The presence of invasive cell clusters known as tumor budding and the closely related epithelial mesenchymal transition (EMT) have a prognostic impact on cancer patients' overall survival. Interestingly, data quantitatively analyzing and correlating the amount of tumor buds and patient overall survival as well as the impact of expression of epithelial phenotype markers are missing.Periampullary carcinoma samples of 171 patients were immunohistochemically stained for E-Cadherin (ECad). Tumor cell clusters  were manually quantified comprising tumor cell number and subcellular localization of ECad expression . Data analyses were performed using elastic net feature selection. Hereby, five distinct intervals of TCC sizes and corresponding fractions of cells with distinct ECad expression were identified. Prognostic features of the defined budding categories were entered into a subsequent Cox regression model together with standard clinicopathological parameters and, based on the model prediction, cases were categorized into “low and high budding” grades.p<0.001) in a multivariable clinicopathologic Cox model.Overall median TCC size was 16 cells (range: 2-36 cells). The median number of TCCs per tumor was 42 (range: 3-283). Elastic net feature selection identified TCCs of 6-10 and 31-35 cells as prognostically most relevant negative and positive features, respectively. Regarding ECad expression, cytoplasmic ECad expression in TCCs of 11-15 as well as of 26-30 cells revealed prognostic relevance. Combining TCC numbers and ECad expression, budding grade qualified as independent prognostic factor for patient overall survival  but tumor cell nests containing 6-10 cells display the strongest negative prognostic relevance. Moreover, we demonstrate that larger TCCs might have a strong positive prognostic impact in periampullary adenocarcinomas, contributing to establishing an advanced grading system. The term “tumor budding” was first introduced by Imai et al. 60 years ago Several studies investigated the prognostic value of tumor budding, focused on tumor cell clusters containing five or less tumor cells The term periampullary carcinoma comprises a group of tumors with a common embryological origin, originating from the same part of the foregut The aim of this study was a quantitative morphomic analysis of tumor cell clusters and their ECad staining pattern in periampullary adenocarcinomas using a well characterized cohort Therefore, we applied a quantitative statistically based approach by linking tumor budding, EMT features and patient prognosis in periampullary adenocarcinomas. Hence, our study characterizes these neoplasms on the basis of several features of the complex morphological appearance of tumor development (morphome) The study protocol was in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the University Medical Center Freiburg (Ref: 13/11). Patient selection, exclusion criteria, handling and processing of clinical data, surgical processes and pathological workup are described in detail by Bronsert et al. All PAMPAC were re-reviewed for tumor budding using standard hematoxylin and eosin (H&E) stained tissue slides. Herein, the slides containing the tumor areas with the highest number of tumor buds were identified. Corresponding tissue blocks were selected and processed further for ECad immunohistochemical staining. Two µm-thick tissue sections were prepared and stained for ECad: Heat-induced antigen retrieval was performed in pH 9.0 antigen retrieval buffer  at 95°C for 40 min. ECad primary antibody , respectively with LINKER reagent  was used on an Autostainer LINK 48 (Dako) device. For the Streptavidin-biotin peroxidase detection, EnVision® Flex Peroxidase-Blocking Reagent , EnVision Flex+ Mouse  secondary antibody and EnVision Flex/HRP solution  were used. Omission of the primary antibody served as negative control. Normal epithelium was used as internal positive controls for ECad. Hematoxylin was used as counterstaining before adding the coverslip.2) was evaluated using CaseViewer 2.0 software . In the studied region, isolated tumor cells and cell clusters comprising ≤50 cohesive tumor cells were the subject of evaluation. Each of these tumor cells was separately investigated and its ECad expression pattern  was determined. Tumor cell number of each cohesive cluster was quantified in a range from 1 cell to 50 cohesive cells.Next, each slide was digitalized using the Mirax Scan Panoramic scanner  with a 20× objective. Previously identified tumor areas with high budding at the invasion front were verified, and one representative field of 200-fold magnification .In all observed areas, cohesive tumor cells (defined as tumor cell clusters) were quantified according to their tumor cell amount and ECad expression. The maximal tumor cell number for one cluster to be defined as cluster and not as main tumor mass was 50 tumor cells, the smallest cluster one cell. Tumor clusters were assigned to ten different cell cluster size categories: 1-5, 6-10 … and 46-50 cells, respectively. Subcellular ECad expression was integrated into the dataset as relative fraction of tumor cells to all cells of the clusters showing membranous, mixed or cytoplasmic ECad staining  in the whole patient cohort as well as in the PDAC subgroup.www.r-project.org;R Core Team 2017). Statistical testing was performed two-sided at a significance level of p<0.05.Differences between the low and high budding grade categories regarding histopathological parameters were compared by Fisher's exact test. Tumor cell cluster size correlations were analyzed using inverse regression. All calculations and plotting were performed using R software version 3.4.4. (Table 1). Gender distribution was 88 males and 83 females. Median age was 67 years (range 30-89 years). The tumors were classified according to their origin /36 ampullary carcinomas (AMPAC)/seven small intestinal carcinomas /18 distal bile duct carcinomas (DBDAC)). Tumors were subdivided into morphological subgroups according to their histological subtype /17 (undifferentiated) according to Albores-Saavedra et al. 171 patients operated between 2001 and 2011 at the University Medical Center Freiburg were included (th TNM classification According to the 7Table 1). Median cell cluster size was calculated individually for each patient and ranged from 2 to 36 cells, with an overall median of 16 cells across all tumors. There was no statistically significant relationship between total cell count, cluster count or cluster size and clinicopathological parameters  in linear regression analysis .In total, n=144395 cells were assessed manually and evaluated according to their E-cadherin (ECad) expression. Considering ECad staining, 36981 (26%) tumor cells demonstrated a membranous, 77535 (54%) tumor cells a mixed and 29879 (21%) tumor cells a cytoplasmic pattern. The median number of tumor cell clusters per field of 200-fold magnification was 42 (range 3-283). The median total cell number of cells in the clusters was 336 (range 16-1108)  revealed a significant inverse relationship , meaning that cell cluster number increases steeply with decreasing cell cluster size.As described above, cell cluster counts were summarized in ten cluster size categories . Relative frequencies of cells in each cluster category for membranous, mixed and cytoplasmic ECad staining were calculated A-E. HereFigure ). Herein, the fraction of cells with cytoplasmic staining increased significantly in smaller cell clusters while the fraction of cells with mixed and membranous pattern decreased with decreasing cluster size. These observations are reminiscent of a previous analysis from our working group Regarding the subcellular ECad expression pattern, similar inverse proportional relationships to cell cluster size could be demonstrated (Table 2) shows that clusters of 6-10 cells and cytoplasmic ECad expression in clusters of 11-15 as well as 26-30 cells are negative prognosticators (hazard ratio >1) for overall survival. Notably, higher counts of tumor cell clusters with 31-35 cells had a positive influence on patients overall survival (hazard ratio <1). All these morphome features were statistically independent prognostic factors (p<0.05). To simplify further analysis, linear predictors derived from this multivariable model were used to stratify patients into two equally sized quantile groups termed “low budding grade” and “high budding grade”, with low and high predicted risk of death during follow-up, respectively. Univariable comparisons of low versus high budding grades are shown in Elastic net feature selection identified the counts of following cell cluster size categories as predictors of overall survival: 6-10 cells, 31-35 cells, and the fractions of cytoplasmic ECad expressing cells in clusters of sizes 11-15 as well as 26-30 cells. A Cox model containing only the selected features (Table 3) including baseline clinicopathological parameters  together with the budding grade (defined above) was performed. Cox proportional hazards model including all PAMPAC patients identified distant metastasis , intestinal histologic subtype , ampullary location  and high budding grade  as independent prognostic factors regarding patients overall survival.As the final step, multivariable survival modelling (p=0.025) as well as metastatic spread to distant organs  and high budding grade  demonstrated an independent prognostic value on patient overall survival (Table 4).As the majority of the included patients  suffered from PDAC, we performed the same analysis in the subgroup of PDACs. In PDAC, metastatic spread to the regional lymph nodes . In the subgroup of PDAC, median survival was 27 vs. 14 months in patients with low versus high budding grade (p<0.001 Table 5).Survival plots highlighting the significant differences in overall survival between the two budding grade groups in the whole PAMPAC cohort are shown in In the current literature, tumor buds are commonly defined as an isolated cancer cell or cluster including less than or equal five Albeit EMT is a pivotal process in cancer biology, the link between the EMT and tumor budding - as a putative histomorphological manifestation of EMT- is still not well clarified. The concept of EMT describes a process in which tumor cells disengage from the main tumor mass by activating transcriptional programs To investigate the prognostic impact of tumor budding and ECad shuttling simultaneously, we performed quantitative assessment of tumor cell cluster size and immunohistochemical ECad expression pattern of the tumor cells from 171 resected periampullary carcinomas. Based on the detailed tumor budding analysis comprising tumor cell quantification, cell cluster assignment and subcellular ECad expression, using elastic net feature selection and multivariable survival modeling, prognostic histologic features were identified from this morphomic dataset.The morphome features with the strongest significant impact on patient overall survival were counts of tumor cell cluster of a size from six to ten cells (poor prognosis) and of a size from 31 to 35 cells (good prognosis). Of note, the final statistical model included all compiled standard clinicopathological parameters as well as the histological subtype (as published previously) Several studies were conducted to identify prognostic subgroups in different carcinomas according to their tumor budding behavior Figure 2) show the membranous and mixed pattern to be more characteristic for the larger clusters and the cytoplasmic ECad staining for the smaller buds. The loss of membranous ECad expression indicates EMT which is related to increased tumor cell dissemination, tumor spread and decreased patient overall survival Table 2). These data are in accordance with our prior study, indicating membranous ECad positivity as a positive prognostic factor in pancreatic cancer. Albeit the strong relationship between EMT and tumor budding is widely accepted, verification of direct quantitative correlations between these phenomena is still scanty. ECad is a transmembrane adhesion glycoprotein It is important to emphasize that tumor budding is a special, incomplete form of EMT since tumor buds will not acquire complete mesenchymal phenotype (for example they lack to express vimentin) Tumor budding is widely associated with bad prognosis. Interestingly, not only the lack of smaller tumor buds, but also the presence of larger tumor cell clusters have a positive prognostic effect on patients overall survival. To our knowledge, the presented study is the first one describing larger tumor cell clusters as positive prognosticators in periampullary tumors. These results should be analyzed in further studies also comprising other cancer entities.Furthermore, our data emphasize that the quantitative concept of tumor grading can be extended and refined by integrating EMT relevant data. Hereby, patients with periampullary carcinomas but without the knowledge of the exact topographical primary tumor can be stratified into prognostically relevant groups and, where applicable, benefit from an adjusted adjuvant treatment. Due to the fact that the digital revolution is more and more entering routine diagnostics and assists pathologists in the evaluation of HE and immunohistological stainings 2 at the invasive front. 2 visible area of the digital slide was available for manual quantification of tumor buds at the recommended 20× objective magnification. Nevertheless, Lohneis et al. demonstrated that, regardless of the applied quantification approach, an increased number of tumor buds correlates with a poor patient survival. The second limitation is the usage of ECad as an immunohistological marker for a) EMT and b) the facilitated detection of epithelial cells . As being a reliable marker for EMT, Nevertheless, two main limitations of our study concept have to be discussed. First, the investigated hotspot area was smaller than the internationally recommended field size. ITBCC guidelines recommend to assess tumor budding in one hotspot measuring 0.785 mmTaken together, we established a multivariable model of tumor budding and ECad shifting in periampullary adenocarcinomas. We first investigated their relationship in the complex morphomic context, confirming prognostic relevance of this process. Our results also call attention to the fact that the prognostic value of the larger tumor cell clusters can be as high as that of the small tumor buds. We can conclude that tumor budding of periampullary cancers can be used in prognostic stratification of these tumors and may assist therapeutic decision making in the future.Supplementary figures and tables.Click here for additional data file.


Caenorhabditis elegans. With advances in massively parallel sequencing, multiple whole-genome sequenced (WGS) strain collections are now available to the research community. The Million Mutation Project (MMP) for instance, analyzed 2007 N2-derived, mutagenized strains. Individually, each strain averages ∼400 single nucleotide variants amounting to ∼80 protein-coding variants. The effects of these variants, however, remain largely uncharacterized and querying the breadth of these strains for phenotypic changes requires a method amenable to rapid and sensitive high-throughput analysis. Here we present a pooled competitive fitness approach to quantitatively phenotype subpopulations of sequenced collections via molecular inversion probes (PhenoMIP). We phenotyped the relative fitness of 217 mutant strains on multiple food sources and classified these into five categories. We also demonstrate on a subset of these strains, that their fitness defects can be genetically mapped. Overall, our results suggest that approximately 80% of MMP mutant strains may have a decreased fitness relative to the lab reference, N2. The costs of generating this form of analysis through WGS methods would be prohibitive while PhenoMIP analysis in this manner is accomplished at less than one-tenth of projected WGS costs. We propose methods for applying PhenoMIP to a broad range of population selection experiments in a cost-efficient manner that would be useful to the community at large.Whether generated within a lab setting or isolated from the wild, variant alleles continue to be an important resource for decoding gene function in model organisms such as  C. elegans haploid genome is compact, containing just over 100 Mb, and yet is capable of generating a complex organism with a defined cell lineage . The identification of causal variants across this space can be a laborious process although a variety of methods now exist to aid in the sequencing and mapping of mutant genomes .Caenorhabditis elegans Natural Diversity Resource (CeNDR) there are now many sequenced alleles across the C. elegans genome that could prove informative to deciphering the roles in organismal fitness of uncharacterized and characterized genes alike  mimic the allelic diversity of a forward genetics screen but with a smaller solution space much like a reverse genetics screen and 2) generate quantitative data regarding population fitness to assess potential gene function. We exploited the self-fertilizing hermaphroditic nature of C. elegans to grow multiple strains in pools with little or no genetic mixing. We hypothesized that distinct mutations in each strain could be treated as a barcode to identify and quantify the representation of each strain within the pool. To assay the mutations and thus the representation of each strain in these pools, rather than use cost-prohibitive whole genome sequencing, we adapted molecular inversion probes (MIPs) to identify strain-specific variants (C. elegans mutant strains (phenotype via MIPs (PhenoMIP) by quantifying the proportion of each strain in a pool. As a proof of principle, we utilized the Million Mutation Project as a source for our strains. The MMP library of 2007 N2-derived mutant strains harbors a variety of coding alleles including potential null alleles across 8150 protein-coding genes, and coding or splice site-altering SNVs across 19,666 genes  and appropriate MIP pools as previously described in 6 and 32.7x106 combined reads with an average 1566 reads per probe .MIPs were pooled based on worm pools being tested and generated as previously published  E. coli strains. Pools were grown at 20° for 96-120 hr which allowed for a brief period of starvation of less than 24 hr to synchronize any remaining eggs to the L1 stage. Worms were then washed off with 10-15 ml M9, pelleted and aspirated to 5-6 ml before population density was assessed. 50-100 ul of pellet was frozen as a representative sample of the initial pooled population. Pools were then redistributed in equal-sized populations between 5000 and 10000 animals on 15 cm 8X peptone NGM plates that were seeded with bacteria based on the reported experimental conditions. RNAi pooling experiments were carried out on 8X peptone NGM plates supplemented with IPTG to 4mM and carbenicillin at 25 µg/ml. RNAi plates were seeded with ∼500 ul of saturated overnight cultures grown with carbenicillin at 25 µg/ml. For each batch of plates generated, dpy-11 RNAi control plates were used to ascertain the effectiveness of the RNAi feeding. Populations were grown for ∼96 hr before being transferred to replicate condition plates either by chunking or washing again. At this point, the transferred populations were in a “starvation” state for less than 24 hr, and most remaining eggs hatched and synchronized to approximately the L1 stage. Any remaining animals were washed from plates with double-distilled water, pelleted, and frozen as samples for later analysis. Each cycle of transfer approximately followed a single generation and pooling experiments were propagated for 6-10 generations. Heavily contaminated plates/conditions were terminated from propagation and removed from analysis.Worms were maintained at 20° on standard nematode growth media (NGM) seeded with VC20019 mapping strain or DM7448 . Briefly, mapping strain males were crossed with mutant hermaphrodites. 15-20 cross progeny L4 hermaphrodites were selected to a single 10 cm OP50-seeded NGM plate and grown to starvation before propagating a subpopulation to a replicate 10 cm plate. Slow growth mutants were mapped on 10 cm NGM plates seeded with OP50 and grown at 20°. ED3052 was mapped on 10cm NGM plates supplemented with 25 µg/mL carbenicillin, 4mM IPTG, and seeded with emb-27 RNAi or HT115 bacteria. Mapping populations were propagated under selection for four to seven generations. Representative samples were chosen to extract genomic DNA as template for MIP-MAP libraries and then sequenced on Illumina MiSeq or NextSeq instruments. MIP-MAP analysis was completed as previously described  before being chunk-transferred to replicate plates for continued selection. Samples were grown in 2 selection series (emb-27 RNAi bacteria) and 2 control series (HT115 bacteria). Plates were propagated in this manner for 11 cycles with samples collected each cycle for subsequent sequencing analysis. Samples from a subset of timepoints were made into genomic DNA and the VC20019 MIP-MAP series of probes were used to create sequencing libraries. Incompatible probes were removed from analyses before determining the mean abundances across all probes within a sample library. This value was used to represent the abundance of VC20019 within each replicate at each timepoint.The head-to-head assessment of For each specific MIP pool, reads were initially analyzed as previously described . In samples with negative trajectories, however, the final generation of growth was calculated as the first instance of abundance at or below the lower limit of 1.0x10−3. Mean fold-change rate was calculated based on the total fold-change abundance in the final generation of growth divided by the expected number of generations passed. Each experimental condition may have had multiple biological replicates (referred to in text as replicates), each with a time-series of samples. Each fold-change rate calculated for a single time-series represented a single replicate. Mean fold-change rate for each strain was therefore a combination of multiple replicates across multiple experimental conditions .Population structure and fold-change analysis was calculated across each experiment using the amalgamated data from above. Strains with a starting abundance value below 2.5x10Comparison of intra-strain growth differences were completed by identifying pairs of conditions from pool M10 and M11 strains for which the mean fold-change rate had a difference of more than 20% between conditions. All pairs of data sets meeting the threshold difference were then statistically analyzed using Wilcoxon rank-sum test with p-values adjusted for multiple testing by the Benjamini-Hochberg method.Principal component analysis (PCA) of pool M11 was completed by separating the data set by generation or growth condition. PCA analysis was completed in R using the prcomp function (stats library) with data centered and scaled. Centroids represent each group as indicated by the legends, colored with information provided after PCA was completed.https://github.com/camok/PhenoMIP). Raw sequence files for pools M1 to M11 and the wild isolate pooling are available from the NIH Sequence Read Archive bioproject PRJNA595923. File SD4 contains all wild isolate pooling information including strains pooled, fold-change rates by replicate, and ED3052 mapping interval information. File SD5 contains all read depth information for each MIP within each sequencing library separated by pool and the mean abundance of each strain within each replicate separated by pool. Supplemental material available at figshare: https://doi.org/10.25387/g3.12869570.MMP and wild isolate strains are available through the Caenorhabditis Genetics Center (CGC) and the mapping strain DM7448 is available upon request. File SD1 contains molecular inversion probe sequences and data for all 2007 MMP strains and 40 wild isolates of the Million Mutation Project. Four candidate probes for each strain were designed and listed in this file. File SD2 contains all information used in the false positive and precision analysis of PhenoMIP. File SD3 contains all mean FCR data for each strain on each replicate in each experimental pool and summaries of each strain based on pool and treatment. Custom scripts used to analyze sequencing data are available at GitHub . We generated five different sets of genomic DNA mixtures composed of subsets from 46 of the 48 MMP target strains in different proportions (two strains failed to yield adequate amounts of DNA) and used these as template samples for the generation of MIP sequencing libraries . From these libraries we observed the expected composition and proportion of genotypes for the original genomic templates, suggesting that overall cross-MIP interference from multiplexing was negligible  and that the variant information from sequencing was correct. We analyzed the total number of UMIs for each MIP to gauge the efficiency of each probe. We observed eleven MIP targets that, across all libraries, consistently produced UMI counts below 20% of the mean number of UMIs per MIP in an individual library; these were removed from further analyses . To investigate the read distribution of this adjusted dataset, we normalized the UMI counts for each MIP against the minimum read number within its sequencing set. The normalized distribution of reads spanned across a ∼ninefold range with an inter-quartile range of twofold to sixfold suggesting that our distribution was relatively unimodal and ranged within a single order of magnitude .−4 across five MiSeq-generated data sets for which the mean UMI count per MIP was 1630 with 1.2x106 unique capture events across the total set. We also compared two sequencing runs of the same PhenoMIP library with false positive rates of 1.49x10−4 at 3.9x105 total capture events vs. 1.18x10−4 at 5.14x106 total capture events. Combining all data sets we confirmed a total false positive rate of 1.25x10−4 across all MIPs. We estimated the mean false positive rate per individual MIP to be 1.29x10−4 ± 1.38x10−4, which compares well with our prior observations .From our analyses, we concluded that relatively consistent and balanced pools of MIPs could be generated for future analysis on complex populations; that our false positive rates remained in line with previous observations; and that overall variance among MIPs for a specific target strain was low, especially in the lower ranges of abundance. In combination with our MIP-MAP data  were used in different experiments and in one experiment two different methods of transfer were used (see below). The proportion of each strain in the pool was assayed at the start, terminal and various intermediate points. To ensure that a similar number of animals was present at the start and in each of the replicates (and different conditions in experiments where more than one condition was assayed), we hand-picked 20 animals from each strain at either the L1  or L4  stages to duplicate E. coli seeded plates. Strains observed to have extremely poor population growth, or a high incidence of spontaneous males were excluded from the pooling process. We chose to grow these “starter” pools until a brief starvation of less than 24 hr  suggesting they harbored potentially strong deficits to population fitness . We classified the remaining 202 strains using 393 sequencing libraries across seven competitive fitness pooling experiments on 95 replicate conditions to generate profiles for 170 strains grown on the bacteria HT115, 149 strains grown on NA22, and 105 strains grown on OP50 . While we observed more subtle differences within some strains for growth on different bacteria and even for methods of transfer , we observed pronounced differences in growth profiles between strains and focused further analysis on this feature. We observed strains that exhibited poor growth with steep population decline suggesting fitness defects as well as strains with enhanced growth when compared to our reference strain VC20019. Based on these observations, we classified each strain into one of four classes as determined by its mean FCR across all experimental replicates (10) based on the log2-transformed mean fold-change rate . In particular, we observed that the MMP strain VC20019, which we had previously reported as having a rate of growth similar to the lab reference strain N2 . The higher VC20019 in pool M8 may be a result of over-representation in the seeding population by double as this strain was also conspicuously absent from the M7 seeding population, which was pooled in parallel to M8. Our observations of the Confident that the assay was behaving well overall, we next assessed each strain separately for relative changes in its abundance over multiple generations across multiple replicates. For each replicate condition within a pooling experiment, this effectively created a growth profile for each strain consisting of the total fold-change and the mean fold-change rate (FCR) per generation. For example, neration . Comparist pools , which sVC20019. We used our MIP-MAP protocol . The primary candidate mutation for VC40788 is a G405R mutation in the mitochondrial protein B0303.3, which is predicted to have multiple functions including an acetyl-CoA C-acyltransferase activity. B0303.3 has no reported hypomorphic or null mutant alleles but is reported to have an embryonic lethal phenotype by RNAi  and its human ortholog HADHB is implicated in trifunctional protein deficiency phenotype . These changes encompass 6 protein-coding variants across 5 genes . In addition, there is a 66kb copy number variant deletion within this interval that encompasses 17 protein coding genes and 1 non-coding RNA. There may also be variants that were not positively identified by the MMP sequencing data due to high divergence from the N2 reference genome  that maximized genomic diversity while reducing SNV redundancy and pooled these with VC20019 . The embon HT115 . This maC. elegans genome is our ability to identify the phenotypes associated with gene function (With advances in sequencing, genome-editing, and imaging, one remaining bottleneck in the characterization of the function . The abi−4 which translates to better than 1 in 1000 genomes per sample. As a demonstration of this method, we pooled MMP strains into groups and dissected population composition over multiple generations. Our observations suggest that this form of population barcoding is indeed capable of identifying specific Million Mutation Project strains with differing levels of relative fitness. Our analysis demonstrates that PhenoMIP identifies reproducible condition-dependent population stratification among populations that have been separated for multiple generations. Based on the strains tested thus far, we estimate upwards of 82% of MMP strains may harbor alleles contributing to fitness phenotypes in the range of class 0 to class 2. Given the mutagenized and inbred nature of the MMP strains (To use MIPs as a means of barcoding strains for population analysis, we designed a series of probes for the 2007 MMP strains and tested a subset on the MMP collection. We observed that we could accurately gauge a strain’s relative abundance within a sample. By sequencing multiple genomic mixtures, we confirmed a low false positive rate, suggesting we could use MIPs to accurately identify subpopulations with abundance as low as 8x10VC20019 animals in the initial pooling also affected the population structure and mean fold-change rate of VC20019 itself. A potential solution to mitigate “seeding” variation would be to synchronize all of the target strains with sodium hypochlorite in the L1 larval stage (OP50 replicates may have experienced premature starvation or uneven food distribution among populations, leading to lower population sizes and possibly affecting the consistency of the OP50-grown replicates. For an auxotrophic food source such as OP50, it would be best to concentrate cultures to generate a higher density bacterial lawn for nematode populations to consume. Lastly, the method and timing of population transfer is a potential source of selective influence. Our data suggested that chunking vs. washing populations to propagate them did introduce technical variation within a small subset of strains. A method of population transfer that was not addressed in this work is the sodium hypochlorite synchronization method (The observed population-level phenotypes presented in this work are a readout of relative fitness in a multi-strain competitive environment. Depending on selection and pooling method, weaker changes to relative fitness may be attributed to the population mixture rather than the selection variable itself (al stage  and thenn method , which wCaenorhabditis elegans Natural Diversity Resource (et al., utilized RAD-seq techniques to assess starvation resistance on a multiplexed pool of 96 wild isolate strains (C. elegans genome.Looking to the future, given the wide range of sequenced strains available from the Million Mutation Project and C. elegans populations for relative fitness. With PhenoMIP, we analyzed 217 MMP mutant strains across 95 replicate conditions and 29 timepoints for a total of 393 genomic samples. A similar analysis of our experimental data by whole genome sequencing with at least a 2000X read depth across the genome on 393 genomic samples would require between 430 and 650 300-cycle Illumina NextSeq runs. In contrast, our data were generated on the equivalent of a single 75-cycle NextSeq run. Methods such as harp can calculate the maximum likelihood estimates of pooled sequence data (harp results are most optimal when the divergence between founder genomes is 5–33% and the MMP genomes are much less divergent at an average of 400 SNVs per strain. The wild isolate strains, while more diverse, may be eligible for such methods. To match the sensitivity of PhenoMIP, samples would still require at least 200X coverage per sample or greater than 40 times the sequencing burden of PhenoMIP itself (To our knowledge these experiments are the first to use molecular inversion probes to analyze PhenoMIP has the potential to be applied beyond the MMP and wild isolate strains to the quantitative analyze of genomic variants in many contexts. Coupled with genome-editing techniques, PhenoMIP could be useful in studying allelic series or mutants of entire pathways for subtle phenotypic effects. The assay format could be converted to look at the selection of phenotypes occurring within a single event or generation, as in a bulk taxis assay or as a method for targeted genome monitoring under selective conditions. The fundamental leverage of this method is the use of MIPs to reduce the sequencing burden while maintaining informative parity with WGS formats in identifying subpopulation frequency. In doing so, we can vastly increase the throughput of population-level experimentation while minimizing costs.


Theobroma cacao L.) is part of the global cocoa and chocolate industry valued at 44 billion US dollars in 2019. Cocoa pod borer (CPB), Conopomorpha cramerella is a major pest of cocoa in Malaysia and Indonesia that is responsible for the decline for cocoa production. They have been detected since 1980s. Unfortunately, current control strategies are inefficient for CPB management. Although biotechnological alternatives, including RNA interference (RNAi), have been proposed in recent years to control insect pests, characterizing the genetics of the target pest is essential for successful application of these emerging technologies. We generated a comprehensive RNA-seq dataset  for larva and adult stages of CPB by using the Illumina HiseqTM 4000 system to increase the understanding of CPB in relation to molecular features. The CPB transcriptome was assembled de novo and annotated. The final assembled produced 249,280 unigenes, of which 75,929 unigenes annotated against NCBI NR database and were distributed among 156 KEGG pathways. The raw data were uploaded to SRA database and the BioProject ID is PRJNA553611. The transcriptomic dataset we present are the first reports of transcriptome information in CPB that is valuable for further exploration and understanding of CPB molecular pathways.Cocoa bean ( This will lead to identification of differently expressed genes between the developmental stages that will reveal putative developmental pathways and mechanisms for further exploration.•This data will benefit molecular biology researchers of CPB in gene discovery, characterization and cloning works.•Transcriptome data of CPB is a solid foundation for studies related to CPB development. The data is valuable for further studies on putative genes and proteins discovery that controls the development of CPB. Understanding the molecular mechanisms of CPB may lead to novel control methods of the pest.1RNA-seq transcriptome data from CPB larvae and adult stages with three biological replicates have been obtained using the Illumina HiSeq™ 4000 sequencing platform. Raw reads from the sequencing have been uploaded to the NCBI Sequence Read Archive (SRA) database. Links and accession number to each sample fastq file is listed in 22.1Cocoa pod borer (CPB) adult and larvae were collected from cocoa pod in the Malaysian Cocoa Board Bagan Datuk Plantation, Perak, Malaysia (3.894131 N 100.8642093 E). Cocoa pods showing premature yellowing of the husk, a characteristic symptom of CPB infestation were collected in the field.2.2free™ DNA Removal Kit (Invitrogen), according to the manufacturer's instructions. RNA quality was assessed using Nanodrop 1000 spectrophotometer . The OD260/280 values of each RNA sample were between 1.8 and 2.0, indicating sufficient quality. Finally, the integrity of the total RNA sample was evaluated using an Agilent 2100 Bioanalyzer , with an expected RNA integrity number (RIN) threshold of 7.0. Poly(A) RNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module and libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina, both following the protocol of the manufacturer. Library construction and sequencing was performed by a commercial service provider Novogene Med NGS Clinical Laboratory  using Illumina HiSeq4000 at 150-bp paired-end (PE) reads (Each sample was homogenized with liquid nitrogen in a mortar and dissolved in 1 ml of TRI Reagent (Thermo Fisher Scientific) per 100 mg tissue. Total RNA was purified using RNeasy mini kit  following the manufacture's protocol RNA extraction with modification step. Residual genomic DNA was removed using DNA-E) reads .2.3Weak signals and low-quality sequences were removed; read ends were also screened and trimmed for Illumina adaptor sequences by sequencing company. Approximately 142 million  reads were obtained, resulting in over 42.7 Gb of paired-end data. Raw reads containing ambiguous ‘N’ nucleotides (with ration of ‘N’ greater than 10%) and low quality sequences  were removed using an in-house software (service by Novogene) in order to obtain clean read sequence . Next, a2.4Trinity RNA-Seq assembly software package version 2.9.1 The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Hevea brasiliensis is exploited for its latex production, and it is the only viable source of natural rubber worldwide. The demand for natural rubber remains high due its high-quality properties, which synthetic rubber cannot compete with. In this paper, we present transcriptomic data and analysis of three H. brasiliensis clones using tissue from latex and bark tissues collected from 10-year-old plant. The combined, assembled transcripts were mapped onto an H. brasiliensis draft genome. Gene ontology analysis showed that the most abundant transcripts related to molecular functions, followed by biological processes and cellular components. Simple sequence repeats (SSR) and single nucleotide polymorphisms (SNP) were also identified, and these can be useful for selection of parental and new clones in a breeding program. Data generated by RNA sequencing were deposited in the NCBI public repository under accession number PRJNA629890. Specifications TableValue of the Data•These datasets are important to obtain the information from biological processes and pathways occur in the formation of natural rubber.•These datasets can be useful for plant breeders to get the information for improving rubber breeding program as well as researchers which involved in the transcriptomic analysis and RNA dataset.•The data will be of practical use in the development of genetic SNP and SSR markers as a tool in rubber breeding programmes.•The data will facilitate further analysis aimed at identifying genes and pathways related to latex yield for the development of new rubber clone with improved performance.1Hevea brasiliensis) collected in Malaysia . Rubber trees have been planted on a large scale in Malaysia since the 1920s. The transcriptome dataset generated from RNA-Seq can be used to identify high-expression genes that could be manipulated in order to improve desirable agronomy traits. Currently, there are more than 150 transcriptome datasets and bioproject of rubber tree available in the various online databases including NCBI , EMBL (The European Bioinformatic Institute) and DDBJ (DNA Database of Japan). Previous transcriptome studies were focusing among others in phylogenetic relationships The data presented here contain a combined transcriptome assembly of two different types of tissue (bark and latex) from three rubber clones , Kedah, Malaysia. Three replicates were collected for each tissue type.2.2The total RNA from the bark and latex tissue samples was extracted following the Qiagen RNAeasy Plant MiniKit protocol . The resulting RNA quality and integrity was then estimated using standard Qubit Nanodrop spectrophotometry (O.D. ∼ 2.0) and Agilent 2100 Bioanalyzer  protocols. The paired-end Illumina mRNA libraries were generated using an Illumina TruSeq Kit from 1 ug of total RNA, following the manufacturer's protocol. Each sample was sequenced in multiple HiSeq 2000 lanes using the TruSeq SBS 36 Cycle Kit  to obtain 2 × 101 bp reads.2.3Q > 20. The sequences with Ns were removed before downstream analysis using Prinseq-Lite, version 0.2.0.4 H. brasiliensis genome Hevea reference sequences.Raw reads generated in FASTQ format obtained from Illumina platforms were analysed using FastQC, version 0.10.1 2.4−5. The transcriptome analysis was annotated using the BLAST2GO program The clone transcripts were searched against UniProtKB/Swiss-Prot protein databases using BLASTX (version ncbi-blast-2.2.29+), with a cut-off e-value of 1e2.5Expression profiling for each tissue sample was calculated according to the relative abundance of transcripts, aligned to the assembled reference sequence by Bowtie2 2.6Identification of contigs containing microsatellites (SSRs) was performed using the MISA program This article does not contain any studies with human participants or animals performed by any of the authors.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Until some years ago, studies carried out on in vitro models investigating the action of Aβ/tau on proliferation and differentiation of hippocampal neural stem cells led to contrasting results, mainly due to discrepancies arising from different experimental conditions . To date, studies investigating in situ adult hippocampal neurogenesis indicate severe impairment in most of transgenic AD mice; this impairment precedes by several months cognitive dysfunction. Using experimental tools, which only became available in the last few years, research in humans indicated that hippocampal neurogenesis is altered in cognitive declined individuals affected by either mild cognitive impairment or AD as well as in normal cognitive elderly with a significant inverse relationship between the number of newly formed neurons and cognitive impairment. However, despite that such information is available, the question whether impaired neurogenesis contributes to AD pathogenesis or is a mere consequence of Aβ/pTau accumulation is not definitively answered. Herein, we attempted to shed light on this complex and very intriguing topic by reviewing relevant literature on impairment of adult neurogenesis in mouse models of AD and in AD patients analyzing the temporal relationship between the occurrence of altered neurogenesis and the appearance of AD hallmarks and cognitive dysfunctions.Adult hippocampal neurogenesis is a physiological mechanism contributing to hippocampal memory formation. Several studies associated altered hippocampal neurogenesis with aging and Alzheimer's disease (AD). However, whether amyloid-β protein (Aβ)/tau accumulation impairs adult hippocampal neurogenesis and, consequently, the hippocampal circuitry, involved in memory formation, or altered neurogenesis is an epiphenomenon of AD neuropathology contributing negligibly to the AD phenotype, is, especially in humans, still debated. The detrimental effects of Aβ/tau on synaptic function and neuronal viability have been clearly addressed both in  The hippocampus is recognized as a brain area primarily involved in memory formation, e.g., pattern separation, emotional memory, and cognitive flexibility  Selkoe, . Alzheimin vitro and in vivo experimental AD models.In the last 15 years, many studies investigated hippocampal neurogenesis  in In vitro experimental paradigms usually consisted of NSC incubation with Aβ. However, researches carried out in various laboratories led to contrasting results about the effects of proliferation/differentiation of hippocampal NSCs, mainly because of different (i) Aβ preparations ; (ii) peptide lengths (40 vs. 42 amino acids); and (iii) working concentrations, which are often irrelevant from a physiological point of view. For example, in 2004 López-Toledano and Shelanski reported that in vitro treatment of hippocampal NSCs with micromolar concentrations of Aβ42 oligomers dose-dependently increased their neuronal differentiation  mouse model  along with the PS1 M146L mutation  phenotype  with respect to age-matched WT mice. In agreement with Demars's results, in the same experimental model Unger et al.  did not survive during maturation resulting in a reduced number of BrdU+/DCX+ and PCNA+/DCX+ cells, thus indicating impaired adult hippocampal neurogenesis. Similar findings were also obtained in Tg2576 mice, which showed increased NSC proliferation in the SGZ of the hippocampus DG but reduced integration of newly formed neurons in the DG at an age at which these mice exhibited neither amyloid extracellular deposits nor major cognitive impairment  AD hallmarks accumulation and (ii) appearance of learning and memory dysfunction suggests that the former might have a causal role in cognitive decline characterizing prodromal AD.In support of this hypothesis, experimental evidence indicates that ablation of hippocampal neurogenesis in APPswe/PS1ΔE9 mice alters hippocampal circuitry and excitability exacerbating performance deficits with respect to age-matched non-ablated animals  reduced proliferation of NSCs residing in the SGZ and (ii) decreased differentiation toward the neuronal phenotype. These changes were statistically significant before the onset of memory deficits, i.e., after 2 cycles of thermal stress. Specifically, in the brain of infected mice the number of proliferating NSCs (identified through BrdU and Ki67 labeling) in the SGZ of the DG was significantly reduced with respect to mock-infected mice, and the percentage of cells acquiring glial phenotype  vs. neuronal one [doublecortin (DCX)-positive] was significantly higher than in mock-infected cells  may play a major role in modulating adult hippocampal neurogenesis. As extensively reviewed in Lazarov and Demars  did not exhibit alterations in neurogenesis following HSV-1 infection and recurrent virus reactivations  to HSV-1-induced impaired neurogenesis is negligible. However, we cannot exclude, in vivo, that other APP metabolites generated after HSV-1-induced APP amyloidogenic cleavage may participate in the alteration of neurogenesis.Although HSV-1 infection in cultured hippocampal NSCs determined increased APP processing with the consequent intracellular accumulation of Aβ peptides and intranuclear accumulation of AICD, we found that the 4G8 antibody (recognizing the 17–24 sequence of Aβ) was able to completely revert the effects of HSV-1 on in vivo silencing of APLP2 in an APP/APLP1 double knockout mouse keeps cortical progenitors much longer in their undifferentiated state, which is consistent with the view that APLP2 plays a key role in the commitment of neuronal progenitors to neuronal differentiation  occur before the appearance of memory impairment and that stimuli, increasing hippocampal neurogenesis, ameliorate cognitive functions of AD mice. To date, a correlation between altered hippocampal neurogenesis and AD has been suggested in humans, although the cause–effect relationship between these two processes has not been ascertained yet. Therefore, strategies aimed at restoring and/or boosting adult hippocampal neurogenesis in both normal elderly people and subjects at high risk of AD  could emerge as effective strategies to prevent the onset and/or counteracting the progression of the disease. In this perspective, it is worth mentioning that in mouse models exposed to extremely low-frequency electromagnetic fields a significantly enhanced adult neurogenesis at both hippocampal DG and the subventricular zone has been reported along with memory improvement (Cuccurazzu et al., Manuscript was written and revised by RP, DDLP, and CG. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although emergence of keratin 17 (K17) and reciprocal loss of K13 are immunohistochemical hallmarks for oral mucosal malignancy, we report here findings of K17-positive (+) speckles, possibly equivalent to Civatte bodies, in benign oral lichen planus. Sixty-two biopsy samples from oral lichen planus cases were subjected to immunohistochemical examinations to analyze the distribution as well as histopathogenesis of Civatte bodies. K17 was irregularly positive among oral lichen planus-affected epithelial cells, and K17-positive (+) filamentous structures were irregularly distributed within the cytoplasm in confocal images. K17+ speckles were identified as Civatte bodies, and they were mainly distributed in the interface between epithelial cells and lymphocytic infiltrates , followed by distribution within the epithelial layer  or within the lamina propria with lymphocytic infiltration . Apoptotic figures were often engulfed by macrophages and clearly distinguished from Civatte bodies by the presence TUNEL signals. These results indicate that K17 is a sensitive immunohistochemical marker for Civatte bodies and useful for differential diagnosis of oral lichen planus from other oral mucosal lesions. Civatte bodies are generated from denucleation of K17+ epithelial cells during the process of cell death via dyskeratosis, which is possibly related to blood capillary collapse. Emergence of K17 has been shown to be required for cell proliferation of cancer cells and their larger size3. Additionally, our recent proteome analysis findings confirmed that loss of K13 and emergence of K17 were more exclusively specific to oral CIS and squamous cell carcinoma (SCC) as compared to other keratin subtypes4. We have also noticed the presence of K17-positive (+) cell-debris-like speckles scattered in tissue specimens obtained from oral lichen planus cases, which were obviously benign and not candidates for differential diagnosis from any malignant lesions.In previous studies, we have emphasized that reciprocal immunohistochemical profiles between keratin (K) 13 (loss) and K17 (emergence) are valuable for histopathological differential diagnosis of oral carcinoma in-situ (CIS) from epithelial dysplasia, which is challenging when only using hematoxylin and eosin (HE) stained sections5. Historically, they were first clearly described and illustrated as “corps hyalins” by Achille Civatte in the lichen planus section of his dermatopathology atlas published in 19576, though the presence of Civatte bodies themselves in lichen planus was first described by Sabouraud in 19127, according to the review by Burgdorf and Plewig8. Following presentation of Civatte’s atlas6, the presence of colloid/hyaline bodies has come to be recognized as one of the five classic and important histopathological findings of oral lichen planus, in addition to (1) hyperkeratosis or parakeratosis, (2) saw-toothed rete pegs, (3) superficial infiltrate of lymphocytes, and (4) basal cell liquefaction9. Hyperkeratosis, especially hyperorthokeratosis with hypergranulosis, are typical of skin lesions, but not always features of oral lichen planus11. The term “Civatte body” itself as replacement for colloid/hyaline body seems to have become widely accepted in the late 1960s, as seen in Montgomery’s textbook of dermatology, though that eponym is not specifically referred to in the text12.Civatte bodies, also known as colloid bodies or hyaline bodies, are regarded as a diagnostic hallmark for lichen planus13. However, Jean Civatte presented the initial immunohistochemical finding of immunoglobulin (Ig) M localized in Civatte bodies14. Thereafter, Ig-absorbed Civatte bodies were shown to be epithelial cell debris based on immunohistochemistry findings of keratin subtypes in skin lesions, including lichen planus17. Contrary to skin lesions, there has only been a small number of immunohistochemical investigations of keratin (wide spectra) presented and no keratin subtypes have been clarified for Civatte bodies of oral lichen planus20, though the immunohistochemical profiles of oral mucosal epithelial cells are known to be different from those of skin epidermal cells.Ebner and Gebhart had previously disclosed in their ultrastructural study conducted in 1972 that Civatte bodies are of epithelial origin, based on evidence showing that 8- to 10-nm sized filaments and cell organelles, but no nuclear components, were packed inside themBased on our findings noted above showing that K17+ speckles in oral lichen planus were identical to Civatte bodies, we speculated that K17 immunohistochemistry would be useful to demonstrate Civatte bodies and might also be effective for histopathological differential diagnosis of oral lichen planus. In the present study, we investigated tissue samples obtained from oral lichen planus patients to determine the exact distribution profile of Civatte bodies and elucidate the background of K17 emergence in oral lichen planus-affected epithelial cells. From those results, we discuss the possible histopathogenesis of Civatte bodies as well as their mode of cell death, which is different from apoptosis.Biopsy specimens from a total of 62 cases of oral lichen planus were collected for the present study from the surgical pathology files of the Division of Oral Pathology, Niigata University Graduate School of Medical and Dental Sciences, that had been obtained during the 12-year period from 2008 to 2019, as well as from the files of Fukuoka Dental College Hospital from 2018 to 2019, following a histopathological review. As controls, 10 surgical excision samples of CIS of the oral mucosa containing normal mucosal components were subjected to immunohistochemical examination. The specimens were fixed in 10% formalin and routinely embedded in paraffin. Serial sections were cut into 4-µm slices from paraffin blocks and subjected to hematoxylin and eosin (HE) staining and immunohistochemistry. The present experimental protocol for analyzing surgical materials was reviewed and approved by the ethics review boards of Niigata University Graduate School of Medical and Dental Sciences  and Fukuoka Dental College . All experiments were performed in accordance with the relevant guidelines and regulations. The need for written informed consent was waived by the institutional ethics committees with informed consent obtained in the form of an opt-out form available on the web-site of each hospital.3. For K10, K13, K17, and K19, sections were pretreated as previously described21. For cleaved caspase 3 (caspase 3) and CD68, sections were autoclaved at 121 °C for 10 min in Tris–EDTA buffer (pH 9.0) and citric acid buffer (pH 6.0), respectively. For CD31, sections were pre-treated with 0.2% trypsin  in 0.01 M Tris–HCl (pH 7.6) containing 0.1% CaCl2 for 30 min at 37 °C. For the control experiments, the primary antibodies were replaced with pre-immune IgGs. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed as described elsewhere4. K17+ speckles were compared with Civatte bodies seen in serial HE-stained sections.Immunohistochemistry was performed using the ChemMate Envision system , as described elsewhere22. The slides were examined with an LSM710 confocal laser scanning microscope . Z-stack images were obtained at constant Z intervals of 0.5–1.0 µm. Images and montages were generated using Zeiss Zen software (Zeiss) and Adobe Photoshop .For double-immunofluorescence examinations of caspase 3 and CD68, Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody  and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen), respectively, were used as the secondary antibodies. For caspase 3 and hemoglobin, the anti-caspase 3 was pre-labeled with Alexa Fluor 568 , while Alexa Fluor 488-conjugated goat anti-rabbit IgG was used as the secondary antibody for hemoglobin. Labeled sections were covered with Immunoselect Antifading Mounting Medium DAPI . K17 immunofluorescence was performed for confocal microscopy to investigate K17+ keratin filament distribution within oral squamous epithelial cells on tissue sections by an indirect method using Alexa Fluor 568-conjugated goat anti-mouse IgG (H + L) (Invitrogen) as the secondary antibody, as described elsewhereP-value < 0.01 was considered to indicate significance.A chi-squared test was used to determine whether there was a difference for Civatte body distribution among the three tissue zones using SPSS 22 . A As a control, we performed immunohistochemistry to compare K13 and K17 under two different conditions of the oral mucosa; normal epithelia .In addition to K13 and K17, we also performed immunohistochemistry for K19, and K10, known to be specific for basal cells, and dysplastic orthokeratotic cells, respectively, of the oral mucosaP < 0.01). Civatte bodies within the epithelial layer (type A and B) were round/ovoid shaped, while type C bodies did not always have a smooth outline, but rather showed a rough margin, irregular shape, and size variations. We did not analyze the relationship between distribution modes of Civatte bodies  and clinical characteristics of the patients.Immunohistochemical results revealed round or ovoid-shaped eosinophilic cell debris without nuclei, Civatte bodies, that were strongly positive for K17. Among all cases (62 biopsy specimens) of oral lichen planus investigated, 56 (90.3%) contained Civatte bodies. Furthermore, K17-immunohistochemistry enabled visualization of Civatte bodies in three different locations; the interface between the epithelial layer and underlying lymphoid cell infiltrates 14, Biermann and Rauterberg reported that Ig-coated Civatte bodies of skin lesions were simultaneously immunopositive for K10/11, K8/18, and K13, with the latter two termed fetal keratins because of their transient expressions in fetal epidermis and results showing that skin Civatte bodies were also immunolabeled with the basal cell-specific monoclonal antibody BL716. Gürbüz et al. showed altered keratin expression profiles including mild K17 immunoreactivity in keratinocytes in lichen planus of the skin, though they did not investigate Civatte bodies15. Prior to this article, no detailed immunohistochemical investigation of keratin subtypes in Civatte bodies in oral lichen planus has been reported, though the immunohistochemical profiles for keratin subtypes of oral mucosal epithelial cells are different from those of the epidermis31. Pihlman et al. only demonstrated simultaneous immunolocalization of fibrin, IgM, and keratin (wide spectra) within Civatte bodies19.Following the first report of IgM+ Civatte bodies by Jean Civatte20. The present results showed that the former phenomenon also occurs in oral lichen planus, though switching was incomplete, as shown in Fig. 15. Therefore, lack of K10 expression in oral lichen planus-affected epithelial cells as well as Civatte bodies demonstrated in the present study indicates that epithelial cell resources for Civatte bodies are not related to orthokeratotic dysplasia cells or dyskeratotic cancer cells, both of which are positive for K10 but not for K13 or K1921.In our previous studies, we confirmed reciprocal switching from K13 to K17 and the emergence of K10 in oral malignancy16, though K13 is expressed in normal oral mucosa. Fetal epidermal K13 is now known to be expressed in the epidermis of patients with psoriasis, in which autoantibodies against K13 are also present32. In addition to K13, ectopic K17 is also expressed in the epidermis of individuals with psoriasis, in which epidermal cells are considered to be damaged and activated by external stimuli such as infection, oxidative stress, and drugs. Damaged or activated keratinocytes are monitored by dendritic cells, which mediate recruitment of Th1 or Th17 lymphocytes. Secretion of TNF-α, INF-γ, IL-17, or IL-22 from those T cells induces epidermal keratinocytes to express K17 via STAT1/STAT3 pathways33. Thus, some similar cell machineries may mediate the incomplete reciprocal switching to K17 from K13 in oral lichen planus, though no direct evidence for that has yet been shown34. In oral malignancies, the collaboration between K17 and 14-3-3 sigma modulates proliferation of oral SCC cells3, while it remains unknown whether this is also true for oral lichen planus.It should be emphasized that K13 is regarded as fetal keratin and K10 as postnatal keratin in skinLactococcus lactis subsp. lactis22, one of the resident members of bacterial flora on the oral mucosa35. Such abnormal cytoskeletal arrangement may also be considered to be due to keratinocyte damage or activation22, though the stimulating factors in cases of oral lichen planus are unknown. Previous electron microscopy studies have disclosed increased thickening and granularity of tonofibrils in oral lichen planus30. In findings presented by Griffin et al., clumped tonofilaments were shown to be engulfed by macrophages and some of the phagocytosed epithelial material could be released into the lamina propria27, likely because they are not digestible. This indigestible nature of K17+ filamentous conglomerates would enable Civatte bodies to have extended survival and be easily recognized by pathologists. However, in the present investigation, we did not find any Civatte bodies phagocytosed by macrophages, which were instead shown to frequently engulf apoptotic bodies. Although apoptotic oral SCC cells are often engulfed by neighboring SCC cells36, Civatte bodies are not able to phagocytose apoptotic cells because they are just denucleated cell ghosts/debris.As for the alternative change of keratin subtypes occurring in damaged or activated epithelial cells, our confocal microscopic study is the first to demonstrate that aberrant K17+ filaments characteristically show an irregular arrangement in oral lichen planus-affected epithelial cells. We previously reported that K17+ filaments were found to be distributed in a limited area in ring- or net-like shapes in the HaCaT cell line, which are immortalized keratinocytes, following exposure to nisin, a product of 11, we found no evidence in that article that they are derived from apoptotic cells. In a further search of relevant literature, there was no documentation showing apoptotic evidence for Civatte bodies, while the present results are the first to demonstrate that Civatte bodies are not apoptotic products. Our findings indicating absence of TUNEL signals within Civatte bodies and their K17 positivity are identical to the first electron microscopic studies conducted by Ebner and Mehregan, which demonstrated that Civatte bodies contained no nuclear components, but were exclusively composed of 8- to 10-nm (intermediate keratin) filaments13. Based on the present results, it is possible to consider that Civatte bodies are not in an apoptotic process but rather generated as a part of an unknown cell death process different from conventional apoptosis or necrosis, as an irregular process of single cell keratinization expressing K17 during which nuclei are deleted. It is difficult to explain the caspase 3 positivity in Civatte bodies shown in the present study, but it is unlikely that is simply due to its retention in filamentous aggregates, even after the autophagocytotic processes of macrophages.It is important to note that Civatte bodies do not represent apoptotic figures because they lack TUNEL signals, which must be a natural condition because of their lack of nuclei. Although Cheng et al. described Civatte bodies as representing keratinocyte apoptosis in a position paper of the American Academy of Oral and Maxillofacial Pathology for oral lichen planus diagnosis16. In addition to Ig, hemoglobin caused by hemolysis of extravasated erythrocytes from collapsed vessels must spontaneously enter Civatte bodies, which have lost their cell membranes13, and be condensed within them. In other words, Civatte bodies are soaked in soluble blood contents. We speculate that vascular collapse is due to lymphocytic infiltration, though the detailed cellular mechanism of lichen planus remains largely unknown. Nevertheless, as we previously showed in an examination of oral SCC cell lines, oxidation by extravasated erythrocytes or hemoglobin induces keratinization of carcinoma cells23. Thus, exposure of prickle cells to hemorrhage may be the general background for their K17 expression, eventually leading to K17+ Civatte bodies. We also consider that hemorrhage and hemosiderosis following blood capillary collapse are important histopathological events in oral lichen planus that have not yet attracted attention as possible histopathogenesis factors.Hemoglobin labelling in Civatte bodies suggests a possible histopathogenesis triggered by collapse of blood capillary loops in the lamina propria. Based on their results of Ig+ Civatte bodies that had absorbed Ig, Biermann and Rauterberg suggested that Civatte bodies can naturally absorb soluble molecules that are retained in the local milieuCivatte bodies of oral lichen planus are clearly demonstrable by their strong K17 immunopositivity, and they are not apoptotic products but rather the result of an unknown type of single cell keratinization process that includes denucleation and loss of cell membranes from K17+ epithelial cells. This unusual course of keratinization is likely attained in a process different from normal, starting from K13+ prickle cells of the oral mucosa. Squamous epithelial cells affected by oral lichen planus contain irregularly arranged K17+ filaments and have lost K13+ filaments. However, the switch from K13 to K17 in oral lichen planus is only halfway completed, whereas oral malignancy shows full switching.


Patients newly diagnosed with lung adenocarcinoma with bone metastases (LABM) have poor survival rates after treatment with conventional therapies. To improve outcomes, we retrospectively investigated whether the application of a more comprehensive genetic test of tumor biopsies samples from LABM patients could provide the basis for treatment with more effective tyrosine kinase inhibitors (TKIs) regimens.EGFR and ALK fusion mutations. More detailed genetic profiles of somatic exon SNVs and CNVs in 457 cancer-related genes were retrospectively derived using capture single molecule amplification and resequencing technology (capSMART).Fine needle biopsies were taken from the primary tumor (PT) and a secondary bone metastasis (BM) of 17 LABM patients before treatment. Simple genetic profiles for selecting therapies were initially obtained using an ARMS-PCR test for EGFR positive, 3 EGFR negative and 1 ALK fusion positive patient. A therapy regimen incorporating TKIs Gefitinib and Crizotinib was offered to the EGFR and ALK fusion positive patients, respectively. With the exception of two patients, molecular profiling of matching PT and BM biopsies identified a highly shared somatic variant fingerprint, although the BMs exhibited additional genomic instability. In six of 13 EGFR positive patients and in all three EGFR negative patients, examination of the genetic profiles identified additional clinically significant mutations that are known or experimental drug targets for treatment of lung cancer.ARMS-PCR identified 14 Our findings firstly suggest that treatment regimens based on comprehensive genetic assessment of newly diagnosed LABM patients should target both the PT and secondary BMs, including rogue clones with potential to form new BMs. Second, the additional information gained should allow clinicians to design and implement more personalized treatment regimens and potentially improve outcomes for LABM patients. Lung cancer is one of the most common types of cancer worldwide with an estimated 1.8 million new cases diagnosed annually  domain hyperactivity which then drives tumorogenesis  that are effective in the down-regulation of TK activity  can arise . Between January 2017 to December 2018, 17 patients diagnosed with LABM were recruited to the study (Table 7–008. BeFine needle biopsies were taken as directed by the clinician from the primary lung tumor (PT) and the most accessible bone metastasis (BM). Biopsy tissue was washed twice in phosphate buffer saline (PBS), formalin fixed and paraffin-embedded (FFPE), and then sent for histology and genetic analysis.EGFR/ALK/ROS1 Mutations Detection Kit, a one-step real-time PCR test (combined reverse transcription and PCR amplification) designed for qualitative detection of 18 EGFR mutations (exons 18–21), 5 ALK gene fusions and 9 ROS1 gene fusions.For assessing immediate treatment options after diagnosis of LABM, primary tissue was analyzed by ARMS-PCR for common hotspot mutations. Rapid genetic profiling was performed using the Chinese FDA (cFDA) approved AmoyDx® Comprehensive exome analysis of primary and metastasis FFPE biopsy tissue samples for somatic variants as well as matching blood samples for identifying background germline variants was performed by capture single molecule amplification and resequencing technology (capSMART) for a panel of 457 genes. Genes were selected mainly from the Catalogue of Somatic Mutations in Cancer , and then used the circular binary segmentation (CBS) algorithm to segment the log ratio profile into segments of equal copy number , p is the tumor purity calculated by Facets  diagnosed with LABM are summarized in Table EGFR TK activity variants and ALK/ROS gene fusions , L858R (n = 5), G719X (n = 1) and L861 (n = 1) and, one patient (P14) was positive for an EML4-ALK gene fusion. The remaining three patients (P15–17) were mutation negative.For rapid evaluation of targeted therapy options, PT and BM samples were initially screened for ns Table , Fig. 3.EGFR mutation positive patients, a variety of TKIs such as Gefitinib, Erlotinib and Osimertinib , standard chemotherapy and/or targeted radiation therapy regimens were administered. Disease was progressive in P15 and stable in P16 after treatment for 5 and 3 months, respectively. P17 succumbed to the disease 2 months post-treatment.Based on disease severity, tumor assessments and genetic evaluation, the managing clinicians initiated personalized treatment regimens for each patient Table . DiseaseWe applied the capSMART assay that surveys somatic exonic variants in 457 tumor-related genes (Supplementary Table EGFR (13 patients) and TP53 (12 patients). The remaining 97 variants in 78 genes were sporadically distributed in the tumor samples. The most common CNVs involved EGFR mutation amplifications (5 patients), followed by TP53, CDKN2A, RAC1, FGFR1, SDHA, SDHC, RECQL4 and STK11 deletions/duplications (> 8 of the 17 patients for each CNV). All the somatic EGFR variant types and the EML4-ALK gene fusion originally detected by ARMS-PCR were also confirmed by capSMART analysis , were shared between the PT and BM. In the vast majority of cases, the copy numbers of the shared variants were also very similar. This suggests that in these patients, the secondary BM was most likely derived from a dominant clonal line present in the PT. The remaining non-shared variants presumably represent independent de novo passenger variants that subsequently arose by random events associated with ongoing tumorigenesis. In contrast, the PT and BM from patients P10, P11 and P17 showed very few somatic variants, and of these, none were shared genetic variants. For patients P10 and P11, the EGFR driver mutation L858 was found exclusively in the secondary BM by both ARMS-PCR and capSMART analysis. While low levels of EGFR mutations cannot be completely excluded in the PT, the divergent genetic profiles suggest that the BMs most likely originated from a less dominant L858 positive clonal line present in the PT.Overall, for 14 patients , > 50% of the somatic variants identified  and P10 and P11 (only BM), all had low level EGFR mutation amplifcations (< 5 copies) and thus no additional treatable drug was indicated. In contrast, for patients P3 P9, P12 and P13 (both tumors) and P6 (only BM), all had clinically significant EGFR mutation amplifications (> 5 copies). Thus, these patients may have benefited from the level B drug Erlotinib which is known to be more effective than Gefitinib for EGFR driver amplifications  above and beyond those initially identified by the simple ARMS-PCR test.EGFR, the genetic PT and BM profiles of somatic variants identified in other affected genes were very different, indicating divergent clonal evolution in each patient which is a common feature of LA as well as many other cancers  in these patients.Using the simple ARMS-PCR test, 14 of the 17 newly diagnosed patients were initially treated with TKIs for either s Bender . It is iOur capSMART test also enabled quantitative assessment of the copy numbers of treatable mutations and deletions/duplications within the PT and the BM tissues which could be an aid to gauge the clinical significance of these variants. Thus for clinicians managing these LABM patients, it could have been possible to use this information to design and tailor a more personalized TKI treatment regimen with or without chemotherapy or radiotherapy that potentially would have been more effective in targeting both the PT and BM tissues harboring these tumorigenic mutations. On this basis, further studies in a larger cohort of newly diagnosed LABM patients may assist to apply the knowledge gained from using a more comprehensive gene test and determine whether the disease can be more effectively stabilized, and importantly, extend survival rates above current median values.While the main strength of this study was access to PT and secondary BM biopsy samples from a valuable set of patients with LABM, there were some clinical and technical limitations. Firstly, tumor analyses were restricted to biopsy samples from the PT and the most accessible secondary BM. Thus, due to clinical risk, we were unable to obtain biopsy samples from other secondary tumors that had additionally formed in some of the patients. Secondly, we did not collect biopsies from multiple tumor sites to assess intratumor heterogeneity. Such an approach may have revealed additional treatable somatic variants against other clonal cell lines, particularly for P10 and P11 where there was strong evidence of discordant genetic profiles between the PT and the secondary BM. Thirdly, while genetic profiles were focused on a preselected panel of 457 genes, a more comprehensive whole exome sequencing approach may have yielded additional treatable somatic variants in other tumor related genes.LABM is an aggressive disease whereby effective treatment needs to be rapidly administered to slow disease progression and improve survival rates. We suggest that based on our findings, the new standard of care should involve an initial comprehensive screen of the PT biopsy together with any available BMs that are accessible for biopsy. This approach will ensure that the treating clinician is provided with more personalized genetic information to tailor effective targeted therapy options and develop a more effective treatment regimen with or without chemo or radiation therapy. Further, with the advent of liquid biopsy as a means to survey the ctDNA released from clonal variants of PT and BM tissues (Cohen et al. Additional file 1.Additional file 2.Additional file 3.


Field resistance is often effective and durable as compared to vertical resistance. The introgression line (INGR15002) derived from O. glumaepatula has proven broad spectrum field resistance for both leaf and neck blast.Quantitative Trait Loci (QTL) analysis of INGR15002, led to the identification of two major QTL - qBL3 contributing about 34% and 32% phenotypic variance towards leaf and neck blast resistance, respectively and qBL7 contributing about 25% of phenotypic variance for leaf blast. Further, qBL3 was fine mapped, narrowed down to 300 kb region and a linked SNP maker was identified. By combining mapping with microarray analysis, a candidate gene, Os03g0281466 , was identified in the fine mapped region and named as Pi68(t). The nucleotide variations in the coding as well as upstream region of the gene was identified through cloning and sequence analysis of Pi68(t) alleles. These significant variations led to the non-synonymous changes in the protein as well as variations (presence/absence) in four important motifs  at promoter region those are associated with resistance and susceptible reactions. The effect of qBL3 was validated by its introgression into BPT5204 (susceptible variety) through marker-assisted selection and progeny exhibiting resistance to both leaf and neck blast was identified. Further, the utility of linked markers of Pi68(t) in the blast breeding programs was demonstrated in elite germplasm lines.This is the first report on the identification and characterization of major effect QTL from O. glumaepatula, which led to the identification of a putative candidate gene, Pi68(t), which confers field resistance to leaf as well as neck blast in rice. Magnaporthe oryzae, is one of the most destructive diseases that affects the crop, right from the vegetative stage (leaf blast) to the reproductive stage (neck blast)  were identified from wild species i.e. O. minuta and O. australiencis. However, a total of 350 QTL were identified for blast resistance, which includes QTL for field resistance  derived from the cultivar Gumei-2 is reported to confer resistance to both leaf and neck blast  and the best progeny lines exhibiting blast resistance was identified. Sequence analysis of alleles (promoter and its coding part) of candidate gene from resistant and susceptible lines revealed the presence of unique SNPs at coding as well as promoter region. The candidate gene linked markers identified along with improved lines possessing QTL for blast resistance developed through this study are novel genomic and genetic resources, which can be effectively used for imparting blast resistance in rice improvement programs.Wild species of O. glumaepatula (IRGC 104387)//2*PR114, was identified and registered as a donor for field resistance to leaf and neck blast , INGR15002/BPT5204/*BPT5204 (BC1F2:3), INGR15002/CO39 (F2)  were sown on uniform blast nursery beds at ICAR-Indian Institute of Rice Research (ICAR-IIRR), Hyderabad. After 15 days of germination, they were screened for leaf blast using a mixture of seven virulent isolates (to simulate the field resistance) collected from India as well as at field conditions at North East India  were evaluated under natural conditions at Barapani (Manipur) in North Eastern region of India. Sowings were done in two sets - one set was used for leaf blast and another set for neck blast. To obtain a natural high neck blast incidence, three panicles for each F2 and BC2F2 plant were syringe inoculated with a mixture of four isolates collected from the hotspot region of NE India  and BC1F2  population were used for QTL analysis. To detect the QTL, the software, inclusive composite interval mapping (ICIM) version 4.0 with additive and dominant QTL (ICIM-ADD) methods was chosen (http://www.isbreeding.net). Parameters like LOD score of > 5.0, window size of 1 cM, 1000 permutations and type I error set at 0.05 were applied for the QTL identification.A linkage map was constructed using the genotyping data of 150 polymorphic SSR markers. Phenotypic data collected from different crosses at two different locations  was used for the fine mapping studies. Also, additional polymorphic SSR and SNP markers (Table SA modified version of RAD sequencing known as ddRAD was performed in two parents (INGR15002 and PR114) using Illumina TrueSeq chemistry on Illumina Hi Seq 2000  as described by Sinha et al. (http://genotypic.co.in/Products/4/Biointerpreter.aspx).The resistant (INGR15002) and susceptible (PR114) lines were grown in a growth chamber maintained at 26 °C with 70% relative humidity with 14 h day and 10 h night regime. The mock samples were inoculated with water while the test samples were spray inoculated with blast inoculums as described earlier. The leaf tissues were collected before and after infection at 10 dpi. Total RNA was isolated using the protocol described by Chomczynski . Labelina et al. . DiffereqRT-PCR was performed among INGR15002; PR114 and BPT 5204 [was added as a susceptible check for comparisons and the tissue samples were collected as discussed earlier]. cDNA was synthesized using Thermo cDNA synthesis kit as per manufacturer’s instructions. Triplicate quantitative assays  were performed using 1 μl cDNA dilution samples with SYBER Green Master Mix  in Light Cycler® 96 Real-Time PCR. The primers for different genes were designed using Primer 3 (Table STaq polymerase (MBI Fermentas) and cloned separately into pGEM-T-Easy vector of 3 kb using JM109 competent cells . The positive transformants were selected and confirmed through PCR and restriction digestion with Not I enzyme. About 3–4 confirmed clones per amplicon were sequenced through Sanger sequencing . Based on the reference gene (Nipponbare) sequence and cDNA sequence information, promoter and genic regions were identified. For gene prediction, the online tools such as Genescan (http://genes.mit.edu/GENSCAN.html), Fgenesh (http://www.softberry.com) and Megante (https://megante.dna.affrc.go.jp/) were used. Conserved domains were identified through NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Nucleotide level polymorphisms among the alleles were identified by using Clustal Omega; MEGA 4.0 (http://www.megasoftware.net/) and DnaSP 5 (http://www.ub.edu/dnasp/) as reported earlier  and PLACE (http://www.dna.affrc.go.jp/PLACE/).The candidate gene  in the fine mapped region was cloned from four parents  using gene-specific primers. cDNA was cloned using another set of primers (Table Shttp://www.softberry.com/berry.phtml?topic=proteinloc&prg=ProtCompA) with default parameters. Phosphorelation sites among the four sequences were analyzed using the online tool Musite (http://musite.net/).Protein localization was predicted using the Protcomp software (1F2 line (IL-31–2-425) possessing the qBL3 locus in homozygous condition selected based on the foreground selection using the linked markers was crossed with BPT5204 so as to develop BC2F1 and subsequently BC2F2. About 800 BC2F2 plants were used for phenotyping and subsequent development of BC2F3 homozygous progenies. Foreground selection was carried out with linked markers as shown in Table 2F3 families that were resistant to leaf blast at IIRR and neck blast at NE were transferred to field and evaluated for agronomical traits. Background selection was performed using 125 SSR markers to determine the recovery of recurrent parent genome. Morphological features such as plant height, number of productive tillers, grain type, days to 50% flowering and average panicle length were recorded for the lines possessing the QTL in field condition for two seasons.A BCqBL3 and qBL7 were used to evaluate the polymorphism among the nine elite varieties, which are being used regularly in the rice breeding programs but susceptible to leaf / neck blast. The varieties tested are; Akshyadhan is a medium duration variety released for south India. Jaya (long bold) and Rasi  are medium duration popular high yielding varieties released for North India. RP Bio-226 is a bacterial leaf blight resistant indica rice cultivar widely cultivated in South India and known for its high yield, premium grain quality, and excellent cooking qualities. Jarava has good yield traits and released for shallow lowland, coastal saline and irrigated ecosystem. MTU 1010 is one of the very popular mega-rice varieties of India, which is extensively cultivated across India. It has high yield, short duration and desirable long slender grain type. MTU 1081 and MTU 1153 are also short duration, non-lodging and high yielding varieties. Swarna is the popular rice variety mostly grown in the low land areas with high production potential.The flanking markers of Our earlier studies, have demonstrated that the introgression line (INGR15002) exhibited resistance to leaf and neck blast  for blast resistance showed continuous distribution, indicating the resistance in INGR15002 is quantitative in nature . Out of these, 150 polymorphic markers distributed equidistantly (~ 1 marker per 5 Mb) across the genome were selected for single plant analysis. Genotyping of 188 F2 individuals with 150 polymorphic markers resulted in a linkage map with a total length of 1364.99 cM and an average genetic distance of 9.09 cM between two markers  score > 5.0  was used  and consistent results were observed  derived from a cross between INGR15002 and BPT5204  region of India, which is a hotspot for neck and leaf blast, was chosen as the second location for the determination of QTL. Phenotypic data of leaf and neck blast resistance was generated from Barapani (NE India) with two sets of same FqBL3 were BLAST-searched against the reference genome (Nipponbare) resulting in the location of the linked marker RM14635 on 6.8 Mb position and another linked marker RM14761 on 9.7 Mb position on chromosome 3, thus corresponds to 2.9 Mb genomic region of Nipponbare. Forty SSR markers from the 2.9 Mb region were used for polymorphism assay between the parents and found ten markers were polymorphic. Hence, ddRAD sequencing was employed for the identification of SNPs. A total of 6649 SNPs were identified between the parents (INGR15002 and PR114)  selected for genotyping which resulted in to the identification of polymorphism of six SNPs between INGR15002 and BPT5204. These polymorphic SNPs and SSRs together genotyped in the large population   before and after infection, a microarray analysis was carried out with various combinations. A total of 722 genes were up-regulated in INGR15002 after infection as compared to PR114  (Table STop three up-regulated genes from the coarse mapped region of ) Table S.qPCR data, Os03g0281466 has been identified as a promising candidate gene in imparting leaf and neck blast resistance in INGR15002 and the gene is designated as Pi68(t). Sequence of the candidate gene (Os03g0281466) from the resistant parent (INGR15002) was used for the prediction of gene structure through various software’s as discussed in Pi68(t) is a plasma membrane localized protein with trans-membrane domain as predicted by the Protcomp software.By integrating fine mapping, transcriptome and All the susceptible alleles have five common SNPs  leading to a non-synonymous amino acid (AA) change in the protein. These amino acid change in malectin and kinase domain in the susceptible protein  might play a role in conformational changes, which might be responsible for lack of resistance. For instance, the presence of Threonine (T) at position 165 in malectin domain is involved in formation of the kinase-specific phosphor-serine-threonine (Protein kinase B), which was replaced by Alanine (A) in the susceptible protein. In addition to the common non-synonymous amino acid changes, there were sequence-specific non-synonymous amino acid changes present in BPT5204 (3), PR114 (2) and CO39 (2) proteins (numbers in parenthesis indicate the number of non-synonymous amino acid changes in each sequence). Furthermore, two specific variations at 5′ UTR [162 (C/T) and 402 (G/A)] were also identified among the susceptible alleles. The complete details of the SNPs and the corresponding synonymous and non-synonymous changes are tabulated in Table SPi68(t)BPT5204 indicating that this allele has least evolutionary deviation compared to the other two susceptible alleles. The complete sequence of the gene along with promoter of all the alleles studied were submitted to NCBI with accession numbers MG742320; MG742321; MG742322; MG742323.Key variations (five SNPs) were found at the promoter regions of all susceptible alleles, of which, four SNPs led to the absence of four important TFBMs i.e. W-Box element, MYC element; BIHD1OS (benzothiadiazole-induced homeo domain protein); and TCP element. The resistant allele has all the four motifs which might play an important role in the resistance mechanism Table S. Among tqBL3 was introgressed into Samba Mahsuri  through marker-assisted backcross breeding (MABB) coupled with phenotyping. The complete details of the number of F1, BC1F1, BC2F1 plants raised and the corresponding positive plants identified at each stage were tabulated in Table S2F2 plants, about 400 were screened at IIRR for leaf blast, of which, 100 plants were homozygous positives. Among the other 400 BC2F2 plants screened at NE region, about 100 plants were homozygous positive for both leaf and neck blast resistance. Thus, a set of 200 homozygous BC2F2 plants were identified through foreground selection using markers linked to qBL3 as well as through phenotyping. Initially, foreground selection was performed using linked markers  were selected for background selection analysis and the recovery of recurrent parent genome ranged from 93% to 97%  and landraces  as donors for leaf and neck blast resistance , pb1 and Pi64  near telomere (Wang and Pan indica accessions reported 20 candidate genes on chromosome 3 (Chr03_1170958 locus) for rice blast  is identified to be associated with blast resistance. Interestingly, qBL3 is nearer to one of the loci associated with field blast resistance (LAFBRs) identified by Zhu et al. qBL3 is a novel QTL associated for leaf and neck blast resistance. In addition, Wang et al.  was used for identification of QTL and simultaneous introgression in to elite variety, similar strategy was reported Tanksley and Nelson qBL3 derived from the wild species O. glumaepatula for leaf and neck blast resistance.The consistency and stability of any identified QTL was determined by use of alternate mapping populations and evaluating them at diverse environments as reported by Mishra et al. qBL3 region was primarily targeted for fine mapping, since it was contributing resistance for leaf as well as neck blast. This facilitated in the rapid identification of putative candidate gene(s) associated with blast resistance. qBL7 locus is quite larger, its contribution to leaf blast resistance is less compared to qBL3 and it was not identified in the second environment, hence not chosen for further studies. To accelerate the fine mapping, ddRAD sequencing was employed between INGR15002 and PR114 since both are isogenic lines. Interestingly, a greater number of SNPs were observed on chromosomes 3 and 7 as compared to all other chromosomes, enabling fine mapping of the qBL3 region from 2.9 Mb to 300 kb and identified SNP5 as tightly linked marker for qBL3. Our previous study  are expected to have less variation since they are isogenic, except at few loci, hence the variation observed at transcript level would be primarily due to the wild genome introgression in INGR15002. Although BPT5204 was used as a susceptible parent in developing mapping populations, this parent was not included in the transcriptome analysis to confiscate obvious variations between INGR15002 and BPT5204. However, while performing the qPCR, BPT5204 was chosen as an additional susceptible check. Though many genes were up-regulated in INGR15002 after infection, only one gene (Os03g0281466) encoding malectin serine threonine kinase was up-regulated in the qBL3 fine mapped region. In addition, the peak marker SNP5 also lies very near (50 kb) to the gene Os03g0281466, hence it was identified as the putative candidate gene. Up-regulation of transcript derived from this gene was also reported in Nipponbare leaf infection with blast fungus [GSE18361]  for qBL7. However, the results obtained would certainly help in the fine mapping of qBL7 locus in future.To identify putative candidate genes for Os03g0281466 was annotated as a hypothetical gene in the RAPDB database (coding for 213 amino acids). Cloning cDNA as well as predicting the ORF with three different algorithms confirmed the gene has single ORF and deducing the protein containing 839 amino acids with two domains viz, malectin and kinase domain. Since the origin of this gene is from wild species (O. glumaepatula), the change in the gene structure as well as protein is obvious. Serine threonine kinases were known to be involved in blast resistance mechanisms  may trigger the downstream signal transduction pathways leading to resistance. However, this hypothesis needs further experimentation. The phylogenetic analysis of protein sequences of Pi68(t) clustered with the Pid2 as a separate clade, which signifies that they were grouped together as both genes encode for lectin (carbohydrate binding proteins). Nevertheless, Pi68(t) encode for malectin with kinase domain whereas Pid2 encode for AB-lectin receptor kinase.The gene Pi68(t) led to the absence of key TFBMs like W-box element, MYC element, TCP element and BIHD1OS, which may play a vital role in altering the gene expression. It is evident from previous studies that these elements are known to involve in defense mechanisms  in 5’UTR as well as promoter region of all the susceptible alleles of 2F2 plants), best five lines with high recurrent genome recovery (97%) were identified and these lines showed resistance to leaf and neck blast at two hotspot locations. Further, these lines were nominated to All India Coordinated Research Project (AICRP) for studying their reaction towards field resistance at various locations across India. The simultaneous introgression and identification of superior lines with improved resistance would accelerate in releasing the variety, and reaching the farmers in a short time. The usefulness of identified linked markers in the present study were also assessed in the popular varieties (became susceptible to blast) so as to use them effectively in marker-assisted breeding programs  through MABB approach. Through evaluation of large  designated as Pi68(t) conferring resistance to leaf as well as neck blast. The SNPs located in the promoter, 5′ UTR and coding region might play a key role in the resistance mechanisms. QTL-NILs derived from Samba Mahsuri possessing the QTL, qBL3 were identified, which exhibited high level of resistance to leaf and neck blast. The QTL linked functional markers and the MAS derived lines identified in this study will eventually aid in the building the durable broad-spectrum field resistance in race non-specific manner.The major outcomes of this investigation include the identification and high-resolution mapping of a major effect field resistance QTL from the wild species Additional file 1.Additional file 2.