Plasmodium falciparum causes a spectrum of malarial disease from asymptomatic to uncomplicated through to severe. Investigations of parasite virulence have associated the expression of distinct variants of the major surface antigen of the blood stages known as Pf EMP1 encoded by up to 60 var genes per genome. Looking at the population genomics of var genes in cases of uncomplicated malaria, we set out to determine if there was any evidence of a selective sweep of specific var genes or clonal epidemic structure related to the incidence of uncomplicated disease in children. By sequencing the conserved DBLα domain of var genes from six sentinel sites in Uganda we found that the parasites causing uncomplicated P. falciparum disease in children were highly diverse and that every child had a unique var DBLα repertoire. Despite extensive var DBLα diversity and minimal overlap between repertoires, specific DBLα types and groups were conserved at the population level across Uganda. This pattern was the same regardless of the geographic distance or malaria transmission intensity. These data lead us to propose that any parasite can cause uncomplicated malarial disease and that these diverse parasite repertoires are composed of both upsA and non-upsA var gene groups. Plasmodium spp1.Children living in many regions of Africa are at risk of febrile episodes of malaria until they eventually acquire immunity that protects them against the development of clinical disease. This immunity is non-sterilizing and occurs after repeated exposure to Plasmodium falciparum Erythrocyte Membrane Protein 1 (Pf EMP1) has been proposed as the major variant surface antigen (VSA) of the most virulent malaria parasite known as P. falciparum. This highly polymorphic antigen is encoded by up to 60 diverse var genes present in each parasite genome4. Differential expression of these genes leads to switching of Pf EMP1 variants in a single cell7. This mechanism of clonal antigenic variation occurring in a hierarchical manner allows the parasite to evade the host immune response to Pf EMP1 thereby establishing a chronic infection. High levels of diversity have been observed in the var gene repertoire in a single parasite11 as well as in populations15. Importantly, Pf EMP1 is also a virulence factor as it mediates adhesion of infected erythrocytes to host endothelial cells and expression of specific variants is associated with the pathogenesis of uncomplicated, severe and placental malaria (reviewed in e.g. ref.16).var genes has revealed a conserved domain architecture consisting of an N-terminal segment (NTS), followed by multiple Duffy-binding like (DBL) and cysteine-rich interdomain region (CIDR). This is despite the sequences being highly polymorphic11. Var genes can be further classified into four main sub-groups based on semi-conserved upstream promoter sequences (ups): groups A (upsA), B (upsB), C (upsC), and E (upsE) with group E consisting of only var2csa, a gene associated with placental malaria18. Recombination events occur mostly between var genes from the same group, generating high sequence diversity while still preserving the var domain architecture19. Despite this considerable diversity, comparisons to distantly related primate parasites (e.g. P. reichenowi) have shown that balancing selection has maintained ancient var sequence fragments over millions of years20. Where malaria transmission is high, multiple-clone P. falciparum infections are common22 leading to increased rates of meiotic recombination resulting in extensive repertoire diversity24. Moreover, high levels of mitotic recombination have been observed within cloned laboratory lines25. Whether this mitotic diversification process occurs as frequently in nature remains an open question.Characterization of the structure of var genes are preferentially expressed in children with severe and/or cerebral malaria, group B var genes are associated with clinical malaria (uncomplicated and severe) and cerebral malaria, and group C transcripts are present in larger proportions in children with chronic asymptomatic malaria infections34. Sequencing of seven P. falciparum genomes has shown that all parasites have the group A and B var genes11 so expression of these genes rather than the presence of these genes per se is associated with disease severity.Evidence from case-control studies have shown that group A var genes have focused on sequencing the highly conserved DBLα domain and demonstrated by limited sampling that extensive diversity in DBLα sequences and repertoires exist in endemic areas of South America, Africa and Papua New Guinea36. Recently, we investigated the population structure of var DBLα sequences in parasites from asymptomatic children from an area of high malaria transmission in Gabon, West Africa15. Strikingly, in this first study of deep sampling of var genes in the asymptomatic reservoir in Africa we found that every parasite isolate had a distinct var DBLα repertoire with minimal sharing of DBLα types among 200 isolates. The absence of parasite genomes with high sharing of types was consistent with immune selection structuring a large effective parasite population size, as defined by DBLα diversity, into repertoires as different as possible in a transmission system favoring outcrossing. The case was made that these data provide evidence for a novel type of “strain structure” in a system where recombination is the key driver of diversification of the major genes under immune selection. These important observations regarding the var population genomics of asymptomatic infections in children lead us to ask the question: what is the population structure of var genes in uncomplicated malaria cases of P. falciparum in an area of high transmission in Africa? We wanted to explore whether they exhibit the same population structure or whether there is epidemic transmission of parasites with related repertoires of var genes causing uncomplicated malaria. Moreover, we wanted to explore the population structure of different groups of var genes  to see if they had distinct patterns of genome evolution under variable conditions of transmission.Population genetic studies of var DBLα diversity of the parasite population causing uncomplicated P. falciparum cases in children under five. We present results for six sentinel health sites in Uganda and demonstrate that P. falciparum parasites causing uncomplicated cases in children were highly diverse with distinct var DBLα repertoires despite varying levels of transmission intensity across Uganda. In our study we found no evidence of epidemic expansion or clonal propagation of P. falciparum parasites with highly related var DBLα repertoires in the population.Specifically, our experiments describe the var gene groups in proportions expected from whole genome sequencing11.These data led us to propose that any parasite can cause uncomplicated disease. Furthermore, these highly diverse parasite repertoires contain both upsA and non-upsA DBLα amplicons were pooled and sequenced from 517 of the 600 isolates from the six sentinel sites.A total of 51,401 DBLα sequence reads were obtained from the 517 isolates and clustered based on average linkage using a 96% sequence identity threshold resulting in 21,156 unique DBLα sequences, or DBLα types. Sixteen (3%) isolates with less than 20 DBLα types were excluded from analyses due to limited DBLα type counts. From the 501 isolates utilized for this analysis, a total of 50,624 DBLα sequence reads were obtained  and we observed a total of 21,134 DBLα types   with upsA representing 7.7%  and upsB/upsC representing 92.3%  of the total number of DBLα types sequenced in Uganda (Table p < 0.001). This pattern of increased abundance of the upsA DBLα types being more conserved was only statistically significant within the high transmission sites of Arua (p = 0.026), Tororo (p < 0.001), and Apac (p < 0.001). For the low to moderate transmission sites  there was no significant repeat occurrence of specific upsA DBLα types. It is important to note that the conserved upsA DBLα types represented a minority (13.1%) of the total upsA DBLα types observed.We further classified the 21,134 DBLα types into upsA or upsB/upsC by translating the DBLα types and then characterizing them as upsA or upsB/upsC according to their domain class  in comparison to the curve for upsB/upsC DBLα types, indicating an approximation of the total upsA DBLα types in the total Ugandan population  by Chao2 statistics, 44,092 types by ICE statistics and 42,207 types by Jackknife2 statistics  to a maximum of 262 DBLα types per isolate  the varying transmission intensities across Uganda and (ii) the number of DBLα types per isolate (i.e. DBLα repertoire size) Fig. . The meaP. falciparum genome, PTS is a robust estimator of relatedness between isolates12. The presence of highly related genomes that share many DBLα types would be evidenced by high PTS scores between isolates. PTS among the 501 isolates resulted in 125,250 pairwise comparisons, of which 13,245 (10.6%) shared no DBLα types  comparisons to examine the overlap of DBLα repertoires between isolates within and among the sentinel sites. Briefly, the DBLα repertoire in one isolate was compared to the DBLα repertoire in all other isolates within a site and among all sites. As mentioned above, 75.0% of isolates contained more than one parasite genome. Previous studies have shown that even when a parasite isolate contains more than one var DBLα types we examined repertoire overlap based on the upsA and upsB/upsC DBLα types separately, as upsA DBLα types are expected to be more conserved relative to upsB/upsC DBLα types24. It is important to note that as expected the repertoire sizes being compared were smaller for the upsA comparisons as the upsA DBLα types comprised, on average, only 16% of the repertoires. For the upsA DBLα types the median and mean PTS scores were 0.06 and 0.06 respectively among all sites , with a maximum PTS score of 1 among all sites   by bird’s flight and −0.72 (p = 0.003) by road , and indicate a negative correlation between distance and DBLα type sharing. Increasing the distance between sentinel sites decreases the sharing of DBLα types. Despite these trends, there was conservation of a large number of DBLα types, as defined by 96% sequence identity, across Uganda with 1,995 DBLα types seen in at least three sentinel sites and 277 DBLα types seen in all sites.To assess the structuring of DBLα types on a geographic scale, we investigated the number of shared DBLα types between the sites. There was substantial sharing of DBLα types between sites regardless of the distance between them Fig. . The PTSoad Fig. . The resp < 0.001). This is not altogether surprising, since upsA DBLα types have been reported to be more conserved, possibly due to balancing selection24.To take into account the patterns observed related to the upsA or upsB/upsC DBLα types, we investigated the number of shared DBLα types between sites independently for DBLα types classified as upsA and upsB/upsC. The same trend was observed for both upsA and upsB/upsC DBLα types with a negative correlation between distance and type sharing between sites, with sites farther apart sharing fewer DBLα types  with defined disease outcomes, especially severe disease, in case control studies34. Unlike previous studies we have asked a different question: looking at the population genomics of var genes in cases of uncomplicated malaria, we set out to determine if there was any evidence of a selective sweep of specific var genes or epidemic structure related to the incidence of uncomplicated disease in children.var gene sequences encoding the Pf EMP1 DBLα domain of P. falciparum causing uncomplicated malaria. This was examined in six sites across Uganda with varying malaria transmission intensities, from low  to high . Surprisingly, even in low transmission sites we found no evidence of a selective sweep of any specific var DBLα types in the parasites causing uncomplicated malaria, even after stratifying by both the upsA and upsB/upsC DBLα types. In fact, a key genetic feature of these parasites was that they all showed minimal overlap with respect to their DBLα repertoires suggesting that there is selection for var diversity and heterogeneity at the DBLα repertoire level in this population. Indeed, there was no particular common genetic signature indicative of clonal propagation or epidemic expansion of related P. falciparum parasites causing uncomplicated malaria as has been reported in Senegal45 where, in comparison to our study sites, malaria control has been intense.We investigated the diversity and population structure of var genes, consistent with Chao estimates of DBLα diversity from previous limited sampling that we completed in African sites with high transmission13. Paradoxically, we found high diversity at the DBLα type level yet conservation of individual DBLα types with 40.9% of the DBLα types identified being conserved in Uganda . These striking results would not be predicted by the extremely high rates of mitotic recombination that have been reported from in vitro experiments with culture adapted P. falciparum lines25. Based on the conservation of DBLα types between isolates and across sites, we propose that there are forces structuring the parasite population to maintain these DBLα types in the transmission system. This would be consistent with a mechanism such as balancing selection24.Our analysis of 21,134 unique DBLα types from 501 uncomplicated cases revealed extensive diversity of the DBLα domain of 24. This is likely due to their chromosomal orientation and reduced levels of recombination with other var gene groups to maintain domain architecture47. Interestingly, when looking at the frequency of the DBLα types across all sites, the upsA were ~9x more likely to be seen in 10 or more isolates when compared to the upsB/upsC. This significant pattern was only observed in the high transmission sites (EIR >300) where exposure to malaria is intense. Thus, the assumed increased rates of meiotic recombination in high transmission sites have led to the dispersal of the same upsA DBLα types into a greater proportion of P. falciparum repertoires.Although we observed extensive diversity at the DBLα repertoire level, the proportional representation of upsA and upsB/upsC DBLα types within each repertoire was maintained as expectedet al.23. They explored the population structuring of var genes by simulating the dynamics of all possible var gene combinations given a large and diverse pool of Pf EMP1 variants as well as patterns of immunity in the host population. They showed that despite high levels of outcrossing  there is a defined parasite population structure that exhibits “strain” structuring23. This is consistent with our observations as the P. falciparum parasites causing uncomplicated malaria have distinct, minimally overlapping DBLα repertoires despite varying transmission intensities across Uganda. Furthermore we observed the same pattern locally  and nationally  as predicted by the immune selection model where the same var repertoire structuring would be expected despite different host population sizes23. This is also the same pattern of “strain” structure that we observed in isolates from asymptomatic children in Bakoumba where a clear var population structure with limited overlap in the DBLα repertoires was described15. This structure would enhance the parasites’ ability to evade the host immune response under conditions of high transmission and high recombination rates.Every DBLα repertoire was unique in the 501 uncomplicated malaria cases with minimal overlap of the DBLα repertoires within, between and across all sites. The observed parasite population structure further supports the immune selection model described by Artzy-Randrup 24. They proposed an evolutionary model where parasite phenotypic plasticity could explain population structuring such that there is a trade-off of evolutionary strategies: conservation of var gene domain architecture  serves to optimize parasite fitness and diversity at the var repertoire level maximizes immune evasion. Sequencing results from our study show for the first time both conservation of individual DBLα types (at 96% sequence identity) and yet high repertoire diversity in uncomplicated P. falciparum cases. The evolutionary forces maintaining this pattern of diversity need to be explored further with models that consider the large number of DBLα variants we have seen in nature. There is no question that the need to prolong infection so as to enable transmission requires evasion of the host immune system, which provides a strong selection force to diversify var genes as well as repertoires within a host.A trade-off between maintaining parasite fitness in the host while still achieving successful transmission between hosts , has been described by Buckee and ReckerPf EMP1 variants49. Although we define sequences of the same DBLα type as sharing 96% sequence identity, epitopes can be shared between sequences with a lower identity . Nonetheless, the prevalence of the host variant-specific anti-Pf EMP1 response will be dependent on the extent of Pf EMP1 variant diversity in the parasite population. Hence, acquisition of immunity to the less diverse upsA DBLα types would be expected to occur faster than the ~12x more diverse upsB/upsC DBLα types. Serological network studies by Buckee et al. (2009) propose that immune selection occurs at different levels within the upsA or upsB/upsC groups of the same var multigene family51 and this could influence the acquisition of immunity to specific var gene groups.Many studies have shown that individuals with uncomplicated malaria exhibit a broad range of antibody responses and harbor parasites that express diverse When considering geographic structuring of conserved DBLα types there was significantly higher sharing of upsA DBLα types (range 37–58%) between sites compared to the upsB/upsC DBLα types (range 18–29%) over the large geographic distances sampled in Uganda (~100 km to ~600 km). Paradoxically given this significant conservation of the upsA DBLα types across sites, we still report minimal PTS overlap of DBLα repertoires between isolates. This pattern of PTS was consistent no matter how we partitioned the repertoires per isolate: (i) the entire DBLα repertoire, (ii) only the upsB/upsC DBLα types, and perhaps most interestingly, (iii) even when exclusively examining the upsA DBLα types. This highlights the fact that there was no detectable evidence of linkage between the DBLα types at the isolate repertoire level.var DBLα repertoire level within a host, yet conservation of 40.9% of DBLα types. This overall pattern was observed repeatedly by our sampling at multiple sites in Uganda and was the same regardless of geographic distance or malaria transmission intensity. Whether demographic forces or immune selection are maintaining this pattern remains to be answered. Importantly, the low PTS among var DBLα repertoires show that any parasite with a diverse repertoire of var genes, rather than an epidemic of parasites with related var repertoires, can cause uncomplicated disease.We observed a paradoxical pattern of high diversity and minimal overlap at the parasite The study was reviewed and approved by the Uganda National Council of Science and Technology and by the institutional review boards/human ethics committees of Makerere University , University of California , New York University School of Medicine , and The University of Melbourne . Written informed consent was obtained in the local language from the parent/guardian(s) for the children enrolled in this study and for the future use of the biological specimens. Parent/guardian(s) of the children were asked to volunteer and were not coerced. The researchers in the study had no professional relationship with the individuals recruited for this study. Biological specimens collected consisted of dried blood spots on filter paper collected by trained personnel at the hospital. The consent process was consistent with the ethical expectations at the time of enrollment and the ethics committees approved these procedures.52. Briefly, after obtaining informed consent from all individuals and/or their parents/guardians, the participants’ age and sex were recorded and a finger-prick blood isolate was obtained for a thick film blood smear, a rapid diagnostic test (RDT) and a dried blood spot (DBS) for molecular testing. From the 1000 outpatients who were enrolled at each of the six sentinel sites across Uganda (N = 6000), isolates from 100 children between 6 months and 5 years from each of the six sentinel sites (N = 600) with a microscopy confirmed P. falciparum infection were included in this analysis. For the purposes of this study all children who tested positive for P. falciparum by microscopy, were febrile (≥37.5 °C), and showed no additional symptoms to indicate severe disease when enrolled were defined as having an “uncomplicated P. falciparum infection”.The study was performed in six independent sentinel health centers established by the Uganda Ministry of Health. Malaria endemicity is widely variable across Uganda and the six sentinel study sites were chosen to represent the geographic diversity of malaria transmission intensity: based on the annual entomological inoculation rates (EIR) Kanungu, Jinja and Kyenjojo experience a low to moderate transmission  whereas the study sites Arua, Tororo and Apac experience relatively higher transmission intensities . A cross-sectional survey was used to consecutively enroll 1000 outpatients per sentinel site who had been referred to the laboratory for a diagnostic malaria blood smear in accordance with the standard of care for fever case management between May 2006 and February 2007. Details on the study population and data collection procedures have been published elsewhereGenomic DNA from each DBS isolate was extracted using the QIAamp DNA Mini Kit  according to the manufacturer’s instructions.P. falciparum var genes from genomic DNA were amplified using the DBLα domain as previously described with modifications13. From each isolate of genomic DNA, a ~550–700 bp region of the DBLα domain was amplified using a degenerate primer set  designed against the semi-conserved blocks B and H of DBLα8. Each of the DBLα primers were designed by adding a GS FLX Titanium primer sequence 10 bp multiplex identifiers (MID) published by Roche53. These MID primers were used to ‘barcode’ and distinguish the DBLα sequences amplified from a unique isolate once all isolates were pooled and sequenced together54. The PCR conditions for the DBLα amplification were as follows: 2 μl of isolate genomic DNA, dNTPs at a final concentration of 0.07 mM, each primer (forward and reverse with same MID combination) at a final concentration of 0.375 μM, MgCl2 at a final concentration of 2 mM, 1x reaction buffer, and 3 units GoTaq Flexi polymerase (Promega) in a 40 μL total reaction volume. PCR cycling was carried out on an Eppendorf thermal cycler and involved an initial denaturation step of 95 °C for 2 min, 30 cycles of 95 °C × 40 sec, 49 °C × 90 sec, and 65 °C × 90 sec, followed by a final extension step of 65 °C for 10 min. Finally the isolate amplicons were pooled and sequenced using next generation 454 sequencing (Roche) performed at NYU School of Medicine at the Center for Health Informatics and Bioinformatics and the Memorial Sloan-Kettering Cancer Center Genomics Core Laboratory. The 454 sequencing provides average read lengths of 400 bp, therefore lending itself to the assembly of the individual var DBLα amplicons of 550–700 bp lengths using the forward and reverse sequence reads from each direction.The http://www.cbs.dtu.dk/services/MultiPass-1.0, and the following cleaning steps described below are implemented in a python script available here: https://github.com/454data/postprocess. The sff-files obtained from each region on the 454-plate were divided into smaller isolate specific sff-files by identification of reads with exact matching MID sequences in both ends using BioPython v1.57. Ambiguous primer sites were then identified (exact match) and trimmed off the flowgrams, reverse reads were reverse complemented, and a dat-file (AmpliconNoise format) with the resulting flowgrams was created for each isolate, using BioPython v1.55. By combining the forward and reverse reads this method takes advantage of bi-directional amplicon sequencing, since the forward reads will have highest quality in the 5′-end of the target sequence, and the reverse reads will improve the 3′-end quality. Flowgram clustering was performed using PyroDist, FCluster and PyroNoiseM from the AmpliconNoise package v1.2556. The flowgram clusters produced by AmpliconNoise were base called using Multipass to obtain the most likely var DBLα sequences given the flowgrams and a high open reading frame likelihood, as described in ref.57. The nucleotide sequences generated by Multipass were clustered by 96% identity using Usearch v5.2.3258 with seeds (cluster member with support from highest number of reads after de-replication) as output. Chimeras were removed using Uchime implemented in Usearch v5.2.3259, first in de-novo mode where chimera detection is based on read abundance, all parents are expected to be present in the sequence set, and candidate parents must be at least 2x more abundant than the chimera candidate sequence; subsequently in database mode, where sequences are searched against self and chimeras are found irrespective of the abundance of the parents. To increase overall quality of the sequences remaining at this point, a minimal coverage threshold of three reads per sequence type was applied to remove the least supported sequences. Next, we screened for and removed non-target amplified human sequences by local alignment search against the BLAST human genomic databases (http://ftp.ncbi.nlm.nih.gov/blast/db/) using the blastn feature of BLAST+ 2.2.25 (NCBI), with expectation value criteria of 1e-50. Sequences were also searched using a DBLβ HMM with HMMer v3.1 (hmmer.org). After the human and non-target P. falciparum check, a small number of sequences remained that had no similarity to a DBLα-tag HMM and these were removed. The pipeline was validated and optimized on experimental sequence data generated on the laboratory clones  for which published genome sequence is available. More than 90% of the sequences obtained from the control samples had no errors when compared to the known reference, and the deviating sequences had maximally 5 errors. To subsequently determine DBLα types shared between isolates, the cleaned DBLα reads were clustered using a pipeline based on the USEARCH software suite version 8.1.183158. Initially duplicate reads were removed and the remaining reads were sorted by how many duplicates were present using the derep_prefix command. The remaining reads were then clustered at 96% pairwise identity using the usearch cluster_fast command. Finally, the original unfiltered reads were aligned back to the centroids of the clusters and an OTU table was generated using the usearch_global command before a binary version of the table was generated. The code for the pipeline is available on GitHub at https://github.com/UniMelb-Day-Lab/clusterDBLalpha.A custom pipeline was developed to de-multiplex, de-noise and remove PCR and sequencing artefacts from the DBLα domain reads. The first part of the pipeline is available as the Multipass web server: 60 to estimate var DBLα richness by sampling all DBLα types from each sentinel site and among all sites without replacement. All sampling depths between one and the number of DBLα types obtained from the study site were repeated 100-fold, whereupon the mean number of var DBLα types obtained for each sampling depth was calculated. The cumulative diversity curve was plotted using R v3.1361 to plot the number of var DBLα types as a function of the number of var DBLα sequences sampled.The cumulative diversity curves, analogous to species accumulation, were generated using EstimateS v9.1Frequency plots were tabulated using the data generated and plotted using Microsoft Excel.60 the diversity of DBLα types within and among all sites in Uganda were calculated by estimating the total number of DBLα types and the proportion of DBLα types shared between isolates. For each sentinel site two different statistical estimates of richness were used, non-parametric statistical Chao2 and incidence-based coverage estimator (ICE). Chao2 calculations were used to estimate the total number of DBLα types in a particular sentinel site based on singletons and doubletons63. ICE statistics were used to estimate the total number of DBLα types in a study site based on all the observed types by dividing them into two groups: rare and abundant types64. Jacknife2 calculations were used to estimate the total number of DBLα types in a particular sentinel site by resampling the unique and duplicate types, thereby reducing bias41.Using EstimateS v9.113 and is a useful statistic to analyze diversity and determine the number of DBLα types shared between isolates. Briefly, PTS is calculated as follows:For each sentinel site, pairwise type sharing (PTS) statistics were calculated to quantify the relatedness between the DBLα repertoires identified from two isolates. This methodology has been published elsewhereto estimate the mean DBLα type sharing between two sentinel sites. PTS scores between sites were calculated as follows:https://www.google.com/maps/preview) and transit/road distance was calculated using the Via Michelin Maps and Routes application (http://www.viamichelin.com). Spearman’s rank correlation coefficient was calculated to evaluate the trend and was performed using IBM SPSS (Version 22) software.PTS statistics were calculated et al.11 were aligned to the translated sequences using HMMER v3.1b1 with an e-value of 0.01. Both the bias composition correction and composition filter were turned off as suggested in Rask et al. (–nonull2 —tobias)11. The most significant match to any of a reads six translated frames was then taken as the domain assignment for that read. Reads were further classified into upsA if their most significant match was with a DBLα1 domain and upsB/upsC  if that matched with either DBLα0 or DBLα2 domains. The domain models did not allow for an accurate distinction between var groups B and C. Cross validation was used to ascertain the success of this approach with the classification accuracy of upsA and upsB/upsC combined found to be 96.2% and 99.8%, respectively  software. A test was deemed to be statistically significant if the p-value was less than 0.05. For all analyses the sentinel sites  were categorized as defined in the study design. Chi-squared test (χ2) was used for univariate analyses of discrete variables to compare proportions.Statistical analyses were carried out using IBM SPSS Statistics (Version 22) software and the open source EpiInfo v7 .Supplementary Information


Scientific Reports7: Article number: 4113310.1038/srep41133; published online: 01242017; updated: 02242017This article contains typographical errors in the ‘Methods’ section under subheading ‘Stimuli’.“All face images measured approximately 7 × 10 cm and were shown in full-face view with a neutral expression . A complementary set of 120 inverted face arrays were created for the control condition by turning the stimuli upside-down .The stimuli for the object-matching task were adapted from the Matching Familiar Figures Test (MFFT). This test consists of 20 line drawings of common objects, which act as targets and are presented above six variants of the same object, only one of which is exactly identical to the target image ”.should read:“All face images measured approximately 7 × 10 cm and were shown in full-face view with a neutral expression . A complementary set of 120 inverted face arrays were created for the control condition by turning the stimuli upside-down .The stimuli for the object-matching task were adapted from the Matching Familiar Figures Test (MFFT). This test consists of 20 line drawings of common objects, which act as targets and are presented above six variants of the same object, only one of which is exactly identical to the target image ”.


Photocatalytic activity of semiconductor nanostructures is gaining much importance in recent years in both energy and environmental applications. However, several parameters play a crucial role in enhancing or suppressing the photocatalytic activity through, for example, modifying the band gap energy positions, influencing the generation and transport of charge carriers and altering the recombination rate. In this regard, physical parameters such as the support material and the irradiation source can also have significant effect on the activity of the photocatalysts. In this work, we have investigated the role of reduced graphene oxide (RGO) support and the irradiation source on mixed metal chalcogenide semiconductor (CdS–ZnO) nanostructures. The photocatalyst material was synthesized using a facile hydrothermal method and thoroughly characterized using different spectroscopic and microscopic techniques. The photocatalytic activity was evaluated by studying the degradation of a model dye  under visible light (only) irradiation and under natural sunlight. The results reveal that the RGO-supported CdS–ZnO photocatalyst performs considerably better than the unsupported CdS–ZnO nanostructures. In addition, both the catalysts perform significantly better under natural sunlight than under visible light (only) irradiation. In essence, this work paves way for tailoring the photocatalytic activity of semiconductor nanostructures. Many ofllutants –8. Theseal acids –10. Thusal acids , ozonizaal acids  and adsoal acids , as thesacids [2 –16.Zinc oxide is a well-known semiconductor with a band gap energy of 3.37 eV and has been widely explored as photocatalytic material due to its non-toxic nature, high exciton binding energy (60 meV), photosensitivity and stability on exposure to high energy radiation . Due to We have focused our study on the coupling of ZnO nanorods with nanoparticles of CdS, a semiconductor active under visible light, to form coupled CdS–ZnO heterojunction nanostructures. The CdS nanoparticles are an attractive photocatalytic material for visible-light harvesting due to the narrow band gap (2.42 eV) . But fas2-hybridized carbon atoms with hexagonal packed lattice structure [2·g−1) [−1·K−1) [2·V−1·s−1), which makes it an ideal material for photocatalyst support [2–RGO support to improve the photocatalytic performance of ZnO nanoparticles. However, the role played by RGO support in enhancing the photocatalytic performance of the nanocomposites has not been fully explored. Furthermore, the photocatalytic activity is also influenced by the irradiation source. With regard to this, we focused our studies on determining the role of the RGO support and the irradiation source on the photocatalytic activity of CdS–ZnO semiconductor nanostructures. Another unique aspect of this work is the formation of efficient binary and ternary heterojunctions having nanoparticles (NP), nanorods (NR) and nanosheets (NS), comprising of CdS, ZnO and RGO, respectively. In this work, , the preparation and detailed characterization of binary and ternary nanocomposites are presented and their photocatalytic activity have been demonstrated with respect to the degradation of methyl orange (MO) dye, both under visible light (only) irradiation from a solar simulator and natural sun light. The obtained results have been discussed in detail, and the role of RGO support and irradiation source on the photocatalytic activity of CdS–ZnO nanostructures has been elucidated.The CdS–ZnO semiconductor nanostructures can be further supported on graphene/reduced graphene oxide (RGO) materials to improve their photocatalytic properties. Ideally, graphene is a single layer carbon sheet, which consists of a two dimensional (2D) network of sptructure . Graphen [2·g−1) , chemica[−1·K−1) . Its opt support . Several support –35. In o support , we have3), sulfuric acid (H2SO4), potassium permanganate (KMnO4) and hydrogen peroxide (H2O2) were purchased from Merck. Zinc chloride (ZnCl2), sodium hydroxide (NaOH), cadmium acetate dihydrate (Cd(OOCCH3)2·2H2O), sodium sulfide (Na2S), ammonia solution and methyl orange were also supplied by Merck. Polyvinyl pyrrolidone (PVP) used in synthesis was purchased from Sigma-Aldrich. All chemicals were used as received without further purification. Deionized water (18.2 MΩ·cm) used in synthesis was obtained from a double-stage water purifier (ELGA PURELAB Option-R7).For the synthesis of GO graphite powder  was purchased from Alfa Aesar, whereas sodium nitrate , which increased the temperature to 95–98 °C and resulting suspension was maintained at this temperature for 15–20 min; subsequently, the suspension was then diluted to about 250 mL by the addition of deionized water. This is followed by the addition of 10 mL H2O2 to remove unreacted KMnO4 in the reaction mixture. In order to remove the ions of oxidant origin, the mixture was washed with 10% HCl and then with deionized water until pH value of the filtrate was neutral. Obtained graphite oxide was subjected to ultrasonication for its exfoliation followed by centrifugation at 4500 rpm for 15 min. The final product was obtained by drying with rotary evaporator at 40 °C followed by vacuum drying overnight at same temperature.Graphene oxide (GO) was synthesized from natural graphite flakes using a modified Hummers’ method . As desc2) solution in ethanol was added into 70 mL of 0.5 M sodium hydroxide (NaOH) solution dropwise under vigorous stirring. This was followed by ultrasonic treatment of the solution for 30 min for homogenization. Then, this homogenous solution was transferred to a 100 mL teflon-lined stainless steel autoclave, sealed tightly and maintained at 180 °C for 12 h. White precipitates of ZnO NR were collected by centrifugation and washed several times with deionized water and ethanol and finally dried at 60 °C.ZnO nanorods (NR) were synthesized through a previously reported solvothermal method . In brie3)2·2H2O) (0.2 M) was prepared in deionized water. To this solution, 20 mL of sodium sulfide (Na2S) solution (0.2 M) was added dropwise under continuous stirring. After 10 min of stirring, 0.5 g of PVP was added as capping agent under vigorous stirring. The pH value of the solution was maintained at around 10 by adding ammonia solution. The resultant solution was refluxed for about 1 h at 70 °C. Upon completion of the reaction, the product was washed with deionized water and ethanol thrice and finally yellow colored CdS NP were formed after drying in an oven at 80 °C for 2 h.CdS nanoparticles were synthesized as per a previously reported method . In a ty2 was dispersed in 40 mL deionized water, followed by the addition of 0.5 M NaOH solution dropwise with continuous stirring. Aqueous ammonia was added to maintain the pH value around 8. Finally, Cd(OOCCH3)2·2H2O (0.2 M) and 4 mL of thioglycolic acid (0.2 M) was added into 40 mL of above solution under vigorous stirring. Subsequently, this homogenous suspension was transferred to 50 mL teflon-lined stainless steel autoclave and kept at 140 °C for 48 h. After the completion of reaction time, the product was collected by centrifugation and washed with ethanol and deionized water thrice, and dried in vacuum. Based on the molar ratio of the precursors used in the synthesis, the ratio of CdS to ZnO is expected to be 1:1 in this binary nanocomposite.CdS–ZnO binary nanocomposite was prepared by employing a reported hydrothermal strategy . In shorFor the synthesis of the CdS–ZnO–RGO ternary nanocomposite, about 0.01 g of as prepared GO was dispersed in ethanol by ultrasonication. Then, 0.2 g of previously prepared CdS–ZnO was added to the GO solution under vigorous stirring for 2 h to obtain homogenous suspension. Once homogenization is achieved, the suspension is transferred to teflon-lined stainless steel autoclave and kept at 120 °C for 24 h. The desired product was obtained after washing with water and dried at 60 °C. Based on the molar ratio of the precursors used in the synthesis, the ratio of CdS to ZnO is expected to be 1:1 while having 1 wt % of RGO in this ternary nanocomposite.−5 M). Initially, the suspension was magnetically stirred in the dark for 30 min to attain adsorption–desorption equilibrium. Subsequently, the suspension was continuously stirred under visible light irradiation. For one set of experiments, a solar simulator  having a UV cut-off filter (λ > 420 nm) was used for visible light illumination. Another set of experiments were performed under natural sunlight and the light intensity was measured using a LX-101A digital luxmeter. At periodic time intervals, the photo-reacted suspension (ca. 1 mL) was analyzed by recording the absorbance using a UV–vis spectrophotometer.Photocatalytic activities of as prepared photocatalysts were evaluated by monitoring the decomposition of MO dye at room temperature. Typically, 10 mg of photocatalysts (CdS–ZnO or CdS–ZnO–GO) was added to 50 mL of the aqueous solution of MO (10−1.The UV–visible absorption spectra of the samples were recorded using Shimadzu UV-2450 spectrophotometer in the wavelength range from 200 to 800 nm. Fourier transform infrared (FTIR) spectra were collected using Agilent K8002AA Carry 660 FTIR instrument. Optical properties were analyzed by UV–vis diffuse reflectance spectroscopy (DRS) using a Perkin Elmer UV/VIS/NIR Lambda 750 spectrophotometer, in which polytetrafluroethylene (PTFE) polymer was employed as internal reflectance standard. Morphology of the samples was characterized using a field-emission scanning electron microscope (FESEM) JFEI Nova Nano SEM-450 and a high resolution transmission electron microscope (HRTEM) FEI Tecnai G2 20 S-twin microscope operating at 200 kV. Energy dispersive X-ray spectroscopy (EDAX) was obtained using the same HRTEM instrument. X-ray diffraction (XRD) measurements were done using the Agilent Supernova X-ray diffractometer using Ni-filtered Cu Kα irradiation (λ = 0.1542 nm) at 45 kV and 40 mA in 2θ ranging from 5 to 80° with a scan rate of 2°·minIn order to investigate the crystalline phase of as prepared nanocomposites, powder X-ray diffraction (XRD) analysis was performed. The optical properties of all prepared samples were analyzed by using UV–vis diffuse reflectance spectroscopy (DRS), the results of which are presented in The surface morphology of all pristine materials as well as the binary (CdS–ZnO) and ternary (CdS–ZnO–RGO) nanocomposites was investigated by scanning electron microscopy (SEM). Transmission electron microscopy (TEM) analysis was performed to obtain further information on the surface morphology and microstructures of all prepared nanostructures . The TEM−1 and 1404 cm−1, which could be attributed to the O–H stretching vibrations and deformation vibrations of intercalated water [−1, 1229 cm−1 and 1725 cm−1 are the characteristic stretching vibrations of C–O, epoxy C–O, and C=O of carbonyl groups, respectively, which are present at the edges of the GO sheets [−1 could be attributed to the aromatic C=C stretching vibrations in GO [−1 and 1424 cm−1 could be attributed to O–H stretching and deformation of C–OH groups of water molecules. Zn–O bond stretching vibrations appears at 504 cm−1 [−1 is attributed to the C–N stretching vibration of the PVP monomer, which was used as capping agent [−1 peak corresponds to the C–H bond of PVP [−1 range and Zn–O stretching vibrations between 500 and 600 cm−1. The peak at 1394 cm−1 in the binary composite is assigned to C–H bonds of the capping agent. It is clear from the FTIR spectrum of CdS–ZnO–RGO nanocomposite that characteristic peaks of oxygen containing functional groups particularly at 1725 cm−1 are weakened and the O–H stretching peak decreases with some red shift. This is mainly attributed to the loss of oxygen containing functional groups and the reduction of GO to RGO after the hydrothermal treatment [FTIR spectra for all samples are presented in ed water . AbsorptO sheets . The absns in GO . Similar504 cm−1 . FTIR spng agent . The 140d of PVP . CdS–ZnOreatment .max value and shows two absorption peaks with maximum at 227–230 nm due to aromatic π→π* transition and a small shoulder at 303 nm due to the n→π* transition of carbonyl groups [l groups . The absl groups . UV–vis l groups . The UV–l groups . EnhanceThe photocatalytic performance of CdS–ZnO binary and CdS–ZnO–RGO ternary nanocomposites is evaluated by measuring the photodegradation of methyl orange (MO), as model dye, under visible light irradiation from a solar simulator or under natural sun light. Prior to illumination, the suspension was equilibrated in dark for 30 min. During this period adsorption and desorption equilibrium was achieved between the photocatalyst and MO. This is followed by illumination either under solar simulator or under natural sun light. The degradation of MO was studied by measuring the concentration of MO with a UV–vis spectrophotometer at regular intervals of time. The UV–vis spectra are shown in The degradation efficiency of both the photocatalysts was evaluated on the basis of initial and final concentration of the dye by monitoring the main absorption peak (λ = 454 nm) of MO. So degradation rate of the MO can be calculated by applying following equation :At and A0 are the absorbance at reaction time t and t = 0, respectively.where 4 lux. Substantial improvement in the photocatalytic activity can be observed for both binary and ternary nanocomposites as shown by the histogram in In accordance with the above equation, with CdS–ZnO–RGO ternary photocatalyst about 98% of dye was degraded in 90 min, under visible light illumination from a solar simulator, but with CdS–ZnO binary photocatalyst, only about 70% of dye was degraded. The degradation of the same concentration of MO was also investigated under natural sunlight illumination having an intensity of 9.5 × 10The possible mechanisms of the photocatalytic activity of CdS–ZnO–RGO ternary nanocomposite for degradation of MO under visible light irradiation from a solar simulator and natural sun light are illustrated pictorially in ECB = −0.66 eV vs NHE, which is more negative than the ECB of ZnO [2/O2− (−0.13 V vs NHE) but more negative than the redox potential of O2/H2O2 (+0.695 V vs NHE) [2 and H+ ions to produce H2O2, which further decomposes in the presence of light to generate hydroxyl radicals (•OH) [•OH/OH− (2.38 eV vs NHE) and •OH/H2O (2.72 eV vs NHE), •OH cannot be generated by reacting with H2O molecules. The •OH radicals are responsible for the dye degradation into CO2 and H2O [Under visible light illumination, electron–hole pairs are generated in conduction band (CB) and valence band (VB) of CdS. The CB potential of CdS are B of ZnO –63. ThisB of ZnO . The chaB of ZnO . The worB of ZnO . Thus ells (•OH) . Also Zn and H2O –69. The  and H2O –71. The Overall, the improved photocatalytic activity of the ternary nanocomposites could be mainly attributed to the better adsorption capacity of graphene, rapid charge transfer at the semiconductor interface and then to graphene, which degrade the adsorbed dye on its surface. This whole proposed mechanism can be formulated as ,73–74,Furthermore, In this work, we have prepared and thoroughly characterized CdS–ZnO semiconductor nanostructures both with and without RGO support. Their photocatalytic activity towards the degradation of methyl orange dye, has been investigated both under visible light irradiation from a solar simulator and under natural sunlight. The obtained results show the significant role played by the RGO support and the source of irradiation on the photocatalytic activity of the mixed metal chalcogenide nanocomposites. The RGO-supported CdS–ZnO nanocomposites exhibits considerably better photocatalytic activity compared to its unsupported counterpart, which could be attributed to the enhanced photo-generated charge separation, facile charge transfer and strong adsorption of dye on to RGO. In addition, superior photocatalytic activity was observed for the nanocomposites irradiated under natural sunlight than visible light from solar simulator. This could be ascribed to the higher generation of electron–hole pairs, their effective separation and rapid transport to the reaction site. In this case, both the semiconductors are active, in their respective wavelength domains, as sunlight is comprised of both UV and visible light regions. This work not only demonstrates the role of the RGO support and irradiation source on the activity of photocatalysts, but also paves way for tailoring the photocatalytic activity of semiconductor nanostructures in general.


Patients were divided into two groups (HF and non-HF) based on left ventricular ejection fraction and plasma proBNP level. After CABG, 67% of patients developed HF. The mean distance 1 month after CABG in HF was 397 ± 85 m versus 420 ± 93 m in non-HF. PPARγ mRNA expression was similar in both HF and non-HF groups. 6MWT distance 1 month after CABG was inversely correlated with PPARγ level only in HF group. Higher PPARγ expression was related to smaller LVEF change between 1 month and 1 year , especially in patients with HF. Higher initial levels of IL-6 in HF patients were correlated with longer distance in 6MWT one month after surgery and lower PPARγ expression. PPARγ expression is not related to LVEF before CABG and higher PPARγ expression in the myocardium of patients who are developing HF following CABG may have some protecting effect.Activation of PPARs may be involved in the development of heart failure (HF). We evaluated the relationship between expression of PPAR Heart failure (HF) is characterized by reduced reserve of the cardiac output. Impaired functional capacity in patients with heart failure is common and results from inability to achieve sufficient oxygen and nutrients delivery and altered washout of metabolites from working muscles.α or IL-6, which is characteristic for chronic HF, may be responsible for altered muscular structure and function . Informed consent was obtained from all participants.p < 0.05 was considered statistically significant. Analysis was performed using Statistica 9.0PL.Data are presented as mean ± SD for quantitative variables or percent of study group for qualitative variables. Specific parameters of both groups (group with and without heart failure at baseline) and change in parameter values during follow-up were compared using chi square test and ANOVA with post hoc analysis. Correlations between variables were tested using Pearson's method. A value of p = 0.002) and 24 months after CABG it was significantly longer than in HF group . However the distance of 6MWT one month after CABG was inversely correlated with myocardial PPARγ level  only in patients developing HF during the follow-up and was not present in those without HF (γ gene in the myocardium was not correlated with left ventricular ejection fraction (LVEF) before or after CABG . The relation described was more pronounced in those patients, who developed HF during the follow-up  and was not significant in patients without HF (p < 0.0001 for the trend). Before CABG there was negative correlation between concentration of IL-6 in plasma and PPARγ expression . In patients who developed heart failure higher initial levels of IL-6 were correlated with longer distance in 6MWT one month after surgery .157 patients were qualified to the study. All patients did not have heart failure before CABG. After 1 month of CABG, 67% of patients developed heart failure. During 2-year follow-up the number of patients has been reduced in 1 year to 124 and in 2 years to 86 because of the loss of connection. In the HF group one patient died because of myocardial infarction in 1 year after CABG. The baseline characteristics of the study group were presented in the previous paper . One monthout HF . Althougter CABG , higher thout HF . The conThe six-minute walk test (6MWT) is a submaximal exercise test for evaluating physical functional capacity. Fiorina et al. suggest that 6MWT is feasible and well tolerated in adult and older patients shortly after uncomplicated cardiac surgery and provides reference values for distance walked after cardiac surgery . In our γ expression and cardiac function or exercise capacity after CABG is very scant. In addition existing experimental data present contrasting results about the influence of PPARγ on cardiac function. On one hand, overexpression of PPARγ in transgenic mice was reported to evoke accumulation of lipids and glycogen and distortion of mitochondrial architecture leading to dilated cardiomyopathy [γ with its agonist reduced lipotoxicity and improved cardiac function [γ in the myocardium of anthracycline-induced cardiomyopathy in rodents, and treatment with an angiotensin converting enzyme inhibitor restored normal level of PPARγ but was not sufficient to restore normal cardiac function [Literature describing potential links between myocardial PPARmyopathy . On the function . Expressfunction . Cerneckfunction .γ expression at the time of CABG and left ventricular size or ejection fraction at any time point. The level of PPARγ in the myocardium was not able to predict development of heart failure after CABG, as was shown in our previous work [γ level seems to preserve the systolic function of the left ventricle. On the other hand, also only in patients developing HF, PPARγ expression was inversely correlated with exercise capacity during the follow-up. It should be emphasized that left ventricular systolic function, if not significantly depressed, is not related to the exercise tolerance, but abnormalities of its diastolic function are independently associated with exercise capacity [γ in the myocardium at all. Possible protective effect of PPARγ on the cardiac function may be related to suppression of the excessive inflammatory process and inhibition of apoptosis [α, ET-1, and IL-6, plays a pathogenic role in heart failure [γ expression and IL-6 level only before CABG, when none of patients had features of heart failure. A subgroup of patients who developed HF and had higher initial levels of IL-6 had also better exercise capacity at one-month follow-up, which may reflect the protective role of IL-6 on skeletal muscle physiology, which is more important for skeletal muscle function and exercise capacity than weak PPARγ-related phenomena affecting cardiac muscle.Our data showed no clear relation between PPARous work . Howevercapacity . In our poptosis , 26. Per failure , 28. In  failure , 30 and  failure  that res failure . We founγ in myocardium of patients who developed HF after CABG were correlated with smaller attenuation of LVEF, reduced plasma level of IL-6, and worsening of exercise tolerance.Higher levels of PPARγ expression in the myocardium was not related to left ventricular systolic function before CABG. However higher levels of PPARγ gene transcript in the myocardium of patients who develop heart failure following CABG may have some protecting effect on cardiac contractility, which seem not to be directly related to exercise capacity.These results indicate that PPAR


In this work, a chitosan–gelatin–ferulic acid blend was used in different ratios for preparing novel films that can be used in biomedical applications. Both acetic and formic acid were tested as solvents for the chitosan–gelatin–ferulic acid blend. Glycerol was tested as a plasticizer. The thickness, mechanical strength, static water contact angle and water uptake of the prepared films were determined. Also, the prepared films were characterized using different analysis techniques such as Fourier transform infrared spectroscopy (FT-IR) analysis, X-ray diffraction (XRD), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Acetic acid produced continuous compact surfaces that are not recommended for testing in biomedical applications. The plasticized chitosan–gelatin–ferulic acid blend, using formic acid solvent, produced novel hexagonal porous films with a pore size of around 10–14 µm. This blend is recommended for preparing films (scaffolds) for testing in biomedical applications as it has the advantage of a decreased thickness. Recently, many natural polymeric materials have been recommended for use in treating the loss or failure of organs and tissues . In a tyN-acetyl glucosamine with a β-(1–4) link. It can be obtained from naturally occurring chitin through N-deacetylation  [3+) depends on its content: the amount of free amino and N-acetyl groups. Chitosan is known for its antimicrobial properties against fungi and bacteria. This is attributed to the cationic nature of the amino groups in chitosan that are associated with the anions in the bacterial cell walls. Their association suppresses biosynthesis and disrupts mass transport across the cell wall leading to the death of bacteria cells [Chitosan is a polysaccharide of marine origin consisting of glucosamine and with a β-–4 link. ia cells ,5. Otheria cells  proposedwith a β-–4 link. ia cells . Therefoia cells ,9, synthia cells , blendinia cells ,12, or bia cells  and/or tia cells  have beeia cells ,16, whicia cells ,18,19,20ia cells , solventia cells , gelatioia cells ,24, wet ia cells , particlia cells , compresia cells , melt spia cells ,29 and fia cells ,31,32.Crosslinking is an effective tool for amplifying the characteristics of chitosan-based biomaterials. Typically, the mechanical strength and biostability can be enhanced by crosslinking treatment ,2,33. ThGelatin is a protein that is obtained through a controlled denaturing of collagen, the major component of skin, bone and connective tissue . HoweverA chitosan–gelatin polyelectrolyte complex hydrogel membrane that shows improved tensile strength and elongation in the swollen state has been reported . These i3∙g−1) [The post-treatment (stabilization) of chitosan polymer/fibers using an ethanol–water mixture is recommended for several reasons. First, an ethanol–water mixture works as a non-solvent for charged polysaccharides . For exa3∙g−1) . This di3∙g−1) .In this study, chitosan was selected to be the base material due to its known antimicrobial properties. However, to mitigate its drawbacks mentioned earlier, gelatin was selected both as a blending material to decrease the content of chitosan and as a porogen to create pores by being dissolved out in ethanol–water solution (70–30 wt %). To ensure formation of stable films, ferulic acid was selected as a crosslinker. It was also selected for its natural antioxidant properties, which make it very safe for biomedical applications. Moreover, glycerin was selected to be a natural plasticizer for the prepared nature (green) film/membrane. The aim of this study is to investigate a novel, simple method for preparing a chitosan–gelatin film/membrane (polymer matrix) suitable for biomedical applications using acetic or formic acid solvents. The effect of the solvent (acetic or formic acid) used in polymer solution and its effect on the shape and properties of the produced films/membranes will be highlighted. Ferulic acid was used as a crosslinker in the chitosan–gelatin blend. Thermal, spectroscopic and mechanical properties of the chitosan–gelatin–ferulic acid plasticized and non-plasticized film/membrane (polymer matrix) were evaluated to determine if they are suitable for future testing for biomedical applications.Mw 100,000–300,000), ACROS ; Gelatin from bovine skin, type B, Sigma Aldrich ; Ferulic acid (≥99.5%), Fluka ; Glycerin (>98%), Oxford Laboratory Reagent ; acetic acid (99.9%), formic acid (85%) and ethyl alcohol (95%), Laboratory Chemicals . Chemicals were used as received. Distilled water was used in all the experiments. The chemical structure of chitosan, gelatin and ferulic acid is shown in Chemicals were obtained from the following sources: Chitosan (w/w) polymer (chitosan or gelatin) in 1 wt % acetic acid or 1 wt % formic acid solution at 50 °C for 1 h. Of each solution, 50 mL were mixed at 50 °C for 1 h. Ferulic acid was added as a crosslinker in specific amounts (1.5–6 mM) and mixed at 50 °C for 1 h. The film-forming solutions were left standing for 2 h at 40 °C, then were poured on to preheated glass petri-dishes and left to dry for 24 h at 40 °C. To plasticize the film, glycerin  was added to the film-forming solution and mixed at 40 °C for 1 h, before pouring as previously described. The dried films/membranes were peeled off the plate by soaking in ethanol (80%) for 30 min, then the films were left to dry overnight in the atmosphere for characterization.The chitosan films were prepared using the solvent evaporation technique. Chitosan and gelatin solutions were prepared separately by dissolving 2% , the average value was calculated from 10 measurements of two different films/membranes (5 measurements per film) that were prepared from two independent polymer solutions for each condition.The water uptake (wt %) of the prepared films (scaffolds) was determined as follows: the weights of the samples were determined after drying for 24 h in an oven at 50 °C. The samples were then completely soaked in a specific amount of deionized water for the absorbing process for 3 h. The swollen films were taken out of the water and the excess water on the surface was wiped off with tissue paper until a constant weight was obtained. The water uptake, wt %, of films was calculated as the difference between the wet and dry weights relative to the dry weight (see Equation (1)). Four samples from two films/membranes, which were prepared from two independent polymer solutions, for each condition were used.The static angle of water molecules on each prepared film was determined using a Krüss DSA 100 apparatus . Drops of demineralized water (7 µL) were deposited on different spots on each film. Four different spots on each of the two different independently prepared films/membranes were measured and results were averaged. All the tested membranes were kept in desiccators for 48 h before measuring.r = 0.5) with and without the ferulic acid crosslinker. The FTIR spectra of the prepared films/membranes were recorded with an attenuated total reflectance (ATR) accessory. The IR spectra were recorded in the wavenumber range of 4000–400 cm−1.A Fourier transform infrared spectrometer  was used to characterize the prepared pure 2 wt % chitosan and chitosan–gelatin , with and without 3 mM ferulic acid crosslinker and also with added glycerin plasticizer) were obtained on a Shimadzu XRD-7000 X-ray  diffractometer using a CuKα radiation source operating at 40 kV and 30 mA.X-ray diffraction patterns of the polymeric films/membranes made with 1 wt % formic acid  with and without 3 mM ferulic acid crosslinker using either 1 wt % acetic acid or 1 wt % formic acid was carried out using a Thermal Gravimetric Analyzer . The samples were scanned over a temperature range from 20 to 650 °C at a temperature gradient of 10 °C·min−1 under nitrogen flow.TGA analysis of the pure 2 wt % chitosan and the chitosan–gelatin films  with 3 mM ferulic acid crosslinker films/membranes were analyzed with differential scanning calorimetry  at a heating rate of 10 °C·min−1. The samples were kept in a desiccator before analysis.The pure 2 wt % chitosan and chitosan–gelatin , at a constant crosshead speed of 0.1 mm·sr = 0.5) with 3 mM ferulic acid crosslinker using 1 wt % acetic acid and the pure 2 wt % chitosan and the chitosan–gelatin films/membranes  with 3 mM and 6 mM ferulic acid crosslinker using 1 wt % formic acid solvent, were imaged using a Scanning Electron Microscope . The film/membrane (polymer matrix) samples were coated with Au and were imaged at a voltage of 15 KV; both 1000 and 2000 times magnification were used. The pore size of the different tested films/membranes was determined.The pure 2 wt % chitosan and chitosan–gelatin films/membranes  and chitosan–gelatin (r = 0.5) with different concentrations of ferulic acid crosslinker  were prepared using acetic acid solvent (1 wt %) and were used for characterization. As a primary observation, the prepared chitosan–gelatin films/membranes  were homogenous, pale yellow, transparent and slightly brittle in the dry state; whereas, the pure 2 wt % chitosan was rigid and less transparent. The thickness of the prepared pure chitosan increased with increasing chitosan content (The pure chitosan (1 wt % and 2 wt %), chitosan–gelatin . The addition of ferulic acid crosslinker caused a recovery in the strength of chitosan–gelatin  film/membrane to a level that is comparable to the strength of the 2 wt % chitosan film (28 ± 4 N∙mm−2). Although the addition of glycerin improves the elasticity of the prepared films (result not shown here), the prepared films/membranes were still relatively rigid and are, therefore, not suitable for biomedical applications such as scaffolding. Films/membranes that are appropriate for medical purposes should have a softness comparable or similar to soft biological tissues [The water uptake (%) was small (66%) and was gradually increased up to 100% as a result of both increasing the chitosan (2 wt %) content and the addition of ferulic acid crosslinker see . On the  tissues . The fil tissues ,4.r = 0.5) film/membrane with and without 3 mM ferulic acid are shown in 2OH groups [r = 0.5) blend showed similar decomposition ranges but with higher remaining weights at each temperature. About 22% remaining weight at 650 °C may be considered as an indication of the formation of a stronger network due to the reaction between the chitosan and gelatin [r = 0.5).Thermogravimetric analysis (TGA) on pure gelatin film/membrane was previously published ; the weiH groups ,70,71. T gelatin ,71. The r = 0.5), with 3 mM ferulic acid, films/membranes has illustrated the formation of typically extended nonporous (solid) surfaces without any observed difference  relative to the case of using the acetic acid solvent. Most probably, the crosslinking occurs as a result of the ionic interaction between the carboxylic groups of the ferulic acid and the amino groups of chitosan . Also, sThe water uptake (%) was 100% for all the prepared films/membranes as shown in r = 0.5) from 17.7 N∙mm−2 to around 24 N∙mm−2. However, increasing the used amount of ferulic acid did not cause a significant change in the measured strength; the lowest used concentration in this study had a strength of 1.5 mM F  blend films/membrane without and with ferulic acid crosslinker using formic acid solvent are shown in r = 0.5) blend films/membranes without ferulic acid crosslinker, r = 1) film/membrane with and without ferulic acid crosslinker is stronger than the pure 2 wt % chitosan film/membrane (r = 1) film/membrane without ferulic acid crosslinker is weaker than the pure 2 wt % chitosan film/membrane and becomes stronger than the pure 2 wt % chitosan film/membrane after adding the ferulic acid crosslinker. This weakness may be due to the formation of less connected proposed complex/polymer coacervation systems before the solidification process, relative to the solid extend chitosan or the chitosan–gelatin blend when acetic acid solvent was used [TGA of 2 wt % chitosan, chitosan–gelatin  without plasticizer is shown in The DSC analysis is shown in −1 for the hydroxyl group stretching and 3560 cm−1 for the C–N stretching primary amine. The 1602 and 2144 cm−1 peak may be related to the amide II and III bands of chitosan, respectively. The spectrum at 1070 cm−1 can be attributed to the saccharide band in the chitosan film/membrane  of the chitosan and the carboxylate ions (–COO−) of the gelatin. However, the produced pores were not orientated as is the case in the produced films in this study. In another study [r = 1) was comparable to the strength of the film/membrane produced using acetic acid solvent in our study (around 37.91 ± 4.42 MPa). Also, chitosan–gelatin hybrid polymer network scaffolds were prepared via the freeze-drying technique by using the ice microparticles as a porogen [In a previous study , freeze-er study , chitosaer study  and thei porogen . The bilr = 0.5) films/membranes with ferulic acid crosslinker produced solid (nonporous) films/membranes that are not suitable for biomedical applications. However, the prepared films/membranes, in this study, using acetic acid solvent may be recommended for other applications such as packaging purposes.Using acetic acid solvent for preparing chitosan–gelatin –ferulic acid films/membranes made using formic acid solvent produced novel hexagonal porous films/membranes with a thickness of around 60 µm, 10–14 µm pore size and a tensile strength of around 20 N∙mm−2. The produced films/membranes using formic acid solvent are recommended for biomedical application. Testing the produced films/membranes for cell growth suitability and/or the application of a cytotoxicity assay should be considered in future.Formic acid solvent is about 10 times stronger than acetic acid: acidity (pKa) of formic acid is 3.77, whereas for acetic acid it is 4.76. The interactions between gelatin and chitosan are produced by both electrostatic and hydrogen bonding. The gelatin acts as a porogen to create a well-defined size network. The chitosan–gelatin–ferulic acid films/membranes created using formic acid solvent could form a polymer coacervation system. The ferulic acid crosslinks the chitosan . Glycerin was used as a plasticizer to improve the softness of the produced films/membranes. The chitosan–gelatin (1:1 g;


Trans-3WJs can influence the final shape of one or both of the RNA molecules and can thus provide a means for modulating the availability of regulatory motifs including potential protein or microRNA binding sites. Regulatory 3WJs generated in trans represent a newly identified regulatory category that we call structurally interacting RNA or sxRNA for convenience. Here we show that they can be rationally designed using familiar cis-3WJ examples as a guide. We demonstrate that an sxRNA “bait” sequence can be designed to interact with a specific microRNA “trigger” sequence, creating a regulatable RNA-binding protein motif that retains its functional activity. Further, we show that when placed downstream of a coding sequence, sxRNA can be used to switch “ON” translation of that sequence in the presence of the trigger microRNA and the amount of translation corresponded with the amount of microRNA present.RNA-based three-way junctions (3WJs) are naturally occurring structures found in many functional RNA molecules including rRNA, tRNA, snRNA and ribozymes. 3WJs are typically characterized as resulting from an RNA molecule folding back on itself in  The 3WJ is an important structural component of a wide variety of non-coding RNAs including most notably, ribosomal RNAs, which contain numerous examplestrans and that many have the potential for significant regulatory importance45trans, of a microRNA to a mRNA producing a stem-loop structure that could be targeted by an RBPtrans-3WJs could serve as the foundation for designing regulatable RNA “switches” that would form only in the presence of a particular microRNA and would recruit a regulatory RBP that controls protein translation7The established RNA three-way junction 3WJ) structural element results from a single RNA molecule folding back on itself in a WJ structMany RBPs associate with their RNA targets by binding to motifs with structures that contain one or more stem-loops910121312131414trans-3WJ structures that may result from the atypical binding of microRNAs to the flanking region at the base of the HSL-motif and termed these complexes, structurally interacting RNAs or “sxRNA” for shortcis-3WJ structurestrans HSL-sxRNA into a reporter message, with a regulatable, HSL-sequence (the bait) that requires a targeted cellular microRNA to act as a “trigger” to form a functional trans-HSL motif, which can now associate with endogenous SLBP. We show that an HSL-sxRNA can be used to regulate the translation of an upstream coding sequence and that expression correlates with the amount of triggering microRNA present in the cell.Using an informatic-based approach, we previously predicted potential naturally occurring cis-3WJs to form in trans has been demonstrated318192021trans-3WJs interactions. Existing definitions of 3WJs categorize structures into three general families based on characteristics relative to the two roughly coaxially stacked helices central to the 3WJ. While it is difficult to accurately predict which of the trans formed 3WJ helices will orient in a defined manner, generally we have sought to produce “Family A” type 3WJtrans-RNA. The intent behind this approach was to produce a structure where no two helices would be forced into such close proximity that one might block the availability for protein binding.The ability to re-engineer trans interaction  before being run on TBE-Urea denaturing gels. Trans-binding of sxRNA bait and trigger was readily observed 242526in-vitro binding assay described earlier. SLBP binding to the HSL-sxRNA was evident, and co-precipitation of complexed trigger microRNA was observed . Transcripts were nucleofected into mammalian cell lines in combination with its paired, triggering microRNA and then cells were assessed for luminescence activity.To determine the potential for the sxRNA technology to be used to regulate the translational activity of gene expression, we designed various plasmids containing HSL-bait sequences in the 3′- UTR of a luciferase gene and downstream of a T7 promoter so the sxRNA could be transcribed into a functional mRNA . Plasmidtrans-3WJ to be selected for further testing. Specific sequence and nomenclature information is detailed in Multiple HSL bait sequences were designed to target the same microRNA trigger sequence, each of which was expected to recruit cellular SLBP when bound to microRNA. We started with a bait sequence that incorporated a stable HSL (predicted to form as part of MFE structure and not considered to be a “switched” construct) with flanks that could base pair to a targeted miRNA and form a 3wj . AdditioIt should be understood that, while the MFE folds for bait alone and bait complexed with trigger are shown (and used in sxRNA design), these specific conformations are at extremes of a spectrum of potential sub-optimal structures that any given RNA might adopt as it accepts and releases energy from the surrounding system. Single RNA molecules have been shown to reach their most energetically stable conformation by traversing a “rugged energy landscape”, often passing through somewhat stable alternative folds firstIt is worth noting that even minimal (1 or 2 base) changes in the bait sequences have substantive effects with regard to expression profiles. For instance, the transition from BR-1c to BR-1d replaces a G-U pairing in the trigger binding region with a G-C and introduces a U to form a predicted G-U pairing in the destabilized “off” structure. The predicted MFE of the “off” state structure changes by only ~1.3 kcal/mol but the ΔG from “off to on” changes by ~3 kcal/mol in favor of the transition. ectopically introduced into cells, we assessed the ability of an sxRNA bait-reporter mRNA to be regulated when encountering an endogenously produced microRNA. To do this, we designed an sxRNA bait (BR-5a) to target a highly expressed viral microRNA called BART10-3p produced uniquely by the Epstein-Barr virus (designated as T-5 here). This microRNA is produced in abundance by EBV during latency, particularly in the EBV+ Burkitt lymphoma cell line, HH514-16. Conversely, the BART10-3p microRNA (T-5) is absent in the lymphoblastoid cell line (LCL), which was generated using the B95.8 strain of EBVtrans-3WJ interaction for proper HSL motif formation  and in a highly predictable manner. By incorporating the sxRNA concept into the HSL regulatory motif, we demonstrated that the binding and therefore activity of an RBP (SLBP in this case) could be modulated in a microRNA dependent manner. In this case, the translational level of a tethered upstream reporter gene was regulated. We have also shown that our sxRNA approach can be used to modulate gene expression using an ectopic or endogenous microRNA as the trigger to regulate the availability of a switched RBP motif such as the HSL. Because the HSL is so well characterized and plays such an important role in histone mRNA metabolism and translation, it served as an ideal RBP motif for development of the sxRNA technology. However, its cell cycle regulation could limit its utility and other RBP-regulatory motifs are actively being investigated. Alternatively, the design of sxRNAs based on synthetic aptamers for RBPs may prove more robust. This approach is also more flexible, as it removes the constraint of solely targeting RBPs via their natural motifs and associated recognition domains. Additionally, this strategy could allow targeting of anything a structural RNA aptamer can be developed for .In the present study, we demonstrated the ability to rationally design an sxRNA-based regulatory switch, in which an mRNA and microRNA , bind to each other in a predictable structural and functional manner. Specifically, we were able to design 3WJs that form and function in Current informatic tools used for predicting RNA structure, unfortunately often lack the ability to accurately predict either non-canonical base pairings beyond the GU wobble, or resulting tertiary structure. Additionally, our ability to design a switched sxRNA construct currently depends primarily on consensus definitions of known functional structures. Alternatively, optimization of sxRNA switches could be generated using a combinatorial approach such as that provided by SELEXThe expression patterns of microRNA have been shown to be distinctively expressed in normal tissuestrans-RNA interactions, like the 3WJ as well as the manner in which RBPs interact with these structures, would be beneficial for fully exploiting sxRNAs as a technology. While optimization of a single sxRNA construct may be sufficient, in some cases, better optimization of a switched mRNA might be achieved through the incorporation of multiple discrete sxRNA switches. These switches could redundantly target a single RBP-microRNA interaction or some combination of various RBP-microRNAs. In the case of an aberrantly overexpressed microRNA, a single microRNA could be used as the trigger, or it may be more advantageous to target multiple microRNAs that reflect a particular cell type profile.It is clear that a better understanding of trans-interactions, such as sxRNA, represent a powerful new approach with broad applications, especially when coupled with recent advances in RNA delivery methods3536We are only beginning to appreciate the vast RNA-RNA interactome and its functional relevance45cis 3WJsRepresentative human HSL sequences were selected  and incoWhile some stability of the HSL leading to greater SLBP binding might be conferred to this sequence, we believed a true “switch” with greater dynamic range could be developed. To promote a more reliable “OFF-state” in absence of target microRNA, introducing a G-U pairing at its lowest position was used to weaken the HSL stem and decrease the potential for optimal HSL-motif formation in the absence of the trigger microRNA. Additionally, we added a “destabilizing sequence” at the 5′ of the existing sequence  which co2 in RPMI 1640 (Flow Laboratories) containing 10% fetal bovine serum , 50 units/mL penicillin, and 50 μg/mL streptomycin.Human myeloid leukemia K562 cells (ATCC), EBV-positive Burkitt lymphoma cells HH514-16 and EBV-positive lymphoblastoid cell line (LCL) were maintained at 37 °C under 5% CO® PCR Purification Kit (Qiagen) and analyzed for size/integrity and quantity via agarose gel electrophoresis and spectrophotometry (NanoDrop) respectively. 1ug of the purified product was then used as a template for T7 transcription using the MEGAScript® T7 Transcription Kit (Invitrogen) according to the recommended protocol using a 2 hour incubation at 37 °C. The resulting mRNA was TURBOTM DNase treated (Invitrogen) for 15 minutes at 37 °C followed by purification with the MEGAclearTM Transcription Clean-up Kit (Ambion®). Concentration of purified message was measure by spectrophotometry (NanoDrop). 60 ug aliquots of purified mRNA were then capped using the ScriptCapTM m7 g Capping System (CELLSCRIPTTM) following the recommended protocol with a 1 hour incubation at 37 °C followed by another round of purification via the MEGAclearTM Transcription Clean-up Kit (Ambion®).DNA template molecules (see ® TBE (10%) Pre-cast Urea Gel (BioRad) for 1 hour at 150 volts. The gel was then stained with SYBR® Gold Nucleic Acid Gel Stain (InvitrogenTM) and analyzed using the VersaDocTM Imaging System (BioRad).sxRNA baits sequence and microRNA triggers  were des® PCR Purification Kit (Qiagen) and analyzed for size/integrity and quantity via agarose gel electrophoresis and spectrophotometry (NanoDrop) respectively. 1 ug of the purified product was then used as a template for T7 transcription using the MEGAshortscriptTM T7 Kit (InvitrogenTM) according to the recommended protocol using a 1-hour incubation at 37 °C. The resulting mRNA was TURBOTM DNase treated (Invitrogen) for 15 minutes at 37 °C followed by phenol/chloroform extraction and ethanol precipitation. Trigger-microRNA was designed and purchased commercially (TriLink BioTechnologies) and concentration was analyzed using the spectrophotometer (NanoDrop). 3 ug of microRNA-trigger and 1 ug of the sxRNA bait were used for each RIP sample and the total volume was brought up to 10 uL with low salt solution (10 mM Tris/100 mM NaCl) as necessary.Plasmid templates containing for the sxRNA-Bait sequences (GenScript) were linearized using Hind-III (New England Biolabs Inc.) following the standard protocol. Linearized DNA was purified using the QIAQuick2, NP-40, Di H2O). During this incubation, the DYNABEADS® Protein G magnetic beads (Novex by Life Technologies) were washed and tumbled with 5 ug of SLBP monoclonal antibody (Abgent) for 1 hour at room temperature. The beads were then washed with NT-2 buffer, re-suspended in NET-2 buffer  and mixed with the RNA/protein mix. The tubes were then allowed to tumble overnight at 4 °C. Each sample was washed with cold NT-2 buffer and then re-suspended in Proteinase K buffer  followed by an incubation at 55 °C for 30 minutes. A phenol/choloroform extraction followed by ethanol precipitation  was then performed on each tube. Samples were re-suspended in 10 uL low salt solution (10 mM Tris/100 mM NaCl) and mixed with Gel Loading Buffer II (Ambion®). Tubes were heated at 95 °C for 5 minutes and spun briefly before loading warm into a Mini-Protean® TBE-Urea denaturing Gel (BioRad) and run for 1 hour at 150 volts. The gel was then stained with SYBR® Gold Nucleic Acid Gel Stain (InvitrogenTM) and analyzed using the VersaDocTM Imaging System (BioRad).SLBP-RIPs were performed using previously published methods24252638TM, and brought to 1 mM DTT. 1.25 ug of Recombinant SLBP were added where noted. All samples were incubated at room temperature for 1 hr, mixed with native loading dye, and run on a native 8% polyacrylamide TB gel with 1x TB buffer for 1 hr at 100 volts. Gel was stained with SYBR® Gold and analyzed with the VersaDocTM System.Individual and mixed samples of 500 ng T-1 RNA (synthezised by Trilink) and 1 ug B-1a or B-1b sxRNA (T7 transcribed from linearized plasmid template as previously described) were heated to 85 °C in 40 uL volume of 1x NT2 buffer, then allowed to slow cool. Volumes were then brought to 50 uL, retaining 1x NT2 concentration, adding 10 units of RNaseOUTAliquots (10 ug) of the T7 transcribed mRNA (sxRNA luciferase reporter) were used for nucleofection (Amaxa 4D) either alone or mixed with 1 ug of the microRNA trigger. All samples were heated in a hybridization oven at 85 °C for 5 minutes followed by a slow cool back down to room temperature. During this time, the K562 cells were spun down and old media was removed. The SF nucleofection solution (Lonza) was added to the cell pellet per the standard protocol and then aliquots containing 1 million cells were added to the RNA samples before being place in the nucleofection cuvettes. After nucleofection (program FF120), fresh media was immediately added to the cuvette to dilute the nucleofection buffer. All samples were then plated out in 6 well plates containing pre-warmed media. 4-hours post nucleofection, samples were spun down in tubes, media was exchanged, followed by the addition of the two buffers associated with the luciferase assay (ABI). Samples were loaded into a 96-well plate and allowed to incubate, protected from light for 15 minutes. During this incubation, the plate reader (TECAN) was warmed up to the recommended temperature (25 °C) and the program was loaded. Post incubation the plates were analyzed for luciferase signal.6 cells by nucleofection using an Amaxa Nucleofector IIHH514-16 cells and LCL (lymphoblastoid cell lines) were transfected with sxRNA as previously described5 cells/ml and 5 × 105 cells/ml, respectively 24 h prior to transfection and washed twice with phosphate-buffered saline (PBS). sxRNA was introduced into 1 × 106 cells in 100 μl of electroporation solution . Cells were electroporated using an Amaxa Nucleofector II (A024 program) and then diluted to 3 × 105 cells/ml in complete RPMI 1640. Cells were harvested 6 hours later and assayed in triplicate for luciferase expression using a Lumat LB 9507 (Berthold Technologies).For HSL mutation analysis studies, HH514-16 cells and LCL were subcultured at 3 × 10How to cite this article: Doyle, F. et al. Engineering Structurally Interacting RNA (sxRNA). Sci. Rep.7, 45393; doi: 10.1038/srep45393 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.