Subclinical psychotic experiences at the level of the general population are common, forming an extended psychosis phenotype with clinical psychosis. Persistence of subclinical experiences is associated with transition to later mental disorder. Increased daily life stress reactivity is considered an endophenotype for psychotic disorders. We examined, in a longitudinal framework, whether baseline momentary assessment markers of stress reactivity would predict persistence of subclinical psychotic experiences over time. In a general population sample of female twins (N = 566), the Experience Sampling Method  was used to assess  daily life stress reactivity. Persistence of subclinical psychotic experiences was based on the Community Assessment of Psychic Experiences (CAPE), assessed three times over 14 months post-baseline. It was investigated whether baseline daily life emotional and psychotic stress reactivity predicted persistence of psychotic experiences over time. Higher levels of emotional stress reactivity (a decrease in positive and an increase in negative affect in response to stress), and increased psychotic reactivity to daily stress was found in individuals with persistent psychotic experiences over time compared to individuals with transient psychotic experiences. The results suggest that markers of daily life stress reactivity may predict “macro-level” persistence of normally transient expression of psychotic liability over time. Linking daily life markers of altered reactivity in terms of emotions and psychotic experiences to longitudinal persistence of psychotic experiences, associated with increased risk of transition to overt mental disorder, may contribute to earlier and more accurate diagnosis of risk. Psychiatric research is complementing its previous focus on categorical, heterogeneous disease entities with dimensional approaches towards psychopathology. For example, there is evidence that subclinical psychotic experiences at the level of the general population are common, and represent an extended psychosis phenotype outside the boundaries of clinical disorder High levels and persistence of psychotic experiences is influenced by both environmental and genetic factors Earlier work has shown that markers of  sensitization can be identified at the level of everyday life experience. For example, in patients with clinical psychotic disorder In a longitudinal study of young adult female twins from the general population, we examined the following questions:Do individuals with stable high levels (i.e. persistence) of subclinical psychotic experiences over time (macro-level) at baseline show increased emotional stress reactivity in daily life (micro-level) compared to those with stable low levels of psychosis?Do individuals with stable high levels (i.e. persistence) of subclinical psychotic experiences over time (macro-level) at baseline show increased psychotic stress reactivity in daily life (micro-level) compared to those with stable low levels of psychosis?We hypothesized that stable high levels (i.e. persistence) of subclinical psychotic experiences would be associated with increased emotional and psychotic reactivity to stress in daily life at baseline.This study forms part of a general population twin study that investigates gene – environment interactions in vulnerability for mental disorders, as described previously The positive item scale of the Community Assessment of Psychic Experiences (CAPE) (20 self-reported items) was used to assess subclinical psychotic experiences The Structured Clinical Interview for DSM-IV Axis I disorders (SCID) was administered at T0 (and T4). To measure baseline psychotic symptoms, the sum score of the subscales delusions (consisting of 15 items) and hallucinations (consisting of eight items) of the SCID was used.In a previous paper, growth mixture modeling was used to identify two different developmental trajectories of the CAPE items, assessed at the three time points of the Flanders twin study The Experience Sampling Method (ESM) is a random time-sampling self-assessment technique that has been shown to be feasible, valid, and reliable in general and patient populations very unpleasant, 0 =  neutral, 3 =  very pleasant). For the current analyses, all positive responses were recoded as 0, and the negative responses were recoded so that high scores reflect more unpleasant and potentially stressful events  Event stress was conceptualized in terms of subjective appraisals of events and minor disturbances that continually occur in the natural flow of daily life. After each beep, participants were asked to report the most important event that had happened between the current and the previous report and then to rate this event on a 7-point, bipolar Likert scale .Social stress was measured by asking subjects whether they were alone at the time of the beep. If not alone, they were asked whether they liked the company they were in at that moment. This was rated on a 7-point Likert scale. The scale was reversed so that higher scores represent higher disliking of being in that company.“I feel suspicious” rated on a 7-point Likert scales Paranoid ideation was assessed with the ESM item : “I feel insecure”, “I feel lonely”, “I feel anxious”, “I feel down” and “I feel guilty” Negative affect (NA) was assessed as the mean score on 5 ESM items, rated on 7-point Likert scales : “I feel happy”, “I feel cheerful”, “I feel relaxed” and “I feel satisfied” Positive affect (PA) was assessed as the mean score on 4 ESM items, rated on 7-point Likert scales Momentary ESM data were analysed using multilevel linear regression techniques, which take the hierarchical structure of the data (repeated measurements clustered in persons) into account. In addition, the sample consisted of twin pairs, resulting in a further level of clustering. Thus, in the current study, repeated momentary measurements (level 1) were clustered within subjects (level 2), some of whom were members of the same twin pair (level 3). Data were analyzed using the XTMIXED multilevel random regression routine in STATA 11.0 ANOVAs (for continuous variables) and Chi-square  tests were conducted to investigate group differences in demographic characteristics and ESM variables.NA and (b) PA as the dependent variables and group (low versus persistent),  stress and their interaction as the independent variables To investigate whether stress  elicited differential emotional reactions in the two groups, two multilevel analysis was conducted with (a) paranoid ideation as the dependent variable and group (low versus persistent),  stress and their interaction as the independent variables To investigate whether stress  elicited differential psychotic reactions in the two groups, a multilevel analysis was conducted with 2 (1)  = 0.06, p = 0.80, and educational level (χ2 (3) = 1.29, p = 0.73). Group differences on ESM variables are summarized in The final sample included 529 participants, of whom 62 (12%) were in the persistent group and 467 (88% of the sample) in the low psychosis group, who completed a total of 21270 valid ESM observations . The groups did not differ in mean number of ESM reports (low group: mean (SD)  = 37.05 (6.92); persistent group: 36.58 (5.87); F = 0.26, p = 0.61). The two groups did not differ in age  = 0.55, p = 0.46, marital status (χ2 (1)  = 21.54; activity: b = .06; 95%CI .04, .08, p = 0.000; χ2 (1)  = 42.4; p = 0.0000; social: b = .05; 95%CI .03, .07, p<0.001; χ2 (1)  = 21.49; p<0.001). For all stressors, stratified analyses showed a significantly greater increase in NA following stress in the Persistent group compared to the Low group   = 5.08), however not in the models with event stress and social stress predicting PA   = 0.25; social: b = −.03; 95%CI −.07, .01, p = 0.10; χ2 (1)  = 2.65). For activity stress, stratified analyses showed a significantly stronger decrease in PA following stress in the Persistent compared to the Low group   = 13.83). The Persistence group  reported significantly more momentary paranoid ideation when experiencing stressful activities compared to the Low group   see .  = 0.95). Similarly, there was no significant interaction between group status and social stress in the model of momentary paranoid ideation   = 0.15).No significant interaction was apparent between group and event stress in the model of momentary paranoid ideation Additional analyses were carried out, investigating whether baseline psychosis psychopathology influenced the results. All analyses were repeated with both exclusion of participants who reported delusions or hallucinations at baseline  and controlling for baseline psychosis CAPE scores. Apart from some small effect size alterations, all results remained comparable .The current study shows that sensitivity to daily life stress is associated with stable high levels of psychotic experiences over time. Although a causal relationship cannot be established, more stress reactivity may predict more persistence of psychosis over time. Thus, sensitivity at the micro-level is associated with persistence of psychotic experiences at the macro-level. More specifically, higher levels of emotional sensitivity (i.e. less expression of positive and more expression of negative affect in response to stress), and psychotic reactivity to daily stress were found in individuals with persistent subclinical psychotic experiences compared to individuals with persistently low levels of psychotic experiences.Individuals with persistent subclinical psychotic experiences were more emotionally responsive to all types of daily stressors . This was more consistently found in models of NA reactivity compared to PA reactivity. Earlier ESM studies have found increased emotional stress reactivity in patients with psychotic disorders The findings indicate that individuals who are more responsive to stress are more likely to pertain to a group of individuals with persistently high levels of psychotic experiences. One important factor underlying these findings may be type and amount of coping. Thus, individuals who are liable to develop psychosis are more likely to (i) be more sensitive to stress An innovative aspect of the study is that it introduces a multilevel approach, linking different levels of manifestation of psychosis liability, namely “micro-level” daily life interactions and “macro-level” psycho(patho)logical experiences. This may represent an important step forward in complementing the current, diagnosis-oriented approach in psychiatry. Moreover, the results are in line with earlier propositions that liability to develop psychosis is distributed in the general population The results of the current study should be interpreted in the light of its strengths and limitations. First, use of ESM booklets instead of electronic devices means that the exact timing of participants' self-reports cannot be established with 100% certainty


Ridgeia piscesae, in high-pressure aquaria and examined global and quantitative changes in gene expression via high-throughput transcriptomics and quantitative real-time PCR (qPCR). We analyzed over 32,000 full-length expressed sequence tags as well as 26 Mb of transcript sequences from the trophosome (the organ that houses the endosymbiotic bacteria) and the plume . R. piscesae maintained under conditions that promote chemoautotrophy expressed a number of putative cell signaling and innate immunity genes, including pattern recognition receptors (PRRs), often associated with recognizing microbe-associated molecular patterns (MAMPs). Eighteen genes involved with innate immunity, cell signaling, cell stress and metabolite exchange were further analyzed using qPCR. PRRs, including five peptidoglycan recognition proteins and a Toll-like receptor, were expressed significantly higher in the trophosome compared to the plume. Although PRRs are often associated with mediating host responses to infection by pathogens, the differences in expression between the plume and trophosome also implicate similar mechanisms of microbial recognition in interactions between the host and symbiont. We posit that regulation of this association involves a molecular “dialogue” between the partners that includes interactions between the host’s innate immune system and the symbiont.The relationships between hydrothermal vent tubeworms and sulfide-oxidizing bacteria have served as model associations for understanding chemoautotrophy and endosymbiosis. Numerous studies have focused on the physiological and biochemical adaptations that enable these symbioses to sustain some of the highest recorded carbon fixation rates ever measured. However, far fewer studies have explored the molecular mechanisms underlying the regulation of host and symbiont interactions, specifically those mediated by the innate immune system of the host. To that end, we conducted a series of studies where we maintained the tubeworm,  Deep-sea hydrothermal vents host highly productive ecosystems based on microbial chemoautotrophy  as our study organism because it shares many of the attributes of the better-studied Riftia pachyptila, including gross morphology and symbiont type Ridgeia physiology Ridgeia’s “short” morphotype exhibits sulfide uptake rates comparable to those of R. pachyptilaRidgeia during numerous expeditions to the Juan de Fuca ridge system. We examined gene expression in two different tissues to understand how the plume, which serves as the direct interface between the host and free-living microorganisms in the environment, and the trophosome, which maintains the highly specific intracellular symbiont population, respond under in situ conditions. We present gene expression data from these tissues to identify host biochemical and biomolecular mechanisms that are involved in cell cycle, signaling, stress response and innate immunity. We observed expression of a number of putative pattern recognition receptors (PRRs) involved with recognition of microbe-associated molecular patterns (MAMPs). Via quantitative real-time PCR (qPCR), we quantified and characterized the abundance and pattern of eighteen genes involved with innate immunity, cell signaling, and metabolite uptake. Although studies of the innate immune systems of polychaete worms are limited, there is evidence that this group has an immune response similar to those described for other invertebrates, such as the use of PRRs, reactive oxygen species and the production of antimicrobial compounds To further our understanding of the biomolecular mechanisms used by siboglinid tubeworms to maintain symbiostasis, we conducted a series of studies of Ridgeia piscesae is not an endangered or protected species.No specific permits were required for the described field studies. Ridgeia piscesae tubeworms were collected during a research expedition to the Mothra (47°55.4′N by 129°06.4′W) and Main Endeavour (47°57.0′N by 129°5.82′W) vent fields along the Juan de Fuca ridge from August to September 2006 and 2007 as previously described Ridgeia tubeworms were collected via the DSV Alvin from approximately 2300 m, and placed in thermally insulated boxes prior to returning to the surface. On the surface, tubeworms were assessed for health and select worms were immediately placed in flow-through, high-pressure respirometry aquaria Ridgeia were maintained at conditions previously shown to support high primary productivity by R. pachyptila . Between 50–100 mg each of both trophosome and plume from six separate worms maintained in the HPRS system between 50–72 h were used to isolate total RNA according to manufacturer’s guidelines . After extraction, total RNA was quantified on a microscale UV spectrophotometer (Nanodrop Inc.), quality checked by gel electrophoresis or a Bioanalyzer RNA Nano chip , and stored at −80°C.One cm pieces of flash-frozen tissues of trophosome and plume were excised on a dry-ice-cooled stainless steel dissecting tray and immediately placed into pre-chilled (−20°C) RNAFirst-strand cDNA was synthesized using the SuperScript II Reverse Transcription kit  according to manufacturer’s guidelines. A mixture of 5 ml of total RNA, 1 ml of 250 ng random hexamer primers, 1 ml of dNTP mix (10 mM each), and sterile distilled water to a total volume of 12 ml were incubated for 5 min at 65°C and quick-chilled on ice. After adding 4 ml of 5x First-Strand Buffer and 2 ml of DTT (0.1 M), the mixture was incubated for 2 min at 25°C. One ml (200 units) of SuperScript II RT was then added to the reaction, then incubated for another 10 min at 25°C before 50 min at 42°C, and finally inactivated by heating at 70°C for 10 min. Storage of cDNA was maintained at −80°C.Quantitative Real-Time PCR (qPCR) was performed on Stratagene Mx3005P Real-Time PCR System  using SYBR Green PCR Master Mix (Applied Biosystems Inc.). The PCR reaction consisted of 10 µl of SYBR Green PCR Master Mix, 0.75 µl each of forward and reverse primers (10 mM), and 5 µl of template cDNA in a final volume of 20 µl. The thermal cycle profile was 15 min at 95°C, followed by 40 cycles of 94°C for 15 sec, 58°C  for 30 sec, and 72°C for 30 sec, and a final segment of 95°C for 15 sec, 58°C (or 55°C) for 20 sec, and 95°C for 15 sec. A dissociation curve and subsequent capillary sequencing (Applied Biosystems Inc.) verified that the used primer pair produced only a single product. The assay included a no-template control and each of the test cDNAs from twelve tissues  for each target gene. Each reaction was run in technical duplicates.T) measured at fluorescence dRn  = 0.05 was utilized to calculate the fold expression using the 2–ΔΔCT method T  =  CT:target – CT: actin) and the relative quantification was obtained by comparing the expression of the same RNA in the target organ with the reference organ, plume (ΔΔCT  =  [CT:target – CT: actin]sample – [CT:target – CT:actin]reference). The mean and standard deviations of the 2–ΔΔCT equation were then calculated from the duplicate samples, with the mean value of the plume samples equating to approximately one (20 = 1).Threshold cycle  using the 1st and 3rd quartiles (Q1 and Q3) of each gene for the upper and lower bounds of the box, respectively. Thus, 50% of the values  are within the box, with the median drawn as a solid black line. Whiskers are depicted at the maximum and minimum values or 1.5*IQR , whichever value is smaller. A dotted line indicates the fold expression of plume samples (y = 1). Outliers were those values lying more than the IQR beyond those quartiles and are not represented in the analysis.–ΔΔCt method to determine whether they yielded similar results with respect to consistency within a single organism, consistency between the two tissues, and consistency among technical replicates. The program NormFinder was used to assess the stability values of actin and Ef1α from six plume and trophosome samples We examined three potential housekeeping genes, actin, elongation factor-1 alpha (Ef1α), and 18S ribosomal RNA, by qPCR. These data were also analyzed using the 2Ridgeia tubeworms used in our studies had to exhibit sulfide and oxygen uptake rates that were indicative of high chemoautotrophic activity. While sulfide uptake for these individuals has been previously reported 2 uptake rates , including carbon dioxide, bicarbonate and carbonate) that are a direct indicator of chemoautotrophy (net chemoautotrophy occurs when there is a net ΣCO2 uptake). Both the DIC and O2 uptake rates indicate that Ridgeia sustained very high carbon fixation rates  and the trophosome  under E-values <10–06 when analyzed using the National Center for Biotechnology Information (NCBI) databases (www.ncbi.nlm.nih.gov/). From the plume, 454 pyrosequencing generated ∼26 Mb of sequence data. 108,717 single sequences were used to assemble 20,757 isotigs, which again were analyzed using the NCBI databases with the aforementioned E-value  for eukaryotes . CompariCloser examination of expression in each tissue revealed a number of putative genes involved with host cell cycle, innate immunity, signal transduction, cell stress response and apoptosis . We idenqPCR primers were designed to target a total of eighteen genes, including seven genes involved with MAMP recognition ; other humoral-associated genes, including an alpha-2-macroglobulin receptor-associated protein and a bacteriocidal lipobinding protein; immune signaling genes, including a NF-κB inhibitor, a macrophage inhibitor factor and a LPS-induced TNFα transcription factor (LITAF); and cell stress mediators, including heat shock protein 70 and a reactive oxygen species modulator See –S2. For The majority of the PRRs analyzed, including all five of the PGRPs and a Toll-like receptor (TLR) showed between five and one hundred-fold higher expression in the trophosome compared to the plume . The NF-R. piscesae differentially expresses a suite of genes in the trophosome and the plume that are involved with detecting and responding to bacteria, cell stress and the regulation of cell cycle. The substantial expression of putative receptors in the trophosome that are involved with recognizing bacterial MAMPs suggests that elements of the host’s innate immune system are involved with mediating interactions with the endosymbiont population. A suite of other genes associated with innate immunity, as well as cell signaling and stress, were differentially expressed in the trophosome. When considered in the context of previous research on the structural morphology within the trophosomes of siboglinid tubeworms The main objective of this study was to examine gene expression in the trophosome (the organ where symbionts are housed in bacteriocytes), and the plume (the site of metabolite exchange and where the host encounters free-living microorganisms), in order to better understand how this association remains stable and highly productive. Our results indicate that, during high chemoautotrophic productivity, R. pachyptila hosts a tremendous density , bacterial lipoproteins, and peptidoglycan and its derivatives, lead to the activation of innate immunity effectors through interactions with specific host PRRs. This activation involves well-regulated signaling cascades and the induction of host functions such as innate immunity activation, apoptosis, mucus secretion, and cell regression Ridgeia (Ridgeia PGRPs (Rpi1) has homology (42% at the amino acid level) to a putative PGRP (EsPGRP3) from the Hawaiian bobtail squid Euprymna scolopes. In that system, four homologues of the PGRP family have been implicated in playing a role in the development of the association between the host and its bioluminescent bacterial symbiont Vibrio fischeriRidgeia, Rpi2 and Rpi5, have homology to PGRP-SC3 and PGRP-LE. In Drosophila, these PGRPs have been implicated in regulating gut homeostasis of ingested bacteria and the mediation of host response to intracellular pathogens Ridgeia suggest that they are found both intracellular and at the membrane surface, with three of the five  also having the potential of being secreted  in Ridgeia –3. Evidesecreted , suggestsecreted . At leasRidgeia PGRPs showed markedly higher expression in the trophosome relative to the plume  receptor and a serum protein . HomologRidgeia also revealed a number of genes known to be involved with regulating cell cycle, apoptosis, and cell growth, including known mediators of cell cycle and apoptosis in Drosophila  in a number of different systems Ridgeia are likely occurring within bacteriocytes and/or with the surrounding vasculature , regulation of bacteriocyte and symbiont cell density and division is likely required to maintain a stable association. Previous studies suggest that symbiont digestion, as well as carbon translocation, may be the primary modes of regulating symbiont numbers Figure S1Dissolved inorganic carbon (DIC) and oxygen uptake by R. piscesae. ∑CO2  and O2 uptake rates (µmol·g–1·h–1) by Ridgeia were recorded over 70 hours under either quasi-in situ conditions  or sulfide limiting conditions. All rates are expressed in terms of wet mass. During all respirometry experiments, tubeworms were maintained in the high-pressure respirometry system  and O2 concentrations, 30 ml of effluent was added to N2-purged 50-ml serum vials containing 3 ml of concentrated NaOH. ∑CO2 concentrations were measured by adjusting the pH to 3 using degassed HCl, and measuring PCO2 in the headspace using a blood gas analyzer . O2 concentrations were measured using a gas chromatograph with a thermal conductivity detector Click here for additional data file.Figure S2KOG analysis and comparison of Ridgeia piscesae trophosome and plume ESTs and 454-generated contigs. Significant matches to sequences in the KOG database were determined using the tblastx BLAST program (NCBI), and KOG categories were accordingly assigned. Percentages are based on the number of sequences successfully assigned to categories for each database.(TIF)Click here for additional data file.Table S1Quantitative RT-PCR primer sequences for target genes. F  =  Forward primer, R  =  Reverse primer, TA  =  Temperature used for annealing step of qPCR reaction. The gene abbreviations are as follows: Actin; β-actin: EF1α; elongation factor 1-alpha: TLR2p; toll-like receptor 2 precursor: NFκBic; NF-kappa-B inhibitor (cactus): A2MRAP; alpha-2 macroglobulin receptor associated protein: HSP70; heat shock protein 70: LBPIP; LPS induced bactericidal permeability increasing protein: LITAF; lipopolysaccharide-induced tumor necrosis factor-alpha: MMIF; macrophage migration inhibitory factor: MR; macrophage mannose receptor 1-like protein: CAtr; carbonic anhydrase (trophosome): CAbr; carbonic anhydrase : ROSm; reactive oxygen species modulator: PGRPrpi1–5; peptidoglycan recognition protein 1–5: hbB1; hemoglobin B1.(DOC)Click here for additional data file.Table S2Putative identities of Ridgeia genes found in ESTs and used in quantitative RT-PCR.(DOC)Click here for additional data file.


Solanum melongena L.). The former benefit from an enhanced frequency of marker polymorphism, but their relevance to marker-assisted crop breeding is limited. Combining the restriction-site associated DNA strategy with high throughput sequencing has facilitated the discovery of a large number of functional single nucleotide polymorphism (SNP) markers discriminating between the two eggplant mapping population parental lines ‘305E40’ and ‘67/3’. A set of 347 de novo SNPs, together with 84 anchoring markers, were applied to the F2 mapping population bred from the cross ‘305E40’ x ‘67/3’ to construct a linkage map. In all, 415 of the 431 markers were assembled into twelve major and one minor linkage group, spanning 1,390 cM, and the inclusion of established markers allowed each linkage group to be assigned to one of the 12 eggplant chromosomes. The map was then used to discover the genetic basis of seven traits associated with anthocyanin content. Each of the traits proved to be controlled by between one and six quantitative trait loci (QTL), of which at least one was a major QTL. Exploitation of syntenic relationships between the eggplant and tomato genomes facilitated the identification of potential candidate genes for the eggplant QTLs related to anthocyanin accumulation. The intra-specific linkage map should have utility for elucidating the genetic basis of other phenotypic traits in eggplant.Both inter- and intra-specific maps have been developed in eggplant ( Solanum melongena L.) is the third most important solanaceous crop , with a global production level in 2010 of ∼41.8Mt. It is an important vegetable in south Asia, the Middle and Near East, Africa and Southern Europe http://solgenomics.net/genomes). The eggplant is an autogamous diploid (2n = 2x  = 24), with a haploid DNA content of 1.2pg 2 population bred from the interspecific cross S. linneanum x S. melongena2 populations using a mixture of microsatellites and gene-based markers, mostly derived from putative orthologs among eggplant, tomato and potato The eggplant  discovery exercise, achieved by combining the “Restriction-site Associated DNA”  for field trials for phenotypic characterization of eggplant mapping populations.2 plants was bred from the cross ‘305E40’ x ‘67/3’, breeding lines which differ from one another with respect to a number of key agronomic traits. The highly homozygous female parent (‘305E40’) forms a pink corolla and produces long, highly pigmented dark purple fruit. The line was derived from a somatic hybrid between the cultivar ‘Dourga’ and S. aethiopicum. Its pedigree also includes cvs. ‘DR2’ and ‘Tal1/1’ 8 selection from the intra-specific cross cv. ‘Purpura’ x cv. ‘CIN2’. DNA samples were extracted from young leaves, using the GenEluteTM Plant Genomic DNA Miniprep kit , following the manufacturer's protocol.A population of 156 FFusarium oxysporum f.sp. melongenaeRfo-sa1 gene, which confers resistance to F. oxysporum f.sp. melongenaeThe mapping population was genotyped with respect to 472 markers, comprising 388 SNPs, 43 microsatellites, three CAPS,11 RFLPs and 27 COSII markers. Of the SNPs, 384 were those selected by Barchi et al. 2 test. Only markers associated with a χ2 value ≤ χ2α = 0.1, or slightly deviating from expectation (χ2α = 0.1< χ2 ≤ χ2α = 0.01) were considered, provided that their inclusion did not alter the local marker order. Loci suffering from significant segregation distortion , and any for which 30 or more of the 156 progeny were not successfully genotyped were excluded. JoinMap v4.0 software Differences between observed and expected segregation ratios were assessed using a χhttp://solgenomics.net/); for the RAD-derived SNPs, a 0.6 ratio between the number of identities and the query length was used as a cut off. A blastX search was carried out against the NCBI protein database, adopting a threshold of e–15. Finally, a blastN search was conducted of all the markers represented on the Fukuoka et al. A blastN search of SNP, RFLP and COSII markers was made against the SL2.40 genome build published by the International Tomato Annotation Group  and Monsampolo del Tronto ] in 2009. In both trials, the material was arranged as a set of two randomized complete blocks with four replicate plants per entry per block . The traits assayed were: adaxial leaf lamina anthocyanin (adlan), stem anthocyanin (stean), abaxial leaf lamina anthocyanin (ablan), calyx anthocyanin , corolla colour (corcol), leaf venation anthocyanin (lvean) and fruit peduncle anthocyanin (pedan). Six of the seven traits were scaled from 0 to 3, while corcol was scored on a 1–5 scale, with “1” representing pink, “2” dark pink, “3” light violet, “4” violet-pink and “5” violet. Statistical analyses were performed using R software iYj  =  μ + gi + bj + eij, where μ, g, b and e represented, respectively, the overall mean, the genotypic effect, the block effect and the error. Based on the F2, the broad-sense heritability (2BSh) values were calculated as σ2G/( σ2G + σ2E/n), where σ2G represented the variance in g and σ2E the residual variance and n the number of blocks. Correlations between traits were estimated using the Spearman coefficient, and normality, kurtosis and skewness assessed with the Shapiro-Wilks test (α = 0.05). Segregation was considered as transgressive where at least one F2 individual recorded a trait value higher or lower by at least two standard deviations than, respectively, the higher or lower scoring parental line.The mapping population, along with both of the parental lines and their FBoth interval 2 segregants. Segregation was skewed (χ2 > χ2α = 0.01) for only 13 markers , but in no case was the distortion enough (χ2 > χ2α = 0.01) to discard the data. The genotype data relating to the 431 informative markers were assembled into 12 major and one minor (5 markers) LG, comprising 415 loci  in ML, and for adlan (six plants), stean (two plants) and lvean (two plants) in MT. In each case the transgression involved a lower level of pigmentation than in ‘305E40’. 2BSh was overall high, ranging from 0.71 (lveanML) to 0.99 (pedanML) (2  = +0.23), and the most highly correlated were stean and calan in ML, and stean and lvean in MT (+0.86). The performance with respect to each trait was highly correlated between the two locations.A summary of the phenotypic performance and the derived pedanML) . SignifipedanML) . The leaSeparate QTL analyses were performed for each location, resulting in the detection of 21 QTLs in ML and 18 in MT distributed over eight chromosomes . QTL clul-sane et al. A blastX search of the NCBI non-redundant protein database carried out for the seven marker loci on E10 and the two on E05 linked to the QTLs , failed Lycopersicon esculentum genome sequence build 2.40; http://solgenomics.net/organism/Solanum_lycopersicum/genome) in the tomato syntenic regions to search for genes and transcription factors known to be involved in anthocyanins pathways . With respect to corcol, Doganlar et al. To identify candidate genes underlying the eggplant E10 and E5 QTLs for anthocyanin accumulation identified in the present work, we investigate the available tomato sequence . The global genetic length defined by the map was 1,390 cM, a length not dissimilar to that obtained by both Wu et al. The positions of the homologues of the SNP, RFLP and COSII loci in the tomato genome agreed well with previous analyses Solanaceae1 being very similar in each of the two environments. The high 2BSh value associated with all the traits along with the correlated trait performances between the two sites can be taken as evidence that soil and climate variation – to the extent that the two sites differed for these – had little influence on the phenotypic outcome. The 2BSh values were higher than 80% for all the traits; only for lvean it was markedly higher in ML (0.93) than in MT (0.71) as in the latter location an higher phenotypic variation was detected. Heritability values for some of the traits in study have been recently reported The genetic basis of anthocyanin synthesis and accumulation has been widely explored in the Transgression was rare, and always in the direction of the less pigmented parent (305E40). As previously reported Collard et al. Anthocyanin pigmentation in eggplant, at least in the cross used for mapping, is controlled by loci on chromosomes E05, E06 and E10. The region between 68 and 70 cM of E10 (defined by seven marker loci) is particularly prominent for anthocyanin production and accumulation throughout the plant (except in the corolla). The major site for corcol was within a ∼13 cM region of chromosome E05 (defined by two marker loci) which harboured both major QTLs expressed in both locations and minor QTLs responsible for lvean (in both locations) and pedan (just in MT). The implication is that the corcol trait is controlled by one or more genes which are not directly responsible for pigmentation of the leaf, stem or calyx. Since both parental lines produce anthocyanin pigmented fruits, no detectable variation in fruit pigmentation was observed in the F2 progeny.QTLs linked to related traits have a tendency to co-localize Solanaceae has been carried out to date in potato, pepper and tomato. In the former, van Eck et al. P, I, R and F ) required for anthocyanin synthesis in the tuber's skin and flowers. In pepper, the gene A (which is a component of the anthocyanin synthesis pathway) was shown by Borovsky et al an2. In tomato, 13 genes related to anthocyanin synthesis have been described by De Jong et al. Solanaceae family. These data enabled both candidate orthologues for three of the above potato genes to be proposed, and for predictions to be made with regard to orthologue identity in tomato  and its corresponding tomato physical chromosome (in yellow) are connected by solid lines (in green). Distances on the eggplant chromosomes are given in cM, and on the tomato chromosome segments in Mbp.(PDF)Click here for additional data file.Figure S2Eggplant maps alignment. Alignment of the current genetic map  with that constructed by Fukuoka et al (PDF)Click here for additional data file.Table S1Parameters associated to the 261 loci used for synteny and collinearity between the eggplant and tomato genomes.(XLSX)Click here for additional data file.


Follicular B cell survival requires signaling from BAFFR, a receptor for BAFF and the B cell antigen receptor (BCR). This “tonic” BCR survival signal is distinct from that induced by antigen binding and may be ligand-independent. We show that inducible inactivation of the Syk tyrosine kinase, a key signal transducer from the BCR following antigen binding, resulted in the death of most follicular B cells because Syk-deficient cells were unable to survive in response to BAFF. Genetic rescue studies demonstrated that Syk transduces BAFFR survival signals via ERK and PI3 kinase. Surprisingly, BAFFR signaling directly induced phosphorylation of both Syk and the BCR-associated Igα signaling subunit, and this Syk phosphorylation required the BCR. We conclude that the BCR and Igα may be required for B cell survival because they function as adaptor proteins in a BAFFR signaling pathway leading to activation of Syk, demonstrating previously unrecognized crosstalk between the two receptors. ► Inducible loss of the Syk tyrosine kinase results in death of follicular B cells ► Syk transduces survival signals from BAFFR to the ERK and PI3 kinase-PDK1 pathways ► BAFFR signaling results in phosphorylation of Igα and Syk ► BAFFR transduces signals via the BCR to activation of Syk B lymphocytes play a critical role in the adaptive immune response, in part by producing high affinity antibodies to pathogens. There are at least three main lineages of mature B cells. Recirculating follicular B cells reside in the follicles of secondary lymphoid organs and traffic between them through the blood and lymphatic circulations; marginal zone (MZ) B cells are located in the periphery of the splenic white pulp and are largely nonrecirculating; B1 cells are found predominantly in the peritoneal and pleural cavities. The total number of mature naive (unactivated) B cells remains largely constant despite continuous production of new B cells in the bone marrow as well as recruitment of naive B cells into antigen-activated compartments, such as germinal center cells, plasma cells, and memory B cells. This homeostasis of mature B lymphocytes is known to depend on at least two receptors: BAFFR (TNFRSF13C) and the B cell antigen receptor (BCR).Mice deficient in BAFFR or its ligand BAFF (TNFSF13B) have substantially reduced numbers of follicular and MZ B cells, but unaltered numbers of B1 cells . FurtherOn mature B cells, the BCR is found in the form of surface-bound immunoglobulin M (IgM) and IgD. These proteins are both associated with the nonpolymorphic Igα and Igβ (CD79a and CD79b) transmembrane proteins, which are required for BCR signal transduction . InducibSyk gene in mice results in a partial block in B cell development at the pre-BCR checkpoint and complete arrest at the BCR checkpoint. Consequently, no mature B cells develop at all, consistent with a key role for Syk in transducing pre-BCR and BCR signals required for developmental progression  and an allele of ROSA26 (Rosa26MerCreMer) expressing a tamoxifen-inducible MerCreMer fusion protein consisting of the Cre recombinase fused to two mouse estrogen receptor hormone-binding domains  or conditional Syk-deficient mice . Both strains of mice expressed the Cre recombinase and had one loxP-flanked allele of Syk but differed in the second Syk allele, with the control mice having a wild-type allele (Syk+) and the Syk-deficient mice having a nonfunctional deleted allele (Syk−). This allowed us to control for the activity of the recombinase and any potential effect of deleting and recombining genomic DNA. Treatment of conditional Syk-deficient mice with tamoxifen resulted in the loss of Syk in virtually all B cells, with the amount of Syk falling below detection by 10 days following start of tamoxifen treatment  and either control or conditional Syk-deficient bone marrow cells (Ly5.2+). Eight weeks later, once the mice were fully reconstituted, they were treated with tamoxifen and B cell numbers were followed for 8 weeks. Results showed that there was a selective loss of B cells that had lost Syk, even in the presence of wild-type B cells . As before, we found that 3 weeks after the start of tamoxifen treatment pre-B and immature B cells were lost from the bone marrow, all transitional and marginal zone B cells and around 80% of follicular B cells were lost from the spleen, whereas most B1 cells persisted in the peritoneum  or conditional Syk-deficient mice (Sykfl/-RMCM), which were used to reconstitute irradiated mice. Once reconstitution was complete (around 8 weeks posttransplant), the chimeric mice were treated with tamoxifen and analyzed 6 weeks later. B cells from the conditional Syk-deficient mice infected with the retrovirus (GFP+) expressed about 40% the amount of Syk found in control B cells (−) Syk-deficient B cells, as seen by a higher ratio of GFP+ to GFP− conditional Syk-deficient B cells compared to the ratio of GFP+ to GFP− T cells in the same animal  to increase receptor amounts on both control and conditional Syk-deficient B cells. We found that increased expression of BAFFR did not promote increased survival of Syk-deficient B cells in vivo, had no effect on the increased amounts of surface IgM and did not rescue the defect in BAFF-induced survival in vitro C–2G. In A critical signaling pathway from BAFFR required for B cell survival involves phosphorylation of NF-κB2 p100 by IKK1, its processing into p52 and subsequent nuclear translocation of the p52 transcription factor . It has To explore this pathway further, we made use of a mutant form of p100 (p100ΔC), which is converted more efficiently to p52 A Liao a. Once agStimulation of B cells with BAFF leads to the upregulation of several antiapoptotic members of the Bcl-2-family of proteins, including Bcl-2, Bcl-xL, Mcl1, and A1, and to the downregulation of the proapoptotic Bim and Bad proteins . To inveAnother critical pathway controlling B cell survival is one leading to the activation of the ERK MAP kinases . Because3). Previous work has shown that the death of B cells that have lost the BCR can be reversed by ectopic activation of the PI3 kinase pathway, leading to the proposal that the BCR survival signal uses this pathway  and subsequent phosphorylation of Akt on Thr308 by PDK1 and on Ser473 by mTORC2 or other kinases (Pdpk1 (Pdpk1fl) crossed to a tamoxifen-inducible Cre recombinase expressed from the ROSA26 locus  to reconstitute irradiated mice. Analysis showed that 6 weeks after tamoxifen treatment there was a small reduction in pre-B and immature B cells in the bone marrow, but a large decrease in mature recirculating B cells in the marrow, as well as transitional, follicular, and marginal zone B cells in the spleen  in which a rearranged VDJ segment flanked by loxP sites is placed in the Ig heavy-chain locus in such a way that the B cells predominantly express this one prerearranged heavy chain  for 5 days. Radiation chimeras were generated using standard protocols. For retrovirus-mediated gene transfer, bone-marrow cells were infected in vitro with retroviruses prior to reconstitution of irradiated recipients. If required, chimeric mice were treated with tamoxifen 6–8 weeks after reconstitution.Splenic B cells were purified by depletion with antibodies to CD43 and CD1d, and lysates were prepared using RIPA buffer. Immunoblotting was performed using standard procedures.Purified splenic B cells were cultured for 4 days with or without BAFF. Absolute numbers of live B cells were assessed using ToPro3 exclusion.+CD93−CD23+IgM+) were sorted from spleens 10 days after start of tamoxifen treatment. RNA was isolated using RNEasy mini kit (QIAGEN) and used for microarray or RNASeq analysis.Follicular B cells (B220∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.All statistical comparisons were carried out using the nonparametric two-tailed Mann-Whitney test. Statistically significant differences are indicated on the figures:


Geomyces destructans (Gd)) that invades the skin of torpid bats. During hibernation, arousals to warm (euthermic) body temperatures are normal but deplete fat stores. Temperature-sensitive dataloggers were attached to the backs of 504 free-ranging little brown bats (Myotis lucifugus) in hibernacula located throughout the northeastern USA. Dataloggers were retrieved at the end of the hibernation season and complete profiles of skin temperature data were available from 83 bats, which were categorized as: (1) unaffected, (2) WNS-affected but alive at time of datalogger removal, or (3) WNS-affected but found dead at time of datalogger removal. Histological confirmation of WNS severity  as well as confirmation of presence/absence of DNA from Gd by PCR was determined for 26 animals. We demonstrated that WNS-affected bats aroused to euthermic body temperatures more frequently than unaffected bats, likely contributing to subsequent mortality. Within the subset of WNS-affected bats that were found dead at the time of datalogger removal, the number of arousal bouts since datalogger attachment significantly predicted date of death. Additionally, the severity of cutaneous Gd infection correlated with the number of arousal episodes from torpor during hibernation. Thus, increased frequency of arousal from torpor likely contributes to WNS-associated mortality, but the question of how Gd infection induces increased arousals remains unanswered.White-nose syndrome (WNS), an emerging infectious disease that has killed over 5.5 million hibernating bats, is named for the causative agent, a white fungus ( Myotis lucifugus), which was once the most common hibernating bat in the American Northeast (NE), has incurred an average of 91% mortality in sites that have been affected for at least two years Geomyces destructans (Gd) grows on the muzzle, wings, and ears of bats suffering from WNS starting in late January/early February White-nose syndrome (WNS) is estimated to be responsible for the deaths of at least 5.7 to 6.7 million hibernating bats in the eastern United States and Canada b) and metabolic rate are both greatly reduced. Because their energetic costs in the subsequent spring are greater than those of males, female little brown bats enter hibernation with higher body mass indexes (BMI) and manage their energy stores during hibernation more efficiently than males b close to ambient temperature (2° to 8° for bats) bFor insectivorous bats that live in northern temperate zones, such as those affected by WNS, food is primarily available from late spring to early autumn and absent during winter. Bats survive this winter energetic bottleneck by building stores of body fat (depot fat) in late summer and early autumn and by conserving metabolic energy through hibernation. In little brown bats, body fat increases from approximately 7% of total mass (∼6 g) during summer to 27% of total mass (∼9 g) prior to hibernation, an increase of 3 g or more in body mass bAlthough euthermic periods account for approximately 1% of the total time budget during winter, about 80–90% of the energy (depot fat) used during hibernation is consumed during these periodic arousals from torpor, because metabolic rate greatly increases with increased Tsk), which correlates well with Tb in small insectivorous bats, and which has been used extensively to study mammalian hibernation We tested the hypothesis that WNS reduces the length of torpor bouts during hibernation in free-ranging little brown bats. We predicted that a primary cause of the increased mortality/disease state associated with WNS is abnormally shortened torpor bouts, due to more frequent arousal episodes, as was shown previously for one affected free-ranging bat in late hibernation This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at Bucknell University (protocol number DMR-02). In the states of VT and WV, research was conducted by state wildlife officials  on non-endangered bats; thus numbered permits were not required or issued. In Michigan, research was conducted each year under MI Scientific Collector’s Permit SC620 from the Michigan Department of Natural Resources to AK. In PA, research was conducted each year under PA Game Commission permits to DMR , in collaboration with GGT, a wildlife biologist for the state of PA. In accordance with the permits and with state wildlife policies, research was either conducted on state land or on private property, with the explicit permission of private landowners.sk) every 30 min and were attached to 504 bats over the course of three winters at six different hibernacula using standard methods Temperature-sensitive dataloggers were programmed to read skin temperature  were collected from a subset of freshly euthanized animals (N = 26). Nucleic acid was extracted from each skin sample using the Gentra Puregene genomic DNA purification kit  per the manufacturer’s instructions (solid tissues protocol), with the following modifications: proteinase K was added to a final concentration of 0.5 mg/ml during the cell lysis procedure and no RNase treatment was performed. To determine presence/absence of DNA from Gd on each sample of wing skin (within the defined sensitivity limitations of the technique used), extracted nucleic acid was analyzed by PCR as previously described by Lorch et al. Wing membrane from these same animals was also analyzed by histology Usable data for our analyses were recovered from 99 of the 504 loggers deployed see . Althougsk was 10°C or more below its highest temperature (Tmax). Duration of an arousal episode (when Tsk was within 10°C of Tmax) was calculated to the nearest 30 min. Although recording Tsk every 30 min was sufficient to detect arousal episodes, it did not provide sufficient resolution to describe precisely the true length of an arousal bout, as arousal episodes averaged less than 90 min in length (see results). Thus, we did not attempt to determine if there were significant differences in arousal episode length by WNS status. Torpor bout length  was defined as the period between two arousal episodes. For both arousal bout length and TBL, values were first averaged for each bat and then averaged across all bats. Data on TBL were log(10) transformed to achieve normality and homogeneity of variance, as determined by Shapiro-Wilk’s test for normality and examination of skew and kurtosis and by Levene’s test for equality of variances. BMI data were normally distributed. TBL data from multiple years are combined in our analysis, which is supported by the lack of a year-to-year difference in TBL in bats from a given hibernaculum when the WNS status did not change between years  = 3.091, p = 0.098; partial eta squared  = 0.162, power  = 0.380). For all analyses, power and effect size are reported for non-significant results. All data are presented as the mean ± standard deviation (SD).Torpor was defined as when a bat’s Tm site 6 : 10.52±1 = 1.111, p = 0.297; partial eta squared  = 0.020, power  = 0.179). Effects of WNS status on TBL were tested with ANCOVA, with BMI (random), site identity (fixed), and sex (fixed) as covariates. Post-hoc examination of sex differences in BMI was conducted using a Student’s t-test .For the initial analysis, bats for which we had data on TBL, BMI, and sex were grouped into three ‘WNS status’ categories: (1) unaffected [N = 57], (2) WNS-affected (as determined by histology and/or visible fungus) and alive at time of datalogger removal [N = 14], and (3) WNS-affected and found dead at time of datalogger removal [N = 12]. Bats were assigned to the ‘unaffected’ category either when the presence of fungal infection with Gd was not detected with PCR or histology [N = 10] or when they were from a hibernaculum presumed to be unaffected and not located in the WNS zone at the time of study [N = 47] . Combinisk <0°C for the first time, since the Tsk of little brown bats always remains above 0°C during torpor 2) was calculated by squaring significant correlations.Within the WNS-affected bats that were found dead at the time of datalogger removal, the relationships between TBL and BMI and date of death were analyzed using Pearson Product Moment Correlations (PPMC) . Date of death was measured as the date on which TUsing a subset of animals for which a ‘WNS severity score’ could be calculated and for which BMI at the start of hibernation was available (N = 26), the effects of severity score, BMI, and site on TBL were examined with ANCOVA. A significant relationship between severity score and TBL was examined using the Gamma Correlation Statistic, which allows for multiple ‘tied rankings’ During the course of this study, when bats aroused from torpor, they remained at euthermic temperatures for a short period, averaging 78.3±27.3 min. The range of average arousal bout length per bat was from 38.18 to 180 min (N = 83 bats), while the shortest recorded arousal bout lasted 30 min (the shortest period that could be discerned by our methods) and the longest 330 min. We were unable to test for differences in arousal bout length in relation to WNS status (or severity score) due to the limited data storage capacity of our dataloggers . = 0.031, p = 0.861; partial eta squared  = 0.000, power  = 0.053). Likewise, we did not detect a relationship between BMI at the start of hibernation and TBL  = 0.140, p = 0.710; partial eta squared  = 0.000, power  = 0.066). Our BMI analyses were not biased by recapture dynamics as there was no significant difference in BMI at the time of datalogger attachment between bats for which loggers were retrieved and bats that were not recovered . However, both WNS-status and site identity significantly influenced TBL. Site identity heavily influenced the model  = 25.027, p<0.001) as two of the sites contained only one category of bat . Despite the strong influence of site identity, a significant WNS status main effect was still apparent  = 7.569, p = 0.007).Although female bats were in significantly greater body condition than males at the start of hibernation (BMI: 0.2284±0.0283 g/mm (N = 32) vs. 0.2073±0.0210 g/mm (N = 51); t = −3.633, adjusted df  = 52.2, p = 0.001), there were no detectable influences of sex on TBL  = 1.491, p = 0.226, partial eta squared  = 0.021, power  = 0.226). However, these affected but alive bats had significantly longer TBLs than WNS-affected bats that were found dead at the time of datalogger collection  = 17.191, p<0.0001). Limited data collected from an additional four WNS-affected bats found dead from a field site where dataloggers were deployed for only eight weeks late in the hibernation season in 2010 are similar with a mean TBL of 6.17±1.79 days , TBL significantly predicted the date of death, explaining 58% of the variance. Similar to the findings of our full ANCOVA, we did not detect a relationship between BMI at the start of hibernation and TBL  or between BMI at the start of hibernation and date of death . While the power to detect significant differences at these low effect sizes (correlation coefficients of 0.178 and 0.026) is extremely low (<0.05), even if they were statistically significant, they are not biologically significant. In each bat, mortality was observed immediately after the last arousal to euthermic temperatures. While several bats  = 0.111, p = 0.743, partial eta squared  = 0.005, power  = 0.062) or site identity  = 2.515, p = 0.104, partial eta squared  = 0.186, power  = 0.045), but was related to severity score  = 6.509, p = 0.018). Bats with more severe fungal infections had significantly shorter torpor bouts , TBL was not related to BMI  Our analysis of WNS severity based upon histological confirmation of the degree of fungal invasion and infection further supported and strengthened our conclusion – as the severity of infection increased, so did the frequency of arousals from torpor. Our data mirror the independently derived mathematical model of Boyles and Willis We also predicted that relationships between WNS and torpor patterns would be influenced by the amount of energy stores available to the bat. In a previous study of little brown bats, BMI significantly influenced hibernation energetics such that bats with lower body masses at the beginning of hibernation selected colder roosting sites, which allows for decreased metabolic rates and thus lower energy expenditure These energetic arguments underlay the model of Boyles and Willis Although statistical analyses confirmed the significance of our findings, studies of behavior and physiology in free-ranging animals are often fraught with unknowns and potential biases, which likely underlie the significant site effects in our statistical models. One potential source of bias in our dataset is BMI at the start of the hibernation season. While one could predict that bats in poorer body condition would find datalogger attachment more physiologically stressful than bats in greater body condition (and thus be less likely to be recaptured), there was no difference in starting BMI between bats that were recaptured and those that were not. Another source of bias in our WNS-affected bats could have been ambient temperature of hibernacula, because TBL generally decreases with increased ambient temperature Collectively, our data indicate that one proximate mechanism of the mortality associated with WNS is decreased TBL. Warnecke et al. In other mammalian hibernators, mechanisms associated with immunity are reduced during hibernation, when the conservation of energy is critical A detailed understanding of the mechanism(s) by which infection with Gd causes mortality in hibernating bats may provide insights to develop interventional strategies to mitigate this unprecedented wildlife disease. Insectivorous bats perform significant ecosystem services because they are primary predators of nocturnal insects Appendix S1Instructions for producing temperature sensitive dataloggers for attachment to bats, including figures.(PDF)Click here for additional data file.Appendix S2Description of WNS histopathology and assignment of wing damage severity scores (SS), including figures.(PDF)Click here for additional data file.


Here, we aimed to evaluate this procedure regarding: i) viability of the rectal specimens obtained by biopsy forceps for ex vivo bioelectrical and biochemical laboratory analyses; and ii) overall assessment  of the rectal forceps biopsy procedure from the patients perspective to assess its feasibility as an outcome measure in clinical trials.Measurements of CFTR function in rectal biopsies We compared three bowel preparation solutions , and two biopsy forceps (standard and jumbo) in 580 rectal specimens from 132 individuals (CF and non-CF). Assessment of the overall rectal biopsy procedure (obtained by biopsy forceps) by patients was carried out by telephone surveys to 75 individuals who underwent the sigmoidoscopy procedure.Integrity and friability of the tissue specimens correlate with their transepithelial resistance  and are influenced by the bowel preparation solution and biopsy forceps used, being NaCl and jumbo forceps the most compatible methods with the electrophysiological analysis. The great majority of the individuals (76%) did not report major discomfort due to the short procedure time (max 15 min) and considered it relatively painless (79%). Importantly, most (88%) accept repeating it at least for one more time and 53% for more than 4 times.Obtaining rectal biopsies with a flexible endoscope and jumbo forceps after bowel preparation with NaCl solution is a safe procedure that can be adopted for both adults and children of any age, yielding viable specimens for CFTR bioelectrical/biochemical analyses. The procedure is well tolerated by patients, demonstrating its feasibility as an outcome measure in clinical trials. Moreoex vivo assessment of CFTR-mediated Cl- secretion channel in freshly collected rectal biopsies [in vivo efficacy on CFTR. Indeed, there is also great potential to exploit this method to pre-clinically assess compound efficacy directly on human tissues ex vivo, as we previously showed [One of the most useful and sensitive laboratory parameters used for the diagnosis and prognosis of CF, is biopsies ,9,12-19.y showed  or as a y showed -23. MoreFor such disseminated usage of this method, standardized operational procedures (SOP) for bowel preparation and biopsing are essential to ensure good tissue viability for the quantitative assessment of the bioelectric parameters . MoreoveOur two-fold aim of the present study was: a) to evaluate the technical procedure regarding the quality for bioelectrical/biochemical laboratory analyses of 580 rectal specimens from 132 individuals (CF and non-CF), namely by comparing different bowel preparations and different biopsy forceps sizes as well as regarding the safety of the procedure to the patient; and b) to determine individuals’ assessment regarding the rectal biopsy procedure feasibility to be possibly used as an outcome measure in clinical trials, as successfully described in other studies ,25.vs standard size) biopsy forceps allowing collection of larger specimens without disrupting tissue integrity. Obtaining rectal biopsies with a flexible endoscope and jumbo forceps is also shown here to be a safe procedure for use in both children and adults (age range was 6 months to 52 years). Data collected on patient’s comfort show that the great majority of individuals (76%) did not report major discomfort due to the short procedure time (max 15 min) and considered it relatively painless (79%), regardless of sedation. Moreover, most individuals accept repeating the procedure for at least one more time (88%) and 53% for more than 4 times, supporting the feasibility of the current approach as an outcome measure in clinical trials.Our results demonstrate that best tissue viability for Ussing chamber measurements results after bowel preparation with isotonic solution (NaCl 0.9%) and usage of jumbo , in accordance with the Helsinki Declaration of the World Medical Association. Signed informed consent was obtained from all individuals . Altogether, 580 freshly excised rectal biopsies were analysed at Unicamp from 132 individuals, including CF patients (n = 67) with previously established diagnosis  (genotypi) saline solution ; or ii) glycerinated solution , for individuals undergoing sigmoidoscopy; or iii) oral mannitol solution  for individuals undergoing colonoscopy  by applying an enema of Superficial 4–6 rectal mucosa specimens (2–4 mm diameter-Ø) were obtained by colon forceps with visual examination Figure , avoidinFrom the 132 individuals enrolled in this study, 63 performed endoscopic procedures under sedation and 69 without. Intravenous sedation with midazolam (associated or not with meperidine) was performed for all individuals undergoing colonoscopy (n = 28) for other reasons than the rectal biopsy procedure .Sigmoidoscopy was done with or without sedation depending if the individual was already performing other procedures  or depending on individuals’ will, collaboration or anxiety . For children under 9 years old, the parents together with the paediatric gastro-endoscopists decided whether it was preferable to have sedation; while the individuals above 10 years old could choose to have sedation or not by themselves, because they had already a better understanding of the overall procedure. For those 35 individuals, sedation was performed as follows: a) intravenous sedation with midazolam (n = 9); b) intravenous anaesthesia with propofol (n = 2); c) intravenous anaesthesia with propofol + alfentanil (n = 5); d) sevoflurane (with nitrous oxide 1:1) inhalation (n = 8); and e) intravenous anaesthesia with propofol + sevoflurane inhalation (n = 11). In general, individuals were monitored for blood pressure, pulse, oxygen saturation and ECG tracing by an anaesthologist. Children could also choose if they wanted their parents with them during the procedure or not.Prior to mounting in Ussing chamber, tissues were macroscopically evaluated regarding bleeding, mucus, biopsy thickness (presence of submucosa or not), and also for friability (tissue breakdown from manipulation), in a scale from 0 to 3 (from absence to clear presence of the descriptor), by two different technicians who were blinded for the individual condition (CF/non-CF), bowel preparation method and biopsy forceps used Figure . RegardiOne out of the 4–6 rectal specimens collected per individual was fixed in 4% formaldehyde, embedded in paraffin and cut in thin sections (2–3 μM) for histological observation and data were obtained for 78 individuals. Sections were then deparaffinized dehydrated in xylene , re-hydrated with absolute ethanol , 95% ethanol (2 min), 70% ethanol (2) min and briefly rinsed with distilled water and stained with hematoxylin-eosin (HE) and Tricome’s Masson as previously . Slides 2PO4 0.4, K2HPO4 1.6, D-glucose 5, MgCl2 1, Ca2+-gluconate 1.3, pH 7.40, at 37°C) as previously described [te were used to assess tissue viability, after appropriate correction for fluid resistance  of two monoclonal anti-CFTR antibodies, M3A7 and MM13-4  recognizing distinct epitopes to increase sensitivity as CFTR has low expression levels in native tissues ,28. TotaIndividuals were approached by telephone by two researchers that did not have contact with the rectal biopsy procedure to evaluate the overall procedure from bowel preparation to collection of the biopsy . Pearson coefficients (r) were used to find correlations and partial correlations between descriptors for macroscopic evaluation and Rte of biopsies. ANOVA, with Bonferroni post hoc correction when appropriated, was used to find differences between groups’ means with a 90% confidence interval. For Crosstabs regarding patients questionnaires, Pearson’s Chi-Square tests were used to determinate independence among variables analysed. Monte Carlo estimates of the exact p-value are provided whenever the data are too sparse or unbalanced for the asymptotic results to be reliable.Statistical analyses were performed with SPSS software  and a A flow-chart summarizing the technical biopsing aspects assessed in the present study is shown in Figure The bowel cleaning procedures, consisting in administration of an oral laxative (for colonoscopy) or by enema (for sigmoidoscopy) allowed equally good visualization of the rectal mucosa and forceps during the sigmoidoscopy/colonoscopy procedure. Expectedly, jumbo biopsy forceps (3.4 mm Ø) generated larger specimens than standard forceps (2.5 mm Ø), thus facilitating the mounting of the tissue in Ussing chamber inserts.Data referring to bowel preparation Table .vs Standard = 2.04 ± 0.18) and also for Rte (Jumbo = 20.97 ± 0.81 Ω.cm2vs Standard = 14.01 ± 0.86 Ω.cm2) are statistically different between the forceps used  and between Rte and friability , which is supported by partial correlations with bowel preparation and biopsy forceps. Data concerning presence/abundance of blood and mucus do not correlate with tissue viability-Rte  or bowel preparation Figure A.te) to assess tissue viability  under a dissecting microscope by two different technicians in blinded way Table . As showty Table .- secretion. As shown in Figure We have also used these native colonic tissues to further look at maturation pattern and localization of wt- and mutant-CFTR protein, which can serve as a valuable tool to evaluate the effects of CFTR-modulators together with Ussing chamber measurements of CFTR-mediated ClNo major complications  were reported following NaCl 0.9% bowel preparation or jumbo biopsy forceps , thus making this a safe procedure. There was only one CF patient complaining about abdominal pain who was observed for 4 h after the procedure, but had no other complications and recovered by then from such pain. We also report other minor complications that cannot be fully related to the sigmoidoscopy procedure, (see Discussion).i) procedural pain/discomfort and sedation requirement ; ii) comparison with other clinical/diagnosis procedures (question 4); and iii) acceptance towards the possible introduction of this method as an outcome measure in clinical trials (question 7). In addition, there was also a question regarding preconceptual concerns or discomfort and pain associated a priori with this procedure (question 6).Patients were asked by telephone to assess the rectal biopsy procedure by posing several questions targeting several aspects of patient assessment . The questions were divided into 3 broad categories comfort show that 57/75 (76%) of the interviewed individuals did not report high levels of discomfort, independently of sedation or age, but shows statistically significant differences p = 0.032) regarding gender , the reported answers were similar: 46.7% for preconception/taboo and 41.3% for discomfort/pain. The remaining 12% did not answer (data not shown).Measurements of CFTR function in rectal biopsy specimens have proven its value in the fine-diagnosis of patients with milder or “non-classical” forms of CF, in particular when sweat test results are equivocal or borderline and/or if CFTR-disease causing mutations are not readily identified by DNA mutation analysis ,14,16. TMoreover, this technique has even potential for a much wider scope, namely to monitor diseases, affecting epithelial ion transport -31. AlthBecause functional and biochemical data are not available for most rare mutations, these specimens can also be used to establish functional and biochemical correlations with rare CFTR genotypes Figure B, 3C, asSince good tissue viability is critical for quantitative assessment of bioelectric responses our recommendation is that specimens are maintained in appropriate medium on ice until used in functional measurements, which should be performed immediately . To miniThe present study aimed to determine whether and how bowel preparation for sigmoidoscopy and choice of biopsy forceps influence tissue viability for subsequent laboratory analyses, in particular, bioelectric measurements. We compared two commonly used solutions for bowel preparation at our endoscopy unit, namely glycerol and isotonic saline enema (for sigmoidoscopy) as well as oral mannitol (for colonoscopy). Our data show that isotonic saline solution, which we speculate is less harmful for the mucosa, is superior to glycerol-based preparation, but there are no significant differences between NaCl-enema and oral mannitol. Indeed, differences were detected between mean values for the tissue integrity parameter between biopsies obtained after NaCl- and glycerol-based bowel preparations, the former showing better results  are comparable with those found previously using similar (forceps) [2 - or even different (suction) techniques [2, and 24–39 Ω × cm2 . That approach is thus expected to be equally tolerated by the patients’ while also providing adequate specimens for electrophysiology studies. Here, we did not compare suction and forceps biopsy, but just the forceps size (standard vs jumbo forceps) in terms of how sample size might affect its viability and also regarding safety of the procedure. Overall, mean values for tissue integrity (jumbo = 1.07 ± 0.09 vs standard = 2.04 ± 0.18) and also for Rte (jumbo = 20.97 ± 0.81 Ω.cm2vs standard = 14.01 ± 0.86 Ω.cm2) were shown to be statistically different   - 24 Ω ×chniques ,37: 27 Ωte measurements) and are influenced by bowel preparation and biopsy forceps  with tissue viability (assessed by Rps Table . Our datte), but interestingly there is a trend positive correlation between mucus and Rte (r = +0.078), indicating that presence of mucus (i.e. probably resulting from a less “aggressive” bowel cleaning) could somehow serve to preserve tissue viability (Table Data concerning presence/abundance of bleeding and mucus do not correlate with tissue viability (Rty Table .te (21.82 ± 1.03 Ω.cm2) and the best for tissue integrity (1.00 ± 0.11). On the other hand, usage of 12% glycerol enema and smaller (standard) forceps produce the less viable tissues, namely (Table te (13.57 ± 2.70 Ω.cm2), and highest friability (1.81 ± 0.22), thus rendering quantitative determination of CFTR-mediated Cl- secretion less reliable. It is likely that usage of the glycerol-based enema, acting as an osmotic laxative and thus increasing the luminal volume, which stretches the mucosa, makes it more susceptible to friability and disruption, compromising tissue integrity. Alternative for bowel preparation in children could be sodium citrate + sodium lauryl sulfoacetate, together with glycerol + sorbitol (“Microlax®”) or dioctyl sulfosuccinic acid sodium salt + sorbitol (“Clyss-go®”), which we had previously experienced to yield viable specimens if done the day before sigmoidoscopy (data not shown), probably allowing rectal mucosa to recover from this enema procedure. However, as these procedures were not rigorously assessed, we cannot compare them we the ones used in the present study.Therefore, performing bowel preparation with isotonic saline and obtaining the rectal biopsies with jumbo forceps are demonstrated to constitute the best combination for the procedure Table  with theThe easy access to the patients rectum and the low innervation of this area minimizing pain, makes this approach to be expectedly well tolerated ,14. MoreRegarding safety of the patients, one CF patient who complained of abdominal pain (see Results) was the only patient with inappropriate bowel cleaning, where more insufflation had to be used in order to carry out the procedure. This patient also had a clinical history of surgical interventions for meconium ileus and colonic adhesions, which might be related to the abdominal pain. We also report minor complications that cannot be fully related to the sigmoidoscopy procedure: one patient who vomited  and another patient who underwent both upper endoscopy and sigmoidoscopy also reported pain, but it is likely that the former procedure was the source of pain.Thus, the present study also shows that this procedure with jumbo forceps is safe to be applied from young children to adults (age range was 6 months to 52 years), which application is relevant also for other disorders as Hirschsprung or inflammatory bowel disease. Although we have no experience, it also expected that it can be safely applied to newborns, namely those identified in increasing numbers as asymptomatic CF patients by the recently implemented extensive newborn screening programs, merely based on elevated serum concentrations of immunoreactive trypsinogen (IRT). Indeed, these patients, posing new challenges to the CF diagnosis and prognosis are likely candidates to undergo this procedure to find evidence of CFTR (dys)function.One of the major limitations usually related to patient surveys, is that we can only rely on the responses of the individuals who agree to undergo certain procedures. In this case, we are relying on the perspectives of the patients who agreed to participate on the sigmoidoscopy/rectal biopsy procedure, so there is a selection bias that leads to underscoring the fraction of individuals who do not tolerate this kind of procedure. Nevertheless, we can only rely on the responses of the individuals who agreed to participate, as those who have not undergone the procedure can only have preconceptions and not real experience to assess it. Importantly, patient enquiries demonstrate that for the majority of the individuals (76%) the rectal biopsy procedure is not associated with high levels of discomfort due to the short procedure time  of rectal biopsies vs. biopsy forceps. Table S3 – Summary of macroscopic evaluation data and bioelectrical measurements (Rte) of rectal biopsies vs. presence of sub-mucosa. Table S4 – Summary of macroscopic evaluation data and bioelectrical measurements (Rte) of rectal biopsies vs. sedation. Table S5 – Comparison of the rectal biopsy procedure with other clinical/diagnosis procedures (n = 75). 6 Table S6 – CF patients and CFTR genotypes (n = 67). Figure S1 Rectal biopsy procedure patient assessment questionaire. Summary of macroscopic evaluation data and bioelectrical measurements. (Rte) of rectal biopsies vs. bowel preparation. Click here for fileSupplementary Methods.Click here for file Rectal biopsy procedure.Click here for file


In the present study we aimed to analyze the bacterial community structure of oral biofilms at different maturation stages in young healthy adults. Oral biofilms established on membrane filters were collected from 32 human subjects after 5 different maturation intervals  and the respective phylogenetic diversity was analyzed by 16S rDNA amplicon sequencing. Our analyses revealed highly diverse entire colonization profiles, spread into 8 phyla/candidate divisions and in 15 different bacterial classes. A large inter-individual difference in the subjects’ microbiota was observed, comprising 35% of the total variance, but lacking conspicuous general temporal trends in both alpha and beta diversity. We further obtained strong evidence that subjects can be categorized into three clusters based on three differently occurring and mutually exclusive species clusters. High-throughput sequencing approaches have revolutionized our prospects and knowledge on microbial communities. For instance the human microbiome with its high variability between host individuals and distinct species assemblies on different parts of the human body was extensively examined To evaluate potential shifts in bacterial biofilm composition during maturation and due to different bacterial colonizer pools in various hosts, we investigated oral bacterial colonization profiles close to the teeth in 32 young healthy adults  after 5 different maturation intervals between 1 and 14 days, by 16S rDNA amplicon sequencing using a high-throughput sequencing approach.All procedures related to subjects were approved by ethics committee of Hannover Medical School, registered at the International Clinical Trials Register Platform of the WHO (ID: DKRS00003366) and follow the guidelines of the Declaration of Helsinki. All participants gave written informed consent and were recently involved in our earlier study that investigated the systemic effects of experimental gingivitis.Human subjects agreeing following criteria were selected: (a) 20–30 years of age, (b) non-smokers, (c) no clinical signs of gingival inflammation, (d) no probing pocket depth >3 mm at any site and (e) no alveolar bone loss. Criteria for exclusion were: (a) systemic diseases (e.g. diabetes mellitus), (b) pregnancy or breastfeeding, (c) history of drug abuse, (d) allergic diathesis, (e) medications (e.g. antibiotics) within 3 months before entering the study, (f) untreated carious lesions and/or insufficient restorations, implants, crowns and (g) mouth breathing.In order to obtain sufficient plaque material for sequencing maxillary and mandibulary impressions were obtained and used to fabricate individual acrylic splints. Membrane filters (MilliporeExpress® PLUS) were attached to the splints next to the teeth and gingiva by surgical sutures. In each case four membranes on the upper and four on the lower jaw on buccal and oral sides of the teeth rows. Participants were advised to carry these splints for the time intervals 1, 3, 5, 9 and 14 days and to store them in provided humidified chambers during the meals and daily oral hygiene procedures. On every sampling day all 8 filters were removed from splints, washed with sterile PBS and immediately frozen in liquid nitrogen until further processing.Genomic DNA of dental plaque was isolated from a pool of 2 different membrane filters using the RTP® Bacteria DNA Mini Kit  following the manufacturer`s instruction (protocol 4).CTATGCGCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG3′) and barcoded reverse primer 338R (5′CGTATCGCCTCCCTCGCGCCATCAGXXXXXXXXXXCATGCTGCCTCCCGTAGGAGT3′). The primer contained the 454 Life Sciences Adaptor B (forward) and A (reverse) denoted by italics and the underlined sequences represent the broadly conserved bacterial primers 27F and 338R. A two-base linker sequence (TC/CA) and four-base key (TCAG) were added as recommended by Roche (454). A unique 10mer multiplex identifier (designated X) was added to every reverse primer to tag each PCR product. Template DNA (100 ng) was added to a 25 µL PCR reaction mix including Phusion Hot Start DNA Polymerase . Cycling conditions started with an initial denaturation step for 30 s at 98°C, followed by 35 cycles of 9 s at 98°C, 30 s at 55°C and 30 s at 72°C and ended with a final extension for 10 min at 72°C. All reactions were performed in duplicates and combined after PCR. Amplicons were size-checked, purified with the MiniElute Gel Extraction Kit  and quantified with the Quant-iT dsDNA Broad-Range Assay Kit  using a NanoDrop 3300 fluorometer. Equimolar amounts of purified PCR product were pooled and further purified using Agencourt AMPure XP . A sample of each library was run on an Agilent Bioanalyzer prior to emulsion PCR and sequencing as recommended by Roche. Amplicon libraries were sequenced on a 454 GS-FLX using Titanium sequencing chemistry .The hypervariable regions 1 to 2 (V1–2) of the 16S rRNA gene was amplified from isolated genomic DNA using universal primer Pyro_27F (5′trim.flows). Data were denoised (shhh.flows) and kept if their mean phred score trim.seqs). Sequences were aligned  to the SILVA reference alignment based on the SSURef database (v102) of bacterial sequences http://www.mothur.org/wiki/Silva_reference_files. The alignment was optimized by deleting the shortest and longest 2.5% of the sequences, respectively (screen.seqs), followed by removal of gap-only columns as well as columns containing missing data at both ends of the alignment (filter.seqs). The resulting alignment had 696 positions with sequence lengths ranging from 239 to 315 nucleotides. In order to further reduce PCR/pyrosequencing errors, reads with one mismatch to a more abundant sequence were merged with the latter (pre.cluster). Chimeras were eradicated employing mothur’s implementation of Perseus chimera.perseus, remove.seqs). Initial sequence classification was conducted by a Bayesian approach classify.seqs). The RDP http://www.mothur.org/w/images/4/4a/Trainset7_112011.pds.zip) was used with reference sequences trimmed to the V1–V2 primer region to improve accuracy of the classification [cf. remove.lineage) as these sequences would obviously have originated from vegetable food rather than from bacteria. A random subset of 1,042 sequences per sample  was generated (sub.sample) to eliminate bias due to unequal sampling effort. A coverage of about 1,000 sequences per sample is suggested as a good balance between number of samples and depth of sampling dist.seqs) for binning sequences into operational taxonomic units (OTU) by average neighbor clustering (cluster.split). Based on a 97% similarity threshold (roughly corresponding to species level distinction) 1,779 OTUs were identified. A sample-by-OTU table was generated (make.shared) which for each sample states the number of sequences belonging to a certain OTU. This table was the basis for subsequent bioinformatic analyses. OTUs were classified according to the CORE reference database, a phylogenetically curated 16S rDNA database of the core oral microbiome classify.seqs, followed by classify.otu).All steps of sequence processing were conducted with the program mothur v1.27.0 All downstream computations were performed in R v2.15.2 1D) 1D was fitted to “Day” in a generalized linear mixed model using function gamm of R package mgcv et al.Effective OTU richness  and consisted of the fixed effect “Day” and the “Subject” effect as conditioning term: OTU count data ∼ “Day” + Condition (“Subject”). The model was tested for significance with function anova.cca using 1,000 random permutations stratified within subjects. Homoscedasticity of the fixed effect was assessed using vegan’s betadisper function, followed by permutest with permutations stratified within subjects.Redundancy Analysis (RDA) was used to explore the extent of change in OTU composition  in oral microbial communities within subjects over time. OTU abundances were subjected to Hellinger transformation in order to make them compatible with RDA. This transformation downweights highly abundant OTUs, providing a good compromise between linearity and resolution wascores) function for projection of species points into the PCA space of samples . Consistent clusters of species were determined by average-linkage clustering based on Spearman rank correlations of relative species abundances. Cluster number and robustness were assessed by consensus clustering OTU abundances were normalized by total counts per sample and summarized at species level . Taxa that could not be distinguished at the species level or with terms “uncultured” or “unclassified” added to their names were removed from the dataset. Species relative abundances were used for the resulting sample-by-species table which was subjected to Principal Component Analysis (PCA) after Hellinger transformation. Based on this ordination configuration, weighted-average scores of species were calculated by vegan’s  was subjected to consensus clustering as described above with 10,000 random subject subsamples, using Partitioning Around Medoids A 3D plot of combined clustering and ordination data was produced in kinemage format Sequence data were submitted to the NCBI  Sequence Read Archive under accession no. SRP027013. Table S1 of Oral biofilm formation on membranes close to the gingiva was monitored in 32 healthy adults over 14 days. Aiming to examine the bacterial biofilm composition during the establishment, five different time points in biofilm formation were collected . A total of 160 biofilm samples were analyzed after DNA extraction and amplifying the hypervariable region V1–V2 of the 16S rDNA by 454 pyrosequencing. 1,042 sequences per sample were retained after random subsampling, binned into a total of 1,779 operational taxonomic units (OTUs) of which about only 15 were highly abundant across all samples  exhibited a conspicuously low level of subjects . Since m1D. Initial data visualization did not reveal conspicuous general trends in 1D with time  as the best choice. While the model was valid , the effect of variable “Day” was not significant (p = 0.13), thus no time-related change in alpha diversity of oral microbiota was observed.As a measure of alpha diversity, we employed effective OTU richness 2 (a measure for the variance explained by the RDA model) was only 0.5%. Hence there was no obvious and consistent time-related change in relative OTU abundances within subjects. In contrast, a comparatively large portion of variance (35%) was attributed to the conditioning term “Subject”, arguing for a large inter-individual difference in microbial composition.In beta diversity analysis, Hellinger-transformed OTU counts were subject to RDA with “subjects” as conditioning term to account for inter-individual variation. The effect of “Day” on the change of individual OTU abundances between samples of the same subject was not significant (p≈0.1). Accordingly, adjusted R−5). The magenta-colored species cluster was dominated by different Prevotella species (“Prevotella cluster”), the green one contained β- and γ-Proteobacteria (“Proteobacteria cluster”) and the orange-colored cluster mainly contained Streptococcus species (“Streptococcus cluster”). The “Prevotella cluster” and the “Streptococcus cluster” showed a strong multivariate correlation  and mutually exclude each other. A weak but still significant multivariate correlation  was observed between the “Proteobacteria cluster” and the contrasting “Prevotella cluster”. No significant correlation could be observed between the “Proteobacteria cluster” and the “Streptococcus cluster”. Prevotella cluster” (60%) and “Streptococcus cluster” (40%). In the second subject cluster the “Streptococcus cluster” dominated (66%), followed by members of the “Prevotella cluster” (32%) and a minority of the “Proteobacteria cluster” (2%). The third subject cluster exhibited the largest occurrence of the “Proteobacteria cluster” (27%), while the “Prevotella cluster” represented about half of the counts (54%) and the “Streptococcus cluster” was least abundant (20%).Since alpha and beta diversity analysis did not show any significant time-related response in our data, we concentrated on evaluating correlations between individual species across all data points. For this purpose, we employed Spearman rank correlation for species abundances combined with consensus clustering - a simple and straightforward method of inferring robust clusters by resampling - to obtain robust results. A combined consensus clustering and unconstrained ordination analysis  revealed three clusters of species mutually excluding each other across subjects and time points . These tin vivo studies Oral microbial profiles in health and disease have been investigated in numerous Within the 16S rDNA amplicon sequencing approach of oral biofilms we analyzed a highly diverse dataset with more than 1,700 different OTUs at the 97% similarity level, although only 15 of them were highly abundant across all samples. The number of OTUs within each sample ranged from only 7 up to 130 consequently already suggesting a large variation between individuals. Further evidence for large inter-individual differences in microbial composition was obtained by RDA. This is in accordance with previous findings that the oral community is especially diverse in contrast to other body habitats, e.g. the vagina We did not detect any systematic time-related patterns, neither in alpha nor in beta diversity. This suggests that both, the cumulative number of OTUs as well as their individual turnover in a subject, is influenced more by random (or unaccounted/unknown) effects than by time in the course of biofilm formation between 24 h and 14 days. These findings are in agreement with previous reports that propose a high variability in the oral microbiome between individuals The most remarkable result obtained from our data set is the clustering of species into three groups that mutually exclude each other and differ in their relative abundances in three corresponding subject clusters . To our Streptococcus cluster” and “Prevotella cluster”. Mutualistic interactions as well as interspecies competition among microbial partnerships are often triggered simultaneously by several members of oral biofilms and are in the focus of current research Porphyromonas gingivalisP. gingivalis has not been detected in our dataset, this species is known to coaggregate with Prevotella intermediaPrevotella cluster”. Moreover, the “Prevotella cluster” included other Porphyromonas spp. Hence, our study provides additional evidence for the reciprocal exclusion of streptococci and Porphyromonas spp.In particular, the strongest exclusionary relationship was observed between the “et al. S. sanguinis and S. anginosus, on P. intermedia. Further studies of Stingu and collaborators showed that the development of periodontitis is associated with an increased colonization of two Prevotella species (P. intermedia and P. nigrescens) as well as a reduced establishment of S. sanguinisOur results of cluster analyses are further in agreement with previous findings obtained by a co-culture experiment with cluster-representative species. Herein, Stingu Streptococcus ssp. and Parvimonas micra), as well as numerous Gram-negative species . Remarkably, all those bacteria are present in the identified “Prevotella cluster”. Moreover, another study Capnocytophaga ssp., which is also a member of the “Prevotella cluster”, on the onset of gingivitis and identified Prevotella spp. in areas with established gingivitis. On the other hand, species that are associated with periodontal health - such as primary or early colonizers  Streptococcus cluster”. Since we know from our earlier study with the same subject cohort Despite of the high inter-individual microbial variation in oral biofilms, our results revealed the existence of various colonizer pools in different hosts. We assume that even in health, the oral bacterial community features a broad potential compositional spectrum. The obtained composition of the oral biofilm of a certain individual may be indicative for this person’s ability to resist pathogens or disease susceptibility. Although all species in the identified clusters are members of the oral commensally community, there is evidence that some of these microorganisms are associated with gingivitis Prevotella cluster”. The role of this cluster consisting primarily of aerobic β- and γ-Proteobacteria remains to be elucidated. However, in sufficient quantity it appears to replace the “Streptococcus cluster”.Another conspicuity is the third subject cluster that consists of only three test persons. This cluster was shown to host a substantial proportion of the “Proteobacteria cluster”, which exhibits another - albeit weaker - contrasting relationship with the “Due to the high inter-individual variability in microbial composition, an optimized study design is crucial to observe a potential time effect in oral biofilm formation. Even though our subject group was quite homogenous in terms of age and health status, several systemic and external factors such as diet, gender, hormone status and stress known to have an impact on the oral microbiome were not controlled for in the present study. This contributed to the observed highly diverse colonization profiles. A subsequent investigation with a stricter control of the subject set-up, optimally over periods longer than 14 days, may reveal clearer temporal trends in alpha diversity. Since a potential correlation between the oral microbiome and inflammatory diseases such as gingivitis might exist at the strain or even at the genomic/metabolic level, this correlation may not be reflected by 16S rDNA based phylogenetic analysis even after optimization of the experimental set up. Consequently, more advanced tools such as shotgun metagenomics or metatranscriptomics In summary, our results provide evidence that, despite high microbial diversity in individuals, different consortia composition of oral microbiota exist in healthy hosts. External factors as well as physical predisposition of the host might be responsible and determine the structure of these consortia. Moreover first indications were obtained that community structures might be indicative for host’s disease susceptibility.File S11D is shown within individual subjects over time. Fig. S2: Kinematic image representation of the combined consensus clustering and ordination (PCA) of robust species and human subjects shown in http://kinemage.biochem.duke.edu/software/king.php) is needed to view the image.Table S1: Multiplex identifiers and corresponding information on samples in the respective sequence libraries. Table S2: OTU counts in subjects over time. Table S3: OTU taxonomy. Table S4: Bacterial abundances at class-level in subjects over time. Table S5: List of species names in species clusters. Fig. S1: Alpha diversity analysis. Effective OTU richness (ZIP)Click here for additional data file.