PI-II in tomato plants was repressed by virus infection. These findings suggest that JA-related resistance plays an important role in the tripartite interactions between whitefly, begomovirus and tomato plant.The indirect interactions between insect vectors, such as whiteflies, and the viruses they transmit, such as begomoviruses, via host plants may produce a range of outcome depending on the species/strain of each of the three organisms involved, and the mechanisms underlying the variations are not well understood. Here, we observed the performance of whiteflies on three types of tomato, which vary in level of jasmonic acid (JA)-related resistance and were either uninfected or infected by a begomovirus, to investigate the role of JA-related resistance in mediating whitefly-begomovirus interactions. Compared to the performance of whiteflies on plants of the wild type, the performance was elevated on plants deficient in JA-related resistance but reduced on plants with a high level of JA-related resistance. Further, on plants with a high level of JA-related resistance, the whitefly performed better on virus-infected than on uninfected plants; however, on tomato plants deficient in JA-related resistance, whitefly performance was less affected by the virus-infection of plants. Additionally, the expression of the JA-regulated defense gene  Many case studies have shown that the effects of the tripartite interactions may constitute major determinants of the population dynamics of the organisms in the field4. Both direct and indirect interactions between the three organisms may operate in a complex network resulting in various consequences. For example, the indirect interactions between insect vectors and the viruses they transmit via their host plants may produce positive, neutral to negative effects on the performance of the insect vectors and in turn on the transmission of the viruses and the performance of the plants6.In recent decades, increasing attention has been given to the research on the tripartite interactions between insect vectors, viruses and plantsBemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a species complex composed of at least 35 morphologically indistinguishable cryptic species, some of which are invasive and important crop pests worldwide11. As the vectors of begomoviruses, which are the largest and economically the most important group of plant viruses in tropical and sub-tropical regions, the outbreaks of whiteflies in many regions are often accompanied by epidemics of plant diseases caused by begomoviruses, such as the virus diseases of tomato and other crops caused by Tomato yellow leaf curl virus (TYLCV) that have spread around the world in the past three decades16.The whitefly 19. These interactions are often complicated. For example, the effects of virus infection of the host plants on whitefly performance can be positive, negative or neutral depending on the combinations of whiteflies, begomoviruses and plants18. Thus, in some cases, a begomovirus may cause infected plants to promote the performance of its transmitting vector, which accelerates the spread of the pathogen, while in other cases the infection of the host plants by a begomovirus may have no or negative effects on the performance of the whiteflies21. The mechanisms underlying these variations remain largely unknown.Like in other systems that have been investigated, plant-mediated whitefly-begomovirus interactions exert important influences on the population dynamics of the whiteflies and the epidemiology of the virus diseasesIn this study, we firstly observed the performance of two cryptic species of whiteflies on three types of tomato plants that are known to vary in the level of jasmonic acid (JA)-related resistance. Next, we compared the performance of whiteflies on uninfected and begomovirus-infected plants for each type of the tomato. Finally, we examined the relative expression levels of genes related to JA signaling pathway between uninfected and virus-infected tomato plants. Our objectives were to investigate whether JA-related resistance participates in the tripartite interactions between whiteflies, begomoviruses and tomato plants.mtCO1 GenBank accession no: KM821540) and MED whitefly (mtCO1 GenBank accession no: GQ371165) of the B. tabaci species complex were reared on cotton Gossypium hirsutum L. cv. Zhemian 1793 in insect-proof cages at 27 ± 1 °C, 70 ± 10% RH and a 14 L:10D light cycle in climate-controlled rooms. The purity of the cultures was monitored every three generations using PCR-RFLP (PCR-restriction fragment length polymorphism) and mtCO1 sequencing analysis as described before22.The MEAM1 whitefly (Clones of TYLCV isolate SH2 (GenBank Acc. No.:AM282874) were provided by Professor Xueping Zhou, Institute of Biotechnology, Zhejiang University.olanum lycopersicum, the cv. CastleMart was used as the wild type, and JA-deficient spr2 mutant plants and JA-overexpression 35S-prosystemin transgenic plants, both in the CastleMart background, were used. To obtain virus-infected tomato plants, each of the test plants at the 3–4 true-leaf stage was inoculated with 0.2 ml of the TYLCV culture by agroinoculation as previously described23. All virus-inoculated and un-inoculated plants were grown in a greenhouse under natural lighting supplemented with artificial illumination (light 05:00–18:00 hours) and controlled temperature at 25 ± 3 °C, and were used for whitefly bioassays when they reached the 6–7 true-leaf stage (approximately 25 days after agroinoculation of the virus). Virus infection of plants was determined by typical disease symptoms and further conformed by PCR24.For plants of tomato SCastleMart, spr2 and 35S-prosystemin, to investigate the effect of JA-regulated defence of the plants to whiteflies  were sampled. Total RNA was isolated using TRIzol Reagent . RNA purity and quantity were determined using Nanodrop (Nanodrop Technologies), and 1000 ng of total RNA for each sample was reverse transcribed using the PrimeScript RT reagent Kit with gDNA Eraser  according to the manufacturer’s instructions. qRT-PCRs were performed on the Bio-Rad CFX96TM Real-Time System  using the SYBR Premix Ex TaqTM II . Three technical qRT-PCR replicates were analysed for each biological replicate. The relative expression levels were calculated with the 2−ΔΔCT method and further log2-transformed as described by Yuan et al.25 and Luan et al.26. The actin gene was used as reference. Primers for qRT-PCRs were shown in Table Following our observation that whitefly performance was better on TYLCV-infected Statistical significance was evaluated using ANOVA at a 0.05 level followed by LSD tests for whitefly performance. Data of percentages of adult survival were transformed by arcsine square root before analysis. Differences in gene expression levels were analyzed by nonparametric tests. All the data analyses were performed using IBM Statistics SPSS 20.0.35S-prosystemin compared to those on either CastleMart or spr2, and in females of MEAM1 survival was significantly higher on spr2 than that on CastleMart  can promote the performance of whiteflies on infected tobacco plants through inhibiting JA-related defence34. The pathogenicity factor ßC1 of TYLCCNV was found to be interacting with MYC2, and the interactions drive the down-regulation of JA signaling pathway and in turn suppression of terpenoid synthesis and release30. However, in this study, the begomovirus TYLCV infection did not inhibit the expression of the four JA-biosynthesis genes  in the 35S-prosystemin transgenic plants, but the transcript level of the JA-regulated defense gene (PI-II) was down-regulated (Fig. JA-induced defenses in B. tabaci complex were included in the experiments. Statistical analyses of the data indicated that in some cases whitefly species had a significant role in the interactions (Tables 21. In another study, we found that TYLCV infection of tomato plants had little effects on the performance of both the invasive MED and the indigenous Asia II 1 whiteflies20. These results are not surprising but do help to show the complexity that can occur in the tripartite interactions between whiteflies, begomoviruses and host plants. The data of this study indicate that the effects of virus infection of the plants on the performance of whiteflies vary with the intrinsic level of JA-related resistance (Tables 35.In this study, two species of whiteflies of the s Tables –4. For eAll in all, we show that begomovirus infection may suppresses JA-related resistance in tomato plants and in turn promote the performance of whiteflies. However, this benefit effect conferred to the whiteflies via infection of the host plants by the virus becomes significant or substantial only when JA-related resistance plays a major role in the plants against whiteflies. Our findings confirmed the general role of JA-related resistance against whiteflies, and further show that the effects of suppression of JA-related resistance in plants by begomoviruses on whitefly performance may vary depending on the intrinsic role of JA in that plant.


Temporal lobe epilepsy (TLE) is a common chronic neurological disease in humans. A number of studies have demonstrated differential expression of miRNAs in the hippocampus of humans with TLE and in animal models of experimental epilepsy. However, the dissimilarities in experimental design have led to largely discordant results across these studies. Thus, a comprehensive comparison is required in order to better characterize miRNA profiles obtained in various post-status epilepticus (SE) models. We therefore created a database and performed a meta-analysis of differentially expressed miRNAs across 3 post-SE models of epileptogenesis  and human TLE with hippocampal sclerosis (TLE-HS). The database includes data from 11 animal post-SE studies and 3 human TLE-HS studies. A total of 378 differentially expressed miRNAs were collected (274 up-regulated and 198 down-regulated) and analyzed with respect to the post-SE model, time point and animal species. We applied the novel robust rank aggregation method to identify consistently differentially expressed miRNAs across the profiles. It highlighted common and unique miRNAs at different stages of epileptogenesis. The pathway analysis revealed involvement of these miRNAs in key pathogenic pathways underlying epileptogenesis, including inflammation, gliosis and deregulation of the extracellular matrix. The contribution of pathological changes within the brain to the process of epileptogenesis in TLE cannot be easily addressed in human hippocampal specimens, since brain tissue is usually resected at the end-stage of the disease. Hence, a variety of experimental models have been developed to study epileptogenesis. The most widely used models that resemble the features of TLE, including hippocampal pathology, are the post-status epilepticus (SE) rodent models in which epilepsy develops after a chemically or electrically-induced SE3. The large-scale transcriptomic studies of such animal models have identified changes in the expression of hundreds of genes shortly after SE and during the course of epileptogenesis6. These changes have been found to underlie major pathological processes associated with human TLE, including the occurrence of spontaneous recurrent seizures, neuronal loss, aberrant growth and neurogenesis in the hippocampus, gliosis, neuroinflammation, reorganization of the extracellular matrix (ECM) and blood–brain barrier (BBB) dysfunction10.Epilepsy is a common neurological disease characterized by recurrent seizures. The complex of epilepsy-associated comorbidities is a major burden for health care systems all over the world. Temporal lobe epilepsy (TLE) is one of the most common forms of focal epilepsy in adults11. miRNAs have been shown to be involved in the regulation of numerous biological processes within the central nervous system13. Differential expression of miRNAs in the temporal lobe of TLE patients and animals during the course of experimental epileptogenesis has been extensively studied over the past decade18. A variety of techniques including microarray, qPCR-array and RNA-sequencing have been utilized to profile the changes of miRNA expression in the rodent brain following the induction of SE evoked by electrical stimulation (ES)23, administration of pilocarpine29 or kainic acid (KA)32. These studies have produced vast amounts of data, but their outcomes are often discordant, with each study producing a unique array of differentially expressed miRNAs.The regulation of gene expression is not only confined to the transcriptional level. miRNAs are small non-coding RNAs that are crucially involved in the post-transcriptional control of gene expression. miRNAs act in association with the RNA-induced silencing complex (RISC), predominantly through binding to the 3′-untranslated regions (UTR) of the mature messenger RNA (mRNA) transcripts, which leads to repression of a target gene translation35, suggesting that the source and quality of sample preparation are important factors for miRNA profiling studies in the brain. Furthermore, the miRNA expression patterns alter dramatically over the time course of epileptogenesis19. Finally, technical dissimilarities in the study design, such as the choice of profiling platform, sample size, RNA extraction method, application of empirical cut-off thresholds and different statistical tests influence the study outcomes strongly.Obtaining a clear profile of miRNA changes in animal post-SE models is confounded by many dissimilarities in experimental approaches. There are fundamental differences between models, which are characterized by different SE-triggering events and the subsequent induction of multiple pathological pathways, associated with unique patterns of transcriptional activation and miRNA deregulation. This is further complicated by differential miRNA expression between various brain regions and cell types37 and neurological diseases38. Finally, we compare biological pathways associated with altered miRNA expression in post-SE models during the chronic phase  and human TLE-HS profiles.Given its complexity, the field of epilepsy lacks the level of standardization necessary to address the role of miRNA regulation in the pathology. In the present study we integrate the existing large-scale profiling knowledge on differential miRNA expression in epilepsy, produced by the efforts of several laboratories, to create a database of differentially expressed miRNAs. We review and standardize various parameters that are crucial for comparison of profiling studies. Using a meta-analysis approach, we seek to identify consistently differentially expressed miRNAs across different studies. Such an approach has been successfully used for inter-study comparisons in cancer) and 3 profiling studies in human TLE-HS (Table ). The RNA-Seq studies were included in the database, however, due to the limited number of studies they were excluded from any subsequent analyses. From the 11 post-SE array-based studies 21 distinct miRNA gene expression profiles were extracted based on different animal species, post-SE models and time points. In total 378 miRNAs were found to be differentially expressed in at least one study. The expression of 274 miRNAs was increased (p < 0.05) and the expression of 198 miRNAs decreased (p < 0.05) as compared to control in at least one study. About 48% of all miRNAs (182) were found to be differentially expressed only in a single profile. Only the minority of all miRNAs were repeatedly found as differentially expressed across all profiles . It showed that different studies produced vastly different expression profiles. Then we extended the analysis to identify the clusters of miRNAs specific for each parameter. In order to do so, we analyzed only the miRNAs that appeared in a single model or a single time point. Several clusters of miRNAs were specific for each model with the largest cluster corresponding to ES model . The largest cluster of miRNAs specific for a particular time point corresponded to the acute stage  to estimate how well the miRNA profiles grouped by various parameters, such as model , time point after SE and species . No clear clustering by any parameter was found for both up-regulation and down-regulation sets of profiles We further assessed the differences and commonalities of differentially expressed miRNAs across post-SE models. The number of differentially expressed miRNAs was not uniform both between studies, in which the same post-SE model was used and between studies of different post-SE models Fig. . For theNext, all miRNAs reported as differentially expressed in each animal model were overlapped Fig. . In totaNext, we assessed the differences between expression profiles depending on time point following SE, but regardless of model and species. The largest number of differentially expressed miRNAs belonged to the acute stage (296), which was almost double the number of miRNAs seen at the chronic stage (169) and 4 times the amount at the latent stage (71) Fig. . There wNext, the differential expression of miRNAs was assessed across different species, but regardless of post-SE model or time point. We combined miRNA expression data from 7 rat and 5 mouse studies at any stage following SE and compared it to the data obtained from 3 human TLE-HS profiles Fig. . The num; Supplementary Table . Using a Fisher’s exact test we assessed the human TLE-HS profile for an enrichment of miRNAs from a particular stage or model. No significant enrichment for any stage or models was seen amongst human TLE-HS miRNAs .We further investigated the representative value of miRNA profiles obtained in animals in regard to human TLE-HS by comparing human and rodent miRNA profiles with respect to different models and stages of SE. The human set shared approximately equal portions of miRNAs with each different model . We performed the analysis for the data from the post-SE profiles at 3 stages. The human datasets were not used in this analysis. For the acute stage, 9 mature miRNA sequences were identified as consistently differentially expressed: 8 were up-regulated  and one was down-regulated (miR-302b-5p)  method was applied to identify consistently differentially expressed miRNAs in similar studiesmiRNAs identified as consistently differentially expressed at each stage of epileptogenesis were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using Diana miRPath3. The pathway analysis was done for mouse and rat species. At the acute stage “MAPK signaling” and “Cytokine-cytokine receptor interaction” were the only common pathways for both rat and mouse species Fig. . Among tThe latent stage was accompanied by activation of a variety of signaling pathways and, similar to the acute stage, showed the involvement of miRNAs in the MAPK signaling Fig. . HoweverFor the miRNAs at the chronic stage of epileptogenesis, common pathways for both rodent species were found only by the “Gene union” function . Besides, we found a high variability in the number of differentially expressed miRNAs between the profiles at the acute stage, which may be associated with the larger entropy of changes at this time point. The ES model showed less inter-study variability in the number of up-regulated miRNAs at the acute stage compared to the two chemoconvulsant models. The use of different dosages of chemoconvulsants and the drugs for the cessation of SE may result in different courses of epileptogenesis21. Thus a single seizure episode prior to sacrifice may significantly alter the outcome profile and that must be thoroughly controlled in order to get reproducible results.Despite a very small overlap between the 3 stages, the acute and chronic stages of epileptogenesis shared a considerable amount of miRNAs. The miRNA profiles at the chronic stage may be affected by the acute seizure-related changes if a period of time between the last spontaneous seizure and the sacrifice is short. It has been demonstrated that miRNA expression patterns were clearly different between the acute seizure model and the chronic model39. While this method drastically reduced the number of differentially expressed miRNAs it highlighted a handful of miRNAs for further study. A relatively unique profile of miRNAs accompanied each stage. Combined, these miRNA signatures may be characteristic of the major pathogenic processes underlying epileptogenesis, such as cell death, gliosis and neuroinflammation52. The only 3 common miRNAs that were found between all 3 stages of epileptogenesis were miR-21–5p, miR-132–3p and miR-212–3p. Both miR-21 and the cluster of miR-212/132 have been implicated in epilepsy and various neurologic disorders54. Deregulation of these miRs may represent the interplay between neuronal cell death and neuroprotection, as both miR-21 and miR-212/132 have been previously implicated in these processes in the brain55. The role in inflammation has also been attributed to both miR-2156 and miR-13257. Our pathway analysis showed that miRNAs at the acute stage were strongly involved in the cellular pathways associated with innate immune response and inflammation. At the latent stage, up-regulated miRNAs miR-20a, miR-17, miR-23a, miR-27a and miR-146a have been previously shown to be enriched in mouse microglia58. Noteworthy, both the latent and chronic stages were marked by the up-regulation of miR-146a, a key regulator of inflammation in astrocytes59.Considering the differences between the profiles, the involvement of miRNAs in epilepsy can hardly be concluded based on the observations made in one single profile, but rather the relevance of a miRNA to some pathogenic pathways activated in a chosen model in particular sub-region of the brain and at given time point may be assessed. In order to identify consistently differentially expressed miRNAs at different stages of post-SE epileptogenesis we applied the RRA method62. There are also certain limitations of using human TLE tissue, like the quality of RNA and the use of autopsy control samples, heterogeneity of epilepsy cases and limited supply63. Patients undergoing hippocampal resection in most cases have a long clinical history of various anti-epileptic drug therapies, altering the molecular profile. Besides, SE is rarely an initiation event for epilepsy in humans, while it is characteristic for animal post-SE models.In addition to the observed differences in miRNA profiles between different post-SE models and time points, there were differences associated with species. We found a large amount of miRNAs that were identified only in rats, mice or humans. That raised a question of how relevant the findings in rodents are to human TLE. The comparison of the results obtained in animal post-SE profiles and in human TLE profiles is hampered by many obstacles. The amount of identified miRNAs, their predicted targets and functions may vary considerably between different species67. Neither of TLE-HS profiles included in the meta-analysis has reported significant up-regulation of miR-21 or miR-132. However, both these miRNAs have been found to be overexpressed in human TLE-HS when their expression was assessed individually68. In order to identify the biological pathways associated with the differentially expressed miRNAs in human TLE-HS we combined the results of the TLE-HS profiles and subjected them to the pathway analysis taking into account the combinatorial effect of all miRNAs69. Since the chronic stage in post-SE models has been considered to be representative of the neuropathological features of human TLE-HS, we performed the pathway enrichment analysis for the 100 most consistently differentially expressed miRNAs in chronic post-SE profiles and compared the results with the enriched pathways associated with the 100 most consistently differentially expressed miRNAs in TLE-HS. The pathways associated with the differentially expressed miRNAs in chronic profiles and in TLE-HS largely overlapped. The most enriched pathways were related to the regulation of the ECM and fatty acids.The profiles of miRNA expression in TLE-HS have been reported by several research groups4. Consistent with our observations a recent transcriptome analysis in 129 TLE patients has revealed the cell-to-extracellular matrix adhesion processes  as one of the two major clusters of differentially expressed genes along with the inflammation70. The changes in expression of the ECM genes cause alterations in the molecular environment surrounding neurons, which is a hallmark of epileptogenesis4. The components of the brain ECM produced by both neurons and glial cells are involved in multiple physiologic processes, which are disturbed as a result of the ECM remodeling, leading to further epileptogenic events, such as mossy fiber sprouting, granule cell dispersion and astrogliosis71. Interestingly, the miRNAs associated with the “ECM-receptor interaction” pathway in our analysis were different between the chronic post-SE profiles and the TLE-HS profiles. These observations suggest that deregulated miRNAs and their target genes may differ between the rodent and human species, but the pathogenic pathways, that the deregulated miRNAs are associated with, are generally conserved across species and can be indicative of the TLE neuropathological features. Thus, the deregulated pathways, rather than the individual miRNAs may be of more interest when translating results from rodent models to TLE. Of note, there was little overlap in identified differentially expressed miRNAs across the 3 TLE-HS profiles and we acknowledge that the low consistency across these studies does not allow a proper statistical evaluation of these data. Thus, there is a pressing need for more profiling studies on differentially expressed miRNAs in TLE-HS, which will allow a better characterization of the translational validity of miRNA expression data from rodents to humans.The ECM is an important player in the epileptogenic network and the involvement of miRNAs in neuroinflammation, reactive gliosis and BBB breakdown may be directly or indirectly associated with the ECM remodelling, which can be evident already during the latent stage following SE75. Further, the differential expression of miRNAs in a microarray experiment is defined by the fold change of expression. The fold change may be low if a miRNA is highly expressed at basal level and may be high if the miRNA is not abundantly expressed. Thus, the fold change value will be practically meaningless for the low-expressed miRNAs, while the changes in highly expressed miRNAs will be missed if a fold change cut off is applied76. This issue is somewhat alleviated in RNA-Seq, as an RNA-Seq output can provide a quantitative expression output, such as raw or normalized read count or counts per million (CPM) for each miRNA. This can allow lowly expressed miRNAs to be filtered out, or for an exploration of the nature of the changes in expression. Overall, RNA-Seq offers a higher resolution, more comprehensive view of the small RNA transcriptome that will not only increase confidence in the role of previously identified miRNAs in epileptogenesis but also potentially identify new key players.Currently, the majority of miRNA profiling in post-SE rodent models and human TLE have utilized microarrays or qPCR arrays. Moving forward high-throughput RNA-Seq will become the standard technique used for the identification of differentially expressed miRNAs. RNA-Seq offers a relatively unbiased, discrete, and digitized read count from a cDNA library input. When compared to microarrays, RNA-Seq has lower levels of background noise, produces less false positives, has a higher dynamic range, requires a lower amount of input RNA and can be used to detect novel short non-coding RNA species and isomiRsIn this work we combined data from various profiling studies and created a database of differentially expressed miRNAs in epilepsy. The analysis of these data has demonstrated that the outcomes of different profiling studies are vastly different due to heterogeneity of input parameters. The profiles differ depending on the type of post-SE model, time point, region of the brain and species. Despite many differences between the profiles we identified the most common differentially expressed miRNAs across the studies. For each stage of epileptogenesis there was a certain combination of consistently differentially expressed miRNAs, associated with the key pathogenic pathways underlying such processes as neuroinflammation, gliosis, cell death and deregulation of the ECM. We identified only the most consistently differentially expressed miRNAs, but many model- and process- specific miRNAs should exist that are associated with all the variety of biological processes. The major pathways, associated with deregulated miRNAs in post-SE models may be conserved across mammalian species and be characteristic of epileptogenesis in human TLE. The analysis of the pathways associated with the differentially expressed miRNAs has revealed a remarkable similarity between the human TLE-HS and chronic profiles in post-SE rodent models with a cluster of the ECM-related pathways among the most enriched. Thus, a better understanding of miRNA regulation during the course of epileptogenesis in rodent models has the ability to contribute to the development of preventive treatment for TLE.The studies containing profiles of miRNA expression in rodent models of epileptogenesis and in human TLE with hippocampal sclerosis (TLE-HS) were searched through PubMed database using the keywords “miRNA”, “epilepsy”, “status epilepticus”, “profile”, “micro-array” in various combinations. The last search was performed in May 2017. The content of each study was carefully analyzed. Only studies published in peer-reviewed journals were included in the analysis. Table We created a database of differentially expressed miRNAs in post-SE rodent models and human TLE-HS. Mature miRNA sequences were arranged in a table with respect to species (mouse or rat), anatomical region  and time point of assessment , however, all species indicators were abandoned to simplify the comparison of miRNA profiles between different species. The comparison of miRNAs between different species was done based on total sequence homology between orthologues. If the miRNA ID obtained from the selected studies had outdated nomenclature, it was substituted with the new one. The miRNA signatures of species other than human, rat or mouse and non-miRNA probes were excluded from the analysis.The IDs were assigned to miRNAs according to the nomenclature recommended by miRbase . The lists of extracted miRNAs from each animal post-SE study were split into up-regulated and down-regulated miRNAs. The human TLE-HS profiles were not analyzed by the RRA. The miRNAs in each list were ranked by p-value, when possible. If no p-value was available, the miRNAs were ranked by fold change value. To identify miRNAs that were ranked consistently better than expected by chance, we used the RRA method as implemented in the R package39. This method assigns a p-value to each element in the aggregated list indicating how much better it is ranked compared with a null model expecting random ordering. All statistical and clustering analyses were carried out using the statistical software R (www.r-project.org).The lists of extracted miRNAs were prioritized based on statistical test adjusted http://snf-515788.vm.okeanos.grnet.gr/), a web-based computational tool that identifies potentially altered molecular pathways by the expression of multiple miRNAs. This program performs an enrichment analysis of miRNA target genes, and compares each set of miRNA targets to all known KEGG pathways79. We used the microT-CDS 5.0 algorithm for target prediction. The lists of differentially expressed miRNAs were analyzed separately for each species . The common pathways enriched for both mice and rats were united based on the geometric mean of their p-values. The non-common pathways between the rodent species were merged together and ranked based on their p-values. The pathway analysis was done using both “Gene union” and “Pathway union” algorithms69. The pathways were considered to be enriched if they passed the p-value threshold 0.05. All p-values were FDR-corrected.The miRNAs identified by the RRA analysis as consistently differentially expressed were submitted to DIANA mirPath v.3 .Supplementary Figures and Tables S1, S7Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6


Mobile-phone-based smoking cessation intervention has been shown to increase quitting among smokers. However, such intervention has not yet been applied to secondhand smoke (SHS) reduction programs that target smoking parents of newborns. This randomized controlled trial, undertaken in Changchun, China, assessed whether interventions that incorporate traditional and mobile-phone-based education will help create smoke-free homes for infants and increase quitting among fathers. The results showed that the abstinence rates of the fathers at 6 months  and 12 months  were both significantly increased in the intervention group compared to the control. Mothers of the newborns in the intervention group also reported reduced exposure to SHS at 12 months . The findings suggest that adding mHealth interventions to traditional face-to-face health counseling may be an effective way to increase male smoking cessation and reduce mother and newborn SHS exposure in the home. Evidence suggests that exposure to SHS, mostly occurring in the home, threatens the health of roughly 40% of children worldwide8.Second-hand smoke (SHS) exposure is a major, yet preventable, threat to infant and child health. It puts newborns at risk of developing sudden infant death syndrome, and increases susceptibility to lower respiratory infections, middle ear infections, childhood cancers, asthma, allergies, reduced physical development, and decreases in cognition and behavior for infants and children9 yet a large number of infants are still exposed to SHS in the home due to the high adult male smoking rate (>50%)10. Estimates of the prevalence of household SHS exposure among non-smoking pregnant women in China range from 58% to 73%11; levels of exposure remain high after babies are born12. These high exposure rates among pregnant women underscore the fact that at least half of all newborns in Chinese families are regularly exposed to SHS in the home. The risks associated with exposure to SHS among infants and the high exposure rates in the home due to the father’s smoking status creates an urgent need to create smoke-free homes for the health of infants in China.In China, most women do not smoke (<3%)13. According to the Global Adult Tobacco Survey (GATS), Chinese smokers were among the most resistant to quitting in the world, with more than 80% reporting having no intention to quit smoking14. Chinese women have traditionally accepted the social norm of men smoking at their presence15, and have had limited success with stopping in-home smoking due to the desire to maintain household harmony16. Studies have found that only a small portion of families in China have strict home smoking restrictions (<15%). For households with regular smokers, usually husbands and/or fathers, the rate of complete smoking bans is as low as 3%17. Without interventions targeted towards reducing in-home smoking, children and infants will remain exposed to high levels of SHS.For Chinese families with infants and children to maintain a smoke-free home, fathers would have to quit smoking or smoke only outside of the home. However, the quit rate among men in China is low22. In these studies, standard health advice was usually provided by health professionals and supplemented with printed health education materials; one study utilized follow-up telephone support19. A recent meta-analysis found that traditional interventions were not effective in reducing smoking rate among parents of children under the age of 421. Many other shortcomings of traditional methods are apparent as well. For instance, the amount of educational information, motivational messages, and behavior-change guidance that can be conveyed during physician consultations is limited23. The time when and the location where the participants can be reached by health professionals are restricted too, and hence, it is costly to maintain routine communication with the participants. New technology is clearly needed for improved delivery of existing health services.Previous smoking cessation and SHS reduction intervention programs that targeted smoking parents have relied heavily on face-to-face counseling and distribution of self-help materials23. Mobile phones are a flexible, accessible, and low cost method for delivering health promotion interventions. mHealth interventions allow for the conveyance of information, triggers, and support whenever clients carry a mobile phone, and embrace the element of anonymity24. In addition, these interventions can be scaled to large populations and personalized to meet individual needs25. The application of mHealth interventions, in particular short message service (SMS), has shown improved outcomes in smoking cessation interventions28. According to a recent review of 13 randomized controlled trials, smoking quit rates were significantly higher for the text messaging intervention group than the control group 26.The rapid increase in the number of people owning a mobile phone has led to the incorporation of mobile health  interventions into traditional health practicesTo our knowledge, mobile-phone-based health interventions have not yet been applied to household SHS reduction programs that target smoking parents of young children. We also found an absence of any experimental research studies aimed at creating smoke-free homes for infants in China. To fill these knowledge gaps, we designed a randomized controlled trial (RCT) that aimed to assess whether or not interventions incorporating traditional and mobile-phone-based education help create smoke-free homes for infants and increase quitting among fathers in Chinese households. In addition, our study included non-smoking spouses (wives/mothers) based on the hypothesis that spousal pressure and support could better help convince male smokers to quit for the health of their children.29. The recruitment of participants was completed over a one-month period . The protocol and survey questionnaires of the study were approved by Jilin University Second Hospital Institutional Review Board (IRB). All methods included in the study were consistent with the principles of the Declaration of Helsinki.The Smoke-free Homes mHealth Intervention Project was a single-blind, randomized controlled trial of an SHS reduction and smoking cessation service enhanced with mobile phone text messages. The intervention was implemented in Changchun, China and managed by Changchun Health Education Institute and the China Tobacco Control PartnershipFamilies were eligible for inclusion if they met the following criteria: nonsmoking mothers and their newborns were currently exposed to SHS in the home; fathers currently smoked cigarettes in the home; the parents both owned a mobile phone that could receive text messages; and the family was able to provide informed consent. There were no restrictions for trial participants on the use of other smoking cessation services or additional support.The screening process took place in 15 local maternal-child health centers that were selected as cooperating organizations of our intervention project. Trained health workers in those centers asked all mothers attending their initial post-delivery visit (1 month after birth) to complete a short health questionnaire with questions related to tobacco use and household SHS exposure. Among all eligible families, 342 were recruited as participants for the trial.Participant families were randomly allocated to one of the following three groups: Intervention Group A (I-A), Intervention Group B (I-B), or the Control Group. Randomization was fully computerized, using no blocks or strata, and each participant was allocated a number 1 (then assigned to I-A), 2 (then assigned to I-B), or 3 (then assigned to control group) with equal probability.th and February 22nd in 2015. The control group received only standard care for their initial postnatal visits, which did not include any tobacco control and cessation counseling service.Three home visits were conducted with all participants. The initial visit, conducted in December 2014, assessed baseline SHS levels in the home. The parents were asked to complete short surveys on smoking, exposure to SHS, and knowledge of the harms of smoking and SHS. The husband was asked additional questions about his smoking habits and quit history. At this visit, participants allocated to I-A received in-person counseling from the trained health care workers on the harms of SHS to infants; education on establishing a smoke-free home, including a manual with step-by-step instructions; and table tents and posters to display in the home to encourage fathers and other visitors not to smoke. The smoke-free homes manual provided a 5-step plan for creating a smoke-free home with information on: (1) deciding to create a smoke-free home; (2) talking to family members; (3) setting a date for going smoke-free; (4) actually creating a smoke-free home; and (5) keeping the home smoke-free. Participants allocated to I-B received the same educational intervention and materials as I-A at this visit, and also received a text message intervention in the coming months. The text message intervention included messages to the mother and her husband on the harms of SHS to the mother and the infant. The husband received additional cessation text messages to encourage him to quit smoking. Text message examples are listed in Table Follow-up home visits were performed at 6 and 12 months. Follow-up self-reported data on SHS levels and smoking behavior were collected on-site. Participants in I-A and I-B received additional counseling at the six-month follow-up visit if they had been unable to successfully create a smoke-free home since the initial visit.Newborn’s parents were required to complete an onsite questionnaire survey at baseline, 6 and 12 months. The questionnaire was initially developed in English, and translated into Chinese to onsite survey. Both questionnaires were tested among Emory University students who were fluent in both languages. The questionnaire included questions on demographic information, smoking status, exposure to SHS at home, and perceived health risks of smoking and SHS exposure. Major outcomes included fathers’ abstinence rate and mothers’ SHS exposure at home at baseline, 6 months and 12 months. Fathers’ self-reported smoking status at home and mothers’ exposure to SHS at home in the last 6 month were compared and matched to determine fathers’ current smoking status and infants SHS exposure at home. If mothers reported exposing to SHS due to fathers smoking at home in the last 6 month, the father will be classified as smoking regardless of his self-reported status.Based on 80% power and 5% level of significance, we calculated that 300 participants (100 in each group) were needed to detect a moderate to large effect size at the end of the program, given an expect dropout rate of 20%.The primary outcomes of the study were self-reported smoking status among the fathers and self-reported SHS exposure at home among the mothers at the 6- and 12-month follow-up periods. Other outcomes included: fathers’ self-reported intent to quit, knowledge of SHS and tobacco smoking, and smoke-free home policy enforcement at the 6- and 12-month follow-up periods. For each outcome, a multivariate logistic regression was performed to determine how each intervention affected the outcome variable, controlling for demographic and socio-economic variables including age, ethnicity, education level and occupation. Data were analyzed using SAS 9.3 . Estimation of relative risks, 95% confidence intervals, and two-sided p values at 6 and 12 months were reported.The protocol of the study was approved by the Changchun Institutional Review Board (IRB).The written consent forms were distributed to all participants. The study participants provided written consent to participate.This study has been registered as a randomized clinical trial (RCT) at the Chinese Clinical Trial Registry (ChiCTR) with registration number as ChiCTR-OIC-17010803 on March 6, 2017.The first round of recruitment included 342 mothers who were randomly assigned to I-A, I-B, or the control group Fig. . Three hFathers of the newborns (n = 342) were also assigned to the three groups, corresponding to the assignment of their wife; 103 (90%), 100 (88%), and 96 (84%) were allocated to I-A, I-B, and the control group, respectively, to complete the baseline surveys. At the 6-month follow-up, 298 fathers completed the surveys. The I-B group lost one participant. At 12 months, 292 fathers completed the follow-up surveys .We conducted a t-test and found no significant differences in the characteristics between those who lost to follow up and those who remained in the sample (the results are available upon request), and we therefore handle the missing data with listwise deletion in the analysis.All baseline characteristics are shown in Table The average age of participant mothers was 29.6 (SD: 3.8). Almost seventy percent had completed community college education or above, and 25.3% were unemployed. When asked about household SHS exposure, 185 (61.7%) participants reported exposed to SHS in the home almost every day. No significant differences existed between groups for any baseline characteristics.The primary outcomes assessed were self-reported smoking cessation among fathers and self-reported exposure to household SHS among mothers of the newborns at 6 and 12 months post-randomization. Results showed that the crude odds ratios are similar to adjusted odds ratios in all tables, which is typical in a randomized controlled trial study. We hereafter report adjusted odds ratios only. Table Regarding other outcomes, participants in I-B group are more likely  to report “smoking never permitted inside home” compared to participants in control group at 12 months ; the risk difference remained high (13.0%) at the 12-month follow-up visit. Meanwhile, the self-reported SHS exposure among non-smoking mothers whose husbands received text message interventions was significantly reduced at the 12-month follow-up. These findings suggest that the addition of an mHealth element to interventions with in-person counseling and provision of educational materials effectively aided in creating smoke-free homes and promoted smoking cessation among fathers of newborns.26, including two recent RCTs conducted in China. One RCT included a large smoker population in China and found a significant increase in the self-reported past-7-day abstinence rate among the group that received a high-frequency of text contacts compared to the group that received low-frequency text contact, although the magnitude of the difference was relatively small30. The other RCT targeted a subpopulation of Chinese smokers, adolescent smokers aged 16 to 19 years, and found that the text messaging intervention group had a significantly higher rate of smoking reduction compared to the control group31. Our study adds novel insight to the current research on mHealth smoking cessation interventions in China because it targeted a unique subgroup of Chinese smokers (i.e. fathers of newborn babies) and supported current evidence that mHealth is a promising intervention for future practice. In China, more than 90% of population now have mobile phones32, allowing for a more accessible and efficient means of delivering health information. Based on this study and the findings from other mHealth interventions, text-messaging interventions should be promoted more aggressively as a tool to protect women and children from second-hand smoking exposure. In addition, based on past and present evolution of mobile phone-based applications, it may be useful to consider other mobile platforms  for future mHealth interventions33.The differential increase in smoking cessation rates among the mHealth intervention group in our current study confirms the findings of earlier research on smoking cessation interventions using text-based mobile phone services36. Although complete cessation is not the norm for Chinese smokers, when the health of unborn or newborn babies is at risk, smoking fathers usually become more susceptible to opinions from their spouses who are primarily responsible for caring for their children. For instance, a RCT study conducted in Guangzhou, China found that simple health advice provided by obstetricians to non-smoking pregnant women was effective in helping their partner reduce consumption or quit smoking completely37. A similar study design was applied to parents of sick children in Hong Kong and found that health education provided by nurses to mothers of sick children triggered quit attempts by the husband in the short term and aided with increased cessation38. Thus, women should be mobilized in interventions designed to protect children, especially at the most vulnerable stages of child growth and development.Our study implies the important role of non-smoking women in helping their spouses quit smoking by focusing the major concerns with smoking on the health of their children. Unlike a number of previous smoking cessation studies in China targeted only to male smokers, our study also targeted non-smoking mothers and encouraged them to be change agents. Non-smoking mothers of the newborns and their smoking husbands received the counseling at the same time. This arrangement promoted spousal interaction and support, which has been found to be effective in helping smokers quit or reduce smoking39. Second, our study is based on a relatively small sample, which may have limited the ability to detect a true effect even when it existed. As the statistical power analysis suggested, the sample size of this study only allow us to detect association with odds ratio larger than 2.0 or smaller than 0.5 at 80% statistical power and 5% significance, which may explain why we failed to detect any significant difference in the outcomes neither between control group and traditional intervention group, nor the difference between traditional intervention group and mHealth intervention group. Future research with a larger sample is merited. Third, the participants were recruited from two urban districts of Changchun, where residents were considered to maintain higher social economic status, education level and health status. Therefore, the sample may not be representative of the whole city. Finally, the content of the text messages sent to the participants in our trial lacked diversity. The vast majority of the messages were related to the health effects of smoking and SHS exposure and the health benefits of quitting. Cessation advice, such as making a public declaration, asking for social support, tips to cope with cravings, and using distraction techniques; and personalized messages that address individual needs were not included in our trial. A recent trial conducted in China found that text messages including cessation advice were more effective than messages that addressed health effects alone30. Future mHealth interventions should consider sending participants an increased variety of smoking cessation messages.Our study faces limitations and the results should be interpreted with caution. The first limitation is the reliance on self-reported data in our approach to evaluate outcomes. Such self-report may be biased and lead to inaccurate measures of smoking abstinence and nicotine exposure. Biochemical verification with cotinine or carbon monoxide tests is desirable for future studies, even though bio-samples, such as hair, blood, saliva, and urine, can be difficult to obtainIn summary, mHealth interventions with a text message component are an effective way to increase male smoking cessation and reduce mother and newborn SHS exposure in the home in China. Including female spouses as a supportive aid in these interventions can increase the success of cessation for husbands. Future studies should include measured nicotine consumption and exposure, other potential messaging platforms, an increased variety of smoking cessation messages, and cost-effective analysis of the mHealth intervention.


Subsequently, the carcinoma cells acquire chemoresistance and stem-cell properties.2 Downstream to these cues, the molecular machinery involves dynamic epigenetic, transcriptional, and post-translational control of the EMT effectors, such as the E-cadherin gene; those machinery plays part in maintaining the mesenchymal trait and executing the EMT.1 However, the gain of the mesenchymal trait is perhaps not the most dominant effect of EMT. Rather, the loss of the epithelial trait is more frequently observed. Therefore, to achieve mesenchymal–epithelial transition, which is the reversal of EMT, two approaches would be required to either abolish the mesenchymal or to restore the epithelial features.Epithelial–mesenchymal transition (EMT) is a mechanism in which differentiated epithelial cells can lose their epithelial features. Some features include apicobasal polarity and cell adhesion system. Also, epithelial cells can acquire certain mesenchymal traits that are usually indicative of increased migratory and invasive properties.3 we described a promoter-reporter-based bioluminescent assay platform, which aimed to discover compounds that could restore the epithelial gene expression. This epithelial marker promoter induction (EpI) screen utilizes a sequence at the promoter region of the prototypic epithelial gene, E-cadherin, that is coupled in a firefly luciferase reporter system. The EpI screen was first performed in an ovarian cancer cell line with an intermediate EMT state. Later, the EpI screen was validated in other carcinoma cell lines with similar EMT states. We further demonstrated that promoter sequences of other epithelial markers, such as the epithelial-specific epidermal growth factor receptor family member ERBB3, can be incorporated into this EpI screen. Selecting from a compound library, we identified a class of compounds that manifests consistent and robust EpI effects, the histone deacetylase inhibitors (HDACi). Among the 41 HDACi screened, Mocetinostat was found to be the most potent compound that demonstrated the lowest concentration required for 50% of EpI (EpIC-50).In our recent publication,5 The philosophy of EMT reversal is to eliminate the mesenchymal cells that might have acquired therapeutic resistance and cancer stem-cell-like properties.1 Since EMT is a spectrum of intermediary states,6 it would be crucial to know at which threshold these aggressive features diminish. The reversal of EMT is not without consequences, because metastatic colonization is known to require mesenchymal–epithelial transition to occur.7 Therefore, it is important to carefully define and position the application of EMT reversal. In designing a readout assay for EMT, one practical and philosophical consideration is how much and how far should one reverse EMT. The key to success for assay development relies heavily on robust quantitative measurements of designed end point readouts with high reproducibility and tight sample variations. Any drug discovery assay for EMT would be required to fulfill these considerations.Currently, there is limited high-throughput screening (HTS) drug discovery platform designed for EMT. Contemporary platforms utilize EMT-related phenotypic functional assays, targeting the colony scattering or migration and invasion.8 Because of their low backgrounds in the mammalian system, the dynamic linear range of theses assays can be very wide. The firefly and Renilla luciferases are the most versatile luciferases that act as genetic reporters in HTS. By incorporating the upstream gene regulatory elements (RE) with the luciferase gene, these assays can be associated with the regulation of gene transcription. The direct transcriptional control of EMT is achieved by the binding of EMT inducing transcription factors (EMT-TFs), such as the SNAIL and ZEB family, to their target epithelial genes.1 The SNAIL and ZEB family EMT-TFs act as transcriptional repressors and recognize the palindromic enhancer-box (E-box) DNA sequences, CANNTG, via their zinc-finger DNA binding domains.9 Therefore, the E-box at the promoter sites of epithelial genes is pivotal for EMT execution. This provides the rationale to utilize the E-box as the RE in developing luciferase reporter assay for EMT drug screening. Within the short 233 base pair (−108/+125) of E-cadherin promoter sequences, three E-boxes are present. In cells characterized by intermediate EMT states, the high expression of SNAIL and ZEB family EMT-TFs occupy these E-boxes and suppress the transcription, resulting in low luciferase activities. Therefore, compounds, that could ‘lift' these transcriptional suppressions at the E-box, would induce luciferase activities downstream. The dose-dependency of HDACi for EpI activities not only suggests that the transcriptional repression at the E-box is ‘lifted', but also indicates that there is dynamic linear control of the epigenetic and transcriptional regulation of epithelial differentiation. Thus, the derivation of EpIC-50 provides a useful tool, in a quantitative manner, to assess the degree of restoring epithelial differentiation. Moreover, this EpI platform can be applied to other epithelial differentiation genes. Grainyhead-like 2 (GRHL2) has been demonstrated as an EMT suppressor that forms a negative regulatory loop with ZEB1 and miR200 family.10 GRHL2 binds to the enhancer site at the second intron of the E-cadherin gene; furthermore, GRHL2 regulates its promoter activity via local DNA looping.11 Since GRHL2 is an important epithelial gatekeeper, GRHL2 target genes could be candidates for the EpI screen. For example, the gene encoding an epidermal growth factor receptor family member, ERBB3, could be considered as a candidate. Within the 1.2 kb length of ERBB3 promoter, in addition to two E-boxes, there are two GRHL2 binding sites.3 As the direct transcriptional target for GRHL2, ERBB3 expression would be downregulated during EMT, and GRHL2 is lost.10 Thus, ERBB3-EpI activity could be utilized as a secondary validation,3 and it could be applied at the initial phase of EpI screen.Bioluminescent assays have been utilized for HTS in drug discovery.12 the identification of hybrid epithelial–mesenchymal clones of circulating tumor cells has given us a glimpse of future possibilities in the context of real-time monitoring of EMT. Patient-derived three-dimensional spheroid, or organoid cultures, would be a promising platform to incorporate phenotypic and EpI screens. However, one can expect that several technical hurdles, including capturing, preserving, and amplifying minute quantity of in-transit cells in culture conditions, would need to be overcome. It requires the incorporation of technology, such as droplet microfluidics,13 to encapsulate in-transit cells and to develop robust reporter assays at the single-cell level14 for downstream screening. Yet, recapitulating the microenvironment in this kind of system would be another challenge. The future of EMT drug discovery requires much creativity in combining multidisciplinary science at the forefront and to go beyond the binary view of EMT.1One can envision the incorporation of various EpI reporters covering different REs to be essential for EMT drug discovery. EMT is a mechanism that converges the diverse upstream signaling pathways with dynamic control of various EMT effectors. Thus, EMT drug discovery approaches must evolve from existing pathway-centered paradigms. The functional difference between epithelial and mesenchymal cells provides the basis for phenotypic screens. The transcriptional regulation between EMT-TFs and EMT effectors further provides the basis for a screen-like EpI. A drug discovery screening-like EpI might not appeal to skeptics of the clinical relevance of EMT. Despite of such skepticism, the EpI screen strategy integrates our understandings of transcriptional regulation in epithelial differentiation and modifications of epithelial plasticity. Hence, we might be able to give new meanings to numerous leads that have been dropped from the traditional drug screening for cytotoxicity, and we could rescue them for further clinical development with revised positioning. It might sound naïve, but the EpI screen could be an effective strategy amidst the drug drought and patent cliff.


Alternative splicing is a critical event in the posttranscriptional regulation of gene expression. To investigate whether this process influences radiation-induced gene expression we defined the effects of ionizing radiation on the generation of alternative transcripts in total cellular mRNA (the transcriptome) and polysome-bound mRNA (the translatome) of the human glioblastoma stem-like cell line NSC11. For these studies, RNA-Seq profiles from control and irradiated cells were compared using the program SpliceSeq to identify transcripts and splice variations induced by radiation. As compared to the transcriptome  of untreated cells, the radiation-induced transcriptome contained 92 splice events suggesting that radiation induced alternative splicing. As compared to the translatome (polysome-bound RNA) of untreated cells, the radiation-induced translatome contained 280 splice events of which only 24 were overlapping with the radiation-induced transcriptome. These results suggest that radiation not only modifies alternative splicing of precursor mRNA, but also results in the selective association of existing mRNA isoforms with polysomes. Comparison of radiation-induced alternative transcripts to radiation-induced gene expression in total RNA revealed little overlap (about 3%). In contrast, in the radiation-induced translatome, about 38% of the induced alternative transcripts corresponded to genes whose expression level was affected in the translatome. This study suggests that whereas radiation induces alternate splicing, the alternative transcripts present at the time of irradiation may play a role in the radiation-induced translational control of gene expression and thus cellular radioresponse. The development of molecularly targeted approaches for enhancing the efficacy of radiotherapy, a primary treatment modality for most solid tumors, requires a comprehensive understanding of the processes comprising cellular radioresponse. Along these lines, fundamental determinants of radioresponse, such as DNA repair and cell cycle checkpoint activation, have been best described in terms of the post-translational modification of existing proteins. Modifications in gene expression, however, are also a consequence of irradiation suggesting a role for de novo protein synthesis in determining cellular radioresponse. Towards understanding the biological significance of radiation-induced gene expression, studies initially focused on defining the changes in the transcriptome  in irradiated cells -6. HowevOne of the critical events in the post-transcriptional regulation of gene expression is the splicing of introns from precursor mRNA (pre-mRNA) . SplicinRadiation has been shown to induce AS in the evaluation of individual genes -19 and aWe recently described the radiation-induced transcriptomes  and translatomes (polysome-bound mRNA) for the NSC11 glioblastoma stem-like cell (GSC) line along with 2 additional GSC lines using microarray analysis . In that+ selected RNA was subjected to paired-end sequencing with mapping to the human hg19 reference genome  or total cellular RNA, polyAnome see  corresponome see . The splnome see . Genes cTo investigate the effects of radiation on AS, total cellular mRNA or polysome-bound mRNA was collected from NSC11 cells 1h after irradiation (2 Gy) or after mock irradiation (control) and subjected to RNA-Seq. An advantage provided by using NSC11 cells is that the majority of changes in the translatome occur at 1h after irradiation , which rThe presence of the radiation-induced splice events was validated using qRT-PCR. The 6 genes evaluated involved radiation-induced splice events enriched in polysome-associated RNA only , total cellular RNA only , and splice events common to both . These genes were selected for evaluation because they contained among the largest RPKMs for alternative exon skip events, the most common radiation-induced splice event detected in the transcriptome and translatome; were predicted to be modified in only the translatome, only the transcriptome or both and for which there were commercially available primer assays. Primers were selected based on sequence analysis to flank a splice junction of the transcript as predicted by SpliceSeq. This allowed detection of the changes in each transcript predicted to be alternatively spliced after irradiation and thus to reflect changes in the splice event rather than transcript abundance. The dPSI (change in percent spliced in and a measure of splice frequency) for each of these splice events in the individual replicates from each treatment condition show the change in splicing after irradiation as well as a high reproducibility across the 6 replicates Figure . The qRTDNA Double-Strand Break Repair by Homologous Recombination, Ceramide Degradation, Sphingosine and Sphingosine-1-phosphate Metabolism and L-carnitine Biosynthesis. The networks enriched in total RNA and polysome-bound RNA after irradiation are shown in Tables DNA Replication, Recombination, and Repair”, processes critical in determining cellular radioresponse. This network, which is diagramed in Figure The radiation-induced splice events in total cellular RNA 92) mapped to 89 genes and the radiation-induced splice events in polysome-bound RNA (280) mapped to 262 genes  and 4. T mapped tBecause RNA-Seq data allows for not only evaluation of alternative transcripts (SpliceSeq), but also changes in gene expression, we compared radiation-induced alternative transcripts to radiation-induced gene expression as detected in total RNA and polysome-bound RNA. In terms of the radiation-induced gene expression substantially more genes were affected by radiation in the translatome 3929) versus the transcriptome 284) Figure , consist4 Figure 29 versusIn the study reported here we used RNA-Seq and the mRNA isoform identification tool SpliceSeq to investigate the contribution of AS to radiation-induced gene expression. At the whole genome level, the effects of radiation on AS have previously been reported for human lymphoblasts, fibroblasts  and periWhereas radiation-induced changes in the transcriptome may provide an indicator of radiation exposure , 21, it An additional difference between the alternative transcripts in the radiation-induced transcriptome and translatome involved the relationship to gene expression. In contrast to the transcriptome, a substantial percentage of the alternative transcripts detected in the radiation-induced translatome corresponded to genes whose expression was also modified, which suggests that the selective polysome association of the existing alternative transcripts has a quantitative effect on radiation-induced gene translation. The alternative transcripts in the radiation-induced translatome that do not overlap with gene expression, while perhaps not affecting translational efficiency, could qualitatively influence other aspects of protein function such as location and activity. With respect to the mechanism through which alternative transcripts are recruited to and away from polysomes, Sterne-Weiler et al. proposed that AS not only provides a process for increasing protein diversity, but also modifies the cis-regulatory structure of mRNAs resulting in an altered susceptibility to post-transcriptional regulatory processes . This scThe data presented here suggest that whereas radiation induces AS, it is the alternative transcripts present at the time of irradiation that may play a role radiation-induced translational control of gene expression and thus cellular radioresponse. Although the studies described here were conducted using a single human tumor cell line, given that radiation-induced translational control of gene expression is a fundamental component of cellular radioresponse, and that many of the changes in isoforms were also observed in another GSC line, the selective modification of the polysome-binding of existing alternative transcripts is likely to be a common response to radiation. This is supported by the data in Figure 2/5%O2. CD133+ NSC11 cells were isolated from neurosphere cultures by FACS and used as a source for the described experiments . After 15 minutes on ice, lysates were centrifuged , and the resulting cytosolic supernatant was layered onto a 10% to 50% sucrose gradient. Gradients were then centrifuged at 35,000 × g for 3 hours at 4°C and polysome-bound fractions were collected using an ISCO Density Gradient Fractionation System  with continuous monitoring based on A254. Polysome-bound RNA was collected corresponding to pooled fractions 4-12 obtained from sucrose gradient fractionation [Isolation of polysome-bound RNA was performed on 6 biological replicates initiated from different frozen stocks following the procedure described by Galban et al  with sliionation  and extrTotal RNA, from 6 biological replicates corresponding to the same replicates of polysome-bound RNA, was extracted from irradiated (2 Gy) or mock irradiated cells using TRIzol (Invitrogen) followed by the RNeasy Mini Kit (Qiagen). The integrity of the RNA was assured using a Bioanalyzer (Agilent).RNA-Seq was performed by the Center for Cancer Research Sequencing Facility in Frederick, Maryland. Briefly, between 100ng to 1μg of total RNA was used as the input for mRNA capture with oligo-dT coated magnetic beads. The mRNA was fragmented followed by random-primed cDNA synthesis. The resulting double-strand cDNA was used as the input to a standard Illumina library prep with end-repair, adapter ligation and PCR amplification to generate a sequencing ready library. The final library was then quantitated by qPCR before cluster generation and sequencing on the Illumina HiSeq2500 sequencer. The HiSeq Real Time Analysis software (RTA 1.18) was used for processing image files, the Illumina CASAVA_v1.8.4 was used for demultiplex and converting binary base calls and qualities to fastq format. The sequencing reads were trimmed of adapters and low quality bases using Trimmomatic (version 0.3), the trimmed reads were aligned to human hg19 reference genome (GRCh37/UCSC hg19) and Ensembl annotation version 70 using TopHat_v2.0.8 software. Given the aligned sequencing reads and a list of genomic features, counts of mapped reads for each gene were calculated using HTSeq . , and then data is log2 transformed .For gene expression analysis, RPKM reads were used for pairwise comparison between untreated control and irradiated groups for polysome-bound RNA or total RNA samples. This analysis was performed in R  with theribution . In all ribution . We set ribution  and are t method using 18s  as an internal control. Statistical significance (p ≤ 0.05) was determined using Student’s t-test with data expressed as the mean ± standard error.Complementary first-strand DNA was generated from irradiated and mock-irradiated total and polysome-bound RNA isolated separately from the RNA used for RNA-Seq, using the Applied Biosystems High-Capacity RNA-to-cDNA Kit  according to the manufacturer’s protocol. TaqMan Primers and probes for the specified genes , selected based on sequence homology to exon flanking regions, were used and PCR was performed using TaqMan RT-PCR kits , according to the manufacturer’s protocol. All assays (probes and primers) used are listed in ®, QIAGEN Redwood City, www.qiagen.com/ingenuity) for core expression analysis and evaluated using canonical pathway and network analyses with IPA default settings and a p-value ≤ 0.01.Genes exhibiting significant changes in alternative splicing or gene expression were submitted to Ingenuity Pathway Analysis (IPA


Top-down processes conveying contextual information play a major role in shaping odor representations within the olfactory system, yet the underlying mechanisms are poorly understood. The hippocampus (HPC) is a major source of olfactory top-down modulation, providing direct excitatory inputs to the anterior olfactory nucleus (AON). However, HPC-AON projections remain uncharacterized. In an effort to understand how hippocampal inputs are distributed within the AON, we systematically outlined their organization using anterograde and retrograde tracing methods. We found that AON-projecting hippocampal pyramidal neurons are located mostly in the ventral two-thirds of the HPC and are organized topographically such that cells with a ventral to intermediate hippocampal point of origin terminate, respectively, at the medial to lateral AON. Our neuroanatomical findings suggest a potential role for the HPC in the early processing and contextualization of odors which merits further investigation. Olfaction is the primary sensory modality employed to perceive chemical signals in the environment. Within the olfactory bulb (OB), neural input arriving from olfactory sensory neurons terminate in glomeruli onto the dendrites of mitral and tufted cells . Our results demonstrate a topographic gradient in the organization of hippocampal outputs to the AON, such that a ventral-to-intermediate point of origin in the CA1/Subiculum resulted in a medial-to-lateral point of termination at the AON.ad libitum access to food and water. All procedures were performed in accordance with the guidelines of the Canadian Council on Animal Care (CCAC) and the University of Toronto Animal Care Committee. Surgical procedures were performed on mice under general anesthesia with isoflurane and mounted onto a stereotaxic frame. The atlas of the mouse brain by Paxinos and Franklin  were used. Prior to surgery, mice were group housed in a temperature-controlled room on a 12 h light/dark cycle with Franklin  was used12 GC/ml) purchased from the Vector Core at the University of Pennsylvania and a total of 18 mice were used. Two groups of six mice received unilateral infusions of 0.3 μL of channelrhodopsin-2 (ChR2) into the dorsal HPC  or ventral HPC . One group of six mice received a simultaneous co-injection of AAV2/5-hSyn-hChR2-mCherry (~1012 GC/ml) and AAV2/5-hSyn-hChR2-eYFP into the dorsal (AP: −2.70 ML: ±2.20 DV: −2.00) and ventral HPC using a counterbalanced combination.For anterograde tracing experiments, AAV2/9-hSyn-hChR2(H134R)-eYFP , or lAON , respectively. A lower injection volume was used compared to anterograde tracing experiments to limit spread of the infusion outside the boundaries of the AON and neighboring AON subregions. All infusions were made by means of pressure ejection at a rate of 0.1 μL/min through a cannula connected by Tygon tubing to a 10 μL Hamilton syringe  mounted onto an infusion pump. Following each infusion, the cannula was left in place for an additional 20 min before retraction to limit the spread of the tracer.For the retrograde labeling experiments, a total of twenty four mice were used. Three groups of eight mice received a unilateral injection of 0.2 μL of red fluorescent retrobeads  into the mAON , dAON , followed by 4% paraformaldehyde in phosphate buffer. Brains were extracted and postfixed overnight at 4°C and subsequently cryoprotected with PBS containing 30% sucrose. Coronal 40 μm thick sections were obtained using a cryostat . The sections were slide-mounted, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, and coverslipped with Aquamount . The wide-field fluorescent images were captured with a 4× objective on a fluorescent microscope . eYFP and red fluorescent retrobeads signals were captured using an U-MWIBA3 filter cube  and an U-MWIG3 filter cube , respectively. Confocal images were captured through a Quorum spinning disk confocal microscope (Zeiss) using a 20× objective lens and were subsequently analyzed with Volocity Software (Perkin Elmer). eYFP and red fluorescent retrobeads signals were excited with 491 and 561 nm laser, respectively. Adobe Photoshop CS6  was used to adjust the brightness and contrast of representative sections.Retrobead-positive cell counting was performed using the cellSens software . The HPC was divided into three portions such that the dorsal one-third consisted of the anterior portion of the HPC prior to its transition into its longitudinal form in coronal sections (approximately 2.46 mm posterior to Bregma). The remaining ventral two-third of the HPC was further divided into intermediate and ventral HPC separated by the rhinal fissure (rf). Calibration parameters were established using randomly chosen tissue sections and were selected such that the threshold for cell diameter was high enough to exclude the majority of falsely identified cells (noise). Calibration parameters were maintained for consistent counting of all tissue sections. Tissue sections from three animals in each of the medial, dorsal, and lateral-AON injected groups were analyzed. The density of retrobead-positive cells found across nine coronal sections (number of labeled cells in region of interest/area of region) for each subject, and mean densities for each group were calculated.To examine the hippocampal innervation pattern of the AON, we first performed antero- grade tracing of hippocampal projections using virally delivered-ChR2 as an anterograde tracer . The spread of injected retrobeads was restricted to the targeted AON subregions  that are uptaken at presynaptic axon terminals. Retrobeads were microinjected unilaterally into the medial, dorsal, or lateral aspects of the AON in separate cohorts of mice ( Figures , which a Figures . In all Injection of retrobeads into the mAON yielded labeled cells found in their greatest density near the ventral-most part of the CA1/Subiculum subregion Figures , 3. ThesThe present study has revealed a topographic gradient in HPC-AON projections such that the ventral-most part of the HPC innervates most heavily the mAON, and progressively more dorsal parts of the HPC innervate increasingly more lateral positions at the AON as illustrated in our model Figure . This paOur tracing results are largely consistent with previous studies which examined the downstream targets of CA1 projection neurons (Swanson and Cowan, The functional connection between the HPC and the olfactory system is growing increasingly apparent. The HPC has been implicated in different aspects of odor processing, including olfactory pattern separation, the formation of context-odor associations, coding of the temporal sequence of a series of odors, and olfactory learning and memory (Eichenbaum, The functional implication of the gradient in the HPC-AON connection remains unclear. Notably, a similar topographic organization is known to exist in other extrinsic targets of the HPC including the amygdala, nucleus accumbens, and lateral septum (Groenewegen et al., Ultimately, the topographic organization of the HPC-AON pathway offers a unique model for investigating how information originating from distinct hippocampal subregions contribute to olfaction. In an initial effort to elucidate the role of hippocampal inputs to the AON, we recently showed that the vCA1-mAON pathway can modulate olfactory sensitivity (Aqrabawi et al., AJA and JCK carried out the study conceptualization and experimental design and wrote the manuscript. AJA performed the surgical procedures.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.