Fetal alcohol spectrum disorders (FASDs) are associated with abnormal social behavior. These behavioral changes may resemble those seen in autism. Rats acutely exposed to ethanol on gestational day 12 show decreased social motivation at postnatal day 42. We previously showed that housing these ethanol-exposed rats with non-exposed controls normalized this deficit. The amygdala is critical for social behavior and regulates it, in part, through connections with the basal ganglia, particularly the ventral striatum. MicroRNAs (miRNAs) are short, hairpin-derived RNAs that repress mRNA expression. Many brain disorders, including FASD, show dysregulation of miRNAs. In this study, we tested if miRNA and mRNA networks are altered in the amygdala and ventral striatum as a consequence of prenatal ethanol exposure and show any evidence of reversal as a result of social enrichment. RNA samples from two different brain regions in 72 male and female adolescent rats were analyzed by RNA-Seq and microarray analysis. Several miRNAs showed significant changes due to prenatal ethanol exposure and/or social enrichment in one or both brain regions. The top predicted gene targets of these miRNAs were mapped and subjected to pathway enrichment analysis. Several miRNA changes caused by ethanol were reversed by social enrichment, including mir-204, mir-299a, miR-384-5p, miR-222-3p, miR-301b-3p, and mir-6239. Moreover, enriched gene networks incorporating the targets of these miRNAs also showed reversal. We also extended our previously published mRNA expression analysis by directly examining all annotated brain-related canonical pathways. The additional pathways that were most strongly affected at the mRNA level included p53, CREB, glutamate, and GABA signaling. Together, our data suggest a number of novel epigenetic mechanisms for social enrichment to reverse the effects of ethanol exposure through widespread influences on gene expression. Prenatal ethanol exposure can cause fetal alcohol spectrum disorders (FASDs). With 30% of all women reporting drinking alcohol at some time during pregnancy , FASD prBehavioral deficits can also be seen in animal models of prenatal ethanol exposure. Acute exposure on gestational day 12 (G12) in rats leads to decreased social investigation and play fighting, as well decreased social motivation in late adolescence and adulthood , 5. To dThe amygdala is critical for normal social behavior. Lesions of the amygdala alter social functions in human beings and experimental animals , and devAt the cellular level, the amygdala is composed of a group of 13 sub-nuclei located in the medial temporal lobe . These nmicroRNAs (miRNAs) are a class of short, hairpin-derived RNAs that repress gene expression at the post-transcriptional level. Mature miRNAs of ~20 nt in length canonically bind to complementary sequences found in the 3′ untranslated region of messenger RNAs (mRNAs), thereby repressing translation by ribosomes. In neurons, miRNAs also play a role in compartmentalizing specific mRNA translation in subcellular components, including axons  and synaIn this study, we extend our previous characterization  of selecTreatment of animals, as well as behavioral and mRNA expression outcomes, were described in Middleton et al. . BrieflyAnimals were exposed to ethanol prenatally as described previously . On G12,Our previous behavioral study on the same cohort of rats, described in Middleton et al. , examineThe present study was designed to determine the potential molecular substrates of decreased social motivation following prenatal ethanol exposure and its reversal by social enrichment. We first dissected the whole amygdala and ventral striatum from a total of 72 42-day-old male and female rats, using established anatomical landmarks, as described previously . After iHigh-resolution quantification of miRNA expression was performed using small RNA-sequencing from 1 μg of each pool of total RNA according to the TruSeq Small RNA Sample Prep kit . Subsequent purification methods including gel purification of small RNAs 20–30 nt in length, resulted in more than 90% of all reads in the sample attributed to miRNAs. Libraries were indexed and multiplexed in sets of 8  prior to sequencing  using Reagent Kit v3 reagents on a MiSeq Benchtop Sequencer . Raw sequence FASTQ files were imported into Partek Flow software for initial analysis. Base calls below a phred score of 20 were trimmed from the reads. These were then aligned to the Rn5 version of the rat genome using the Bowtie algorithm . The aliIn order to validate the changes seen by RNA-Seq, we also examined the samples using the GeneChip miRNA 2.0 array . Samples from the same pooled RNA were prepared using the FlashTag Biotin HSR RNA Labeling Kit . Arrays were hybridized, washed, stained, and scanned according to manufacturer protocol and the data exported and normalized using RMA in Partek Genomics Suite.® Pathway Analysis (IPA) software.We also examined the relationship of the miRNA data to our previously described mRNA dataset generated from the same pooled rat brain RNA samples; see Ref. . Those dAll of the raw and normalized microarray and RNA-Seq expression data generated in this study have been deposited in the NCBI Gene Expression Omnibus .p < 0.1 for both RNA-Seq and microarray analyses) due to prenatal ethanol exposure and social enrichment. These ANOVAs were followed by Fisher’s post hoc testing to compare specific groups within each brain region. Notably, after identifying the miRNAs with the most robust main effects within each brain region, we also performed exploratory four-way ANOVAs, using the previous three factors plus brain region, and examined the top miRNAs for any evidence of significant interactions . The results of this exploratory analysis are provided as Supplementary Material.The major finding from our previous behavioral study was that social motivation was significantly decreased in male and female rats prenatally exposed to ethanol at G12, and that this was reversed by social enrichment. The focus of the present study was to identify the molecular substrates underlying the social motivation deficit and its reversal. To accomplish this, our primary analysis utilized a 3 way ANOVA  for each brain region to identify miRNAs with highly consistent changes (1-tailed p < 0.05) according to the RNA-Seq analyses, but were not probed by the Affymetrix GeneChip miRNA 2.0 array.The top findings from the analysis of individual miRNAs were displayed in table format Tables  and 2. W® Pathway Analysis (IPA) software. We also examined the entire mRNA dataset for specific canonical neuronal pathway effects using IPA software, using a threshold of p < 0.1 from the ANOVA post hoc testing.A combined analysis of miRNA data and mRNA data was then performed. The mRNA targets of the most robustly affected miRNAs were mapped using the miRNA target filter workflow of QIAGEN Ingenuityde facto threshold for significance throughout most of our analyses because of the combined use of multiple platforms for cross-validation or the combined use of multiple genes within networks as well as miRNAs and their target mRNAs.We note that 0.1 was chosen as the We performed a global screen of all known, curated miRNA molecules. To ensure full coverage, a conservative cross-platform approach employing both miRNA microarrays and RNA-Seq was used for identification of potential miRNA of interest. Quantification of miRNAs was based primarily on small RNA-Sequencing, which has increasingly emerged as the gold standard of miRNA quantification technologies, owing to its greater sensitivity and dynamic range compared to other techniques. Orthogonal validation was performed using Affymetrix miRNA GeneChips. The application of these two complementary technologies improved our capacity to discover relevant miRNAs that may have been overlooked had a single quantification method been employed. On the other hand, because miRNA microarrays are limited to the interrogated content of the arrays at the time of manufacture, we also included in our analyses those miRNAs that were found only by small RNA-Seq.Rattus norvegicus precursor and mature miRNAs . The RNA-Seq analysis that we employed identified 1063 precursor and mature miRNAs listed in the miRBase 21 annotation  (Table p < 0.05 level) due to ethanol effects in non-enriched rats (Table p < 0.1) in both platforms and 10 additional miRNAs significantly changed (p < 0.05) that were only found by RNA-Seq  due to ethanol in non-enriched animals representing 48% directional concordance. Of these, 12 miRNAs were changed in both platforms (at the p < 0.1 level) in both platforms and 11 additional miRNAs significantly changed that were only found by RNA-Seq (Table p < 0.1) in both platforms, and 13 additional miRNAs significantly changed (p < 0.05) that were only found by RNA-Seq  showed concordant directional changes due to ethanol in non-enriched animals, with 3 changed  miRNAs showed directional concordance with 9 miRNAs changed in both platforms at the p < 0.1 level) in both platforms and 9 additional miRNAs changed (at the p < 0.05 level) by RNA-Seq  showed concordant directional changes due to enrichment, with 11 changed ] and the social enrichment effect in ethanol-exposed rats [E (SvN)], as these groups exhibited a striking reversal in social motivation . HierarcThis comparative analysis also revealed several notable individual miRNAs Table . First, p-values within the same comparisons. We note that the identical results between members of the same miRNA family likely reflect the fact that our RNA-Seq analysis could not distinguish the two isoforms using the standard read count quantification algorithm that we employed. Additional experiments on precursor forms of these miRNAs would be needed to elucidate the effects of individual miRNAs within such families. This would be particularly interesting for the mir-8 family, whose members have been implicated in synaptic development  and mir-8 (miRBase family accession # MIPF0000019) had very similar fold changes and Most importantly, we also noted several miRNAs that appeared to significantly reverse their expression levels in the amygdala after social enrichment in ethanol-exposed animals. These include precursor miRNAs mir-204 and mir-299a as well as mature miRNAs miR384-5p, miR-222-3p, and miR-301b-3p. Because these molecular changes parallel the behavioral changes, it is possible that they may be more directly related to the primary mechanisms underlying each phenotypic effect.® IPA software. The IPA core analysis workflow determines key gene regulatory networks that are overrepresented in any given set of molecules. Additionally, the activation or inhibition of upstream and downstream molecules can be predicted based on existing data and overlaid on any network to show how the overall network is affected.In order to elucidate the large-scale functional changes being affected by ethanol and social enrichment, we performed functional network enrichment analysis using QIAGEN IngenuityWe first performed this analysis using only the specific miRNAs validated using both RNA-Seq and array that were altered by ethanol in non-social animals and by social enrichment in ethanol-exposed animals Tables  and 2. Tp < 0.1) derived from the same tissues   were also found to be reversed in the amygdala after social enrichment Table . AdditioOn the other hand, 7 miRNAs  have reported significant changes in miRNAs that only show directional changes opposite to what we observed , 37, 38.Rather than focusing on individual genes, our large-scale analyses of ethanol-induced changes in miRNA and mRNA expression focus on whether effects on functionally related pathways are consistently seen across studies. From this perspective, our results add substantial support to the concept of a systems-level disruption of major gene regulatory pathways by a common environmental insult. We show in our study that prenatal alcohol exposure imposes a long-lasting effect on neuronal and, ultimately, behavioral function in adolescents. Notably, our results also extend our previous molecular analyses by incorporating the vast post-transcriptional regulatory aspects embodied by microRNAs. By evaluating concurrent changes in miRNA and mRNA levels, this work shines light on an additional layer of complexity to the gene expression changes occurring in the amygdala and ventral striatum. This is critical because miRNAs are thought to respond greatly to environmental stressors and are thought to mediate global gene expression changes .Notably, the functions represented by the pathways represented in Figures To our knowledge, this is the first report of miRNA-directed gene expression changes brought about by environmental interventions in any FASD model. The potential reversal of abnormal changes in miRNA and mRNA expression by a relatively simple intervention  is consistent with data from other disorders, where specific changes in miRNA levels have been seen to result from an enriched environment, corresponding with slowing of the disease progression and improvement in hippocampal synaptic transmission using an Alzheimer’s disease model . Given tIt is beyond the scope of the present report to fully examine the evidence for regionally specific changes in expression. However, we note that both at the individual miRNA level and target mRNA level, many of the changes appeared to be region specific. This was even more evident for some of the comparisons made for specific functional pathways. Taken together, these patterns reinforce other recent findings, such as those by Tapocik et al. , who shoThere are several limitations to note in the present study. First, it is important to acknowledge that the resulting gene expression effects we have observed in P42 rat brains reflect the cumulative effect of all life experiences to that age. That is, everything that the animal has experienced could alter miRNA and mRNA expression patterns; consequently, some of these alterations could interact with variables that were outside our ability to control. We tried to minimize the differences due to random noise that might exist between treatment groups. Nonetheless, in our study, pregnant animals ware received in the lab on G4, handled and injected on G12, potentially causing gestational stress. Exposure of the offspring to the anesthetic agents ketamine and xylazine immediately prior to decapitation may also have altered gene expression in some manner. It is possible that the treatment itself (ethanol exposure) interacted with the pre- or postnatal stressors, which include shipment, handling, and anesthetic administration. We note that the time course by which the anesthetics acted (ketamine/xylazine) is extremely brief (lasting only a few minutes prior to decapitation and dissection) and thus is not likely to create any large-scale biases in gene expression differences.per se also helps reduce concern about type 1 error. The seemingly low concurrency of RNA-Seq and array data may be the result of several factors, most notably the use of 2 different miRBase databases in our high-throughput quantification . In addition, we used very stringent concurrency criteria because it was based on exact sequence homology between the array probe and the gene annotation against which the RNA-Seq data were quantified upon.Another limitation in the present study is the lack of correction for multiple testing. We contend that our use of two independent quantification methodologies somewhat mitigates this concern. Furthermore, our focus on functional gene network analysis, rather than individual miRNAs and mRNAs In conclusion, despite some limitations, our data strongly demonstrate that prenatal ethanol exposure has the capacity to impart long-lasting gene expression changes at both the miRNA and subsequent target mRNA level. Some of these changes clearly impact large-scale functional pathways in the brain that are involved in synaptic function and intracellular signaling, as well as cell cycle regulation, and brain development. Further studies are necessary to determine the extent to which changes in these pathways represent points of no return, or novel therapeutic opportunities for intervention.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.http://www.frontiersin.org/Journal/10.3389/fped.2014.00103/abstractThe Supplementary Material for this article can be found online at Datasheet S1p-values for main and interaction effects in miRNAs of interest. These include miRNAs with significant ethanol effects in non-enriched rats [N(EvC)] and social enrichment effect in ethanol rats [E(SvN)] from both brain regions.Click here for additional data file.Datasheet S2Messenger RNA targets from group comparisons. Targets were filtered on opposing expression pairing between miRNAs and corresponding mRNA targets. Genes with functions related to neurological disease are highlighted in green. Microarray and RNA-Seq expression data have been deposited in the NCBI Gene Expression Omnibus .Click here for additional data file.


P<0.003) or squamous cell carcinoma (P=0.023) in the TCGA data set. We validate the survival prediction framework with the TMA cohort (P<0.036 for both tumour types). Our results suggest that automatically derived image features can predict the prognosis of lung cancer patients and thereby contribute to precision oncology. Our methods are extensible to histopathology images of other organs.Lung cancer is the most prevalent cancer worldwide, and histopathological assessment is indispensable for its diagnosis. However, human evaluation of pathology slides cannot accurately predict patients' prognoses. In this study, we obtain 2,186 haematoxylin and eosin stained histopathology whole-slide images of lung adenocarcinoma and squamous cell carcinoma patients from The Cancer Genome Atlas (TCGA), and 294 additional images from Stanford Tissue Microarray (TMA) Database. We extract 9,879 quantitative image features and use regularized machine-learning methods to select the top features and to distinguish shorter-term survivors from longer-term survivors with stage I adenocarcinoma ( Diagnosis of lung cancer through manual histopathology evaluation is insufficient to predict patient survival. Here, the authors use computerized image processing to identify diagnostically relevant image features and use these features to distinguish lung cancer patients with different prognoses. Lung cancer is the most prevalent cancer and the leading cause of cancer-related deaths worldwide, resulting in more than 1.4 million deaths annually1346711121416κ=0.48–0.64)κ=0.41–0.46 among community pathologists, κ=0.64–0.69 among pulmonary pathology experts and κ=0.55–0.59 among all pathologists under study)422242627Currently, lung cancer samples are manually evaluated for their histological features by light microscopy. However, qualitative evaluation of well-established histopathology patterns alone (such as the classification of tumour grades) is insufficient for predicting the survival outcomes of patients with lung adenocarcinoma or lung squamous cell carcinoma18Computerized image processing technology has been shown to improve efficiency, accuracy and consistency in histopathology evaluations, and can provide decision support to ensure diagnostic consistency3135In this study, we aim to improve the prognostic prediction of lung adenocarcinoma and squamous cell carcinoma patients through objective features distilled from histopathology images. We design a fully automated informatics pipeline to extract objective quantitative image features, assess the diagnostic utility of the feature sets, build classifiers to distinguish lung cancers with different survival outcomes, discover novel image features that predicted patient prognosis and validate the results in an independent data set. Our methods may ultimately provide prognostic information for the patients, and contribute to precision medicine of lung cancer.We obtained 2,186 haematoxylin and eosin (H&E) stained whole-slide histopathology images from The Cancer Genome Atlas (TCGA)37To extract objective morphological information from thousands of images, we built a fully automated image-segmentation pipeline to identify the tumour nuclei and tumour cytoplasm from the histopathology images using the Otsu method42P value=0.8514)) in distinguishing between adenocarcinoma and adjacent dense benign tissue when using the top 80 quantitative features . In general, the top quantitative features were Haralick features of the nuclei , radial distribution of pixel intensity and intensity mass displacement of the cytoplasm.To determine if the quantitative image features were biologically relevant, we first examined if they could distinguish malignancy from normal adjacent tissue  for the TCGA cohort. We used seven classifiers: naive Bayes, support vector machines (SVM) with Gaussian kernel, SVM with linear kernel, SVM with polynomial kernel, bagging for classification trees, random forest utilizing conditional inference treesfeatures . When clfeatures . The perP value=0.08). The top quantitative features selected by information gain ratio included Haralick texture features of the nuclei , edge intensity of the nuclei, texture features of the cytoplasm and intensity distribution of the cytoplasm. Some of the feature groups overlapped with those that were used to distinguish between benign and malignant lesions. For instance, Haralick texture features such as sum entropy and difference variance were among the top features in both classification tasks.To further validate the biological relevance of the quantitative features, we applied our classifiers to distinguish between adenocarcinoma and squamous cell carcinoma using the same set of fully automated features in both TCGA and TMA data sets. Our results showed that using 240 features selected by their utility in this task (assessed through the information gain ratio measurement), our best classifiers, including SVMs with Gaussian kernel and random forest classifiers, attained an AUC of above 0.75 in the TCGA data set ), indicating the robustness of our informatics method .Our approach for survival prediction was validated with images from an independent data set (the Stanford TMA database). The same image processing workflow with elastic net-Cox proportional hazards model selected a similar set of features, which also successfully distinguished longer-term survivors from shorter-term survivors in the stage I adenocarcinoma cohort . Similarly, Zernike shape, texture and radial distribution of intensity were among the top prediction features. 24Our prognostic methodology for squamous cell carcinoma was also confirmed in the independent Stanford TMA cohort. Elastic net-Cox proportional hazards model successfully distinguished longer-term survivors from shorter-term survivors with lung squamous cell carcinoma  and more global patterns  of the tumour cell and tumour nuclei. As a benchmark for the utility of our objective features, machine-learning models with selected features successfully identified images with tumour cells and classified tumour types, showing that our image features could recapture the important image labels annotated by trained pathologists.Patients with lung adenocarcinoma or squamous cell carcinoma are known to have very diverse survival outcomes. Even patients with the same stage and pathology grade can have very different survival times1844849We validated our informatics framework for survival prediction by an independent TMA data set, demonstrating the generalizability of our approach. We leveraged elastic net-Cox proportional hazards models, which are computationally efficient, and are capable of reducing the number of parameters in the models effectively and handling right-censored survival data. This method is well-suited for analysing large amounts of data and large number of features in our analysis. Accurate prognostic prediction generated by our models can guide clinical decision making and enhance precision medicine.We also investigated the top features associated with prognosis in lung adenocarcinoma and squamous cell carcinoma. In the adenocarcinoma group, the primary prognostic features that distinguished longer-term survivor from shorter-term survivors included Zernike shape features of the nuclei and cytoplasm and nuclei texture features. For each tumour cell, Zernike shape features of the nucleus were generated first by identifying the circle of the smallest diameter that covers the tumour nucleus, setting all pixels within the tumour nucleus to one and background to zero, and then decomposing the resulting binary image into Zernike polynomials, where the coefficients serve as features. Texture features quantified the correlations between nearby pixels within the regions of interest. This showed that nuanced patterns of nuclear shape are important determinants of patient prognosis. In the squamous cell carcinoma group, the most important features also included Zernike shape features of the nuclei. This showed that both local anatomical structures  and global patterns of the tumour cell nucleus  are associated with survival outcomes.Machine-learning techniques have previously been shown to be useful in predicting patient prognosis in several cancers and pre-cancerous lesions313234One limitation of this study is that cases submitted for TCGA and TMA databases might be biased in terms of having mostly images in which the morphological patterns of disease are definitive, which could be different from what pathologists encounter at their day-to-day practice. For instance, pathologists reviewed many slides and microscopic views, and only uploaded the most representative views to the TMA database. Although histopathology images with typical pathological patterns might be helpful in generating machine-learning models, how these diagnostic models performed in the actual clinical settings remain to be explored. In addition, certain semi-quantitative pattern assessments of adenocarcinoma, such as acinar or papillary, were not available in either databases. Future research could integrate quantitative image features along with a richer set of qualitative and semi-quantitative annotations. In addition, as the universal standard for digitalizing histopathology images is not yet established, retraining of prediction models is required for data sets with different levels of magnification. Another limitation is that this study only focused on H&E stained images. The clinical utility of integrating quantitative features from immunochemical stained images or molecular data remain to be established.In summary, we demonstrate that histopathology image classifiers based on quantitative features can successfully predict survival outcomes of lung adenocarcinoma and lung squamous cell carcinoma patients. This capability is superior to the current practice utilized by pathologists who assess the images in terms of tumour grade and stage. Investigating the objective features associated with survival also provides insights for histopathology studies. Similar approaches may be applied to the pathology of other organs. Our methods could facilitate prognostic prediction based on the routinely collected H&E stained histopathology slides, thereby contributing to precision oncology and enhance quality of care.A total of 2,186 whole-slide H&E stained histopathology images were obtained from TCGA37The whole-slide images with × 40 magnification were tiled into overlapping 1,000 × 1,000 pixels using bftools in the open microscopy environmentTo ensure the extensibility of the developed methods, tissue microarray (TMA) images from Stanford Department of PathologyInformed consent of the TCGA and TMA participants were obtained by the TCGA consortium37The pathology reports and clinical profiles of each lung adenocarcinoma and lung squamous cell carcinoma patient were acquired from TCGA as well as the Stanford TMA Database. Pathology grade , stage, and pathology diagnosis for each patient were manually curated from the pathology reports. Demographic information, such as age, gender, ethnicity, survived days and survival status for the same set of patients were also obtained. All patients with missing stage were excluded from the survival analyses.A segmentation and feature extraction pipeline was built using CellProfiler42Naive Bayes classifiersTwo automated classification tasks were designed to evaluate the utility of the extracted features: (1) to classify images of malignancy from images of adjacent benign tissues; and (2) to distinguish lung adenocarcinoma from lung squamous cell carcinoma. The inputs to the classification algorithms were the quantitative features extracted from the images as described in the previous section, and the outputs were the predicted diagnoses groups. For tumour-type classification, the prediction results for image tiles of the same patient were aggregated.Elastic net-Cox proportional hazards models (R package ‘glmnet') were built to calculate the survival index of each patientFor diagnostic classification and distinguishing malignancy from adjacent dense normal tissues, a held-out test set from each database was utilized to evaluate the performance of each of the different classifiers. Receiver operator characteristics curves were generated and AUCs of each classifier were calculated using ‘ROCR'2-tests were employed to determine if there were any treatment  differences in the predicted survival groups.For survival prediction, patients in the test set were classified into good or poor prognostic groups based on their survival indices as described above. Since there were only about 100 patients in most prediction tasks, leave-one-out cross-validation was utilized to assess the performance of our prediction models. Log-rank test was employed to examine the survival difference between different predicted groups. χBoth the diagnostic and prognostic prediction methods were validated by the TMA data set with the same evaluation methods.https://tcga-data.nci.nih.gov/tcga/). Those from the Stanford Tissue Microarray database are available at https://tma.im/cgi-bin/home.pl. All other data supporting the findings of this study are available within the article and its The histopathology images, pathology reports, and clinical information of the TCGA data set are available in a public repository from the TCGA Data Portal .Supplementary Figures 1-3 and Supplementary Tables 1-3Features and Associated Weights in the Prognosis Prediction Model for Stage I Lung Adenocarcinoma Patients in the TCGA DatasetFeatures and Associated Weights in the Prognosis Prediction Model for Lung Squamous Cell Carcinoma Patients in the TCGA DatasetA Comprehensive List of Quantitative Features Extracted By the Fully Automated Histopathology Image Processing PipelineStage I Lung Adenocarcinoma Patient Survival Prediction with Quantitative Image FeaturesLung Squamous Cell Carcinoma Patient Survival Prediction with Quantitative Image Features


Miichthys miiuy, is a representative Sciaenidae known for its exceptionally large otoliths. This species mainly inhabits turbid aquatic environments with mud to sandy mud bottoms. However, the characteristics of the immune system of this organism and its specific aquatic environment adaptations are poorly understood. Thus, we present a high-quality draft genome of miiuy croaker. The expansions of several gene families which are critical for the fish innate immune system were identified. Compared with the genomes of other fishes, some changes have occurred in the miiuy croaker sensory system including modification of vision and expansion of taste and olfaction receptors. These changes allow miiuy croaker to adapt to the environment during the long-term natural selection. The genome of miiuy croaker may elucidate its relatively well-developed immune defense and provide an adaptation model of the species thriving in turbid deep aquatic environments.The miiuy croaker,  The Sciaenidae, known for their exceptionally large otoliths , are economically important marine fishes and commonly called drum fishes or croakers because of the sounds that these organisms make with their well developed swim bladdersMiichthys miiuy, one of the representative Sciaenidae, mainly inhabits in the Zhoushan Fisheries located in the estuary of Yangtze River with mud to sandy mud bottoms356The miiuy croaker This study presented a high-quality genome sequence and annotation of the miiuy croaker by a whole-genome shotgun approach. Comparative genomic analyses provide insights into the characteristics of the immune system and evolutionary sensory adaptations to the turbid-deep aquatic habitats.de novo by employing the assembler Allpaths-LGWe performed a whole-genome shotgun strategy to sequence the genome of a wild female miiuy croaker by using an Illumina Hiseq 2000 sequencing platform. We obtained 100.79 Gb high-quality reads from seven pair-end libraries and four mate-pair libraries with various insert sizes ranging from 180 bp to 20 kb after low-quality and duplicated reads were filtered −3)−2)Good annotation was obtained because of the accurate assembly of the miiuy croaker genome. The ab inito, transcriptome-based and homology-based prediction methods. All of the predicted gene structures were integrated with GleanAfter screening out the repetitive contents, we predicted the miiuy croaker genes with In previous studies, the phylogenetic analysis with a mitochondrial genome and multiple nuclear genes suggested that Sciaenidae exhibit a closer affinity to Tetraodontiformes21P < 0.05) were involved in calcium ion binding , eye morphogenesis , and muscle cell development , innate immune response , and immune effector process The miiuy croaker is a typical benthic predatory fish living in mud to sandy mud bottoms that seems to have evolved an effective visual system apart from a pair of large eyes. Compared with other fish, certain visual-related genes have been lost, mutated or duplicated in the miiuy croaker. Five classical types of visual pigment genes were identified in fish, including SWS1 , SWS2 , LWS , RH2 (green opsin), and RH1 (rhodopsin); however only four types were identified in the miiuy croaker, and SWS1 was lost . In addi27Because taste is an important factor for food selection in dietary habits, we searched the miiuy croaker genome for taste receptors and found the expansion of T1R2 gene with seven copies . T1R2 an30Olfaction is an indispensable physiological function for detecting food, mates and predators, and is mainly dominated by olfactory receptors (ORs) and vomeronasal receptors (VRs). We identified 113 ORs and 46 VRs in the miiuy croaker, which are more than those of most teleosts  and the expansion of innate immune genes such as TNF and NLR-C were identified. We also found that the genes encoding for other innate immune genes such as TLRs, IFNs, IRFs and ILs were present in the miiuy croaker genome. These results illustrated that the miiuy croaker may have a more developed innate immune system than adaptive immune system, which needs further study.New insights into the genetic diversity and evolutionary mechanisms may explain the adaptation of the miiuy croaker to its specific aquatic environment. A major sensory adaptation to turbid living conditions includes the loss, mutation of vision-related genes. The loss of SWS1 and RH2B in the miiuy croaker may have resulted from its long-time survival in deep and turbid environments with less exposure to UV and green lights. In addition, the mutation of SWS2 helps the miiuy croaker broaden its field of vision by shifting the wavelength of absorption toward the long-wave region, which may help effectively measure its distance to prey or predators. Moreover, the expansion of T1R2 may have helped the miiuy croaker develop its dietary habits through food selection. Additionally, the miiuy croaker seems to have been equipped with a developed olfactory system with abundant ORs and VRs. Overall, the miiuy croaker has evolved an effective sensory system for better basic activities in muddy habitats.Our analyses of the miiuy croaker genome provide new insights into its adaptation to the muddy aquatic environment. At the same time, further studies on molecular functions are needed to better understand the survival of the miiuy croaker, which has great significance for aquaculture industries.The study involving live vertebrates was approved by the Ethics Committee of Zhejiang Ocean University. The methods were carried out in accordance with the approved guidelines.de novo assembly. Assembly of the miiuy croaker genome was carried out using the software program Allpaths-LGA wild female miiuy croaker was caught from the East China Sea area of the Zhejiang Province and was selected for the extraction of DNA from the abdominal muscle for sequencing. Seven pair-end libraries with short insert sizes  and four mate-pair standard libraries with long insert sizes  were constructed according to the Illumina standard protocol. Then the libraries were sequenced using an Illumina Hiseq2000, producing 101 bp or 151 bp reads. Finally, 136.29 Gb of raw data were generated. After filtering, 100.79 Gb of data that had more than 90% of bases with base quality greater than or equal to Q20 remained for the de novo prediction using RepeatModeler. The tRNAs in the genomic sequence were predicted by tRNAscan-SETandem repeat sequences in the miiuy croaker genome were identified using the program TRF (Tandem Repeats Finder)ab initio prediction methods were combined. Homology-based prediction was performed by searching against eight related species of proteins using TBLASTN. Then, homologous genome sequences were aligned against the matching proteins using GeneWise to generate gene model structuresAb initio prediction was performed by Augustusab initio, transcriptome-based and homology-based prediction by using GleanTo predict the protein-coding genes in the miiuy croaker genome, homology-based, transcriptome-based and Annotation of the predicted genes were assigned with the best matched alignment to a number of nucleotide and protein sequence databases, including NT, NR, SwissProt, KOG, and InterPro using BLASTP with an E-value threshold of 1E-5. InterProScanMiiuy croaker and 20 other chordate proteomes were selected to identify the gene families that descended from a single gene in a common ancestor using OrthoMCL 2.0.9p < 0.01).BLASTN and TBLASTN  were used to search for sensory genes in the miiuy croaker genome. Genes were predicted by GeneWise based on a homology prediction methodAccession codes: The whole-genome sequencing project for the miiuy croaker has been deposited in DDBJ/EMBL/GenBank Bioproject database under the accession code JXSJ00000000. The data set of transcript and protein sequences have been deposited in Figshare: ( http://dx.doi.org/10.6084/m9.figshare.2059896).How to cite this article: Xu, T. et al. The genome of the miiuy croaker reveals well-developed innate immune and sensory systems. Sci. Rep.6, 21902; doi: 10.1038/srep21902 (2016).


The Gerstmann syndrome of dyscalculia, dysgraphia, left-right confusion, and finger agnosia is generally attributed to lesions near the angular gyrus of the dominant hemisphere. A 68-year-old right-handed woman presented with sudden difficulty completing a Sudoku grid and was found to have dyscalculia, dysgraphia, and left-right confusion. Magnetic resonance imaging (MRI) showed a focus of abnormal reduced diffusivity in the left posterior insula and temporoparietal operculum consistent with acute infarct. Gerstmann syndrome from an insular or peri-insular lesion has not been described in the literature previously. Pathological and functional imaging studies show connections between left posterior insular region and inferior parietal lobe. We postulate that the insula and operculum lesion disrupted key functional networks resulting in a pseudoparietal presentation. Since Gerstmann asserted in the early 1900s that a syndrome of finger agnosia, left-right confusion, dysgraphia, and dyscalculia has localizing value, this complex of symptoms has generally been attributed to lesions near the angular gyrus of the dominant hemisphere . The impA 68-year-old right-handed woman with history of migraine headaches and patent foramen ovale (PFO) on clopidogrel presented with sudden onset of difficulty in completing a Sudoku grid.  Proficient at baseline, she abruptly found herself unable to fill in the squares . She repAt the hospital, she had good attention and could speak fluently and name, comprehend, repeat, read, and identify her fingers accurately. Her difficulty in writing persisted. For example, intending to write “5,” she instead wrote “15” and she wrote “hong” instead of “long.” She also had difficulty in calculations and could not produce more than one correct answer in a task of serial subtraction by sevens or calculate amount of money in nine quarters. There was one error in three left/right discrimination tasks (incorrectly stated left for right). She also initially reported being left-handed instead of right-handed. There was otherwise no visual or sensory neglect of the left side, and there were no acute changes in muscle strength, skin sensation, or coordination in her limbs. Magnetic resonance imaging (MRI) performed eleven hours after onset of symptoms showed a focus of abnormal reduced diffusivity within the left posterior insula and temporoparietal operculum consistent with acute ischemic infarction .Over the next two days, her ability to calculate improved although she continued to have difficulty in serial subtraction tasks and accurate completion of Sudoku grids. In poststroke follow-up after three weeks, she reported twice daily sensation of a rising sense of heat in her body followed by period of confusion and sense of doom. Electroencephalography (EEG) showed bitemporal slowing worse on the left side. Topiramate was initiated for presumed focal onset seizures, and the periodic sensation of rising warmth abated though difficulty with calculations persisted. Stroke workup including 24-hour continuous electrocardiographic monitoring, transthoracic echocardiography, and cervical and intracranial MR angiography revealed only a known patent foramen ovale. Subsequent imaging of the lower extremity and pelvic veins showed no thrombosis, but May-Thurner anatomy was noted. For presumed paradoxical thromboembolism, she was started on anticoagulation five days after her stroke with plan to continue indefinitely.Insular and peri-insular infarctions are known to produce a wide range of motor disturbances, somatosensory syndromes, and language deficits. In a meta-analysis of clinical symptoms associated with peri-insular strokes from institutional case series and published cases (23 patients), the predominant symptoms were dysarthria , somatosensory deficits , and aphasia  .Our patient, however, presented with profound dyscalculia accompanied by milder dysgraphia and left-right confusion from a left posterior insular and temporoparietal operculum ischemic infarction. None of these deficits have been traditionally attributed to insular or peri-insular infarcts. The ability to perform calculations—the patient's most impaired faculty—has been studied in both pathological and physiological contexts. Lesion studies in patients with acquired dyscalculia have primarily implicated the left parietal cortex, particularly near the angular gyrus , 7. FuncAdditionally, dyscalculia often occurs in the context of Gerstmann syndrome . Our patOur patient consequently had a pseudoparietal lobe presentation from a posterior insular and temporoparietal opercular infarct. Based on animal and human pathological studies, the insula is known to have extensive connections both to the cortex and deep structures . With thAside from the frontal and parietal connections described above, the insula also has rich connections with the temporal lobe, cingulate cortex, limbic structures, and multiple thalamic nuclei . To explThis patient's poststroke course was complicated by presumed insular or peri-insular seizures manifesting as autonomic sensations of warmth followed by feelings of doom and confusion. The initial autonomic signs are consistent with a peri-insular epileptic focus though similar ictal sensations can be caused by lesions in the amygdala, anterior cingulum, and supplementary sensorimotor areas . Her autThis unique presentation of pseudoparietal syndrome and “dys-sudokia” resulting from a left posterior peri-insular infarct raises a few final points. The patient presented to the hospital about eleven hours after the onset of symptoms. She already had abnormal T2 hyperintensity in the region of the infarct suggesting that the infarct had been present for at least several hours. She may have had additional symptoms that were not noticed or reported. Furthermore, her MRI also had scattered abnormal T2 hyperintensities throughout the subcortical and periventricular white matter, without reduced diffusivity. These abnormalities could represent prior subclinical pathological changes which influenced the presentation of this infarct. Finally, without functional imaging data we are not certain of her hemispheric dominance with respect to language. Even in extreme right-handed healthy subjects, right brain language dominance is present in about 4% when increased blood flow is used as a surrogate for brain activity . Altered


Danio rerio embryos during development and in mature, differentiated tissues. We found that embryonic and terminal tissues have unique methylation signatures in CpG islands and repetitive sequences. Fully differentiated tissues have increased CpG and LTR methylation and decreased SINE methylation relative to embryonic tissues. Unsupervised clustering analyses reveal that the embryonic and terminal tissues can be classified solely by their methylation patterning. Novel analyses also identify a previously undescribed genome-wide exon methylation signature. We also compared whole genome methylation with genome-wide mRNA expression levels using publicly available RNA-seq datasets. These comparisons revealed previously unrecognized relationships between gene expression, alternative splicing, and exon methylation. Surprisingly, we found that exonic methylation is a better predictor of mRNA expression level than promoter methylation. We also found that transcriptionally skipped exons have significantly less methylation than retained exons. Our integrative analyses reveal highly complex interplay between gene expression, alternative splicing, development, and methylation patterning in zebrafish.DNA methylation is a dynamic process through which specific chromatin modifications can be stably transmitted from parent to daughter cells. A large body of work has suggested that DNA methylation influences gene expression by silencing gene promoters. However, these conclusions were drawn from data focused mostly on promoter regions. Regarding the entire genome, it is unclear how methylation and gene transcription patterns are related during vertebrate development. To identify the genome-wide distribution of CpG methylation, we created series of high-resolution methylome maps of  The recognition of cytosine methylation in DNA predates the discovery of the structure of DNA by Watson and Crick . By the Classically, CpG methylation analysis was performed using methylation-sensitive enzymatic reactions or with bisulfite conversion coupled to Sanger sequencing . These mWe used an alternative to the techniques mentioned above, methyl-CpG binding domain protein-enriched genome sequencing (MBD-seq). MBD-seq uses methyl-CpG binding proteins or anti-methylated CpGs antibodies to purify methylated DNA from the genome. This isolated DNA is then assayed using high-throughput sequencing (Danio rerio) are a popular organism for the study of vertebrate development and gene function. The zebrafish genome contains approximately 26,000 genes, most of which contain several exons with large intronic space . SAM is a product of the one-carbon metabolic pathways that obtains its substrates from dietary, folate, and choline . The metTübingen/AB fish were used for all methylome analyses and cared for using standard practices . GenomicWe applied 400 µM methotrexate  to one-cell zebrafish in E3 embryo medium. One hundred of these MTX-treated zebrafish were raised to 3dpf. Genomic DNA was extracted from these embryos as reported above.tert and sox2 loci, previously reported to be methylation-rich and methylation-poor, respectively .Genomic DNA was sonicated to 300-bp fragments with a Covaris S2 with the following settings: 10% duty cycle; 4 intensity; and 200 cycles/burst for 90 sec in 130 µl Tris-EDTA. No more than 10 µg gDNA was used per sonication reaction. DNA was concentrated in a Speed Vac SC110 A. Methylation enrichment was performed using EpiMark DNA Enrichment kit (New England Biolabs). Both the captured (methylated) DNA and flow-through (unmethylated) DNA were retained for downstream analysis. The degree of enrichment was assessed with SYBR quantitative PCR on the Applied BioSystems 7900HT with both the DNA Methylation control package  and custom primers designed to amplify the zebrafish Multiplex libraries were created using ChIP-Seq DNA Sample Prep Kit (Illumina) and sequenced with 101-bp paired-end reads with an Illumina HiSeq 2000.Table S2 and Illumina Fastq files were aligned to the zebrafish Zv9 build using BWA . See TabIllumina-based sequencing reads were aligned with STAR 2.1.2 . Briefly, ensembl68 zebrafish gene annotations were used to identify exons, introns, and UTRs. In cases where there were multiple overlapping features, i.e., overlapping exons with slightly different stop and start positions, the feature was expanded to encompass all annotations. Promoters were defined as 2000 bp 5′ of the transcriptional start site. Any promoters that overlapped another annotated gene were removed. The upstream and downstream regions were defined as 10,000 bp upstream and downstream of the promoter and 3′ UTR, respectively. Any upstream or downstream region overlapping an annotated gene was removed. Exons and introns were split into quarters and thirds by position in the gene, respectively. Methylation ratio scores were calculated in 20 windows per element (see below). The features with their subdivided windows were then averaged across the entire genome and plotted.The meta-gene was created using custom Unix, Python, and R scripts as well as BEDTools and ggplot2  / . The windows are 5% of the length (in bp) of the feature. We found that correcting for CpG content did not improve the concordance of methylation scoring between biological or technical replicates.The aligned reads were used by ChromHMM to determine two states: methylated or unmethylated for 200-bp windows across the genome . The ChrSpliced-out exons were identified by using splice junctions detected by STAR and intersecting them with Ensembl gene models. More specifically, custom Unix and Python scripts were used with BEDTools to identify cases where the 5′ and 3′ ends of the splice junctions are in different exons. If there is a single annotated exon between the aligned exons, then this is declared a skipped exon. Peaks were intersected against the identified splicing exon/intron classes. Enrichment was calculated by taking the results of bootstrapping 1000 fold with position-randomized peaks and comparing against the original peaks. The SD was calculated from the distribution of the 1000 fold bootstrapping.Peak files and raw sequencing files are available under GEO accession GSE52110.Table S1).To study the methylomes of developing and developed zebrafish, we purified DNA from cells and tissues by first independently pooling the sperm of 10 males, 20,000 one-cell embryos, more than 10,000 mbt embryos, and 100 3dpf embryos, and the eyes, brain, hearts, and livers of 10 females. Methylation-rich DNA regions were selected and the methylation-enriched fraction (with no NaCl extraction) and the methylation-poor flow through were sequenced with an Illumina HiSeq 2000. More than 10 million unique properly paired reads were generated for each sample  from these data were compared with our MBD-seq data from the same developmental stage. We found a substantial concordance between the two techniques. At least one read from our methylation-enriched regions covered 70% of called methylated CpGs and the methylated CpGs called by Figure S1).To independently assess the MBD-seq technique, we compared our data with those obtained from whole-genome bisulfite sequencing. We downloaded whole-genome bisulfite sequencing data derived from mbt stage embryos (GSM1133397) . This confirmed that peak regions are highly methylated, relative to non-peak regions. However, we noted that there was no relationship between the peak “score” generated by MACS2 and the amount of CpG methylation.The MACS2 peak caller was used to identify highly methylated regions . These pMaterials and Methods). In all tissues, peaks are highly enriched across CpG islands, in contrast to the findings of The distribution of peaks across the zebrafish was overlaid on genomic features taken from UCSC Zv9 build and Ensembl-annotated genes. Enrichment was calculated by using a bootstrapping approach  . We found 14 clusters to create the most stable divisions of the data .To assess whether methylation was differentially positioned in the “average” gene, we overlaid our data onto a representative “meta-gene” . The metThis visualization of methylation reveals patterns shared across all of the tissues . First, Materials and Methods). We reprocessed the raw RNA-seq data to quantify gene transcription and to identify splicing events . We divided the meta-gene into deciles by gene transcription levels to determine if differentially expressed genes had unique methylation patterns .MTX is a potent inhibitor of the dihydrofolate-reductase enzymatic conversion of dihydrofolate to tetrahydrofolate. The block of this reaction prevents the conversion of homocysteine to methionine, a necessary molecule for the creation of the universal methyl donor s-adenosyl methionine. Application of MTX to early zebrafish embryos results in shortened anterior–posterior axis, cardiac defects, perturbed cell cycles, and highly increased mortality  (There are three probable outcomes from the MTX treatment in genome-wide methylation patterning. First, there may be no significant difference in methylation. Second, methylation could be reduced globally. Third, methylation intensity could be different at specific genomic positions. We found that genome-wide methylation patterning is largely identical between MTX-treated 3dpf and untreated 3dpf zebrafish . There aDespite a long history of studying DNA methylation, only recently has it become possible to analyze, in high-resolution, DNA methylation across the genome. Here, we successfully performed MBD-seq covering the entire zebrafish genome in eight different embryonic tissues and stages and examined the influence of a drug that interferes with the methyl donor producing pathway. At the macro level, methylation is evenly distributed across the 25 chromosomes of the zebrafish. While appearing to be uniform when averaged over long distances, methylated CpGs are not randomly spread across the genome. As expected, methylation density is concentrated in CpG islands and moderately enriched in LTRs. In contrast, LINEs and promoters have lower methylation signals. We also note that terminal tissues and embryonic tissues have different proportions of methylation across the genomic elements, which can be seen most clearly in LTRs, SINEs, and CpG islands.Our unsupervised clustering analysis allows us to distinguish between embryonic and terminal tissues using global methylation patterns alone, which demonstrate that each tissue or time point carries a unique methylation signature. In addition, the distribution of methylation across the clusters produced highlight two trends. First, the majority of the genome-wide methylation signal assayed is shared in a common set of genomic coordinates in the embryonic tissues and a majority in the terminal tissues. Second, the embryonic tissues have more diverse and widespread methylation signal, suggesting that as tissues differentiate CpG methylation is selectively removed or lost.Publications by We created the meta-gene to represent the methylation anatomy of the “average” gene. These analyses reveal a previously undescribed pattern of methylation signals across the average gene. Specifically, last exons appear to contain a disproportionate amount of methylation. The meta-gene also demonstrates that across the tissues and time points assayed, that gene-body element methylation patterning is largely stable. The only notable difference between embryonic and terminal tissues is that exon methylation is a bit lower compared to intron methylation as the tissues develop.Because CpG methylation is classically reported as influencing gene transcription, we merged published RNA-seq datasets with our genome-wide methylation data. This allowed us to probe the relationship between gene-body methylation and transcriptional levels. In contrast to expectation, we found that promoter methylation is not the strongest determinate of transcription levels. Methylation signal in the last exon of a gene is a better predictor than promoter methylation, with increasing methylation associated with higher transcript levels. We note that regions upstream and downstream of genes show stronger correlation between methylation and transcriptional levels than promoter methylation. We also examined the relationship between DNA methylation and patterns of alternative exon splicing. This revealed that methylation status of alternatively spliced exons differs from that of constitutively expressed exons. This observation held true for embryonic and terminal tissues, suggesting that different processes are used to mark spliced out exons in differentiated tissues.MTX is used in humans as a chemotherapeutic agent and in low doses to treat rheumatoid arthritis. It is a strong inhibitor of the DHFR, which catalyzes the conversion of DHF to THF. This has a major effect on the nucleotide pools available for DNA synthesis. This inhibition is also predicted to reduce the number of methyl donors potentially available for DNA methylation reactions. Previous research has demonstrated that this drug also disrupts zebrafish development. We found that it appears to reduce methylation across the genome, with the strongest effects happening at CpG islands and exons. This suggests that a downstream effect of this drug, besides its known role in nucleotide metabolism, may be to disrupt the methylation patterning of important genome elements and proteins, potentially disrupting transcriptional and protein activity.Our analysis of methylation patterning in zebrafish reveals that this important model organism has similar canonical methylation patterning as other vertebrates and mammals.In this study, we leverage our datasets against other ChIP-seq and RNA-seq datasets to demonstrate the complexity of methylation patterning in relation to histones, alternative splicing, and transcription. Analysis of disparate genome-wide datasets allows us to make generalizations about the relationships between epigenetic modifications and gene expression, which especially demonstrates the need to re-evaluate the emphasis on promoter methylation being important in modifying gene expression. Our novel meta-gene analysis provides a useful template for analyzing the effects of methylation changes on the gene-body across the genome and highlights how methylation changes across the gene. Further research is needed to determine whether methylation is driving these processes or is a passenger mark. Our work confirms that methylation is a dynamic process. It also highlights the power of using genome-wide analyses, without which the importance of DNA methylation outside of gene promoters would remain hidden from view.


A standardized imaging proposal evaluating implanted left atrial appendage (LAA) occlusion devices by cardiac computed tomography angiography (cCTA) has never been investigated.rd generation dual-source CT system and reconstructed with a slice thickness of 0.5 mm. An interdisciplinary evaluation was performed by two interventional cardiologists and one radiologist on a 3D multi-planar workstation. A standardized multi-planar reconstruction algorithm was developed in order to assess relevant clinical aspects of implanted LAA occlusion devices being outlined within a pictorial essay.cCTA datasets were acquired on a 3LAA Occluder View for post-implantation Evaluation (LOVE). Aligned true axial, sagittal and coronal LOVE planes offer a standardized and detailed evaluation of LAA occlusion devices after percutaneous implantation.The following clinical aspects of implanted LAA occlusion devices were evaluated within the most appropriate cCTA multi-planar reconstruction: (1) topography to neighboring structures, (2) peri-device leaks, (3) coverage of LAA lobes, (4) indirect signs of neo-endothelialization. These are illustrated within concise CT imaging examples emphasizing the potential value of the proposed cCTA imaging algorithm: Starting from anatomical cCTA planes and stepwise angulation planes perpendicular to the base of the LAA devices generates an optimal This pictorial essay presents a standardized imaging proposal by cCTA using multi-planar reconstructions that enables systematical follow-up and comparison of patients after LAA occlusion device implantation.The online version of this article (doi:10.1186/s12880-016-0127-y) contains supplementary material, which is available to authorized users. The left atrial appendage (LAA) represents the main origin of thrombus formation in atrial fibrillation, where the principles of Virchow’s triad, such as dysfunction and structural changes of the endothelium as well as abnormal blood stasis and homoeostasis are present –3. The iThe WATCHMAN (WM)  and Amplatzer Cardiac Plug (ACP)  represent the most commonly implanted LAA occlusion devices with valuable scientific evidence being available at present , 15, 16.Therefore, this pictorial essay aims to develop this standardized imaging proposal by cCTA in order to analyze relevant clinical aspects of LAA occlusion devices after percutaneous implantation. This cCTA imaging proposal outlines relevant clinical aspects such as device positioning in relation to the varying morphology and topography of the LAA as well as functional device aspects such as peri-device leaks, lobe coverage and neo-endothelialization of the device and demonstrates illustrations of these conditions.All participants of the presented patient-related data within this pictorial assays gave written consent to this analysis. The analyses were carried out according to the principles of the declaration of Helsinki and was approved by the medical ethics commission II of the Faculty of Medicine Mannheim, University of Heidelberg, Germany.Based on our experience, cCTA protocols for the evaluation of LAA occlusion devices do not require significant protocol adjustments, when compared to standard cCTA protocols being performed for the evaluation of coronary artery stenosis –24. OptiReconstruction of cCTA raw data was performed with a slice thickness between 0.5 and 0.6 mm using a sharp convolution kernel that is also used for the reconstruction of cCTA images in patients with coronary artery stent grafts or heavily calcified plaques. If available iterative reconstruction techniques should be used to lower image noise and blooming artifacts originating from metal components of the devices –28. Tabl0.6 mm thick images reconstructed with a medium convolution kernel are uploaded on a simple 3D multi-planar rendering workstation with the 3 planes locked at a 90° angle Figs.  and 2. TSpecifically for the ACP device, move the coronal axis within the transverse window perpendicular to the disc of the ACP device  images with a slab thickness of approximately 10 mm allows visualization of the whole nitinol frame of the WM device. Afterwards, move the coronal axis within the transverse window perpendicular to the coves of the parachute of the WM device   Figs. , 3 and 4Imaging reports about percutaneously implanted LAA occlusion devices should focus on the following important clinical aspects:Global positioning of the device including rotation around the entry axis to the LAA. This can be best measured on LOVE axial and LOVE sagittal views.LOVE axial views allow optimal evaluation of global compression of both devices:WM device: compression of 10–20 % referred to the original device size is recommended.ACP device: concave disc; device lobe positioned 2/3 distal to the LCX inside and engaged within the LAA; ACP device axis in line with the LAA neck axis.LOVE axial views allow optimal evaluation of impairment to neighboring structures by the LAA occlusion device: including positioning in relation to the mitral valve annulus, pulmonary artery, left pulmonary veins as well as LCX that is best visible in LOVE axial views Fig. .Fig. 3LOPeri-device leaks:These can be seen best on LOVE sagittal views. The presence of a peri-device leak should be measured in all reports Figs. , 5 and 6 Coverage of all lobes:The LAA often consists of different lobes not corresponding necessarily to the fixed shape of the LAA occlusion device. Therefore, complete coverage of all LAA lobes is not always achievable. Assessment of lobe coverage should consider the angle between the sealing part of the device  and the plane of the LAA ostium. This angle corresponds to the incompletely covered LAA lobe. Accordingly, Fig.  Complete neo-endothelialization:Absence of contrast enhancement within the LAA without any peri-device leak suggests complete neo-endothelialization. Accordingly, contrast enhancement in the LAA of less than 50 Hounsfield units compared to the left atrium suggests incomplete neo-endothelialization. Equal contrast enhancement in both LAA and left atrium suggests no or very early stages of neo-endothelialization Figs.  and 8.This pictorial essay presents a standardized imaging proposal by cCTA using multi-planar reconstructions that enables systematical follow-up and comparison of patients after LAA occlusion device implantation. As described above and being accompanied by a case series of striking illustrations this imaging proposal intends to cover the most relevant clinical challenges of implanted LAA occlusion devices including morphologic, topographic as well as functional device-related aspects. The presented imaging proposal generates novel hypotheses, which have to be proven by ongoing prospective, randomized imaging studies.Based on the so far available literature on safety and patient outcome, it is likely that the number of patients undergoing percutaneous LAA occlusion device implantation will raise significantly within the near future similar to the growing number of patients undergoing transcatheter aortic valve replacement (TAVR). However, in order to identify patients with poor outcome and/or post-procedural complications, such as a residual LAA larger than a specific device size that still leads to thrombus formation, accurate, reproducible and reader-independent imaging is crucial. This is of particular importance in order to generate more evidence about optimal placement of LAA occlusion devices.For instance, the presence of relevant peri-device leaks or incomplete coverage of residual LAA lobes might be associated with individually impaired neurologic outcome of the patients. These mal-appositions of LAA occlusion devices have been attributed to clinical overt neurologic disability and silent cerebral ischemia being caused by cerebral micro-embolism and visualized by cerebral magnetic resonance imaging (cMRI) . ImportaLittle is known regarding incidences of device related complications after successful percutaneous implantation of LAA occlusion devices, such as device compression, peri-device leaks, lobe coverage and progress of neo-endothelialization related to study-specific antithrombotic treatments. The latter were evaluated for DAPT (i.e. aspirin and clopidogrel) lasting 6 months, as within the present study, or for warfarin for 45 days, followed by clopidogrel for 4.5 months and lifelong aspirin , 10, 16.Furthermore, accurate differentiation between thrombus formation and blood stasis within the LAA is challenging using standard cCTA. An additional delayed cCTA approximately 70 s after the start of the contrast injection is recommended . ImagingAs outlined, post procedural image analysis with cCTA might bear the potential of a standardized imaging using defined multi-planar reformations that best display the complex 3-dimensional shape of the LAA and the device. In our opinion, cCTA has the main advantage that the images can be very standardized analyzed and reconstructed and make quantitative measurements reliable. This is of particular importance to generate more evidence on relevant clinical questions: (1) What size of a residual LAA is acceptable with different devices? (2) What size of peri-device leaks still leads to a complete occlusion over time? However, the advantages of cCTA for post-implantation LAA occlusion device evaluation have to outscore the patients’ individual risk being associated with an additional radiation dose and additional administration of iodinated material.Cardiac CTA allows the comprehensive non-invasive visualization of the whole heart including coronary arteries as well as all important neighboring structures of the LAA and thus accurate evaluation of post-implantation evaluation of LAA occlusion devices by the described LOVE view. The main arguments for the use of cCTA in this context, besides its non-invasiveness, are firstly, the whole volume coverage with a high spatial resolution and secondly, the high reproducibility of the technique that allows to angulate the isotropic dataset retrospectively after the procedure in all desired projections. Thereby generating an investigator’s independency, cCTA becomes particularly attractive for clinical studies that require a high standardization of follow-up examinations. Thirdly, image artifacts do not hamper cCTA image quality because all available LAA occlusion devices do not lead to metal artifacts such as other metal implants. Based on our experience, cCTA allows assessing all relevant clinical questions including indirect visualization of LAA occlusion device neo-endothelialization if no peri-device leak is present. For instance, no contrast enhancement within the LAA suggests complete neo-endothelialization, whereas contrast enhancement in the LAA of less than 50 Hounsfield units compared to the enhancement within the left atrium suggests incomplete neo-endothelialization. Contrast enhancement within the LAA that is equal to the left atrium suggests no or very early stages of neo-endothelialization.The main concerns with cCTA in the context of LAA occlusion device evaluation are the additional radiation dose that is given to the patient. However, with low radiation dose techniques of state-of-the-art CT systems the radiation dose has been reduced significantly over the past 5 years . Thus, tThe second relevant concern of cCTA is the need for iodinated contrast material, which is crucial in patients with impairment of renal function. Although the amount of contrast material for the evaluation of LAA occlusion devices can be reduced to a minimum of 30–40 cc of contrast in patients with elevated serum creatinine levels, the indication should be checked strictly in patients with serum creatinine levels above 1.5 mg/dL. Additionally, cCTA is more expensive and not as widely available compared to echocardiography.This pictorial essay describes a standardized imaging proposal including examples of (ab)normal findings after percutaneous implantation of LAA occlusion devices. Whether the proposed cCTA imaging instructions might facilitate future clinical follow-up, cost effectiveness and therapeutic decisions needs to be evaluated further in larger, prospective and randomized imaging studies evaluating this specific subset of cardiac patients.


While drinking water biofilms have been characterized in various drinking water distribution systems (DWDS), little is known about the impact of different DNA extraction methods on the subsequent analysis of microbial communities in drinking water biofilms. Since different DNA extraction methods have been shown to affect the outcome of microbial community analysis in other environments, it is necessary to select a DNA extraction method prior to the application of molecular tools to characterize the complex microbial ecology of the DWDS. This study compared the quantity and quality of DNA yields from selected DWDS bacteria with different cell wall properties using five widely used DNA extraction methods. These were further selected and evaluated for their efficiency and reproducibility of DNA extraction from DWDS samples. Terminal restriction fragment length analysis and the 454 pyrosequencing technique were used to interpret the differences in microbial community structure and composition, respectively, from extracted DNA. Such assessments serve as a concrete step towards the determination of an optimal DNA extraction method for drinking water biofilms, which can then provide a reliable comparison of the meta-analysis results obtained in different laboratories. Biofilm growth in drinking water distribution systems (DWDS) is of public health concern. To better understand how potable water can be maintained and distributed to consumers, it is important to monitor changes in microbial communities with respect to the environmental conditions in the DWDS. For instance, several studies have investigated the effects of disinfectants , 29, nutMany of the widely used methods have been developed for extracting DNA from soil. These methods involve procedures such as incubation during enzyme lysis and phenol/chloroform extraction, which are laborious, time-consuming, and generate hazardous wastes. Moreover, the amount of DNA obtained during phenol/chloroform extraction can differ among analysts. Unlike soil samples, which can be obtained relatively easily in large quantity, drinking water biofilms can be limited by biomass availability. The presence of humic substances and corrosion in the DWDS can also interfere with the DNA extraction process, which can inhibit the downstream PCR. Thus, methods developed for soils may not be suitable for drinking water biofilms. It is therefore necessary to determine the impact of DNA extraction biases on the analysis of microbial communities in drinking water biofilms.The objective of this study was to determine a suitable DNA extraction method for drinking water biofilms in order to enable reproducible and reliable comparisons in subsequent meta-analysis results amongst different laboratories. We evaluated five widely used DNA extraction procedures, each with different physical, chemical, and enzymatic approaches, based on the following criteria: DNA yield, DNA purity, and the molecular weight of the extracted DNA. We then selected three out of the five DNA extraction procedures and used terminal restriction fragment length polymorphism (T-RFLP) analysis and the 454 pyrosequencing technique to demonstrate the impact of different DNA extraction procedures on microbial diversity and composition.Aeromonas caviae (ATCC 14486), Aquabacterium parvum (ATCC BAA-207), and Sphingomonas sp. RO2 ; the Gram-positive Bacillus subtilis (ATCC 23856) and Gordonia hirsuta (ATCC 700255); the acid-fast Mycobacterium smegmatis (ATCC 19420); and Escherichia coli (ATCC 4157) as a positive reference strain. More importantly, these bacterial strains were selected as their related species have been isolated from drinking water and are either biofilm producers or are part of the biofilm in the DWDS, and some are also opportunistic human pathogens  and the FastDNA Spin Kit for Soil , and three standard phenol/chloroform methods, which included DNA extraction procedures for soil and sediment samples , 39 and 260/A280) and the quantity of DNA. DNA is assumed to be free from protein contamination when the A260/A280 ratio is higher than 1.7. Since spectrophotometer measurements can be affected by contaminants  and are not sensitive to low DNA concentrations, a fluorescent-based quantitation, Q-bit Quantitation Platform , was also used to complement values obtained from the spectrophotometer. The quality of the extracted DNA was evaluated by observing the size of the extracted DNA fragments via agarose (0.8%) gel electrophoresis with a DNA/HindIII fragment ladder (Promega) as a size standard.A Nanodrop 1000 spectrophotometer  was used to assess both the purity of DNA  in order to obtain PCR amplified products. The PCR products were treated with mung bean nuclease  at 37°C for 1 h and purified via the Wizard SV Gel and PCR Clean Up System (Promega) according to the manufacturer’s instructions. Enzyme digestion with MspI (New England Biolabs) was carried out overnight at 37°C and DNA fragment analysis was performed on the ABI 3730xl Analyzer . To determine the effects of DNA extraction methods on the resulting bacterial community composition and structure, cluster analysis was performed with the Bray-Curtis matrix using the Primer 6 (version 1.0.3) computer program .The bacterial community structure of each sample was assessed by performing terminal restriction fragment length polymorphism (T-RFLP) using the primers 47F (5′-6 FAM-CYTAACACATGCA AGTCG-3′) and 927r (5′-ACCGCTTGTGCGGGCCC-3′). Briefly, the reactions contained 12.5 μL Bullseye Taq 2.0 Master Mix , 1.0 μL of each primer , approximately 1 ng μLhttp://pyro.cme.msu.edu/) and the NAST alignment tool from Greengenes .Based on the results of cluster analysis, duplicate samples that were clustered more closely together from each triplicate extraction were subjected to pyrosequencing. The extracted DNA was amplified with bacterial specific forward 515F (5′-Fusion A-Barcode-CA linker-GTGYCAGCMGCCGCGGTA-3′) and reverse 907R (5′-Fusion B-TC linker-CCCCGYCAATTCMTTRAGT-3′) primers. PCR products were gel purified according to the manufacturer’s instructions (Promega). The 454 pyrosequencing was carried out on a 454 Life Science Genome Sequencer GS FLX . The sequences were trimmed (resulting sequence length was an average of 375 bp), and merged alignments of the sequences aligned via the Infernal aligner from the Ribosomal Database Project (RDP) pyrosequencing pipeline (eengenes  were obthttp://pyro.cme.msu.edu/). The sequences (OTUs defined at genus level) were analyzed with DCA, performed via CANOCO version 4 , to examine the similarity of the microbial community profiles. Diversity indices (Shannon Index and Chao1 estimator), at a 3% cut-off for species-level identification, of the samples were estimated via the analytical tools available from the RDP pyrosequencing pipeline (http://pyro.cme.msu.edu/).To determine the bacterial composition of the samples, an RDP Classifier was used for taxonomical assignments of the aligned 454 pyrosequences at 95% confidence level , which indicated that the methods were efficient in removing protein contamination.Typically, the DNA content in a bacterial cell is ~3–4% of the total mass (dry weight). acterium . To inteNA yield . Thus thThe extent of DNA shearing as an indication of DNA quality was evaluated via gel electrophoresis . The DNAe.g. DNA extracted from FastDNA kit typically had a maximum absorbance spectrum at around 230 nm (data not shown) due to inherent kit properties, DNA concentration determined by direct spectrophotometric measurement may not be accurate. Our results showed that spectrophotometric-and fluorescent-based DNA measurements indeed gave varied DNA quantifications. DNA concentration measured by Q-bit gave a lower yield than that measured by the Nanodrop  again yielded higher DNA concentrations from DWDS samples than the FastDNA kit . DNA conNanodrop , which cproducts .T-RFLP analysis was first used to compare the molecular fingerprinting patterns that resulted from the different DNA extraction protocols and to compare whether there were differences between samples before and after DNA purification. Cluster analysis of T-RFLP molecular fingerprinting patterns showed that the three different sample sets grouped more closely with each other. Moreover, the results also showed that samples extracted from their respective protocols were more often grouped together, which indicated that each method yielded slightly different molecular fingerprints . FastDNAAs mentioned previously, some DNA extracted from Schmidt’s and Zhou’s protocol required DNA purification in order to obtain PCR products. Thus, DNA obtained before and after purification from some sample sets was also subjected to T-RFLP analysis in order to compare the effect of purification on T-RFLP profiles. The results showed that while purification did not affect the T-RFLP profiles in samples extracted from both FastDNA and Schmidt’s protocol, it did affect those from Zhou’s protocol as samples after purification often became less clustered with the samples before purification . This shFirmicutes (i.e. predominantly Bacillus-like sequences) and the Actinobacteria (i.e. predominantly Mycobacetrium-like species) phyla was observed (Actinobacteria and Firmicutes phyla (data not shown). Compared to Zhou’s protocol, the DNA extracts from FastDNA and Schmidt’s protocols gave a higher proportion of unclassified Bacteria and Planctomycetes, and Gemmatimonadetes in the second set of brass and plastic samples, respectively  and the extraction methods affected the taxanomic composition of DNA extracts, which was apparent at the phylum and OTU levels. In the first set, the samples looked similar at the phylum level, except for the purified DNA extracts from Zhou’s protocol where an increase in the proportion of observed . A similobserved . In contectively , and a h samples .Proteobacetria  of the relative abundance of the sequences at the genus level indicated significant variance associated with the sample sets . The surDifferent diversity indices have been criticized for the assumptions made on the relative importance of spread of abundance amongst species; therefore, estimations of total species richness may be a reliable alternative to provide the best approximation of actual total species richness . Here, be.g. fecal samples  had higher DNA yield for Gram-positive bacteria than Gram-negative bacteria, 454 pyrosequencing analysis results indicated that regardless of the protocols used, the proportion of Gram-negative bacteria was higher than Gram-positive bacteria in the DWDS. Moreover, 454 pyrosequencing indicated that FastDNA’s protocol yielded a similar proportion of Gram-positive bacteria in the DWDS to the phenol-chloroform-based methods even though it was not efficient in extracting DNA from Gram-positive bacterial cultures. This showed that results from bacterial monocultures may be different when applied to actual environmental samples. Here, FastDNA was able to generate comparable results to the two phenol-chloroform-based methods when applied to DWDS samples. In addition, studies have shown that the FastDNA kit was efficient in extracting DNA from samples spiked with bacterial spores  and the overall objective  of the experiment.The results presented here suggested that the DNA extraction method of choice for DWDS samples was the commercial kit, FastDNA, despite its relative high cost in comparison to the phenol-chloroform-based methods. FastDNA’s protocol gave nucleic acids of higher purity than the phenol-chloroform-based methods and also did not generate hazardous wastes. Although phenol-chloroform-based methods gave higher DNA yields, they may require DNA purification, which became another factor in the selection of the optimal protocol for DWDS samples, especially when our results indicated that DNA purification could affect microbial community profiles. While DNA extracted from the FastDNA kit gave lower species richness estimates than Schmidt’s protocol, the microbial community composition obtained from FastDNA kit was relatively similar to that of Schmidt’s protocol and the sequences were reflective of those typically found in the DWDS. To address species diversity and richness representation, other studies suggested the use of several different validated methods in parallel and pooling the extracted nucleic acids to capture greater biodiversity , 22; howet al. also sug


Hypoxic–ischemic encephalopathy induces secondary brain injury characterized by delayed energy failure. Currently, therapeutic hypothermia is the sole treatment available after severe intrapartum asphyxia in babies and acts to attenuate secondary loss of high energy phosphates improving both short- and long-term outcome. In order to develop the next generation of neuroprotective therapies, we urgently need to understand the underlying molecular mechanisms leading to cell death. Hypoxia–ischemia creates a toxic intracellular environment including accumulation of reactive oxygen/nitrosative species and intracellular calcium after the insult, inducing mitochondrial impairment. More specifically mitochondrial respiration is suppressed and calcium signaling is dysregulated. At a certain threshold, Bax-dependent mitochondrial permeabilization will occur leading to activation of caspase-dependent and apoptosis-inducing factor-dependent apoptotic cell death. In addition, hypoxia–ischemia induces inflammation, which leads to the release of TNF-α, TRAIL, TWEAK, FasL and Toll-like receptor agonists that will activate death receptors on neurons and oligodendroglia. Death receptors trigger apoptotic death via caspase-8 and necroptotic cell death through formation of the necrosome , both of which converge at the mitochondria. •Hypoxic-ischemic encephalopathy induces secondary brain injury characterized by delayed energy failure and excitotoxicity.•Hypoxia-ischemia triggers accumulation of reactive oxygen species andintracellular calcium, which induces mitochondrial dysfunction.•Mitochondrial impairment can cause Bax-dependent mitochondrial permeabilization, which triggers release of pro-apoptotic proteins and cell death.•During the recovery phase, Inflammation is produced leading to death receptor activation and induction of necroptosis. In the developing brain, lack of oxygen availability results in an initial depletion of high energy phosphates, in particular ATP and phospho-creatine. These levels return transiently to baseline but are followed by a second more prolonged depletion of cellular energy reserves accompanied by progression of brain injury 2Activation of NMDA and AMPA receptors after HI (hypoxia–ischemia), in response to excitotoxic amino acid release, results in cellular uptake of calcium. Indeed, we have found increased deposits of intracellular calcium in the endoplasmic reticulum, cytosol, nucleus and more significantly in the mitochondrial matrix of neurons 3Mitochondria determine cell fate in many different ways. They can induce cell death due to their ability to release proapoptotic proteins, which occurs following MP. MP can occur either through selective opening of the outer mitochondrial membrane, mitochondrial outer membrane permeabilization (MOMP), or be the result of opening of the mitochondrial permeability transition pore, which permeabilizes both the outer and inner mitochondrial membranes Mitochondrial permeabilization results in the release of key proapoptotic proteins cytochrome c, apoptosis inducing factor (AIF), endonuclease (endo) G and Smac/Diablo from the mitochondria to the cytosol 3.1The induction of MOMP in the immature brain after HI depends on Bcl-2 family proteins; Bax translocates from the cytosol to the mitochondria and in association with Bak forms pores in the outer membrane resulting in the subsequent release of proapoptotic proteins. In the immature brain, Bax and its associated proteins are highly expressed, with further upregulation of expression occurring following neonatal HI One of the proposed regulators of Bcl-2 proteins includes the tumor suppressor p53, which has been shown to stimulate mitochondrial permeabilization and apoptosis thereby regulating cell death. Following activation, p53 accumulates in the nucleus and can upregulate proapoptotic genes such as Bax in vivo models of immature brain injury without adverse effects The initiator caspase caspase-2, also triggers Bax-mediated MOMP 4−/− mice are unable to undergo necroptosis Necrosis is defined as accidental uncontrolled cell death. On the other hand, necroptosis or programmed necrosis is a form of highly regulated cell death that morphologically resembles necrosis Necroptosis and apoptosis are fundamentally linked as certain ligands can trigger both pathways. In this situation, caspase-8 activation state sits at the divergence point. RIP1 and RIP3 are substrates for cleavage by active caspase-8 and therefore necroptosis is inhibited 4.1in vitroThere is increasing evidence that death receptors are involved in immature brain injury Caspase-8 inhibition reduces HI brain injury in the neonate in some studies 5A variety of drugs targeting cell death pathways have been tested in animal models of perinatal brain injury. The amplitude of neuroprotection observed in these studies has been quite variable, and sometimes the results are inconsistent between models and research groups. However, several compounds  have shown promising neuroprotective properties The clinical translation is, however, hampered by several obstacles that have to be overcome. Firstly, it is not known whether all of the candidate drugs can cross the blood–brain barrier, but recent studies using dendrimers hold promise as a means of facilitating transfer across both blood brain barrier and across cell membranes There are two drugs, melatonin and erythropoietin, which have been tested in clinical trials in preterm infants, and in term infants in conjunction with hypothermia. A recent randomized trial based on a relatively small number of patients has shown that preterm infants had a significantly better cognitive outcome after erythropoietin vs. placebo 6Mitochondria are center stage in the response to HI in the neonatal brain. Mitochondrial impairment leads to bioenergetic failure, generation of reactive oxygen species and dysregulation of calcium homeostasis culminating in Bax-dependent mitochondrial permeabilization and apoptotic cell death. In addition, death receptors are activated that could lead to caspase-8 dependent cell death or triggering of necroptosis through the formation of a necrosome, composed of RIP1 and RIP3. Recruitment of MLKL targets the necrosome to associate with plasma and mitochondria/endoplasmic reticulum membranes and RIP3 induces mitochondrial fission, excessive reactive oxygen species production and cell death with a predominately necrotic phenotype.


Implantable cardioverter-defibrillators (ICDs) have a proven role in primary and secondary prevention of sudden death from ventricular arrhythmias. Transvenous ICD systems despite this proven benefit, have been plagued by lead problems including fracture, insulation breaks and recalls; and issues with transvenous access including pneumothorax and venous obstruction. Infected or fractured leads require full extraction which may require laser-assisted removal which carry a mortality and morbidity risk. Subcutaneous ICDs (S-ICD) are an exciting recently approved technology providing the advantages of effective shock rescue without need for transvenous leads. They function as essentially shock boxes without pacing capability except for a short period of post shock transcutaneous pacing. This technology provides an emerging option especially in patient groups who do not have a pacing indication, have no venous access, patients at high infection risk, patients with no structural heart disease and hereditary arrhythmias; and patients with congenital heart disease that precludes transvenous leads. Patients with slow monomorphic ventricular tachycardia consistently terminated by antitachycardia pacing are also not candidates for this therapy in evolution. In addition, S-ICDs do not have remote monitoring capability.The S-ICD system uses modified subcutaneous electrocardiography through either a primary, secondary, or alternate vector, which detects changes in ventricular rate. The heart rate is measured as the rolling average of 4 consecutive sensed intervals. Ventricular fibrillation (VF) is diagnosed when 18 of 24 consecutive sensed events exceed the shock zone limit.In this issue of the Indian Pacing and Electrophysiology Journal, Tekkatte et al  describeOf note, the S-ICD device in this case was always programmed with one zone. Discriminator functions on the S-ICD are however possible only with dual zone programming into a shock zone and a conditional shock zone. There are no discriminators in the shock zone. Discrimination has 3 steps: 1- comparison of current tachycardia complex waveform with stored template acquired at rest (>50% = supraventricular tachycardia). 2- beat to beat analysis evaluates for polymorphic or monomorphic relationship . 3 - QRS width analysis .There is early data now available regarding usefulness of these discriminators. In the START Study, Gold et al  showed 1This discrimination algorithm however would not have helped in the reported case because it relies on comparing R wave to R wave. In the case study, flutter waves were oversensed as R waves and it is unlikely that they would match the template. The problem here was the far-field sensing vectors (8 cm shock coil separating sensing electrodes) and the large flutter waves.Analogous to this case, in another patient with hypertrophic cardiomyopathy, we have seen flutter waves sensed as ventricular fibrillation signals in a transvenous ICD. The lead was repositioned which is not an option with the S-ICD. In addition, chances of cross chamber sensing are likely to be less due to bipolar near - field sensing in both atrium and ventricle with transvenous system. Pre- implant ECG assessment is primarily for T wave to R wave ratio and not atrial versus ventricular ratio and importantly the patient was not in flutter at time of assessment.Anecdotal case reports now demonstrate compatibility with transvenous and epicardial pacemakers which will increase the clinical utility of S-ICD despite being limited in not having the capabilities of bradycardia pacing, anti-tachycardia pacing and cardiac resynchronization therapy. Superior discrimination algorithms in future iterations of this device may help expand implant indications further. However, there may remain situations like in the reported case where it may not be possible to tackle oversensing of large atrial electrograms by programming changes in a S-ICD in its current form.