Acquisition of the M1 or M2 phenotypes by microglia has been shown to occur during the development of pathological conditions, with M1 activation being widely involved in neurotoxicity in relation with the anatomical localization and the reactivity of subtypes of microglia cells. On the contrary, little is known on the ability of microglia to undergo M2 polarization by interleukin-4 (IL4), the typical M2a polarization signal for peripheral macrophages.Fizz1, Arg1, and Ym1 genes, known to be up-regulated by IL4 in peripheral macrophages, together with additional polarization markers, were evaluated in the striatum and frontal cortex at different time intervals after central administration of IL4; in parallel, M2a protein expression was evaluated in tissue extracts and at the cellular level.Recombinant mouse IL4 was injected in the third cerebral ventricle of mice to induce brain alternative polarization. The mRNA levels of Fizz1 mRNA induction reached 100-fold level, underscoring the potency of this specific IL4 signaling pathway in the brain. In addition, immunochemistry assays demonstrated the localization of the M2 response specifically to microglia cells and, more interestingly, the existence of a subpopulation of microglia cells amenable to undergoing M2a polarization in the healthy mouse brain.Our results show that the potency and temporal profile of IL4-mediated M2a gene induction vary depending on the gene analyzed and according to the specific brain area analyzed, with the striatum showing a reduced M2a response compared with the frontal cortex, as further substantiated by assays of polarization protein levels. Of notice, These results show that the responsiveness of brain macrophages to centrally administered IL4 may vary depending on the gene and brain area analyzed, and that M2a polarization can be ascribed to a subpopulation of IL4-responsive microglia cells. The biochemical pathways that enable microglia to undergo M2a activation represent key aspects for understanding the physiopathology of neuroinflammation and for developing novel therapeutic and diagnostic agents.The online version of this article (doi:10.1186/s12974-014-0211-6) contains supplementary material, which is available to authorized users. Microglia are myeloid cells that populate the parenchyma of the central nervous system (CNS); their physiological activity includes most of the biological properties that are typical for peripheral macrophages, although their developmental origin and anatomical distribution allows these cells to perform distinctive immune and neuromodulatory functions in the CNS. Through their physical and biochemical interaction with neurons, microglia are able to sense and remodel neuronal activity, support neurogenesis, and maintain CNS homeostasis .in vivo [Microglia also possess the striking ability to rapidly react to endogenous or exogenous signals with a variety of physiological responses . Like pein vivo -6. The nin vivo ,8. Furthin vivo . This fuin vivo , neural-in vivo -14, and in vivo  and lipoin vivo ,17.in vitro or ex vivo studies, showing the responsiveness of microglia to IL4 and the subsequent activation of gene expression programs related to neuroprotection, tissue remodeling, and angiogenesis [Conversely, macrophages and microglia undergo an alternative ‘M2’ phenotype as a consequence of parasite invasion and in response to the endogenous immune signals, interleukin-4 (IL4) and interleukin-13 (IL13), in order to provide tissue repair and resolution of inflammation. A more detailed analysis led to the identification of two distinct alternative activation states, M2a and M2c, which reflect the actions of IL4 and IL13, on the one hand, and interleukin-10 (IL10) and TGFβ, on the other hand; importantly, these responses are associated with specific panels of regulated genes and a distinct range of effects . Howeverogenesis . As a coogenesis . Furtherogenesis .in vivo and evaluate the responsiveness and functional heterogeneity of microglia to undergo M2 polarization among and within selected brain areas. We thus stimulated microglia by central administration of IL4 in the third cerebral ventricle of the mouse brain. Interestingly, our data show that the induction of microglial M2a phenotype varies among the two brain areas analyzed, with M2a gene expression being more potent in the frontal cortex than in the striatum, and that only a subpopulation of microglia cells within these areas is amenable to undergoing an M2a response. Finally, we show that, among the M2a genes tested, Fizz1 reached the highest induction levels, underscoring the potency of this IL4 signaling pathway in the brain.The aim of this study was thus to investigate microglia M2 activation Recombinant mouse IL4 was obtained from PeproTech . Unless otherwise specified, chemicals were purchased from Merck .C57BL/6− male mice 4 months of age were supplied by Charles River Laboratories . All animals were allowed free access to food and water and were kept in temperature-controlled facilities on a 12-hour light and dark cycle. Animals were housed in the animal care facility of the Department of Pharmacological and Biomolecular Sciences at the University of Milan, and experiments were performed in compliance with regulations approved by the Institutional Animal Care and Use Committee of the University of Milan and in accordance with European legislation.Bone-marrow-derived macrophages were prepared as previously described . BrieflyIntracerebroventricular (icv) injections were made as previously described . BrieflyThe striatum and frontal cortex were homogenized using steel beads and tissue Lyser  at 28 Hz, for three cycles of 20 s followed by 30 s, on ice and in RLT buffer and total RNA was purified using RNeasy minikit (QIAGEN) according to the manufacturer’s instructions including a step with deoxyribonuclease incubation. One μg RNA was used for cDNA preparation using 8 U/μl of Moloney murine leukemia virus reverse transcriptase  in a final volume of 25 μl; the reaction was performed at 37°C for 1 h, and the enzyme inactivated at 75°C for 5 min. Control reactions without the addition of the reverse transcription enzyme were performed (data not shown).arginase-1 , chitinase-like 3 , Found-in-the inflammatory zone , IL4 receptor-α , CD206 , TGFβ , IL1β , TNFα , and for the reference genes 36B4  and complement component 1qA . The reactions were carried out according to the manufacturer's protocol using a 7900HT fast real time PCR system , and the data were analyzed using the 2-ΔΔCt method.A 1:16 cDNA dilution was amplified using SYBR technology. The PCR was carried out in triplicate or duplicate on a 96-well plate using GoTaq®qPCR Master Mix technology (Promega) according to the manufacturer’s protocol using 7900HT fast real time PCR system  with the following thermal profile: 2 min at 95°C; 40 cycles, 15 s at 95°C, 1 min at 60°C. Gene expression of target genes was assessed for 2, 20 mM HEPES (pH 7.9), 420 mM NaCl, 0.1 mM EDTA, 20% glycerol, 0.1% Triton, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 10 μg/ml aprotinin, and 1 μg/ml leupeptin. The lysates were frozen on dry ice for 5 min and then thawed at 37°C for 5 min for three times. The samples were centrifuged at 13,000 rpm at 4°C for 20 min and the supernatants were collected and stored at −20°C. Protein concentration was estimated by Bradford protein assay using BSA as standard. Equal amounts of protein (20 μg) were dissolved in Laemmli’s sample buffer, boiled for 5 min, and separated with a SDS-polyacrylamide minigel  and then transferred overnight at 15 mA into 0.45 μm Hybond-ECL membrane . Membranes were incubated for 1 h with blocking solution containing 5% (w/v) nonfat milk in Tris-buffered saline (TBS) and subsequently probed for 1 h at room temperature with a rabbit anti-mouse YM1 antibody  in incubation solution (TBS containing 5% (w/v) nonfat milk and 0.1% Tween 20. After extensive washing in TBST (TBS + 0.1% Tween 20), or goat anti-mouse MMR/CD206 polyclonal antibody . Blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG  or horse anti-goat IgG  in incubation solution, for 1 h at room temperature. After extensive washing in TBST, immunoreactive bands were visualized using a chemiluminescence assay detection system according to the manufacturer’s instructions . To ascertain that blots were loaded with equal amounts of protein lysates, they were also incubated in the presence of the antibody against β-actin protein . Subsequently, for semiquantitative analyses, the densities of the protein bands of YM1 (45 kDa), CD206 (175 kDa), and β-actin (42 kDa) were measured by densitometric scanning of the membrane with Gel Doc™ XR Imaging Densitometer  and a computer program . Western blotting images were arranged in the final figures using Microsoft software.Brain tissues were homogenized using steel beads with a tissue Lyser (QIAGEN) at 28 Hz, for 3 cycles of 20 s followed by 30 s, on ice and in a buffer for total cellular extracts, containing 5 mM MgClAll immunohistochemical analyses were performed on animals treated for 16 h with IL4. Right brain hemispheres were removed and fixed overnight at 4°C by immersion in 4% formalin solution.n = 3).Brains were immersed at 4°C in 30% sucrose solution until they sank, embedded in optimal cutting temperature compound, and stored at −80°C until analysis. Coronal sections of brain 20 μm thick were collected using a cryostat . Free-floating sections were washed five times with TBS + 0.01% Triton and incubated with blocking solution (TBS + 10% goat serum + 0.4% Triton) for 1 hour at room temperature. Next, sections were incubated overnight with the following antibodies, diluted in TBS with 1% goat serum: rabbit anti-mouse antibody against Ym1 , rat anti-mouse antibody against macrophage antigen complex-1 Mac-1 , mouse anti-mouse antibody against feminizing locus on X-3 , mouse anti-mouse glial fibrillary acidic protein . Sections were washed five times with TBS + 0.01% Triton and incubated for 2 h with secondary antibodies  at room temperature. Sections were washed five times with TBS + 0.01% Triton and then incubated for 15 minutes with Hoechst stain (Sigma-Aldrich). In parallel, some sections were tested for antibody specificity by omitting primary or secondary antibodies. After five washings in TBS + 0.01% Triton, sections were mounted on slides and observed using a Zeiss Axioskop microscope equipped with a digital camera ; images were captured at 40,000× and 63,000× magnification. Quantification of Ym1-positive microglia cells was performed by counting the number of cells showing a red-labeled Ym1 signal and a green-Mac-1 signal; cells were scored as positive on the basis of nuclear DAPI staining in close proximity. Four counting fields of 50 × 50 μm were analyzed in two sections from three different levels, at least 120 μm apart, of the striatum and frontal cortex (n = 3).Brains were trimmed using a brain matrix  and sections were routinely processed, paraffin embedded, and sectioned in 4 μm serial sections. For Iba1 and Arg1 immunohistochemistry, sections were immunostained with rabbit polyclonal anti-Iba1 antibody , and goat polyclonal anti-Arg1 antibody ; sections were than incubated with biotinylated goat anti-rabbit (Iba1) and rabbit anti-goat (Arg1) secondary antibodies , labeled by the avidin-biotin-peroxidase procedure with a commercial immunoperoxidase kit . The immunoreaction was visualized with DAB  substrate and sections were counterstained with Mayer’s hematoxylin . Digital image analysis was performed by scoring the number of Iba1- and Arg1-positive cells (microglia) in three 400× microscopic fields in the frontal cortex of both vehicle and IL4 treated mice  for 15 min at 4°C; after washings, cells were suspended in 500 μl of PBS + 0.05% BSA and applied to a magnetic column to purify CD11b+ cells, namely microglia. Immediately after isolation of microglia, cells were fixed with 4% paraformaldehyde, extensively washed with 125 mM glycine in PBS and permeabilized overnight in PBS containing 0,1% Triton X-100, 5% normal goat serum, and 2% BSA, at 4°C. Cells were incubated with rabbit anti-mouse Ym1 antibody (Stem Cell Technologies) diluted 1:50 in Incubation Solution  at room temperature for 1 h. After extensive washes in incubation solution, cells were incubated with Alexa633-conjugated anti-rabbit secondary antibody  for 1 h at room temperature. Cells were extensively washed with PBS and then analyzed with a flow cytometry system . Isotype IgG controls were also used to evaluate nonspecific signals. Incubations with FITC-antiCD11b antibody (Miltenyi Biotec) and flow cytometry analyses were performed separately on dissociated brain cells as well as on CD11b-immunosorted cells to calculate recovery and purity; our protocol allowed us to obtain 80 to 85% recovery of CD11b-positive microglia cells, that is, 12% of the total brain cells population after tissue dissociation and myelin removal, with 90 to 95% purity after magnetic immunosorting.After 16 h of IL4 or vehicle treatment, brains were dissected and washed in Hank’s Balanced Salt Solution ; after removing the meninges, cortices from five mice were pooled as a single experimental group. Enzymatic cell dissociation was performed using Neural Tissue Dissociation Kit P , following a modified version of the protocol supplied by the manufacturer. Briefly, after enzymatic digestion with papain, samples were dissociated mechanically, homogenized, and filtered through a 40-μm cell strainer. After extensive washes in HBSS, myelin was removed by centrifuging the dissociated brain cells, which had previously been suspended in 10 ml of cold 0.9 M sucrose solution, at 850 n observations. The results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons using GraphPad Prism 5 software [P <0.05 was considered significant.Unless otherwise stated, all values are expressed as mean ± standard error of the mean (SEM) of software . A valueFizz1, Arg1, and Ym1 genes, known to be induced by IL4 in peripheral macrophages, were analyzed in a time course experiment following icv IL4 treatment. As shown in Figure Fizz1 mRNA levels in the frontal cortex were significantly elevated following 8 h treatment and further increased after 16 h, while after more prolonged time intervals the effect of IL4, although not statistically significant, could still be observed and remained around 20-fold higher than vehicle-injected mice 48 h after the injection of IL4. Analogously, a significant induction of Fizz1 mRNA levels was observed in the striatum at the earliest time points analyzed, with a temporal profile of mRNA induction that is superimposable to that observed in the cortex, except for the 8 h treatment; interestingly, at this time point the effect of IL4 is significantly different in the two brain areas, with the cortex resulting in a significantly higher gene induction than the striatum. The increased variability in Fizz1 mRNA levels that is observed in both brain areas along with treatment duration could probably account for loss of statistical significance in the IL4 effect observed at 30 and 48 h after injection.To evaluate whether microglia populating different brain regions are able to attain a similar M2 response, we analyzed M2 gene expression in the frontal cortex and striatum, in which M2 polarization was shown to occur under neurodegenerative conditions ,24. The Arg1 mRNA levels in the brain; also, for this M2 gene, the IL4 activity is significantly more pronounced in the frontal cortex than in the striatum following 16 and 30 h treatment, although the response after 8 h treatment is statistically significant only in the striatum; unlike Fizz1 induction, no difference between vehicle or IL4-treated mice was observed at the last time point analyzed. Analogously, the temporal profile of Ym1 induction also supports diversity in the response to IL4 among the frontal cortex and striatum. In fact, Ym1 mRNA levels are increased for up to 30 h in the frontal cortex, although never reaching statistical significance, while the effect of IL4 is weaker and faster in the striatum  mRNA levels are significantly increased in the frontal cortex following 8 and 16 h treatment, while no effect is observed after longer treatments. This effect was expected based on the fact that CD206 is also associated with the M2a phenotype induced by IL4; however, the induction level of this gene is much lower than those observed for the M2a genes, reported in Figure CD206 mRNA induction is observed in the striatum following 8 h, while IL4 activity is lost afterwards; this is consistent with a reduced IL4 response of this brain area as compared to the frontal cortex. The mRNA levels of TGFβ, as an M2c marker, as well as IL1β and TNFα, which are M1 response genes associated with inflammation, showed no alteration at any points of our time course experiment, as shown in Figure Based on recent knowledge suggesting that microglia or macrophage activation exists as a spectrum of combinations of activation markers that are strictly dependent on experimental conditions , we evalCD206 Mrc mRNA levFizz1 is a more reliable and targetable marker of IL4-induced activation in the brain.Altogether, these data show that activation of the M2 response in the striatum is less efficient when compared with the frontal cortex in the experimental conditions utilized. Furthermore, our data show that the response to icv IL4 is gene-specific, suggesting that per se does not alter Ym1 expression, at least in the experimental condition assayed. The potency of the IL4 induction of Ym1 protein is in agreement with that related with Ym1 mRNA levels, which are shown in Figure We next analyzed whether IL4 activity in the brain correlated with an increase in M2 protein levels and whether the temporal profile of protein expression reconciled with that observed for the mRNA levels of M2 genes. To this aim, M2 protein levels were evaluated by Western blot analyses in the striatum and frontal cortex of mice injected with IL4; we were unable to assay Fizz1 and Arg1 proteins, owing to low specificity of the antibodies tested. As shown in Figure Importantly, the induction of CD206 protein by IL4 is also restricted to the frontal cortex, with no effect occurring in the striatum , mRNA levels of IL4Rα were evaluated in the striatum and frontal cortex of control and IL4-treated mice following 48 h treatment. Similar levels of IL4Rα mRNA were detected in these experimental groups (see Additional file Altogether, the difference in the Ym1 and CD206 levels between the striatum and frontal cortex demonstrates a region-specific difference in M2 polarization in response to IL4.We then asked whether the observed induction of M2 gene expression by icv IL4 could be ascribed specifically to microglia. Double-labeling immunohistochemistry was used to localize Ym1 protein expression within specific brain cell types using antibodies against cell type-specific proteins, such as Mac-1, NeuN, and GFAP, known to be exclusively expressed by microglia, neurons, and astrocytes, respectively, each one was assayed together with an antibody against Ym1. As expected, the Ym1 signal was detected in microglial cells of the frontal cortex and striatum following 16 h of IL4 treatment, as shown in Figure in vivo.To assess more precisely the percentage of IL4 responsive microglia cells, fluorescence-activated cell sorting analyses were performed on microglia purified from the cortex of adult mice 16 h after icv IL4 injection. Data show that the dissociated brain cells population before immunosorting contains CD11b-positive microglia that are about 12% of the total cell number and show a specific morphological and scattering profile, as expected (see Figure To extend our observation on the existence of subsets of IL4 responsive microglia, we analyzed the percentage of microglia expressing Arg1. As shown in Figure Altogether these results demonstrate that the M2 polarization induced by IL4 in brain can be ascribed specifically to a subset microglia cells.in vivo. We thus developed a method that makes use of centrally administered IL4 to induce the M2a phenotype in brain. This experimental model allowed us to estimate the different ability of microglia populating the striatum and cerebral cortex to induce M2a gene expression and to identify the existence of subpopulations of microglia that support the IL4 response in brain.The aim of this study was to evaluate the extent to which microglia undergo M2 polarization Phenotype and functional plasticity is a characteristic of microglia; the presence of M1 and M2 phenotypes and a wide spectrum of intermediate and concomitant activation programs has been documented in animal models of cerebral diseases although with different ratios, temporal involvement and functional outcomes ,24,27-30per se, might be exaggerated or unrestrained as a consequence of both acute CNS damage, such as after traumatic and ischemic reperfusion injuries, or under chronic neuroinflammation, as in Parkinson’s and Alzheimer’s diseases or multiple sclerosis; under these pathologic conditions several studies have shown that the anti-inflammatory and reparative response driven by alternative microglia polarization is dampened, as a consequence of the increased, self-propagating proinflammatory phenotype, revealing that the loss of an appropriate M2 response harms the dynamic and heterogeneous nature of microglia response and thus contributes to neurodegeneration [Activation of the brain M2 phenotype has been previously observed in animal models of neurological diseases and associated with suppression of inflammation, tissue remodeling and matrix deposition in a time- and environment-specific manner ,24,27-30neration . HoweverIntriguingly, our data show that the M2a response in the striatum is less efficient than that observed in frontal cortex, in terms of M2a gene expression both at the mRNA and protein levels (see Figures Besides the nature, intensity, and persistency of the trigger, the responsiveness of microglia at a given time and within a specific anatomical location plays a central role in neuroinflammation. Activation of a specific phenotype is dictated by the number of responsive microglia and the ability of these responder cells to undergo polarization; it is believed that such properties are influenced by the specific environment in which microglia reside ,9. AddinFizz1 reached the highest induction levels in both the striatum and the frontal cortex. Expression of all M2a genes analyzed in this study has been shown to be dependent upon the IL4 signaling and coordinate action of IL4-inducible transcription factors [Fizz1 expression in the brain is highly efficient and, although its role in neuroinflammation is not clear [Fizz1 may be considered as a reliable marker of IL4-induced M2a activation in brain.In this perspective, icv IL4 administration represents a valuable experimental procedure to study the signaling pathways that control microglia M2 polarization. The use of this experimental model allows induction of the M2a response all over the brain, with no signs of inflammatory reactions in vehicle-treated subjects. Along with distribution, the rapidity, potency, and persistency of the M2a response observed following central administration of IL4 represent advantageous features in estimating the capability of microglia cells to acquire the M2a phenotype among and within different brain areas, in physiological as well as pathological conditions. Among the genes analyzed,  factors -38; yet,ot clear ,40, suggFizz1 being the most inducible gene among those tested. Our results show that the responsiveness of brain macrophages to centrally administered IL4 might change among brain areas and that microglia M2a polarization can be ascribed to a subpopulation of IL4 responsive cells. Therefore, the biochemical pathways that instruct and enable microglia to undergo M2a activation represent key aspects in the physiopathology of microglia and challenging opportunities for the development of novel therapeutic and diagnostic agents.The results presented here show that central administration of IL4 induces a specific temporal pattern of M2a gene expression in striatum and frontal cortex, with


Cold-acclimation genes in woody dicots without winter-dormancy, e.g., olive-tree, need investigation. Positive relationships betweenOeFAD8, OeOSM, andOeLIP19and olive-tree cold-acclimation exist, and couple with increased lipid unsaturation and cutinisation.FAD8, coding a desaturase producing α-linolenic acid, is activated by temperature-lowering, concomitantly with bZIP-LIP19 genes. The research was focussed on finding OeLIP19 gene(s) in olive-tree genome, and analyze it/their expression, and that of OeFAD8 and OeOSM, in drupes and leaves under different cold-conditions/developmental stages/genotypes, in comparison with changes in unsaturated lipids and cell wall cutinisation. Cold-induced cytosolic calcium transients always occurred in leaves/drupes of some genotypes, e.g., Moraiolo, but ceased in others, e.g., Canino, at specific drupe stages/cold-treatments, suggesting cold-acclimation acquisition only in the latter genotypes. Canino and Moraiolo were selected for further analyses. Cold-acclimation in Canino was confirmed by an electrolyte leakage from leaf/drupe membranes highly reduced in comparison with Moraiolo. Strong increases in fruit-epicarp/leaf-epidermis cutinisation characterized cold-acclimated Canino, and positively coupled with OeOSM expression, and immunolocalization of the coded protein. OeFAD8 expression increased with cold-acclimation, as the production of α-linolenic acid, and related compounds. An OeLIP19 gene was isolated. Its levels changed with a trend similar to OeFAD8. All together, results sustain a positive relationship between OeFAD8, OeOSM and OeLIP19 expression in olive-tree cold-acclimation. The parallel changes in unsaturated lipids and cutinisation concur to suggest orchestrated roles of the coded proteins in the process.Olive-tree is a woody species with no winter-dormancy and low frost-tolerance. However, cold-tolerant genotypes were empirically selected, highlighting that cold-acclimation might be acquired. Proteins needed for olive-tree cold-acclimation are unknown, even if roles for osmotin (OeOSM) as leaf cryoprotectant, and seed lipid-transfer protein for endosperm cutinisation under cold, were demonstrated. In other species, The online version of this article (doi:10.1007/s00425-016-2490-x) contains supplementary material, which is available to authorized users. Arabidopsis and winter cereals  are among the early responses to low temperatures, ceasing when cold acclimation is acquired  subfamily members of the basic–domain leucine zipper (bZIP) transcription factors  in olive-tree genome, and analyze it/their expression, and those of OeFAD8 and OeOSM, under different cold-conditions/drupe-developmental stages, in comparison with possible changes in unsaturated lipids and cell wall cutinisation, in drupes and leaves of genotypes traditionally known for a different cold-tolerance, with the goal of establishing whether and how these genes were involved in cold-acclimation.The present research aimed to find OeLIP19 gene was present in olive tree genome, and that, together with OeFAD8 and OeOSM, was involved in cold-acclimation starting from its induction. Changes in unsaturated lipids and cutinisation paralleled the changes in the transcription of these genes, suggesting orchestrated roles of the coded proteins in the cold acclimation process.Results showed that an Olea europaea L. cv. Canino, endemic of the volcanic areas of Northern Lazio, cv. Leccino from Northern Tuscany, cv. Frantoio from Southern Tuscany, cv. Moraiolo from Central Italy, and cv. Taggiasca from Ligury . The experiments were planned in the way that each year 6-year-old specimens were used per genotype. Each year, the genotypes synchronously showed full blooming (last week of May), and drupe development during the weeks after flowering (WAFs).Trees of In the first 2 years (2009–2010), specimens from all genotypes were used, whereas in the lasting 3 years (2011–2013) only those from cv. Canino, cv. Moraiolo and cv. Frantoio.−2 s−1), and exposed to cold-treatments of various intensity/duration. In detail, plants at the beginning of oleogenesis in the drupes, i.e. at WAF 10 , or for 24 h (A2-type cold treatment), or for 72 h (A3-type cold treatment). Alternatively, the plants were exposed at 0 °C for 2 h, and then at 10 °C for 14 days (B-type cold treatment). The controls were kept at 25 °C. In some cases A3-type cold treatment was also applied at WAF19 , under 16 h/8 h (light/dark) photoperiod (300 μmol photons mFull winter plants (WAF26) were also put in the growth-chamber, and progressively exposed to a temperature decrease from 7 °C (open-air temperature) to −10 °C into 35 h, maintained at −10 °C for 2 h, and then exposed to a temperature increase up to 7 °C into other 35 h (C-type cold treatment). The controls were kept at 7 °C. WAF 26 was chosen because it was several weeks after the end of oleogenesis  . Other three WAF19-plants per genotype, coming from the open air stocks, were used for the test. Discs (5-mm-in-diameter) were excised from the leaf lamina and the drupe epi-mesocarp, and one gram/fresh weight per organ type was frozen at a rate of 2 °C/h up to −10 °C, and maintained at −10 °C for 30 min  were selected randomly at WAF19, and 50 mg/dry weight (DW) of each sample extracted. The samples were split in two aliquots. One aliquot was used for quantitative analysis of oxylipins by LC–MS/MS, as reported by Ludovici et al. , and theTen drupes and leaves per year and genotype  were randomly collected at WAF19 and WAF22, hand-cut, and oil bodies and cutin detected by SUDAN IV staining. Epi-mesocarp and leaf samples were either fixed, dehydrated, embedded, sectioned, and stained with toluidine blue, or attached on a scanning electron microscopy stub, and treated as in Matteucci et al. . MoreoveTotal RNA was isolated from ten drupes (epi-mesocarp) and leaves of Canino and Moraiolo, collected from specimens either grown under open-air, or exposed to A- to C-types of cold-treatments, as specified in the Results. Moreover, at WAF26 ten leaves per genotype were excised from open-air grown plants and incubated in 5 μM solution of A23187 calcium ionophore . For the construction of the phylogenetic tree, various accessions of plant b-ZIP proteins (including LIPs) were obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/nuccore/). The multiple sequence alignment was performed by ClustalW2.1 program  and the relative similarity matrix visualized, according to Neighbor–Joining method , according to Matteucci et al.  and D’Ani et al. , 2013. OCry2 was used as housekeeping gene for qPCR based on the stability of its mRNA expression in different plant tissues and developmental stages, and in presence of temperature variations, in accordance with previously reported housekeeping gene selection in the same species . Data from the third year shown.P < 0.05), followed by Tukey’s post test , used to compare mean values (±SE) of the same organ, either within the same genotype or between different genotypes. Alternatively, means (±SE) in pairs were compared by Student’s t test. Results were similar in all the crop years.A normality test was applied before analysis of variance , and one-way/two-way ANOVA , showing acclimation acquisition during the interval between WAF19 and WAF22, in accordance with previous results for the drupes  lower than from the drupes in both genotypes, however it was significantly (P < 0.0001) reduced in Canino in comparison with Moraiolo independently of the organ.Based on these results, the following analyses were focussed on Canino and Moraiolo because representative of the cold responsiveness of the other genotypes, after having confirmed the acquisition (Canino), and the non-acquisition (Moraiolo) of cold acclimation at WAF19 by the application of the ion leakage test see “”. In fac1- and A2-types of cold treatment resulted into the persistence of [Ca2+]cyt-rises, i.e., presence of cold-responsiveness, in both genotypes . An A3-type cold treatment to the plant was still not-sufficient to cause cold-acclimation in Moraiolo, but sufficient in Canino, because, under the cold-shock, only the drupe and leaf protoplasts of Moraiolo continued to show calcium rises   reduced in comparison with Moraiolo under the same A3-type cold treatment to the plant . Also when the plants were exposed to the B-type cold treatment before protoplast cold-shocking, Moraiolo remained cold-sensitive, differently from Canino, because only the cold-shocked protoplasts of the former showed the [Ca2+]cyt-rises  higher in Moraiolo than in Canino . Collectively, results from cytosolic calcium response in the protoplasts, and ion leakage from cell membranes after the B-type cold treatment to WAF10-plants, demonstrate that the sudden exposure of Canino plants to 0 °C for 2 h, coming from an open-air temperature of 25 °C, followed by a long exposure to a higher temperature, which however was fifteen degrees lower than the open-air temperature, is sufficient to induce and maintain a sudden cold acclimation in the genotype. In accordance, perennial plants without winter dormancy, as olive tree, are known to acclimate rapidly and maintain cold hardiness even under periods of unseasonably higher temperatures  mean value, Fig. To verify whether Moraiolo might become cold-acclimated later than WAF19, the cytosolic-calcium response induced by the cold-shock in the leaf protoplasts  was evaluated at WAF26 (full winter) in the open-air grown specimens. Calcium rises continued to be present, sustaining that Moraiolo had remained cold-sensitive . When, at WAF26, the plants were exposed to a C-type cold treatment before protoplast isolation, the inability of Moraiolo to acquire cold-acclimation was confirmed, because its protoplasts still showed presence of .The presence of the oxylipin 13S-hydroxy-9Z,11E,15Z-octadecatrienoic acid (13-HoTre), a C18:3 oxidation-product, was also evaluated, because it is a stress-induced compound and a possible cutin component , a significant (P < 0.01) reduction in OeFAD8 transcripts occurred in the drupes of both genotypes , increase in OeFAD8 levels at day 2 in comparison with day 0, there was a P < 0.0001 rise at day 7, but no further significant variation  increase at day 2, a strong decrease at day 7 , and a new increase (P < 0.01) at the treatment-end , and stable, in Canino  increased in Canino, and decreased in Moraiolo, as in the drupes . However, the gene transcripts were highly lowered in comparison with WAF19 , whereas it did not change significantly in Canino .The application of the Caino Fig. c. The pob-Zip LIP genes of other species was investigated. Total cDNA, synthesized starting from RNA samples of Canino, was used as template for PCR amplifications of partial regions of OeLIP, as described in “OeLIP nucleotide succession  registered in GenBank. Multiple sequence alignment clearly indicated that OeLIP presented the typical features of b-ZIP proteins: the basic domain , and the leucine zipper  . The similarity matrix produced by the comparisons of the b-ZIP protein successions (Table S1) revealed that OeLIP showed a percentage identity of its sequence with the other b-ZIP proteins ranging between 63.37 %  and 30.39 % . According to this matrix, a phylogenetic radial tree was also constructed; OeLIP branch was perfectly inserted in the genetic structure among the dicot b-ZIP proteins  occurred at WAF12/16, and were followed, at WAF19, by a slight decrease in level, which however, remained higher than WAF10 (P < 0.001)  Fig. a.Fig. 7E2-type cold-treatment, a P < 0.01 reduction in OeLIP transcripts occurred in the drupes of both genotypes in comparison with the control plants  OeLIP levels were reached in Moraiolo at days 2–7, but were followed by a strong (P < 0.0001) decrease (day 14)  at WAF19 in comparison with WAF10, whereas decreased (P < 0.01) in Moraiolo , and 72 h (P < 0.0001 difference with 0 h), whereas did not change significantly in Canino  and cold- (winter) drought , known to be triggered by the cold stress  were present in Canino in comparison with Moraiolo, when acclimation occurred in the former and not in the latter (WAF19). Because the expression of the OeFAD8 exhibited lower levels during advanced acclimation in comparison with early acclimation, suggesting a lower necessity of OeFAD8-mediated C18:3 production to maintain acclimation during advanced winter. Conversely, in Moraiolo, OeFAD8 levels continued to be low and fluctuating, but increased in full-winter leaves under the calcium-ionophore-treatment. Because Ca2+-ionophores induce [Ca2+]cyt-transients similarly to cold-shocks in the absence of acclimation  present on OeLIP-target genes, and that should be involved in the interactions with this TF during cold acclimation, remain to be determined. In Arabidopsis, the activation of Rab18, a Cor (cold-responsive)/Lea (Late embryogenesis abundant) gene, coding a dehydrin, is strongly associated to low-temperature stimuli and cold acclimation  were similar under prolonged cold-exposure  is cleaved and goes to the nucleus to induce PR-5 genes .In OeFAD8, OeOSM, and OeLIP19, participate to olive tree cold acclimation starting from its induction. They code proteins with different activities, i.e., a FAD, a PR-5, and a TF, respectively, but orchestrated for causing and maintaining cold acclimation, possibly through an increase in dehydration tolerance.In conclusion, as proposed in Fig. All Authors contributed to the writing of the manuscript and approved it.Supplementary material 1 (DOCX 559 kb)Below is the link to the electronic supplementary material.


Human endogenous retroviruses HERV-W encode a pro-inflammatory protein, named MSRV-Env from its original identification in Multiple Sclerosis. Though not detected in various neurological controls, MSRV-Env was found in patients with chronic inflammatory demyelinating polyradiculoneuropathies (CIDPs). This study investigated the expression of MSRV in CIDP and evaluated relevant MSRV-Env pathogenic effects.50 CIDP patients, 19 other neurological controls (ONDs) and 65 healthy blood donors (HBDs) were recruited from two different countries. MSRV-env and -pol transcripts, IL6 and CXCL10 levels were quantified from blood samples. MSRV-Env immunohistology was performed in distal sensory nerves from CIDP and neurological controls biopsies. MSRV-Env pathogenic effects and mode of action were assayed in cultured primary human Schwann cells (HSCs).In both cohorts, MSRV-env and -pol transcripts, IL6 positivity prevalence and CXCL10 levels were significantly elevated in CIDP patients when compared to HBDs and ONDs . MSRV-Env protein was detected in Schwann cells in 5/7 CIDP biopsies. HSC exposed to or transfected with MSRV-env presented a strong increase of IL6 and CXCL10 transcripts and protein secretion. These pathogenic effects on HSC were inhibited by GNbAC1, a highly specific and neutralizing humanized monoclonal antibody targeting MSRV-Env.The present study showed that MSRV-Env may trigger the release of critical immune mediators proposed as instrumental factors involved in the pathophysiology of CIDP. Significant MSRV-Env expression was detected in a significant proportion of patients with CIDP, in which it may play a role according to its presently observed effects on Schwann cells along with previously known effects on immune cells.Experimental results also suggest that a biomarker-driven therapeutic strategy targeting this protein with a neutralizing antibody such as GNbAC1 may offer new perspectives for treating CIDP patients with positive detection of MSRV-Env expression.Geneuro-Innovation, France. •A protein from endogenous retroviruses (8% of human genome) is found in chronic inflammatory demyelinating polyradiculoneuropathies (CIDP).•Identified in multiple sclerosis, MSRV-Env induces inflammation and autoimmunity.•It is now shown in peripheral nerve lesions, serum and blood cells of CIDP patients.•In Schwann cells, it induces pro-inflammatory factors; the same is detected in serum.•Antibody neutralization of MSRV-Env effects raises therapeutic perspectives in CIDP.Since the 1980's, a new horizon in the search for pathogenic players in complex human disease arose from studies in which human endogenous retrovirus families (HERV) were identified. HERVs represent 8% of the human genome and may express pathogenic proteins as shown here in a chronic inflammatory demyelinating polyradiculoneuropathies (CIDPs). The present study shows that MSRV-Env, an HERV protein, is detected in CIDP lesions and blood. It triggers inflammation in immune and nervous cells. A specific antibody revealed efficient in experimental conditions. This offers new therapeutic and diagnostic perspectives for this disabling disease without curative treatment. HoweverBeyond this fundamental research context, independent studies confirmed an association of MSRV expression with MS . Its envChronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a rare immune disease of the peripheral nervous system (PNS), with multifocal inflammatory and demyelinating lesions in nerve roots also expanding to distal regions . Its cliIn a previous study on MS, MSRV-Env was not detected in healthy controls and in various other Neurological Diseases except for CIDP cases (5/8) . This ob22.1The overall study group consisted of 51 CIDP patients, from the Neurology Departments of Henri Mondor University Hospital  and of Vaudois University Hospital . CIDP patients fulfilled criteria of the EFNS and PNS Joint Task Force guidelines . The majIn a first cohort (Study 1), 20 CIDP patients from Creteil and 21 HBDs from Annemasse, were enrolled. A second cohort (Study 2) included 11 additional CIDP patients and 19 OND patients from Créteil, 20 CIDP patients and 18 HBDs from Lausanne, and 26 HBDs from Annemasse, for a total of 31 CIDPs, 19 ONDs and 44 HBDs in study 2. MSRV transcript levels in PBMC were assessed separately in Studies 1 and 2. In the light of results obtained in human Schwann cells,IL6 and CXCL10 serum levels were determined retrospectively, at the same time for both cohorts .For serum, 6 mL of blood was collected on a dry tube and 500 μL of serum aliquots were frozen at − 80 °C. For peripheral blood mononuclear cells (PBMCs), 4 mL of blood was collected in a Cell Preparation Tube  and treated according to the manufacturer's instructions. PBMCs in heat-inactivated Fetal Calf Serum with 10% dimethyl-sulfoxide were stored at − 80 °C.2.2− 1 with RNase-free water. 50 ng of cDNA was used with iQ supermix  and corresponding sets of primers/probes for qRT-PCR. The internal control was glucuronidase beta gene, GUS B  and specific sets of primers and probes for MSRV-env as described (Thawed PBMCs were washed with PBS (1700 g for 20 min at 10 °C). Total RNA was isolated with QIAamp RNeasy Mini Kit  and treated with Turbo DNA-Free™  according to the manufacturer's instructions. RNA concentration was assessed with a Nanodrop 2000  before adjustment to 10 ng/μL with RNase-free water. First-strand cDNA was synthesized with i-script select cDNA-synthesis kit using oligonucleotide dT (18)  at 42 °C for 60 min, inactivated at 85 °C for 5 min and adjusted to 10 ng μLescribed . MSRV-po2.3IL6 and CXCL10 protein levels were respectively quantified with Human IL6 ELISA Ready-SET-Go!® and BD OptEIA™ Human IP-10 ELISA Set according to the manufacturer's instructions . The absorbance was read at 450 nm with Biotek EL800 device .2.4Frozen superficial peroneal nerve biopsies from 7 patients with CIDP and 2 ONDs, one axonal inflammatory neuropathy with perivascular inflammatory changes and one diabetic neuropathy, were collected from the Biological Resource Platform of Henri Mondor University Hospital (to French Ministry of Research #DC-2009-930). Biopsy samples were obtained independently from prospective studies 1 and 2, and used for research purposes according to French regulation (#AC-2014-2056). 15% PFA fixed and paraffin embedded 3 μm-sections were prepared. After paraffin removal, they were washed with Tween-TBS solution. Endogenous peroxidase activity and avidin/biotin proteins were quenched by Biotin blocking system  according to the manufacturer's instructions. Sections were then washed in Tween-TBS solution, incubated with anti-MSRV-Env mouse monoclonal antibody GN-mAb_04  or an isotype control  diluted at 5 μg/mL in PBS with 10% normal human serum (NHS) for 1 h at room temperature. After washing with Tween-TBS, sections were incubated with a secondary biotinylated antibody  diluted at 10 μg/mL in PBS with 10% NHS for 30 min at room temperature. Sections were washed again in Tween-TBS, streptavidin complex was added for 20 min at room temperature, followed by DAB for 7 min . Then, after washing with water and counterstaining with Hemalun, MSRV-Env specific and isotype control staining were examined by light microscopy on two successive sections of each biopsy.2.5HSCs were purchased from ScienCell Research Laboratories  and cultivated according to the manufacturer's instructions.2.5.1l-lysine coated  labtek , fixed in PBS with 4% paraformaldehyde, washed 3 times with PBS and incubated in PBS with 0.01% Triton X-100 for Schwann cell markers , or in PBS alone for extracellular TLR4. Nonspecific binding sites were saturated by incubation in PBS with 10% Fetal Bovine Serum for 1 h at 37 °C. They were then incubated with primary antibodies diluted in Fetal Bovine Serum 10% in PBS overnight at 4 °C . After 3 PBS washes, HSCs were incubated with secondary antibody solutions for 1 h at 37 °C . Following 3 PBS washes, plastic chambers were separated from the slides and mounted with Vectashield® mounting medium containing DAPI  before examination by fluorescence microscopy.HSCs were grown for 48 h on poly-2.5.2HSCs were cultured for 24 h in 6-well plates. At 60% confluency, HSC medium was replaced by serum-free OPTIMEM medium . Lipofectamine 2000 was used according to the manufacturer's instructions  to transfect HSC with empty pHBB control plasmid or pHHB plasmids expressing either the full length envelope protein (MSRV-Env-T) or its extracellular domain only (MSRV-Env-SU). After 4 h, transfection medium was replaced by HSC medium and cells were cultured for 48 h with GNbAC1 or GNbAC1 vehicle before collecting culture supernatant and cells for analyses.2.5.3Escherichiacoli and purified as endotoxin-free protein by PX'Therapeutics  from plasmid pV14 encompassing the complete env orf cloned from MSRV virion RNA . MSRV Env solubilization buffer  was provided in parallel.HSCs were grown on 6-wels plates and incubated with endotoxin-free recombinant MSRV-Env or its solubilization buffer (negative control), together with LPS-RS , GNbAC1 or GNbAC1 vehicle, at 37 °C for 1 h (IL6) or 4 h (CXCL10) before RNA isolation from cells. Recombinant MSRV-Env was produced in 2.5.4After appropriate treatments, HSCs were washed with PBS and total RNA extracted with QIAamp RNeasy Mini Kit. Relative expression of IL6 and CXCL10 to GUS B was performed with Taqman gene expression assays for IL6, CXCL10, and GUS B  according to the manufacturer's instructions.2.5.596-well microplates were coated overnight at 4 °C with an anti-MSRV-Env capture antibody  diluted at 5 μg/mL in 50 mM bicarbonate buffer, with a 0.05% Tween in PBS, saturated with 1% BSA PBS and washed 4 times. Culture supernatants diluted 1/2 in PBS were then incubated for 2 h at 37 °C, plates washed 4 times and incubated with HRP-coupled anti-MSRV-Env detection antibody  for 1 h at 37 °C. After 6 washes, revelation of antigen-bound HRP-antibody used 3,3′,5,5′-tétraméthylbenzidine  and absorbance at 450 nm wavelength was measured with Biotek EL800 device .2.6post hoc test were used when data passed the normality test, otherwise Spearman rank order correlation test, Mann–Whitney rank sum test, and Kruskal–Wallis one-way analysis of variance on ranks followed by Dunn's post hoc test were used. Chi-square or Fisher Exact tests were used to compare rates and proportions. Statistical analyses were performed with SigmaStat 3.5  and data plotted with Prism 5.04 .Kolmogorov–Smirnov normality test was applied to all data sets. Pearson product moment correlation test, Student's t-test, and one-way analysis of variance followed by Bonferroni's 2.6.1The sample collection and the experimental study were financially supported by Geneuro-Innovation, France. The funders had no role in the study design, nor the data collection and analysis, nor in their interpretation or in the writing of the manuscript.33.1Demographical and clinical characteristics are presented in 3.2Results are illustrated in In Study 1, PBMC mRNA from 20 HBD and 15 CIDP patients passed quality criteria (Cf. Methods). MSRV-env  was calculated (Cf. Methods). In Study 1, 6 CIDP patients had HE for MSRV-env (40%) but none in HBDs . 4 s (27%) and 1 HBD (6%) had HE for MSRV-pol (p = 0.161). 7/15 CIDPs  had HE for at least one MSRV transcript. A dual MSRV env/pol HE profile was observed in 0/17 HBDs and 3/15 CIDPs .In Study 2, 2 HBDs (7%) versus 6 CIDP (53%) presented HE for MSRV-env (p < 0.001). 3 ONDs (25%) had MSRV-env HE, which was not significantly different from HBD or CIDP groups. 8 CIDP (57%) had HE for MSRV-pol, which was significantly different from both HBD  and OND  groups. 9/14 CIDP  presented a HE profile for at least one MSRV transcript. A dual HE profile was observed in 2/26 HBDs (8%), in 0/6 OND and in 6/14 CIDPs  and -pol  expressions in CIDP patients are inversely correlated to the disease duration in Study 2 .3.3We analyzed IL6 cytokine and CXCL10 chemokine levels in sera from Studies 1 and 2. Statistics of comparisons between groups are presented in IL6 values above the limit of detection were more frequent in CIDP (30%) than in ONDs (11%) and HBDs (8%), which was statistically significant (p < 0.01). 2 CIDP patients presented highly elevated IL6 (230 and 387 pg/mL) whereas the highest IL6 levels in ONDs and HBDS were 10 and 14 pg/mL respectively. Similarly, CXCL10 levels were significantly higher in serum of CIDP patients (115 ± 10 pg/mL) than in HBDS  and ONDs . No correlation existed between IL6 and CXCL10 expressions, nor with the age of subjects, the age at disease onset, the gender, the treatment regimen, nor even with MSRV-env and -pol mRNA levels.3.4Distal sensory peripheral nerve (PN) biopsies were examined with an MSRV-Env specific monoclonal antibody or an isotype control antibody. MSRV-Env was detected in biopsies from five CIDP patients out of seven tested (71%), in the absence of staining with the isotype control. As shown in 3.5We then studied the effects of MSRV-Env expression on in vitro transfected HSC, mimicking MSRV-Env expression in HSC as observed in CIDP nerve biopsies. Primary cultured HSCs were transfected with plasmids encoding the complete MSRV-env protein (Env-T) or encoding its extracellular domain (Env-SU). Immunocytofluorescence analyses showed that HSCs strongly express toll-like receptor 4 (TLR4), the pharmacological target of MSRV-Env, at their surface . HSC canAs presented in 3.6We developed experimental conditions appropriate for a pharmacological evaluation of MSRV-Env and GNbAC1 by stimulating HSC for 4 h with purified and endotoxin-free recombinant MSRV-Env protein before HSC transcript isolation. A low concentration of MSRV-Env (3 nM) induced a significant increase of IL6  and CXCL10  transcript levels in HSC A–B. The 4After initial detection of HERV-W protein, MSRV-Env, in CIDP cases within a control group of inflammatory neurological diseases for a study on MS , the preMSRV-Env has for long been shown to induce innate immune dysregulation, autoimmunity and inflammation in cellular and animal models . TLR4 acUnlike available previous studies having investigated IL6 in sera of CIDP patients , we founIt therefore appears that an autoimmune reaction in CIDP may result from a TLR4-driven activation of innate immunity by MSRV-Env protein in immune and neuroglial cell types with potential downstream superantigen-like effects when T-cells are recruited and exposed to MSRV-Env . A mechaThus, MSRV-Env cannot simply represent a peripheral biomarker. Nonetheless, as MSRV-env and MSRV-pol expressions were inversely correlated to disease duration, MSRV activity may peak at early stages of the disease and its early quantification could be of value for CIDP phenotyping or diagnosis. As it may also reflect some efficacy of long-term treatments on its recirculation or expression in the bloodstream, longitudinal and/or transversal studies of accurately representative cohorts should now be envisaged.Finally, the present study also showed that the strong pro-inflammatory upregulation of IL6 and CXCL10 induced by MSRV-Env in HSC was significantly inhibited by a specific neutralizing antibody targeting MSRV-Env, GNbAC1. As this antibody is a humanized therapeutic IgG4, now in phase II clinical trials in MS , this inConcluding from these original findings, we propose that MSRV-Env is a potential therapeutic target in CIDP, at least in a significant proportion of patients, and that it may become a useful blood biomarker along with CXCL10 and IL6. This also provides arguments in favor of the humanized monoclonal antibody GNbAC1, as a potentially innovative treatment to be evaluated in CIDP.RF, AM, NG, IB, MB, CB, FC, ABL, and HP are employees of GeNeuro Innovation SAS or GeNeuro SA.AJS and TK perform consultancy work for Actelion Pharmaceuticals Switzerland.AC gave expert testimony for CSL Behring, Novartis, received grants from Biogen, Novartis, CSL Behring, Geneuro, Octapharma, and gave lectures for Genzyme.RF, AM: developed, supervised, and interpreted all protocols and experiments, except clinical and sample recruitment, interpretation of clinical data and immunohistological analysis of biopsies. They contributed to the text of the manuscript and to its successive reviews.NG, IB, MB: developed and performed all experiments except biopsies analyses, reviewed the manuscript.JA: biopsies analyses, reviewed the manuscript.FC, ABL, AJS, interpreted data and reviewed the manuscript.PAP, CL, CB, TK, AC: set-up of the clinical studies, patients' recruitment, data interpretation, reviewed the manuscript.HP, AC, FC and TK initiated and supervised the study.HP: reviewed contributions, corrected and finalized the text of manuscript as well as that of the revised version.


The four Chineseherbal medicines were intragastricallyadministered to Sprague-Dawley rats,respectively, to prepare drug-containing serumsof corresponding Chinese herbs. BMSCs wereisolated, cultured, and exposed to culturesolution containing 1%, 5%, 10%,and 15% (v/v) Radix Astragali-, Salvia-,Herba Epimedii-, and SaussureaInvolucrata-containing serum, respectively.TGF-β1-induced BMSCs were addressed in thesame manner. Collagen type II protein wasassessed by immunofluorescence methods. Toassess whether the drug-containing serums hadpositive effects on the proliferation of BMSCsand TGF-β1-induced BMSCs, MTT method wasassessed. The proliferation of BMSCs wassignificantly enhanced when exposed to culturesolutions containing 1% and 5% RadixAstragali-, 1% and 5% Salvia-, 5%Herba Epimedii-, and 1%, 5%, and10% Saussurea Involucrata-containing serum.The proliferation of TGF-β1-induced BMSCswas significantly enhanced when exposed to1%, 5%, and 15% Radix Astragali-,10% and 15% Salvia-, 5%, and15% Herba Epimedii-, and 1%, 5%,and 10% Saussurea Involucrata-containingserum.Traditional Chinesemedicine can promote the proliferation of bonemarrow-derived mesenchymal stem cells (BMSCs).We chose four “Kidney-tonifying”Chinese herbal medicines, Radix Astragali, Salvia,Herba Epimedii, and Saussurea Involucrata, toevaluate whether they had positive effects onthe proliferation of BMSCs andTGF- Bone marrow-derived mesenchymal stem cells (BMSCs) are derived from the mesoderm and within bone marrow . BMSCs hβ (TGF-β) family members are potent stimulators of bone formation and have been shown to be positioned in the bone repair site as well as embryonic bone and cartilage formation site [β family members played important roles in promoting the expression of cartilage-specific gene [Osteoarthritis is one of the most common progressive diseases, which affects at least one-third of adults over the age of 55 years . And theion site . Recent fic gene –11. Atractylodis macrocephalae, a tonic traditional Chinese herb, could enhance chondrogenic differentiation in BMSCs and the mechanism was dependent on the Sonic Hedgehog (Shh) pathway [β1 [According to the theories of TCM, the “Kidney” controls the bone metabolism and generates the marrow. Therefore, the use of “Kidney-tonifying” TCM herbs can contribute to bone health. He at al.  reported pathway . A lateshway [β1 .β1-induced chondrogenic differentiation of BMSCs. All the four TCM herbs were often applied to treat osteoarthritis, articular cartilage injury, and osteoporosis with a long history and significant curative effect.Here, we choose four kinds of Chinese herbal medicine, including Radix Astragali, Salvia, Herba Epimedii, and Saussurea Involucrata, to analyze whether they have positive effects on the proliferation of BMSCs as well as TGF-Sprague-Dawley rats  were obtained from the animal center of Nantong University . All rats were housed in the Nantong University animal care facility and were maintained under pathogen-free conditions. Rats were reared in strict accordance with standard operating procedures. Ambient temperature was kept at 22–25°C, humidity of 45% to 55%, light cycle was 12 h light, 12 h darkness. Timely water addition and feeding were managed daily and litter was cleaned once every two days. All experimental procedures were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China. The experimental procedures were approved by the Ethics Committee of the Institute of Wuxi Hospital of Traditional Chinese Medicine.Radix Astragali, Salvia, Herba Epimedii, and Saussurea Involucrata were provided by the traditional Chinese medicine pharmacy of Wuxi Hospital of TCM . Radix Astragali 90 g, Salvia 45 g, and Herba Epimedii 27 g (these amounts were three-day doses for an adult) were immersed in 500 mL distilled water for 40 minutes, boiled, decocted for 1 hour, and condensed to 60 mL, respectively. Saussurea Involucrata 5.5 g (this amount was a three-day dose for an adult) was cut into pieces and immersed in 500 mL 75% ethanol for 30 days. Then the liquid was filtered out, volatilized at 74°C, and condensed to 60 mL. Crude concentrations of Radix Astragali, Salvia, Herba Epimedii, and Saussurea Involucrata in the Chinese herbal solutions were 1.5 g/mL, 0.75 g/mL, 0.45 g/mL, and 0.09 g/mL, respectively. Chinese herbal solutions were placed in a bottle, sealed, sterilized, and stored at 4°C.μm micro porous membrane, and stored at −20°C.The Radix Astragali, Salvia, Herba Epimedii, and Saussurea Involucrata groups (5 rats each group) were intragastrically administered with corresponding Chinese herbal solutions  for 3 consecutive days . The control group was intragastrically administered with normal saline (NS). One hour after the final oral administration on the third day, rats were anesthetized by intraperitoneal injection with 3 mg/100 g sodium pentobarbital . Blood was harvested from the heart and centrifuged at 2500 r/min for 25 minutes. The supernatant was harvested, deactivated in a water bath at 56°C for 30 minutes, filtered through a 0.22 2 environment. The media were changed at 24 hour for the first time and then changed after each 72 hours. The cells were subcultured at a ratio of 1 : 3, and the third generation of cells was prepared for downstream-related experiments. Cell morphology and proliferation were analyzed daily with inverted phase contrast microscopy .4–6-week-old SD rats were sacrificed by cervical dislocation. Both femora and tibias were isolated and rinsed with phosphate-buffered saline . Whole bone marrow cells were collected by repeated washing the bone marrow cavity with low-glucose Dulbecco's modified Eagle's medium . The flushed mixture was centrifuged, washed, and resuspended in low-glucose DMEM containing 15% fetal bovine serum  and 1% penicillin-streptomycin . The resuspended cells were seeded into flasks and incubated in a 37°C, 5% COμg/L transforming growth factor β1 . Medium was replaced twice a week. Cell morphology and proliferation were analyzed daily with inverted phase contrast microscopy .BMSCs of three passages were used for chondrogenic differentiation. Cells were cultured for 2 weeks in chondrogenic medium, consisting of low-glucose DMEM supplemented with 2 mg/L insulin , 3 mg/L transferring , 1 mmol/L sodium pyruvate , 100 nmol/L dexamethasone , and 10 β1-induced BMSCs were fixed with 4% paraformaldehyde  for 30 minutes and pretreated with triton-X100  for 15 minutes. Rabbit polyclonal antibody against collagen type II  and fluorescein isothiocyanate- (FITC-) conjugated anti-rabbit antibodies  were used for fluorescent detection of the specific collagen. Fluorescence images were observed and recorded with a fluorescence microscope . Uninduced BMSCs were used as negative control.Immunocytochemistry was also carried out after 2 weeks of chondrogenic differentiation. For an immunofluorescence stain, TGF-β1-induced BMSCs was treated with the same manner above.The third generation of BMSCs were seeded and exposed to culture solution containing 15% FBS plus 1%, 5%, 10% and 15% (v/v) Radix Astragali-, Salvia-, Herba Epimedii-, and Saussurea Involucrata-containing serum, respectively, serving as experimental groups. 15% FBS plus serum of NS group served as control groups. Freshly prepared DMEM containing 15% FBS was used as blank group. Each experimental group and control group were accompanied by a corresponding blank group. The culture media were replaced every 48 hours. TGF-4/mL cells per well. And then 20 μL MTT 2,5-diphenyltetrazolium bromide; 5 mg/mL; Sigma, USA) was added to each well and the cells were incubated at 37°C for 4 hours. After that, the supernatant was carefully discarded, and 150 μL dimethyl sulfoxide  was added to each well with gentle shaking for 10 minutes. After the blue crystals were dissolved in DMSO, the optic density (OD) values were measured with an enzyme linked immunosorbent assay reader  at wave length of 492 nm. Five parallel wells were set in each group. An average value of the five wells was calculated and used in following formulas: value of Chinese herbal − enhanced cell proliferation = OD of experimental group − OD of blank group; value of control cell proliferation = OD of control group − OD of blank group.On the 7th day after intervention with drug-containing serums of Chinese herbal, the culture fluid was removed. Cells were seeded in 96-well plates at a density of 1 × 10t-test was used to analyze the differences between the values of Chinese herbal-enhanced cell proliferation and control cell proliferation in each group. All data were statistically analyzed using SPSS 16.0 software . A probability value of P < 0.05 was considered statistically significant.All experiments were conducted with a sample size of five and values were reported as mean ± standard deviation. The Student's β1-induced BMSCs exhibited larger cell bodies and multiple cytoplasmic projections and appeared as a polygonal-shape. After 5–8 days the cell bodies increased and the projections extended, exhibiting flattened morphology with the nucleolus split  concentrations of Radix Astragali-, Salvia-, Herba Epimedii-, and Saussurea Involucrata-containing serum, respectively. OD values of experimental, control, and blank groups were detected with MTT assay after 7 days. The results showed that cell proliferation of BMSCs was significantly enhanced when exposed to culture solution containing 1% and 5% Radix Astragali-containing serum, 1% and 5% Salvia-containing serum, 5% Herba Epimedii-containing serum, and 1%, 5%, and 10% Saussurea Involucrata-containing serum .β1-induced BMSCs were exposed to culture medium containing 1%, 5%, 10%, and 15% (v/v) concentrations of Radix Astragali-, Salvia-, Herba Epimedii-, and Saussurea Involucrata-containing serum, respectively. OD values of experimental, control, and blank groups were detected with MTT assay after 7 days. The results showed that cell proliferation of TGF-β1-induced BMSCs was significantly enhanced when exposed to culture solution containing 1%, 5%, and 15% Radix Astragali-containing serum, 10% and 15% Salvia-containing serum, 5% and 15% Herba Epimedii-containing serum, and 1%, 5%, and 10% Saussurea Involucrata-containing serum  of Chinese herbal-containing serum, 1% and 5% Radix Astragali-containing serum, 1% and 5% Salvia-containing serum, 5% Herba Epimedii-containing serum, and 1%, 5%,and 10% Saussurea Involucrata-containing serum could significantly enhance the proliferation of BMSCs. There was no dose-dependent manner between the proliferation of BMSCs and Chinese herbal drug-containing serum concentrations in our study. Moreover, with respect to the higher concentration of 15%, a lower concentration of 1% or 5% of Chinese herbal drug-containing serum could be more effective in increasing BMSCs proliferation, especially a concentration of 5% in our study.Actually, our result was consistent with some previous findings that the influence of Chinese medicine on the proliferation of BMSCs was indeed not dependent on the concentrations. A previous study investigated the proliferative effect of Astragaloside IV (AS-IV) in MSCs. AS-IV was one of the major and active components of the Radix Astragali. They are found in the concentration range of 10 to 200 mM AS-IV; the proliferation rates demonstrated optimal effect at 50 mM, much higher than the 200 mM . AnotherκB induction [β1-induced chondrogenic differentiation of BMSCs. Perhaps it could provide a new research idea for Salvia in the treatment of bone diseases. Actually, a Korean study has reported that tanshinone IIA could enhance the commitment of C2C12 cells into osteoblasts and their differentiation through synergistic cross talk between tanshinone IIA-induced p38 activation and BMP-2-induced Smad activation. They hypothesized that the osteogenic activity of tanshinone IIA might lead to the development of therapeutics that benefit bone regeneration and fracture healing [Salvia was a well-known Chinese herb used in the treatment of diseases including osteoporosis. The precise mechanism of Salvia in treating osteoporosis was unclear. Tanshinone IIA was one of the major active phytochemicals isolated from Salvia. It has been reported that tanshinone IIA could inhibit osteoclast differentiation and suppresses bone resorption through disruption of the actin ring, and the precise inhibitory mechanism of Tanshinone VI on osteoclast differentiating might be through inhibiting RANKL expression and NFnduction . Kwak etnduction  reported healing . Gjb2, Adam17, Adar, Alp1, Bmp10, Bmpr1a, Btrc, Cdh1, Cdkn2a, Fgf21, Fgfr2.Herba Epimedii is a commonly used traditional Chinese herb for “strengthening the kidney” and providing nutrition to bone. The result of our present study confirmed that Herba Epimedii could increase the proliferation of BMSCs and improve the chondrogenic differentiation of BMSCs in an appropriate circumstance. However, the mechanism was not clear. Interestingly, previous studies have demonstrated that Herba Epimedii could improve osteogenic differentiation in BMSCs , 24. Icaβ1-induced BMSCs. Perhaps this finding could expand the application of Saussurea Involucrata in terms of bone regeneration and fracture healing. Of course, this requires more in-depth study of the molecular mechanisms.Saussurea Involucrata is a rare and precious Chinese herb, most of which grow in 4000 meters above sea level in Tianshan mountain in China. As a traditional Chinese herbal medicine, Saussurea Involucrata has been used in the treatment of rheumatoid arthritis, impotence, irregular menses, and so forth . The finβ1-induced BMSCs in vitro. Nevertheless, one limitation existed in our study was that the mechanisms had not been explored. It is the content of our future research. TCM has a long history and a notable curative effect. We suggest that analyzing TCM using modern western research methods can provide a direction for future TCM investigation and contribute to the health of people worldwide.In conclusion, our study revealed that Radix Astragali, Salvia, Herba Epimedii, and Saussurea Involucrata could enhance the proliferation of BMSCs and TGF-


MUC5B rs35705950 variants and determine new plausible candidate variants for IPF among four different European populations. We genotyped 26 IPF candidate loci in 165 IPF patients from four European countries, such as Czech Republic (n = 41), Germany (n = 33), Greece (n = 40), France (n = 51), and performed association study comparing observed variant distribution with that obtained in a genetically similar Czech healthy control population (n = 96) described in our earlier data report. A highly significant association for a promoter variant (rs35705950) of mucin encoding MUC5B gene was observed in all IPF populations, individually and combined . Another non-coding variant, rs7934606 in MUC2 was significant among German patients  and combined European IPF cases . The network analysis for these variants indicated gene–gene and gene–phenotype interactions in IPF and lung biology. With replication of MUC5B rs35705950 previously reported in U.S. populations of European descent and indicating other plausible polymorphic variants relevant for IPF, we provide additional reference information for future extended functional and population studies aimed, ideally with inclusion of clinical parameters, at identification of IPF genetic markers.Idiopathic pulmonary fibrosis (IPF) affects lung parenchyma with progressing fibrosis. In this study, we aimed to replicate  Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive form of fibrosing interstitial pneumonia of unknown cause that predominantly affects lung parenchyma, leading to progressive worsening of dyspnea and lung function . In pathMUC5B and MUC2) or of pro-inflammatory cytokines  and also in cell signaling and innate immunity processes (TLR3 and TOLLIP) involved in inflammatory and profibrotic pathways (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585032/table/T1/), we have described the approach for their simultaneous investigation using a novel mass spectrometry based matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) multiplexed genotyping assay and reported their gene frequencies in healthy Czech (European) population  of 26 IPF candidate loci. Here, we have performed a comparative study for association of these IPF candidate genetic variants among four different European  populations and have ammended it by network prediction for gene–gene/gene–phenotype interactions in IPF and lung biology. We suggest that future extended and replicative studies following hereby described approach could enable better understanding of IPF pathogenesis, and if further supported by patient laboratory and clinical data, it could help to nominate novel disease markers.In this study, 165 IPF patients from four European populations comprising 41 Czechs , 33 Germans , 40 Greeks , and 51 French , representing Central, Southern, and Western Europe, were enrolled (Table CO), and/or restrictive pulmonary deficit, exclusion of other known causes of interstitial lung disease (ILD). For comparisons of genotype, allele frequency, and carriage rate (phenotype frequency) of analyzed genomic variants and case–control association study, we have utilized the data on distribution of these variants in 96 Czech healthy controls  based MALDI-TOF MS assay. The assay consists of an initial locus-specific PCR amplification followed by SBE using mass-modified dideoxynucleotide terminators of an oligonucleotide primer that anneals immediately upstream of the target polymorphic site. The distinct mass of extended primer traces the alternative alleles using MassARRAY Typer 4.0.20. For quality control (QC) step, we determined data missing rate per individuals and missing rate per SNP. Also, for QC of SNP genotyping, positive and negative template control samples were included in each assay plate. Any assay found as positive in negative template control were removed from the study.The details of the panel comprising 26 IPF candidate loci and genotyping procedure in IPF cases have been described previously . In brie2) test or Fisher exact test, as applicable. SNPs within HWE (p > 0.05) and sufficiently common [minor allele frequency (MAF) > 5%] in studied population were included. Carriage rate (phenotype frequency) was calculated as number of individuals carrying one (or two) copies of a particular allele on one or both  chromosomes. Association of SNPs minor alleles with IPF susceptibility were evaluated by Fisher’s exact test providing odds ratio (OR), 95% confidence interval (CI), and level of significance (p). For Bonferroni correction of multiple comparison , a stringent approach with p-value < 0.05/100 (5 × 10−4) was considered as significant.Each SNP was tested for Hardy–Weinberg equilibrium (HWE) by Pearson’s Chi-square (χp < 0.05) and their interaction with IPF and other phenotypes, such as lung disease, lung injury, and lung function was performed using Phenolyzer, a tool for phenotype-based prioritization of candidate genes in human diseases (Prediction of gene–gene network for plausible candidate variants (diseases . The candiseases .PRKCE rs628877 (p = 0.02 in combined IPF cases with F_MISS = 0.008), IL-4 rs2243250 , IL-4 rs2070874 , IL-4Rα rs1801275 , and MAPT rs1981997 . Further, with MAF threshold check, TP53 rs12951053 was found with MAF = 0.04, F_MISS = 0 in Greek IPF cases. Thus, keeping the QC stringent conditions, these six SNPs were removed from further analysis, for which 20 variants remained  using allelic (multiplicative) genetic model revealed a total of nine SNPs for IPF susceptibility. Among these, three SNPs were shared among different IPF populations: first, rs35705950*T within promoter region of Mucin5B (MUC5B) was highly significant among all the IPF populations – Czech ; German ; Greek ; French ; and combined IPF cases  (Table Mucin2 (MUC2) was significant for German , Greek , French , and combined IPF  cases. The third variant, rs1799899*A located in exon region of Transferrin (TF) gene was significant for Germans , French , and in combined IPF  cases. Prediction of loss-/gain-of-function for functional variant TF rs1799899 suggested it as a probably damaging mutation (Polyphen score: 0.869 and SIFT score: 0.07).The primary analysis (Tumor protein 53 (TP53) ; (ii) rs4277405*C in Angiotensin converting enzyme (ACE) ; and (iii) rs4459609*C in ACE  and one each in Greek: Telomerase reverse transcriptase (TERT) rs2736100*G ; French: ATPase, type 11A (ATP11A) rs1278769*A ; and in total IPF cases: Interleukin-1 α (IL-1α) rs1800587*T   rs12602273*G in 2 test and additive test of logistic regression analysis with similar panel and population distribution/stratification of significant risk variants, and an exceptional addition of rs11191865*A (p = 0.046) in Oligonucleotide/oligosaccharide-binding fold containing 1 (OBFC1) exclusively in total IPF set under χ2 test.These findings of allelic model were in concordance with Pearson’s χMUC5B and MUC2 in same gene family , transcriptional interaction among MUC2 and TP53 , and all identified candidate genes belonged to the same biosystem . Further prediction based on their records in several databases , gene–gene interaction and interaction with phenotypes  has , German (1.55 × 10−5), Greek (1.13 × 10−6), French (5.28 × 10−10), and in combined IPF population (1.80 × 10−11); whereas MUC2 rs7934606*A was significant in German (4.03 × 10−4) and in combined IPF (3.73 × 10−5) . Among IPF cases, the allele frequency and carriage rate for rs35705950*T were lowest in Czech  and highest in French ; while rs7934606*A has lowest frequencies in Czech  and highest in Germans .p < 5 × 10−4), we report a high association between MUC5B rs35705950*T and IPF susceptibility in all the four analyzed populations among Czechs: [OR (95% CI) p] 3.77 (7.47–1.9) 1.62 × 10−4, Germans: 4.83 (9.79–2.39) 1.55 × 10−5, Greeks: 5.46 (10.82–2.76) 1.13 × 10−6, and French: 6.77 (12.65–3.62) 5.28 × 10−10 along with all patients from this study as a whole: 5.23 (8.94–3.06) 1.80 × 10−11. Thus, we replicate previous findings and provide an insight for predominant association of gel-forming mucin-encoding gene variants with IPF in Europeans and populations of European descent. The role of mucin variants was also supported with high significance of MUC2 rs7934606 among Germans  and combined European IPF cases . The identified nine variants among MUC5B, MUC2, TF, TP53, ACE, TERT, ATP11A, and IL-1α (significant at p < 0.05) were interactive at gene–gene and gene–phenotype level in network analysis. The present study for inter-population comparison is imperative with respect of comparing the distributions of plausible IPF-associated gene variants and, thus, may provide starting point(s) for further investigations of biological  and translational  aspects.This study is a first report of comparative distribution of genotypes and alleles of 26 candidate gene variants implicated in mucin production, cell-cycle regulation, pro-inflammatory, and profibrotic signaling pathways pertinent to IPF pathobiology among patients from four populations across Europe . Employing stringent statistical approach  on chromosome 11q15.5, MUC5B is the most predominant in the normal distal airway epithelium and is widely associated with sporadic IPF and familial interstitial pneumonia (MUC5B promoter-variant rs35705950 SNP predicted change in transcription factors binding sites (disruption of E2F and creation of HOX9 and PAX2) and the risk T-allele has been strongly associated with increased MUC5B expression in the lung tissue. The relative high frequency of rs35705950*T risk allele  observed in this study are in concordance with several reports, including populations of European descent . To explain the mucin-expressing structures in IPF pathogenesis, Seibold et al. analyzed ciliated, basal, and alveolar type II cells in lung tissue and reported predominant expression of MUC5B in pseudostratified mucociliary epithelium comprised of basal epithelial cells and mucus cells in distal airway. Further, it is suggested that mucociliary dysfunction in the distal airway causing honeycomb cyst may play a role in the development of progressive fibroproliferative lung disease (MUC2 rs79834606 as significantly associated in IPF (p = 3.8 × 10−6) with MAF  0.54 in IPF cases and 0.41 in controls . The SNP rs1799899 marks a Gly/Ser change located in TF gene encoding a glycoprotein involved in iron ion transport and removal of certain organic matter and allergens from serum. As this is the first implication of TF gene in context of IPF, in parallel to the studies of its possible functional role, this association requires replication  rs4277405 and (iii) rs4459609 located in promoter region of ACE-II suggested with critical profibrotic role in IPF (TERT and (v) rs1278769 in 3′-UTR of ATP11A were significant. Several studies have reported the telomerase gene mutation causing short telomerase as risk factor and poor survival in IPF  reported in this study must be replicated in accordance to the guidelines for conductance of genetic association studies  do increase IPF risk among the four European populations, two of which (Czech and Greek) have not been studied before in this regard at all. Additionally, the nominated variants in TF and other variants of TP53, ACE, TERT, ATP11A, and IL-1α may also contribute to IPF susceptibility. Despite our panel of 26 gene variants was designed across pertinent pathobiological pathways, it did not include others from the wide range of plausible IPF-associated SNPs regulating immune and fibrotic functions, such as master regulator TGF-β, TNF-α, full spectrum of TLRs, MHC (HLA) variants, and also SNPs in regulatory microRNAs (miRNAs) (Our findings provide evidence that gene variants involved in mucin production ((miRNAs) , 33, 34.MUC5B promoter region variant (rs35705950) with IPF disease among Europeans. In addition, it suggests further IPF-associated polymorphisms: MUC2 (rs79834606) and TF (rs1799899) variants in general, and the other six  in individual European populations. Their further investigation for disease association among extended patient cohorts is, therefore, warranted. At the same time, findings of our present study represent reference information to be utilized for future extended functional and population studies as well as for translational research of the nominated variants aiming at characterization of biomarkers and/or novel therapeutic targets.The present study confirms and further extends strong association of The study was approved by the ethical committee of the University Hospital, Olomouc; University Medical Center, Freiburg; Medical School University, Athens; and University Hospital, Paris. After approval of the study by ethical committee, the consent of human participants visiting the centres was taken in written for usage of biological sample for research purpose in future.MP, GZ, JM-Q, JP, VK, DB, and DV conceived and designed the work; AK, VZ, LK, JP, EB, HN, VL, GZ, VK, and MP contributed to data acquisition; AK performed data analysis and interpretation; AK and MP drafted the manuscript after its revision for important intellectual context by all authors; MP and AK finalized the article. All authors have read and approved the final manuscript and agreed to be accountable for all aspects of the work.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.


Biological functions are carried out by groups of interacting molecules, cells or tissues, known as communities. Membership in these communities may overlap when biological components are involved in multiple functions. However, traditional clustering methods detect non-overlapping communities. These detected communities may also be unstable and difficult to replicate, because traditional methods are sensitive to noise and parameter settings. These aspects of traditional clustering methods limit our ability to detect biological communities, and therefore our ability to understand biological functions. To address these limitations and detect robust overlapping biological communities, we propose an unorthodox clustering method called SpeakEasy which identifies communities using top-down and bottom-up approaches simultaneously. Specifically, nodes join communities based on their local connections, as well as global information about the network structure. This method can quantify the stability of each community, automatically identify the number of communities, and quickly cluster networks with hundreds of thousands of nodes. SpeakEasy shows top performance on synthetic clustering benchmarks and accurately identifies meaningful biological communities in a range of datasets, including: gene microarrays, protein interactions, sorted cell populations, electrophysiology and fMRI brain imaging. Molecules, cells and tissues carry out biological processes through physical interaction networks124568310111213101516182021a priori community titles. In this context, labels are unique bits of information that are assigned randomly and used to track cluster membership. SpeakEasy differs from previous label propagation algorithms, because nodes update their labels on the basis of their neighbors’ labels, while subtracting the expected frequency of these labels, based on their popularity in the complete network. This process combines a bottom-up approach to clustering (using neighboring information) with a top-down approach (using information from the whole network). This dual approach facilitates accurate community detection in many types of biological networks  clusters in biological networks. SpeakEasy is related to earlier label propagation algorithms2324networks  because not require manual tuning of clustering parameters for good results. Second, it can cluster networks with any type of links  or any type of network structure . SpeakEasy is highly scalable and can quickly cluster networks with hundreds of thousands of nodes. Third, because it is very efficient, the stochastic clustering process can be repeated many times to detect robust clusters that are not generated by data artifacts or noise. The repeated clustering process also allows SpeakEasy to identify multi-community nodes, whose membership tends to oscillate between different clusters. Finally, users can select overlapping or non-overlapping output, as is appropriate for their applications.In addition to accurate cluster detection (see Results section), community detection via SpeakEasy has several practical advantages for biological applications. For instance, since the number of communities in a dataset is rarely known in advance, SpeakEasy automatically predicts the number of communities and does For an intuitive example of how SpeakEasy identifies communities, we illustrate the clustering process on a demonstration network . This neWe use three approaches to determine the accuracy of SpeakEasy community detection. First, we test its performance on a large set of synthetic networks with carefully controlled characteristics, wherein the true clusters are known. Then we apply it to real-world networks, wherein the true clusters are unknown . In thisds)  benchmarks, which are widely used to test overlapping and non-overlapping clustering methodsds) . SpeakEa 25303132. Additio 25303132. Thus, Sm). When multi-community nodes are tied to many communities , community detection becomes more difficult The LFR benchmarks accurately represent certain aspects of social and biological networks, but are limited in other aspects. For example, networks in the LFR benchmarks have low transitivity and null assortativity (propensity for hubs to connect to hubs)ds metric that was shown to be more consistent with other metrics than original Q metric38ds values from SpeakEasy  indicate that it tends to detect more small and highly dense clusters than GANXiSds scores for the two biological networks in this test set (‘dolphins’ and ‘c.elegans’). These modularity values are approach those of methods that directly attempt to maximize modularityWe compare modularity values from SpeakEasy to those from another label propagation algorithm, GANXiS, because that method showed the best overlapping clustering performance in a recent comparison of clustering methodset al.et al.42433233Because a single protein may be part of more than one protein complex (set of bound proteins that work as a unit), Discovery of protein complexes directly benefits from development of methods which detect overlapping communities. We test SpeakEasy community detection of overlapping protein complexes, using two well-studied high-throughput protein interaction networks 46We find that applying SpeakEasy once again, to each of these broad categories of cell types, identifies sub-communities with higher correspondence to the tissue of origin and cell type, considered together . Thus, sSeveral cellular or molecular processes can generate correlated gene expression , including cell-type variation, transcription factors, epigenetic or chromosome configuration21Therefore, we use SpeakEasy to detect overlapping and non-overlapping coexpressed gene sets in two datasets that are commonly used to address many biological questions: The Human Brain Atlas (HBA)We also generate overlapping clusters from both the HBA and CCLE datasets. Overlapping coexpressed gene sets may be useful in biological studies because gene coexpression is driven by overlapping mechanismsExtracellular neuronal recording with single electrodes, tetrodes, or high density multichannel electrode arrays can detect the activity of multiple nearby neurons. However, these combined responses must be separated into responses of specific neurons. This blind source separation process is known as “spike sorting”, because each spike is assigned to a particular theorized neuron. Single neurons often generate relatively unique signatures (i.e. spike waveform shapes and amplitude distributions on multiple adjacent electrodes), and emerge as clusters in the matrix of waveform correlations.To realistically test spike sorting, it is important to match noise levels in real brain recordings. Therefore, we use real depth-electrode recordings generate a simulated time-series of spikes in which the true spike times and unique neuronal sources are known see . CompariFunctional magnetic resonance imaging (fMRI), obtained while a subject is at rest (rs-fMRI), is a valuable tool in understanding of systems-level changes in a variety of domains, including neurodegenerative diseaseCommunities identified in control and PD groups contain biologically similar sets of brain regions  from repeated runs with different initial conditions. The ability to generate many partitions is useful because it can quantify the stability of each cluster . It is a1858How to cite this article: Gaiteri, C. et al. Identifying robust communities and multi-community nodes by combining top-down and bottom-up approaches to clustering. Sci. Rep.5, 16361; doi: 10.1038/srep16361 (2015).