Alcohol consumption and cigarette smoking are associated with an increased risk of breast cancer (BC), but it is unclear whether these associations vary by a woman’s familial BC risk.Using the Prospective Family Study Cohort, we evaluated associations between alcohol consumption, cigarette smoking, and BC risk. We used multivariable Cox proportional hazard models to estimate hazard ratios (HR) and 95% confidence intervals (CI). We examined whether associations were modified by familial risk profile (FRP), defined as the 1-year incidence of BC predicted by Breast Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA), a pedigree-based algorithm.pinteraction = 0.01) for smoking status in women who also consumed alcohol, but not in women who were non-regular drinkers.We observed 1009 incident BC cases in 17,435 women during a median follow-up of 10.4 years. We found no overall association of smoking or alcohol consumption with BC risk , but we did observe differences in associations based on FRP and by estrogen receptor (ER) status. Women with lower FRP had an increased risk of ER-positive BC associated with consuming ≥ 7 drinks/week (compared to non-regular drinkers), whereas there was no association for women with higher FRP. For example, women at the 10th percentile of FRP (5-year BOADICEA = 0.15%) had an estimated HR of 1.46 (95% CI 1.07–1.99), whereas there was no association for women at the 90th percentile (5-year BOADICEA = 4.2%) . While the associations with smoking were not modified by FRP, we observed a positive multiplicative interaction by FRP . For women with a high FRP (5-year BOADICEA ≥ 6.5%) who also consumed alcohol, being a current smoker was associated with increased BC risk. Alcohol consumption is an established breast cancer (BC) risk factor, with a 7–10% increase in BC risk for each standard alcoholic drink consumed daily  . The assBRCA1 or BRCA2 mutation carriers . A receview see .Estimates of relative risks from cohorts with large variation in absolute BC risk are important to understand associations with modifiable factors on an absolute risk scale . For exaBRCA1 and BRCA2 mutations was conducted, as previously described [The Prospective Family Study Cohort (ProF-SC)  includesescribed , 26, 27.N = 17,780). We excluded 191 women without sufficient pedigree data to allow calculation of a lifetime BC risk score using the Breast Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA), and 236 women for whom we did not have complete data on alcohol consumption  and cigarette smoking . From the 17,780 unaffected women in the original cohort, this left 17,435 (98.1%) available for analysis.In the current analysis, we studied women unaffected with breast cancer, aged 18 to 79 years at recruitment (baseline), who had at least 2 months of follow-up, and did not to have a bilateral risk-reducing mastectomy at baseline  consumed per week.We classified women as ever smokers if they answered “yes” and as never smokers if they answered “no” to the following question: “Have you smoked at least one cigarette per day for 3 months or longer?”. For ever smokers, we defined additional exposure variables, including smoking status (former or current), age at smoking initiation , smoking duration , and smoking intensity . We classified women as regular drinkers if they answered “yes” and as non-regular drinkers if they answered “no” to the following question: “Have you ever consumed any alcoholic beverages, such as beer, wine, or liquor, at least once a week for 6 months or longer?”. For regular drinkers, we defined additional exposure variables, including age at drinking initiation  and number of alcoholic drinks consumed per week (< 7 or ≥ 7), a common cut point in the literature. We defined one drink as a 12 oz. serving of beer, one medium glass of wine or wine cooler, or one shot of liquor, and calculated alcoholic drinks per week as the sum of the intake of each of the three different types of alcoholic beverages consumed.BRCA1 and BRCA2 mutation testing was also used to calculate risk. We hereafter refer to this continuous risk score as the familial risk profile (FRP). A previous validation study of family cancer history information communicated within families in the BCFR found high sensitivity and specificity for family history of breast cancer [For each participant, we calculated a 1-year, 10-year, and lifetime (from birth to age 80 years) risk of invasive BC from multigenerational pedigree data on breast and ovarian cancer in relatives using the BOADICEA version 3 , 29. Thit cancer . Additiot cancer .BRCA1 and BRCA2 mutation carriers. We assessed the proportional hazards assumption by evaluating the Schoenfeld residuals. All statistical tests were two sided, and p values < 0.05 were considered statistically significant. All statistical analyses were performed using SAS software version 9.4 .We used Cox proportional hazard regression models with age as the time scale to estimate hazard ratios (HR) and their 95% confidence intervals (CI) for BC associated with FRP and each smoking and alcohol variable. We calculated person-years from 2 months after the age at completion of the baseline questionnaire to the age at diagnosis of BC or the earliest of the following events: age at risk-reducing mastectomy, age at death, age 80 years, or age last known to be alive. We used a robust variance estimator to account for the family structure of the cohort. We incorporated left-truncation in all models to avoid potential survivor bias. All models were stratified by birth cohort  and adjusted for race/ethnicity  and study center. We considered the following variables measured at baseline as potential confounders: age at baseline (continuous), body mass index , education , age at menarche (continuous), parity and breastfeeding , age at first birth (continuous and centered at mean), oral contraceptive use , menopausal hormone use , and menopausal status . We also assessed for confounding by alcohol consumption (regular or non-regular drinker) in the smoking models and confounding by cigarette smoking  in the alcohol models. We included as confounding variables in the final models any variable that changed the smoking or drinking parameter estimate of interest by more than 10%. We assessed multiplicative interaction with FRP using the 1-year BOADICEA risk score and each smoking and alcohol variable of interest by including a cross-product term in the model and assessing the corresponding beta coefficient using the Wald test. We also estimated associations by estrogen receptor (ER) status (positive or negative), where the alternative ER subtype was censored at diagnosis. For example, ER-positive cases were censored at diagnosis in models examining ER-negative breast cancer. For ease of interpretation, we also present HRs by high and low FRP, using 0.34% as a categorical cutoff for absolute 1-year risk because it is the 1-year equivalent to the 5-year risk cutoff of 1.67%. We performed the following sensitivity analyses: including only those with confirmed invasive BC based on pathology reports or cancer registry linkages , where unconfirmed cancers were censored at diagnosis, excluding those with a prior diagnosis of any cancer (except non-melanoma skin cancer) at baseline, and excluding n = 2602) reported currently smoking at baseline, 27% (n = 4675) reported formerly smoking, and 58%  reported being never smokers. Current smokers smoked more intensely (mean cigarettes/day 15.1 vs 13.1) and for a longer period (mean duration 23.9 vs 14.5 years) than former smokers (Table n = 8618) of women reported being regular drinkers of alcoholic beverages at baseline and reported consuming an average of 7.7 total alcoholic drinks/week. Compared with never smokers, current and former smokers were more likely to be regular drinkers  and consumed more alcoholic drinks per week .We followed 17,435 women from 6948 families in the BCFR and kConFab with an average age at enrollment into the cohorts of 46.7 years. During the 181,062 person-years of follow-up , there were 1009 incident cases of BC with an average age at diagnosis of 56.2 years. Of the 17,435 women, 15% , the individual HRs at different percentiles of FRP were not, with HRs of 1.04 (95% CI 0.86–1.25) and 0.92 (95% CI 0.74–1.15) for women in the 90th and 10th percentile of 1-year BOADICEA risk score, respectively . We also observed no statistically significant associations between smoking status and risk of ER-positive BC  or risk of ER-negative BC  , we did find differences by ER subtype. We observed a negative multiplicative interaction by FRP for higher alcohol intake (≥ 7 drinks/week) compared with non-regular drinkers for ER-positive BC. As illustrated in Fig. Overall, there was no statistically significant association between alcohol consumption and overall BC  . When we stratified models examining smoking and BC risk by alcohol consumption, we found no overall significant association for current or former smoking by regular and non-regular alcohol drinking  for smoking status in women who also consumed alcohol, this was primarily driven by women with very high FRP , and cigarette smoking   cancer prior to baseline (results not shown). The results in Table We did not observe a statistically significant increase in BC risk associated with alcohol or tobacco consumption when considering the cohort as a whole, but we did observe some differences by FRP and ER status. Specifically, alcohol intake was associated with increased risk of ER-positive BC for women at lower predicted absolute risk. For women with a high FRP (above 95th percentile or a 5-year BOADICEA of 6.55%) who also consumed alcohol, smoking was associated with increased overall BC risk.BRCA1 and BRCA2 mutation carriers [BRCA1 and BRCA2 mutation carriers aged less than 50 years [Five previous studies have reported increased BC risk associated with smoking only for women with a family history of BC or in carriers –36. Thiscarriers . Similarcarriers . A seconcarriers . Additio50 years , 19, 32.We observed evidence for a negative interaction between FRP and alcohol intake in association with ER-positive disease. As previously reported, our family-based cohort is comprised of women across a large range of familial risk . In addiWe were limited to active smoking exposure and did not have information on exposure to environmental tobacco smoke (ETS). Some studies have reported an association with ETS and elevated BC risk, as reviewed elsewhere . AnotherOur study is strengthened by the comprehensive definition of family history that incorporates pedigree information and age at diagnosis of the relatives, extending beyond the conventional binary variable to cover the entire familial risk profile. The heterogeneity of the cohort with respect to family history allowed us to evaluate women across the full spectrum of risk and, in particular, women at high familial risk.Findings from this large prospective cohort including high-risk women indicate that there is an elevated risk of ER-positive BC associated with alcohol consumption for women at average population risk (5-year BOADICEA < 1.25%). Additionally, while smoking is not strongly associated with BC, and this association is not modified by underlying FRP, there is an increased overall BC risk for women at very high familial risk (BOADICEA > 6.55%) who also regularly consumed alcohol. These findings can have implications in terms of absolute risk reduction, as alcohol and smoking are modifiable risk factors and present risk reduction opportunities for women across the spectrum of familial risk.Additional file 1: Table S1. Adjusted hazard ratios and 95% confidence intervals from Cox proportional hazards modeling of smoking and alcohol variables. Table S2. Adjusted hazard ratios and 95% confidence intervals from Cox proportional hazards modeling of smoking and alcohol variables, excluding BRCA1 and BRCA2 mutation carriers.


Cenangium-dieback” has been frequent and devastating. Cenangium-dieback is caused by an endophytic fungus Cenangium ferruginosum in stressed pine trees. Progression of the disease in terms of molecular interaction between host and pathogen is not well studied and there is a need to develop preventive strategies. Thus, we simulated disease conditions and studied the associated transcriptomics, metabolomics, and hormonal changes. Pinus koraiensis seedlings inoculated with C. ferruginosum were analyzed both under drought and well-watered conditions. Transcriptomic analysis suggested decreased expression of defense-related genes in C. ferruginosum-infected seedlings experiencing water-deficit. Further, metabolomic analysis indicated a decrease in the key antimicrobial terpenoids, flavonoids, and phenolic acids. Hormonal analysis revealed a drought-induced accumulation of abscisic acid and a corresponding decline in the defense-associated jasmonic acid levels. Pathogen-associated changes were also studied by treating C. ferruginosum with metabolic extracts from pine seedlings (with and without drought) and polyethylene glycol to simulate the effects of direct drought. From RNA sequencing and metabolomic analysis it was determined that drought did not directly induce pathogenicity of C. ferruginosum. Collectively, we propose that drought weakens pine immunity, which facilitates increased C. ferruginosum growth and results in conversion of the endophyte into the phytopathogen causing dieback.Recently, the occurrence of “ Cenangium ferruginosum, Mycosphaerella pini, Gremmeniella abietina, and Shaeropsis sapinea, among others1. Recently, the outbreak of Cenangium-dieback (caused by C. ferruginosum) has been frequent and devastating2. C. ferruginosum is reportedly an endophytic fungus that lives as a biotroph in healthy pine trees and is considered a primary decomposer of weakened and dying trees2. However, under stressful conditions such as drought, extreme cold, unusual warm winters, and wounding, the endophytic fungus turns pathogenic, leading to detrimental consequences in pine trees3. The disease is first marked by the appearance of needle necrosis. Subsequently, it spreads causing damage to twigs, branches, and eventually the whole tree (hence the name “dieback”)3. Currently, Cenangium-dieback cases are not frequently reported worldwide; however, a number of pine species were seriously affected by this disease in different regions of Korea in the spring of 2007 and 20093. C. ferruginosum infects and damages different pine species in Korea, namely Pinus densiflora, P. thunbergii, and P. koraiensis2, but strong pathogenicity of the fungus was observed on P. koraiensis. Importantly, a recent outbreak of Cenangium-dieback was reported in early spring 2012 from several regions of Slovakia1, suggesting a wide geographical spread in the occurrence of the disease, which is alarming.Pine species are distributed in most regions of the northern hemisphere. Many species have been introduced to temperate and subtropical regions of the southern hemisphere as well. Pines have high economical value and are grown as timber. As pines are evergreen, they are also commonly cultivated as ornamental plants in parks and gardens. There are a number of pine diseases that affect the growth and survival of different pine species. One of the deadliest diseases is pine-dieback, which is caused by a number of fungal pathogens, e.g., 4. Once activated, these pathways lead to the production of pathogenesis-related (PR) proteins and induction of flavonoid biosynthetic pathways, thereby inducing the synthesis of secondary metabolites that act as antimicrobial agents in plant-microbe interactions5. Needles of pine trees are well known to have antimicrobial effects by virtue of different terpenoids, flavonoids, and phenolic acids that kill microbial cells. The major pine defenses against fungi are regulated by specialized chemicals namely terpenoids of oleoresin and phenolics7. The role of terpenoids in tree-fungus interactions and associated biochemical pathways are characterized in conifer species10. Diverse and variable terpenoids of conifers exist as monoterpenes, sesquiterpenes, and diterpenes, including the diterpene resin acids. Oleoresins, composed mostly of monoterpenes and diterpene resin acids, provide both physical as well as chemical defenses against microbes11. In general, pines produce and store large amount of terpenoids. Over 40,000 different terpenoids have been characterized so far in plants12. Among them, several compounds are produced by primary metabolism, including some phytohormones, such as abscisic acid (ABA). However, the majority of them are secondary metabolites, which are known to have defensive roles and are species specific12. Members of the Pinus genus are known to accumulate high levels of terpenoids, especially essential oils and resins, as defense compounds13. Terpenoids quantities and composition in the oleoresin and volatile emissions can vary dramatically according to the plant defensiveness, environmental conditions, and fungal attacks14. Besides terpenoids, the other important groups of bioactive compounds are flavonoids and phenolic acids. Flavonoids show diverse structure and a broad range of biological activities, with free aglycones and glycoside forms15. Phenolic acids, phenylpropanoids with an attached three carbon side chain and aromatic ring, exist in nature and have free and bound forms as esters and glycosides. The flavonoids and phenolic acids have protective roles against both microbial attack and environmental stresses16.The defense system in pine species is well equipped with various molecular arsenals to combat pathogen attack. Most of them are considered to be mediated by phytohormone-dependent pathways. Necrotrophic fungi have been reported to largely activate the jasmonic acid (JA)-dependent defenses in plantsC. ferruginosum are considered to be complex and differ from those that are effective against other endophytic biotrophs. Studies addressing the conversion mechanism of an endophyte into a phytopathogen are largely lacking and thus the scientific knowledge in this area is still in its infancy. In the wake of the severity and continuing trend of the occurrence of Cenangium-dieback, which has been frequent and covered a large geographical area, the development of preventive strategies is urgently required. To do so, however, it is important to first understand the molecular interaction between the host and the pathogen to shed light on the mechanism of disease progression. Necessitated by this, we studied the progression of Cenangium-dieback at the molecular level from the perspective of both the host and the pathogen. Because C. ferruginosum exhibits high pathogenicity in P. koraiensis, making it an important case to study, we chose this pine species as a model in our work. In our study, we investigated the pathogenicity of C. ferruginosum in P. koraiensis with and without drought stress treatment to elucidate the mechanisms of dieback progression. The interaction between the P. koraiensis and C. ferruginosum was studied at the transcriptome level, using an RNA sequencing (RNA-seq) approach. Additionally, the alteration in compounds  related to immunity in P. koraiensis induced by C. ferruginosum and subsequent drought was also examined. The effect of drought on the pathogenicity of the fungus was also taken into consideration. Our results suggested that drought weakens P. koraiensis immunity against C. ferruginosum, by virtue of reduced expression of the pine’s defense genes and secondary metabolites. This facilitates increased fungal growth leading to the development of Cenangium-dieback disease. We believe that our study is a major advance in this field, which will accelerate/facilitate further studies. This will eventually lead to the design of fruitful combative strategies.Defense mechanisms of pines against C. ferruginosum and grown under normal water supply for 42 d visibly appeared to be healthy  treatment. Fungal response to treatments with metabolic extracts of healthy and drought-stressed pine seedlings was also screened. The goal was to develop a de novo RNA-seq assembly and reveal if drought has any effect on the growth and pathogenicity of the fungus. The growth of the fungus was unaffected after treatment with different concentrations of PEG , microfibrillar associated protein 1 (MFAP1), DNA-binding response regulator PrrA (REGA), and ribonucleotide reductase catalytic subunit M1 (RRM1), in fungal samples treated with pine metabolites from healthily growing and drought stressed pine seedlings  growing within the pine seedlings. To generate the RNA-seq de novo assembly of the P. koraiensis, the RNA-seq data underwent two filtering steps  in the drought-exposed infected pine seedlings.Expectedly, the RNA-seq data from infected seedlings contained reads, besides those from the pine trees, including reads from eps Fig. . First, ce Table . The filC. ferruginosum reference assembly , in drought experiencing seedlings  were compared with treated seedlings (infected seedlings experiencing drought)  that mapped to de novo assembly of C. ferruginosum , were identified. Most of these 7,278 unigenes encode secretory proteins that lack clearly characterized homologs in other sequenced organisms. These unigenes showed a distinct expression pattern under drought stress and thus support a major role for the encoded proteins during the interaction with the pine tree. Additionally, a total of 99 differentially regulated C. ferruginosum unigenes overlapped with the PR genes available in the Pathogen-Host Interaction (PHI) database (www.phi-base.org). Among these 99 unigenes, 10 of unknown function were noted to be up-regulated.As previously mentioned, the fungal transcripts expressed in infected seedlings under drought stress, which represented the dieback progression, were filtered out from the infected seedling RNA-seq data utilizing the RNA-seq sum Fig. . The resC. ferruginosum infection. The biological processes broadly affected included stress responses and defense responses. The biosynthetic genes, for example, transaldolase (TALDO2), glucose-1-phosphatase (G-1-P), chalcone synthase (CHS), MYB5, and MYBPA1, were induced by drought stress in infected seedlings , which are among other chemical players in plant defense against pathogens and predators18.Gene ontology (GO) analysis of the differentially expressed genes was performed to have an idea of the biological processes, molecular functions, and cellular components that were affected under the water deficit condition post CA), asparagine synthetase (ASNS), and R (resistance) gene homologue (KR3), were repressed by drought stress. Furthermore, gene involved in biosynthesis of secondary metabolites via the flavonoid pathway  was enhanced. Along with F3′, 5′H, another flavonoid biosynthetic genes CHS was remarkably up-regulated in drought stressed seedling post C. ferruginosum infection. Several genes associated with diverse cellular components, such as the cytoplasm, endomembrane, proteasome complex, and mitochondrion, were significantly induced. In addition, two unknown genes were highly up-regulated by drought stress. Noticeably, a pathogenesis-related marker gene, PR1, also exhibited considerable induced expression. Contrastingly, the expression of the defense-related genes CA, ASNS, and KR3 encoding a TIR-NBS-LRR protein, was down-regulated. CA and KR3 were significantly down-regulated in the drought-exposed infected seedlings as compared to the control.Contrastingly, however, the defense-related genes, for example, carbonic anhydrase  and β-caryophyllene terpenoid, where former was below detection in the infected seedlings experiencing drought  on the fungus, we examined the fatty acid content extracted from C. ferruginosum through GC-MS analysis. The relative percentages of fatty acid esters are presented in Table C. ferruginosum exposed to PEG-mediated drought and distilled water (used as a control). However, C. ferruginosum treated with the metabolic extracts of healthy and drought-stressed pine seedlings showed a noticeable decline in the levels of ethyl palmitate, ethyl heptadecanoate, ethyl linoleate, and ethyl oleate fatty acids as compared to the control. The compound 1,5-diethoxyanthraquinone was only detected in the samples where C. ferruginosum was treated with the metabolic extracts of drought-stressed pine seedlings  in pine seedlings infected with C. ferruginosum and simultaneously experiencing drought. The levels of ABA increased with progressing drought in infected pine seedlings, with highest levels in 56 d drought samples , UV, and MS data  was selected for MS analysis. Extensive structure information about most phenolic acids and flavonoids present in the samples were obtained by the negative and positive ion modes, respectively. The negative ion chromatograms for phenolics present in the extracts of pine trees are shown in Fig. ata Fig. . A clearCenangium-dieback makes understanding the biology of this disease important in the development of preventive strategies. Recently, the high-throughput omics data attributing the plants’ interaction with Botrytis cinerea  has been suggested to be amply useful for genetic improvement of plants against gray mold disease20. This further highlights the usefulness and pressing need of a detailed study revealing the molecular complexities behind the progression of die-back disease in pine. Necessiated by the above arguments, our study focused on developing a profound knowledge of the molecular interaction between C. ferruginosum and P. koraiensis, with an aim to reveal the progression of Cenangium-dieback in plausible molecular terms.The detrimental consequences and increasing frequency of C. ferruginosum, which otherwise is a pine endophyte, turns pathogenic with the advent of stressful conditions, such as drought3. Under drought stress, the asymptomatic biotrophic fungus exhibits a peculiar hemibiotrophic life cycle, which is marked by dieback symptoms – destructive plant necrosis. Under conditions favorable for pine trees, the asymptomatic biotrophic phase of C. ferruginosum lasts for a long time without any dieback symptoms. We thus simulated the disease conditions by infecting healthily growing pine seedlings with C. ferruginosum and withholding water post infection to cause drought stress. Concurrence of pathogen infection and drought for 42 d or more showed necrotic dieback symptoms unlike the watered seedlings, suggesting the successful simulation of the disease progression. After establishing the disease symptoms, we next studied the molecular interaction between the host and pathogen in terms of transcriptomic, metabolomic, and hormonal alterations.21. Consequent to a complex interaction between host and pathogen, the gene expression changes in both organisms are highly regulated and dynamic21. To study such dynamic alterations in transcriptomes associated with both host and pathogen, the concept of ‘dual RNA-seq’ was introduced22 and has already been used comprehensively in several previous studies23. Using a similar approach in this study, we could identify transcripts that were significant during the biotrophic interaction between P. koraiensis and C. ferruginosum: 86 and 99 million reads mapped to plant and fungal gene models, respectively. From the gene expression results, it becomes obvious that under well-watered conditions the pine tree’s defense against C. ferruginosum remained activated, although insufficiently to suppress the growth of C. ferruginosum completely, and the defense-related pine genes CA, ASNS, and KR3, were highly expressed in the control . Similarly, with the advent of drought, where the unaffected fungal gene expression enables the fungus to overgrow, pine tree tries its best to counteract it. The overexpression of MYBPA1, MYB5, belonging to R2R3-MYB transcription factor family, member of which have already been shown to play important role in plant’s tolerance against the necrotrophic plant pathogen Botrytis cinerea25, is an indicative of the triggered defensive mode. However, plant still fails to mount an effective defense against the overgrowing pathogen as the other important defense-related pine genes, including CA, ASNS, and KR3, were down-regulated. CA plays a role in plant immunity through oxidative stress protection26. ASNS plays a key role in nitrogen remobilization, which is important and modulated in parallel to defense responses27. KR3 plays an important/primary role in detection of pathogens and elicitation of defense responses. This might have resulted in an oxidative burst and reduced elicitation of defense responses. Additionally, the various transmembrane and intracellular receptor-like kinases/proteins (putative immune receptors) that remained induced under optimal water supply declined remarkably under drought stress. Altogether, this possibly resulted in decreased immunity of the pine trees, which enabled the unaffected fungus to overgrow and cause dieback disease.The development of a disease largely depends on the molecular interplay between the pathogen and its host, which involves substantial transcriptional changes in both organismsC. ferruginosum in infected pine seedlings exposed to drought , which suggested that more C. ferruginosum genes were expressed when plants experienced drought post infection. Overall, it is amply clear that the interaction between C. ferruginosum biotrophic hyphae and pine tree cells continues for a long time under optimal conditions and the plant succeeds in keeping a check on the fungal growth. In other words, C. ferruginosum and pine trees establish a mutualistic association, which is also marked by the absence of the expression of the marker gene indicative of pathogen attack – the pine PR1 gene. Similar observations are reported previously in several biotrophic pathogens29. However, an episode of drought weakens plant immunity, thereby enabling the endophytic fungus to overgrow, become a pathogen, and cause dieback. This is also marked by the increased expression of the pine PR1 gene in infected seedlings experiencing drought. Finally, our first of its kind transcriptomic analysis thus presents an inventory of possible candidate genes in pine that can be targeted for genetic improvement of pines specifically against Cenangium-dieback disease. Certainly, it first requires extension of this pioneering work through more directed research in the future.Corroboratively, the RNA-seq data showed a clear increase in the percentage of reads mapping to C. ferruginosum and P. koraiensis limited the possibility of more detailed analyses/inferences from the transcriptomics results. We thus extended our study to also reveal related consequences that may account for the susceptibility of pine trees for Cenangium-dieback under drought conditions. Precisely, we chose to screen the alterations in the levels of terpenoids, flavonoids, and phenolics, which are well known antimicrobial compounds synthesized in pine species. Indeed, a significant difference in the terpenoid production/levels was noted in the pine seedlings growing under well-watered and drought conditions post C. ferruginosum infection. The concentrations of most of the terpenoids, such as 3-carene, β-caryophyllene, phytol, α-terpineol, isolongifolene, ethyl heptadecanoate, and ethyl linolenate, decreased significantly in the needle/stem tissues of C. ferruginosum-infected pine seedlings exposed to drought stress. In previous studies, drought stress was noted to increase the concentrations of terpenoids in wood and needles of conifers30. Interestingly, however, our study suggests that the combination of pathogen-infection and drought had a contrasting effect on the terpenoid profiles. We thus hypothesized that the lowered levels of terpenoids weakened plant defenses and made it more prone to disease. Along with terpenoids, the levels of flavonoids and phenolic acids, which constitute the major bioactive compounds in plants and are important in defense against pathogens16, were also significantly reduced in drought-exposed C. ferruginosum-infected seedlings. Overall, a significant decrease in the levels of secondary compounds in the constitutive resin was noted in drought stressed pine seedlings (both non-infected and infected). We thus believe that drought stress triggered a reduction of these compounds and decreased the immunity of pine trees against C. ferruginosum, which lead to the development of Cenangium-dieback in pine seedlings.The unavailability of complete genome sequences of both 31. These effects were attributed to the activation of the ABA pathway under drought stress33, which through antagonism suppresses SA-, JA-, and ethylene-mediated signaling pathways and thus the disease resistance in plants31. In agreement with these reports, we too noted an increment in ABA levels and a corresponding decline in JA levels with progressing drought. JA is believed to play a significant role in plant defenses against necrotrophic and biotrophic fungal pathogens20. We therefore propose that reduced levels of JA weakened the ability of pine seedlings to mount an effective defense against C. ferruginosum, which resulted in the development of Cenangium-dieback disease.Phytohormones play a critical role in plant development against many biotic and abiotic environmental factors. Different phytohormones and associated signaling may interact in a synergistic or an antagonistic manner, which results in a customized developmental response. Many previous studies have strikingly noted that disease resistance and drought tolerance are often antagonistic34. However, in case of Cenangium-dieback, it is important to note that the C. ferruginosum that colonizes the host plant is already present as an endophyte and causes disease only after a drought episode. We thus questioned whether drought stress that reduces the immunity of pine seedlings also affects the pathogenicity of C. ferruginosum. If yes, how does the pathogen develop the disease? Although the gene expression analysis suggested that there was no adverse effect of drought on C. ferruginosum growth and pathogenicity, we confirmed it at the level of fatty acid contents in the fungus. C. ferruginosum grown in metabolic extracts of well-watered, as well as drought-stressed pine seedlings, and PEG were studied to look for any change in fatty acids. Interestingly, no remarkable difference/effect on the contents of the studied fatty acids was noted. This suggested that environmental drought reduces only the pine’s immunity, and does not impose any indirect negative effect on the pathogenicity of the fungus. This unaffected virulent state of the pathogen combined with the drastically reduced ability of the plant to mount a defense facilitated the development of Cenangium-dieback in drought-stressed pine seedlings.Drought stress is well known to decrease the host resistance and/or increase nutritional value of the plants, which accelerates fresh pathogen infections and disease outbreaksP. koraiensis uses to control the development and virulence of C. ferruginosum under optimal conditions, and (2) lead to the development of disease under drought stress  and transcriptomic changes possibly responsible for conversion of an endophyte to a pathogen under drought stress. In so doing, we bring forth the molecular mechanisms that (1) ess Fig. . HithertC. ferruginosum was isolated from infected branches of P. koraiensis growing in wild2. The fungus was then cultured on MSA medium containing 3% malt extract, 0.3% soya peptone, and 1.5% agar  at 20 °C for 4 weeks in the dark before usage in this study.C. ferruginosum and analyzed under both well-watered and water deficit conditions. Twenty 5-year-old pine seedlings were grown in pots under greenhouse conditions. Each of the 20 healthy pine seedlings was spray-inoculated with 10 mL of a C. ferruginosum spore suspension  and then maintained at a humidity of ≥90% for 3 d to induce spore germination and infection. Fifteen seedlings were then subjected to drought stress for 42, 49, and 56 d allowing the use of 5 biological replicates for each condition. The remaining 5 seedlings were irrigated twice a week with normal water supply to serve as a control. A similar setup of 10 seedlings was maintained without inoculation of C. ferruginosum and grown with and without drought stress for 42 d. Twigs and needles were harvested from the second branch from the base of the tree crown and then immediately snap-frozen, ground in liquid nitrogen, and stored at −80 °C until further use.To study the consequences of pathogen infection on the host, pine seedlings were infected with C. ferruginosum was treated with pine extract isolated from drought-stressed, without C. ferruginosum inoculation samples. As a control, pine extract isolated from healthy (well-watered) seedlings without C. ferruginosum inoculation was used. C. ferruginosum was first grown in MSB  for 7 d at 23 °C and 150 rpm. Subsequently, 2.5 mL of MSB culture was replaced with 2.5 mL of 5,000 ppm pine extract. Similarly, to check the effects of drought stress, instead of pine extract, 2.5 mL of 5% PEG 6000  was used. Culture supplemented with 2.5 mL of H2O served as a control. After the aforementioned treatments, all the tubes were maintained at 23 °C and 150 rpm and the samples were harvested after 1 (for RNA-seq) and 6 h . Samples were then frozen in liquid nitrogen and stored at −80 °C until further analysis. For growth rate estimation, hyphae of C. ferruginosum were grown in MSA media supplemented with various concentrations of PEG .To investigate the effects of the metabolic extracts of pine seedlings on the pathogen, 35. Total RNA from mycelia of C. ferruginosum grown in potato dextrose broth (PDB) was extracted using the CTAB method36 and a Qiagen RNeasy Plant Mini Kit . The quality of RNA, which was determined by the RNA integrity number (RIN), was assessed by an Agilent 2100 Bioanalyzer using the RNA 6000 Nano Chip . The quantity of total RNA was determined by Infinite 200 PRO NanoQuant Spectrophotometer .Total RNA from infected pine needles was isolated using the CTAB method+ RNA was isolated using AMPure XP beads  and fragmented with the Ambion Fragmentation Reagents Kit . cDNA synthesis, end-repair, A-base addition, and ligation of the Illumina indexed adapters were performed according to Illumina’s protocol. Libraries were size-selected for 250–300 bp cDNA fragments on a 3% Nusieve 3:1 agarose gel , recovered using QIAEX II gel extraction reagents (Qiagen), and PCR-amplified using Phusion DNA polymerase  for 14 PCR cycles. The amplified libraries were purified using AMPure XP beads. Library quality was measured on an Agilent 2100 Bioanalyzer for product size and concentration. Paired-end libraries were sequenced with the Illumina HiSeq. 2000, (2 × 100 nucleotide read length). Reads that passed the chastity filter of Illumina Base Call software were used for subsequent analysis.Transcriptome libraries were prepared following Illumina’s TruSeq RNA protocol , using 1–2 μg of total RNA. Poly(A)Transcriptome analysis was performed using the Tuxedo protocol. Sequences were mapped against the Human reference genome (Ensembl release 69) using TopHat v2.0.9 with default options for paired-end sequences and transcript expression was estimated using the Cufflinks program v2.1.1 with genome annotation data (Ensembl release 69).2SO4 (v/v) in methanol and 32 µg of internal standard (heptadecanoic acid) were added. Samples were heated to 80 °C for 1 h using a water bath and then cooled down to room temperature. The mixture of 1.5 mL pentane and 4.5 mL 0.9% NaCl (w/v) was added to extract fatty acid methyl esters (FAME). Samples were then shaken vigorously and centrifuged at 14,000 rpm for 5 min at 4 °C to facilitate phase separation. The upper phase (pentane-containing FAME) was then transferred to an injection vial. Fatty acid analysis was performed by GC with a detector. The samples were analyzed using an Agilent 7890 A GC equipped with a Econo-CapTMECTM-Wax capillary column  and flame ionization detector. For GC-MS detection, an electron ionization system with ionization energy of 70 eV; ion source temperature of 250 °C was used. Helium was the carrier gas, at a flow rate of 1 mL min−1; 1:50 split ratio. Injector and detector MS transfer line temperature was programmed to 40 °C, held 2 min, raised to 250 °C at 10 °C min−1, and held for 10 min.Lyophilized needles and twigs (0.3 g) from each pine seedling were placed in a glass vial where 1 mL of 2.5% H37. Lyophilized samples (0.25 g) were extracted with 1.25 mL of extraction solution comprising methanol/water . Extracts were kept at −20 °C for 1 h, and then centrifuged at 20,000 rpm for 15 min at 4 °C. The pellets were re-extracted in an additional 1.25 mL of the same solution for 30 min. Collected supernatants were filtered through Sep-Pak C18 Plus  to remove interfering lipids and pigments, and evaporated at 40 °C using a speed vacuum. Residues were then dissolved in 1 mL of methanol/water  solution using an ultrasonic bath . Samples were filtered through 13 mm diameter nylon syringe filters (0.45 μm) (Sigma-Aldrich) and the volume was adjusted to 1.5 mL using the extraction solution. Calibration curves were constructed using different concentrations of internal standards (Sigma-Aldrich) – ABA  and JA  – and recoveries ranged over 99%. Four biological replicates for each condition/sample were quantified. LC-MS conditions followed for the analyses of phytohormones are described below.Two apical twigs per pine seedling were processed for the phytohormones analysis. Phytohormones were extracted and purified according to the protocol described previouslyet al.38. Four biological replicates were used for each condition/sample. Tissue samples (0.25 g) were extracted in 1.5 mL pure methanol . Subsequently, 1 mL of 0.1% tert-butylhydroquinone and 0.4 mL of 6 M HCl (in methanol solution) were added and the extracts were heated on a heat block at 90–95 °C for 1 h. Finally, after adding 2 mL of pure methanol, the samples were sonicated (50–60 Hz) for 15 min and then centrifuged at 13,000 rpm for 10 min at room temperature. Supernatants were collected and filtered through 0.45 μm inorganic membrane filter (Sigma-Aldrich), fitted into a disposable syringe, and transferred to a glass vial.Secondary metabolites were extracted from pine seedlings according to the protocol described by Moco, −1. The elution gradient consisted of 0.01–2 min, 0–40%; 2–5 min, 40–60%; 5–13 min, 60–100%; 13–15 min. Subsequently, the column was washed with 100% B for 15 min before the next injection. The injection volume was 5 µL. Ionization was performed using an electrospray ionization source, and masses were detected in both positive and negative mode.LC-MS-8040 tandem quadrupole mass spectrometer  equipped with an LC-30AD binary pump was used for the untargeted metabolic profiling. Composition of the mobile phase was degassed formic acid:water  and 100% acetonitrile (eluent “B”). The chromatographic separation was accomplished using an ACE UltraCore 2.5 SuperC18 column  at 40 °C and eluted at a flow rate of 0.5 mL minP = 0.05 using the statistical software SAS . Graphs were prepared using GraphPad Prism 6 .Values were represented as the mean of 3–6 biological replications. An analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) was conducted to determine the significant differences among means at a level of Supplementary Figure S1


The aberrant expression of N-glycolyl GM3 ganglioside (NeuGcGM3) in patients with sarcomas was reevaluated by assessing the relation of this molecule with some clinicopathological features and overall survival (OS) of patients.Fifty formalin-fixed and paraffin-embedded specimens from patients diagnosed with sarcomas were included. For the evaluation of NeuGcGM3, the 14F7 monoclonal antibody followed by a peroxidase avidin-biotin system was used. Clinicopathological features were obtained from patient records. Survival rates were estimated by the Kaplan-Meier method and compared with the log-rank test. For multivariate analyses, the Cox regression model was used to identify independent prognostic factors for OS.p = 0.014), TNM stage (p = 0.022), histological grade (p = 0.013) and proliferation rates (p = 0.012). In addition, a tendency for association with tumor depth (p = 0.070) was evidenced. In univariate survival analysis, TNM stage (p = 0.000), occurrence of metastasis (p = 0.000) and expression of NeuGcGM3 (p = 0.034) were significant prognostic factors for OS, while a tendency for association was evidenced for histological grade (p = 0.091). Among these variables, only the presence of metastasis (p = 0.001) was an independent prognostic factor on multivariate analysis.The majority of samples had high levels of NeuGcGM3 expression (66.0%) that showed statistical correlation with age (The present research suggests the evaluation of NeuGcGM3 expression as a complementary prognostic factor in sarcoma, although our results need to be validated in a larger series and prospective studies. Moreover, our results could support the use of this molecule as a target for immunotherapy. Soft Tissue Sarcomas (STS) are a heterogeneous group of tumors that originate from primitive mesenchymal tissue, which account for about 1% of all human malignancies. In spite of advanced gains with multimodality treatments, more than 40% of cases eventually experience tumor recurrence and metastatic spreading , which r. However, the expression of NeuGc forming part of gangliosides has been found in a variety of human malignancies [Gangliosides are sialic acid-containing glycosphingolipids localized in the plasmatic membrane of vertebrate’s cell. In normal human tissues, the N-acetylneuraminic acid (NeuAc) is the most common variant of sialic acid, while the presence of N-glycolylneuraminic acid (NeuGc) is limited due to an inactivating mutation in the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMP-NeuAc hydroxylase) gene . Howevergnancies –8, suggeIn particular, the aberrant expression of N-glycolyl GM3 ganglioside (NeuGcGM3) was previously reported by immunohistochemistry in a variety of malignant tumors, including pediatric  and adulIn the present study, it was evaluated for the first time, the association of NeuGcGM3 expression with some clinicopathological features of patients with STS. Moreover, the relation of this molecule with the overall survival of patients was assessed.A number of 50 formalin-fixed and paraffin-embedded specimens from patients diagnosed with sarcoma, who underwent tumor surgical resection at the “Hermanos Ameijeiras” General Hospital  between 2006 and 2013, were included. Samples from adult patients of any age, sex, race, histological subtype (except GIST) and any stage of disease were included in the study. Samples from patients who were treated or followed up in other medical institution were excluded.American Joint Committee on Cancer (AJCC) [All cases were staged according to the TNM classification established by the r (AJCC) . Performr (AJCC) . ClinicaThis research was conducted after receiving the approved consent by the institutional Ethical Committee.-1 antitrypsin (A1AT), smooth muscle actin (SMA), muscle specific actin (MSA), myoglobin, myogenin, and Ki-67 (MIB-1). For N-glycolyl GM3 ganglioside, the 14F7 Mab (a highly specific IgG1 against this molecule) produced at the Center of Molecular Immunology  was used [Sarcomas were immunohistochemically evaluated for a panel including but not limited to vimentin, desmin, pan-actin, S-100 protein, epithelial membrane antigen (EMA), cytokeratins, HNK-1 (CD57), protein gene product (PGP 9.5), CD99, CD34, c-kit, platelet-derived growth factor receptor (PDGFR), CD68 (MIC2), myogenic regulatory protein (MyoD1), h-caldesmon (HCD), alphawas used .The method previously described , was useImmunohistochemical results were analyzed for both proportion of stained cells and intensity of 14F7 Mab reactivity. The percentage of positive cells was graded on a scale of 0–3 . The intensity of reaction was graded on a scale of 0–3; 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. Afterward, an immunoreactive scoring (IRS) was obtained by multiplying the two previously mentioned parameters. Finally, the expression of NeuGcGM3 was divided into low level (IRS < 6) or high level (IRS ≥ 6). For Ki-67 antigen, five areas of greater intensity of staining were subjectively selected and positive and negative cells was counted. Subsequently, the percentage of positive cells  was determined and the measure was grouped as follows: low cell proliferation rate (≤ 10%), moderate cell proliferation rate (11–50%), and high cell proliferation rate (≥ 50%). All slides were assessed by two trained observers  who did not have knowledge of clinical characteristics or outcomes.p-value < 0.05 was considered statistically significant.The relation between NeuGcGM3 expression and clinicopathological variables were analyzed using the chi-square test. Survival rates were estimated by the Kaplan-Meier method and compared with the log-rank test. For multivariate analyses, the Cox regression model was used to identify independent prognostic factors for OS . All statistical analyses were performed with the SPSS program  and GraphPad Prism 6.0 . A Table Median follow-up for the entire cohort was 2.21 years (range 0.17 to 9.50). After 3-years of follow-up, 22/50 (44.0%) of patients displayed tumor recurrence and 21/50 (42.0%) died within 5 years after diagnosis. According to the first-line therapy used, 37 (74.0%), 9 (18.0%) and 4 (8.0%) of cases received surgery, chemotherapy and supportive care, respectively. The scheme of chemotherapy used as a neoadjuvant and adjuvant regimen were: Ifosfamide/Doxorubicin, Doxorubicin, Scheme P6, Trabectedin, Gemcitabine/Docetaxel and Ifosfamide/Paclitaxel. Adjuvant radiation therapy was delivered in 19 (38%) patients.Blanco et al.. The staining was detected on both the membrane and cytoplasm of tumor cells with a finely granular staining pattern. The majority of samples had strong intensity (54.0%) and more than 50% of positive cells (92.0%) as shown in Table p < 0.0001). According to the immunoreactive scoring (IRS) 33/50 (66.0%) of cases showed high levels of NeuGcGM3 expression.The expression of NeuGcGM3 was observed in all cases, although a variable intensity and percentage of positive cells was evidenced Fig. a and b. e cells 9.0% as shp = 0.016), while no significant relation was obtained with Disease-free survival (p = 0.346). In addition, univariate analysis showed that TNM stage (p = 0.000), occurrence of metastasis (p = 0.000), and expression of NeuGcGM3 (p = 0.034) . Among these variables, only the presence of metastasis  was an independent prognostic factor on multivariate analysis.The results of univariate and multivariate survival analysis are summarized in Table p = 0.014). Interestingly, the presence of this ganglioside was statistically significant increased in advanced clinical stages (p = 0.022), high histological grade tumors (p = 0.013) as well as in samples displaying an increased index of cell proliferation (p = 0.012) . No significant differences were observed with the rest of the clinicopathological parameters.The relation of NeuGcGM3 expression with clinicopathological characteristics of patients is shown in Table s p = 0.02, high hBousquet et al. confirmed the capacity of hypoxia to induce NeuGcGM3 in HeLa cells [Although the presence of NeuGcGM3 has been demonstrated in human tumors of epithelial, neuroectodermal, and mesodermal origins , the mecLa cells .Blanco et al. in these tumors [In the present study, an increased expression of NeuGcGM3 was detected in 66% of human sarcomas, regardless of the histological subtype. The presence of this ganglioside was mainly detected in the surface of malignant cells, although diffusion to cytoplasm was also evidenced, as reported by e tumors . In preve tumors . Moreovee tumors , suggestLahera et al. published the association of NeuGcGM3 with the reduced overall survival of patients with colon adenocarcinomas [Blanco et al. obtained similar results in NSCLC [Here, the increased expression of NeuGcGM3 ganglioside was significantly associated with an impaired 5-year overall survival of patients in the univariate analysis. To the best of our knowledge, this is the first report concerning the potential prognostic role of NeuGcGM3 in human sarcomas. However, in the multivariate analysis only the occurrence of metastasis was an independent factor in predicting clinical outcome. Previously, rcinomas , while Bin NSCLC . In thisKanduma et al. reported an increase in the amount of serum total sialic acid in older patients with sarcomas when compared with the rest of groups. Interestingly, these patients also displayed a reduction in the level of natural anti-NeuGc antibodies [Lahera et al. obtained a tendency for association between NeuGcGM3 expression and the age of colon adenocarcinoma patients [Rodríguez-Zhurbenko et al. reported a reduction in the production of anti-NeuGcGM3 in older NSCLC patients [The presence of NeuGcGM3 was also related with age of sarcoma patients. In previous research, patients . Furtherpatients , suggestBlanco et al. reported a relation of NeuGcGM3 expression with an increased histological grade of both malignant gliomas and transitional cell carcinoma of the urinary bladder [, occurrence of metastasis, and decreased survival rates [Blanco et al. in NSCLC using flow cytometry [In addition, the expression of NeuGcGM3 was significantly increased in high histological grade tumors. In previous studies,  bladder . It is r bladder , occurreal rates . Moreoveytometry  and coulInterestingly, the presence of NeuGcGM3 was increased in deep sarcomas when compared with superficial tumors, although only a tendency for statistical association was obtained. It is known that superficial sarcomas are located exclusively above the superficial investing muscular fascia, while deep tumors are defined as either deep or involving the superficial fascia . ConsequLahera et al. regarding colon adenocarcinomas [Finally, the presence of NeuGcGM3 was significantly associated with advanced TNM stage, a clinicopathological feature also related with an adverse clinical outcome of patients. Our data is consistent with a previous report of rcinomas . It is wrcinomas , pediatrrcinomas , and NSCrcinomas . AlthougIn summary, this study reports for the first time the association of NeuGcGM3 expression with the aggressiveness of human sarcomas. The present research suggests the evaluation of NeuGcGM3 expression as a complementary prognostic factor in sarcoma, although our results need to be validated in a larger series and prospective studies. Moreover, our data could support the use of this molecule as a target for immunotherapy. Interestingly, specific therapies against NeuGcGM3 alone or combined with an anti-EGFR strategy showed promising clinical benefits in sarcoma patients . Lastly,


Pteropus seychellensis comorensis and Chaerephon pusillus) tested positive; however, 20.1% of bats sampled in Mozambique shed AstVs at the time of sampling and significant interspecific variation in the proportion of positive bats was detected. Many AstVs sequences obtained from a given bat species clustered in different phylogenetic lineages, while others seem to reflect some level of host-virus association, but also with AstVs previously reported from Malagasy bats. Our findings support active circulation of a large diversity of AstVs in bats in the western Indian Ocean islands, including the southeastern African coast, and highlight the need for more detailed assessment of its risk of zoonotic transmission to human populations.Astroviruses (AstVs) are responsible for infection of a large diversity of mammalian and avian species, including bats, aquatic birds, livestock and humans. We investigated AstVs circulation in bats in Mozambique and Mayotte, a small island in the Comoros Archipelago located between east Africa and Madagascar. Biological material was collected from 338 bats and tested for the presence of the AstV RNA-dependent RNA-polymerase gene with a pan-AstV semi-nested polymerase chain reaction assay. None of the 79 samples obtained from Mayotte bats (The online version of this article (10.1186/s12985-018-1011-x) contains supplementary material, which is available to authorized users. Astroviruses (AstVs) are small non-enveloped RNA viruses, transmitted via the fecal-oral route. They have been detected from over 80 vertebrate host species , and repCurrent knowledge on the epidemiology of AstVs in African bats is limited , 9. In aPteropus seychellensis comorensis, and droppings were collected from 58 Chaerephon pusillus. Swabs and droppings were placed in 1.5 mL of Virus Transport Media , in November–December 2014, and in Mozambique (Inhassoro district) in February and May 2015. Bats were captured using mist nets and harp traps. On Mayotte, rectal swabs were obtained with sterile rayon-tipped applicators  from 21 ia , and weRNA extraction was performed with the QIAamp Viral RNA Mini Kit . Reverse transcription was performed on 10 μL of RNA using the ProtoScript II Reverse Transcriptase and Random Primer 6  using a previously published protocol . PCRs weI = 0.10, α = 0.71; [http://tree.bio.ed.ac.uk/software/figtree).PCR products of the expected size were submitted for direct Sanger sequencing . The 31 sequences obtained in this study were aligned with 112 reference AstV RdRp partial nucleotide sequences, with CLC Sequence Viewer version 7.7.1 . A maximum-likelihood analysis was performed Phylogenetic trees were constructed by maximum likelihood with the software PhyML 3.1 . The evo = 0.71; ), and no = 0.71; , and the = 0.71; . A stricNone of the 79 samples collected on Mayotte tested positive for the presence of AstV. Although this negative result may be affected by the relatively small sample size and differences in sampling protocols (swabs vs droppings), it may also suggest temporal variation in AstVs shedding and circulation in bat populations, as previously documented . Additioχ2 = 104, P < 0.001). A high detection rate was found in Triaenops afer (68.6% ± 12.7%), as compared to other species  with a higher detection rate in May (25.3% ± 6.5%) than in February (10.1% ± 6.3%), in particular for T. afer . This variation may be associated with factors related with bat population dynamics facilitating or limiting virus transmission .In Mozambique, 52 of the 259 bats tested positive for the presence of AstV RdRp . This detection rate was similar to other studies using the same PCR assay, including the one we reported on Malagasy bats . For thondition , 19). NoTriaenops afer and Hipposideros caffer, although statistical support was limited. Sequences of AstVs detected in Nycteris thebaica and Mops condylurus were mostly highly divergent and not included in larger genetic lineages comprising viruses of same bat family or the same geographic area , without strong support for host family or species restriction Additional file 2:List of the bat families and species included in the phylogenetic tree. (PDF 44 kb)


We investigated the prevalence of P/LP variants in the 24 ACMG SF v2.0 We used InterVar to classify variants and subsequently conducted a manual review to further examine variants of unknown significance (VUS).cancer phenotype, we observed 8 P/LP unique variants  in controls and 11 P/LP unique variants  in cases, a non-significant difference. We reviewed 115 VUS. The median estimated per-variant review time required was 30 min; the first variant within a gene took significantly (p = 0.0009) longer to review (median = 60 min) compared with subsequent variants (median = 30 min). The concordance rate was 83.3% for the variants examined by two reviewers.In the 24 genes on the ACMG SF v2.0 list associated with a cancer genes, our work establishes a benchmark P/LP variant prevalence rate in a familial cancer cohort and controls.The 115 VUS required database and literature review, a time- and labor-intensive process hampered by the difficulty in interpreting conflicting P/LP determinations. By rigorously investigating the 24 ACMG SF v2.0 The online version of this article (10.1186/s13073-018-0607-5) contains supplementary material, which is available to authorized users. In 2013, the American College of Medical Genetics and Genomics (ACMG) recommended that “laboratories performing clinical [exome or genome] sequencing seek and report mutations of the specified classes or types” in a set of 56 genes associated with a severe phenotype, and for which disease risk may be reduced or managed before symptoms arise [cancer cohorts remains largely uninvestigated, and to our knowledge, prevalence of P/LP variants has not been determined in a large cancer study with ethnicity-matched healthy controls.Although both ACMG SF policy statements used the older “known pathogenic” or “expected pathogenic” variant categorization terminology , a transThe current American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines use conservative methods to classify variants based on numerous criteria including clinical and family history, previous literature, and known population allele frequency . Currentcancer phenotype in a large, family-based heterogeneous cancer research cohort  and in ethnicity-matched controls (n = 982). (The remaining 35 non-cancer ACMG SF v2.0 genes were not fully investigated.) We used InterVar to classify variants, followed by a manual review to further examine variants of unknown significance (VUS). In addition, we estimated the time to resolve VUS and evaluated the concordance rate between reviewers for 30% of the reviewed variants.In this study, we used the most recent ACMG/AMP criteria and determined the prevalence of P/LP variation in the 24 ACMG SF v2.0 gene list associated with a . Families in which a causative gene was identified were not excluded. Data from 982 controls from 2 cohort studies, Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO [Cases were drawn from the NCI Division of Cancer Epidemiology and Genetics (DCEG) Familial Exome cohort, a large, long-term, longitudinal, heterogeneous group of family-based studies with a cancer phenotype and a Mendelian or near-Mendelian pattern of inheritance. The majority of the families lacked a known causative germline genetic variant; the cancer phenotype in the families may or may not overlap with the known cancer phenotype of the 24 ACMG v2.0 cancer genesal (PLCO ) and theal , were aExome sequencing was performed at the Cancer Genomics Research Laboratory, National Cancer Institute , as described , 17. Cascancer phenotype  was annotated using ANNOVAR [cancer gene variants listed in the Human Gene Mutation Database  as “disease mutation” (DM) underwent manual review, regardless of the InterVar assertion and without knowledge of case or control status. In addition, we used Google Scholar to search the published literature for information on variants designated VUS by InterVar which were not listed in HGMD. The primary literature was then reviewed by 17 reviewers . The reviewers were assigned specific gene(s) after variant review training and classified the variants according to the ACMG/AMP guidelines using a pre-populated Excel file that contained needed variant annotation information. Reviewers were also asked to provide comments for each score provided and to estimate the time needed to review each variant. We noted which variant within each gene was the first one evaluated by each reviewer. After initial review, variants were subject to a quality control (QC) process in which the criteria for scoring and reviewer comments were compared for agreement. As a second QC check, 31% (n = 36) of the 115 variants initially reviewed were re-evaluated by a second independent reviewer. If there was discordance between the primary and secondary reviewers on variant classification, discussion was initiated to reach consensus. The ACMG/AMP combining criteria were implemented using the Genetic Variant Interpretation Tool available online (http://www.medschool.umaryland.edu/Genetic_Variant_Interpretation_Tool1.html/) [p values (t test) were calculated using GraphPad Prism 7 , and 95% confidence intervals were calculated using STATA 14 .Variation in the 24 ACMG SF v2.0 genes primarily associated with a  ANNOVAR , which i ANNOVAR . To moreFor the entire DCEG Familial Exome cohort (plus controls), exome sequencing was performed such that 88% of coding sequence from the University of California Santa Cruz (UCSC) human genome (hg) 19 transcripts database had ≥ 15 reads with an average coverage of 61×. After the sample quality control, there were 982 control individuals from the PLCO, EAGLE, and CPSII cohorts and 1173 cases (738 families) from 15 cancer-based studies ; 2 were in MUTYH. MUTYH is the only ACMG SF v2.0 cancer gene in which the phenotype is associated with an autosomal recessive pattern of inheritance and is therefore reportable only for compound heterozygotes or homozygotes [MUTYH variant, we excluded this gene from our prevalence calculation.We used InterVar to classify all filtered variants into 6 categories (pathogenic (P), likely pathogenic (LP), variant of unknown significance (VUS), likely benign (LB), benign (B), and no classification) for cases and controls. Since our cohort includes family members, we performed 2 separate analyses: first, we used all cases, and second, we randomly selected 1 affected individual per family. Table ozygotes . Since an = 297) or “no classification” variants (n = 463) in cases and controls, 115 (15%) had been reported previously, as per queries of HGMD and Google Scholar (VUS variants only). A total of 77 variants were classified as “DM” in HGMD, and an additional 38 VUS variants were identified in the searchable published literature queried through Google Scholar. Of the remaining 645 variants, there was little or no additional published or online information available, and therefore, these variants were not further evaluated. After review by 1 cancer expert, 36 (31%) randomly selected variants underwent review by a second cancer expert. The concordance rate between the primary and secondary reviewers for the pathogenicity category of these 36 variants was 83.3%. Discussion between reviewers led to the resolution of the 6 discrepant variants from the 36 re-reviewed variants(16.7%) in this study. Among the 115 variants reviewed, 2 unique variants were promoted to P from VUS and 5 unique variants were promoted to P from “no classification.” Two unique variants were promoted to LP from VUS, and 1 unique variant was promoted to LP from “no classification” , MSH2 (one), PMS2 (one), and TP53 (one)  BRCA2 (one), PMS2 (one), and TP53 (eight unique)  to review  compared with subsequent variants in the same gene . However, these estimated times did not account for the time required to run InterVar, perform a QC check, conduct secondary reviewer validation, and resolve discordances. Incorporating these additional tasks into the review process would result in a much higher time requirement to classify variants.The allele and total counts of P/LP variants for the 24 ACMG SF v2.0 cancer genes was 1.2%, not significantly different from P/LP variant prevalence in 982 ethnicity-matched controls (0.8%). Our study is notable for the large cohort size, the use of variation-based ethnicity-matching of cases and controls, thorough expert-driven review of variants by ACMG/AMP criteria, and an exclusive focus on the 24 ACMG SF v2.0 cancer genes.In 1173 individuals from a heterogeneous, family-based study of inherited cancer predisposition, the prevalence of P/LP variants in the 24 ACMG SF v2.0 Direct comparison of our results with previous studies is challenging because of the differences in methodology and study populations. We acknowledge that our familial cancer cohort is heterogeneous since it is comprised of individuals drawn from a wide variety of familial tumor-predisposition studies, making comparison difficult. Analyses of the 1000 Genomes and the NHLBI GO Exome Sequencing Project cohorts for P/LP variants in a list of “medically actionable” genes (larger than the ACMG SF v.2.0 list) found a prevalence of 2.2–3.4% , 22. Thecancer genes largely underlie risk in common cancers  and well-known genetic disorders  and are not necessarily associated with the disorders constituting our study cohort  (Table The ethnicity-matched controls (PLCO/EAGLE/CPSII) were on average 70 years of age, healthy adults without a history of cancer (other than non-melanoma skin cancer) at the time of study enrollment and sample collection. Interestingly, 0.8% of this control sample harbored a P/LP variant in one of the 24 ACMG SF v2.0 cancer genes, our work establishes a clinically useful benchmark prevalence rate, especially in controls. Recent studies have shown that pathogenic variation in single genes like DICER1 [TP53 [DICER1 and TP53, the recognition that pathogenic variation in recognized cancer genes is more common than expected is an important, emerging, and unanticipated finding from population-based exome sequencing, one that has significant clinical implications. In this study, we observed P/LP variation in the 24 ACMG SF v2.0 cancer genes  in 0.8% of our 982 controls, who, by a mean age of ~ 70 years, had not developed any malignancy. Thus, in our controls, the prevalence of P/LP germline variation in BRCA2 was 0.5% . In Lynch syndrome genes, the prevalence was 0.4% , and for Li-Fraumeni syndrome, it was 0.1% . These frequencies are comparable to other published estimates  may be greater than for lesser-known (non-ACMG SF v2.0) genes. In addition, our study population was restricted to people of non-Finnish European ancestry. Published work has highlighted the additional challenges in interpreting genetic variation in non-European populations [Reviewers were required to track the estimated amount of time needed to classify variants. Our study is the first to distinguish between the amount of time to review first and subsequent variants within a gene. We found that the first variant took significantly longer to review when compared with subsequent variants, a reflection of the learning curve inherent in applying these new, complex classification algorithms. Although our team was composed of cancer experts, they were not necessarily experts on the specific genes they were reviewing. This could potentially have led to the additional time for familiarization with the gene(s) to be reviewed. Our overall finding that variant review was time-consuming is consistent with previous studies . Howeverulations . Thus, oOur experience with InterVar and the ACMG/AMP guidelines deserves a brief comment. We found that InterVar was a useful tool to start the initial variant classification using the ACMG/AMP guidelines. Despite the use of InterVar and manual review, most variants remain unresolved due to the lack of published literature and for our study and limited clinical information. Proper classification of variants, especially those used in clinical decision-making, is a time-consuming and laborious process that, for now, requires human expertise and judgment. Currently, this process is more subjective, and yields less reproducible results, than is optimal. In the future, this may be streamlined with more extensive, comprehensive electronic databases of definitively classified variants, more sophisticated software  , and artCDH1, colon cancer, PTEN, and TP53).Reviewers in this study frequently noted that gaining a working familiarity with the ACMG/AMP guidelines was demanding. As a quality control procedure, we compared criterion scores (0 or 1) with the respective comments provided by the reviewer; we observed confusion related to multiple criteria. Despite these challenges and after correction of inconsistently scored criteria (based on the comments provided), our secondary review and consensus process showed a concordance rate of 83.3%, which is at the upper limit of previously reported concordance rates (34–79%) , 37, 38.cis/trans status or to assess segregation of a variant with phenotype. Availability of these data may have increased the number of variants that were definitively classifiable, which would have reduced the number of VUS. In addition, we did not review all VUS variants called by InterVar; we only considered the 115 variants that were reported by HGMD as DM or for which sufficient information was found in Google Scholar. Furthermore, this study only examined the white population, potentially limiting the applicability of these findings on P/LP prevalence to other ethnic groups and variant review time. Lastly, although the cases were drawn from a heterogeneous, convenient cohort of families assembled over multiple decades and protocols, the generalizability of our results may be limited, given the broad spectrum of cancer diagnoses.We acknowledge the limitations of our study. Since cases and controls were anonymized, there were restrictions on the depth and detail of clinical information. Thus, we were not able to assess de novo or cancer genes in a cancer cohort (1.2%) and ethnically matched healthy controls (0.8%). Variant review, even with the help of sophisticated software tools, is time-consuming. Newer approaches, perhaps using artificial intelligence tools and neural networks, are needed to simplify and expedite this important task.We found a non-significant difference in the prevalence of P/LP variants from the ACMG SF v2.0 Additional file 1:Supplemental Methods. Detailed methods on DNA preparation, hybridization, exome sequencing, variant calling, use of ethnicity-informative variation, quality control, and capture region matching. (PDF 233 kb)Additional file 2:Figure S1. Population stratification of cancer cases and controls. Figure S2. Principal component analysis of cancer cases and controls. Table S1. Study names and predominant cancer types in DCEG Familial Exome cohort. (PDF 449 kb)Additional file 3:Table S2. InterVar classification of ACMG SF v2.0 cancer genes after expert review. (XLSX 30 kb)


Streptococcus gordonii, Fusobacterium nucleatum and Porphyromonas gingivalis was documented in vitro using growth models and scanning electron microscopy. Analysis of growth rates by species-specific 16s rRNA probes revealed distinct, early advantages to consortium growth for S. gordonii and F. nucleatum with P. gingivalis, while P. gingivalis upregulated its short mfa1 fimbriae, leading to increased invasion of DCs. F. nucleatum was only taken up by DCs when in consortium with P. gingivalis. Mature consortium regressed DC maturation upon uptake, as determined by flow cytometry. Analysis of dental plaques of PD and healthy subjects by 16s rRNA confirmed oral colonization with consortium members, but DC hematogenous spread was limited to P. gingivalis and F. nucleatum. Expression of P. gingivalis mfa1 fimbriae was increased in dental plaques and hematogenous DCs of PD patients. P. gingivalis in the consortium correlated with an adverse clinical response in the gingiva of PD subjects. In conclusion, we have identified polymicrobial synergy in a three-species oral consortium that may have negative consequences for the host, including microbial dissemination and adverse peripheral inflammatory responses.Years of human microbiome research have confirmed that microbes rarely live or function alone, favoring diverse communities. Yet most experimental host-pathogen studies employ single species models of infection. Here, the influence of three-species oral microbial consortium on growth, virulence, invasion and persistence in dendritic cells (DCs) was examined experimentally in human monocyte-derived dendritic cells (DCs) and in patients with periodontitis (PD). Cooperative biofilm formation by  Most notable are P. gingivalis, Treponema denticola and Tannerella forsythia, based on their virulence factors and strong association with diseased sites.14 The early interpretation of these findings was that the transition from health to destructive periodontitis is caused by emergence of specific pathogens within oral biofilm. More recent evidence implicates ecological disruption of the commensal oral biofilm in the pathological transition, i.e., dysbiosis.18 Although the evidence of microbiome impact on the human health and disease conditions is well established, presently unclear is how the oral microbiome influences local and systemic health. P. gingivalis has been termed a keystone pathogen due to its ability to orchestrate a dysbiosis,21 presumably through expression of specific virulence factors21 that modulate innate immunity.23 For example, in macrophages, P. gingivalis subverts nitric oxide synthase (iNOS)-dependent killing in vitro and in vivo by stimulation of cAMP production.24 In dendritic cells (DCs), P. gingivalis employs adhesive fimbriae to manipulate TLR activation, as well as activate cross talk of TLR2 with C-type lectin receptor DC-SIGN on.26 This enables P. gingivalis to evade innate immune recognition and sustain disease progression in a susceptible host. Indeed, P. gingivalis can initiate bone destruction upon inoculation into the oral cavity of animals such as mice and nonhuman primates;28 however, recent studies in a germ-free mouse suggest that the existing flora may be required for P. gingivalis pathogenesis.29 Consistent with this notion, very low inoculum (less than 0.01%) with P. gingivalis induces a dysbiosis and promotes alveolar bone loss in mice.29The human microbiome project has revealed the enormous diversity of the oral microbiome.P. gingivalis has been shown to invade myeloid DCs with its glycoprotein Mfa1 fimbriae, routing it into non-autophagosomal compartments.30 This routing enables P. gingivalis to evade the intracellular degradation by autophagy machinery and increase its survival within DCs.30 Due to early indications that P. gingivalis fimbrial expression is under environmental control,31 combined with the role of mfa1 fimbriae invasion of DCs in vivo45 and survival inside DCs in vitro,30 we hypothesized that polymicrobial conditions would influence the mfa1 expression. We developed a polymicrobial consortium model, involving growth of P. gingivalis with commensal early colonizer Streptococcus gordonii (S. gordonii), and Fusobacterium nucleatum (F. nucleatum). In addition, those three species were analyzed in plaque and circulating blood cells in chronic periodontitis patients. Therefore, the influence of this three-species consortium on biofilm colonization and on DC invasion in vitro and in vivo was investigated. Our results indicate that growth of P. gingivalis with S. gordonii, and F. nucleatum facilitates biofilm formation and increases expression of mfa-1; which subsequently promotes invasion of DCs by P. gingivalis and F. nucleatum and impairs DC maturation. Analysis of subgingival biofilm samples  from PD patients vs. healthy controls indicates an increase in P. gingivalis and its encoded mfa-1 fimbriae in PD plaques. Circulating DCs in PD patients showed increased carriage of F. nucleatum and P. gingivalis, with higher expression of mfa-1. Strikingly, the majority of microbial carriage in these patients was contained within blood DCs compared to the remaining PBMCs. Overall our results support an important function for polymicrobial synergy in invasion and survival of consortia members in dendritic cells in vitro and in vivo, leading to microbial dissemination and disruption of immune homeostasis.S. gordonii, F. nucleatum and P. gingivalis in broth cultures. The ability of these three strains to form biofilm was tested, alone and in consortium, on sterile extracted human teeth : 10 anaerobic: . The three species together formed a well-organized biofilm structure or consortium  for each species was developed. This facilitated an estimation of CFU (eCFU) for each species grown alone or in complete consortia (detailed in the method section). tum Fig. . On the 6 h Fig. .Fig. 2ThP. gingivalis fimbriae play critical roles in colonization and binding to other biofilm species (reviewed in11) we analyzed the level of expression of both the (minor) mfa-1 and (major) fimA fimbriae by P. gingivalis grown alone, in two-species (incomplete) and three-species (complete) consortia in vitro. mfa-1 mRNA expression was quantified by qrt-PCR during consortium maturation, from 0 to 12 to 24 h. Significant increases in mfa-1 were detected at 12 and 24 h in complete consortium  expression was maintained during biofilm maturation with no significant changes , relative to P. gingivalis alone at 12 h (1.79E + 03 ± 1.45E + 03)  within MoDCs  , short rod  and Fn  Fig. . MoreoveDCs Fig. . This waws) Fig. . Shown i Fn Fig. . Persistine Fig. .Fig. 3CoP. gingivalis has previously been reported to inhibit or activate DC maturation, depending on whether Mfa1 or FimA, respectively, was expressed by the isogenic fimbriae mutant Pg strain used.35 Here we looked at whether time in consortium growth would influence immaturity marker DC-SIGN and maturity/costimulatory marker CD86. Control immature MoDCs were 75.57% positive for Pg uptake mediator and immaturity marker DC-SIGN30  and in blood DCs isolated from PD patients. Our previous studies have identified blood DCs as hematogenous carriers of microbes in PD.nts Fig.   was necessary for invasion and survival within human myeloid DCs.30 In the current study, we detected significant increases in the expression of mfa-1 mRNA within complete consortium in vitro. Hence, we quantified mfa-1 and fimA mRNA expression in oral biofilm , however 58% of S. gordonii were detected within PanDCs . S. gordonii, an early colonizer of the human tooth, formed a biofilm on extracted teeth. F. nucleatum was traditionally considered a ‘bridge-organism’ that facilitates colonization of other bacteria by coaggregation-mediated mechanisms and by promoting growth of other anaerobes;43 however, more recent studies using spectral imaging fluorescence in situ hybridization do not support a central role for Fusobacterium in physically connecting consortium members.44F. nucleatum is reported to promote colorectal carcinogenesis by FadA mediated E-cadherin/beta-catenin signaling,45 but very little is known about its uptake by DCs. Fn has been shown to be resistant to phagocytosis by PMN, although the mechanism was not identified.46 Pg and Fn cocultured together or alone with mouse DCs show that Pg is the driving force for whether or not by Fn activates DCs. Although uptake was not assessed, in one study the influence of Pg on Fn was attributed to gingipains47 In a separate study, Fn extracts have been shown to have a suppressive effect on cell-mediated immunity and phagocyte functions, although no mechanism was not identified.48P. gingivalis, a late colonizer, has been called a pathobiont49 and keystone pathogen, the latter due to its ability orchestrate inflammatory disease by remodeling a normally benign microbiota into a dysbiotic one.29 We showed that P. gingivalis alone was sparsely adherent to human teeth, but when grown as a consortium, a thick biofilm resulted, with structural similarities to those in isolated dental plaque in earlier studies.5 Early reports indicate that P. gingivalis biofilm formation is a multiple step process, involving binding of both mfa1 and fimA fimbriae and aided by S. gordonii.32 Activity of the fimA promotor is under environmental control, with decrease in fimA promotor under conditions of increased temperature, decreased hemin, and presence of saliva or serum of environmental conditions.31 Presence of S gordonii and S. cristatus50 promote or inhibit fimbriae expression respectively. While regulation of the fimA gene in P. gingivalis is controlled by a two-component system (FimS/FimR), regulation of mfa1 fimbriae is less well understood. Recent studies indicate that FimR is a transcriptional activator of the mfa1 gene. Unlike the regulatory mechanism of FimA by FimR, regulation of the mfa1 gene is accomplished by FimR directly binding to the promoter region of mfa1.32 Induction of mfa1 in P.gingivalis under conditions of 24 h consortium growth or when inside MoDCs suggests that these conditions may influence of FimR binding to the promoter, but this has yet to be proven. As mfa1 expression promotes survival of P. gingivalis within MoDCs through evasion of canonical autophagy and prevention of lysosomal fusion,30 this may be a factor in increased mfa1 expression in MoDCs.A microbial consortium, defined as two or more microbial groups living symbiotically, was first described by Johannes Reinke in 1872.F. nucleatum and S. gordonii, but not P. gingivalis, at least in the short term. The interrelationships of S. gordonii and P. gingivalis in two-species consortia have been studied before, with cooperative,52 and antagonistic54 relationships reported. Many oral Streptococci, including S. gordonii, produce inhibitory factors such as hydrogen peroxide to inhibit growth of anaerobes.53P. gingivalis is more sensitive to peroxidases than F. nucleatum55 which could explain the difference in consortium growth advantage in the presence of S. gordonii.Our results also suggest that there is an advantage to consortium growth for F. nucleatum does not enter MoDCs unless in a consortium. The simplest explanation is the “Trojan horse” phenomenon, in which F. nucleatum is taken inside DCs by P.gingivalis. P.gingivalis readily invades DCs via its glycoprotein mfa134 by targeting the C-type lectin DC-SIGN.56 Since we show that DC-SIGN expression is upregulated optimally by the three-species consortium, this could further account for increased uptake, although this will have to be tested experimentally. A recent review describes the concept of a “mobile microbiome,” involving the systemic spread of oral commensals and pathogens through the bloodstream to distant body sites, where they cause extra-oral infections and inflammation.57 These sites include human atheromatous plaques, amniotic fluid, cord blood, synovial fluid of rheumatoid arthritis, inflammatory bowel disease, colorectal cancer and respiratory tract infections, to name but a few. Of particular importance in this regard are Streptococcus spp, Fusobacterium nucleatum and Porphyromonas gingivalis. Most studies use non-cultivation-based techniques such as bacterial DNA or most recently, 16s rRNA to identify these species. The present study used 16s rRNA, but is unique in that we quantitated, by CFU-Ct regression analysis, the level of P. gingivalis, F. nucleatum and S. gordonii within blood PBMCs and panDCs, the latter consisting of plasmacytoid and myeloid DC subsets.Among the most surprising and unexpected findings of our study is that P.gingivalis and F.nucleatum within PD patients’ blood. These data may be reflective of the apparent higher carriage capability of DCs over PBMCs due to the diversity of C-type lectins (CTLs) expressed by immature DCs in the bloodstream. Immature DCs, by virtue of these CTLs, are specialized in the recognition and capture of pathogens, and in the polarization of the resultant effector T cell response (reviewed in58). Interestingly, our results also suggest that the consortium inhibits the DC maturation process that normally would downregulate C-type lectins such as DC-SIGN and convert DCs from antigen capture- to antigen-presenting cells, thus further confounding initiation of an adaptive immune response.To estimate the relative carriage rate of oral consortia in panDCs or PBMCs, we calculated total counts of the three species in relation to total numbers of DCs and PBMCs in PD patients. The resultant numbers suggest that panDCs carry more bacteria per cell on average than PBMCs. By our calculation, circulating panDCs carry the majority of In conclusion we have identified a three-species oral consortium that, through polymicrobial synergy, drives microbial growth, invasion and persistence in dendritic cells (DC) and that appears to have negative consequences for the human host. It is important to reiterate in this context that the oral biofilm contains around 700 distinct species that may have other synergistic effects. Moreover, in vivo animal testing is required to confirm our in vitro and ex vivo human findings.Porphyromonas gingivalis: 381 (Pg) and Fusobacterium nucleatum: ATCC 4925659 were cultured anaerobically  in DifcoTM Anaerobe Broth MIC  in a Forma Scientific anaerobic system glove box model 1025/1029 at 37 °C. Growth curves were established for each microbe 1–4 days to determine the log . Streptococcus gordonii DL160 growth curve was established in aerobic and anaerobic condition as consortium was designed to be in anaerobic condition. Hence, all experiments were carried out for each strain in anaerobic condition using DifcoTM Anaerobe Broth MIC  in anaerobic system glove chamber. This allow unified conditions to compare each bacteria growth and gene expression as an individually or within the polymicrobial model. Growth of the three strains together was confirmed on sterile root surfaces of extracted human teeth. Initial layer of Streptococcus gordonii incubated at sterile human root for 2 h and then Porphyromonas gingivalis and Fusobacterium nucleatum were added for 12 h at ratio of 1 aerobes: 10 anaerobes. Samples were prepared for scanning electron microscopy (SEM) as discussed below. Complete consortium consists of the three strains, at a 1:10 aerobes to anaerobes ratio and incomplete consortium consists of two of these strains maintaining the same ratio. Mature consortium where the three strains grown together for 12 h while immature consortium was established by mixing the three strains with no time for maturation (refered to as 0 h immature consortium).P. gingivalis, F. Nucleatum and S. gordonii. Individual TaqMan® gene expression primers were designed to target mRNA 16s rRNA of P. gingivalis (GenBank: AB035455.1), F. nucleatum (GenBank: AJ810280.1) and S. gordonii (GenBank: D38483.1). Forward, reverse and probe for primers were designed based on these gene sequences using online tool (Thermofisher). Each primer was verified for specificity and sensitivity by running qrt-PCR of RNA isolated from each bacterial strain with different dilutions . 5 µl of the RNA sample, 25 µl PCR master mix (2×) and 2.5 µl TaqMan® gene expression assays were used per reaction. All PCRs were performed in triplicate and were carried out on a real-time PCR, StepOne® (Applied Biosystems).mfa-1) and major (fimA) fimbriae were quantified using 16s rRNA as housekeeping. For calculations and statistical analysis, fold changes were calculated using (2−ΔΔCT) method in the experimental samples.61 Statistical analysis for gene expression was performed using the one sample t-test, which estimates the calculated difference (in fold regulation) between experimental and control samples. A p-value of <0.05 is the cut-off for significant differences.Gene expression of minor  for 20 min, then monocyte separation carried out by density Ficoll . Cells were cultured in the presence of GM-CSF  and IL-4  at a concentration (3–4 × 105 cells/ml) for 5-6 days. Flow cytometry analyses were carried out to verify the immature DC phenotype . Cell surface markers of DCs were evaluated by four-color immunofluorescence staining with the following antibodies: CD1a-PE , DC-SIGN-FITC , CD14-PerCP  and CD83-APC .7 CFU. MoDCs were pulsed with single species or consortia at 10 MOI and incubated with the MoDCs for 2, 6, 12, 24, and 48 h and each experimental condition was performed in triplicate. MoDCs were infected with mature and immature as well as complete and incomplete consortia. After MoDCs infection with the single species or consortia for 2, 6, 12, 24, and 48 h, cells were re-suspended in sterile water on ice for 20 min to lyse the cells. RNA isolation was carried out for control (uninfected cells) and experimental (infected cells) samples and eCFUs were carried out as described above.Bacterial suspensions were washed five times in PBS and re-suspended for spectrophotometer reading at OD 660 nm of 0.11, which previously determined to be equal to 5 × 10P. gingivalis, F. nucleatum and S. gordonii, were grown on the lower chamber of membrane-double chamber slide  and the MoDCs were added to the upper chamber of the slide. MoDCs were cultured at the upper chamber either: (1) immediately after culturing the bacteria on the lower chamber to test their interaction with immature consortium, or (2) after 12 h of bacterial culture to test their interaction with mature consortium. Field of interest were chosen and images (at x40 magnification) were captured for 24 h for both groups (mature and immature consortia) using autofocus property. Temperature at 37 °C, humidity, and CO2 gas at 5% for normoxic condition were adjusted for the length of experiment (24 h). After image acquisition time lapse videos . The three species,  either:  immediattium, or  after 12PD was defined as PPD ≥ 5 mm in ≥10 teeth, and BOP in ≥30% of sites. Periodontally healthy controls had PPD ≤ 4 mm and BOP in ≤30% sites. Periodontal assessment was carried out by two calibrated examiners (M.S.R. and A.M.F). RNA isolated from PanDCs and PBMCs then qrt-PCR was performed to detect molecular microbial signatures in each population. In addition, quantifications of Sg, Fn and Pg were carried out in the oral biofilm of healthy and PD patients. For each subject, subgingival biofilm samples were collected from the four deepest sites (one site per quadrant) that did not exhibit suppuration as observed by prior clinical examination. Before sampling, the teeth were isolated with cotton rolls and the supragingival plaque was removed. A mini-five Gracey curette , was gently inserted into the pocket in the most apical portion and the subgingival biofilm was collected with a single stroke. The samples were placed in polypropylene tubes with 1.5 ml of a solution containing 100 ml buffer solution  and stored at −80 °C until use.In vitro monocyte-derived DCs (MoDCs) studies were determined by the Human Assurance Committee at Augusta University to be human subject exempt, due to the use of anonymized peripheral blood samples for monocytes. Human samples in the clinical study were collected between January 2014 and May 2015 following the Helsinki Declaration of 1975, as revised in 2013. The study protocol was reviewed and approved by the Institutional Review Board of the School of Dentistry, University of São Paulo, Brazil (658.998/CEP). All subjects signed an informed consent form.Samples were fixed for 30–60 min in 4% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium cacodylate (NaCac) buffer, pH 7.4, postfixed in 2% osmium tetroxide in NaCac buffer, dehydrated with a graded ethanol series (25–100%), followed by a graded alcohol hexamethyldisilazane (HMDS), and the HMDS was allowed to evaporate overnight in a fume hood. The dried discs were mounted on aluminum stubs with carbon adhesive tabs and sputter coated with gold-palladium. Discs were observed and imaged in a FEI XL30 scanning electron microscope  at 10 kV.After MoDC fixation, the procedures were carried out at the Electron Microscopy and Histology Core, Department of Cellular Biology and Anatomy, Augusta University. The cells were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate (NaCac) buffer, pH 7.4, postfixed in 2% osmium tetroxide in 0.1 M NaCac, stained en bloc with 2% uranyl acetate, dehydrated with a graded ethanol series and embedded in Epon-Araldite resin. Thin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate. Cells were observed in transmission electron microscope (JEM 1230–JEOL USA Inc.) at 110 kV and imaged with a CCD camera and first light digital camera controller (Gatan Inc.).62 buffer (Abcam) supplemented by protease inhibitor cocktail . The lysates were centrifuged at 12,000×g for 10 min at 4 °C. Samples were normalized to the amount of total protein in the supernatant using Pierce™ BCA Protein Assay Kit (ThermoFisher). Protein aliquots (30 μg) were separated by size on a 10% SDS-tricine-polyacrylamide gel and transferred to polyvinylidene difluoride membrane . Nonspecific binding sites were blocked by incubation in 1 × TBS-T  containing 2% bovine serum albumin (BSA) for 1 h at room temperature, followed by incubation overnight at 4 °C with 1:1000 dilution of mouse monoclonal anti-mfa1 antibody (in 1 × TBS-T containing 2% BSA. After membranes were washed three times in 1 × TBS-T (10 min each at room temperature), horseradish peroxidase-conjugated rabbit anti-mouse IgG (Abcam) was added at a 1:2000 dilution and incubated for 1 h at room temperature. After three more washes with 1 × TBS-T, the immunoreactive peptide was detected by Western Lightning ECL Pro Chemiluminescent reagent  and imaged using C-DiGit Blot Scanner (LI-COR).Whole lysates were prepared from bacterial cultures by centrifuging and washing the pellet with ice-cold PBS. Proteins were extracted by adding ice-cold Radio-immunoprecipitation assayAll blots derive from the same experiment and were processed in parallel.Immature consortium and MoDCsmature consortium and MoDCsSupplementary Material.