Texture analysis is an alternative method to quantitatively assess MR-images. In this study, we introduce dynamic texture parameter analysis (DTPA), a novel technique to investigate the temporal evolution of texture parameters using dynamic susceptibility contrast enhanced (DSCE) imaging. Here, we aim to introduce the method and its application on enhancing lesions (EL), non-enhancing lesions (NEL) and normal appearing white matter (NAWM) in multiple sclerosis (MS).We investigated 18 patients with MS and clinical isolated syndrome (CIS), according to the 2010 McDonald's criteria using DSCE imaging at different field strengths (1.5 and 3 Tesla). Tissues of interest (TOIs) were defined within 27 EL, 29 NEL and 37 NAWM areas after normalization and eight histogram-based texture parameter maps (TPMs) were computed. TPMs quantify the heterogeneity of the TOI. For every TOI, the average, variance, skewness, kurtosis and variance-of-the-variance statistical parameters were calculated. These TOI parameters were further analyzed using one-way ANOVA followed by multiple Wilcoxon sum rank testing corrected for multiple comparisons.Tissue- and time-dependent differences were observed in the dynamics of computed texture parameters. Sixteen parameters discriminated between EL, NEL and NAWM (pAVG = 0.0005). Significant differences in the DTPA texture maps were found during inflow (52 parameters), outflow (40 parameters) and reperfusion (62 parameters). The strongest discriminators among the TPMs were observed in the variance-related parameters, while skewness and kurtosis TPMs were in general less sensitive to detect differences between the tissues.DTPA of DSCE image time series revealed characteristic time responses for ELs, NELs and NAWM. This may be further used for a refined quantitative grading of MS lesions during their evolution from acute to chronic state. DTPA discriminates lesions beyond features of enhancement or T2-hypersignal, on a numeric scale allowing for a more subtle grading of MS-lesions. The search for novel imaging biomarkers in multiple sclerosis (MS) has modified the concepts of neuroimaging from identifying imaging sequelae of demyelination on conventional MR-images towards strategies capable of examining functionality and pathophysiology of the MS brain. A recent technique that contributed to a better understanding of vascular changes in MS, is dynamic susceptibility contrast enhanced (DSCE-) MRI. DSCE-MRII identifies various patterns of impaired perfusion, either in non-enhancing MS lesions or NAWM, compared to healthy controls. On the other hand, inflammatory activity is accompanied by increased perfusion in lesions during the acute phase of the disease, compared to NAWM Local perfusion changes in MS lesions are currently interpreted as a consequence of local inflammation-mediated vasodilatation – a phenomenon secondary to hyperemia and blood congestion within the brain parenchyma The contribution of vascular changes to the generation of MS lesions in MS is still a matter of debate. Currently, it remains to be elucidated whether alteration in CNS perfusion in MS is a cause or rather a consequence of disease pathogenesis. From a methodological point of view, current concepts of perfusion imaging have mainly addressed vascular changes that are described either by the time needed for a contrast agent to pass the vasculature (MTT), the total volume of blood within the cerebral vasculature during the passage of a contrast bolus (CBV), or the amount of blood that perfuses the brain per time unit (CBF) A potential alternative to leakage modeling is the analysis of the tissue of interest by means of texture analysis of the MR-images at different time points along with the bolus passage of Gadolinium. Texture parameters Recently a novel approach has been proposed, namely Dynamic Histogram Analysis (DHA) where a simple form of texture analysis is applied to a time series of DSCE-MRII-images Patients with clinical isolated syndrom (CIS) or MS diagnosed according to the 2010 McDonald's criteria, with or without immunomodulatory therapy, were included if at least one EL was present on MRI –1 bodyweight. Patients were positioned comfortably in the head coil and padding on either side of the head was used to help immobilization. Further the intravenous line with a long tube was put before acquisition to avoid unnecessary MRI table moving during examination.DSCE- imaging was performed on our institutes' Siemens MR-scanners using standard echo planar imaging (EPI) sequences at 1.5 T and 3.0 T. For our 1.5T scanner a TE of 47 ms and a TR of 1440 ms was used. For our 3T scanner we had a TE of 32 ms and a TR of 1500 msec. At both field strengths a FOV of 230 mm×230 mm was used with a slice thickness of 5 mm. A total of 19 parallel images with a time resolution of 1500 ms were acquired on our 3.0T system, and a total of 12 parallel images with a time resolution of 1440 ms were acquired on our 1.5T scanner. At 3.0T a total of 80 different time points were acquired, whereas on 1.5T a total of 40 different time points were sampled after bolus application. All patients received Gadobutrol (Gadovist™) 0.1 ml kgdifference images: These difference images are computed from the difference between the first steady state EPI image and images recorded during bolus arrival, passage and reperfusion. In mathematical terms, let The image analysis method we applied focuses on DSCE-tter see . Gray mael range , for eveOur study sample has been recorded on three MR scanners runnung at with 1.5 and 3T of the same vendor using the same DSCE-Perfusion sequence. Differences in field strength, patient head size, and head positioning lead to significant differences in RF-coil-load and thus to changes in MRI-voxel intensities Normalization for these differences was performed by setting a user defined region encircling a specific tissue of interest (TOI) to a certain value. The normalization constant Tissue concentrations rves see  as a funBase line period (BLP): time interval between the first measureInflow period (IFP): time interval between Outflow period (OFP): time interval Reperfusion period (RPP): time interval between For the normalization of inter-patient differences due to differences in cardiac ejection fraction the area-under-the-time-difference-curve The time points demarking the four time intervals  are determined automatically by the developed program by numerical analysis of the time dependent bolus passage function. Due to differences in timing caused e.g. by differences in cardiac function, only separately time averaged values over the four defined time intervals as defined above  can be adequately compared.This section shortly describes the computed histogram based on TPMs. Original EPI MR image time series: TPM-ORIG The original increase during the RPP and OFP in ELs with a disrupted BBB. The leakage effect during the OFP and RPP is illustrated in 1. entified . In caseDifference image time series: TPM-DIFF The brightness of a voxel is proportional to its Gadobutrol concentration for the difference image series decrease during the first passage of the contrast agent and reperfusion  were defined by a board certified radiologist (RV) with 12 years of experience in the diagnostic of MS lesions. The following selection criteria were maintained: (a) a contour was drawn around the complete hyperintense NEL in an image of the BLP; (b) a contour was drawn enclosing the hypointense part of the EL at the end of the RPP, i.e. in the last image of the time series; and (c) the NAWM contour was drawn in the WM at the end of the IFP, i.e. within the time period where the best GM/WM contrast was obtained. For every patient at least one image covering one or more lesions was analyzed, and a maximum of 3 EL and 3 NEL lesions was not exceeded. A total of 37 TOIs were defined in NAWM, 27 TOIs in ELs, and 29 TOIs in NELs. In most cases lesions and NAWM were analyzed on the same slice. Since in a few cases only one lesion and the corresponding NAWM were analyzed, the number of NAWM measurements exceeds the number of EL and NEL measurements. Four out of 27 ELs were surrounded by vasogenic edema see . AdditioData analysis included the following workflow steps: (a) launch DTPA analyzer tool; (b) mount examination and load DICOM DSCE image-series (TPMs are computed and displayed); (c) draw contour in NAWM and press normalize button ; (d) define the TOIs by drawing contours around EL, NEL and NAWM contours and press reporting button; all statistical parameters are written to text files that can be easily imported into a statistical analysis application; and (e) per TPM with three different tissue types defined, six parameters were determined for each tissue type, and for each of the four time intervals separately, making a total of 72 parameters, where 72 independent Wilcoxon-tests were performed in order to test for differences between the tissue types.http://www.rstudio.org/) was used for the statistical analysis of the data. In this explorative study we investigated (a.) which dynamic texture parameters discriminate between NAWM, NEL and EL, and (b.) during which time interval  these tissue types show most discriminative properties. The distribution of all texture parameters was examined in advance, in order to be able to select the correct test statistic. Since the texture parameters that we determined were not normally distributed, we employed a one way ANOVA followed by Wilcoxon sum rank tests. If the p-value of the ANOVAs F-test satisfied the condition p<0.05, the multiple Wilcoxon sum rank tests between NAWM, NEL and EL were performed. The p-values of these tests were corrected for multiple Wilcoxon sum rank testing by setting the “BH” option, which stands for the Benjamini, Hochberg, and Yekutieli method to control for false discovery rate. The false discovery rate of this method is a less stringent condition than the family-wise error rate, so these methods are more powerful than e.g. the Bonferroni test.The statistical software package R-Studio , acute disease exacerbation and symptomatic EL are presented in Overall, the differences in tissue types between between EL, NEL and NAWM are predominantly expressed during RPP (62 out of 144 parameters were discriminative). The TPM types with least significant discrimination between the TOIs are the TPM-SKEW, with only (5 TPs), and TPM-KURT with 10 significant different TPs.−11) during outflow and reperfusion. NEL were discriminated from NAWM by three parameters during outflow . The best discrimination between EL and NAWM and NEL and NAWM was achieved by the TPM-VAVA during reperfusion. For discrimination between NEL and EL, the mean TPM during reperfusion was the best discriminator (p<9.7×10−6), followed by mean TPM during outflow (2.8×10−5). Notably, the TPM-VAVA contributed less to the discrimination of NEL and EL during outflow (p<0.03) and reperfusion (p<2.5×10−5). During the inflow period, the mean TPM was the best discriminator between NAWM and EL (4.4×10−5), while TPM-VAVA most reliably discriminated between NAWM and NEL (2.5×10−5). In summary, the mean TPM could be used as a discriminator between EL, NEL and NAWM during inflow and outflow, while TPM-VAVA discriminated during the outflow and reperfusion period. The TPM-VAR, the TPM-SKEW and TPM-KURT failed to discriminate between the different tissue types.As an example, In In this paper, we suggest a novel strategy towards in-vivo tissue characterization of ELs, NELs and NAWM in multiple sclerosis. The goal of this pilot study was to investigate the time dependent behavior of statistical texture parameters derived from time series of DSCE difference images. While previous studies aimed at either static texture analysis or description of cerebral blood flow, cerebral blood volume and blood-brain barrier permeability, we aimed to analyze specific texture parameter constellations that differentiate between ELs, NELs and NAWM by analyzing DSCE image time series data. We further analyzed whether these texture parameters evolve as a function of time intervals during bolus passage, and whether these differences are sufficiently large to enable a quantitative grading of the perfusion-related texture of MS lesions. Three points stand out from this study: (a) the voxel intensity distributions of tissue types in the DSCE (difference) image series vary as a function of time during bolus passage; (b) histogram based texture parameters reveal characteristic time responses for ELs, NELs and NAWM; and (c) different tissue types can be discriminated by statistically significant difference in histogram based texture parameter signatures.T2 lesion load in MS may thus be influenced by microscopic inflammation that contributes more strongly to disability The key finding of our study – that DTPA detects subtle differences in time dependent contrast agent distributions otherwise obscured by visual inspection of T1w CE images - implicates that quantitative differences in voxel intensity distributions of the examined DSCE difference images are related to alterations in micro-circulation and BBB properties of the investigated tissue types. The BBB constitutes a dynamic interface forming a neurovascular unit that controls the supply of nutrients while shielding neurons and glial cells off from potentially harmful substances. In MS, GD-DTPA diffuses into the nervous system along the leaky BBB, and conventional MRI provides only a gross estimate of tissue damage. Leukocyte infiltration into the perivascular space is mainly driven by BBB disruption and parenchymal inflammation, although some authors demonstrated passive diffusion of hydrophilic molecules and leukocyte recruitment in post-capillary segments quantitative information by post-hoc analysis. Different texture parameters have been suggested in the literature: (a) histogram-based parameters; (b) gradient-based parameters; (c) run length-based parameters; (d) co-occurrence matrix-based parameters; (e) auto regressive model-parameters; and (f) wavelet parameters histogram based texture parameters  is restricted to explore whether differences between ELs, NELs and NAWM can be extracted directly from DSCE (difference) images. To the best knowledge of the authors, texture analysis has not been applied previously to time series of DSCE-MRI (difference) images aiming to characterize tissue response to a bolus of Gd-containing contrast agent as a function of time.Texture analysis applied to MR-images enables such an extraction of different static and dynamic tissue properties) during bolus passage and the difference image time series (TPM-DIFF) and its derived TPMs. In the latter, the different structural pathological components that influence the static signal components of the tissues are canceled out by the subtraction of the raw image at Previous studies have examined the feasibility of texture analysis to differentiate ELs (88%) and NELs (96%) with high sensitivity Potential relevance of TPMs for the clinical evaluation of MS lesions:difference in TPM-DIFF (or TPM-AVE) parameter between IFP and RPP (indicated by ΔTPM-DIFF) obtrudes itself as a surrogate marker to grade the MS-lesion state: the higher the value of ΔTPM-DIFF, the larger is the grade of leakage. The ΔTPM-DIFF-value allows grading of the leakage of the MS-lesion on a continuous numeric scale, instead of an ordinal scale known from T1w imaging (enhancing/non-enhancing). Further clinical studies are necessary to validate the added value ΔTPM-DIFF for the predictive evaluation of disease progression.The TPM-ORIG shows monotonously increasing values for ELs during IFP, OFP, and RPP. Yet, this TPM did not discriminate ELs from NELs due to: (a) hypo- and hyper-perfused ELs; and (b) large variations in leakage among ELs; the latter is demonstrated by the TPM-DIFF in difference in the numeric values obtained for the TPM-VAVA between IFP and RPP, and is indicated by ΔTPM-VAVA. The smaller ΔTPM-VAVA, the more severely inflammatory is the MS-lesion. This also applies to ΔTPM-STDEV and ΔTPM-VAR. Again, further research must be done to find out whether these parameters correlate with disease activity.These TPMs are all measures of heterogeneity of the examined tissues. For IFP, OFP and RPP the values of the ELs were < NELs < NAWM. TPM-VAR/TPM-STDEV values increase slowly from IFP towards RPP for ELs, and strongly decrease for NELs and NAWM. For TPM-VAVA we observe reductions as a function of time for all three tissue types. A second DTPA-derived numeric MR marker is proposed from the Due to their low SNR (see discussion below) the comparison of the mean values between the different tissues did not reach statistical significance. TPM-SKEW decreased over time in ELs, NELs and NAWM. ELs showed the highest variation in its values including a sign reversal during the late stages. TPM-KURT decreases for NAWM and ELs towards RPP, whereas the ELs increased. For the used and kernel size. In this study we choose a kernel size of This study aimed to introduce DTPA and to demonstrate its feasibility in a clinical setting. The female preponderance in our cohort is determined by the epidemiology in MS. The incidence of MS in women is doubling males, and there is still a ongoing disproportional increase Finally, this study only examined a limited number of texture parameters and focused on histogram based texture parameters. In a follow up study we will also incorporate gradient-based, cooccurrence-matrix based and run length matrix based TPMs to explore the differences in TPMs of various subtypes of MS lesions.dynamics of texture parameter evolution in time series of DSCE-images in 18 patients with CIS or MS according to the 2010 McDonalds criteria. A novel software program is introduced here to investigate time dependent texture parameter maps derived from DSCE (difference) images. In order to enable comparison of texture parameters between two patients, a twofold image-normalization was performed. The first normalization, to compensate for variations in coil-load, is obtained by setting the mean NAWM voxel value during BLP to a specific value in the ORIG-TPM. The second normalization, to compensate for differences in cardiac output, is obtained by normalization of the time integral of the TPM-DIFF over the IFP and OFP for the same NAWM. After normalization, comparisons between patients by comparing the time averaged texture parameter values during the BLP, IFP, OFP and RPP are feasible. We detected dynamic texture features that revealed highly statistically significant differences between ELs, NELs, and NAWM. Based on these dynamic TPs, novel grading parameters for MS lesions can be introduced allowing for grading of MS-lesions on a numerical scale instead of an ordinal scale as is the case with pre/post contrast T1w image analysis. Our data support the hypothesis that, dependent on the tissue type, subtle differences in micro-circulation are present in enhancing and non-enhancing MS lesions.This paper investigated the


Platynereis dumerilii is particularly well suited for such studies. Recently a reference template with registered gene expression patterns has been generated for the anterior part (episphere) of the Platynereis trochophore larva and used for the detailed study of neuronal development.Digital anatomical atlases are increasingly used in order to depict different gene expression patterns and neuronal morphologies within a standardized reference template. In evo-devo, a discipline in which the comparison of gene expression patterns is a widely used approach, such standardized anatomical atlases would allow a more rigorous assessment of the conservation of and changes in gene expression patterns during micro- and macroevolutionary time scales. Due to its small size and invariant early development, the annelid Platynereis trochophore and nectochaete larvae based on whole-mount in situ hybridization, confocal microscopy, and image registration. We achieved high-resolution whole-body scanning using the mounting medium 2,2’-thiodiethanol (TDE), which allows the matching of the refractive index of the sample to that of glass and immersion oil thereby reducing spherical aberration and improving depth penetration. This approach allowed us to scan entire whole-mount larvae stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) in situ hybridization and counterstained fluorescently with an acetylated-tubulin antibody and the nuclear stain 4’6-diamidino-2-phenylindole (DAPI). Due to the submicron isotropic voxel size whole-mount larvae could be scanned in any orientation. Based on the whole-body scans, we generated four different reference templates by the iterative registration and averaging of 40 individual image stacks using either the acetylated-tubulin or the nuclear-stain signal for each developmental stage. We then registered to these templates the expression patterns of cell-type specific genes. In order to evaluate the gene expression pattern registration, we analyzed the absolute deviation of cell-center positions. Both the acetylated-tubulin- and the nuclear-stain-based templates allowed near-cellular-resolution gene expression registration. Nuclear-stain-based templates often performed significantly better than acetylated-tubulin-based templates. We provide detailed guidelines and scripts for the use and further expansion of the Platynereis gene expression atlas.Here we introduce and evaluate a method for whole-body gene expression pattern registration for Platynereis trochophore and nectochaete larvae. We anticipate that nuclear-staining-based image registration will be applicable for whole-body alignment of the embryonic and larval stages of other organisms in a similar size range.We established whole-body reference templates for the generation of gene expression atlases for  Three-dimensional digital anatomical atlases represent biological structures within a reference space, and can provide morphological, molecular and functional information for the components of these structures. Atlases are used to represent specimens that possess the same set of morphological features, such as embryos of the same developmental stage or adult brains with stereotypic morphology ,2. Such in situ hybridization [Atlases show their full potential when data can be compiled across several individuals labeled or manipulated in various ways. The mapping of additional features to the reference template requires co-labeling of the specimens with the reference marker and the markers of interest. Markers can include neuron-filling dyes labeling individual neurons -7, fluordization -11, immudization , or trandization ).Drosophila[Platynereis[When the morphology of individual neurons is projected onto a reference template, such a representation can give insights into the organization of neural circuits and their influence on behavior ,5,6,8. Trosophila, mouse [rosophila, chickenrosophila, zebrafirosophila and the atynereis.The integration of imaging data acquired across multiple individuals and experiments requires sophisticated image registration techniques. Simple superimposition would be insufficient due to variations in morphology and experimental conditions. The image registration methods can be classified as ‘voxel value-based’ ,11,15 orDrosophila melanogaster) [Manduca sexta) [Tribolium castaneum) [Schistocerca gregaria) [Apis mellifera) [S. gregaria) [Heliothis virescens) [Image registration protocols usually start with coarse, rigid registration that is further refined by non-rigid registration. For example, the virtual insect brain (VIB) protocol  begins wogaster) , the spha sexta) , the flostaneum) , and theregaria) . Anotherregaria) , starts llifera) , the desregaria)  and the rescens) .Platynereis[A similar protocol was applied for the registration of the gene expression patterns in atynereis,15. Thisatynereis. The refPlatynereis, image registration has to date been restricted to the trochophore  larval episphere, due to the limited depth-penetration achieved by using imaging setups with refractive index mismatches . Here we push confocal microscopy closer to its depth-limit by using the mounting medium 2,2′-thiodiethanol (TDE) with a refractive index matched to that of glass and immersion oil [Platynereis trochophore (48 hpf) and nectochaete (72 hpf) larvae using isotropic voxel size. Isotropic voxel size also alleviated the need for precise body orientation, allowing us to scan and register larvae in any orientation. These whole-body scans were first oriented along their antero-posterior (AP) and dorso-ventral (DV) axes based on anatomical landmarks using a problem-specific algorithm developed in ImageJ [Platynereis, and to facilitate its transfer to other organisms. This work provides the foundation for the development of a near-cellular-resolution whole-body gene expression atlas for Platynereis larvae.In sion oil . The usen ImageJ . The oriIn situ hybridization using nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) staining combined with anti-acetylated-tubulin and DAPI staining was performed as previously described [in situ hybridization, larvae were transferred into 97% TDE diluted with PBS plus 0.2% Tween. Larvae were placed on a glass slide with several layers (three to five) of adhesive tape on both sides, forming a chamber with the coverslip.escribed , with slescribed ). FollowImaging was performed with an Olympus FV1000 confocal system on an IX81 inverted microscope using an UPlanSApo 60× Oil objective with 1.35 N.A. and 0.15 mm working distance . For excitation we used 405, 559 and 635 nm diode lasers. Three-channel excitation was performed simultaneously and emission light passed through a 405/488/559/635 main dichroic beam splitter and through a pinhole of diameter 120 μm before going to the detection module. Fluorescent light was split into three beams directed to three PMTs by the secondary dichroic filters 490 and 640. Channel 1 for DAPI detection had a further 425 to 475 band-pass filter, channel 2 for TRITC detection had a 570 to 625 band-pass filter and channel 3 for the detection of NBT/BCIP had a 780 long-pass filter. Images were scanned with 1.0× zoom and recorded as 512 × 512 pixels corresponding to a pixel size of 0.414 μm × 0.414 μm. The entire volume of the larvae was scanned with a z-step size of 0.41 μm, in order to have an isotropic voxel size. This pixel size is above the X-Y optical resolution (approximately 0.23 μm for our system), but is small enough to provide good resolution, tolerable scanning times and minimal bleaching. We chose to use an isotropic voxel size to allow the scanning of larvae in any orientation. Using a Z-step of 0.41 μm (the Z-resolution is approximately 1 μm), all larvae in all orientations have the same pixel size for all three axes.All average image stacks are available for download . Raw imaPlatynereis[For image registration we used a voxel value-based protocol that sequentially applies rigid, affine and deformable transformations. The method extends the published protocol used for the registration of the genes expressed in the episphere of 48 hours post fertilization (hpf) larvae of atynereis. The proatynereis and the atynereis.Since larvae were scanned in random orientations, we developed two ImageJ algorithms for the rapid coarse orientation of whole-body scans. The scans were oriented based on the features provided by the acetylated-tubulin signal and DAPI stained nuclei. For 48 hpf larvae, we defined the AP orientation based on the prominent acetylated-tubulin signal of the prototroch ciliary band. We subsequently found the position of the ventral nerve cord in the acetylated-tubulin signal to define the DV axis. For 72 hpf larvae, we defined the AP axis by finding the body major axis based on the DAPI signal. For DV orientation we then used the position of the nerve cord. The algorithms were implemented in ImageJ using the Particle Analyzer, the TransformJ , and theRegularStepGradientDescentOptimizer for both affine and deformable image registration steps), the interpolator, that estimates the intensity of the pixels at non-grid positions after transformation (we used the ITK class LinearInterpolateImageFunction for both affine and deformable image registration steps), and the metric, that evaluates the alignment at each optimizer step . The Mattes mutual information metric [The initial coarse orientation was refined using affine and deformable transformations  for both affine and deformable image registration steps. This method first performs registration at low resolution and then stepwise registrations at higher resolution levels. This approach helps to avoid local minima. Fine registration takes approximately 50 min on a PC with 32 Gb RAM and an Intel® Core™ i5-2500 CPU 3.3 GHz × 4 processor.We used the multi-resolution approach  to the template and then applied the obtained transformations to the gene expression channel. We scanned up to ten samples for each gene in both developmental stages and averaged them to avoid bias towards one sample and compensate for natural and technical variation.To evaluate the precision of the registration, we introduced a metric that provides information about the spatial precision of the registration, which cannot be determined based on the Mattes mutual information metric. First we determined the center positions of corresponding cells marked by the same gene expressed in single cells using ImageJ. We thresholded the signal in the image stacks and determined the X and Y coordinates in a Z-projection of the expressing cell. The Z coordinate was defined as the middle of the Z-span of the cell. We first calculated the average cell center coordinates for each group of corresponding cells from the individual registered samples and then calculated the distances of the individual cells to this average coordinate (absolute deviation).etcetera). The ChannelMerger plugin can be downloaded from [To visualize registered gene expression patterns, we developed a ChannelMerger plugin for ImageJ that allows merging multiple channels in one RGB stack. The number of channels that can be merged is not restricted by the plugin. The plugin provides basic options such as color change and visibility setting. The merged image is compatible with ImageJ functionality  on the Based on the whole-body confocal scans we developed averaged anatomical reference templates Figure . We geneFor image registration, we extended the protocol used in previous studies ,15. GiveUsing this procedure we progressively aligned and averaged 40 individual scans from both 48 and 72 hpf larvae  [rhabdomericopsin1 (r-opsin-1)[TrpC). All of these genes show expression in a restricted number of neurons in the episphere and the trunk, allowing quantitative analyses [see Additional file We next registered the expression patterns of test genes to the nuclear-stain and tubulin templates. We used five neuropeptide precursor genes (and WLD) ,28, rhab-opsin-1), and a Cprohormone convertase 2 (phc2), a gene with broad expression in the Platynereis nervous system . We also found that the nuclear-stain templates performed equally well or significantly better than the tubulin templates. We therefore used the nuclear-stain templates for further analyses. These results show that it is possible to register gene expression patterns in both the head and the trunk of Platynereis larvae with near-cellular resolution using the whole-body templates.The average absolute deviation for all cells was lower than the average cell body diameter  for the open source image analysis platform ImageJ ,31. The To further enable fast and unbiased identification of colocalizing gene expression signals, we also developed a pair-wise channel merging macro [see Additional file in situ hybridization).The above methods rely on visual inspection and image thresholding to detect potentially coexpressing genes. Given the accuracy of the registration method, such approaches are reliable for broadly expressed genes that show considerable overlap. The analysis of genes expressed in single cells may require further statistical tests, or eventual experimental validation , then the two measures should be similar. If two genes are expressed in adjacent cells, then the average absolute deviation of the ‘test cell’ relative to the ‘reference cell’ average should be larger than the average absolute deviation of the ‘reference cell’.We tested both of these measures for selected cell pairs expressing different genes. Coexpressing genes were simulated by dividing a set of ten sample images from the same gene into two subsets (a ‘reference’ and a ‘test’ set). We chose the L11_1 and WLD1 cells that are closely adjacent in the map of 48 hpf larvae. Both measures distinguished the L11_1-WLD1 cell pair from coexpressing cells, indicating that these two genes are likely expressed in closely adjacent cells Figure .in situ hybridization or double immunohistochemistry experiments. For example, the gene expression map revealed broad colocalization in 72 hpf larvae of r-opsin-1 and TrpC in several cells of the dorsal larval eyes , as a rule of thumb we recommend five scans per gene.We next analyzed how the number of scans per gene influences the position of the average cell body coordinate. From all cells in Figure Platynereis trochophores [Platynereis gene expression atlas containing expression information for a large number of genes at different stages. Such an atlas will provide an overview of the spatial distribution of gene expression patterns, giving insights into the dynamics of gene interactions during development, and will provide a catalog of cell types, providing the foundation for understanding physiology.Here we extended the gene expression registration protocol that was originally applicable to the episphere of hophores  to the wPlatynereis atlas is to identify genes that coexpress in particular cells, allowing the characterization of the molecular fingerprint and function of cells. Our quantitative analyses showed that the average absolute deviation for any registered cell is approximately half a cell diameter, meaning that gene expression patterns could be registered at or close to cellular resolution. The convincing demonstration of coexpression may nevertheless require experimental validation in some cases. For example, the atlas revealed a strong colocalization in the average signals of ropsin1 and TrpC in the eye photoreceptors, which we subsequently verified experimentally. The coexpression of ropsin1 and TrpC indicates that Platynereis rhabdomeric photoreceptors may also employ a phototransduction cascade similar to insect rhabdomeric photoreceptors [A particularly important aspect of the eceptors  and intreceptors , strengteceptors .Platynereis, the sharp and invariant acetylated-tubulin signal was used for the generation of the first gene expression map [Platynereis larvae. Nuclear stains (TOTO™-3 and Sytox green) have also been used in the recently established registration protocol for zebrafish larvae [in situ hybridization; A Hejnol, personal communication), nuclear stains may also be more suitable as reference markers in other animals. Nuclear stains have several other advantages over antibody markers. By labeling every cell, such stains give the broadest possible label of the anatomy. In contrast, tubulin is absent from large parts of the body, as in Platynereis larvae, and consequently provides less information for the registration. As our templates clearly show, nuclear stain gives a fuller representation of the anatomy, yet it is not a homogeneous label and reveals detailed internal structure . Additionally, nuclear stain is cheap and easy to perform, and is compatible with standard in situ hybridization protocols -containing hybridization solution yielded poor DAPI signal in Platynereis in our hands). We therefore propose nuclear stains as the first markers of choice for future image registration projects.Several different reference markers have been used for the generation of anatomical templates ,3,8-11. h larvae . Given tPlatynereis larvae, imaged by confocal microscopy. We hope that the reference templates and detailed instructions and scripts we provide will facilitate the building of a community resource for Platynereis. The possibility of near-cellular-resolution coexpression analysis makes Platynereis a powerful experimental model for the detailed characterization of cell types. We anticipate that by using optimized confocal imaging and whole-body scans it will be possible to develop reference templates for other developmental stages in Platynereis as well as other animals of a similar size range.Here we introduced whole-body gene expression registration for AP: Antero-posterior; DIC: Differential interference contrast; DAPI: 4’,6-diamidino-2-phenylindole; DV: Dorso-ventral; hpf: Hours post fertilization; ISO: Iterative shape-averaging method; ITK: Insight toolkit; NBT/BCIP: Nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; SDS: Sodium dodecyl sulfate; TDE: 2,2’-thiodiethanol; VIB: Virtual insect brain.The authors declare that they have no competing interests.AA developed the image registration protocol, performed image registration, performed confocal microscopy and wrote the paper. AP and CV performed confocal microscopy. CL advised on microscopy and suggested the use of TDE. GJ designed the project and wrote the paper. All authors read and approved the final manuscript.In situ hybridization protocol for Platynereis larvae. The protocol allows the combination of in situ hybridization with anti-acetylated-tubulin immunostaining and DAPI nuclear staining.Click here for fileCompressed scripts and instructions for the rigid orientation step in ImageJ. The archive contains an instruction file imagej_instructions.txt and a folder with the scripts.Click here for fileCompressed scripts and instructions for the non-rigid registration step using ITK. The archive contains an instruction file itk_instructions.txt and a folder with the scripts.Click here for fileLoss of DAPI signal intensity and sharpness with scanning depth in TDE versus glycerol mounted specimen. Whole-body confocal stacks of a 48 hpf larva scanned in TDE and subsequently in glycerol. The edges detected by FeatureJ Edges in Fiji are also shown.Click here for fileAcetylated-tubulin whole-body reference template for 48 hpf Platynereis larvae (QuickTime movie). Image stack with the whole-body acetylated-tubulin reference template for 48 hpf larvae generated by the iterative registration of 40 individual scans.Click here for fileNuclear-stain whole-body reference template for 48 hpf Platynereis larvae (QuickTime movie). Image stack with the whole-body DAPI reference template for 48 hpf larvae generated by the iterative registration of 40 individual scans.Click here for fileAcetylated-tubulin whole-body reference template for 72 hpfPlatynereislarvae (QuickTime movie). Image stack with the whole-body acetylated-tubulin reference template for 72 hpf larvae generated by the iterative registration of 40 individual scans.Click here for fileNuclear-stain whole-body reference template for 72 hpf Platynereis larvae (QuickTime movie). Image stack with the whole-body nuclear-stain reference template for 72 hpf larvae generated by the iterative registration of 40 individual scans.Click here for fileMaximum projections of raw in situ hybridization data.In situ hybridization (red) for the analyzed neuropeptide precursor genes, counter-stained for acetylated tubulin (white) in 48 (A-E) and 72 hpf (F-J) larvae. Maximum projections of anterior (top rows) and ventral (bottom rows) views are shown. The anterior views only show a maximum projection of the episphere. Scale bar 30 μm.Click here for fileShadowing effect and registration of a broadly expressed gene.  Average expression pattern (red) of phc2, counter-stained for acetylated tubulin (white) in a 72 hpf larva.  Shadowing of the DAPI signal  due to the broad expression of phc2 in situ hybridization signal (red). Superimposed template (green) and sample images (red) before (C) and after (D) deformable registration.  are lateral views, (B) is an anterior view,  are ventral views. Scale bar 30 μm.Click here for fileAffine and deformable registration.  Evolution of the Mattes mutual information metric during the affine (A) and deformable (B) registration steps. The minimization metric converges after approximately 50 steps.  A slice of the superimposed template (red) and the sample images (green) before (C) and after (D) deformable registration. (E) Representation of the 3D deformation field corresponding to the transformation from (C) to (D) visualized in Paraview http://www.paraview.org/. Scale bar: 30 μm.Click here for fileImage stack of registered expression patterns of five genes in 48 hpf Platynereis larvae (QuickTime movie). The image was generated using the ChannelMerger plugin. Color code as shown in Figure Click here for fileImage stack of registered expression patterns of seven genes in 72 hpf Platynereis larvae (QuickTime movie). The image was generated using the ChannelMerger plugin. Color code as shown in Figure Click here for file3D view of registered expression patterns of five genes in 48 hpf Platynereis larvae (QuickTime movie). The movie was generated using Imaris. Color code as shown in Figure Click here for file3D view of registered expression patterns of five genes in 72 hpf Platynereis larvae (QuickTime movie). The movie was generated using Imaris. Color code as shown in Figure Click here for fileFiji macro for generating an all-against-all coexpression montage for several genes. To use this macro, download and install Fiji (http://fiji.sc/). Open the macro in Fiji and run it on a set of image stacks of gene expression averages aligned to the references provided in Additional files Click here for fileAll-against-all coexpression analysis for five genes in 48 hpf Platynereis larvae. The image montage of gene coexpressions was generated with the custom Fiji macro (Additional file Click here for fileAll-against-all coexpression analysis for seven genes in 72 hpf Platynereis larvae. The image montage of gene coexpressions was generated with the custom Fiji macro (Additional file Click here for file


Several conflict processing studies aimed to dissociate neuroimaging phenomena related to stimulus and response conflict processing. However, previous studies typically did not include a paradigm-independent measure of either stimulus or response conflict. Here we have combined electro-myography (EMG) with event-related brain potentials (ERPs) in order to determine whether a particularly robust marker of conflict processing, the N450 ERP effect usually related to the activity of the Anterior Cingulate Cortex (ACC), is related to stimulus- or to response-conflict processing. EMG provided paradigm-independent measure of response conflict. In a numerical Stroop paradigm participants compared pairs of digits and pressed a button on the side where they saw the larger digit. 50% of digit-pairs were preceded by an effective cue which provided accurate information about the required response. 50% of trials were preceded by a neutral cue which did not communicate the side of response.EMG showed that response conflict was significantly larger in neutrally than in effectively cued trials. The N450 was similar when response conflict was high and when it was low.We conclude that the N450 is related to stimulus or abstract, rather than to response conflict detection/resolution. Findings may enable timing ACC conflict effects. Functional magnetic resonance imaging (fMRI) studies have identified brain structures involved in detecting/resolving conflict at the stimulus and response levels ,2. In adIn principle, Stroop conflict can appear at the level of stimulus representations (stimulus conflict) or at the level of motor response organization (response conflict). According to the stimulus conflict view, Stroop conflict appears because the representations of parallel processed stimulus dimensions are incongruent with each other . AccordifMRI studies have pointed to several brain areas involved in conflict processing. The most prominent of these areas is the ACC which has been shown to be active in nearly all studies examining conflict processing. According to a prominent theory, the conflict monitoring view -12, the ERP studies have identified a succession of events in paradigms involving conflict processing ,6,21. Th1 We also detected an N450 ERP effect in the incongruent, relative to the congruent, condition right after the offset of incorrect hand activity. This confirmed that the N450 is probably closely related to conflict detection/resolution  minus [neutrally cued condition] difference potentials.Click here for fileFigure S6. The topography of Cue × Congruency interactions (p < 0.025) in ERPs in the effectively (Eff-C) and neutrally cued (Neut-C) conditions. The amplitude of ERPs in the neutral condition is shown on the left for reference. The amplitude of difference topographies is shown on the right . The upper row shows the effectively cued condition, the lower row shows the neutrally cued condition.Click here for fileFigure S7. The Lateralized Readiness Potential in the cued (A) and non-cued (B) conditions.Click here for file


The epidermal growth factor receptor kinases, or ErbB kinases, belong to a large sub-group of receptor tyrosine kinases (RTKs), which share a conserved catalytic core. The catalytic core of ErbB kinases have functionally diverged from other RTKs in that they are activated by a unique allosteric mechanism that involves specific interactions between the kinase core and the flanking Juxtamembrane (JM) and COOH-terminal tail . Although extensive studies on ErbB and related tyrosine kinases have provided important insights into the structural basis for ErbB kinase functional divergence, the sequence features that contribute to the unique regulation of ErbB kinases have not been systematically explored.cis regulation of the kinase core by the JM and C-terminal tail. ErbB3, and EGFR orthologs in sponges and parasitic worms, diverge from some of the canonical ErbB features, providing insights into sub-family and lineage-specific functional specialization.In this study, we use a Bayesian approach to identify the selective sequence constraints that most distinguish ErbB kinases from other receptor tyrosine kinases. We find that strong ErbB kinase-specific constraints are imposed on residues that tether the JM and C-terminal tail to key functional regions of the kinase core. A conserved RIxKExE motif in the JM-kinase linker region and a glutamine in the inter-lobe linker are identified as two of the most distinguishing features of the ErbB family. While the RIxKExE motif tethers the C-terminal tail to the N-lobe of the kinase domain, the glutamine tethers the C-terminal tail to hinge regions critical for inter-lobe movement. Comparison of the active and inactive crystal structures of ErbB kinases indicates that the identified residues are conformationally malleable and can potentially contribute to the Our analysis pinpoints key residues for mutational analysis, and provides new clues to cancer mutations that alter the canonical modes of ErbB kinase regulation. The epidermal growth factor receptor (EGFR) and related kinases, ErbB2, ErbB3, and ErbB4  Extensive studies on the extracellular ligand-binding domain A key step in ErbB kinase signaling is the activation of the intracellular kinase domain, which is achieved by an intermolecular interaction between two kinase molecules in an asymmetric dimer The catalytic activity of EGFR is also regulated by conformational changes in the JM and C-terminal tail—two sequence segments flanking the kinase domain. The JM segment functions as an activation domain Receptor tyrosine kinases (RTKs) outside of the ErbB family also contain flexible JM and C-terminal tail segments that play important regulatory roles. In c-KIT and Ephrin receptor tyrosine kinases, for example, the JM segment plays an autoinhibitory role, in contrast to its activating role in EGFR, by interacting with the active site cis interaction between the kinase core and the flanking JM and C-terminal tails. Our analysis provides new testable hypotheses regarding the cis regulation of the kinase core by the JM and C-terminal tails, and provides new insights into cancer mutations that alter this mode of regulation.We have shown using several case studies that Bayesian analysis of the evolutionary constraints distinguishing functionally divergent kinases is a viable approach for investigating the functional specificity of kinases in signaling pathways To identify which sequence features most distinguish ErbB kinases from other RTKs, we measured and analyzed the selective constraints imposed on ErbB kinase sequences from diverse organisms see . These cIn addition to the kinase domain, strong ErbB-specific constraints are also imposed on residues flanking the kinase core, namely, the Juxtamembrane segment, the JM-kinase linker and the COOH-terminal tail.The JM segment is conserved across diverse organisms within the ErbB family . HoweverThe C-terminal tail is also a distinctive feature of the ErbB family. In particular, the sequence segment immediately following the kinase domain  within the C-terminal tail [DE]x[DE]xYL motif is conserved as an aspartate (D) in ErbB3. Likewise, ErbB3 and ErbB4 replace a canonical tyrosine (Y764) in the C-helix by a leucine (L) . The strSince ErbB kinases are one of the most frequently mutated gene families in human cancers, we investigated whether any of the identified ErbB conserved residues are among those known to be associated with human cancers. Mapping of somatic mutations identified in the ErbB family see  to availLobe Tether (NLT): residues that tether the JM and C-terminal tail to the kinase N-lobe; (ii) Active Site Tether (AST): residues that tether the C-terminal tail to the ATP binding site; and (iii) C-Lobe Tether (CLT): residues that tether the JM and C-terminal tail to the kinase C-lobe  in the JM segment hydrogen bonds to the side-chain of Y764 and S768 in the C-helix . While tS768I: S768I is a frequently occurring mutation , and parasitic worms contain a glutamine at the D1014 position. Notably, both sponges and parasitic worms replace the glutamine (Q791) in the inter-lobe linker by an isoleucine (I) and glutamate (E), respectively . AlthougAs mentioned earlier, some of the strongest ErbB kinase-specific constraints are imposed on residues in the JM-kinase linker and β2-β3 loop. ErbB-conserved residues in these two regions structurally couple a phosphorylatable tyrosine (Y1016) in the C-terminal tail to the JM and N-lobe regions involved in dimerization . Some ofThe canonical ErbB kinase-conserved interactions between the C-terminal tail, JM-kinase linker, and β2-β3 loop are altered in the inactive dimer Our analysis suggests that the structural coupling between the C-terminal tail and JM-kinase linker (described above) is likely to differ in ErbB3, and EGFR orthologs in sponges and parasitic worms because ErbB3 conserves an aspartate at the Y1016 position, and EGFR orthologs in sponges and parasitic worms lack some of the canonical ErbB residues in the JM-kinase linker and β2-β3 loop . Our anaPKA) in the C-terminal tail moves in and out of the ATP binding pocket to serve as a gate for nucleotide binding The AST is largely formed by a helical segment (residues 997–1001) in the C-terminal tail , also recis regulation of the kinase core by the JM and C-terminal tail can be tested experimentally. Likewise, the hypothesis that the activation mechanism of sponges and roundworms differs from their mammalian counterparts can also be tested experimentally. Finally, by identifying a potential role for the C-terminal tail in ATP binding (AST), our study provides new avenues for designing selective ErbB kinase inhibitors.Bayesian analysis of the evolutionary constraints acting on receptor tyrosine kinase sequences has revealed a co-conserved pattern characteristic of the ErbB family. Analysis of this co-conserved pattern, in light of the wealth of structural and functional data available on ErbB kinases, suggests a model in which the identified residues contribute to ErbB kinase functional divergence by providing a structural framework for the JM and C-terminal tail to uniquely regulate ErbB kinase activity . A compeErbB and related receptor tyrosine kinase (RTK) sequences from diverse organisms were identified within NCBI nr, env_nr, and translated EST databases using PSI-BLAST and motif-based search procedures. These sequences were multiply aligned using the MAPGAPS program The residues that contribute the most to ErbB kinase evolutionary divergence are shown using a “Contrast Hierarchical Alignment (CHA)” ErbB3 is considered an atypical member of the ErbB family. To determine to what extent ErbB3 contributes to the divergence of the ErbB family, we ran the BPPS procedure by removing ErbB3 sequences from our alignments. Removing ErbB3 sequences did not significantly alter the pattern-partitions created by the BPPS procedure.Representative sequences from ErbB3 and ErbB4 (query set) were multiply aligned against mammalian ErbB kinase sequences (∼164 sequences). The BPPS procedure was applied on this alignment to identify sequence patterns that most distinguish ErbB3 and ErbB4 from other ErbB members (ErbB1 and ErbB4). Among other residues, L794 in the C-helix  was idenhttp://www.rcsb.org). Protein hydrogen bonds were added to structural coordinates using the Reduce program http://www.pymol.org). The symmetry related molecules in Crystal structures of ErbB kinases solved in various function states (see below) were obtained from the PDB database ). Conjugate-gradient energy minimization was performed on the solvated protein for 10,000 steps, followed by heating from 0 to 298.15 K. The restraints on the protein backbone atoms over multiple stages of equilibration under NPT ensemble  were released to obtain a relaxed protein. The unrestrained MD productions were run for 10 ns using a time step of 2 fs and the NPT ensemble. Root-mean-square deviation (RMSD) calculations were performed and monitored to ensure that the simulation was stable during the 10ns time scale. Hydrogen bonding analysis was done using the ptaj program in Amber suite of programs.For molecular dynamics studies, water molecules, bound inhibitors, and other heteroatom's were removed. The missing residues were modeled using MODELLER Table S1(0.09 MB DOC)Click here for additional data file.Figure S1(0.07 MB DOC)Click here for additional data file.


Detection of choroidal inflammation in Vogt-Koyanagi-Harada (VKH) disease is still a challenge. Progression to sunset glow fundus has been observed despite apparent good clinical control of inflammation. Indocyanine green angiography (ICGA) permits choroid inflammation detection, though it is invasive, time consuming, and costly. The purpose of the present study is to report a sign indicative of probable inflammation on enhanced depth imaging spectral-domain optical coherence tomography (EDI-OCT): a localized increase in choroidal thickness with bulging of the outer retina  in patients with VKH disease in the non-acute uveitic stage.This is a retrospective observational study. The choroidal bulging was a particular finding observed in four eyes of three patients with VKH disease in the non-acute uveitic stage . This study is part of an ongoing longitudinal study in patients with VKH disease carried out in the Uveitis Service, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil. In all eyes, the choroidal bulging was identified in the presence of anterior chamber cells and/or on fundus angiographic (fluorescein and indocyanine green) findings, indicative of disease activity. Changes in the thickness of the choroidal bulging accompanied the variation in the clinical and angiographic signs of inflammation.The choroidal bulging is a particular finding detected on EDI-OCT that may indicate ongoing inflammation in the posterior segment of the eye. This EDI-OCT feature may assist in the treatment-monitoring of patients with Vogt-Koyanagi-Harada disease in the non-acute uveitic stage. Vogt-Koyanagi-Harada (VKH) disease is an autoimmune disorder, in which the choroid is the main site of disease-related inflammation in the eye . Detectilocalized thickening of the choroid) [Newer imaging techniques, such as enhanced depth imaging optical coherence tomography (EDI-OCT), have been recently evaluated as an alternative modality of monitoring treatment effect. In this context, we describe a unique finding in patients with VKH disease in the non-acute uveitic stage: the ‘choroidal bulging’, which seems to be related to active inflammation. This finding appears as a localized thickening of the choroid that assumes a convex appearance with consequent bulging of the adjacent retina, with no nearby obvious retinal lesion to justify this finding. Although diffuse thickening of the choroid has already been described in some retinal diseases (including VKH disease), we are not aware of other conditions that commonly produce choroidal bulging  ,4.We retrospectively reviewed sequential clinical and imaging data from all eyes noted to have the choroidal bulging on EDI-OCT performed as part of the follow-up protocol in an ongoing longitudinal VKH disease study, which is being conducted at the Uveitis Section, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil. The study protocol followed the statements of the Declaration of Helsinki and was approved by the Universidade de São Paulo Institutional Review Board. In accordance with the Revised Diagnostic Criteria for VKH disease , incomplOne area of localized thickening of the choroid that assumes a convex appearance with consequent bulging of the adjacent retina  was identified in at least one follow-up visit in three patients. In all cases, at least two sequential visits were analyzed. Observation of the same scan area on EDI-OCT revealed appearance/disappearance of the choroidal bulging during the period analyzed. One patient had bilateral involvement and will be described further. At the time of identification of the choroidal bulging, all four eyes presented at least one concomitant inflammatory sign . The changes observed in the thickness of the choroidal bulging along with the sequential follow-up EDI-OCT evaluations coincided with the variation in these inflammatory signs Table .Patient 2, Table A 54-year-old woman with VKH disease diagnosed 3 months ago came for routine evaluation  revised it critically for important intellectual content and had approved the final manuscript.


Listeria monocytogenes is a Gram-positive microaerophilic facultative anaerobic rod and the causative agent of the devastating disease listeriosis. L. monocytogenes is able to form biofilms in the food processing environment. Since biofilms are generally hard to eradicate, they can function as a source for food contamination. In several occasions biofilms have been identified as a source for genetic variability, which potentially can result in adaptation of strains to food processing or clinical conditions. However, nothing is known about mutagenesis in L. monocytogenes biofilms and the possible mechanisms involved. In this study, we showed that the generation of genetic variants was specifically induced in continuous-flow biofilms of L. monocytogenes, but not in static biofilms. Using specific dyes and radical inhibitors, we showed that the formation of superoxide and hydroxyl radicals was induced in continuous-flow biofilms, which was accompanied with in an increase in DNA damage. Promoter reporter studies showed that recA, which is an important component in DNA repair and the activator of the SOS response, is activated in continuous-flow biofilms and that activation was dependent on radical-induced DNA damage. Furthermore, continuous-flow biofilm experiments using an in-frame recA deletion mutant verified that RecA is required for induced generation of genetic variants. Therefore, we can conclude that generation of genetic variants in L. monocytogenes continuous-flow biofilms results from radical-induced DNA damage and RecA-mediated mutagenic repair of the damaged DNA.The food-borne pathogen  Listeria monocytogenes is a food-borne pathogen and the cause of listeriosis, which is a disease that is associated with meningitis, encephalitis, or spontaneous abortions L. monocytogenes is frequently encountered in food processing facilities, where it thrives in the form of biofilms on food processing equipment or in pipelines L. monocytogenes biofilms, it has indeed been shown that various stress mechanisms are activated in different types of biofilms and that some of them are involved in the increased resistance of biofilms against disinfectants L. monocytogenes biofilm formation focus on static conditions. Static biofilms of L. monocytogenes consist of small rod-shaped cells that are attached as microcolonies or homogeneous layers L. monocytogenes biofilms formed under continuous flowing conditions appeared to consist of a dense network of knitted-chains that are composed of elongated cells and surround ball-shaped microcolonies L. monocytogenes has previously been related with contaminated chocolate milk tank draining pipes L. monocytogenes has been encountered in whey transport pipes Streptococcus pneumoniae and Staphylococcus epidermidus, it was shown that biofilms of these organisms produced genetic variants L. monocytogenes that RecA contributes to survival of conditions mimicking the gastro-intestinal tract and translocation across the intestinal barrier recA and/or other SOS response genes of L. monocytogenes are activated during growth in a mouse macrophages cell line in vivo in mouse organs during infection Escherichia coli the SOS response appeared to be an essential mechanism during urinary tract infections Pseudomonas aeruginosa grown in a drip-flow reactor was dependent on the presence and activity of RecA L. monocytogenes biofilms and the possible mechanisms involved. Therefore, we aimed to investigate whether L. monocytogenes biofilms show mutagenesis and what mechanisms might be involved.One of the important phenomena that has been attributed to bacterial biofilms is the generation of genetic variants that might result in the persistence of adapted strains in the industry or in human infections L. monocytogenes biofilms the occurrence of rifampicin-resistant variants in continuous-flow and static biofilms was investigated and compared with planktonic grown cultures. Resistance to the antibiotic rifampicin can be the result of point-mutations in the gene rpoBL. monocytogenes show induced generation of genetic variants.To investigate the formation of genetic variants in Pseudomonas aeruginosa biofilms grown in a drip-flow reactor it was shown previously that the generation of variants was dependent on endogenous oxidative stress-mediated DNA damage and repair Since continuous-flow biofilms specifically show induced generation of variants, the question arises what the specific mechanism is behind this phenomenon. For L. monocytogenes RecA is also required for mutagenesis in planktonic cells recA is specifically activated in L. monocytogenes during continuous-flow biofilm formation and RecA-mediated activation of the SOS response member yneA is required to obtain fully-grown biofilms consisting of ball-shaped microcolonies surrounded by knitted-chains composed of elongated cells recA promoter reporter we verified that recA is indeed activated in cells obtained from continuous-flow biofilms, but furthermore that activation of recA in continuous-flow biofilms is the result of radical-induced DNA damage  in continuous-flow biofilm formation due to the inability to form knitted-chains composed of elongated cells, which is dependent on the activation of SOS response factor yneA , but an incomplete tricarboxylic acid (TCA) cycle because the α-ketoglutarate dehydrogenase is missing Although we now identified the specific oxidants produced in continuous-flow biofilms that are associated with DNA damage and the induced generation of genetic variants, we do not yet know why the formation of these oxidants is increased during continuous-flow biofilm formation. It might be related with the continuous influx of oxygen saturated BHI broth in the flow-cells during continuous-flow biofilm formation. Notably, we observed a fivefold higher rifampicin-resistant fraction for planktonic cultures grown under shaking conditions compared with cultures grown under static conditions , which iL. monocytogenes continuous-flow biofilms it was shown previously that expression of sigB, which encodes the activator of the class II stress response, and hrcA and dnaK, which encode the regulator and major chaperone of the class I heat-shock response, are highly induced L. monocytogenes cells grown in continuous-flow biofilms experience stress. For several other organisms it has been shown that exposure to different stresses might result in oxidative stress on a molecular level. For instance, exposure of E. coli and Staphylococcus aureus to bactericidal antibiotics resulted in the generation of ROS due to perturbation of the ETC and subsequent leakage of electrons from ETC intermediates Bacillus cereus cells to acid and heat resulted in the induction of radical formation, which corresponded with reduced viability L. monocytogenes cells grown in continuous-flow biofilms experience stresses that might perturb the ETC, which subsequently results in the formation of superoxide and hydroxyl radicals.Alternatively, the increased formation of superoxide and hydroxyl radicals in continuous-flow biofilms might be related with the wide variety of different stresses that the bacterial cells experience in the different microniches of the biofilms recA and the SOS response after DNA damage often results in the occurrence of genetic variants due to mutagenic DNA repair yneAL. monocytogenes  Single colonies of the strain 9 cells from the static and continuous-flow biofilms and planktonic cultures (grown for 24 h in BHI broth using a 1% inoculum of an overnight grown culture) were used for serial dilution in PBS and plated on BHI agar with and without 0.05 µg/ml rifampicin . This rifampicin concentration is the MIC for L. monocytogenes EGD-e (results not shown). The plates were incubated for 2 days at 30°C and colonies were enumerated.To detect the generation of genetic variants, approximately 2*10To detect radical formation in biofilm and planktonic cells, the reporter dyes MitoSOX , which is a derivative of hydroethidine, and 3′-(p-hydroxyphenyl) fluorescein  were used. MitoSOX binds to DNA after specific oxidation by superoxide and subsequently gives a red fluorescent signal B software .Phase contrast and fluorescence microscopy experiments were performed on a BX41 microscope . Fluorescence of MitoSOX, HPF, and EGFP was visualized using the U-MWIG3 and MNIBA3 filters  and images were acquired with a XC30 camera  run by Olympus CellDetection of the integrity of genomic DNA was performed as described previously t-test (p<0.05) in SPSS.Significant differences in biofilm formation or the generation of variants were identified using levene's test for equality of variances and the independent-samples Figure S1Induced generation of variants after mitomycin C exposure. The graph presents the average and standard deviation of the rifampicin resistant fraction (0.05 µg/ml) of static planktonic cultures grown for 24 h at 20°C before (dark grey) and after exposure to 2 µg/ml mitomycin C for 1 h (white) or 2 h (light grey). Experiments were performed in three biological independent replicates. *Significantly different from the unexposed condition and the ΔrecA mutant strain .(TIF)Click here for additional data file.Figure S2Generation of variants in planktonic cultures. The graph presents the average and standard deviation of the rifampicin resistant fraction (0.05 µg/ml) of planktonic cultures grown for 24 h at 20°C under static and shaking (180 rpm) conditions. Experiments were performed in three biological independent replicates. *Significantly different from the static condition .(TIF)Click here for additional data file.