chains currently involve substantial drawbacks. Although most of these materials have attractive characteristics , their continuous and extensive disposal has raised considerable concerns about their deleterious effects on the environment ,9,10. InEdible films can be classified as a primary packaging, which is in direct contact with the product, and it is also necessary to use a secondary, external packaging to protect the food from external contamination and, in some cases, from contact with . Since tAllium cepa L.) is a potential source of edible films for applications related to biodegradable packaging [Allium cepa L. has several phytonutrients such as: flavonoids, fructooligosaccharides (FOS) and thiosulfide and other sulfur compounds, recognized as important elements of high nutritional value [More than 35 plant species have been used as primary ingredients in the production of edible films ,11,12. Aackaging . Allium al value . Quercetal value , which hal value . Films oal value . Furtheral value . Thus, cp > 0.05) between storage time and application of the edible onion film for the variables of color and physicochemical composition of the beef burger patties and, therefore, the factors will be discussed separately as a function of each time.There was no interaction (p < 0.05) by onion film application at storage time (p < 0.05) in the control at all storage times, except at day 6, and redness, yellowness and chrome were higher (p < 0.05) in edible onion film at all storage times. The uncoated burger patties (control) suffered the most intense color modifications during the storage. However, all color parameters decreased slightly from 3rd day onwards (p < 0.05).The color parameters L*, a*, b* and saturation index or Chrome (C*) of the burger patties were affected (age time . Luminosp < 0.05) compared to the control treatment at days 3, 6 and 9 (p < 0.05) between day 3 and 6, whereas the control samples increased the pH values at days 3 and 6 and decreased at 9 days.It was observed that the application of edible onion film in beef burger patties promoted lower pH values ( 6 and 9 . Over thThe water holding capacity (WHC) of all beef burger patties decreased as the storage time increased. However, it was observed that the beef burger patties coated with the edible onion film were the ones with the lowest values of WHC and the highest rates of cooking loss (CL) at the end of 9 days compared to the control samples.p > 0.05) between treatments regarding the amount of iron as well as for the lipid oxidation values. However, the quantification of heme iron content present in the beef burger patties showed a gradual decrease in the amount of Fe over the days in both treatments, whereas, through the analysis of lipid oxidation. evaluated through the measurement of TBARS values. It showed an increase in malonoaldehyde values also occurred in both treatments over the storage period.There was no significant difference (p > 0.05) for the hardness, cohesiveness and springiness variables (p > 0.05) degree of springiness as the days of storage increased.Regarding the texture parameter indices there was no effect of the edible onion film in beef burger patties (ariables . Howeverp < 0.05) growth of mesophilic bacteria in the control treatments when compared to the beef burger patties preserved with the edible onion film from 3 days and persisted until the end of the shelf life (p < 0.05) than the treatment in which the beef burger patties were preserved with edible onion film from the 6th day of preservation (There was a greater (elf life a. Regardervation b.p < 0.05) for all analyzed attributes  compared to treatments without using the coating  was better evaluated by consumers and had the highest scores, with a significant difference ( coating .p < 0.05) by beef burger patties with edible onion film and salt, followed by the beef burger patties with edible onion film and without salt (32.5%). Then beef burger patties without edible onion film and with salt (16.25%) and the lowest acceptance for beef burger patties without edible onion film and without salt (5%).The preference ranking  demonstrColor indexes of an edible coating can change the overall appearance of food, since coating color may vary depending on the type of material used for their production. Moreover, in relation to meat, the typical form of myoglobin, the main protein responsible for meat color, associated with low oxygen concentration (deoxymyoglobin—with edible coating) or with oxygenation (oxymyoglobin—without edible coating) can influence meat purchasing decisions , so it wThe a*, or redness values, of burger patties presented better stabilization with the application of the edible onion film, possibly caused by changes in the meat structure related to the highly oxidizing conditions . The comThe b* color index (yellow) reflects the amount of fat present in the beef burger patties. Thus, the tendency to decrease these values in the control (uncoated) is related to the process of lipid oxidation present in beef that generates α- and β-aldehydes (secondary products of lipid oxidation) reducing the stability of myoglobin redox ,23,24. OThe edible onion film showed a higher chroma value than uncoated burger beef patties throughout storage, which may be appealing to consumers at purchase time. Fresh meat typically becomes less red and lighter after a few days. This may also be related to the fact that the coated burgers had less microorganism growth. The inhibition of microbial growth prevented excess oxidation thus, limited the reduction in a* . Thus, aThe pH value of beef burger patties without application of edible onion film increased with storage time, probably as a result of the activity of microbial-based enzymes, which can degrade meat proteins into nitrogenous compounds, such as ammonia and trimethylamine ,31 and tThe lower retention capacity and greater cooking loss at the end of storage at 9 days by the beef burger patties with edible onion film can be explained by the higher water content present in the treated samples due to the inclusion of the edible onion film itself, which has a hydrophilic nature, thus occurring a rapid moisture absorption, which diffuses through the material .The non-heme iron content has capacity to catalyze lipid oxidation ,35, thusEscherichia coli, Pseudomonas fluorescens, Bacillus cereus, and Aspergillus niger fungi, Trichoderma viride and Penicillium cyclopium [The application of the edible onion film in beef burger patties provided, throughout the storage period, less microbial growth and proliferation, demonstrated the antimicrobial potential of onion . The higyclopium . Behbahayclopium  observedyclopium .Allium may have been responsible for positively impacting the sensory characteristics of the beef burger patties. The improvement in color indexes, as well as in other physicochemical aspects, may be associated with a greater preference for beef burger patties preserved with edible onion coating [Processing storage time and cooking are the main factors that determine the sensory proprieties of foods treated with coating from natural sources . In the  coating ,45, as c coating ,47,48.This experiment was conducted at the Federal University of Campina Grande (UFCG), Patos city, Brazil. All protocols were approved by the Research Ethics Committee on of the UFCG, with protocol n° CAAE: 45259321.0.0000.5182. The beef burger parties were randomly distributed in a completely randomized design distributed in a factorial design 2 × 4 with two treatments (use or no use of edible onion coatings) and four beef burger patties storage times  and three and ten replicates  according to the realized analyses.Allium cepa L.) bulbs were obtained at markets in the city of Araraquara, SP, Brazil  [Allim cepa L.) yellow-type and medium size (average moisture content of about 89%) were selected and separated for the production of edible films. Onion bulbs were previously washed with water to remove soil impurities during harvest and after transport. After that, the outer layers that were dry or deteriorated were removed before the bulbs were cut lengthwise into four pieces and washed again. The films were prepared by casting formulations comprising raw onion pulp and hydrothermally treated (1% by weight solids) according to the methodology described by Dias et al. [The films were produced and supplied by Dr. Diogenes dos’ Santos Dias from BioSmart Nanotechnology . The medium-sized onion (′ 36″ W) . Onion  was poured into 90 mm diameter Petri dishes  and dried in an exhaust at room temperature with circulating air for 6 h or until detachment of the films.A total of 100 g raw pulp washed from the onion bulbs were placed in an industrial blender , filtered through a qualitative filter paper , washed with distilled water ten times to eliminate the characteristic odor. Then, a 0.5% aqueous suspension  using 8 mm discs and placed in plastic trays. To each homogeneous mass was added 30 g of salt. The mixing was carried out manually for 20 min and subsequently conditioned to a refrigerator for 12 h at 4 °C.The beef burger patties were prepared using the quadriceps femoris muscle following the method used by Ramos et al. . In the After the resting period, 100 g of the beef burger patties were molded in a manual press with a diameter of 9.5 cm (100 g). And then, the beef burger patties were individually packaged with edible onion films, labelled and cooled at 4 °C , for fur®®, São Paulo, Brazil), calibrated with a buffer solution with between pH 4.0 and pH 7.0 at temperature between 6–7 °C for 40 min.The pH of beef burger patties was obtained immediately after the preparation, with a digital skewer type probe  was determined as weight loss. It was used a 2.0 g beef burger sample, that was placed on a circular filter paper, between two acrylic plates, then a 3.4 kg equivalent force was put on the top of the paper for approximately 5 min. For each sample, results were expressed as a percentage of weight loss relative to its initial weight.The analysis of cooking weight loss (CWL) followed American Meat Science Association  recommenThe TPA was determined in the cooked beef burger patties in a TA-XT plus texture analyzer  and using its own Exponent Gram equipment, version v.51.1.0, based on the method described by Bourne . The parThe total heme iron content was determined as previously described in the study by , where 2g for 10 min. Then 4 mL of the supernatant was mixed with 4 mL of 0.02 M TBA and incubated in a water bath at 100 °C for 60 min. Absorbance was measured at 532 nm and the results expressed as µMoL of MDA/g of beef burger.The lipid oxidation indicator was performed from thiobarbituric acid reactive substances (TBARS), according to the methodology of Witte et al.  in whichw/v) of peptone in 0.9% NaCl solution (w/v)), and then homogenized for 5 min at each proposed shelf life. In a sterile environment, dilutions from 10−1 to 10−5 were made using the maximum recovery diluent. One milliliter of each dilution was added to sterile Petri dishes, then approximately 15 mL of Plate Count Agar culture medium at 40 °C was added. The plates were carefully shaken and incubated at 30 °C and 4 °C for 3 days and 7 days, respectively, for the counting of mesophilic and psychrophilic microorganisms [A total of 25 g of beef burger patties samples (in triplicate) with and without onion coating in all storage times were collected in sterilized bottles containing 225 mL of maximum recovery diluent  at 75 °C only that the temperature of the samples remains between 65 and 70 °C until distribution to the tasters [Then, consumer appeal was assessed using a panel made up of 80 untrained tasters, including 40 women and 40 men (aged between 18 and 54 years) . Two bee tasters .n = 8). The sessions consisted of eight tasting rounds of booths at pre-established times. Each taster received eight samples of beef burger (four treatments in duplicate), randomly distributed and coded with three numerical digits [Sensory evaluations were performed in 10 individual cabins on a single day between 09:00 and 11:00 a.m., with 10 participants per session  and the replicates according to analyses realized. The following statistical model was used:For sensory characteristics, the linear mixed model was used to analyze taste, odor, texture (tenderness), appearance and overall acceptance of each sample to identify the factors influencing the response. The fixed effects in the model included the use of edible onion film with or without salt as a condiment, and the random effects in the model included panelists and sessions.t test. To compare three or more groups, one-way analysis of variance (ANOVA) was used, followed by Tukey using the GraphPad Prisma Software. . Sensory perception was analyzed using Fisher’s LSD test and preference classification was expressed using the GraphPad Prisma software. Statistical data were considered significant when p < 0.05.Results were expressed as mean ± standard deviation. Data were statistically evaluated using the GraphPad Prisma Software. The means of two of the groups were compared using Student’s The edible onion film is recommended for preserving beef burgers patties, as it delays the proliferation of unwanted microorganisms, stabilizing and improving the color parameters and thus improving sensory attributes of beef burger patties and being more accepted by the consumer. This edible film may be used in the meat and meat product packaging industry, replacing conventional packaging, ensuring improved quality and increased shelf life of food.