Streptococcus (GBS) is an opportunistic pathogen that causes preterm birth and neonatal disease. Although GBS is known to exhibit vast diversity in virulence across strains, the mechanisms of GBS-associated pathogenesis are incompletely understood. We hypothesized that GBS strains of different genotypes would vary in their ability to elicit host inflammatory responses, and that strains associated with neonatal disease would induce different cytokine profiles than those associated with colonization. Using a multiplexed, antibody-based protein detection array, we found that production of a discrete number of inflammatory mediators by THP-1 macrophage-like cells was universally induced in response to challenge with each of five genetically distinct GBS isolates, while other responses appeared to be strain-specific. Key array responses were validated by ELISA using the initial five strains as well as ten additional strains with distinct genotypic and phenotypic characteristics. Interestingly, IL-6 was significantly elevated following infection with neonatal infection-associated sequence type (ST)-17 strains and among strains possessing capsule (cps) type III. Significant differences in production of IL1-β, IL-10 and MCP-2 were also identified across STs and cps types. These data support our hypothesis and suggest that unique host innate immune responses reflect strain-specific differences in virulence across GBS isolates. Such data might inform the development of improved diagnostic or prognostic strategies against invasive GBS infections.Group B  Streptococcus (GBS) is a common commensal of the gastrointestinal and genitourinary tracts in approximately 30% of healthy individuals [in utero [Group B ividuals . Althougividuals . Maternain utero . Babies in utero . To reduin utero .In order to develop novel prevention and diagnostic strategies to combat infection, there is a critical need to understand how GBS modulates host immune responses. Indeed, prior studies have shown that inappropriate inflammatory signaling can contribute to adverse pregnancy outcomes such as extraplacental membrane weakening and neonatal sepsis ,4. The min vitro [The GBS strain population is diverse and we and others have shown that GBS strains belonging to multilocus sequence type (ST)-17 are more common in sick neonates than colonized pregnant mothers . Furtherin vitro . We havein vitro  and can in vitro , due in in vitro .Because of the extensive phenotypic and genotypic variation in the GBS strain population, we sought to compare macrophage responses across 15 clinical GBS strains of varying STs and capsule types (serotypes). We hypothesized that GBS strains of different genotypes would vary in their ability to elicit host inflammatory responses, and strains associated with neonatal disease would induce different cytokine profiles than those associated with colonization. Our results show that certain inflammatory cytokines were universally induced in response to the 15 strains, while other responses were unique to specific strains, genotypes, or serotypes. The data generated through this study can be used to identify responses that represent early diagnostic indicators of maternal or neonatal complications or can be targeted for novel therapeutic intervention strategies.600 of 0.4), washed in sterile PBS, and resuspended in RPMI 1640 (Gibco) prior to infection.Fifteen previously characterized GBS strains were used in this study; invasive strains were from neonates with LOD or EOD  and colo6 cells per well in 24 well plates or at 4x106 cells per well in 6 well plates.THP-1 monocyte-like cells (ATCC TIB-202) were cultured in RPMI 1640 medium supplemented with 2mM L-glutamine (Gibco), 10% fetal bovine serum , and 1% penicillin/streptomycin (Gibco) at 37ºC with 5% carbon dioxide. Cells were differentiated into macrophages by incubation with 100nM phorbol 12-myristate 13-acetate  in RPMI medium with 2% FBS for 24hr as previously described [600 nm) were normalized based on experimentally determined CFU to OD600 corollaries for each strain to achieve the desired multiplicity of infection (MOI). All strains used in this study had identical growth rates in the experimental conditions utilized; no statistically significant differences were observed. THP-1 cells were infected with the normalized cultures of GBS at a MOI of 10 bacteria per host cell, which equated to 1x107 CFU/well in 24 well plates or 4x107 CFU/well in 6 well plates. The infected cells were incubated at 37°C with 5% CO2 for 1hr. Extracellular bacteria and media were aspirated, and the cells were washed with PBS and RPMI 1640 supplemented with 2% FBS, 100μg/ml gentamicin (Gibco), 5μg/ml penicillin G (Sigma). The cells were incubated in 100nM PMA at 37°C with 5% CO2 for 24hrs prior to collecting the culture media for cytokine profiling. This time point was selected to allow for the detection of both rapid and delayed cytokine responses.Prior to bacterial infection, PMA-treated THP-1 cells were washed with PBS and fresh RPMI 1640 media without supplements was added. Log phase GBS cultures were centrifuged, washed in PBS, and resuspended in RPMI 1640. Optical densities  (Culture media was collected 24hrs after antibiotics were added and centrifuged (2400 rcf for 10 mins) to remove bacteria and cellular debris; supernatants were stored at -20˚C. Samples were thawed on ice and centrifuged at 16,000 rcf for 5 mins to remove any remaining debris. The Abcam Human Cytokine Antibody Array Kit (ab133998), which allows for the detection 80 human cytokines, was performed in duplicate with two independent biological replicates per condition and used according to the manufacturer’s instructions to determine relative cytokine levels per sample with an Amersham Imager 600 (GE Life Sciences) . Cytokinot.org/) . Densitoot.org/) . Densitoot.org/) . SignifiELISAs were used to confirm a subset of cytokines identified in the antibody arrays using supernatants from additional independent biological replicates for 15 GBS strains. Comparisons were made relative to mock infection. To ensure that inclusion of PMA had no impact on cell signaling responses, we compared responses from two mock infections; one included the addition of PMA throughout the experiment and another included the addition of PMA exclusively during the initial differentiation step. No significant differences in cytokine levels for the six cytokines were observed among all 15 strains between the cells treated once or continuously with PMA (data not shown). Only the PMA-treated results are shown.https://www.genenames.org/). Data from 3 to 5 independent biological replicates were pooled to quantify the average cytokine concentrations (pg/mL) per condition for each of the 15 strains and to calculate the standard deviation of the mean. Significance was determined in each assay by ANOVA and post-hoc Dunnett’s tests relative to mock infection.The following ELISA kits were used according to the manufacturer’s instructions: IL-1β Human ELISA kit: EH2IL1B2 (Thermo-Fisher Scientific), MIG Human ELISA kit: EHCXCL9 (Thermo-Fisher Scientific), IL-6 Human ELISA kit: KHC0061 (Thermo-Fisher Scientific), MCP-2 Human ELISA kit: EHCCL8 (Thermo-Fisher Scientific), RANTES Human ELISA kit: EHRNTS (Thermo-Fisher Scientific), and IL-10 Human ELISA kit: ab100549 (Abcam). MIG is also referred to as CXCL9  and CCL5 (C-C motif chemokine ligand 5), respectively  were pooled to identify significant differences by strain characteristic using ANOVA followed by post-hoc Tukey’s tests. Samples for the additional 10 strains not included in the cytokine array were collected and analyzed separately from the five strains included in the array. Because these additional samples were not part of the same set of biological replicates, a normalization step was performed to account for experimental variability. The average cytokine signal levels for each cytokine tested by ELISA were determined for the two sets of strains . These average signal values were compared to calculate a ratio , and the values for the second set of strains were adjusted based on the difference in the average signal value between the two sets . This adjustment allowed for a more accurate representation of the two data sets together on the same graph for each cytokine.Array data for the five GBS strains found that a subset of inflammatory cytokines were induced in most strains in response to infection. Cytokines GM-CSF, GRO-α, I-309, IL-1β, IL-6, MCP-1, MCP-2, TNF-α, thrombopoietin, BLC, Flt-3 ligand, HGF, IP-10, MIF, and MIP-3α were elevated by ≥1.5 fold relative to mock infection in both independent biological replicates of the cytokine array .In addition to these shared macrophage responses, some changes in cytokine production were unique to strains of specific genotypes or were induced only by strains isolated from infected neonates or from colonized mothers. MCP-2, MIG, BLC, and PARC were more highly elevated in the two ST-17 strains relative to the other STs, while GRO-α and RANTES were more highly elevated in the non-ST-17 strains . CytokinSix representative cytokines were selected for confirmation by ELISA based on the cytokine array results. The cytokine arrays had indicated high protein levels of IL-1β in response to the first five GBS strains tested, with the highest levels being induced by GB00590 and GB00653 . The IL-As predicted by the arrays, increased levels of IL-6 were also observed in response to all 15 strains, with the highest cytokine levels being induced by the four ST-17 strains , and moderate levels being induced by the other 11 strains . StratifThe cytokine array results also showed an increased level of IL-10 across conditions compared to mock infection, with the greatest level being induced by ST-17 strains . The ELISubstantially increased levels of MCP-2 was also observed in response to most GBS strains, which was also confirmed by ELISA. The highest levels of MCP-2 were observed in response to challenge with the ST-17 strains . ST-19 aThe cytokine arrays also predicted enhanced levels of MIG in response to ST-17 infection, which was confirmed by ELISA . All fouThrough the use of an array specific for 80 cytokines, we found that five genetically diverse clinical isolates of GBS induced a range of pro- and anti-inflammatory cytokine responses following infection of THP-1 cells. A subset of the responses identified in the array were strikingly similar and likely represent general or shared cytokine responses that are induced in macrophages by other GBS strains as well . ImportaSome cytokines, particularly those involved in multifunctional pro-inflammatory responses, negative regulation of the inflammatory response or in the recruitment of specific immune cell types, such as B cells and T cells, were strongly induced in response to specific strains , which iAnother study has reported elevated levels of IL-1β, IP-10, I-309, and MCP-2 in blood samples from newborns with sepsis induced by a variety of bacterial pathogens compared to healthy babies ; all of Some of the most unique responses observed by cytokine array occurred in response to the two strains isolated from infants with sepsis (GB00411 and GB00037). Both strains induced responses that could dampen or deregulate inflammatory signaling through induction of the anti-inflammatory cytokine IL-10, and, in the case of the GB00411 strain, induction of the anti-inflammatory cytokine IL-13. This strain also caused production of angiogenin, which regulates tissue vascularization, and induced production of immune cell activators such as ENA-78, Fractalkine and GCP-2. Cytokines that regulate embryonic and placental development, such as PLGF and TGF-β3  were indin vitro and in vivo using different animal model systems. Even though we have identified important differences in cytokine responses across strains, additional strains from different sources or representing other ST/CPS combinations should be examined to classify the range of GBS responses and identify those that are most important for deregulating host immunity, inflammation and neonatal and maternal infections.The notable differences observed between strains coupled with epidemiological data suggest that invasive strains or STs and serotypes associated with severe disease have specific characteristics that can trigger more potent or potentially harmful immune responses. It is likely that these responses vary depending on the timing and duration of infection as well as the primary infection site. Some strains may contribute to more rapid responses, perhaps resulting in miscarriage or EOD, while others take longer to initiate, and may result in complications associated with LOD. In this study, we only examined cytokine responses after 24 hrs post-infection since our aim was to detect both early and delayed responses. We anticipate, however, that other differences in cytokine production might also be observed between strains at earlier time points, which could be indicative of more rapid induction of responses or inhibition. Early inhibition of specific cytokines has been linked to delayed or inappropriate immune responses for several infections , which cBecause it has been shown that faulty inflammatory signaling may lead to extraplacental membrane weakening and premature membrane rupture , we expeAlthough we did not confirm each cytokine by ELISA, the set of six cytokines evaluated showed highly similar trends to those observed in the arrays and provided useful targets for examining these key responses in other GBS strains. We anticipate that most array hits with consistent trends across the two array replicates are likely to represent true positives. However, cytokines that were markedly altered compared to mock infection in only one of the two arrays may still have a role in the macrophage response to GBS infection, but additional verification and study is required.in vitro and in vivo using a murine model of GBS infection. Together, these studies will enhance understanding of how invasive and colonizing strains of GBS differ in their ability to induce host responses that can initiate or exacerbate the disease process. Such discoveries will aid in the identification of new biomarkers and vaccine targets that can guide prevention practices and therapeutic intervention strategies aimed at reducing diseases caused by GBS.Collectively, these data suggest that more virulent strains, STs, or serotypes of GBS elicit different cytokine responses compared to less virulent strains. Some of the differences might be partly due to differences in GBS uptake by or survival within macrophages, or in the differential expression of key virulence factors, which we have demonstrated previously ,16. FutuS1 FigDifferentiated THP-1 cells were infected with GBS strains for 1hr at a MOI of 10 bacteria per host cell. Cytokine levels in the collected cell culture supernatants were determined by cytokine array. Blots from one representative exposure are shown for each condition for each array set along with a key identifying each spot in the array.(PDF)Click here for additional data file.S2 FigDifferentiated THP-1 cells were infected with GBS strains for 1hr at a MOI of 10 bacteria per host cell. Densitometry values for each cytokine are shown from each of the two array replicates. The internal positive control values were averaged for individual arrays and used to normalize each array condition to allow accurate comparisons between mock and infection conditions. Negative control values from each array are also shown to indicate the lower limit of detection for the array.(PDF)Click here for additional data file.S3 FigAverage fold changes for all cytokines included in the cytokine arrays are shown, along with ANOVA p-values based on normalized densitometry values from the two array replicates and ANOVA p-values based on fold changes . Cytokines with increased production relative to mock infection in both cytokine array replicates during infection with GBS strains are shown in red, and cytokines with decreased production are shown in blue. Values represent the average fold change relative to mock infection from the two independent array replicates for each condition. Bolded values represent cytokines that had fold changes of ≥1.5 above or below the mock infection in both array replicates.(PDF)Click here for additional data file.S4 FigTHP-1 cells were infected with GBS at MOI of 10 for 1hr. Data from three to five independent biological replicates were pooled to determine the average cytokine concentrations (pg/mL) produced following each of the infection conditions shown. The average and standard deviation of each condition were plotted for comparison, and significance was determined by ANOVA, followed by post-ANOVA Dunnett’s tests to compare the mean of each condition to the mean of the mock infection condition. All cytokines tested had ANOVA p-values <0.0001.(PDF)Click here for additional data file.S5 FigTHP-1 cells were infected with GBS at MOI of 10 for 1hr. Data from three to five independent biological replicates were pooled to calculate the average RANTES concentration (pg/mL) produced following infection with invasive or colonizing GBS strains. The standard deviation was plotted for comparison, and significance was determined by ANOVA using Tukey’s test of multiple comparisons. p-values <0.0001 are indicated.(JPG)Click here for additional data file.S6 Fighttp://www.uniprot.org/).Cytokines with increased production relative to mock infection in both cytokine array replicates during infection with GBS strains are shown in red, and cytokines with decreased production are shown in blue. Values represent the average fold change relative to mock infection from the two independent array replicates for each condition. Bolded values represent cytokines that had fold changes of ≥1.5 above or below the mock infection in both array replicates. Major cytokine functions were classified via the UniProt Database ((PDF)Click here for additional data file.S1 Tablehttp://www.uniprot.org/). Abbreviations: NKs–Natural Killer Cells; DCs–Dendritic Cells; MMP–Matrix Metalloproteinases; TIMPs–Tissue inhibitors of metalloproteinases; SC–Stem Cells.Major functions were defined by and extracted from the UniProt Database ((PDF)Click here for additional data file.


INDEHISCENT, as well as disrupting the auxin minimum. Both factors influence the formation of the dehiscence zone and consequently reduce pod shattering. Unravelling the mode of action of this unique biostimulant provides data to support its effectiveness in reducing pod shatter and highlights its potential for growers to increase seed yield in a number of OSR varieties.The yield of podded crops such as oilseed rape (OSR) is limited by evolutionary adaptations of the plants for more efficient and successful seed dispersal for survival. These plants have evolved dehiscent dry fruits that shatter along a specifically developed junction at carpel margins. A number of strategies such as pod sealants, GMOs and hybrids have been developed to mitigate the impact of pod shatter on crop yield with limited success. Plant biostimulants have been shown to influence plant development. A challenge in plant biostimulant research is elucidating the mechanisms of action. Here we have focused on understanding the effect of an Ascophyllum nodosum based biostimulant  on fruit development and seed dispersal trait in Arabidopsis and OSR at genetic and physiological level. The results indicate that Sealicit is affecting the expression of the major regulator of pod shattering,  Although agricultural development continues, gains in productivity have plateaued due to abiotic stress, soil degradation, pollution and pressure from pathogens. These factors have made the achievement of a crop’s genetic potential increasingly challenging3.For centuries, the careful selection and crossing of the best performing plants, along with steady improvement of agricultural practices were at the heart of increasing crop productivity. In the twentieth century with the beginning of the Green Revolution, crop production increased rapidly due to the employment of new technologies, high-yielding varieties, chemical fertilisers, agricultural chemicals for crop protection and efficient irrigation5. Recent reports from a number of countries around the globe show that soybean seed loss associated with pod shattering is largely affected by the weather and may vary between 5% to an extreme 100% in susceptible varieties at delayed harvest9. The second largest oil crop OSR, with worldwide production exceeding 74 million metric ton, records on average in UK 15–25% pod shattering associated seed loss and up to 70% in extreme cases, which translates approximately to $70 million loss in the UK alone10. For the entire European production, which consists almost one third of global supply, losses are tenfold higher11. Therefore, the mechanism behind shattering and seed dispersal has been closely studied in the model plant Arabidopsis thaliana as well as OSR (Brassica napus)15. Both species belong to the Brassicaceae family that evolved dry dehiscent fruits derived from cylindrical carpels that encapsulate developing seeds16. The lateral parts of the fruits are developed into valves, and the ovules/seed protecting layers are fused to the central replum through just a few cell files. This, so called dehiscence zone or valve margin, is crucial for seed release upon maturity17. Many of the key regulators of fruit development and particularly valve margin development have been identified. FRUITFUL (FUL) and REPLUMLESS (RPL) genes specify valve and replum formation21, partially by restricting the valve margin identity genes SHATTERPROOF1/2 (SHP1/2), INDEHISCENT (IND) and ALCATRAZ (ALC)26. MADS-box genes SHP1/2 are on the top of the signalling cascade and their elimination leads to the reduction of the lignified layer and separation layer in the dehiscence zone that ultimately results in a more indehiscent fruit12. Acting downstream and in parallel to SHP1/2, IND and ALC valve margin identity. While ALC promotes the separation layer development, IND regulates the formation of the separation and lignification layers24. As demonstrated by Liljegren et al., 200412, the elimination of IND results in no lignified cells throughout the entire dehiscence zone, resulting in extremely shatter-resistant fruits. Next to transcription factors, proper valve margin differentiation and dehiscence zone development is closely related to hormone homeostasis. By regulating AGC3 kinases, IND precisely regulates auxin levels through PIN3-mediated auxin efflux28. On the contrary, auxin levels regulate IND expression leading to indehiscent fruits27.Besides the previously mentioned factors, crop yield is also limited by evolutional adaptations of plants for more efficient and successful seed dispersal. These limitations are particularly evident and problematic in the two most important oil crops, namely soybean and OSR, which evolved dehiscent dry fruits, that shatter along a specifically developed junction at carpel margins14. Currently, the global tendency is to drive productivity by increasing crop yield/quality in an environmentally sustainable manner, which creates an unprecedented opportunity for plant biostimulants to play an important role. Plant biostimulants contain substance(s) and/or micro-organisms whose function when applied to plants or the rhizosphere is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality29. The global biostimulants market is projected by some analysts to reach $4.14 billion by 2025, with Europe projected to be the largest revenue-generating region30. Seaweed extracts are prominent in the biostimulant market, representing the fastest growing biostimulant product category29. The effects of seaweed extracts, in particular Ascophyllum nodosum extracts (ANE), on plants have been reviewed in detail32. ANE biostimulants have been shown to improve plant vigour, increase root development, enhance chlorophyll synthesis, promote earlier flowering, enhance fruit set and uniformity of fruit, delay senescence and enhance tolerance to abiotic stress42. However, the role of specifically formulated ANEs in enhancing the quality of pods in oilseed rape (OSR) has not previously been reported. Demonstration of a beneficial effect would highlight the potential for biostimulants in enhancing crop quality with subsequent productivity gains.In the last decades the prevention of shattering was mostly tackled by classical breeding and genetic modificationsIn this study, multiple experimental approaches were employed to explore the mode of action (MOA) of a novel ANE biostimulant, Sealicit which was developed utilising a targeted plant signal induction (PSI) approach to formulation development. In multi-annual field trials Sealicit was found to increase the yield of a number of OSR varieties. Observed developmental changes indicated that Sealicit has an impact on key genetic players determining fruit development and potentially seed dispersal. In order to test our hypothesis, that Sealicit reduces pod shatter and increases yield, fruits of Arabidopsis and OSR plants treated with Sealicit were analysed using phenotyping, genetic tools and confocal microscopy. Here we show that thoroughly tested and refined biostimulants can be employed to enhance crop quality by providing desirable traits without interference into the genome or time-consuming classical breeding thereby bringing exciting opportunities for the agriculture of the future.Arabidopsis thaliana. Siliques at stage 17b, when fruits are still green but fully extended , were analysed with respect to their length and weight. We observed that fruits of treated plants were significantly longer (p ≤ 0.001) and heavier (p ≤ 0.001), when compared to those from water sprayed controls  in comparison to the control  display a lack of differentiated valve margin and a wider replum due to the ectopic expression and activity of AtFUL and AtRPL transcription factors13. In order to assess the Sealicit MOA and its impact on dehiscence zone formation, we tested the expression levels of genes that determine valve (AtFUL)20 and replum (AtRPL)19 development, as well as a key player involved in valve margin formation . Real-time quantitative PCR (RT-qPCR) of these genes was conducted in reference to three house-keeping genes ACTIN8 (ACT8)45, GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (AtGDPDH)46, UBIQUITIN CONJUGATING ENZYME E2 21 (AtUBC21)47  and AtFUL (p ≤ 0.001) expression and simultaneously upregulate AtRPL (p = 0.31)  Fig. . These rlum Figs  and S2B.p = 0.029) reduced AtIND expression  was analysed. Plants sprayed with Sealicit showed considerably altered fluorescence signal intensity when compared to the control  were performed  and weight , while V5 displayed the opposite effect  after Sealicit application. Treatments also showed a positive effect on pod weight in the case of the naturally shatter-resistant V2 (p ≤ 0.001), whereas V3 and V6 showed negative effects on this parameter   Fig. .44. Among all tested WOSR varieties, V2 and V5 were the most pod shatter resistant when compared to the others , except V4 and V6, which did not show any significant change. These results indicated that Sealicit was able to reduce pod shattering, however the size of the effect was variety dependent.For quantifying and comparing the exact pod shattering potential between individual varieties, the RIT was performed on dry pods at BBCH 99et al., 2016 reported that increased lignification in the whole pod correlates with improved pod shattering in different OSR varieties51. Using a similar approach, the effect of Sealicit on the total lignification of the replum was measured. Interestingly, pods collected from Sealicit treated plants produced increased amounts of lignin in each WOSR variety tested. The largest increases were in varieties V4 (p = 0.008) and V5 (p = 0.009)  Fig. .BnIND were determined to establish if the expression levels were related to reduced pod shatter. Transcript levels were determined using RT-qPCR in different WOSR varieties in reference to three house-keeping genes: BnACTIN2 (BnACT2)45, UBIQUITIN11 (BnUBQ11)52 and, TRANSLATION ELONGATION FACTOR1 (BnEF1a)53. The most consistently expressed house keeping gene was BnEF1a, hence this was used for normalisation of expression levels  except V6 , grown for 45–48 weeks and sprayed with a desiccant three weeks prior the harvest. Tested WOSR varieties showed fluctuations on harvested seed yield in their response to Sealicit, which could be additionally influenced by the type of soil and changing weather conditions . All WOSR varieties mentioned above were sprayed with the appropriate concentration of Sealicit at 6–8 leaf stage .The demand for increased agricultural production in the context of arable land scarcity and climate change means that new agricultural solutions are required to meet these challengesArabidopsis thaliana), we tested whether similar effects can be observed in field conditions in commercial WOSR varieties. The impact of Sealicit on fruit/pod shattering was assessed by diverse approaches; (i) pod morphology; (ii) testing pod shattering by performing RIT; (iii) analysing the expression level of the major transcription factors determining pod development and shattering; (iv) measuring OSR replum lignification; and (v) OSR seed yield assessment at harvest. Morphological analysis indicated that Sealicit MOA affected the biomass. However, while a significant increase in fruit size was observed in Arabidopsis fruits  present in BnIND gene sequences that may affect the primer binding and PCR reaction efficiency. Overall, the interesting correlation between pod firmness as tested by RIT, IND depletion and replum lignin amount suggests that Sealicit was not only able to improve pod shattering resistance, but also stimulate other pathways associated with lignin biosynthesis in the whole replum tissue.Sealicit treated Arabidopsis and five out of the six tested WOSR varieties showed a correlation between the extended RIT half-life time and decreased els Figs  and S1C.BnIND expression) as decreasing the expression levels of V2 and V4 which are already low may lead to pods that are too firm with a resulting impact on yield. Overall, Sealicit improved pod firmness and decreased IND expression, these parameters were shown to correlate well with WOSR yield data. This indicates that part of the Sealicit MOA involves the modulation of IND-mediated dehiscence zone development, resulting in less pod shatter and higher seed yield.Two seasons of field testing of Sealicit in commercially relevant WOSR varieties demonstrated its robustness and efficacy in improving seed yield. The average yield increase was recorded at 4.5% with a maximum of 16% Fig.  which trAtIND expression27, therefore we analysed the fluorescence signal intensity in the auxin signalling reporter line DR5-GFP in Arabidopsis fruits collected from Sealicit sprayed and control plants. Fruits from plants treated with Sealicit showed a disrupted auxin signalling at stage 17b which may suggest that the MOA of Sealicit encompasses the local modulation of auxin homeostasis in the dehiscence zone, interfering with its development and further fruit growth27. Additionally, this effect coincided with a replum widening in the auxin signalling reporter and the transgenic Arabidopsis AtpINDx3VENUS-NLS48, which has also been reported as a direct effect of decreased AtIND in ind knockouts or elevated AtRPL gene expression19. Therefore, our confocal microscopy results were in agreement with the obtained qPCR expression results for AtIND and AtRPL and, consequently, they supported the observation that Sealicit affects the expression of both genes leading to altered fruit morphology and decreased pod shatter 57, transporter genes (PIN3) or phosphorylation-based PIN recruiting machinery 28 in the fruits of plants treated with Sealicit. Another possibility would be to test the effect of Sealicit on their respective genetic mutants. There is also increasing evidence that intracellular alterations in Ca+2 levels, can work as a secondary messenger during auxin-mediated signalling60. In fact, one of the key components of ANEs has previously been shown to influence calcium flux50. In order to gain a better understanding of Sealicit MOA it would be interesting to further explore auxin dynamics in relation to Ca+2 release, perception and Ca+2 responsive proteins in the context of fruit development and pod shattering.It has been previously reported that AtIND can regulate auxin flux and high auxin concentration can inhibit Plant biostimulants and in particular ANEs are emerging as effective crop inputs that improve plant growth and health, mitigate effects of stressful conditions, improve crop quality and, most importantly, increase yield. Although there are many reports on diverse biostimulant formulations and their efficacy in numerous plant systems, the identity of active molecules, their perception and downstream signalling are still poorly documented. This work shows a direct connection between OSR treatment with a specific biostimulant formulation, genetics and plant physiology underpinning the seed dispersal mechanism, which consists a significant leap forward in understanding a biostimulant’s mode of action.BnIND). Our data show that Sealicit improves pod firmness thereby reducing pod shattering and could therefore be an attractive solution for efficient OSR production. The methods utilized here could easily be used in other economically important podded crops. For example, soybean could be an interesting target because it is a member of the Brassicaceae family that employs a similar strategy of seed dispersal for successful reproduction. Although, pod shattering depends on the variety and weather conditions, plant biostimulants such as Sealicit have the potential to activate the crops genetic potential to achieve a high quality crop with enhanced yield omitting time consuming, labour intense breeding and still controversial genetic modifications. ANE biostimulants like Sealicit, that are robust, effective and scientifically validated, offer entirely new solutions for tackling specific developmental aspects or crop traits that can be concealed, like in the case of pod shattering, or apparent such as increased fruit set, size, uniformity or longer shelf life.Confirming the initial hypothesis, our results showed that the biostimulant Sealicit affected the development of the dehiscence zone by disrupting the expression of a crucial genetic player in pod shatter  under light intensity of 100 µmol m−2s−1 and 80% relative humidity. Six commercial WOSR varieties (V1-V6) were tested in variable weather conditions through open-field trials. All the WOSR seeds were kindly provided by farmers. WOSR varieties were selected according to the recommendations of the Irish Department of Agriculture, Food and the Marine (DAFM) and the British Agriculture and Horticulture Development Board (AHDB) as the putatively highest yielding varieties in the seasons 2016–2018 . Fully senescent plants were sprayed with a desiccant three weeks prior to the harvest.0.5 mL of commercial water-soluble Sealicit  solutions were applied by single foliar spray at a dilution of 1/1200; 1/600; 1/300 to 4 to 5-week-old Arabidopsis plants (fully developed rosettes and bolting plants with developed auxiliary branches and first flower buds). Sealicit was applied on WOSR by single foliar spray at a dilution of 1.5 L/Ha at BBCH 16–18. Control plants were sprayed with equal volume of distilled water.63. For optimal plant growth and development, recommended fertilizer, fungicide, insecticide, herbicide and desiccation rates were applied. All agricultural practices and pest control measures were applied according to the Agriculture and Food Development Authority (Teagasc) recommendations64. The density of plants per square meter was 35 and 55 for hybrids and conventional varieties respectively. The plots were harvested at BBCH 99 using a combine harvester and seed yield was recorded. Before harvesting, fifteen plants were picked randomly from each plot and pods were sampled to determine the RIT and phenotypic traits.The open-field trials with six WOSR varieties were laid out in a randomized block design with single plots in two consecutive years from September 6, 2016 to July 25, 2018 at Shanagarry, County Cork, Ireland. The unit plot size for each experiment was 12 m × 4 m and buffer distances between plots were 0.2 m. The soil type was medium loam over limestone with a pH of 7.5 and soil nutrition tests showed sufficient amount of phosphorous (soil at index 4) and medium levels of magnesium, potassium, copper, manganese and zinc (soil at index 3). Previous crop was barley and soil N index was 2et al.44. In principle, six randomly picked oilseed rape pods at BBCH 99 were mixed with 15 metal beads (13 mm in diameter each) and shaken for 11 seconds until half of the pods were broken or damaged. The number of cycles was recorded and the pod shattering resistance was expressed as the half-time of each sample. Regarding Arabidopsis fruits, 10 siliques at stage 18  of each sample were collected and placed with 3 mm metal beads on a 30 mm diameter petri dish installed on the top of a vortex mixer. After shaking them for 30 seconds at speed 3 inside a Petri dish (30 mm diameter) fixed on the top of the vortex, the percentage of open or damaged fruits was recorded to calculate the difference in shattering. All OSR and Arabidopsis varieties sprayed with water or Sealicit were tested at least 40 times.RIT of dried OSR pods was performed according the protocol developed and described by Arnaud et al.43, and dried OSR pods used for the RIT test described above were measured using ImageJ and weighed.Fully developed Arabidopsis fruits at stage 17b, according to classification of Smyth 48 and auxin signalling reporter line DR5::GFP61 were imaged using Laser Scanning Microscopy Leica SP8. Fluorescence signal of AtIND-VENUS3x-NLS was measured using segmented line drawn in the middle of valve margin o respective fruits. Fluorescence of DR5-GFP was registered by measuring signal intensity per area and normalizing the signal between measured boxes by dividing the signal intensity by size of measured area. Abovementioned measurements were performed using the latest version of Fiji (ImageJ). Obtained values were subsequently used to generate Box & Whisker charts . Expression analysis were performed by real time-PCR using a Roche LightCycler 96 System . Quantitative PCR was performed using the LightCycler RNA Master SYBR Green I one-step kit  according to the manufacturer’s instructions. The expression level of genes of interest was expressed in n-fold change and calculated according 266.The quantification of lignin from replum of OSR pods at BBCH stage 99 was assessed according to the acetyl bromide method described by Barnes and Andersonp ≤ 0.05 and the post-hoc analysis from ANOVA were conducted using the Holm-Sidak’s test. All data except the Arabidopsis fluorescence measurements  were used, respectively. The normality and equal variance assumptions were checked. The significance level for both parametric tests was set at nts Fig.  and OSR nts Fig.  were repAscophyllum nodosum  when applied to oilseed rape (OSR) leads to disrupted auxin signalling in the valve margin, downregulation of IND expression and increased pod firmness. These changes stimulated a reduced pod shattering and thereby more harvestable seed yield in a number of varieties of OSR.A plant biostimulant produced from the brown seaweed Supplementary Information


Sc) that accumulate in higher order structures of the brain and other tissues. This conversion has been used for in vitro assays including serial protein misfolding amplification and real-time quaking induced conversion (RT-QuIC). RT-QuIC can be used for the detection of prions and for strain discrimination in a variety of biological tissues from humans and animals. In this study, we evaluated how PrPSc isolated from sheep of different genotypes after inoculation with the scrapie agent influence the fibril formation in vitro using RT-QuIC. We found that reaction mixtures seeded with PrPSc from genotype VRQ/VRQ sheep brains have better conversion efficiency with 132M elk substrate compared to reactions seeded with PrPSc from the brains of sheep with the ARQ/ARQ genotype no matter which strain of scrapie was used to seed the reactions. We also inoculated transgenic mice expressing 132M elk PRNP (Tg12) with the scrapie agent from different genotypes of sheep to compare with our RT-QuIC results. The bioassays support the data showing a significantly shorter incubation period for inoculum from VRQ/VRQ sheep when compared to inoculum from ARQ/ARQ sheep. Thus, we conclude that the genotype of both source and recipient can strongly influence transmission.Scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats. This fatal neurodegenerative disease is caused by misfolding of the cellular prion protein to pathogenic β-rich conformers (PrP C) into a pathogenic form (PrPSc) primarily in the brain and spinal cord. These diseases also are known as transmissible spongiform encephalopathies (TSEs). Prion diseases include scrapie in sheep, chronic wasting disease (CWD) in cervids, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), Gerstmann-Sträussler-Scheinker syndrome (GSS), and kuru in humans. Misfolded proteins accumulate in the central nervous system in all TSEs, and in cases of scrapie in sheep and CWD in cervids, PrPSc also accumulates in the lymphoid tissues , 154 [arginine (R) or histidine (H)] and 171 . Sheep homozygous for the VRQ allele have increased susceptibility, whereas sheep homozygous for the ARR allele are resistant to sheep scrapie –23. In eM or MV) –30.PRNP genotypes to scrapie isolates from sheep of different PRNP genotypes remains unknown. Therefore, investigating how different genotypes of sheep scrapie affect the transmission to cervid hosts with differing prion protein polymorphisms may help us to further understand the risks of transmission of scrapie to cervids and may also provide important information with regard to understanding the origin of CWD. In this study, we utilized the in vitro assay, RT-QuIC, to evaluate how the PRNP genotype of the infectious seed influences the in vitro conversion using sheep scrapie agent of different genotypes and strains as a seed for RT-QuIC based conversion of 132M and 132L elk prion protein. This assessment will determine if different strains or different genotypes of PrPSc present in sheep scrapie may afford a higher risk of transmission to cervids. Where possible, these results were compared to mouse bioassay transmission of the scrapie agent from different genotypes of sheep to transgenic mice expressing elk prion protein.Elk are susceptible to the sheep scrapie agent when inoculated intracerebrally. When challenged with the sheep scrapie agent, elk with MM at residue 132 have a shorter incubation period, and elk with the heterozygous (LM) genotype have a longer incubation period . HoweverAll animal experiments described were reviewed and approved by the National Animal Disease Center’s Institutional Animal Care and Use Committee (protocol numbers: ARS-2017-629). The animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals . Details concerning procedures are included under the section Mouse Bioassay.Brain samples from scrapie-infected sheep were obtained from studies previously conducted at the National Animal Disease Center. Briefly, lambs were inoculated with either x124-infected samples intracerebrally (IC)  or 13–7 Samples of sheep brainstem that had been frozen at -80°C were used to prepare brain homogenate for western blotting. Brain samples were bead homogenized at 20% (w/v) in 1X PBS  and tested shortly after storage at -80°C. PK digestion of whole brain homogenate was performed in 1X PBS for 1 hour at 37°C, at a final PK of 50 μg/ml, and then each sample was separated by SDS-PAGE on 12% polyacrylamide minigels (Invitrogen) and then transferred onto polyvinylidene difluoride (PVDF) membranes  for 45 min at 30 V. The membranes were blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 1 h and incubated at 4° C overnight with mouse anti-PrP monoclonal antibody P4 at a 1:10,000 dilution (0.1 μg/ml) as the primary antibody. Then, a biotinylated sheep anti-mouse secondary antibody at 0.05 μg/ml and a streptavidin–horseradish peroxidase (HRP) conjugate were used in conjunction with a chemiluminescent detection system  and visualized on an imaging system capable of detecting luminescence.The IDEXX HerdChek BSE-Scrapie Antigen EIA test kit was used to selectively detect the presence of disease associated misfolded prion protein in sheep brains. Brain homogenates from scrapie-infected sheep were assessed using the IDEXX HerdChek BSE EIA kit in the absence of proteinase K digestion. EIA was performed as described by the manufacturer. The cutoff value was determined by the negative control sample provided by the manufacturer and the optical density value was around 0.07 ± 0.005. If the optical density value was over 0.15, the samples were considered positive. All brain samples were normalized with EIA kit before analysis by RT-QuIC by diluting to an O.D. around 1.0.E. coli (BL21(λDE3)) was transformed with the pET28a vector containing the wild type elk PrP gene  and the recombinant elk prion proteins were expressed and purified as described by Vrentas et al [-1cm-1 as calculated for mature elk proteins (23–231), 132M and 132L proteins.as et al . The conRT-QuIC reactions were performed as previously described , 33–35. −2 or 10−3) of non-inoculated control or infected sheep brain homogenates . All reactions for each dilution and each sample were performed twice with each individual experiment each consisting of 4 technical replicates for a total of 8 replicates per sample. ThT fluorescence data are displayed as the average ThT fluorescence for an individual experiment consisting of four technical replicates for each time point and, to be considered positive, the ThT fluorescence of at least two out of four replicates must be positive. As previously described for classification of positive samples by RT-QuIC, the positive threshold was calculated as the mean value of non-inoculated control sheep brain homogenates plus 10 standard deviations and the time to the positive threshold is referred to as the lag time [To optimize the conditions of the RT-QuIC reaction, assays using brain samples from negative control or scrapie-infected sheep were performed with 132M and 132L substrates with or without SDS and at different NaCl concentrations . When optimized, RT-QuIC was performed using quadruplicates of two dilutions  [2 chamber by trained personnel if the aforementioned signs suggestive of prion disease were observed or at the end of the experiment (700 days post-inoculation) and tissues were collected for analysis. Positive/negative status of individual mice was determined by EIA (see above). Survival analysis was done using Graph Pad Prism 7  with incubation periods shown in days post-inoculation. In total 10 mice were found dead without observation of clinical signs. Three of those had high levels of detectable PrPSc based upon EIA assay and were likely the result of rapid onset of clinical signs. The remainder died from concurrent disease issues (neoplasms) due to the long term nature of the experiment.Mice expressing the elk prion protein (Tg(ElkPrP-132M)Prnpe; Tg12)  were usee; Tg12)  using 20PRNP genotype was tested. Molecular profiles obtained from western blot of brain homogenates from representative sheep were similar regardless of inoculum used or genotype of sheep or route of inoculation in a comprehensive analysis of these isolates [Brainstem samples (obex) were collected from sheep with clinical signs of scrapie. To confirm that strain specific properties of each inoculum were transmitted to the experimental animals, brainstem samples from selected animals were characterized by western blot using the P4 antibody . These bisolates .Sc were detected in the brainstem of sheep with clinical signs, but not in samples from the negative controls , there is increased ThT fluorescence for both substrates with a longer lag time with lower amounts of fibril formation without spontaneous fibril formation. Optimization with seed from different genotypes of sheep (ARQ/ARQ) was also done with each substrate . ComparThe impact of SDS on the seeding activity with scrapie prion was also tested . For 132Reaction conditions containing 300 mM to 500 mM NaCl and either 0% or 0.002% SDS were determined to be equivalently optimal for x124 scrapie detection when using RT-QuIC assays with recombinant elk prion proteins. For 13–7 scrapie the range extended from 200 mM to 500 mM NaCl, and 0% SDS was found to support better conversion for both substrates by RT-QuIC . Thus thPRNP genotype of the sheep scrapie donor had on the seeded conversion of elk PrP, RT-QuIC reactions containing recombinant elk proteins, 132L  infected with x124 inoculum, two VRQ/VRQ sheep (#3 and #4) infected with x124 inoculum, two VRQ/VRQ sheep (#5 and #6) infected with No. 13–7 inoculum, and two ARQ/ARQ sheep (#7 and #8) infected with No. 13–7 inoculum. Assays utilizing elk substrate 132M showed fibril formation as monitored by ThT fluorescence to be earliest for x124 inoculated VRQ/VRQ sheep (#3 and #4) followed by No. 13–7 inoculated VRQ/VRQ sheep (#5 and #6) then No. 13–7 or x124 inoculated ARQ/ARQ sheep , all within 25 hours. Only assay seeded with x124 ARQ/ARQ sheep (#1) brain homogenate did not produce the positive ThT fluorescence  with brain homogenates from sheep with clinical signs of scrapie. The inoculum genotypes were chosen to be consistent with relevant genotypes of sheep scrapie agent that could be present naturally and to mimic the upper and lower conversion efficiencies by RT-QuIC. More specifically, ARQ/ARQ sheep were not susceptible to x124 by the intranasal route while VRQ/VRQ x124 and ARQ/ARQ No. 13–7 reflect the shortest and longest RT-QuIC lag time. Mice inoculated with x124 from a VRQ/VRQ sheep  had difficulty moving, developed poor motor coordination, and were euthanized with a mean incubation period of 398 days post inoculation. Mice inoculated with No. 13–7 from an ARQ/ARQ sheep had a lower attack rate  with an average incubation period of 635 days. Many of the mice (8/25), however, tested positive at the end of the experiment (700 days post-inoculation) without developing clinical signs  [PRNP sequence, 132L and 132M, when seeded with samples of the scrapie agent from sheep of these two PRNP genotypes. Each substrate showed different seeding activity with brain samples in a genotype dependent manner. We show here that PrPSc from VRQ/VRQ sheep can seed the elk substrate (132M) with higher conversion efficiency than seed from ARQ/ARQ sheep. Also, we inoculated mice expressing elk prion protein (Tg12) with two scrapie isolates, x124 (VRQ/VRQ) and No. 13–7 (ARQ/ARQ). While this is a more limited set of host and recipient genotypes, the mouse bioassay supports the RT-QuIC data by showing a shorter incubation period for Tg12 mice inoculated with the scrapie agent from VRQ/VRQ sheep relative to the incubation period for Tg12 mice inoculated with the scrapie agent from ARQ/ARQ sheep.The first description of chronic wasting disease in deer was in a captive population, but the original source of infection has never been determined . Previous system . The shes system , 43. AnoP (Tg12) . In thesPRNP genotype influences a host’s potential susceptibility to prion agents. For example, the relative susceptibility of sheep to the scrapie agent is influenced by polymorphisms in the amino acid composition of prion protein at residue 136 , 154 (arginine or histidine) and 171 (glutamine or lysine). Sheep with the ARR/ARR genotype are resistant to scrapie, whereas sheep with VRQ/VRQ are highly susceptible [Sc deposition profile was different between ARQ/ARQ sheep and VRQ/VRQ sheep infected with No. 13–7 indicating that different recipient genotypes influence the tissue distribution of scrapie [The host ceptible , 45. Preceptible  and No. ceptible ) that exceptible . In cont scrapie .The difference in fibril stability between VRQ/VRQ and ARQ/ARQ sheep inoculated with x124 scrapie is consistent with either the “cloud” hypothesis of prion strain switching , 48 or dSc from different genotypes of sheep have different conversion efficiencies. For 132M elk substrate this is true for both scrapie isolates evaluated while for 132L elk substrate the PrPSc seed genotype differences are only observed with strain x124. As previously discussed, this is consistent with either cloud hypothesis or deformed templating in the x124 strain in different genotypes sheep by RT-QuIC. The distinct seeding activities from different genotypes of sheep PrPSc were also observed in our mouse bioassays. While a more limited selection of genotypes was assessed, the results of bioassays in transgenic mice expressing elk PRNP (132M) are consistent with the in vitro assay showing that mice infected with the x124 agent from sheep with the VRQ/VRQ genotype have shorter incubation periods than mice infected with the No. 13–7 agent from sheep with the ARQ/ARQ genotype. These differences in the incubation periods for transgenic mouse bioassays are also consistent with the incubation periods observed in the sheep inoculated with x124 inoculum and No. 13–7 [The present study indicates that RT-QuIC reactions conducted in elk rPrP substrates that are seeded with PrPNo. 13–7 . It is wNo. 13–7  making tin vitro conversion based detection methods such as RT-QuIC offer the ability to determine whether the seed genotype influences risk of disease transmission across various combinations of both donor and recipient genotype.Cross species transmission of TSEs is a major concern to animal and human health. Considerable knowledge in this area has been gained utilizing both natural host species and transgenic animals , 42, 44.in vitro conversion through RT-QuIC and compared these results to in vivo transmission experiments. Conversion efficiency with seed derived from the brain of a VRQ/VRQ sheep is greater than that observed for seed derived from an ARQ/ARQ sheep brain. Mouse bioassay also demonstrates a shorter incubation period and higher attack rates in transgenic mice expressing elk PrP when inocula is derived from VRQ/VRQ sheep. When conducting prion transmission studies, the genotype of the prion agent inoculum donor should be given careful consideration.In this study, we addressed the effect of donor genotype with regard to the potential transmission of the sheep scrapie agent to elk by using


Part of the weakness of exome analysis lies in the inability to detect aberrant splicing. An evaluation of the post-splicing mRNA sequence concurrently with genomic variants could improve the diagnostic rate. We aimed to investigate publicly available exome sequencing data and its matching transcriptomics data of phenotypically normal individuals to identify alternatively spliced variants from known genes associated with autosomal recessive disorders under the premise that some of the subjects could be carriers of such disorders.Aberrant splicing events and their triggering genomic variants were detected with the aid of Bayesian network method “SAVNet” which was originally developed for cancer genomics.Forty aberrant splicing events including exon skipping, the creation of a new splice site, and the use of a cryptic splice site in response to the disruption of the authentic site were detected in 1916 genes among 31 of the 179 subjects from the 1000 Genomes Project. The predicted effects on proteins were either frameshift mutations (30) or large in-frame insertions or deletions (10). Five missense mutations and 2 silent mutations were reinterpreted as triggering major changes in transcript sequences. The detection rate of provisionally truncating pathogenic variants increased by 19%, compared with a conventional exome analysis.The coupling interpretation of exome and transcriptome data enhances the performance of conventional exome analyses through the proper interpretation of intronic variants that are outside of the GT/AG splicing consensus sequences and also allows the reinterpretation of “missense” or “silent” substitutions that can indeed have drastic effects on splicing. Currently, the overall diagnostic rate of most clinical exome laboratories is limited to around 30% ,2. This Whole transcriptome sequencing (RNA-Seq), is an effective method for the quantitative estimation of mRNA expression and for the qualitative detection of alternatively or abnormally spliced transcripts within cells, tissues, and organs where a pathogenetic process is in progress. Whole transcriptome sequencing has been successfully applied in several mutation analysis studies . HoweverCoupling the interpretation of personal genomes  with their corresponding whole transcriptomes  provides a valuable clue to the detection of aberrantly spliced transcripts and their neighboring genomic variants that trigger abnormal splicing. Recently, Shiraishi et al. implemented an integrated analytic method of exome-whole transcriptomes data on a personalized level through the development of a novel algorithm/software known as “SAVNet: Splicing-Associated Variant detection by NETwork modeling” and have applied it effectively in the field of cancer genomics . They evIn the present study, we aimed to evaluate how coupling the interpretation of personal genomes with their corresponding whole transcriptomes using the SAVNet algorithm could expand the current diagnostic limitations of an exome alone-approach in terms of detecting abnormal splicing in the genetic studies of rare diseases that are presumably caused by genomic variants that are rare in the general population. We aimed to investigate publicly available exome sequencing data and its matching RNA-Seq/transcriptomics data of phenotypically normal individuals  to ident22.1www.internationalgenome.org) [http://www.internationalgenome.org/data-portal/data-collection/geuvadis) [Publicly available raw exome and RNA-seq sequence data were investigated from the standpoint of splicing abnormalities as they relate to rare genomic variants . We analome.org)  and its euvadis) . We eval2.2http://www.1000genomes.org). The downloaded bam files were converted back to fastq files using the bam2fastq utility [For analyses involving individuals from the 1000 Genomes Project, hg19 aligned exome-sequence data in the bam format was downloaded from the 1000 Genomes Project website  and sjdbOverhang 100 option. Exon counts and gene counts were measured using BEDTools (intersectBed function) and HTSeq, which are implemented in the MAP-RSeq workflow [Raw RNA sequencing data in the fastq format derived from lymphoblastoid cell lines originating from individual subjects who participated in the 1000 Genomes Project was downloaded from the European Nucleotide Archive (ERP001942). Genome indexes were generated using STAR version 2.5  with theworkflow .2.4Splicing abnormalities were correlated with their corresponding rare variants using the.https://github.com/friend1ws/SAVNet) [SAVNet algorithm (/SAVNet) . The SAVSAVNet identifies variants causing splicing alterations from among genome and transcriptome sequencing data focusing on the following two classes: 1) rare variants disrupting authentic splicing donor and acceptor motifs, with this class of variants leading to exon skipping, alternative 5′/3′ splice sites (SS) either within the exon or intron, or intron retentions; and 2) rare variants creating novel splicing motifs, with this class of variants leading to alternative 5′/3′ splice sites within either an exon or intron.2.5software.broadinstitute.org/software/igv/) [http://serialbasics.free.fr/Serial_Cloner.html) and reconstructed so that the resultant sequence would incorporate abnormally spliced segment(s) that had been detected through the SAVNet analysis. We reconstructed the mRNA transcript sequences by knowledge based on junction Walk algorithm by Seok [We restricted our analysis to 1913 genes that are known to cause autosomal recessive disorders  under thre/igv/) . Second, by Seok . In shor2.6Splicing events were visualized as a sashimi plot by drawing a connective element that illustrates the presence of a splice junction between two splice sites . The inf2.7http://provean.jcvi.org/) [The deleteriousness of the abnormally spliced variants causing protein sequence indels was evaluated using the PROVEAN software  , which rvi.org/) .2.8We tested whether the abnormal splicing detected using RNA-seq and the SAVNet software could be predicted by the prototypic software tools MaxEntScan (MES)  and Spli33.1Among the 179 samples, the SAVNet program detected a total of 40 events of abnormal splicing and their associated variants within the known 1916 genes associated with autosomal recessive disorders , exonic alternative 5′SS (n = 8), exonic alternative 3′SS (n = 10), intronic alternative 5′SS (n = 4), and intronic alternative 3′SS (n = 4). None of the SAVs were associated with intron retention. A representative example is illustrated in All 40 abnormal splicing events detected by the SAVNet program, with the exception of GBE1 gene, aberrant splicing patterns associated with two distinctive genomic variants of the same gene were observed in separate individuals. For the GLE1 gene, aberrant splicing patterns associated with two slightly different genomic variants of the same gene were observed in two separate individuals. The net effect of the two genomic variants was deduced to be identical at the transcript and protein levels. In the RARS2 and ERCC8 genes, two distinctive aberrant splicing patterns were associated with the same genomic variant in a single individual, as discussed in the following subsection.Generally, one SAV was associated with one aberrant splicing event and vice versa. The exceptions were as follows. For the GBE1, GYG1, NBAS, NDUFV1, SP110, and TUBGCP3) or the disruption of the acceptor sites . Both the disruption of the donor sites and that of the acceptor sites tended to be associated with exon skipping events .Aberrant splicing events were classified into three categories according to the mechanistic basis of the alterations at the donor/acceptor sites : 1) exonACADVL, ERLIN2, MUTYH, SNX14, and TCTN3) occurred within the authentic coding sequence than within the intron authentic sequence . Six acceptor creation events were observed. Among these 6 events, 4 events  occurred within the coding sequence. As expected, alternative 3′ and 5′ SS were associated solely with the disruption of acceptor and donor sites, respectively. SUCLA2 had a highly complex pattern and was not classifiable according to the categorization outlined above. The SAV NC_000013.10:g.48563118T > G variant led to an abnormal transcript that reflected two independent events: exon skipping of exon 3, and the creation of a completely novel exon within authentic intron 2.Eight donor creation events were observed. Among these 8 events, more events . The variants resulted in protein sequence changes: p.(Ser529ArgfsTer9), p.(His530CysfsTer11), and p.(Ser529ArgfsTer14) .Two of the SAVs, one variant in skipping . These tERCC8 led to the creation of two distinct acceptor sites (data not shown). Two ERCC8 variants were observed in transcripts: one was c.618_629del, and the other was c.618_626del (both were NM_000082.3). The variants resulted in protein sequence changes: p.(Ser206_Ser209del) and p.(Ser206_Ser208del) (both were NP_000073.1).The NC_000005.9:g.60195555C > A variant in 3.3ACADVL, ERLIN2, MUTYH, NIN, and TCTN3 had been interpreted as missense mutations when only the genome sequences were interpreted and annotated . HoweveAn analysis of the expression levels of each gene using MAP-RSeq showed that 1272 of the 1913 currently known causative genes of autosomal recessive disorders had exons with average read counts of 5 or more in the aligned RNA-seq data among the 179 samples. Regarding these 1272 genes, 38 nonsense mutations, 64 frameshift mutations, and 42 mutations involving the GT/AG splicing consensus sequences were identified by the exome analysis alone (data not shown). As shown above, an integrated analysis of the exomes and their matched transcriptomes detected 28 splicing-associated variants outside of the GT/AG splicing consensus sequences. Hence, an integrated analysis, rather than an exome-only analysis, increased the number of putative loss-of-function variants from 144 (38 + 64 + 42) to 172 (38 + 64 + 42 + 28), or 19%.3.4We evaluated the performance of the MES  and SSF Second, we evaluated 14 SAVs that occurred in non-canonical donor/acceptor splice sites according to Cartegni . The non4An integrated analysis of exomes and their matched transcriptomes successfully detected rare genomic variants associated with abnormal splicing using the SAVNet algorithm, which was originally developed for cancer genome research . An analTwenty-nine out of 40 of these variants  led to the disruption of the reading frame, and the remaining 11 variants caused in-frame insertions or deletions that were likely to be pathogenic because of their size.A typical exome analysis kit covers not only the exonic coding regions, but also their flanking intronic regions for up to 20 or 30 bases. Nonetheless, the functional significance of such intronic variants usually remains unknown in conventional exome analyses, except for those occurring in GT/AG splicing consensus sequences. An integrated analysis with SAVNet enabled the direct evaluation of the transcriptional consequences of such non-canonical variants, identifying 16 variants among 179 subjects.Moreover, an integrated analysis allowed the detection of abnormal splicing triggered by variants within the exonic coding sequences that had been annotated as non-synonymous variants or even as synonymous variants in a conventional exome analysis. Our findings regarding the importance of seemingly missense or silent mutations in triggering splicing abnormalities are compatible with recently reported experimental findings by Soemedi et al. , who useThe detection efficiency of pathogenic alleles was significantly enhanced by coupling the interpretation of personal exome sequencing with the corresponding whole transcriptome. The number of SAVs detected through the presently reported study (40) was significantly large, compared with the total number (102) of nonsense variants and frameshift variants. One could argue that 12 of the 40 SAVs were mutations at canonical splice sites and therefore would have been detected by an exome analysis without relying on transcriptome data. Nonetheless, 28 of the 40 splicing mutations would not have been detected as pathogenic variants without the transcriptome analysis. Overall, we expect that the application of SAVNet analyses of lymphoblastoid cell lines in addition to standard exome analyses will increase the efficiency at which pathogenic alleles can be detected by about 20%.Prototypic splicing prediction software , organoid tissues could be generated through the application of regenerative technologies, such blood-induced iPS cells, and these tissues could then be used to perform a combined RNA-seq and exome study .Reassuringly, two thirds of all the genes  had expression levels that were amenable to a SAVNet analysis using mRNA from lymphoblastoid cell lines. Hence, even if mRNA is not available from the affected tissues or from induced tissues, a significant number of genes can be evaluated. The generation of lymphoblastoid cell lines from patient samples is routinely performed in research settings, but not in clinical settings. In view of the strong association between the expression profiles of lymphoblastoid cell lines and that in peripheral blood , when mRThe successful application of an integrated exome-transcriptome analysis that can effectively increase the sensitivity of mutation analyses has therapeutic significance because patients with some classes of splicing abnormalities can potentially be treated using splicing-modulating chemicals such as RECTAS , a rectiIn summary, an integrated analysis that couples the interpretation of personal genomes with their corresponding whole transcriptomes using the SAVNet algorithm successfully detected how genome mutations affect transcripts and identified rare genomic variants associated with abnormal splicing. The application of integrated exome-transcriptome analyses will enhance the sensitivity of conventional exome analyses through the proper interpretation of intronic variants that are outside of the GT/AG splicing consensus sequences and even allow the reinterpretation of “missense” or “silent” substitutions, which can have drastic effects on splicing. Many of the inborn errors of metabolism are autosomal recessive disorders. Successful detection of pathogenic splicing mutations in the recessive genes, as illustrated in the present study, demonstrates that integrated interpretation of the exome and corresponding transcriptome data is a promising approach to significantly enhance the sensitivity of detection of the genetic causes in patients with inborn errors of metabolism. Last but not least, the precise definition of abnormal splicing could be of therapeutic significance in view of the rapid development of splicing-modulating agents.https://gnomad.broadinstitute.org). Allele frequency data on samples from individuals originated from Great Britain and Finland were derived from non-Finnish European and Finnish population, respectively.Forty events of abnormal splicing and their associated variants within the 1916 genes associated with autosomal recessive disorders. Data on allele frequency were derived from the gnomAD database (Supplementary Table 1The following are the supplementary data related to this article.M.Yamada and K.Kosaki designed the study. M.Yamada, H.Suzuki, and K.Kosaki analyzed the data. M.Yamada, H.Suzuki, Y.Shiraishi and K.Kosaki wrote the paper. All authors approved the final version of the paper.The authors declare no conflicts of interest.


Photodynamic therapy (PDT) is a light activated drug therapy that can be used to treat a number of cancers and precancers. It is particularly useful in its topical form in dermatology but improvement of efficacy is required to widen its application.An ester between aminolaevulinic acid (ALA) and CP94 was synthesised (AP2-18) and experimentally evaluated to determine whether protoporphyrin IX (PpIX)-induced PDT effectiveness could be improved. A biological evaluation of AP2-18 was conducted in cultured human primary cells with both PpIX fluorescence and cell viability  being assessed in comparison to the PpIX prodrugs normally utilised in clinical practice (aminolaevulinic acid (ALA) or its methyl ester (MAL)) either administered alone or with the comparator iron chelator, CP94.No significant dark toxicity was observed in human lung fibroblasts but AP2-18 significantly increased PpIX accumulation above and beyond that achieved with ALA or MAL administration +/- CP94 in both human dermal fibroblasts and epithelial squamous carcinoma cells. On light exposure, the combined hydroxypyridinone iron chelating ALA prodrug AP2-18 generated significantly greater cytotoxicity than any of the other treatment parameters investigated when the lowest concentration (250 μM) was employed.Newly synthesised AP2-18 is therefore concluded to be an efficacious prodrug for PpIX-induced PDT in these dermatologically relevant human cells, achieving enhanced effects at lower concentrations than currently possible with existing pharmaceuticals. HoweverPDT uses light to activate a pre-administered drug in the presence of molecular oxygen to kill diseased, precancerous or neoplastic cells without harming healthy cells, so healing occurs without scarring . It also2+ into PpIX by ferrochelatase to form haem) is relatively slow to occur and is thus the secondary rate limiting step of this pathway , where we controlled the amounts of iron supplied to the cells in the tissue culture media, we found that iron chelation had a highly positive effect on PpIX accumulation, which far outweighed any limiting factor that reduced iron availability might have on subsequent free radical production on irradiation, and this led to enhanced PDT effects being produced following the administration of an iron chelating agent. It is clear however, that we are manipulating a highly complex biochemical environment, which can vary depending on cell type and innate iron status.in vitro in human dermatological cells, than existing clinically utilised PpIX prodrugs. With further investigation, the significant increases in cytotoxicity following PpIX-induced PDT conducted with AP2-18, could potentially be translated into clinical PDT settings to produce substantial benefits for patients. This could either take the form of reducing the number of treatment cycles currently required to treat existing licensed indications or alternatively improve outcomes in dermatological applications where a more effective PDT treatment regime is still required.It is therefore concluded that a novel, combined hydroxypyridinone iron chelating ALA prodrug (AP2-18) has been synthesised and demonstrated to be more effective at generating PDT effects when investigated Proximity to Discovery grant from the Medical Research Council, UK and a research grant from Killing Cancer, UK.This work was supported by a A.Curnow, M Wood and A Perry have patent PCT/GB2013/052297 issued.


SMAD6 and TCF12 are highly enriched in patients with non-syndromic sagittal and coronal CS, respectively. Interestingly, both mutations show striking incomplete penetrance, suggesting a role for modifying alleles; in the case of SMAD6, a common variant near BMP2 drastically increases penetrance of sagittal CS. Here, we report a proband presenting with both sagittal and coronal craniosynostosis with the highly unusual recurrence of CS within two months of initial surgery, requiring a second operation to re-establish suture patency at six months of age. Exome sequencing revealed a rare transmitted frameshift mutation in SMAD6 (p. 152 fs*27) inherited from an unaffected parent, absence of the common BMP2 risk variant, and a de novo frameshift mutation in TCF12 (p.E548fs*14). SMAD6 and TCF12 independently inhibit transcriptional targets of BMP signaling. The findings are consistent with epistasis of these mutations, increasing penetrance and severity of CS in this proband. They also add to the list of composite phenotypes resulting from two Mendelian mutations, and support the utility of exome sequencing in atypical CS cases.Non-syndromic craniosynostosis (CS) affects 1 in 2350 live births. Recent studies have shown that a significant fraction of cases are caused by de novo or rare transmitted mutations that promote premature osteoblast differentiation in cranial sutures. Rare heterozygous loss-of-function (LOF) mutations in  The female proband was delivered at term after an uncomplicated pregnancy, and was referred to the pediatric neurosurgical service at birth due to her abnormal head shape. On physical exam, the patient had flattening of the left frontal bone with contralateral frontal bossing and associated harlequin deformity of the left orbit, consistent with left coronal synostosis. In addition, the calvarium posterior to the coronal sutures was elongated and narrow , consistent with sagittal synostosis Fig. . A CT scThe child was subsequently followed biweekly in neurosurgery clinic and by an orthotist for helmet adjustments to shape skull growth. Two months after surgery, the orthotist noted that the child’s left forehead remained flattened and was not rounding as expected. At the child’s subsequent neurosurgical clinic visit, the parietal and occipital regions had rounded and expanded as expected; however, the left frontal region had stopped rounding. A repeat head CT was performed, which demonstrated rapid healing and patency of the sagittal suture and re-fusion of the left coronal suture along with complete fusion of the right coronal suture Fig. . Such ra1. VQSR ‘PASS’ variants with an ExAC allele frequency ≤10−3 sequenced to a depth of eight or greater in the proband and 10 or greater in each parent with Phred-scaled genotype likelihood scores >30 and de novo quality scores (log10(Bayes factor)) >6 were considered. Independent aligned reads at variant positions were visualized in silico to remove false calls. All retained calls had de novo genotype quality scores of 100. Transmitted variants were called as per above, and all variants were annotated using ANNOVAR2 with allele frequencies assigned to each variant from the ExAC database3.To explore potential genetic contributions to her condition, we performed whole exome sequencing of the case-parent trio using DNA prepared from buccal swab samples according to standard protocols. Exome capture was performed using the IDT xGen capture reagent, which was followed by 99 base paired-end sequencing on the Illumina HiSeq 2000 instrument. Sequence reads were aligned to the GRCh37/hg19 human reference genome using BWA-Mem. Local realignment and quality score recalibration were performed using the GATK pipeline, after which variants were called using the GATK Haplotype Caller. A Bayesian algorithm, TrioDeNovo, was used to call de novo mutationsSMAD6 and TCF125. The mutation in SMAD6 was an early frameshift mutation (p. 152 fs*27), which was transmitted from an unaffected parent , which was de novo. Both mutations were absent from the ExAC and GnomAD databases, which contain >240,000 alleles3, and both mutations were confirmed by Sanger sequencing 7. In the case of SMAD6, epistatic interaction with a common risk variant near BMP2, which by itself has modest effect on risk, increases penetrance to >90%7. The BMP2 rs1884302 locus was genotyped in the proband and both parents, and no family members harbored the CS risk allele ‘C’, consistent with the parent harboring the SMAD6 mutation being free of CS  in the proband was particularly interesting8. While SMAD6 has long been known as an inhibitory-SMAD that negatively regulates BMP signaling, TCF12 silencing in mesenchymal stem cells was only recently shown to result in increased phosphorylation of receptor-SMADs, implying that loss of TCF12 function also augments BMP signaling via the BMP/SMAD axis9. This finding suggests that the combination of SMAD6 and TCF12 haploinsufficiency increases BMP signaling to levels substantially greater than those seen with either mutation alone, sufficient to ensure penetrance at both the sagittal and coronal sutures  is offered to craniosynostosis patients, knowing the genetic results for specific patients could prove useful in guiding operative planning for this complex patient population. The goals of cranial vault reconstruction are to obtain an aesthetically pleasing shape of the skull that will allow adequate growth of the brain in ideally one operation. Identification of high risk genotypes prior to surgery—particularly in cases with unusual clinical features—may prove useful in guiding surgical management in the future and may enable more informed discussions with patients’ families.The relevant data from this Data Report are hosted at the Human Genome Variation Database at 10.6084/m9.figshare.hgv.2330 10.6084/m9.figshare.hgv.2333.Supplementary Table 1