P-value < 0.001. Similarly, ORs for POI type 3 and 4 relative to type 2 were 1.99 and 5.83 respectively. A similar relationship was found with tumour size; ORs for T2, T3, and T4 compared to T1 were 2.79, 8.27 and 8.75 respectively. These four histopathological parameters were then used to develop a predictive model for nodal metastasis. This model showed that probability of nodal metastasis is higher among tongue cancers with increasing POI, with increasing T, and with larger depths while other characteristics remained unchanged. The proposed model provides a way of using combinations of histopathological parameters to identify patients with higher risks of nodal metastasis for surgical management.Difficulty in precise decision making on necessity of surgery is a major problem when managing oral squamous cell carcinomas (OSCCs) with clinically negative neck. Therefore, use of clinical and histopathological parameters in combination would be important to improve patient management. The main objective is to develop a model that predicts the presence of nodal metastasis in patients with OSCC.623 patients faced neck dissections with buccal mucosal or tongue squamous cell carcinoma (SCC) were selected from patients’ records. Demographic data, clinical information, nodal status, Depth of invasion (DOI) and pattern of invasion (POI) were recorded. The parameters which showed a significant association with nodal metastasis were used to develop a multivariable predictive model (PM). Univariate logistic regression was used to estimate the strengths of those associations in terms of odds ratios (OR). This showed statistically significant associations between status of the nodal metastasis and each of the following 4 histopathological parameters individually: size of the tumour (T), site, POI, and DOI. Specifically, OR of nodal metastasis for tongue cancers relative to buccal mucosal cancers was 1.89,  The incidence of oral squamous cell carcinoma (OSCC) is increasing globally and is a leading cause of death accounted for 8.8 million deaths in 2015 . TobaccoThe management of clinically negative neck nodes (N0) poses a significant challenge for surgeons, as there are no reliable parameters to predict occult metastasis. In order to identify patients who are likely to have nodal metastases, several parameters like tumour differentiation, perineural invasion, lymphovascular invasion, pattern of invasion (POI) and depth of invasion (DOI) have been previously studied ,5. AccorTumour thickness or the maximum depth of tumour infiltration is a well-established risk factor for many cancers mainly for gastrointestinal tract tumours . HoweverThe aetiological factors for OSCC show significant variation across different parts of the world. In the developed world it is mostly due to smoking, alcohol and human papilloma virus. However, in South and Southeast Asia OSCC is mostly related to betel chewing containing tobacco and arecanut . TherefoMedical records and histological sections were available for 623 patients; of these nodal metastasis was observed for 189 (30%). Majority (76%) were males, 95% were aged 40 or more, 50% had cancer of the tongue, nearly half (47%) were in POI type IV with just one person in POI type I, and 11% were in T1 with near equal numbers in T2-T4 . More thAfter excluding 8 cases due to missing data and a single person in POI type I, 614 complete cases were included in the rest of the analyses. Univariate logistic regression found an odds ratio (OR) of 1.89 (95% CI: 1.33–2.69) for metastasis of tongue cancers relative to buccal mucosa . OR for P-value = 0.28, area under the ROC curve = 0.77). Probability of nodal metastasis predicted by the model for each of the 48 combinations of four clinical parameters are presented in Based on univariate results, age and sex were not used in the multivariable model. The model fit of the multivariable predictive model was acceptable , lymphoid response, tumour budding, degree of keratinisation, nuclear pleomorphism, habits and gene profiling –27. TherWhen reviewing the literature, it was distinct that the tongue was considered as the most common site of oral cancer and exhibits high rates of metastasis outside South Asia while buccal cancers are much more predominant in South Asian countries –30. AnatThe greatest diameter of the tumour surface is used to indicate tumour size in the TNM classification. Controversies exist regarding this parameter where some authors suggest that increased tumour size has a link to cervical nodal metastasis –34, whilThe literature provides strong evidence for association of POI with local and distant nodal metastasis , 36–38. When reviewing the literature, it was noted that lack of standardized method of measuring tumour thickness/ depth of invasion has resulted in different cut off values to decide elective neck dissection for clinically negative neck. Some authors have used a technique developed by Breslow  where thOur descriptive P<0.01,  and univAs mentioned before, different studies have suggested different cut-off values for DOI to predict metastasis for different sites in the oral cavity. Therefore, it is difficult to compare these cut off values due to the discrepancies of the methods used to measure the tumour thickness/ DOI. For example, certain studies which DOI has been measured starting from the adjacent intact mucosa have suggested 4mm –58, 65 aResults of the current study revealed strong associations between cervical nodal metastasis and the four parameters, site, T, POI and DOI. We hypothesised that the use of these parameters together may increase the accuracy of predicting nodal metastasis. Therefore, we used these four parameters to develop a multivariate logistic model which predicts possible presence of nodal metastasis more accurately. For reasons given above, and also to simplify the model, we dichotomised DOI as less than 4mm and equal or more than 4mm. Therefore, the predictive model included two sites, four categories of tumour size, two categories of DOI, and three grades of POI. Probability of nodal metastasis predicted by the model in In order to predict the presence of nodal metastasis and to avoid under treatment the sensitivity of the cut off value must be high. At the same time, it should have a considerable specificity to reduce over-treatment (unnecessary neck dissections). Hence this value requires a compromise between sensitivity and specificity . We do nThe final outcome of the model revealed that T4 tumours in the buccal mucosa that have POI type 3 and has a depth of invasion > 4 mm are more likely to metastasize. It also showed that T3 and T4 tumours in the buccal mucosa with POI type 4 tend to metastasize regardless of its depth of invasion.The model showed that there is a high chance of nodal metastasis when there is a tumour on the tongue that has a POI of type 4 regardless of depth of invasion. T3 tumours in the tongue tend to metastasize when the POI is 2 and DOI is more than 4mm. T3 tumours also has a high chance of metastasis when the POI is type 3 or 4 regardless of its DOI. The model also revealed that T4 tongue tumours have a high chance of cervical nodal metastasis regardless of its DOI and POI.This study proposes a simple predictive model for the risk of nodal metastasis in clinically negative necks. The major drawback of this study was the difficulty in measuring exact DOI in significant number of cases. However, the study has strength that, it is based on a large sample, proposed model being simple, and based on parameters empirically supported as well as established in literature, easy to use in routine clinical practice, and cost effective. Although findings of this retrospective investigation needs required to be validated in a prospective study, it provides a useful model that is practicable to use in developing countries of South and Southeast Asia where OSCC aetiology is similar and advanced facilities are not available.All patients who underwent surgery for OSCC of the tongue or buccal mucosa with neck dissection between 1998 and 2015 were retrospectively studied. The cases were selected from archives of the Department of Oral Pathology, Faculty of Dental Sciences, University of Peradeniya, the only Head and neck Pathology centre which receives most biopsies from almost all Oral and Maxillofacial units in Sri Lanka. Therefore this can be considered as a representative sample of the entire country. Demographic data including age, gender and clinical information such as size of the tumour (T), primary site; tongue or buccal mucosa, and pathological factors  were recorded. Arrangement of tumour cells at the advancing front was graded as large cohesive tumour islands (POI- I), small islands (POI- II), thin strands (POI- III) and individual tumour cells (POI- IV)  Fig 2)..Fig 2). Patient’s histological sections had been prepared primarily for managing with curative intent; therefore full width of the tumour had not been taken to a single section in about one third of the sections. Since the DOI was measured from the basement membrane of the healthy oral mucosa, section containing this may not contained the full depth of the tumour in these cases which prevented from accurate measurement of the depth of the tumour. We recorded the DOI of these sections and used in the analysis as the lower end for their actual tumour depth knowing that these are under-measurements of the actual thickness of these cases. Those measurements are hereafter referred to as censored depths.For the purpose of analysis, age was dichotomised as 40 years or below vs 41 or more years. Depth was initially categorised into five groups; four 1mm groups and one group containing cases with 5mm or more depths . AssociaS1 Dataset(XLS)Click here for additional data file.


Brucella abortus, the causative agent of brucellosis, displays many resources to evade T cell responses conducive to persist inside the host. Our laboratory has previously showed that infection of human monocytes with B. abortus down-modulates the IFN-γ-induced MHC-II expression. Brucella outer membrane lipoproteins are structural components involved in this phenomenon. Moreover, IL-6 is the soluble factor that mediated MHC-II down-regulation. Yet, the MHC-II down-regulation exerted by lipoproteins was less marked than the one observed as consequence of infection. This led us to postulate that there should be other components associated with viable bacteria that may act together with lipoproteins in order to diminish MHC-II. Our group has recently demonstrated that B. abortus RNA (PAMP related to pathogens' viability or vita-PAMP) is involved in MHC-I down-regulation. Therefore, in this study we investigated if B. abortus RNA could be contributing to the down-regulation of MHC-II. This PAMP significantly down-modulated the IFN-γ-induced MHC-II surface expression on THP-1 cells as well as in primary human monocytes and murine bone marrow macrophages. The expression of other molecules up-regulated by IFN-γ (such as co-stimulatory molecules) was stimulated on monocytes treated with B. abortus RNA. This result shows that this PAMP does not alter all IFN-γ-induced molecules globally. We also showed that other bacterial and parasitic RNAs caused MHC-II surface expression down-modulation indicating that this phenomenon is not restricted to B. abortus. Moreover, completely degraded RNA was also able to reproduce the phenomenon. MHC-II down-regulation on monocytes treated with RNA and L-Omp19  was more pronounced than in monocytes stimulated with both components separately. We also demonstrated that B. abortus RNA along with its lipoproteins decrease MHC-II surface expression predominantly by a mechanism of inhibition of MHC-II expression. Regarding the signaling pathway, we demonstrated that IL-6 is a soluble factor implicated in B. abortus RNA and lipoproteins-triggered MHC-II surface down-regulation. Finally, CD4+ T cells functionality was affected as macrophages treated with these components showed lower antigen presentation capacity. Therefore, B. abortus RNA and lipoproteins are two PAMPs that contribute to MHC-II down-regulation on monocytes/macrophages diminishing CD4+ T cell responses. B. abortus establishes a persistent infection inside its intracellular niche, the macrophage  where the surviving bacteria begin to replicate dramatically , suggesting the participation of a Brucella structural component (B. abortus lipoprotein [outer membrane protein 19 (Omp19)], decreased the surface expression of MHC-II molecules (Brucella lipoproteins are capable of inhibiting MHC-II surface expression since Pam3Cys (a synthetic lipohexapeptide that resemble the protein lipid moiety structure) also inhibited MHC-II expression  (Regarding the MHC-II surface inhibition mediated by omponent . In lineolecules . Furtherpression . On the pression . RecentlII genes . More spgulator) .B. abortus, what specially caught our attention was that HKBA or B. abortus lipoproteins were less efficient at reducing IFN-γ-induced MHC-II surface expression than live bacteria  related to pathogens' viability or vita-PAMP , is found in viable bacteria but not in dead bacteria , 16, 17.In this study, human monocytes from adult blood donors in healthy state were utilized in agreement with the guidelines of the Ethical Committee of IMEX. Donors gave their informed consent before the study. With regard to animals, female mice from C57BL/6 strain were maintained under SPF conditions as previously described . All aniB. abortus S2308, Escherichia coli 11105, Staphylococcus aureus 25923, and Klebsiella pneumoniae 700603 strains were cultivated in tryptose-soy agar in which yeast extract was added (Merck). The amount of bacteria on stationary-phase cultures was determined by the comparison of the OD at 600 nm with a standard curve. The experiments that involved infection of cells with live Brucella abortus were performed in biosafety level 3 (BSL-3) facilities, located at the ANLIS-Malbrán  . With regard to T. cruzi, trypomastigotes from the Brazil strain were cultured overnight in Dulbecco's modified Eagle medium , to transform trypomastigotes to amastigotes, as previously described , 100 U of penicillin.ml−1 and 100 μg of streptomycin.ml−1. Cells from the monocytic line THP-1, were purchased from the American Type Culture Collection  and cultured as it was formerly described  for 72 h. In the experiments with primary human monocytes, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient  centrifugation. Monocytes were purified from PBMCs by Percoll gradient  as previously described (6 cell/ml under a humidified atmosphere of 5% CO2 at 37°C in standard medium supplemented with 50 ng/ml recombinant granulocyte-monocyte colony stimulating factor (GM-CSF) (Peprotech) and 10 ng/ml recombinant IL-4 (Prepotech) as described elsewhere  for 48 h. Cells treated with 2% paraformaldehyde (PFA) were included as a positive control of the technique. After 48 h, cells were washed and stained with 7-Amino-Actinomycin D  for 10 min at 0°C in darkness. Immediately after, cells were analyzed on a FACSCalibur® flow cytometer (BD Biosciences) or Sysmex Partec Cytometer  and data were processed using FlowJo® 7.6 software.In order to determine cellular apoptosis, 5 × 108 CFU, 1 × 107 amastigotes or 5–10 × 106 PBMCs were suspended in 1 ml of Trizol Reagent (Invitrogen) and total RNA was isolated with Quick-RNA™ MiniPrep (Zymo Research) according to the manufacturer's instructions. RNA was quantified with OD at 260. The purity of the preparation was determined using a DeNovix DS-11 Spectrophotometer (DeNovix Inc.) with a ratio of absorbance 260/280 >2.0 and a ratio of absorbance 260/230 > 1.8. To exclude the potential effect of B. abortus DNA, some RNA preparations were incubated with DNase I  in a buffer containing 400 mM Tris-HCl (pH 8.0), 100 mM MgSO4, and 10 mM CaCl2 for 30 min at 37°C and the reaction was stopped by addition of 20 mM EGTA and incubation for 10 min at 65°C. Additionally, to rule out the effect of B. abortus proteins in the effects mediated by B. abortus RNA, our preparations were treated with Proteinase K  for 60 min at 37°C, and the digestion was stopped by incubation at 96°C for 10 min.5 × 105.ml−1 were treated with B. abortus RNA, other prokaryotic or eukaryotic RNAs, E. coli RNase I (Life Technologies)-treated B. abortus RNA or B. abortus lipoproteins in the presence of 150 U.ml−1 IFN-γ for 48 h as it was formerly described (B. abortus RNA in presence of 10 ng.ml−1 recombinant murine IFN-γ (PeproTech) for 48 h. Murine MHC-II expression was assessed by flow cytometry.Cells at 5 × 10escribed . MHC-II,5.ml−1 THP-1 were infected with a multiplicity of infection (MOI) of 100 B. abortus S2308 per cell in round-bottom polypropylene tubes . This procedure was performed with 150 U.ml−1 IFN-γ for 2 h in standard medium without antibiotics. Afterwards, cells were extensively washed in order to remove uninternalized bacteria. Infected cells were maintained in culture in presence of IFN-γ, 100 μg.ml−1 gentamicin, and 50 μg.ml−1 streptomycin for other 48 h.5 × 105.ml−1 THP-1 were incubated in presence of 150 U.ml−1 IFN-γ, B. abortus RNA (10 μg.ml−1) and B. abortus L-Omp19 (1 μg.ml−1) in the presence of neutralizing mAb for IL-6  or the isotype control at a concentration of 20 ng.ml−1. After this, MHC-II expression was assessed by flow cytometry.In another set of experiments, 5 × 10B. abortus infection or stimulation of cells were performed, monocytes were stained with PE-labeled anti-human HLA-DR , FITC-labeled anti-human HLA-ABC  or isotype-matched control mAbs. To evaluate MHC-II expression, MHC-II bar graphs were performed on the MHC-II positive cells. In order to evaluate murine MHC-II surface expression, BMDMs were stained with PE-labeled anti-mouse MHC-II (I-A/I-E) . To determine CD40, CD86, and CD80 surface expressions, in another set of experiments cells were stained with PE-labeled anti-human CD40 , PE-labeled anti-human CD86  or FITC-labeled anti-human CD80 . In all cases, monocytes were stained with 7-AAD for 10 min at 0°C in darkness. After that, all markers studied were analyzed on a FACSCalibur® flow cytometer (BD Biosciences) or Sysmex Partec Cytometer , gating on viable cells (7-AAD negative cells). Data were processed using FlowJo® 7.6 software.Once 5 THP-1 cells/well were incubated in chamber-slides (Nunc) with 10 ng/ml PMA (Sigma-Aldrich) for 24 h to promote adherence. Then, cells were stimulated with B. abortus RNA (10 μg.ml−1), RNase I-treated B. abortus RNA, B. abortus L-Omp19 (1 μg.ml−1) or their combination with IFN-γ for 48 h. Afterwards, cells were treated with 2% PFA, permeabilized with 0.1% saponin and incubated with anti-HLA-DR mAb L243 (purified from murine hybridoma culture supernatants) and Alexa 546-labeled secondary Ab (Invitrogen). In order to detect Golgi apparatus, cells were labeled with a mAb specific for GM130 (BD Biosciences) followed by Alexa 488-labeled secondary Ab (Invitrogen). Then, slides were mounted and analyzed with a FV-1000 confocal microscope, as it was previously described  for 3 h at 37°C. After washing, cells were suspended in complete medium at pH 7.3 and BO97.10 cells were added for 20 h. The production of IL-2 by BO97.10 was measured by ELISA (RD) as described previously .Neutralizing monoclonal antibody for human IL-6 (clone MQ2-13A5) was acquired from eBioscience.Human IL-6 was measured in culture supernatants by sandwich ELISA, as it was formerly described .post hoc Tukey test or two-way ANOVA followed by post hoc Bonferroni test with GraphPad Prism software.Results were analyzed with one-way ANOVA followed by B. abortus RNA was a vita-PAMP involved in MHC-II down-regulation. For this purpose, cells from the human monocytic cell line THP-1 were stimulated with B. abortus RNA at several doses and in presence of human IFN-γ for 48 h. Then, the surface expression of MHC-II molecules was assessed by flow cytometry. This PAMP was able to diminish the IFN-γ-up-regulated MHC-II surface expression  were stimulated with B. abortus RNA at several doses. Afterwards, MHC-II surface expression was evaluated by flow cytometry. B. abortus RNA was able to significantly down-modulate the IFN-γ-induced MHC-II expression on both primary cell cultures  was also able to decrease the surface expression of MHC-II  and B. abortus lipoproteins  constitute both Brucella virulence factors which contribute to MHC-II down-modulation in the context of the infection.Taking into account that proteins , 13 and ed cells . As it hr levels . L-Omp19products . Furtherproducts . A simil L-Omp19 . Moreove abortus . Taking  abortus ; and tha ligands , 25. So,aneously . These rB. abortus RNA alone or together with lipoproteins was down-modulating MHC-II expression. Therefore, at first, we evaluated MHC-II down-regulation kinetics. In order to perform this, THP-1 cells were stimulated as previously described, with B. abortus RNA, RNase I-treated B. abortus RNA, the B. abortus lipoprotein L-Omp19 or the combination of each component in the presence of IFN-γ. At 6, 24, and 48 h, the surface expression of MHC-II was evaluated by flow cytometry. At 6 h, there is neither up-regulation of MHC-II on IFN-γ-treated cells nor MHC-II modulation with the different stimuli. However, at 24 h MHC-II expression on IFN-γ-treated cells is induced, being even accentuated at 48 h. At 24 h, L-Omp19 (but not B. abortus RNA and digested B. abortus RNA) significantly down-regulated MHC-II surface expression. Meanwhile at 48 h, B. abortus RNA, digested B. abortus RNA and L-Omp19 were able to significantly inhibit the MHC-II surface expression (B. abortus RNA) plus L-Omp19 induced a higher MHC-II down-modulation than merely RNA, digested RNA or L-Omp19  the greater the degree of MHC-II surface inhibition generated by the components.Next, we wanted to evaluate the mechanism by which pression . Further L-Omp19 . We alsoI on DCs  and downI on DCs . OverallB. abortus decreased MHC-II expression by inhibiting MHC-II genes transcription . At 24 h, IFN-γ-treated cells showed up-regulation of MHC-II surface expression. However, there was not a significant reduction on MHC-II expression mediated by B. abortus RNA, digested B. abortus RNA, L-Omp19 or their combination (data not shown). At 48 h, two populations were observed: cells with MHC-II expression confined to the cellular surface (named MHC-II-positive cells) and cells with no MHC-II expression on the cellular surface (named MHC-II null cells). As expected, the majority of the cells were MHC-II positive in IFN-γ-treated cells  down-modulates MHC-II surface expression is the inhibition of MHC-II inside the cell, we tested whether IL-6 could be a possible mediator in this phenomenon. In order to perform this, THP-1 monocytes were stimulated with B. abortus RNA, RNase I-treated B. abortus RNA, L-Omp19 or a combination of each component in the presence of IFN-γ for 48 h. Afterwards, secreted IL-6 was measured in the supernatants by ELISA sandwich. As previously demonstrated, L-Omp19 stimulates the secretion of IL-6 . Murine BMDM were stimulated with B. abortus RNA, digested B. abortus RNA, L-Omp19 or the combination of each component in the presence of mIFN-γ (murine IFN-γ). After 48 h, BMDM were incubated with OVA peptide and the T cell hybridoma BO97.10. As shown in B. abortus RNA, digested B. abortus RNA, L-Omp19 or the combination of each component plus mIFN-γ showed a significantly reduced capacity of OVA peptide presentation, as evidenced by decreased response of BO97.10 cells, related to BMDM treated only with mIFN-γ . Our results also demonstrate that B. abortus RNA neither delays the kinetic of IFN-γ induction of MHC-II molecules nor it modifies the expression of MHC-II molecules already induced by IFN-γ. On the contrary, B. abortus RNA prevents the correct induction of MHC-II molecules by IFN-γ. Moreover, MHC-II down-regulation was observed on cells of the monocytic line THP-1, on peripheral blood-purified human monocytes, and murine bone marrow-derived macrophages.CD4nfection –9. The s T cells –28. MHC-d spleen , highligrophages . Moreoveproteins , 13. HowB. abortus RNA is also a component involved in B. abortus-mediated MHC-I surface inhibition  were able to mimic the MHC-II down-modulation mediated by HKBA  corroborate in a functional way the involvement of TLR8 in MHC-II down-modulation.As we have previously demonstrated, HKBA was also capable of inhibiting MHC-II surface expression . Further by HKBA , 13. Howiability , 16, 17,iability , 37. Stiiability , 38. Beaiability . Other riability . Anotheriability . It has iability –42. Our B. abortus RNA alone or in combination with B. abortus lipoproteins begins after 24 h post-stimuli, when MHC-II expression was induced by effect of IFN-γ. However, the maximum MHC-II surface down-modulation was observed at 48 h, in the moment of highest induction by IFN-γ. In turn, in agreement with previously published results . Nevertheless, MHC-II down-modulation is more pronounced in the moment of infection in which adaptive immunity begins to be relevant, i.e., when IFN-γ secreted by T lymphocytes is stimulating the presentation of Brucella antigens to MHC-II-restricted CD4+ T cells.Our kinetic studies demonstrated that MHC-II down-modulation by  results , 43, B. nfection . The appt dwells . Th1 celB. abortus RNA alone or in combination with lipoproteins is able to down-modulate MHC-II. We previously demonstrated that B. abortus lipoproteins down-regulate the IFN-γ-induced MHC-II surface expression by decreasing the transcription of MHC-II mRNA with the consequent inhibition of protein synthesis  expression, trough IL-6 . However, the global effect is the reduction of antigen presentation of B. abortus RNA stimulated-macrophages to CD4+ T cells, which is an important functional correlation to our study. Moreover, B. abortus RNA in combination with lipoproteins leads to a lower antigen presentation.Although we demonstrated that B. abortus RNA is a component associated to bacterial viability that along with lipoproteins participates in MHC-II surface down-modulation. Accordingly, through this phenomenon, the bacteria could prevent the CD4+ T cell recognition in order to evade host immune response.Finally, we have elucidated that The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Ethical Committee of the IMEX Institute. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Animal Care and Use Committee of the IMEX Institute.The experiments were designed and conceived by MM, GG, and PB. The experiments were performed by MM, AT, AS, JM, FM, JA, MG, and LB. Data was analyzed by MM. Materials, reagents were facilitated by SO and GG. They provided key suggestions to this work as well. All experiments were supervised by PB. The interpretation of data and the preparation of the manuscript were performed by MM and PB. The manuscript was reviewed by all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The perinatal period carries the highest risk for stroke in childhood; however, the pathophysiology is poorly understood and preventive, prognostic, and therapeutic strategies are not available. A new pathophysiological model describes the development of neonatal arterial ischemic stroke (NAIS) as the combined result of prenatal inflammation and hypoxic–ischemic insult. Neuroinflammation and a systemic inflammatory response are also important features of NAIS. Identifying key players of the inflammatory system is in the limelight of current research.We present four NAIS cases, in whom detailed analysis of intracellular and plasma cytokine levels are available from the first month of life. All neonates were admitted with the initial diagnosis of hypoxic ischemic encephalopathy (HIE); however, early MRI examination revealed NAIS. Blood samples were collected between 3 and 6 h of life, at 24 h, 72 h, 1 week, and 1 month of life. Peripheral blood mononuclear cells were assessed with flow cytometry and plasma cytokine levels were measured. Pooled data from the cohort of four NAIS patients were compared to infants with HIE.At 6 and 72 h of age, the prevalence of IL10+ CD8+ lymphocytes remained lower in NAIS. At 6 h, CD8+ lymphocytes in NAIS produced more IL-17. At 72 h, CD8+ cells produced more IL-6 in severe HIE than in NAIS, but IL-6 production remained elevated in CD8 cells at 1 month in NAIS, while it decreased in HIE. At 1 week, the prevalence of TGF-β + lymphocytes prone to enter the CNS was elevated in NAIS. On the other hand, by 1 month of age, the prevalence of TGF-β + CD4+ lymphocytes decreased in NAIS compared to HIE. At 72 h, we found elevated plasma levels of IL-5, MCP-1, and IL-17 in NAIS. By 1 month, plasma levels of IL-4, IL-12, and IL-17 decreased in NAIS but remained elevated in HIE.Differences in the cytokine network are present between NAIS and HIE. CD8 lymphocytes appear to shift towards the pro-inflammatory direction in NAIS. The inflammatory response appears to be more pronounced at 72 h in NAIS but decreases faster, reaching lower plasma levels of inflammatory markers at 1 month. The perinatal period carries the highest risk for stroke in the entire childhood, with almost similar incidence to that in the elderly population. “Perinatal stroke” is a broad term defining a group of heterogeneous conditions characterized by the focal disruption of cerebral blood flow, of which neonatal arterial ischemic stroke (NAIS) is one of the most common subtypes, with an incidence of 1/8000 live births. NAIS by definition is an arterial ischemic stroke, with clinical symptoms occurring in the neonatal period (within the first 28 days of life) which are supported by radiological evidence .Interestingly, in almost all cases of NAIS, the intra-cranial arteries developing from the carotid arterial tree are affected, i.e., the proximal parts of the anterior cerebral artery (ACA), the middle cerebral artery (MCA), and the posterior cerebral artery (PCA)—while the basilar artery and the extra-cranial arteries are left unaffected –6. The aThe pathophysiology of NAIS is poorly understood, and disease-specific preventive measures, prognostic factors, and therapeutic strategies are not available. The risk factors of NAIS appear to be specific for the perinatal period and can be clearly distinguished from the risk factors of stroke in older children or adults. Major risk factors such as congenital heart disease or bacterial meningitis can be held accountable for approximately 30% of NAIS occurrences; however, in most of the cases, NAIS is thought to be multifactorial, and in 25–40% of cases, no identifiable risk factor is present , 12. OnlAccording to the classic pathophysiological hypothesis, most ischemic lesions are a result of thromboembolic events, where the presumable source of the thrombi is the placenta or the umbilical vessels . While iBased on preclinical findings, a new pathophysiological hypothesis is being raised, which considers the complex and bidirectional relationship of coagulation and inflammatory pathways . It descThe primary aim of current research in the field of perinatal stroke is to gain a better understanding of the pathomechanism of the disease with specific regard to the activation of the inflammatory pathway that could present a possibility for more specific diagnosis, intervention, and even prevention . Human eThe pivotal role of inflammatory pathways in ischemic brain injury is supported by recent years’ research data. T lymphocytes appear to play a central role in ischemic infarct development; they appear in the brain tissue within hours of the hypoxic insult and can be detected for up to a month after the injury. T cell deficiency has been shown to result in smaller infarct size and improved neurological outcome in murine models , 34. SevIn this paper, we present four NAIS cases, in whom detailed analysis of intracellular and plasma cytokine levels are available from immediately after birth until the first month of age. To our knowledge, no such information is available in humans in NAIS; therefore, the data presented could serve as a basis for future, larger-scale case-control studies.All neonates were admitted to the regional cooling center with the initial diagnosis of HIE, requiring therapeutic hypothermia. Eligibility for cooling was assessed according to the TOBY criteria , and hypn = 11) was formed of newborns with moderate-to-severe HIE signs on the MRI scan AND burst-suppression or continuous extremely low voltage or flat tracing background activity on aEEG OR normalization of aEEG after the 48th hour of life or never, OR early death (< 28 days). Neonates that met none of the above-listed criteria constituted the moderate group (n = 17)  [A total of 33 term neonates were enrolled with written parental consent in a study focusing on inflammatory changes in HIE ). Four infants were diagnosed with NAIS after their MRI scans were completed. One infant was excluded due to suspected metabolic disease. In the HIE group, data from 28 neonates were analyzed and divided into two groups based on the severity of the hypoxic-ischemic encephalopathy. The groups were determined based on the initial amplitude-integrated EEG (aEEG) recordings and the recovery time during monitoring  togetherof life) . Pooled Two milliliters of venous blood was collected between 3 and 6 h of life (at admission), as well as at 24 h, 72 h, and 1 week of life, adjusted to blood sampling required by clinical care. A further venous blood sample was obtained at 1 month of age during a routine outpatient follow-up appointment.Plasma was separated from peripheral blood samples by centrifugation. Plasma samples were aliquoted and immediately frozen and stored at − 80 °C for later determination of cytokine concentrations.Remaining cells were resuspended in RPMI -1640 medium . Cells were incubated with PMA (phorbol 12-myristate 13-acetate) (50 ng/ml), ionomycin (1 μg/ml), and BFA (Brefeldin A) (10 μg/ml) for 6 h at 37 °C to allow intracellular accumulation of cytokines. For surface marker staining, samples were then incubated with the following fluorochrome-conjugated anti-human monoclonal antibodies: CD4 PE-Cy7 (phycoerythrin-cyanine 7) and CD8 APC-Cy7  (panel 1) or CD4 APC-Cy7 and CD49d PerCP (peridinin-chlorophyll-protein) (panel 2), according to the manufacturers’ instructions . Red blood cells were lysed and PBMCs were permeabilized using FACSLysing and FACSPermeabilizing solutions . Cells were washed and resuspended in PBS  and divided into two equal aliquots and stained according to the manufacturers’ instructions for intracellular cytokines using the following conjugated anti-human monoclonal antibodies or the appropriate isotype controls: IL-6 PE (phycoerythrin), IL-17A PerCP, IL-10 APC , and IFN-γ FITC (fluorescein isothiocyanate) (for panel 1), or TNF-α PE-Cy7, FoxP3 PE, TGF-β APC, and IL-1β FITC (for panel 2), respectively . Following labeling, cells were washed and resuspended in PBS for flow cytometry analysis. Samples were analyzed immediately on a FACSAria flow cytometer (BD Biosciences) equipped with 488- and 633-nm excitation lasers. Data were processed using the FACSDiVa software (BD Biosciences). One hundred thousand cells were recorded. Evaluators of flow cytometry data were blinded to the clinical status of the neonates.Plasma samples were stored at − 80 °C until analysis. The plasma levels of the following cytokines, chemokines, and growth factors were determined using Bio-Plex Pro Assays : IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IFN-γ, TNF-α, TGF-β, G-CSF, GM-CSF, MCP-1, MIP-1b, and VCAM. Bio-Plex Pro Assays are immunoassays formatted on magnetic beads that utilize principles similar to those of a sandwich ELISA. Capture antibodies against the biomarker of interest are covalently coupled to the beads. A biotinylated detection antibody creates the sandwich complex, and the final detection complex is formed by the addition of a streptavidin-phycoerythrin (SA-PE) conjugate, where PE serves as the fluorescent reporter. Reactions are read using a Luminex-based reader.p values less than 0.05 were considered significant. Outliers were identified using Grubbs’ tests and were excluded from analyses. Statistics were calculated using the GraphPad Prism 5 software .Data are expressed as median and interquartile range. Comparisons between sample populations were performed with Mann-Whitney tests, as a test of normality (performed according to Kolmogorov-Smirnoff) indicated non-normal distribution of data. Comparisons between the paired values (samples collected at different time points) in the same population were made with Friedman tests. The first patient was admitted following an emergency cesarean section performed due to oligohydramnios and fetal tachycardia on the 40th week of gestation. There was no maternal history of chorioamnionitis. However, the mother smoked during pregnancy. The amniotic fluid was stained with thick meconium. The neonate was born with an Apgar score of 5/6/7. She required intubation and ventilation at birth. Her first capillary blood gas showed lactic acidosis , and she became irritable and had generalized increased muscle tone; thus, hypothermic treatment was initiated. On the second day of life, she presented with symptoms characteristic of pulmonary hypertension, which resolved after 1 day of NO inhalation and she was extubated on day 3 of life. Her early neurodevelopmental examination showed mild central hypotonia.Cranial MRI with diffusion-weighted imaging was performed on day 4 of life, which showed a distinct, 5 × 7 mm area of ischemia with decreased diffusion in the left thalamus and no evidence of bleeding. MR spectroscopy showed a decrease in metabolites, correlating to minimal hypoxic-ischemic encephalopathy.. An early neurodevelopmental examination indicated grossly abnormal central and peripheral tone distribution.The second neonate was admitted following an emergency cesarean section due to complete placental abruption on the 39th week of gestation. He was born with an Apgar score of 3/5/6/8. At the time of birth, he had a heartrate of 80/min, had no respiratory effort, and was hypotonic and pale. Following bag and mask ventilation, his heartrate normalized; however, only gasping could be observed, so he was intubated. He later became irritable. His initial blood gas showed severe metabolic acidosis . A chest X-ray was performed, which showed a pneumothorax on the right side, which was drained and hypothermic treatment was initiated. Due to severe anemia and low blood pressure, he required blood transfusion on two occasions and inotropic support for 6 days, respectively. There were no signs of bleeding. TOF MRI angiography showed marked irregularity and significantly decreased blood flow in both, but particularly in the left MCA. No MRI signs of hypoxic-ischemic encephalopathy were present.Cranial MRI was performed on day 4 of life. Diffusion-weighted imaging showed a large area of ischemia with decreased diffusion in the area of the left middle cerebral artery, and several smaller lesions in the area of the right middle cerebral artery. The area of the left parietal cortex was blurred with hyperintensive signal subcorticallyThe third neonate was born from an uncomplicated pregnancy on the 40th week of gestation, with an Apgar score of 5/5. The neonate was hypotonic and had no spontaneous breathing; therefore, she was intubated. The first gasping breaths were observed at 15 min after birth, but she remained generally hypotonic. The first blood gas showed severe lactic acidosis . Therapeutic hypothermia was commenced. The initial aEEG recordings showed abnormal background activity; however, this normalized after a few hours. The early neurodevelopmental examination reported moderate generalized hypotonia.Cranial MRI was performed on day 3 of life. Diffusion-weighted imaging showed a small (3 mm) ischemic lesion with decreased diffusion in the right thalamus. No other signs of ischemia and bleeding or signs of hypoxic ischemic encephalopathy could be observed.The fourth neonate was born from an uncomplicated pregnancy on the 38th week of gestation by emergency cesarean section due to imminent intrauterine asphyxia. He was born with an Apgar score of 3/6/7. He was flaccid with no signs of spontaneous breathing and a heart rate of 80/min. Following bag and mask ventilation, spontaneous breathing was noted after 5 min, but the neonate remained hypotensive and areflexive. He was intubated and hypothermic treatment was initiated. The aEEG recording showed no signs of seizures. The initial blood gas showed mixed acidosis . The initial neurodevelopmental examination demonstrated abnormal central and peripheral tone distribution.Cranial MRI was performed on day 1 of life. Diffusion-weighted imaging showed decreased diffusion indicating ischemia in the area of the right posterior cerebral artery. Smaller lesions were present in the right thalamus and the left parieto-occipital area. No signs of bleeding or hypoxic ischemic encephalopathy were present.At 6 and 72 h of age, the prevalence of CD8+ IL10+ lymphocytes remained lower in NAIS than in the severe HIE group. Although the prevalence of CD8+ IL10+ lymphocytes remained consistently lower in NAIS, the difference did not reach a significant level at the 24-h time point. On the contrary, the prevalence of CD4+ IL-10+ was higher at 24 h in NAIS compared to moderate HIE. The prevalence of IFN-γ + CD4+ cells showed an elevated tendency from 24 h in NAIS; however, this value did not reach significance, which could be due to the small population. At 1 week, the prevalence of TGF-β + lymphocytes prone to enter the CNS (CD4+ TGF-β + CD49d + lymphocytes) was elevated in NAIS compared to both HIE groups, and also compared to all other time points within the NAIS group  of IFN-γ in CD4+ lymphocytes in NAIS was highest at 24 h and decreased significantly by 72 h, when it reached a lower level in NAIS than in either of the HIE groups. The MFI of IL-6 in CD8+ cells was higher at 72 h in severe HIE than in NAIS. Interestingly, by 1 month, the MFI of IL-6 in CD8+ decreased in both HIE groups and rose in NAIS. Data are shown in Table We found no differences between NAIS and HIE in the intracellular production of IL-1β and TNF-α in CD4 or CD8 cells; however, these cytokines appear to be important in differentiating between the mild and severe form of HIE .At 72 h, we found a marked inflammatory response in NAIS, characterized by elevated plasma levels of IL-5, IL-17, and MCP-1 compared to HIE. Plasma MCP-1 level was the highest at 72 h in NAIS. By 1 month, however, inflammatory response appears to be decreased in NAIS compared with HIE, indicated by decreased plasma levels of IL-4, IL-12, and IL-17  cells, was also different in NAIS and HIE. TGF-β is one of the most important cytokines regulating CNS repair. We found increased CD49d expression in this population in NAIS at 1 week compared to 24 h and both HIE groups, reflecting a higher capacity of these cells to enter the CNS. Meanwhile, the prevalence of the entire population decreased by 1 month in NAIS compared to HIE. This appears to be in line with plasma cytokine data, which showed an increased inflammatory response in NAIS in the second phase of neuroinflammation which could be followed by an earlier reparative phase and a decreasing inflammatory response by 1 month of ageTherapeutic hypothermia is currently the standard of care for term neonates with HIE. There is substantial evidence supporting the role of hypothermia in reducing mortality and improving neurological outcome  in infanThe main limitation of these data is the small number of cases; however, they could provide a hypothesis for future, larger case-control studies. Another limitation is the lack of healthy control population; however, due to ethical considerations and the large number of blood sampling, enrolling healthy neonates was not possible. We also saw a notable variation in certain plasma cytokine levels, which is likely a result of the relative instability of plasma cytokine levels, which was the basis of primarily focusing on intracellular cytokine production in the current study. The value of the data from this study could lie in providing incentive for further clinical research on factors enabling differentiation between the two disorders which often present with similar symptoms at birth. Gaining a better understanding of the underlying pathophysiological factors is a main aim at the current stage of research in NAIS.CD8 lymphocytes appear to show a shift in the pro-inflammatory direction in NAIS compared with HIE, with elevated production of pro-inflammatory IL-17 at 6 h, decreased auto-regulatory IL-10 production in the first 3 days, and elevated production of IL-6 at 1 month. Altogether, the inflammatory response appears to be higher at 72 h in NAIS than in HIE, but appears to decrease faster, indicated by lower levels of inflammatory markers at 1 month in NAIS than in HIE. Our data indicate similarities to observations in adult AIS and also support the hypothesis of an ongoing in utero inflammation prior to NAIS. Further clinical research in this area is pivotal, and a better understanding of the pathophysiology could lead to identifying specific factors to distinguish HIE from NAIS early and could open up new pathways for prevention and individualized care.


Carcinoembryonic antigen (CEA) as a diagnostic or prognostic marker has been widely studied in patients with lung cancer. However, the relationship between serum CEA and tumor metastasis in lung cancer remains controversial. This study aimed to investigate the ability of serum CEA to assess tumor metastasis in lung cancer patients.We performed a retrospective analysis of 238 patients diagnosed with lung cancer from January to December 2016 at pneumology department of Dazhou Central Hospital . Serum CEA levels were quantified in each patient at the time of diagnosis of lung cancer. Metastasis was confirmed by computed tomography (CT), and/or positron emission tomography (PET) and/or surgery or other necessary detecting methods.p < 0.001), and the area under the curve (AUC) was 0.724 (95% CI [0.654–0.793]). Subsequent analyses regarding the number and location of tumor metastases showed that CEA also had clinical value for multiple metastases versus single metastasis  and distant metastasis versus non-distant metastasis . In addition, we found that tumor size, histology diagnosis, age and gender had no impact on the assessment performance of CEA.Of the 213 patients eligible for final analysis, 128 were diagnosed with metastasis and 85 were diagnosed without metastasis. Compared to non-metastatic patients, the serum CEA was markedly higher in patients with metastasis (Our study suggested the serum CEA as a valuable marker for tumor metastases assessment in newly diagnosed lung cancer patients, which could have some implications in clinical application. Lung cancer is the most common cause of death among all cancers worldwide . The oveClinically, metastasis is confirmed by using a combination of clinical symptoms and imaging evidence when complete pathological evidence is available for lung cancer diagnosis . The mosBlood-based biomarkers can be accessed easily, quickly, and economically, so they have the potential to greatly improve the efficiency of assessment. Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion, which was first found in 1965 in the blood of patients with colon cancer . PreviouWe retrospectively analyzed the relationship between CEA and tumor metastasis in newly diagnosed patients with NSCLC and SCLC, to investigate the relationship between CEA and tumor metastasis, and assess whether CEA has certain clinical guiding value for organs involved and the number of organs involved in tumor metastasis.We retrospectively reviewed 238 patients with a diagnosis of lung tumors from January to December 2016 at the pneumology department of Dazhou Central Hospital in this study. Clinical information, including gender, age, metastatic locations, histological type and tumor size was retrospectively obtained from electronic medical records. All these patients underwent fiberoptic bronchoscopy biopsy at the initial stage of cancer diagnosis after hospitalization. The specimens needed for pathological diagnosis came from fiberoptic bronchoscopy, percutaneous lung biopsy, or surgical resection. The histological type was confirmed by pathological morphology and immunohistochemistry. The study was approved by the medical ethics review board of Dazhou Central Hospital . The medical ethics review board waived the need for informed consent from participants in this study.n = 7); (ii) patients diagnosed with lung cancer, but without a pathological histology basis (n = 18). Finally, 213 patients were included in the analyses.The exclusion criteria were as follows: (i) patients who lacked serum CEA data  by cobas e601 analyzer . The expMetastasis group: The presence of metastasis was confirmed within the first period of admission diagnosis (no more than one month). Patients diagnosed with lung cancer and with obvious images of metastasis in imaging were included in the metastasis group.Non-metastasis group: Patients with lung cancer but without detected any lymph node metastasis, intrapulmonary metastasis, and any other metastasis within the first hospitalization period and metastases detected beyond the first hospitalization period were included in the non-metastasis group.Single metastasis group: Single metastasis is for a single organ, not for single site. Patients with a single metastasis site and multiple metastases sites in one single organ were all included in the single metastasis group.Multiple metastases group: Patients with multiple organ metastases were included in the multiple metastases group.Non-distant metastasis group: Patients with simple mediastinal/hilar lymph nodes and/or intrapulmonary metastasis were included in the non-distant metastasis group.Distant metastasis group: Patients excluded from the non-distant metastasis group were included in the distant metastasis group.In our study, the diagnosis of metastasis was based on the complete pathological evidence in the diagnosis of lung cancer, combined with the patient’s clinical characteristics and imaging evidence . All metastases, not detected during the first hospitalization period (no more than one month), were not considered to have metastases. CT, PET, surgery, MRI and Fluorodeoxyglucose (FDG)–PET-CT scan are common imaging modalities for metastasis assessment .U test was used to compare CEA levels between patient groups. The relation between CEA level and metastasis was visualized using restricted cubic spline. Receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to represent the overall test accuracy. The optimal cut-off value was determined using Youden’s Index. Sensitivity and specificity of the index test were reported along with the Clopper–Pearson confidence intervals. Covariate adjusted ROC curve was generated using a percentile values approach , while categorical variables were expressed with count and percentage. The Kruskal-Wallis test was used to compare the age difference between patients with and without metastases. The Pearson Chi-squared test was used to compare gender, histology and size of tumor difference between patients with and without metastases. The Mann–Whitney approach . MedCalcapproach  were useThere were 213 patients eligible for the final analyses based on the exclusion criteria, 170 male and 43 female, with a median age of 62 years (range from 35 to 82 years). A cohort diagram was shown in p < 0.001, Among the 213 lung cancer patients with a definite diagnosis of cancer, 128 patients were included in the metastasis group and 85 patients were included in the non-metastasis group . Serum Cp < 0.001, Among the 128 patients in the metastasis group, 60 patients were included in the single metastasis group and 68 patients were included in the multiple metastases group. Serum CEA was significantly increased in the multiple metastases group when compared to patients in the single metastasis group  supported by CEA test kit. Moreover, in patients with tumor metastasis, serum CEA was significantly increased in patients with multiple metastases compared with patients with single metastasis. A cut-off of 7.17 ng/mL could optimally distinguish between single metastasis and multiple metastases. Additionally, our data showed that CEA was also an effective marker for other metastases assessment except for mediastinal/hilar lymph nodes and intrapulmonary metastases with an optimal cut-off of 6.03 ng/mL for this purpose. Tumor size, subtypes, patients’ age and patients’ gender didn’t have any impact on the diagnostic performance of serum CEA levels in assessing metastasis. These results suggested that serum CEA was a valuable marker in assessing tumor metastasis in lung cancer patients.Tumor metastasis, commonly occurred at stage IV, is responsible for majority of lung carcinoma death. Within the different types of lung carcinomas, there is also a preferential metastatic location, such as liver metastasis in SCLC and adenocarcinoma . Most poSeveral limitations in our study should be acknowledged. Firstly, it was a retrospective-design single center study, which may make the results less generalization. Secondly, the time period of patients retrospectively reviewed was only one year, and the sample size was relatively small.Our study revealed the significant qualitative difference of serum CEA in lung cancer patients with or without tumor metastasis and evaluated the diagnostic performance of CEA testing to determine tumor metastasis. In metastasized cases, patients with multiple metastases had significantly higher CEA levels compared to those with single metastasis; distant metastasis patients showed significantly higher CEA compared with non-distant metastasis cases. Furthermore, tumor size, subtypes, patients’ age and gender have no impact on the performance of CEA testing. Taken together, serum CEA has a positive clinical application value in combined with existed techniques for diagnosis of cancer metastasis to timely and comprehensively detect metastasis.10.7717/peerj.7433/supp-1Dataset S1Click here for additional data file.10.7717/peerj.7433/supp-2Figure S1Click here for additional data file.10.7717/peerj.7433/supp-3Figure S2(A) 1: Brain metastasis. (B) 2: Rib metastasis, 3: Massive pleural effusion. (C) 4: Pyramidal bone metastasis. (D) 5: Bone metastasis.Click here for additional data file.10.7717/peerj.7433/supp-4Figure S3Click here for additional data file.10.7717/peerj.7433/supp-5Figure S4(A) Crude ROC curve versus ROC curve adjusted for histological diagnosis. (B) tumor size. (C) age and (D) gender (outcome = metastasis).Click here for additional data file.10.7717/peerj.7433/supp-6Figure S5Click here for additional data file.


Current cellular facts allow us to follow the link from chemical to biochemical metabolites, from the ancient to the modern world. In this context, the “RNA world” hypothesis proposes that early in the evolution of life, the ribozyme was responsible for the storage and transfer of genetic information and for the catalysis of biochemical reactions. Accordingly, the hammerhead ribozyme (HHR) and the hairpin ribozyme belong to a family of endonucleolytic RNAs performing self-cleavage that might occur during replication. Furthermore, regarding the widespread occurrence of HHRs in several genomes of modern organisms , these small ribozymes have been regarded as living fossils of a primitive RNA world. They fold into 3D structures that generally require long-range intramolecular interactions to adopt the catalytically active conformation under specific physicochemical conditions. By studying viroids as plausible remains of ancient RNA, we recently demonstrated that they replicate in non-specific hosts, emphasizing their adaptability to different environments, which enhanced their survival probability over the ages. All these results exemplify ubiquitous features of life. Those are the structural and functional versatility of small RNAs, ribozymes, and viroids, as well as their diversity and adaptability to various extreme conditions. All these traits must have originated in early life to generate novel RNA populations. We know today that RNA is present in all living organisms, in which it performs a variety of structural and metabolic functions. A truly modern “RNA world” exists in each cell, with RNAs in various forms, short and long fragments, single and double-stranded, endowed with multiple roles, informational, catalytic, structural, and regulatory . Certain RNA molecules are even capable of carrying out several of these functions.The “RNA World” hypothesis as an early step in the history of life was so-named by Walter Gilbert  after beIn the past five years, several studies reported the self-assembly of natural ribozymes leading to dimers that modulate the overall catalytic efficiency ,32,33. TThe RNA World hypothesis raises important questions. Could RNA have existed alone? Is there a fossil trail of ancient RNAs? Diener’s hypothesis, proposed in 1989  and re-dViroids are the smallest plant pathogens. From 300 to 500 ribonucleotides long, the single-stranded RNA molecules can pair and form loops and bulges. They were discovered in 1971 by Theodor Diener  followingeometry ,48,49,50geometry .There is an apparent antagonism between two essential properties which are expected for RNAs as primordial molecules from the RNA World: e.g., acting as a template for replication and carrying a catalytic activity that would require a stable fold. One way to escape from this paradox between a stable fold but a poor template  or a poorly folded RNA and a good template (templating ability)  was propIn viroids, some bonded dimeric structures are generated as a result of the fusion of two “single-hammerhead” structures through the merging of both stems III. This typically occurs in the oligomeric forms, corresponding to the (+) or (−) strands, which undergo self-cleavage to generate linear monomers. Such viroids with double-hammerhead structures were reported for a long time in ASBVd , but morBoth strands of viroid in the family Avsunviroidae and similar viroid-like RNAs are by definition self-complementary, but they still maintain a sequence which is compatible with a HHR fold on both strands (+) and (−) . HoweverFrom the current literature, we can classify the viroids and viroid-like RNAs based on three profiles: (1) the “bad catalysts” (inactive) with an unstable HHR fold for both strands (+) and (−) which rely on the formation of double-hammerhead structures for the calytic activity; (2) the “good catalysts” (highly efficient) with a stable HHR fold for both strands (+) and (−); (3) the “intermediate catalysts” with a differential catalytic efficiency between the two strands: an active strand (−) as a single-hammerhead structure and an inactive strand (+) that becomes active only as a double-hammerhead structure. The first profile includes: the satellite RNA of cereal yellow dwarf virus-RPV (satRPV RNA) ,57, the In the RNA World, the dimerization through the formation of double-hammerhead structures could be seen as a strategy to resolve the “biocatalyst/template” conflict, with viroid-like RNAs as good templates for replication and sub-optimal catalysts. By harboring unstable HHR folds, they remain good templates; by merging together inactive single-hammerhead structures into active double-hammerhead structures, they maintain a required catalytic activity. Moreover, the ability to form such bonded dimers probably offered a selective advantage against degradation in a clay mineral environment close to the RNA World conditions .In ribozymes, another mode of dimerization is observed through a reversible self-assembly mechanism. In contrast with the double-hammerhead structures, the two monomers are not covalently bonded but associated in a dimer through weak non-bonded interactions. In this kind of dimer, a loss of catalytic activity was described in some artificial variants of natural ribozymes because of misfolded RNAs or active sites in the dimer  is a well adapted method to study fragile nucleic acid structures and interactions in solution ,64. The Bi-molecular RNA interactions such as linear-linear, loop-loop, loop-linear, or kissing interactions are important in the control of biological activity, and hairpin loops present rich potential for establishing both intra- and inter-molecular interactions through standard Watson–Crick base pairing or non-canonical interactions. In a previous study, we applied SANS with native gel electrophoresis and analytical centrifugation to the structures and self-interactions of two 85 base adenine-dependent hairpin ribozymes (ADHR1 and ADHR2) . SimilarArabidopsis thaliana. In this particular case, the hypothesis of a viroid origin was discarded based on phylogenetic analyses [Considering the absence of an envelope, the fact that viroids are non-coding and the existence of ribozymes, it is tempting to consider them as vestiges of ancient RNAs as Diener proposed in 1989 . As noteanalyses . Among tanalyses . In otheanalyses  , one of the smallest rod-like viroids (247 nts), including a hammerhead ribozyme (HHR) of 42 nucleotides. In vivo, both complementary strands (+) and (−) (harboring a HHR motif: profiles  shows twprofiles .The SANS approach developed in the ADHR study was applied to ASBVd (−) and its derived 79-nt HHR (−). It revealed a reversible temperature-dependent dimer association in both molecules. In The 2D and 3D modeling of monomeric and dimeric HHR (−) suggested that the intermolecular contacts stabilizing the dimer (between HI and HII domains) compete with the intramolecular ones, stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage . Similaratalysis B. This lS. cerevisiae. The process was followed for 25 generations that is the persistence of the viroid in S. cerevisiae, showing unsuspected functional diversity and adaptability and confirming that the ribozyme can fulfill the cleavage/ligation reaction during the rolling circle replication process in a non-plant host [It remained to be determined whether or not ASBVd can replicate in other types of plastids  and in other species than plants. Following this line of thought we showed an efficient cleavage of ASBVd by the HHR and followed the transcription/replication, as well as circularisation of the viroid in the yeast ant host .The RNAs were extracted at different times from 0 to 50 h and then analyzed by Northern blotting A,B. AfteAnabaena (Anabaena sp. PCC7120) is a filamentous cyanobacterium that is a photosynthetic prokaryote . We detected (−) and (+) ASBVd in Anabaena, that is linear transcript (+) was detected by riboprobe (−) when RNAs/ pRLASBVd(−) was loaded as well as linear transcript (−) detected by riboprobe (+) when RNA/pRLASBVd(+) was loaded [Because ASBVd replicates in plant chloroplasts which have their ancestry in cyanobacteria, via endosymbiosis , we askes loaded  , is a particular feature of ASBVd to do more under tight evolutionary constraints for such small genomes that should preserve functional HHR ,75. The Anabaena. We have further showed that ASBVd replicates and persists in S. cerevisiae, a non-photosynthetic eukaryote. It is legitimate to speculate on extant viroids as descendants of “free-living” proviroids that invaded ancient cyanobacteria, which would later become endosymbionts, evolving in chloroplasts by usurping the biochemistry of their hosts. Finally, in a “viroids as living fossils” scenario [ASBVd has adapted to different hosts: it normally replicates in the chloroplast of avocado, but it can also replicate in the cyanobacterium scenario ,38, we mscenario  and RNA scenario . Viroid-scenario .Assuming that viroid-like RNAs and viroids have lost two of their initial catalytic activities, we may propose an evolutionary path from the RNA World to the DNA World . The virPelamoviroid and adopt complex multi-branched secondary structures which are very different from the simple rod-like structure of ASBVd [In this hypothetical evolutionary path, ASBVd would correspond to some half-way state where the catalytic efficiency has been partially optimized. The reason it did not evolve towards a fully optimal catalyst found in other viroids may seem puzzling. However, it might be explained eventually by different sources of evolutionary constraints creating antagonistic epistases  in orderof ASBVd . Thus, tThe existence of another mode of dimerization in viroids through the self-assembly of monomeric RNAs is also a particularity of ASBVd. As mentioned previously, ASBVd exists in two non-symmetrical polarities (+) and (−) with different structures and catalytic activities. A joined thermodynamic analysis of structural and catalytic data indicates that the rate-determining step corresponds to a dimer/monomer transition. Models suggest that the intermolecular contacts stabilizing the dimer (between HI and HII domains) in HHR (−) compete with the intramolecular ones, stabilizing the active conformation of the full-length HHR required for efficient self-cleavage . SimilarThe Avocado Sun Blotch Viroid (ASBVd) is unique since it combines “modern” and “ancient” features that may be attributed to either the RNA or DNA World. Proviroids would have been good templates for replication but poor catalysts . As a sub-optimal catalyst, ASBVd is reminiscent of those proviroids whose dimerization through double-hammerhead structures would have protected them from degradation in a clay mineral environment. ASBVd still has this ability to form such dimers, a trait which is also found in many mobile elements or viroid-like RNAs harboring a HHR motif but absent from the other known viroids of the Avsunviroidae family. On the other hand, the formation of non-bonded dimers, which is unique to ASBVd, tends to decrease the catalytic activity; it could be a recent adaptation and the sign of co-evolution in the DNA World.


This approach could alleviate some of the major caveats of current non-target environmental “omics.” Here, we demonstrate the feasibility of total RNA-SIP in an experiment where hydrocarbon-degrading microbes from a BTEX-contaminated aquifer were studied in microcosms with 13C-labeled toluene under microoxic conditions. From the total sequencing reads (∼30 mio. reads per density-resolved RNA fraction), an average of 1.2% of reads per sample were identified as non-rRNA, including mRNA. Members of the Rhodocyclaceae (including those related to Quatrionicoccus spp.) were most abundant and enriched in 13C-rRNA, while well-known aerobic degraders such as Pseudomonas spp. remained unlabeled. Transcripts related to cell motility, secondary metabolite formation and xenobiotics degradation were highly labeled with 13C. mRNA of phenol hydroxylase genes were highly labeled and abundant, while other transcripts of toluene-activation were not detected. Clear labeling of catechol 2,3-dioxygenase transcripts supported previous findings that some of these extradiol dioxygenases were adapted to low oxygen concentrations. We introduce a novel combination of total RNA-SIP with calculation of transcript-specific enrichment factors (EFs) in 13C-RNA, enabling a targeted approach to process-relevant gene expression in complex microbiomes.While most studies using RNA-stable isotope probing (SIP) to date have focused on ribosomal RNA, the detection of  Unraveling the activity of microorganisms in complex environments is a difficult but essential step toward understanding their metabolism and community interactions in processes like pollutant degradation. Stable isotope probing (SIP) of nucleic acids is widely applied to discover which organisms metabolize a particular substrate, and can provide insights into catabolic pathways used . As micr13CH4 in oxic lake sediment cultures. Their workflow involved both rRNA depletion prior to isopycnic centrifugation and linear amplification of the mRNA recovered from gradient fractions to produce sufficient material for 454 sequencing. With the increased sequencing depth of Illumina platforms, it is now possible to directly investigate total RNA from heavy and light gradient fractions instead, recovering both taxonomic and functional information. This was first accomplished by The majority of RNA-SIP studies to this point have targeted small subunit ribosomal RNA (SSU rRNA) to taxonomically identify microbes involved in substrate metabolism. However, isopycnic gradients have the capacity to resolve both labeled rRNA and mRNA . Still, 13C-toluene in a microcosm study to investigate the microbes involved in biodegradation under microoxic conditions  headspace. This amount of sediment was chosen to prevent redox stratification during incubation. Five microliter of non-labeled (12C) or fully labeled (13C7) toluene were added by injection with a glass syringe after headspace exchange. Abiotic control microcosms were set up in the same manner, but autoclaved 3x before addition of non-labeled toluene. These microcosms controlled for possible abiotic toluene loss during incubation . Isopycnic density gradient centrifugation was carried out according to RNA was extracted from duplicate labeled and unlabeled microcosm sediments after 7 days of incubation using the RNA PowerSoilin vitro transcription kit  from plasmids containing a cloned 16S rRNA gene from Methylobacterium sp., and quantified with the Quant-iT RiboGreen RNA Assay Kit  on an Mx3000p . Each 40 μl RT-qPCR reaction contained: 20 μl 2x AccessQuick Master Mix, 0.4 μl 20 mg/ml bovine serum albumin, 0.2 μl 1/500 diluted SYBRgreen , 0.6 μl 1/500 diluted ROX, 10 μM each primer, and 2 μl of standard or unknown RNA. The temperature program was: 45°C reverse transcription for 30 min, initial denaturation of 95°C for 3 min, then 35 amplification cycles , with a final extension step at 68°C for 5 min. After the run, a dissociation curve from 55 to 95°C was recorded to discriminate products from unspecific amplification products.RNA recovered from fractions was quantified with RT-qPCR using the AccessQuick RT-PCR kit  and Ba519f and Ba907r universal 16S primers on an Mx3000p machine  as described by 12C-light, density 1.778–1.779 g ml-1), unlabeled heavy , labeled light  and labeled heavy . RNA amounts present in the heavy fractions of the 12C gradients were too low for transcriptome sequencing. Instead, the “heaviest” fractions from which sufficient RNA could be obtained from the 12C gradients were chosen, in order to control for possible shifts caused by physical characteristics of RNA such as GC content  using a DNA 1000 LabChip, and quantified with the Qubit®dsDNA HS Assay Kit on a Qubit machine . A 1% PhiX v3 control library spike-in  was added to the library before cluster generation. Cluster generation and sequencing were performed on the NextSeq500 sequencing system  using a high-output paired-end 150 cycle (2x 150 PE) by IMGM Laboratories GmbH . All sequencing raw data generated in this study as well as sequences representing important 16S OTUs and functional transcripts have been submitted to the NCBI sequence read archive (SRA) via the Gene Expression Omnibus under the accession number GSE119644.Linear amplification was performed on a subset of each of the four RNA pools using the MessageAmp II Bacteria Kit  according to manufacturer instructions. RNA was quantified and purity-checked using a NanoDrop ND-1000 spectral photometer  and a 2100 Bioanalyzer  with the RNA 6000 Nano LabChip. Library preparation with input templates ≥20 ng of total RNA was performed with the TruSeqAn in-house bioinformatics pipeline was developed for transcriptome-SIP data handling. Removal of sequencing adapters and merging of paired-end sequences was performed with Adapterremoval  with defp = 0.01), truncate_reverse_primer.py, identify_chimeric_seqs.py (method: usearch61), and filter_fasta.py scripts. Minimum Phred score was 2 .16S OTU assignment was performed using QIIME v1.9.1 . Preprocre was 2 . OTU calre was 2  using thre was 2 . For thire was 2  using thre was 2 . SignifiEnrichment factors (EFs) were calculated as previously done in rRNA-SIP  using th13C” and “light 13C” were the relative abundance of a specific rRNA taxon or mRNA transcript – assigned to the same KEGG/COG category – in heavy and light fractions of the 13C-toluene gradients, respectively, and “heavy 12C” and “light 12C” the same for respective 12C-control gradients. Positive values indicate isotopic labeling of a transcript; negative enrichment indicates more frequent detection in the light fraction of 13C-RNA.where “heavy P. aeruginosa and RT-qPCR of housekeeping genes. Further details can be found in the A direct test of the effect of linear amplification on the proportion of mRNA in the total RNA was conducted in a SIP-independent manner using total RNA from 13C-labeled vs. unlabeled toluene  for sequencing of control fractions . GC-content of SSU rRNA reads appeared unchanged across gradient densities, and was also not significantly affected by linear amplification . Moreover, linear amplification significantly reduced the GC-content of non-rRNA reads by an average of 2.1 ± 0.9% compared to unamplified libraries (p = 0.025).A total of 279 million paired-end reads were obtained by RNA-seq across the eight libraries  with an average of ∼30 million per library. After QC, a total of about 1 million reads could be identified as mRNA using the KEGG or COG databases (Table Proteobacteria (Supplementary Table Bacteroidetes (avg. 1.8%) and other phyla (avg. 0.6%). Most of the transcripts within the Proteobacteria were affiliated to the Betaproteobacteria; of these the majority belonged to members of the Rhodocyclaceae  belonged to the Rhodocyclaceae. All genera within the Rhodocyclaceae were strongly enriched in 13C-labeled RNA, with the exception of reads affiliated to Azoarcus spp.  belonged to the Proteobacteria. Some could be specifically assigned at the species level, and 10–20% of all transcripts linked to fliC were assigned to Azovibrio restrictus.Cell motility was the transcript category most highly enriched in A Figure . Many tr13C-enriched transcripts in this group were identified as genes coding for a catalase-peroxidase (K03782), which averaged at over 1000 copies per library. Labeling observed for the most abundant COG categories was generally consistent with KEGG results , which can be involved in chlorocyclohexane and chlorobenzene degradation, benzoate degradation, toluene degradation, and in the degradation of aromatic compounds in general. Transcripts of the benzoate degradation pathway were the most abundant, though not the most highly labeled , followed closely by Pseudomonadaceae (avg. 6.5%). However, most of these transcripts could not be assigned more specifically than at the phylum level. Transcripts of the catechol-1,2-dioxygenase gene (K03381), on the other hand, were barely present, appearing only in very low read numbers (∼1) per library.Transcripts of catechol-2,3-dioxygenase (C23O), an enzyme previously associated with central pollutant metabolism at the investigated site , 2013, wG Figure . TaxonomG Figure . Of the dmpL; K16243) were highly abundant and labeled, with transcript numbers of up to 160 per library. In contrast, transcripts of genes coding for toluene monooxygenase subunits  were present in low read numbers (∼15), with varying amounts of labeling. Transcripts linked to genes coding for benzylsuccinate synthase  were present in all treatments but not very abundant (∼20 reads) and not labeled.Amongst initial hydrocarbon degradation mechanisms, transcripts of the phenol hydroxylase gene  were transcribed, but the only transcript enriched in 13C-RNA found in these pathways codes for NapA (K02567). We were also interested in whether the putative NO-dismutase genes previously detected at the site (Candidatus Methylomirabilis oxyfera” (between 0 and 62 reads per library) were compared to the NCBI-nr database via nucleotide BLAST. All transcripts annotated as canonical nitric oxide reductases belonging to “Ca. Methylomirabilis oxyfera” actually matched more closely to the putative nitric oxide dismutase (nod). Up to 21% of all transcripts annotated as nitric oxide reductase subunit B  were putative nod transcripts. These nod transcripts appeared in read numbers of ten or fewer, so EF calculation was not robust.Transcripts of genes required for prokaryotic oxidative phosphorylation were present in the microoxic microcosms, but the majority were unlabeled. The full operon of the site  would be12C) microcosm were much more similar, while those of the labeled (13C) microcosm were clearly separated, mainly on NMDS axis 1  decreased, suggesting that the amplification process could leave behind some specific transcripts or over-amplify others. Rare transcripts with copy numbers less than ten appeared with equal frequency in amplified and unamplified libraries  in providing targeted insights into microbial activities in processes such as pollutant degradation, on both taxonomic and functional levels. However, the level of detail clearly depended on the number of mRNA reads obtained from gradient fractions. The amount of non-rRNA reads was between 0.6 and 2.2% in our libraries, but this small proportion was not unexpected for total RNA-seq and is consistent with the percent non-RNA or putative mRNA found in other total-RNA-seq studies  of total RNA recovered after fractionation. To increase yields for low-RNA fractions, several fractions of similar density across duplicate centrifugation gradients were pooled  with metatranscriptomic sequencing provided information on a whole range of functional transcripts produced by active degraders during incubation. Here, we discuss transcript labeling patterns which appeared most relevant to us in understanding degrader activity under microoxic conditions. Transcripts linked to genes coding for proteins involved in cell motility were the most highly labeled group in both KEGG and COG analyses Figure . The imp13C-enriched. Between 10 and 20% of all transcripts of the fliC gene were assigned to Azovibrio restrictus  is an extradiol dioxygenase which has long been used as a conserved functional marker gene for aerobic aromatic degraders , 2013. KRhodocyclaceae were most prominent. This included matches to Azovibrio restrictus, previously found in hydrocarbon-contaminated soil , although Pseudomonadaceae were clearly not isotopically enriched  genes at this site  in databases. In the present study, transcripts from “Ca. Methylomirabilis oxyfera” that were annotated by KEGG as nors more closely matched putative nod sequences (Despite not having added nitrate to microcosms, transcripts of genes coding for all steps of canonical denitrification and DNRA were detected. All related transcripts were unlabeled except for nditions  and has nditions , indictahis site . NO-dismhis site . The enzocarbons . A recendetected . Since tequences  when comRhodocyclaceae and the respective C23O pathways in toluene degradation in microoxic microcosms (In summary, this study builds on the advantage of metatranscriptome sequencing to provide functional details about microbial activities in a given system, which may be hard to delineate with more traditional, targeted techniques like RT-PCR. At the same time, the combination with isotopic labeling adds a new process-targeted handle to the metatranscriptomics pipeline, with a potential to provide functional context even for constituents of the large fraction of unclassified transcripts, which were detected also in our study. Here, we confirm the central role of crocosms . In addiLB, AT, and TL designed the experiments. LB and GV analyzed the data, with specific contributions from BZ and MS. LB and TL wrote the initial manuscript draft with additions and revisions contributed from all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.