Mycobacterium tuberculosis (MTB) within a patient has caused great concern as it might complicate antibiotic treatment and cause treatment failure. But the extent of genetic heterogeneity has not been described in detail nor has its association with heterogeneous treatment response. During treatment of a subject with MDR-TB, serial computed tomography (CT) scans showed this subject had six anatomically discrete lesions and they responded to treatment with disparate kinetics, suggesting heterogeneous MTB population may exist. To investigate this heterogeneity, we applied deep whole genome sequencing of serial sputum isolates and discovered that the MTB population within this patient contained three dominant sub-clones differing by 10 ~ 14 single nucleotide polymorphisms (SNPs). Differential mutation patterns in known resistance alleles indicated these sub-clones had different drug-resistance patterns, which may explain the heterogeneous treatment responses between lesions. Our results showed clear evidence of branched microevolution of MTB in vivo, which led to a diverse bacterial community. These findings indicated that complex sub-populations of MTB might coexist within patient and contribute to lesions’ disparate responses to antibiotic treatment.Genetic heterogeneity of  In this study, taking advantage of the high resolution of deep whole genome sequencing together with molecular typing method, we tracked the population genetic dynamics of MTB population in serial sputum samples and characterized this phenomenon in more detail. Meanwhile, we provided the possible correlation between lesions’ disparate responses and heterogeneous bacterial population.It is now commonly accepted that the MTB population within individual tuberculosis (TB) patients can be more heterogeneous than was traditionally thought1234512363939The subject described in this study had a previous history of tuberculosis and was thought to have been successfully treated as a child about 8 years ago. Upon presenting to the hospital with new signs and symptoms of tuberculosis, computed tomography (CT) images revealed bilateral disease with at least six spatially isolated lesions before treatment recommenced , one of para-amino salicylic acid (PAS). During the treatment, the sputum bacillary load as indicated by sputum smear score was low from week 0 to week 4 but showed a dramatic increase at week 6  were detected in 12 genes/regions, which could be either hitchhiking or compensatory mutations. These results provided a possible explanation for the disparate treatment response of different lesions that MTB sub-populations reacted differentially to the drug treatment. Based on the sub-clone specific SNPs, we estimated the percentage of the three sub-clones at different time points through deep sequencing data  and INH-compensatory mutation (ahpC promoter −74 G-T). Then, sub-clone B acquired an EMB-resistant mutation embB V502A and a mutation in murA-rrs intergenic region, while sub-clone C acquired another mutation also in murA-rrs intergenic region, which was predicted to be associated with drug resistance13As all other 552 SNPs were still presented with frequency of 100% at week 6, the only difference between the three sub-clones were the 19 SNPs identified above . MoleculIn this study, we studied in detail the radiologic response of a single patient with extensive, bilateral TB and showed that the response to treatment in individual lesions was disparate based upon radiologic changes. By deep whole genome sequencing we were able to trace the genetic dynamics of MTB within this subject during treatment. There were three individual cavitary lesions in direct communication with the airways and we identified three branched-evolved sub-clones in this patient with a genetic distance between them of 10 ~ 14 SNPs. Although the three sub-clones were only simultaneously detected with comparable percentages at week 6, they were still likely present throughout the treatment but may have not been identified at the same time due to several potential factors such as limitations imposed by sputum sampling approaches, different degrees of cavitation and communication with the bronchi, etc. A likely explanation for this observation is that the sub-clones resided in different lesions and that sub-clones A and B gained access to the airways as treatment progressed. This hypothesis is supported by the coincidentally observed increase of bacilli load in sputum at week 6 when the three sub-clones were detected simultaneously and previous observations of lesions containing heterogenic populations11in vivo and another is mixed infections/re-infection. We think the former is more plausible based on the following reasons. First, in the previously reported polyclonal mixed infections/re-infection cases, the strains that infected the same patient showed distinct molecular typing patterns or differed in hundreds of SNPs1617181921rpoB mutations and mutations in murA-rrs intergenic region) further suggest the selective pressures on them were quite similar. Thus, considering this patient had a TB history, we speculate the three sub-clones evolved in parallel during the last treatment episode and persisted for years till relapse.There are two possible explanations for the coexistence of the three sub-clones in a single individual. One is microevolution of a single clonal founder strain in vivo might be frequent rather than rare. Tens of MTB genomic regions were newly identified to be under positive selection in drug-resistant strains as the related mutations arise multiple times independentlymurA-rrs intergenic region (annotated as rrs promoter) were statistically associated with drug resistancemurA-rrs mutations identified in sub-clones B and C actually located in the coding sequence of a small regulatory RNA mcr3As sub-clone A and B had actually diverged before sub-clone C and sub-clone C was firstly detected at week 0, we can infer the three sub-clones already existed before treatment started. The last treatment of this subject was more than 8 years ago and we speculate a highly heterogeneous population persisted within the patient after childhood treatment was completed. This case of branched microevolution, and the few others that have been documented389et al. and further reinforces the argument that this phenomenon might not be rare in clinic. Molecular genotyping of the three sub-clones showed the same VNTR pattern, supporting our conclusion that the three sub-clones found in this subject were not due to mixed infections despite the 14 SNPs genetic distance. More recently, phenotypic heteroresistance among HIV-infected patients who are being treated for MDR-TB has been associated with poor outcomes and longer time to culture conversion25in vivo would provide us valuable information for adjusting treatment regimens.Historically, five or fewer SNPs have been considered as a threshold to determine epidemiologically clustered strains, and pairs that differed by 6 to 12 SNPs were thought to be indeterminate for linkagesOver the last 3 years, an unprecedented number of publications focusing on the intratumor genetic heterogeneity revealed that branched evolution was ubiquitous during progression of all tumors2728293334However, our study still has several limitations: 1) Although the observed differential drug susceptibilities of sub-clones might be a possible explanation for the disparate responses of different lesions, there are still some other factors that could contribute to this phenomenon such as the presence of cavities, prior scars, proximity to bronchi, tissue penetration of the drugs, etc. 2) Given that the detection of MTB sub-populations were from pooled sputum samples  and not surgically resected samples, it is not possible to directly determine whether there was a differential distribution of sub-populations in different lesions. It therefore remains possible that the microevolution of MTB within the host took place in a single cavitary lesion.in vivo, which might contribute to the disparate treatment response observed by radiography. The remarkable genetic heterogeneity between the sub-clones provides interesting clues about the molecular events during the evolution of drug-resistance and highlights that branched microevolution of MTB within patient can lead to a diverse community.In conclusion, we provided a detail depiction of the complexity of MTB population The study to investigate the range of tuberculosis presentation and treatment  conducted in Henan Provincial Chest Hospital (HPCH) was approved by the HPCH and National Institute of Allergy and Infectious Diseases institutional review boards and subjects gave written consent for the study. The methods of this study were carried out in accordance with the approved guidelines and written informed consent was obtained from the subjects prior to the study. The female subject in this study was 22 years old when she enrolled in the study and reported that she had been treated for tuberculosis 8 years earlier and that she neither smoked tobacco nor consumed alcohol. Intensive treatment with 7 drugs (HRZE+AMK/LFX/PAS) was prescribed from week 0 to week 8. Drug susceptibility testing (DST) of the initial isolate showed resistance to INH/RIF/SM/AMK but susceptibility to EMB. From week 8, rifapentine (RPT) was added into the treatment regimen after the DST report became available.SRP057886.We collected serial sputum samples from TB patients at 7 time points . For each time point, three sputum samples were collected. Sputum smear microscopy, L-J culture and MGIT culture were preformed from each sputum sample. At each time point, if culture positive, we selected one L-J culture for whole population deep sequencing. That is, we scraped all the colonies on L-J medium and extracted genome DNA through CTAB method described beforescythe (https://github.com/ucdavis-bioinformatics/scythe) and sickle (https://github.com/ucdavis-bioinformatics/sickle) were used for trimming reads, bwasamtools was used for SNP calling. The reference genomes H37Rv (GenBank accession NC_000962.3) and CCDC5079 (GenBank accession NC_021251.1) were used as templates for variants calling. For unfixed SNPs calling, we additionally used lofreqvarscan2A pipeline used for calling fixed/unfixed SNPs was previously introducedHow to cite this article: Liu, Q. et al. Within patient microevolution of Mycobacterium tuberculosis correlates with heterogeneous responses to treatment. Sci. Rep.5, 17507; doi: 10.1038/srep17507 (2015).


Gender and age have been found to affect adults’ audio-visual (AV) speech perception. However, research on adult aging focuses on adults over 60 years, who have an increasing likelihood for cognitive and sensory decline, which may confound positive effects of age-related AV-experience and its interaction with gender. Observed age and gender differences in AV speech perception may also depend on measurement sensitivity and AV task difficulty. Consequently both AV benefit and visual influence were used to measure visual contribution for gender-balanced groups of young (20–30 years) and middle-aged adults (50–60 years) with task difficulty varied using AV syllables from different talkers in alternative auditory backgrounds. Females had better speech-reading performance than males. Whereas no gender differences in AV benefit or visual influence were observed for young adults, visually influenced responses were significantly greater for middle-aged females than middle-aged males. That speech-reading performance did not influence AV benefit may be explained by visual speech extraction and AV integration constituting independent abilities. Contrastingly, the gender difference in visually influenced responses in middle adulthood may reflect an experience-related shift in females’ general AV perceptual strategy. Although young females’ speech-reading proficiency may not readily contribute to greater visual influence, between young and middle-adulthood recurrent confirmation of the contribution of visual cues induced by speech-reading proficiency may gradually shift females AV perceptual strategy toward more visually dominated responses. Behavioral research has reported gender e.g.,  and age Speech-reading may be narrowly defined as the ability to recognize different speech sounds based on visual cues from lip and facial movements. In general, previous research suggests that females are better at speech-reading than males, a difference which has been attributed to females being more active gazers than males e.g., . HoweverResearch has indicated that the ability to identify visual speech  and the influence of visual cues on AV perception should be differentiated e.g., . While IM = 24.5 years) and 17 older adults (M = 68.5 years), where the participants were primarily females (24 out of 34). Interestingly, ERP data revealed that older adults had a more pronounced facilitation of neural responses on AV speech trials than younger adults, interpreted as older adults being more able to benefit from visual speech cues in AV speech perception  and older adults (M = 68.3 years) with normal or corrected-to-normal vision. Whereas the young and older adults had similar scores on speech-reading and AV benefit for clear speech, older adults had significantly lower scores on both measurements for visually degraded speech. Whether these age-related effects are present already in middle-adulthood is not known, but, similar to auditory speech perception in noise  and normal hearing middle-aged adult (51–63 years) for competing speech, music and steady noise maskers . Althougin noise , these fTo the authors’ knowledge no studies have directly investigated the interaction of age and gender on AV speech perception between young and middle adulthood. An influence of age on gender differences would presuppose some experience-dependent flexibility in AV speech perception and research has suggested that both biological  and enviGenerally the behavioral research on AV speech perception reports relatively few and inconsistent findings on age and gender differences in the contribution of visual speech, especially for gender. Although gender differences in speech-reading are quite frequently reported e.g., , few stuThe current study explores the interaction of age and gender using two measurements of visual contribution to AV speech perception: AV benefit and visual influence. AV benefit is calculated as the difference between correct responses in the AV congruent condition and in the corresponding AO condition, whereas visual influence is calculated as the difference between correct responses in the AO condition and the auditory responses in the AV incongruent condition e.g., . Based oA mixed repeated measures design was used to assess speech-reading, AV benefit and visual influence by young and middle-aged males and females using stimuli consisting of AO, VO, AV congruent and AV incongruent stop-vowel syllables produced by eight different talkers, varying in stop place of articulation (POA) and noise type.M = 25 years, SD = 2 years), 10 young females , 10 middle-aged males , and 10 middle-aged females . The study was registered by the Norwegian Social Science Data Services, and all participants gave written consent prior to the experiment. Participants were all highly educated and naive to AV speech perception experiments. Prior to the experiment, hearing was assessed using a standard pure tone audiometry procedure  did not significantly differ from young females , nor did middle-aged males  from middle-aged females . Vision was assessed with a self-report questionnaire and those participants who reported reduced vision wore prescription glasses or contact lenses during the experiment.Forty Norwegian native speakers were recruited at the Norwegian University of Science and Technology (NTNU), including 10 young males . The sound from the external microphone went via a RME FIREFACE 400 soundcard to an Apple Macintosh G5 computer, where Praat version 5. 1  was usedTable 1). Each syllable was repeated eight times. The video file was segmented into separate syllables, using the software MPEG Streamclip 1.9.2 and the audio files from the external microphone were segmented with Praat version 5.1  were selected based on the highest additive audio and visual ratings (see range = 272–537 ms) measured from the consonant release to the end of the vowel.All audio syllable segments were adjusted to the same unweighted sound pressure level in Praat. The average length of the auditory syllables was 400 ms  was first synchronized with the same syllable from the camera microphone in Logic Pro 8.0.2. Then the video clip’s original auditory syllable recording was replaced by the corresponding syllable from the external microphone in AVID Media Composer. The incongruent stimuli were produced in the same manner; except that the video clip’s original auditory syllables were substituted with external auditory syllables that differed in POA. The video clips were cut to a total length of 1520 ms, ensuring that the consonant release of all syllables was initiated during the 16th frame (between 640 and 680 ms). The resulting congruent and incongruent AV syllables constituted the quiet condition of the experiment.As shown in The study employed two types of auditory maskers: babble and white noise. Whereas babble noise occurs more often in natural environments  at ∼70 cm distance, wearing AKG K271 stereo closed dynamic circumaural studio headphones. The sound level was fixed at 68 dBA .Table 1). Each block contained two productions of each syllable by each of the eight talkers. With three audio backgrounds each AV-stimulus block contained 384 stimuli and each AO stimulus block contained 192 stimuli. The VO stimulus blocks contained 64 stimuli. Stimuli were independently randomized for each repetition.Participants were presented six stimulus blocks: two repetitions of an AV block (AV-congruent and AV-incongruent intermixed), two repetitions of an AO block, and two repetitions of a VO block  best corresponded to the syllable perceived. Because of possible ambiguity with incongruent AV signals, the participants were told that no answer was wrong. To ensure that the participants received both auditory and visual input, frequent between-trial reminders instructed the participants to look at the talker’s face throughout the duration of each clip. The experiment took ∼1 h.Table 2, in the AO condition no significant age  or gender  differences were found. Overall in the AO condition, the percentage of correct responses by young males  and females  was similar to middle-aged males  and females . As expected a main effect was found for stop consonant , where females  had significantly more correct POA responses than males . No significant effect was obtained for age  or interaction between age and gender . As expected a significant effect of stop consonant was obtained , and in line with previous research, labials resulted in more correct responses than velars , between subject variables were age and gender and the dependent variable was percentage correct POA responses regardless of voicing, since visual discrimination is difficult for consonants belonging to the same viseme class . As illurs e.g., . Stop coTable 2, no significant age  or gender  differences were obtained for AV congruent stimuli. Overall, the percentage of correct responses for young males  and females  was almost the same as for middle-aged males  and females . Main effects were found for stop consonant  and background , but neither interacted significantly with age or gender.Audio-visual congruent data were analyzed with a repeated measures ANOVA where within subject variables were background  and stop consonant , between subject variables were age and gender and the dependent variable was percentage correct responses. As shown in The high percentage of correct responses for the AO stimuli in the quiet condition implies that the auditory stimuli are good tokens of their respective categories. The percentage of correct responses in the AO condition declines sharply as noise is introduced, but with the visual cues offered in the AV-congruent condition, participants have near perfect responses in noise. Along with the high percentage correct in the VO condition, this indicates that the visual stimuli are good tokens of their respective categories. Furthermore, the high percentage of correct responses found for the AV congruent stimuli makes it unlikely that differences for the AV incongruent stimuli are due to chance responses.Table 2, a significant main effect was obtained for gender , where females  had significantly more correct POA responses than males . No significant effect was obtained for age  or interaction between age and gender . As expected a significant effect of stop consonant was obtained , and in line with previous research, labials resulted in more correct responses than velars , between subject variables were age and gender and the dependent variable was percentage correct POA responses regardless of voicing, since visual discrimination is difficult for consonants belonging to the same viseme class . As illurs e.g., . Stop coTable 2, no significant age  or gender  differences were obtained for AV congruent stimuli. Overall, the percentage of correct responses for young males  and females  was almost the same as for middle-aged males  and females . Main effects were found for stop consonant  and background , but neither interacted significantly with age or gender.Audio-visual congruent data were analyzed with a repeated measures ANOVA where within subject variables were background  and stop consonant , between subject variables were age and gender and the dependent variable was percentage correct responses. As shown in The high percentage of correct responses for the AO stimuli in the quiet condition implies that the auditory stimuli are good tokens of their respective categories. The percentage of correct responses in the AO condition declines sharply as noise is introduced, but with the visual cues offered in the AV-congruent condition, participants have near perfect responses in noise. Along with the high percentage correct in the VO condition, this indicates that the visual stimuli are good tokens of their respective categories. Furthermore, the high percentage of correct responses found for the AV congruent stimuli makes it unlikely that differences for the AV incongruent stimuli are due to chance responses.Audio-visual benefit implies the ability to correctly encode and integrate visual speech cues during AV speech perception, resulting in improved identification scores compared to unimodal identification scores e.g., . AV beneF = 1.44, p = 0.24, η2 = 0.04, power = 0.22] or gender  effects were obtained for AV benefit. Young males  had similar AV benefit as young females  and middle-aged males  had similar AV benefit as middle-aged females .Audio-visual benefit was operationalized as the difference between percentage correct POA match responses for AV congruent stimuli and percentage correct POA match responses for AO stimuli with the corresponding auditory syllable . The datVisual influence denotes the degree to which a perceiver relies on input from the auditory and visual modalities in AV speech perception. Visual influence can be described by the difference between the auditory accuracy in the AO condition and the percent auditory responses in the AV incongruent condition e.g., . ContrarTable 1) and the participants, responded with the syllable alternatives /ba/, /da/, /ga/, /pa/, /ta/ and /ka/. Because members of the same viseme class are difficult to discern visually , young females , middle-aged males  and middle-aged females .For the incongruent Ap-values from all post hoc analyses were collectively adjusted using Bonferroni–Holm corrections  and background, the between subject variables age and gender, with percent of visually influenced responses as the dependent variable. The analysis revealed significant main effects for AV incongruent structure , background , and gender . Although no significant main effect was found for age , a significant interaction effect between age and gender was obtained . As Figure 1 depicts, post hoc analyses of the interaction between age and gender revealed that middle-aged females had significantly more visually influenced responses than middle-aged males , young males , and young females . Middle-aged males’ visual influence was similar to that of young males and young females, for which no significant gender differences were obtained. Because no gender differences were obtained for the young adults the following analyses focus on the middle-aged adults.The visual influence data calculated using Formula [1] and [2] were analyzed with two repeated measures ANOVAs and rections . A firstpost hoc comparisons between middle-aged males and females for the different AV incongruent structures and different backgrounds were conducted. As Figure 2 shows, with the exception of the visual influence of /ka/  in white noise, middle-aged females consistently had more visually influenced responses than middle-aged males for all AV incongruent structures and all backgrounds. Related to the effect of noise, the gender difference in visually influenced responses is comparable across backgrounds for the AbaVga stimuli, slightly more pronounced in noise for the AgaVba and AkaVpa stimuli, and most pronounced in quiet for the ApaVka stimuli. Hence, although there is a tendency toward more significant gender effects in noise across the different AV incongruent structures, the notion that gender differences in AV speech perception would be more observable when the AV task difficulty is increased by auditory noise is generally not substantiated by the data.To assess the consistency of the gender effect for middle-aged adults, F = 21.72, p < 0.001] and perceiver gender , and as Figure 3 depicts, middle-aged females consistently gave more visually influenced responses than males for all talkers. Post hoc comparisons only revealed significant perceiver gender differences for female talkers. To follow up on this finding, talker intelligibility in the AO and VO conditions were analyzed, and in accordance with previous research  were more auditorily intelligible than male talkers , whereas no significant difference was obtained for visual intelligibility . Further, the middle-aged male and female perceivers had very similar auditory identification scores for both the male  and the female talkers . Most importantly, results revealed the clear general pattern across the different talkers, with middle-aged females consistently giving more visually influenced responses compared to middle-aged males.To further investigate the resilience of the perceiver gender differences in visual influence, a second repeated measures analysis assessed middle-aged males and females’ percentage visually influenced responses in AV perception for the eight different talkers, collapsed across AV incongruent structure and background. Significant main effects were obtained for talker [research  results 2 < 0.25; Table 3, the current study could lack statistical power for detection of small effects (0.01 < η2 < 0.09), that is, effects of variables that explain between 1 and 9 percent of the variation in the dependent variable. Since the study employed a relatively small sample size (N = 40), post hoc power analyses  gender differences in the use of visual cues in AV speech perception are negligible in young adulthood and/or (2) sufficiently sensitive measurements of visual contribution and sufficiently demanding tasks are necessary for observable gender differences to emerge. The current findings are more in line with the notion of negligible gender differences in the use of visual cues in AV speech perception in young adulthood, since inclusion of talker variability as well as babble and white noise did not lead to observable gender differences for either AV benefit or visual influence for young adults. These findings are inconsistent with the results by The most notable observation across different talkers and consonants is a clear general pattern showing that middle-aged female perceivers gave more visually influenced responses than middle-aged males. Those middle-aged females consistently showed greater visual influence for AV speech could not simply be explained by gender differences in speech-reading proficiency coming to the fore as hearing acuity decreases. Differences in hearing levels between young and middle-aged adults were relatively small and both clearly inside the boundaries of normal hearing . One mayFor all groups, the percentage of AO syllables correctly identified in babble and white noise suggests that syllable identification could be substantially improved with visual speech cues available. Whereas these AO scores suggest that in noise all groups have similar perceptual incentive to shift toward more visually influenced responses, the results obtained for the AV stimuli show that middle-aged females gave considerably more visually influenced responses than the other groups. Whereas this special pattern of visual influence results for middle-aged females is difficult to explain by group differences in hearing acuity, AO performance, or in the noise induced perceptual incentive to make a shift toward more visual responses, the difference between young and middle-aged females may suggest that age-related AV experience may contribute to altering AV perceptual strategy. However, the lack of main effects of age in the contribution of visual cues in AV speech perception suggest that age-related changes in AV speech experience alone are insufficient to change the AV perceptual strategy. The study did not reveal main effects of age for speech-reading, AV benefit, or visual influence, even when the AV task difficulty was increased by noise. Whereas, the power analyses revealed that increasing the sample size might have rendered some of the small effects related to age significant, the means and effect sizes suggest that such small age effects are of negligible scientific interest. However, it must be stressed that power analyses may not be fully sensitive for a population that is known to be heterogeneous and that a comparatively larger sample size would be needed to ascertain the conclusions. Nevertheless, for visual influence in particular, such a small potential age difference seems to be a result of the performance of the middle-aged females exclusively, as the middle-aged males performed similarly to the young adults. The general conclusion related to age therefore remains that the age-related differences in the use of visual speech previously observed between young and older adults (over 60 years; e.g., That the current study found gender differences in visual influence for middle-aged adults and replicated the frequently reported finding that females, independent of age, are better speech-readers than males e.g.,  suggestsThe 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.


Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd  to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80–90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads , current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis.Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus  Batrachochytrium dendrobatidis (hereafter Bd) in the late 1990s [Natural history collections are becoming increasingly important for ecological and conservation research , 2, facite 1990s  providedte 1990s , 12); hote 1990s . The comte 1990s  remains The “spreading pathogen” hypothesis  posits tHistopathology has been used successfully to identify chytridiomycosis in live and fresh-dead amphibians , 10, 31 The development of PCR  and quanBefore 2011, attempts to detect Bd DNA from formalin-fixed specimens were relatively unsuccessful (reviewed in Richards-Hrdlicka ). Cheng The ability to detect Bd DNA using qPCR can be moderated by several factors, including individual pathogen load. The infection intensity, or Bd load, on an amphibian is commonly measured by taking a skin swab following a standard swabbing protocol . Using qIn addition to the importance of understanding Bd detection success under a variety of infection loads, both length of time in formalin and the pH of the solution can affect the ability to detect DNA after fixation of a specimen in formalin . DespiteHere, we used qPCR to characterize Bd loads on live frogs then fixed them in formalin, preserved them in ethanol, and re-swabbed them three years later to assess the ability to successfully detect Bd on frogs of known infection status using qPCR. We also compared the efficacy of the PM , 39 and Lithobates catesbeianus) is not a legally protected species in the state of California.All specimens were collected in adherence with protocols approved by the University of California, Santa Barbara Institutional Animal Care and Use Committee (266) and permits issued by the California Department of Fish and Wildlife (SC-4436). The non-native American bullfrog  for Bd in the wild  using sterile dacron swabs  following Hyatt et al. , with fiBecause Bd loads detectable on moribund frogs are the most accurate within 24 hours of death , bullfroTo assess Bd detection after formalin fixation and ethanol storage, frogs were re-sampled multiple times for qPCR. We used five separate swabbing events to test the following optimizations, as outlined in To compare the efficacy of subsequent Bd DNA detection using qPCR, DNA from individual swabs were extracted by two separate extraction methods, PM and MN. Although phenol:chloroform may be the extraction method of choice in museum specimens when full animal tissues are being sampled, phenol:chloroform is not demonstrated to be superior when extracting low copy number DNA  in samples with a high DNA background  , so it wIn the first three PM extractions  were extracted with MN following Richards-Hrdlicka , with miA sample was considered positive when any replicates in the sample exhibited a logarithmic curve in the amplification profile that crossed the ΔRn threshold (set at 0.1 ). When sWe used generalized linear mixed models (GLMMs) with a binomial response and a logit-link function to test whether pre-preservation Bd load, DNA extraction method (PM or MN), and individual live frog mass were significant predictors of Bd detection (success or failure) after formalin fixation using duplicate results from swab events D and E. For this analysis, we used only data for individual frogs that we had individual mass recorded (n = 52). We square root-transformed both the live ZE and post-preservation ZE data to facilitate data analysis. We treated initial Bd load, individual mass, and extraction protocol as fixed effects and the identity of each individual frog as a random effect. Treating individual frogs as a random effect allows us to test for differences among extraction protocol and initial load (the main predictors of interest), while accounting for the fact that individual frogs were necessarily swabbed multiple times to collect samples for separate DNA extractions. We used the “MuMIn” package in R  to avera10-transformed ZE data . To compare results of Bd detection success and post-preservation ZE (log10-transformed) across all swab events and a subset of them , using log2 = 0.067, df = 1, p = 0.796). Within Event D, there was no significant difference in ZE values  or recovery rate  when the Genereleaser was used. Within Event C, ZE was significantly reduced when extracts were treated with Genereleaser ; yet there was no significant difference in recovery rate with the treatment . When swabs were rehydrated with TE during the extraction step (Event C), neither ZE  nor recovery rate were affected .None of the optimizations conducted on PM-extracted swabs increased Bd detection success or ZE values. Reducing the PM extract dilution from 1:10 to 1:5  prior to and after formalin fixation for either the MN or the PM extractions  in cases of equivocal or enigmatic results. Yet, because of the uneven distribution of chytridiomycosis on amphibian skin , histoloBased on the results of this study, we make the following recommendations to maximize Bd DNA recovery rates from formalin-fixed museum specimens using qPCR: 1) Use MN or a similar spin-column based DNA extraction method, and/or one specifically designed for formalin-fixed sample types. Like previous research , 61, 62,DNA detection from formalin-fixed tissues is challenging . Establi


Stromal microenvironment increases tumor cell survival, proliferation and migration, and promotes angiogenesis. In order to provide comprehensive information on the stromal heterogeneity of diverse tumors, here we employed the microarray datasets of human invasive breast and prostate cancer-associated stromals and applied Gene Set Enrichment Analysis (GSEA) to compare the gene expression profiles between them. As a result, 8 up-regulated pathways and 73 down-regulated pathways were identified in the breast tumor stroma, while 32 up-regulated pathways and 18 down-regulated pathways were identified in the prostate tumor stroma. Only 9 pathways such as tryptophan metabolism were commonly up or down regulated, but most of them (including ABC transporters) were specific for these two tumors. Several essential tumors stromal marker genes were also significantly identified. For example, CDH3 was significantly up-regulated in the stromals of both breast and prostate tumors, however EGFR was only significantly down-regulated in the stromal of breast tumor. Our study would be helpful for future therapeutic and predictive applications in breast and prostate cancers. Cancer is not a single disease but includes numerous subtypes, each of which has its distinct histopathological and biological features . Cancer Although certain genetic similarities are shared between the primary and metastatic tumor cells, there are also some additional mutations in metastases. For both prostate cancer , 7 and bIt is now widely recognized that cancer progression is not exclusively regulated by intra-tumor heterogeneity but also depends on the heterogeneity of tumor microenvironment, in which the stroma compartment plays an important role . This suThe tumor stroma is the complex arrangement of various stromal cells and extracellular matrix, which act in a coordinated manner to regulate cell function and maintain overall tissue homeostasis. In order to maintain homeostasis, the host tissue stroma interacts with carcinoma in a process similar to the tissue remodeling in wound repair , 13. ThiOne important reason why gene expression of cancer stroma has potential for cancer treatment and prediction is that cancer stroma maintains a normal genotype, because stromal reactions in response to cancer epithelium mostly only alter gene expression, without drastic and stochastic genomic changes , 16. In In the present study, we employed and reanalyzed the published microarray datasets of human breast and prostate cancer stroma from the public database library of GEO. Gene Set Enrichment Analysis (GSEA) was applied to compare the gene expression profiles between these two different invasive cancer types, especially in an effort to indicate the degree of stromal heterogeneity between them and identify candidate stromal gene expression signatures relevant to cancer progression. GSEA has the advantage to highlight genes weakly connected to the phenotype through pathway analysis which may be difficult to detect by using classical univariate statistics -23. ThroAccording to the approach of differentially expressed gene analysis (DEGA) studied by Planche et al., 643 and 319 differentially expressed genes between tumor and normal stroma were identified for breast cancer and prostate cancer, respectively shown in  24]. Th. Th24]. Firstly, the common GSEA method was applied to the stromal regions of human breast and prostate tumors. For individual analysis, we obtained the significant pathways in each dataset, which were summarized in Figure In addition, there were several tissue-specially identified pathways. 2 pathways were up-regulated in breast tumor stromal and down-regulated in prostate tumor stromal, such as the pathways of taste transduction and ABC transporters Table . ATP-binMoreover, 10 pathways were down-regulated in breast tumor stromal and up-regulated in prostate tumor stromal, such as oxidative phosphorylation. Cancer is a heterogeneous class of diseases, each of which has its own metabolic characteristics, even if each of the tumor, which includes different cell constituted a difference in the mode of metabolism . Owing tOur analysis revealed that 447 up-regulated genes and 449 down-regulated genes were common in the stromals of both tumor types, which were shown in Figure P-cadherin, a classical cadherin encoded by the CDH3 gene , are engIt has been long established that the epidermal growth factor receptor (EGFR) and the EGF-family of peptide growth factor play an important role in the pathogenesis and progression of different carcinoma types. The proteins of EGF ligand/receptor system are found to be frequently expressed in the majority of human carcinomas. In this study, EGFR was identified to be heterogeneously regulated in breast and prostate cancer. The expression of EGFR was significantly down-regulated in breast tumor stromal (p=2.70E-03), but remained the same level in prostate tumor stromal compared to the normal stromal Figure . The binThe critical roles played by protein ubiquitination in various biological processes including cell proliferation, cell cycle, apoptosis, signal transduction, while its deregulation contributes to tumor initiation and progression , 48. UbiThe claudin (CLDN) genes encode a family of highly related proteins important in tight junction formation and function. Recently, it has become obvious that CLDN gene expression is frequently altered in various cancers , 57. Spewww.ncbi.nlm.nih.gov/geo/). Data sets were reanalyzed if they met the following conditions: (1) the data were genome-wide, (2) comparison was conducted in the stromal regions between breast tumors and prostate tumors, and (3) complete microarray raw or normalized data were available. The data set of GSE26910, contributed by Paolo Provero, was finally chosen for our re-analysis [The gene expression proﬁling studies related to stromal regions of human breast and prostate cancer were searched in GEO . The annotation of signiﬁcant genes in each pathway was performed by using the biomaRt package (http://www.biomart.org/) BioMart ver. 0.8 rc3 (version 0.8 of release candidate 3). Next, for each signiﬁcant pathway, through hierarchical clustering with Euclidean distance, clustering on groups or genes was performed based on the identiﬁed genes' expression.Here, our gene set enrichment analysis was performed on each study above to identify signiﬁcantly related pathways and genes to either stromal regions of breast cancer or stromal regions of prostate cancer by using Category package in Bioconductor ver. 2.6.0 . GSEA ishttp://dire.dcode.org/) were also used, which is based on the Enhancer Identiﬁcation (EI) method, to predict common regulatory elements (REs) for our input genes that have a cofunction in each identiﬁed signiﬁcantly related pathway [A web server called DiRE (Distant Regulatory Elements of coexpressed genes,  pathway . It pred


Aedes aegypti mosquitoes by allowing them to feed on DENV2-infected patients. We then performed whole-genome deep-sequencing of human- and matched mosquito-derived DENV samples on the Illumina platform and used a sensitive variant-caller to detect single nucleotide variants (SNVs) within each sample. >90% of SNVs were lost upon transition from human to mosquito, as well as from mosquito abdomen to salivary glands. Levels of viral diversity were maintained, however, by the regeneration of new SNVs at each stage of transmission. We further show that SNVs maintained across transmission stages were transmitted as a unit of two at maximum, suggesting the presence of numerous variant genomes carrying only one or two SNVs each. We also present evidence for differences in selection pressures between human and mosquito hosts, particularly on the structural and NS1 genes. This analysis provides insights into how population drops during transmission shape RNA virus genetic diversity, has direct implications for virus evolution, and illustrates the value of high-coverage, whole-genome next-generation sequencing for understanding viral intra-host genetic diversity.Dengue virus (DENV) infection of an individual human or mosquito host produces a dynamic population of closely-related sequences. This intra-host genetic diversity is thought to offer an advantage for arboviruses to adapt as they cycle between two very different host species, but it remains poorly characterized. To track changes in viral intra-host genetic diversity during horizontal transmission, we infected  Aedes aegypti mosquitoes. Virus populations experience significant drops in size and are subject to differing selection pressures as they cycle between human and mosquito hosts. Subsequent changes in viral intra-host genetic diversity may have consequences for the adaptability and fitness of the virus population as a whole but are poorly understood. To study the impact of human-to-mosquito transmission on DENV populations, we allowed mosquitoes to feed directly on patients with acute dengue infections, then deep-sequenced DENV populations from patient plasma samples and from the abdomens and salivary glands of corresponding mosquitoes. These matched samples allowed us to estimate the size of the population drop that occurs during establishment of infection in the mosquito, track changes in viral intra-host variant repertoires at different stages in transmission, and investigate the possibility of host-specific immune selection pressures acting on the virus population. These novel insights improve our understanding of DENV population dynamics during horizontal transmission.Dengue virus (DENV) is transmitted between humans through the bite of infected female  Flaviviridae, genus Flavivirus. The ~10.7 kb DENV genome encodes three structural proteins  and seven non-structural (NS) proteins .With 3.6 billion people at risk and nearly 400 million infections annually ,2, denguAedes aegypti and Aedes albopictus. which acquire the virus by taking a bloodmeal from an infected human. Once ingested, DENV first infects and replicates in the mosquito midgut epithelium. It subsequently disseminates through the hemolymph to infect other organs such as the fat body and trachea, finally reaching the salivary glands, where it is secreted into mosquito saliva and injected into a human host during a subsequent blood-feeding event [DENV is transmitted between humans by the mosquitoes ng event .As is typically the case for RNA viruses, the DENV RNA-dependent RNA polymerase  operates at low fidelity, resulting in the accumulation of a population of closely-related but genetically-distinct viral genomes, organized around a consensus sequence, within each individual human or mosquito host. Sometimes termed a quasispecies, these intra-host variants are thought to interact cooperatively on a functional level, and to collectively contribute to the overall fitness of the virus population (reviewed in ). This hCulex-West Nile virus  system [Adaptability is especially important for mosquito-borne viruses, which encounter distinct selection pressures when cycling between vertebrate and invertebrate hosts. Even within a single host, intra-host variants generated through multiple rounds of virus replication are subject to evolutionary mechanisms such as bottlenecks, drift, and positive and negative selection pressures. In human-derived DENV populations, highly immunogenic E protein domains also display higher levels of intra-host genetic diversity, suggesting that selection pressures on low frequency population variants operate even during acute infection . In mosq) system , where h) system ,10.Host alternation also subjects arboviruses to frequent and significant drops in population size. Within the mosquito and vertebrate hosts, these drops occur during initial establishment of infection and subsequent spread through various tissues and organs, as well as during the process of blood-feeding itself, where only a small percentage of the total virus population circulating in the human actually seeds infection in the mosquito host. It is unclear how such drops shape the diversity and repertoires of intra-host virus populations, and the potential effect they have on transmission.Studies on intra-host genetic diversity in DENV have focused on virus populations in the human, and with few exceptions , have beIn this study, we used whole-genome amplification and next-generation Illumina sequencing to characterize DENV intra-host genetic diversity in both patient- and matched mosquito-derived virus populations. Because mosquitoes in our study were infected by allowing them to feed directly on patients, we were able to track changes in viral populations during human-to-mosquito transmission. We used a rigorous variant-calling algorithm to identify low frequency single nucleotide variants (SNVs) within a viral population. This unique study design provides important insights into how population drops shape viral genetic diversity, as well as into the differing selection pressures between human and insect hosts.Study protocols were reviewed and approved by the Scientific and Ethical Committee of the Hospital for Tropical Diseases (CS/ND/09/24) and the Oxford Tropical Research Ethical Committee (OxTREC 20–09). Written informed consent was obtained from all participants or from the parent or guardian of any child participants on their behalf.et al. [Ae. aegypti mosquitoes were allowed to feed on acute dengue cases. Just prior to blood feeding, venous blood was drawn and the plasma fraction stored. Blood-fed mosquitoes were reared until day 14 when the abdomen and salivary glands were dissected and stored.The human viremic plasma and DENV2-infected mosquitoes used here originated from the study by Nguyen et al. , in whicet al. [Out of the 83 DENV2-infected patients described by Nguyen et al. , we seleet al. . Hence tet al. .Next-generation whole-genome sequencing of human- and mosquito-derived DENV samples was performed as described in . Viral RFor each sample, equal amounts of all PCR-amplified fragments were combined and sheared on the Covaris S2 sonicator (Covaris) to achieve a peak size range of 100–300 bp . Samples were purified with the Qiagen PCR Purification Kit (Qiagen) and quality-checked on the Agilent 2100 Bioanalyzer with a DNA 1000 Chip (Agilent Technologies).Library preparation was performed with the KAPA Library Preparation Kit (KAPA Biosciences). After end-repair, A-tailing, and adapter ligation, ligated products in the 200–400 bp range were gel-extracted with the Qiagen Gel Extraction Kit (Qiagen). Samples were subjected to 14 PCR cycles to incorporate multiplexing indices, quantified with the KAPA SYBR FAST qPCR Master Mix (KAPA Biosciences) on the LightCycler 480 II real-time thermocycler (Roche Applied Science), and pooled.Paired-end, multiplexed sequencing (2 x 101 bp reads) of libraries was performed on the Illumina HiSeq (Illumina) at the Genome Institute of Singapore. Base calling was done with CASAVA 1.7; reads that did not pass Illumina’s chastity filter (CASAVA 1.7 user guide) were removed.https://github.com/CSB5/vipr), which automates the following steps: Iterative mapping of reads against the DENV-2 reference sequence NC_001474 using the Burrows-Wheeler Aligner [Illumina-generated FASTQ files were put through the Viral Pipeline Runner   were preTo identify mutational hotspots, a scanning window approach was used to scan the DENV genome  for an excess of SNVs in a window compared with the genome-wide average . This was done on a per-sample basis. For coldspots, SNVs from all samples were pooled and scanned for windows (minimum size of 40) with a depletion of SNVs  .Since haplotype reconstruction is a notoriously difficult problem, we resorted to a simple approach that estimates a lower bound of the number of haplotypes  present in a sample. This was done by greedily clustering SNVs based on their allele frequency confidence intervals (Agresti-Coull at the 0.05 level). SNVs were sorted by their allele frequency and the SNV with highest allele frequency seeds the first cluster. Variants were added to an existing cluster if their upper confidence interval limit was greater than the cluster minimum, otherwise they form a new cluster. The number of clusters represents a lower bound of the number of distinct viral genomes present in a sample. This clustering approach is implemented as one of the tools that come bundled with LoFreq .The size of the infecting virus population was estimated by simulating 1000 samplings of varying size from the virus population in the human, following a normal distribution with mean SNV frequency matching that in the human. This sampling was carried out for a range of mean SNV frequencies. The sampling error rate was calculated from the simulated SNV frequencies, and p-values were computed using a two-tailed t-distribution .et al. [Ae. aegypti mosquitoes with DENV2 by allowing them to blood-feed directly on patients at the acute stage of infection. To track changes in viral intra-population genetic diversity during human-to-mosquito transmission, we performed whole-genome Illumina sequencing of DENV2 populations from 12 patient plasma samples and 36 infected mosquitoes (three per patient) derived from that study [Nguyen et al.  infectedat study . We succSingle nucleotide variants (SNVs) in each DENV2 population were called with the LoFreq variant calling algorithm . At a coWe examined four measures of intra-sample diversity, calculated on a per sample basis : a) the All four diversity measures varied widely among individual samples . No statAlthough we cannot experimentally follow transmission of DENV2 from mosquitoes into humans, we had access to patient plasma samples from the Early DENgue (EDEN) study carried out in Singapore from 2005–2007 . DENV1 aAlthough levels of intra-host diversity remain unchanged, horizontal transmission may alter the SNV repertoire in a viral population if existing SNVs are lost and new ones generated. To examine this, we tracked individual SNVs during transmission from human to mosquito abdomen to mosquito salivary glands . Of 267 We observed relatively few instances of SNVs being maintained across transmission stages . These SSNVs may also be maintained because they play roles in increasing virus fitness, and selection for these SNVs may result in an increase in their frequencies over the course of transmission. We did not, however, observe this—the frequencies of most maintained SNVs (both synonymous and non-synonymous) remained similar in human-, mosquito abdomen-, and mosquito salivary gland-derived DENV populations , suggestIn the EDEN human-human transmission pairs, 15 of 130 SNVs (11.5%) present in the first member of the pair were observed again in the second member . The maj2 to 107 infectious virions / ml, we estimated the maximum size of the virus population infecting the mosquito for which loss of SNVs could be attributed to random chance. This was done by simulating 1000 samplings of varying sizes from the viral population in the human.Given that ~90% of SNVs detected in the human were lost, presumably due to chance, during transmission to the mosquito, we wanted to estimate the size of the population drop at which this would occur. For an assumed range of plasma viremia levels from 106 RNA copies / ml [For SNVs with frequencies from 0.01 to 0.03 (the vast majority of SNVs in our dataset), the maximum infecting virus population size for SNV loss to be attributable to chance was 100 virions . This reies / ml  would beLow frequency DENV variants that arise during virus replication are subject to immune selection pressure. To examine differences in selection pressures between hosts, we compared the ratio of the number of non-synonymous to synonymous SNVs (#NS/#S) for each gene in human- versus mosquito-derived virus populations. Because of the small numbers of human plasma-derived DENV2 SNVs from this study, we pooled these with SNVs detected in DENV1 and DENV3 populations from the EDEN study plasma samples . We are Human #NS/#S ratios were significantly higher than mosquito ratios for the prM, E, and NS1 genes . While nWe found no significant differences in #NS/#S ratios between mosquito abdomen and salivary gland for any of the viral genes , suggestWe also used a scanning window approach to identify mutational hot- and coldspots in the DENV genome; i.e. regions containing a statistically significant excess or lack of SNVs compared with the genome-wide average . We idenTogaviridae; it will be intriguing to test this hypothesis in DENV, a flavivirus.Hotspots in NS3, which encodes the viral serine protease and helicase, were detected in two mosquito-derived samples. While not significantly different between humans and mosquitoes , the NS3The coldspot in prM detected in mosquito-derived samples corresponds to a conserved region of the gene, which has been reported to be important for the association of prM with E during viral assembly ,28. We a27 SNVs  were found to encode low frequency premature stop codon mutations. These were spread across the coding region of the DENV genome in both human- and mosquito-derived DENV populations . PreviouTo determine how SNVs were distributed into distinct viral genomes, we clustered SNVs in each sample based on frequency, reasoning that SNVs present on the same viral genome would be present at similar frequencies. This gave us an estimate of the minimum number of distinct variant viral genomes in each sample ; the actWe next used network diagrams to visualize transmission patterns of viral genomes . In thisAe. aegypti mosquito counterparts.Here we report the use of next-generation, whole-genome DENV sequencing to follow within- and between-host differences in viral populations in naturally-infected humans and their infected Togaviridae) resulted in a relatively small number of midgut cells being infected, on the order of 15 for WNV [We observed that the vast majority (>90%) of SNVs in a population were lost upon virus transmission from human to mosquito. Based on this, we estimated that a maximum of ~100 virions infect the mosquito midgut, with the actual number varying with the level of viremia in the human. This number is consistent with previous studies that used fluorescent reporter viruses to show that, even with high titer bloodmeals, oral infection with WNV or Venezuelan equine encephalitis virus  of tper cell ,41, suggWe observed that SNVs that were maintained between transmission stages were present at significantly higher frequencies than SNVs that were not maintained. A previous next-generation sequencing analysis of human DENV clinical samples from Nicaragua found that intra-host variants that were also observed at the inter-host level (i.e. in consensus sequences of circulating Nicaraguan DENV strains) were present at higher frequencies than variants that were only observed at the intra-host level . This suWe were unable to follow transmission from the mosquito back into the human, but the EDEN human-human transmission pairs (thought to be separated by a single mosquito) allowed us to make hypotheses about this process. Similar to the human-mosquito pairs, paired human-human DENV clinical samples also showed predominantly different SNV repertoires. We estimate that the majority of SNVs present in a human sample will fall below our detection limit during passage through the mosquito, where most SNVs appear to be neutral. However, once transmitted to a second human host, selection pressures there may once again drive up the frequencies of SNVs that impact virus fitness in the human. Our observation that several viral genes display higher #NS/#S ratios in human- than in mosquito-derived samples is consistent with this idea, but SNVs would have to be followed over a chain of multiple linked transmissions in order to further investigate this.Over multiple rounds of replication in the human or mosquito, viral populations grow exponentially and generate a range of low frequency variants that are subject to selection pressures. Our data suggest that variants in the prM, E, and NS1 genes experience stronger immune pressures in the human than in the mosquito, consistent with the fact that these gene products are known targets of the human antibody response (reviewed in ), which The mosquito immune response lacks an adaptive arm, but is able to mount potent responses against virus, bacteria, and fungi through the Toll, IMD, and JAK/STAT immune signaling pathways (reviewed in ). ComparCulex mosquitoes [Culex mosquito host for WNV [Ae. aegypti system.In addition to the classical immune signaling pathways, RNAi is also a major antiviral defense mechanism in the mosquito ,46. WNV squitoes . Althoug for WNV ,48, thesWe found no evidence for differences in selection pressures between the mosquito abdomen and salivary gland. Still, because the SNV sample size was small, this does not rule out differences in immune pressures between body compartments, and #NS/#S ratios did diverge for several genes. A potential driver of differing immune pressures is the midgut microbiota, which has been shown to impact mosquito physiology and vector competence for human pathogens (reviewed in ). Deplet-4 [After phasing SNVs into distinct viral genomes, we observed that in most cases, only one SNV from a predicted variant genome was maintained across transmission stages, with a maximum of two being maintained as a unit. This suggests that SNVs in a sample are spread out across many variant genomes instead of being clustered together on one genome, and is consistent with the RdRp error rate of 1x10Despite the growing number of studies characterizing intra-host genetic diversity, the impact of this diversity on virulence or disease severity for arboviruses in vertebrate hosts is not well understood. One study unexpectedly found that mouse morbidity and mortality was negatively correlated with WNV intra-host genetic diversity . The autS1 Fig(TIF)Click here for additional data file.S2 Fig1116 SNVs and their frequencies are displayed here.(TIF)Click here for additional data file.S3 Fig(A) Number of SNVs; (B) Sum of SNV frequencies; (C) Average SNV frequency; (D) Standard error of the mean SNV frequency; all calculated on a per sample basis.(TIF)Click here for additional data file.S4 Fig(A) DENV1 and (B) DENV3 clinical samples from the EDEN study. Red, hotspots; each hotspot was found in a single sample; blue, coldspots, indicating a depletion of SNVs across 33 DENV1 and 27 DENV3 samples.Circos plots  of mutat(TIF)Click here for additional data file.S1 TableDOI, day of illness at time of mosquito exposure; Fever day, day (at the time of mosquito exposure) compared to the defervescence day, which is defined as fever day 0. Defervescence day is the day when patient's temperature returns to less than 37.5°C and remains below this threshold until discharge. One day before defervescence day is fever day -1; one day after defervescence day is fever day +1.(DOCX)Click here for additional data file.S2 TableHuman-human pairs of EDEN clinical DENV samples predicted to be separated by one mosquito are shown, along with the degree of SNV overlap between members of a pair.(DOCX)Click here for additional data file.S3 Table2 to 2x107 infectious virions / ml or 1x106 to 1x1011 in 5 liters of blood.The maximum infecting virus population size for which SNV loss could be attributable to chance (p > 0.05) was calculated by simulating 1000 samplings of varying size from the virus population in the human, which was assumed to range from 2x10(XLSX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


To present a method for image segmentation and generation of enface thickness maps and reflectance images of retinal layers in healthy and diabetic retinopathy (DR) subjects.High density spectral domain optical coherence tomography (SDOCT) images were acquired in 10 healthy and 4 DR subjects. Customized image analysis software identified 5 retinal cell layer interfaces and generated thickness maps and reflectance images of the total retina (TR), inner retina (IR), outer retina (OR), and the inner segment ellipsoid (ISe) band. Thickness maps in DR subjects were compared to those of healthy subjects by generating deviation maps which displayed retinal locations with thickness below, within, and above the normal 95% confidence interval.In healthy subjects, TR and IR thickness maps displayed the foveal depression and increased thickness in the parafoveal region. OR and ISe thickness maps showed increased thickness at the fovea, consistent with normal retinal anatomy. In DR subjects, thickening and thinning in localized regions were demonstrated on TR, IR, OR, and ISe thickness maps, corresponding to retinal edema and atrophy, respectively. TR and OR reflectance images showed reduced reflectivity in regions of increased thickness. Hard exudates appeared as hyper-reflective spots in IR reflectance images and casted shadows on the deeper OR and ISe reflectance images. The ISe reflectance image clearly showed the presence of focal laser scars.Enface thickness mapping and reflectance imaging of retinal layers is a potentially useful method for quantifying the spatial and axial extent of pathologies due to DR. Spectral domain optical coherence tomography (SDOCT) has become an essential tool in the management of patients with diabetic retinopathy (DR) and has improved clinicians’ abilities to detect anatomical alterations in the retinal tissue as compared to conventional ophthalmoscopy or fundus photography.–10 SpeciSDOCT imaging visualizes the retinal tissue in axial cross-sections, permitting quantitative assessment of central submacular thickness, which is correlated with visual acuity in clinical treatment studies for DME.–19 The dRecently developed image segmentation algorithms permit depth-resolved enface SDOCT imaging for viewing the retinal layers in the coronal plane. These enface SDOCT “C-scans” allow for quantifiable evaluation of individual retinal layers separated in depth. Several manual and semi-automated techniques have been developed for evaluation of the retinal layers in diabetics with the objective of improving the prognostic capability of SDOCT imaging.–26There is considerable evidence that the continuity of individual retinal layers has prognostic significance. Continuity of the inner segment ellipsoid (ISe) layer was found to correlate with visual acuity in several retinal conditions including DR.–40 ContiWe have previously reported a method for generation of enface images of retinal layers from high-density SDOCT B-scans., 43 SeveSubjects were recruited from the ophthalmology clinics at the Illinois Eye and Ear Infirmary . The research study was approved by an Institutional Review Board at the University of Illinois at Chicago. Prior to enrollment, the research study was explained to the subjects and written informed consents were obtained from each subject according to the tenets of the Declaration of Helsinki. SDOCT images were acquired in 10 healthy control subjects (age: 55 ± 8 years) and 4 DR subjects (age: 59 ± 12 years). The inclusion criterion for control subjects was no clinical history of diabetes or other retinal disease. SDOCT imaging was performed in one eye of each subject. Clinical data were obtained from the charts of DR subjects.A high density SDOCT raster volume scan of the macula was obtained using a commercially available instrument. . The volume scan consisted of 145 raster horizontal B-scans with a depth resolution of 3.9 μm and 768 A-scans per B-scan. The instrument’s eye tracking function allowed 9 B-scans to be averaged at each location. The SDOCT raster scan covered a retinal area of 15° x 15° centered on the fovea with approximately 31 micron spacing between SDOCT B-scans. Scanning laser ophthalmoscope (SLO) images were acquired with the same instrument.Semi-automated image segmentation software was developed in MATLAB  for identification of five interfaces between retinal cell layers in the SDOCT B-scans. Retinal cell layer interfaces were detected using graph theory and dynamic programming, based on a previous described method. Briefly,As shown in After automated segmentation of the retinal interfaces, the operator was able to scroll through all 145 SDOCT B-scans in the volume scan to review the segmentation results and, if necessary, manually correct errors in the detected interfaces. This process was necessary for images obtained in DR subjects due to the presence of irregular layer boundaries and intraretinal fluid. To correct segmentation errors, the operator would first select a segmentation path that required modification and then manually draw a revised line corresponding to the visualized cell layer interface. The search area of the graph was then restricted to include only a small vertical image region around the manually drawn line, and a revised path for the cell layer interface was obtained by determining a new graph cut solution.Enface thickness maps and reflectance images were generated based on segmentation of the retinal interfaces in the SDOCT B-scans. Total retinal (TR) thickness was calculated as the depth separation between the vitreous/ILM and RPE/choroid interfaces. Inner retinal (IR) thickness was calculated as the depth separation between the vitreous/ILM and INL/OPL interfaces. Outer retina (OR) thickness was calculated as the depth separation between the INL/OPL and RPE/choroid interfaces. ISe thickness was calculated as the depth separation between the ONL/ISe and ISe/RPE interfaces. Enface reflectance images of TR, IR, OR, and ISe were generated based on pixel values averaged vertically within the segmented layers in each SDOCT B-scan to create rows of the corresponding enface images.Thickness, standard deviation (SD), and 95% confidence interval (CI) maps for each segmented layer were generated from data in healthy subjects. Thickness maps obtained in DR subjects were compared to the normal CI maps using deviation maps. The deviation maps were generated by categorizing each pixel as below the lower limit, within the upper and lower limits, or above the upper limit of the normal CI and color coded as blue, green, and red, respectively.Reflectance images displayed homogenous patterns without significant variations. The normal retinal vasculature including the major retinal arcades are marginally visible within the IR reflectance image, but appeared more prominently in the OR and ISe reflectance images due to shadowing effects. The foveal center appeared darker, relative to the surrounding parafoveal retina in reflectance images, but more prominently in the IR reflectance image due to the absence of inner retinal cell layers.A 42 year old female with a history of PDR and DME presented for follow up evaluation. She had previously received PRP for PDR, focal laser for DME, and injections of anti-vascular endothelial growth factor (anti-VEGF) agents in her left eye. Her vision was 20/25 and stable from previous visits. She had no new visual complaints. Fundus examination was notable for a blunted fovea, focal laser scars, and prominent macular edema.Both the TR and OR reflectance images displayed regions of reduced reflectivity that corresponded to regions of increased retinal thickness. The IR reflectance image showed a region of reduced reflectivity near the distorted foveal depression. Hard exudates were visible in the IR reflectance image as hyper-reflective spots that cast shadows on the deeper OR and ISe reflectance images. Multiple focal laser scars were visible in the ISe reflectance image.A 62 year old female with a history of PDR with DME presented for evaluation. She had previously received focal laser for DME in addition to several anti-VEGF injections in her left eye. Visual acuity was 20/100. Fundus examination was notable for the presence of significant retinal thickening supero-nasal to the fovea, consistent with clinically significant DME.A 66 year old male with a history of PDR and DME in his left eye presented for follow up evaluation. He had a history of PRP treatment and had undergone focal laser therapy in the left eye for DME 3 months prior to this visit. Visual acuity was 20/40 and he had no new visual complaints.A 61 year old male with a history of NPDR and DME in his left eye presented for follow up examination. His visual acuity was 20/20 and he had no new visual complaints. Fundus ophthalmoscopy initially identified an area of increased retinal thickening nasal to the fovea.TR and OR reflectance images showed a diffuse area of reduced reflectivity in the region of increased thickness. The IR reflectance image demonstrated scattered areas of hyper-reflectance from hard exudates. The ISe reflectance image showed hypo-reflective spots that correspond to shadows from hard exudates in the inner retina.The current study reports a method for generating enface thickness maps and reflectance images of retinal layers using a commercially available SDOCT instrument. The method established normal retinal anatomical features in TR, OR, IR, and ISe thickness maps and reflectance images, and detected pathologic alterations in selected cases of DR. In subjects with DR, pathologic alterations, including thickening and thinning of TR, IR, OR, and ISe across the macular area, were quantitatively displayed on the enface thickness and deviation maps. Furthermore, reflectance images of TR, IR, OR, and ISe displayed regions of reduced reflectivity that corresponded with areas of increased thickness and disruption of ISe integrity. Hard exudates were consistently observed as hyper-reflective regions on IR reflectance images and hypo-reflective regions on OR and ISe reflectance images.Several previous studies have reported methods for segmentation of different retinal layers in DR., 43, 56 Studies employing segmentation techniques to investigate reflectance changes of individual, depth resolved retinal layers in an enface manner are lacking. Murakami et al investigIn the current study, DR subjects who maintained good visual acuity had ISe reflectance at the fovea similar to that of control subjects. Although additional subjects are needed to establish a correspondence between enface ISe reflectance images and visual acuity, this finding is in agreement with previous studies that showed ISe layer continuity is correlated with visual acuity. –33, 57 BEnface OCT imaging has several practical advantages compared to standard cross-sectional OCT imaging. Enface OCT images generated from a high density raster of OCT B-scans enable detection of subtle retinal pathology that may fall between standard OCT B-scans. Additionally, enface OCT imaging allows succinct visualization of the spatial extent of pathologies in different retinal layers compared to scrolling through individual OCT B-scans. This feature is particularly useful for accurate monitoring of pathological changes over time. Enface OCT imaging may also be an effective method to study changes in choroidal thickness, which have been associated with disease severity in DR, although this was not assessed in the current study. Based on individual OCT B-scans, choroidal thickness was reported to be reduced in eyes with moderate-to-severe DR and DME compared to age-matched controls., 59 AddiIn summary, the reported method for segmentation of SDOCT B-scan images and generation of enface thickness maps and reflectance images of retinal layers has the advantage of quantifying the spatial and axial extent of DR pathologies. Future studies are necessary to investigate the clinical utility of enface thickness mapping and reflectance imaging in DR and other retinal diseases.


In the context of performance optimizations in multitasking, a central debate has unfolded in multitasking research around whether cognitive processes related to different tasks proceed only sequentially (one at a time), or can operate in parallel (simultaneously). This review features a discussion of theoretical considerations and empirical evidence regarding parallel versus serial task processing in multitasking. In addition, we highlight how methodological differences and theoretical conceptions determine the extent to which parallel processing in multitasking can be detected, to guide their employment in future research. Parallel and serial processing of multiple tasks are not mutually exclusive. Therefore, questions focusing exclusively on either task-processing mode are too simplified. We review empirical evidence and demonstrate that shifting between more parallel and more serial task processing critically depends on the conditions under which multiple tasks are performed. We conclude that efficient multitasking is reflected by the ability of individuals to adjust multitasking performance to environmental demands by flexibly shifting between different processing strategies of multiple task-component scheduling. Growing demands on information processing due to increasing multimedia interactions call for higher efficiency. Does the human mind have the structural and functional means for efficient multitasking? The architectural properties of the nervous system allow for widely distributed simultaneous neural processing in billions of neurons. However in multitasking, severe performance costs occur when combining even the simplest cognitive tasks for simultaneous execution. Based on the current literature, we answer two key questions of multitasking: serially . Such a conceptualization of multitasking excludes the possibility of parallel  cognitive processing of multiple task components1. Others argue that parallel processing is generally possible  between the stimulus of T1 (S1) and the stimulus of T2  effect  effect . The PRP) effect . Importalocus of slack logic and the backward crosstalk logic.Within the PRP paradigm, the assumption of parallel processing means that central cognitive processing in T2 can proceed in parallel to central capacity-limited stage processing in T1 Figures ,C. StrucIn the locus of slack logic , the poscrosstalk. Central T2 task processing affects central T1 processing prior to completion of Task 1 bottleneck-stage processing  and verbal T2 responses to letter identity  produced substantial crosstalk interference (RT1 prolongation), when the verbal answer right coincided with the left manual response (response-category mismatch). T2 processing facilitated T1 response activation (RT1 decrease), when the verbal answer right was accompanied with the right manual response (response-category match). S-R translation processes of T1 and T2 were not serial and discrete but proceeded in parallel, when dimensional overlap between tasks was provided  onto the correct responses in each task . To solve the binding problem and therefore to minimize the risk of response reversals and/or confusion, a strategy of serial processing is adopted . Accordingly, result patterns that speak for serial processing are not surprising. Yet, when specific priority instructions are lifted, participants freely choose a moderate parallel processing strategy , resulti(1) How is the shift between these complementary dual-task processing modes realized? (2) Under which conditions are parallel and serial processing the more adaptive and thus favorable choice? As a reply to the first question, we propose that optimized and efficient multitasking reflects the ability to flexibly adopt an either parallel or serial task-processing mode, depending on situational demands. Multitasking requires an individual to maintain a balance between two antagonistic types of performance optimization, i.e., minimizing between-task interference  and minimizing mental effort . Flexibly adjusting the degree of more serial versus more parallel dual-task processing to changing task and context requirements reflects high levels of adaptability in dynamic environments including the online analysis of contextual features, their translation into performance requirements, implementation of the optimal processing mode, and continuous monitoring of both the environmental demands and performance outcome as basis for further tuning, where required  influences the balance between parallel and serial task processing . Using cWe further demonstrated that the balance between complementary dual-task processing modes is determined by internal situational features such as variations in an individual’s mood, task-preceding cognitive control state, or acute stress levels. First, we administered a mood-induction procedure  prior to a dual task with crosstalk. Participants that underwent a negative mood-induction protocol displayed higher levels of serial task processing  than participants that underwent positive mood induction . Second,These examples illustrate that the adoption of a more parallel or a more serial task-processing mode largely depends on the conditions under which multiple tasks are performed. With this, the situation-dependent implementation of complementary task-processing modes in multitasking can be integrated into a general framework posting the regulation of cognitive control as fundamental basis underlying adaptive goal-directed behavior . AdaptivSuch extreme biases and their behavioral consequences have been primarily investigated in healthy populations. For example, an experimentally induced bias toward high stability comes at the cost of reduced cognitive flexibility , which, continuum from serial to parallel task processing. Such a conception also has direct consequences for defining efficient multitasking, and it raises the question of how to optimize the flexible and context-sensitive adoption of complementary task-processing modes.Applying this framework, multitasking in itself constitutes a prime control dilemma, in which a crosstalk-avoiding serial processing strategy counters an effort-saving parallel processing strategy. Complete T1 shielding and thus T2 blocking is dysfunctional, as successful multitasking requires the attendance and processing of T2 components. Yet, the efficiency of adaptive multitasking might be seen in the flexible selection of a situation-adequate processing strategy within a individual difference perspective on multitasking ability in general and the adaptive adjustment of task-processing strategies in particular might hold promise for gaining novel insights into factors of optimized multitasking performance. For example, individuals frequently engaging in multitasking are not necessarily the ones displaying efficient multitasking performance . In the second part of the review, we highlighted that evidence for parallel processing critically depends on the theoretical and methodological basis under which multitasking performance is assessed.While serial task processing appears to be the most efficient multitasking processing strategy, participants are able to adopt parallel processing. Moreover, parallel processing can even outperform serial processing under certain conditions. Based on these highlighted insights into multitasking performance, future research aiming to further understand the nature of parallel versus serial processing of multiple tasks to unveil the secrets of multitasking efficiency needs to take into account the preconditions and environmental constraints under which multitasking is performed. We believe that a flexible and context-sensitive recruitment of a more serial or more parallel processing strategy enables the agent to flexibly adjust to environmental demands, providing important mechanisms for adaptive intelligent behavior .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.


N-Acetyl cysteine (NAC) and Sulforaphane are commonly used supplements to prevent diseases including cancers. We studied the effect of these antioxidant supplements on the non-coding intronic RNA expression using publicly available datasets from a mouse model for lung cancer and prostate cancer cell lines. Although high throughput polyA-enriched RNA-Seq data characterize spliced coding mRNA regions, recent studies reveal the expression of reads from the non-coding intronic regions. Our analyses indicate that cancer cells have higher expression of introns compared to that of normal cells and that treatment with antioxidant supplements reduces the increased expression of introns of several genes. However, we did find high expression of introns of multiple genes including many oncogenes in the supplement treated groups compared to that of the control; this effect was distinct depending on the cell type and the supplement studied. Using RT-PCRs, we validated the expression of introns of two oncogenes, DLK1 and LRG1, known to be key players in lung cancer progression, and demonstrate changed intronic expression with supplement treatment in cancer cells. With regard to the antioxidant system, supplements did not change the intronic RNAs for endogenous antioxidant enzymes except for a significant decrease in the expression of superoxide dismutase (SOD) intronic RNA. Concurrently, we also found that a prolonged (48 h) exposure to Vitamin C, Vitamin E and Green tea extract reduced the enzymatic activity of SOD in lung cancer cells. The results from this study reveal that the antioxidant supplements have a significant effect on the intronic RNA expression of many genes including cancer genes that are not directly linked to the body’s antioxidant system. It is important to study this novel effect of antioxidant supplements in detail as it may have a significant role in disease progression.The notion that dietary antioxidants can help fight cancer is popular. However, the mechanism(s) behind the effect of antioxidants in cancer is still unclear. Previous studies indicate that supplements can influence gene expression; however, all of these studies were focused on the coding/exonic gene expression. Studies are now emerging to highlight critical functional roles for RNAs expressed from the non-coding regions. This project was designed to study the effect of antioxidant supplements on non-coding intronic RNA expression in human cancers. Vitamin E,  Reactive oxygen species (ROS) are generated in the body as byproducts of several cellular metabolic reactions; they consist of radical and non-radical oxygen species formed by the partial reduction of oxygen  Low leveet al. [et al. reported that low-dose antioxidant supplementation for 7.5 years lowered total cancer incidence and all-cause mortality in men but not in women [Consuming dietary antioxidant supplements to fight diseases, especially cancer, has become popular among the general public. However, the exact mechanism(s) for the effect of antioxidants in cancer is unclear. In addition clinical trials have reported inconsistent outcomes. Hennekens et al.  reportedin women .et al., antioxidant supplementation up-regulated the cardiac endothelial cell gene expression of the ras homolog gene family member A, which has a role in cardiovascular disease progression [Several studies including state of the art transcriptomic expression analysis using RNA-Sequencing (RNA-Seq), have reported on the effects of antioxidants on gene expression ,6,7,8. Agression . In anotgression .et al. experimentally validated the repression of the human dihydrofolate reductase gene by a non-coding interfering transcript [et al. reported that in renal cell carcinoma, the expression of non-coding intronic RNA discriminates tumor from adjacent non-malignant cells [et al. showed that the expression of six antisense intronic non-coding RNA transcripts correlated with the degree of tumor differentiation in prostate cancer [Until the late 2000s, the focus of gene expression studies was mainly on coding mRNA expression. It was previously believed that RNA transcribed from the intronic regions was destined to be degraded and hence did not have a biological function. However, in recent years, studies have highlighted critical functional roles for these non-coding regions . Martiananscript . Subsequnt cells . Similare cancer . Howevern-Acetyl cysteine and Sulforaphane. The rationale for choosing these data sets was that the datasets were of good quality and that the antioxidant supplements studied are commonly used, while research on the effects of these supplements has yielded contradictory results [The hypothesis for this study was that antioxidant supplements would influence the expression of intronic RNAs in genes linked to ROS system. We utilized two publicly available RNA-Seq datasets for studying the effect of antioxidant supplements on intronic RNA expression. The antioxidants studied in these datasets were Vitamin E,  results ,18,19,20et al. show that most intronic reads arise from nuclear RNA and changes in intronic read counts accurately predict changes in transcriptional activity [For both RNA-Seq datasets, prior to sequencing, the RNA samples were enriched for polyadenylated RNA fractions containing processed spliced RNA transcripts. Polyadenylated RNA-Seq data should generate reads that map to exons. However, it has been shown that RNA-Seq data also capture intronic sequences . The intactivity . In the activity ,25.The analysis approach used in this study is shown in Murine lung cancer dataset (Accession: E-GEOD-52594): RNA-Seq data were downloaded from ArrayExpress. The objective of the original study was to study the impact of Vitamin E and NAC supplementation in murine models of KRAS-induced lung cancer . AntioxiHuman normal and prostate cancer cells dataset (SRP027258): RNA-Seq data were downloaded from NCBI SRA. Normal prostate epithelial cells and androgen-dependent and androgen-independent prostate cancer cells were treated with 15 µM sulforaphane (SFN), a phytochemical derived from cruciferous vegetables, and the transcriptome was determined at 6 and 24 h time points .RNA-Seq is a relatively recent approach to transcriptome profiling that uses deep-sequencing technologies which provide a more precise measurement of levels of transcripts and their isoforms than traditional microarrays . The RNAThe mouse and human genome (Ensembl version) files were downloaded from iGenomes . The RNAp values of the differentially expressed genes were done using p.adjust function in stats, R package [The intronic counts were calculated from BAM files using the multicov function in samtools . The int package . Geneset package  was usedin vitro analyses to experimentally validate the effect of antioxidants on superoxide dismutase activity. The cell lines used in this study were obtained from ATCC (www.atcc.org). We used A549 (lung cancer), MCF-7 (breast cancer), MDA-MB231 (breast cancer) and 293  cell lines for studying the effect of antioxidant supplements including green tea extract, Vitamin C, Vitamin E and n-Acetyl cysteine. The antioxidants were obtained from and manufactured by the Vitamin Shoppe. The non-toxic dosages for the antioxidants: 2 mM Vitamin C, 25 µM Vitamin E2, 50 µg/mL Green tea extract and 0.2 mg/mL N-acetyl cysteine were selected from previously published studies [The results from the bioinformatic analyses indicated that the antioxidants decreased the activity of one of the major group of antioxidant enzymes, superoxide dismutases. We conducted  studies ,38,39,40 studies . Superox studies . The calin vitro study of antioxidant enzyme activity, we had treated NAC and Vitamin E on A549 lung cancer and MCF7 breast cancer cells. We used the cell lysates from antioxidant treated and untreated A549 lung cancer and MCF7 breast cancer cells for RT-PCR validations.To validate intronic RNA expression, we examined DLK1and LRG1 using RT-PCR. For the The primers for DLK1 and LRG1 were designed using the software Primer3 . QIAGEN’The summary of genes with significantly differentially expressed (FDR < 0.01 and fold change >2) intronic regions in the antioxidant supplement treated tumor groups compared to the untreated tumor group is given in Differentially expressed intronic RNA: The summary of the results from the analyses of murine lung cancer dataset is given in Antioxidant enzymes: Since we wanted to know if the supplements would have an effect on the ROS-system, we also studied the changes in expression in the major antioxidant enzymes including, catalase (CAT), glutathione peroxidases (GPXs) and superoxide dismutases (SODs). The exonic expression of these antioxidant enzymes was generally reduced after supplement treatments and a significant decrease was observed after Vitamin E treatment. Furthermore, we found significant decrease in intronic RNA expression of SOD2 after Vitamin E supplementation.NAC supplement: According to Gene set enrichment analyses, out of a total 86 genes with significantly increased intronic RNA expression in the NAC supplemented murine model group, 16 overlapped with the gene-expression signature of KRAS2 mutation in human lung cancer [g cancer   did not show any significant change after SFN treatment. However, significant decreases in SOD2 expression of introns were observed in LNCAP and PC3 prostate cancer cells.Differential Expression analyses: The results from the differential expression analyses of the three prostate cell types after SFN treatment are shown in The top canonical pathways of the genes with differentially expressed introns after SFN treatment in the three prostate cell types are shown in p < 0.3). In vitro analyses of antioxidant supplements on superoxide enzyme activity:i.e., not all introns were differentially expressed and (b) distinct signaling pathways were enriched for the genes with increased expression of introns after Vitamin E and SFN treatments.In recent years, owing in part to increased media attention, the popularity and use of antioxidant supplements as health adjuvants has been rising. However, studies showing both beneficial and harmful effects of antioxidant supplements have been reported ,46,47. TSeveral recent studies clearly demonstrate the identification of functional intronic RNAs from RNA-Seq data ,14,23. TIntronic RNA has been shown to play roles in development , transcret al. identified exonic and intronic tissue-specific expression signatures for human liver, prostate and kidney [Gene Set enrichment analyses of the genes that exhibited changes only in the expression of introns yielded cell cycle, lipid metabolism, and immune system as the top ranking pathways. Similar to our observation, Nakaya d kidney . These iGene Set enrichment analyses of genes with differential expression of introns from our analyses on cancer datasets yielded significant enrichment in the immune system. All three antioxidants analyzed in this study, NAC, SFN and Vitamin E influenced immune pathways. Antioxidant supplements are generally considered to boost immune responses and to prevent diseases . Howeveret al., LRG1 was overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases [et al. identified LRG1 as a candidate biomarker in urine for non-invasive NSCLC [The expression of introns of several genes that are linked to lung cancer was significantly increased with NAC and Vitamin E treatments . Figure hy cases . Li et ave NSCLC . Increaset al. [Delta-like 1 homolog (DLK1) gene is another example where antioxidant treatment increased intronic RNA expression; the increase was significant with NAC supplement treatment. Furthermore, we validated the intronic RNA expression using RT-PCR. DLK1 gene encodes a transmembrane protein containing six Epidermal Growth Factor repeats; the protein is involved in the differentiation of several cell types. Aberrant expression of DLK1 has been found in various types of human cancers, including lung cancer . Li et aet al.  suggest In this study antioxidant supplements reduced the exonic expression of ROS enzymes including catalase, GPXs and SODs in the murine lung cancer dataset. This was further supported by cell culture experiments showing decreased SOD activity after exposure of cancer cell lines to Vitamin C, Vitamin E and Green tea extract. The observed decrease in endogenous SOD activity could be the result of the antioxidant supplements exerting a negative feedback on the endogenous antioxidant enzyme systems and could negate any potential beneficial effect of the antioxidant supplements.In conclusion, this study makes the observation that antioxidant supplements differentially alter the intronic RNA expression of genes including several oncogenes. Furthermore, amongst the cadre of genes comprising the ROS system the change in expression of introns was observed only in SOD gene. The differential intronic RNA expression after supplement treatments, especially the increase observed in oncogenes suggests a possible effect on disease progression. However, further studies are warranted to establish the precise biological significance of this effect of antioxidants on expression of introns.