Seismological data from recent subduction earthquakes suggest that megathrust earthquakes induce transient stress changes in the upper plate that shift accretionary wedges into an unstable state. These stress changes have, however, never been linked to geological structures preserved in fossil accretionary complexes. The importance of coseismically induced wedge failure has therefore remained largely elusive. Here we show that brittle faulting and vein formation in the palaeo-accretionary complex of the European Alps record stress changes generated by subduction-related earthquakes. Early veins formed at shallow levels by bedding-parallel shear during coseismic compression of the outer wedge. In contrast, subsequent vein formation occurred by normal faulting and extensional fracturing at deeper levels in response to coseismic extension of the inner wedge. Our study demonstrates how mineral veins can be used to reveal the dynamics of outer and inner wedges, which respond in opposite ways to megathrust earthquakes by compressional and extensional faulting, respectively. Recent megathrust earthquakes have been documented to cause large-scale stress changes, although this has not been identified in a fossil system. Here, the authors present data that establish a link between the observations at active subduction zones and the structural record preserved in ancient mountain belts. Mw 8.8 Maule and the 2011 Mw 9.0 Tohoku earthquakes, and revealed that coseismically induced stress changes activate splay and normal faults in the upper plate1011Accretionary prisms form along convergent margins when sediments are scraped off from the subducting plate and transferred to the upper plate. Sediments accreted near the toe of the wedge are commonly transported towards greater depths and experience diagenetic to low-grade metamorphic conditions before being exhumed to the surface234678Here we present a detailed field study that documents the long-term conditions of brittle rock failure in a domain of a fossil accretionary complex that evolved from an outer into an inner wedge, overlying the aseismic and seismogenic part of the basal detachment, respectively. Our combination of structural information with isotope data  and predictions of a Mohr–Coulomb wedge model enables us to better understand how accretionary wedges respond to megathrust earthquakes. We conclude that coseismically induced stress changes can trigger wedge internal deformation and leave a discernable signature in the seismic record of rocks.The study area is located in the Palaeogene accretionary complex of the central European Alps, which is one of the best documented fossil systems worldwide. The high relief of ∼2,600 m provides unique insights into the internal architecture of this orogenic wedge. The area comprises an exhumed ∼4-km-thick sequence of hemipelagic shales, marls and limestones, as well as syn-orogenic turbidites (flysch) including volcanic detritus131413171) consists of calcite shear veins that are widespread in fine-grained lithologies, such as slates, marlstones and siltstones, but are absent in coarse-grained sandstones. The majority of G1 veins were formed incrementally along bedding planes during reverse faulting, documenting horizontal contraction within the wedge  cross-cut G1 veins, folds and cleavage , carbon  and oxygen  isotope systematics of mineral veins and host rock to further constrain the setting and relative timing of vein formation during the prograde evolution of the Alpine wedge  C and O isotopes bear information on carbonate diagenesis, fluid sources, open- vs closed-system behaviour and temperature-controlled isotope fractionation. All samples were taken from a homogenous, ∼300-m-thick rock unit (Globotruncana marl) to minimize potential variations in the isotopic composition of the host rock. The unit was deposited in a calcite sea  and has a stratigraphic age of Santonian–CampanianWe studied the strontium  and to bulk carbonate leachate obtained from a limestone bed intercalated within the Globotruncana marl . This documents the precipitation of G1-vein carbonates from a pore fluid that was dominated by a Sr isotopic signature of contemporaneous sea water. Such a signature is commonly found in pore fluids of marine sediments subjected to carbonate diagenesis, that is, the recrystallization of biogenic carbonates to secondary inorganic calcite222387Sr/86Sr ratio as contemporaneous sea water, but their Sr concentration is 1–3 orders of magnitude higher than in pore fluids or sea water222387Sr/86Sr ratios of G1 veins, limestone beds and contemporaneous sea water suggests vein formation during carbonate diagenesis, whose conditions will be constrained in the following.Calcite of G18O values of calcite cement from host rock and adjacent calcite from G1 veins cluster tightly between 20.5 and 25.6‰ and 22.1 and 26.3‰, respectively . Similar to G1 veins, the δ18O and δ13C values of calcite from G2- and G3 veins lie between 22.2 and 23.2‰ and 1.5 and 2.2‰, respectively, indicating that also during later stages of vein formation the C and O-isotopic composition of the pore fluid was buffered by the host rock , which grew coevally in equilibrium with the fluid , which indicates the progressive interaction of the pore fluid with the host rock and the formation of G2- and G3 veins during the prograde evolution of the wedge.Analyses of whole-rock samples and leached silicate fractions of the Globotruncana marl yield relatively high ectively . The porost rock . This enhe fluid . The δ18ectively . The temTaken together, our combined structural and geochemical data reveal two distinct modes of brittle deformation within the Alpine wedge: (1) contractional faulting within the outer wedge, and (2) extensional faulting and fracturing within the inner wedge.σ1) and the stress conditions under which the wedge fails. This enables us to constrain the timing of vein formation within the subduction earthquake cycle. The model is constrained by the wedge geometry, the strength of the wedge and the basal detachment, as well as the pore fluid pressure ratio λ (where λ is the ratio of pore fluid pressure and lithostatic pressure). The parameters were estimated from previous wedge analyses33μ′b). The outer wedge overlies the  stable zone of the basal detachment, which shows velocity-strengthening behaviour and is modelled by a coseismic increase in μ′b. The division of the basal detachment into a velocity-strengthening and a velocity-weakening part assumes a common behaviour for the subduction fault. Exceptional situations, in which the earthquake rupture propagates through the upper aseismic zone, suggesting that the shallow part of the detachment experience only little strengthening or even weakeningTo investigate how the two modes of brittle deformation are related to the large-scale tectonic setting at convergent margins, we apply the dynamic Mohr–Coulomb wedge theoryλ>0.92 and >0.96 for the outer and inner wedge, respectively). For the outer wedge, we consider this as unlikely, as such high fluid pressures are not supported by seismological and borehole data37σ1 rotates slightly towards the horizontal , rock failure occurs at an even lower increase of the basal friction. The inner wedge behaves in a converse manner to the outer wedge. Coseismic weakening of the basal detachment brings the wedge towards an extensionally critical state, and σ1 rotates towards the vertical  originate from coseismic compression of the outer wedge, whereas normal faults and steep extension fractures (G2 and G3) were formed during short periods of extension within the inner wedge  σn is about 270 MPa and τF∼11 MPa during the interseismic period. A coseismic decrease of μ′b to 0.01–0.02 corresponds to stress drops of 5–8 MPa, indicating that the apparently high stress drops fall within the range of average values. Moreover, stress drops can vary significantly along the fault surface. While some patches experience complete stress drops others may even undergo stress increases50In contrast, the formation of Ger wedge  and 5. FWe conclude that faulting and vein formation in accretionary complexes can be triggered by megathrust earthquakes, which shift the wedge into an unstable state. This implies a coseismic increase in fracture permeability within the hanging wall of megathrusts, followed by a stable phase of interseismic fault sealing. Our work helps to understand how fractures are generated throughout the subduction earthquake cycle, which is essential to better-constrain the nature of postseismic fluid flowThe study area is located in the UNESCO World Heritage Swiss Tectonic Arena Sardona, in the northeastern part of the central European Alps . StructuQs in the study area is ∼80 mW m−2 (ref. The modern average surface heat flow m−2 ref. . For them−2 ref. Ts is the surface temperature (273 K), k the thermal conductivity (2.5 Wm−1 K−1) and Auc the radiogenic heat production rate within the upper crust. Following Pollack and ChapmanAuc in the upper crust accounts for 40% of the surface heat flow, that is, Auc=0.4Qs/Duc, where Duc is the thickness of the upper crust (taken to be 20 km). Solving equation (1) gives an average geothermal gradient of ∼27 °C km−1 for the upper 15 km of continental crust. As the surface heat flow may have been lower before the onset of continental collision and associated thickening of the continental crust, we assume a conservative range for the geothermal gradient in our study area of 20–30 °C km−1.where α and the basal dip β, is related to the strength of the wedge and the strength of the basal detachment33μ, the cohesion gradient η and the pore fluid pressure ratio λ. The strength of the basal detachment is given by the effective coefficient of basal friction μ′b=μb(1–λb), where μb and λb denote the coefficient of basal friction and the basal pore fluid pressure ratio, respectively. The model is divided into an outer and an inner wedge, to account for differences in wedge strength and in seismogenic behaviour of the basal detachment. The outer wedge represents the near-trench part of accretionary wedges, which overlies the updip velocity-strengthening (aseismic) segment of the basal detachment and comprises young and weak sediments. The landward inner wedge overlies the velocity-weakening (seismogenic) part of the detachment and consists of well-consolidated older accreted unitsμ′b. For the interseismic period μ′b is set to a fixed reference value, which is equal for both parts of the basal detachment. Megathrust earthquakes are considered by increasing and decreasing μ′b along the aseismic and seismogenic part of the detachment fault, respectively. Given the strength and geometry of the wedge, the model calculates the effective stress ratio m for every value of μ′b, determining whether the wedge is stable (elastic deformation) or critical (at Coulomb failure). The results are pictured in a stability field diagram , as well as the angle of σ1 with the wedge surface Ψ as a function of basal friction μ′b  than the inner wedge . The reference pore fluid pressure ratios for the outer and inner wedges were set to intermediate pore fluid pressures (λ=0.8) and high pore fluid pressures (λ=0.9), respectively . The extracted O2 was collected on a molecular sieve (0.5 nm) and subsequently expanded into the mass spectrometer. Replicate oxygen isotope analyses of the standards used during the runs (n=19) yielded an external reproducibility of ≤0.20‰.δ13C and δ18O values of vein and host rock calcites were measured at the University of Bern with a GasBench II connected to a Finnigan MAT Delta Plus XL mass spectrometer, using a He-carrier gas system3PO4. All results are normalized using an in-house standard calibrated against δ13C and δ18O values of NBS-19 . The external reproducibility for the analyses estimated from replicate analyses of the in-house standard (n=16) is ±0.05‰ for δ13C and ±0.04‰ for δ18O.δ2O and Cc-H2O, which were determined for a temperature range of 200–500 °C and 0–500 °C, respectively31σ error on the vein formation temperature was calculated by assuming a total 1σ error of ±0.24 in the δ18O measurements, that is, the sum of the external reproducibilities (±0.20‰ for δ18OQz and ±0.04‰ for δ18OCc).The temperature-dependent O isotope fractionation between quartz and calcite was calculated using empirically derived fractionation equations for Qz-H87Rb–84Sr spike and dissolved in concentrated HF-HNO3. Insoluble residuals of organic matter were oxidized with concentrated H2O2.Rb–Sr isotopes were obtained for three samples of Globutruncana marl, comprising two samples with 50–60% calcite and one sample of shale containing no calcite. Aliquots of the two calcite-rich samples were treated with 1 M HCl for 5 min and subsequently centrifuged for 10 min at 3,000 r.p.m. to gain the solution and the residual fraction. Residuals, solutions and powdered bulk rocks were spiked with a mixed 87Sr/86Sr was measured for calcite from mineral veins and for bulk carbonate leachates of pure limestone. The bulk carbonate leachates were obtained by submerging small rock chips for 30 s in 0.1 M HNO3. The solutions were centrifuged for 10 min at 3,000 r.p.m. The vials were checked under the stereo microscope for eventual residuals, but no particles could be found.2O5 activator. The measurement commenced when a signal intensity of 5 V on mass 88 was achieved. The 87Sr/86Sr ratios of the samples were normalized to a 86Sr/88Sr ratio of 0.1194  at the mass spectrometer facility of the Institute of Geological Sciences at the University of Bern. The samples were loaded on rhenium ribbon single filaments in combination with a Ta194 ref.  using th024 ref. . The ext87Rb/85Rb value of the in-house standard and its nominal value (87Rb/85Rb=0.38581). The external reproducibility (2 s.d.) estimated from the replicate analyses of the standard (n=8) is ±0.00012. The age-dependent 87Sr/86Sr ratios of the three host rock samples given in the text and in 87Rb decay constant of 1.42 × 10−11 a−1 .Supplementary Figures 1-5, Supplementary Tables 1-4 and Supplementary References


All six microarray-identified genes were confirmed to be CM responsive, as was tgfb and mmp13. Histopathological analyses identified signs of inflammation in the DI of salmon fed CM-containing diets, including lamina propria and sub-epithelial mucosa thickening, infiltration of eosinophilic granule cells, increased goblet cells and decreased enterocyte vacuolization. All of these were significantly altered in 24 % CM compared to all other diets, with the latter two also altered in 16 % CM compared with 8 % CM and control diet groups. Significant correlation was seen between histological parameters as well as between five of the qPCR analyzed genes and histological parameters. These molecular biomarkers of inflammation arising from long-term dietary CM exposure will be useful in the development of CM-containing diets that do not have deleterious effects on salmon growth or physiology.The inclusion of plant meals in diets of farmed Atlantic salmon can elicit inflammatory responses in the distal intestine (DI). For the present work, fish were fed a standard fish meal (FM) diet or a diet with partial replacement of FM with solvent-extracted camelina meal (CM)  during a 16-week feeding trial. A significant decrease in growth performance was seen in fish fed all CM inclusion diets  contains supplementary material, which is available to authorized users. Fish products are an important component of many human diets, and the demand for marine products is increasing as the global population has grown past seven billion FAO . The incSalmo salar) is a carnivorous species and, as such, aquaculture feeds for farmed Atlantic salmon typically include marine animal-based protein and oil sources (such as fish and crustacean) that rely heavily on the inputs of unsustainable fishery resources . Camelina is a flowering oilseed crop of the Brassicaceae family with many characteristics that are favorable to agriculture. These include low fertilizer and nitrogen requirements and high resistance to biotic and abiotic stress . The solvent-extracted CM used in this study contained 39 % crude protein and had a gross energy content of 4,320 kcal/kg  was grown and harvested by the staff of the Dalhousie Agricultural Campus in Canning, NS, Canada. The seeds were cold pressed using a KEK 0500 screw press at Atlantic Oilseed Processing . The resulting meal was ground with a hammer mill (screen size 8 mm) into a pre-pressed meal cake at Atlantic Oilseed Processing, and solvent extracted with petroleum ether at a concentration of 3 mL gAll diets in this trial were formulated at the Dalhousie Agricultural Campus to be iso-nitrogenous, iso-energetic practical diets and were formulated to meet the nutritional requirements of Atlantic salmon  and all fish were kept on a photoperiod of 12-h light and 12-h dark. Fish were fed a commercial diet  during a 1-week acclimation period. Once the fish were acclimated, they were gradually moved onto the control diet over a period of 3 days. Initial sampling was done 1 week after the fish were completely weaned from the commercial diet. Subsequently, fish were weaned onto their assigned experimental diet  over another period of 3 days. Fish were fed the experimental diets to apparent satiety twice daily in triplicate tanks over a period of 16 weeks.A feeding trial experiment was conducted in accordance with the regulations set out by the Canadian Council of Animal Care for the ethical treatment of animals . This feeding trial was carried out in the Dr. Joe Brown Aquatic Research Building (JBARB) at the Ocean Sciences Centre (OSC) of Memorial University of Newfoundland (Canada) using Atlantic salmon smolts  from the Saint John River stock. The fish were received from Cooke Aquaculture , where they were held in freshwater, and were transferred to seawater at the JBARB to undergo smoltification. Six hundred fish were randomly distributed into twelve 500-L tanks (50 fish per tank) with flow-through water supply at a rate of 12 L min−1 tricaine-methane-sulfonate bath . Three fish per tank were dissected by removing the total viscera, and the DI was removed. For each fish, an ~2-cm-long section was removed from the middle of the DI; the distal-most ~0.5-cm portion was flash-frozen for RNA isolation , and the remainder was fixed for histological analyses . Further details on the rearing conditions and sampling can be found in Hixson et al. , samples were further homogenized using QIAshredders  and RNA was extracted following the manufacturers’ instructions. Twenty micrograms of total RNA were treated with 6.8 Kunitz units DNaseI  to remove genomic DNA. DNaseI-treated RNA samples were column-purified using the RNeasy MinElute Cleanup Kit (QIAGEN) following the manufacturer’s protocol. RNA integrity and purity were assessed using 1 % agarose gel electrophoresis, and A260/230 and A260/280 NanoDrop spectrophotometry, respectively.n = 9) from the control diet (no CM inclusion) and from the 24CM (high level CM inclusion diet) were used for microarray analysis with the 4x44K Atlantic salmon oligonucleotide array -designed Agilent 4x44K salmonid oligonucleotide array (GEO accession: GPL11299)  at a resolution of 5 μm. Photomultiplier tube (PMT) settings were adjusted for each array individually with the laser power set at 90 % for both channels (Cy3 and Cy5). Scanned array images were saved in TIFF format to be used for data extraction.2), print-tip Loess normalization and removal of control and flagged  spots were carried out. This was performed using the Bioconductor and mArray packages for R, using scripts adapted from those reported in Booman et al.  using the cGRASP 4x44K GAL file to extract probe intensities. The extracted data were imported into the R statistical computing software where background correction, data transformation  terms were manually obtained using the putative ortholog from Homo sapiens  from the UniProt Knowledgebase (http://www.uniprot.org).Prior to significance analysis of microarrays (SAM) and rank products (RP) analyses, missing data points for the 14,897 probes were imputed using the EM_array method from LSimpute  terms mapped to each microarray probe in Blast2GO were collected.In order to confirm the results obtained using SAM and to potentially identify additional transcripts that were differentially expressed between 24CM and control groups, the microarray data were also analyzed using the RP method  was synthesized from 1 μg of column-purified total RNA in 20 μl reactions using 250 ng of random hexamers (Life Technologies), 1 μL of dNTPs (10 mM each), and Moloney-Murine Leukemia Virus (M-MLV) reverse transcriptase (Life Technologies) with the first strand buffer and Dithiothreitol (DTT) supplied by the manufacturer. Samples were incubated as follows: 65 °C for 5 min after the addition of the random hexamers and dNTPs, then 25 °C for 10 min and 37 °C for 50 min after the addition of the first-strand buffer, DTT, and M-MLV. cDNA was diluted 1:10 using nuclease-free water . Negative controls were performed by omitting the reverse transcriptase.E = 10[−1/slope]; Pfaffl Candidate genes from the microarray analysis were subjected to qPCR to validate transcript levels. cDNA was synthesized from nine individuals (three from each replicate tank) from each of the four experimental diets  for a total of 36 individuals. Primers used for qPCR analysis can be found in Table elongation factor 1 alpha (ef1a) and 60S ribosomal rna subunit L32. Five candidate normalizer genes were tested across 18 samples using the GeNorm software . Expression analyses were carried out in 384-well format with each sample run in triplicate. The respective CT values were determined using the ViiA7 Gene Expression Study Application (Applied Biosystems) with automated threshold determination and walking baseline. The relative starting quantity (RQ) was determined for each transcript using the 2−ΔΔCT method  with the following conditions: 50.0 °C for 5 min, pre-incubation at 95.0 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Each amplification was performed using 13 μl reactions with 4 μl of input cDNA (representing 10 ng of input RNA), 1.46 μl of PCR-grade water (Life Technologies), 6.5 μl of 2x SYBR Green Mastermix (Applied Biosystems) and 0.52 μl each of the forward and reverse primers (1.25 μM concentration). Expression levels were normalized to DI tissue was prepared from fish from the week-16 time point using standard histological techniques. Briefly, samples were dehydrated in ethanol, cleared using xylene and infiltrated with paraffin. Samples were embedded in paraffin blocks and 4 μm cross-sections were taken . All slides were stained using hematoxylin and eosin, and blinded microscopic evaluations were carried out .n = 9). All quantitative measurements were made using the ImageJ image analysis software  were carried out for pairwise comparisons.Statistical analysis of growth and histology data was carried out using Minitab 16 . A two-way nested ANOVA was performed using the general linear model with a term that accounts for the effect of diet on the response variable and a term that nests tanks within diets to negate any tank effect or individual variance that may be present. Tukey’s post hoc tests for multiple comparisons at a significance of 5 % . Data were tested for normality using the Shapiro–Wilks test and analyzed using a one-way ANOVA and Tukey’s multi-comparison pairwise post hoc test. Statistically significant difference in RQ values between test groups was taken at 5 %.Correlation between histopathological parameters (except villus height), or between histopathological parameters and transcript expression data, was evaluated using Pearson’s correlation coefficients using MiniTab 16.p = 0.003 and p = 0.001, respectively). The apparent feed intake was decreased by the CM-containing diets compared to the FM control (p = 0.001). However, fish fed all diets had comparable feed conversion ratios (p = 0.299). The visceral somatic index (VSI) of fish fed the CM-containing diets also increased when compared to the fish fed the control diet (p = 0.001) but there was no significant difference between the CM diet groups. The growth performance of the fish, as measured by the specific growth rate, remained unchanged by the CM-containing diets (p = 0.107). Full growth performance results are reported in Table The growth performance of the fish involved in this feeding trial was previously reported in Hixson et al. , and theA microarray experiment utilizing nine biological replicates was carried out to identify transcripts responsive to a CM-containing diet 24CM) compared to a control diet in the DI of Atlantic salmon. SAM analysis (FDR 6.25 %) identified 16 differentially expressed features, with all being more highly expressed in the fish fed the 24CM diet; the fold change values of these microarray-identified genes ranged from 1.57 to 3.94. In total, eight different transcripts were represented by the SAM-identified features and six were annotated through BLASTx searches against the NCBI nr protein database (Table CM comparES1 protein homolog (es1), which was identified by one common  feature that was 3.3-fold upregulated (Table Transmembrane protease serine 9 (tmprss9) was identified by one common differentially expressed feature (2.6-fold upregulated) and GO annotation included “proteolysis” and “integral component of plasma membrane”. Thioredoxin (txn) was represented by five common upregulated features (1.9- to 2.5-fold) and had many functional annotations, including “cell proliferation”, “innate immune responses”, “cell redox homeostasis”, and “negative regulation of hydrogen peroxide induced cell death”. Pirin (pir) was represented by one common upregulated feature (3.9-fold) and was associated with GO terms including “myeloid cell differentiation”, “monocyte differentiation” and “oxidoreductase activity”. Ependymin (epd) was represented by one common upregulated feature (2.0-fold). The functional annotation for this gene included the GO terms “cell matrix adhesion” and “calcium ion binding”. The last gene identified by both SAM and RP with a significant BLASTX hit was N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D (napepld), which was represented by one common upregulated feature (2.8-fold) and included the GO terms “phospholipase activity” and “zinc ion binding”. Two features were identified by both SAM and RP that did not have significant BLASTX hits, meaning that their identities could not be determined for this study. These genes were represented by one common feature each and had fold changes of 2.0 and 3.9  were validated to be responsive to the 24CM diet. All six genes were significantly upregulated in the fish fed the 16CM diet compared to the fish fed the control diet, and five out of the six genes  were upregulated in the fish fed the 8CM diet compared to those fed the control diet.The expression of the six transcripts identified by both SAM and RP, and with significant BLASTX hits, was quantified using qPCR Fig. . This wagamma-interferon-inducible lysosomal thiol reductase (GILT), matrix metalloproteinase 13 (mmp13), and transforming growth factor beta (tgfb)  compared with fish fed the 8CM diet  Fig. . Of thesiet Fig. , i.p = 0.001). There was a significant increase in the number of GCs per square millimeter in the DI of the fish fed the 24CM diet compared to all other diets and in fish fed 16CM diets compared to the 8CM diet and control (p = 0.027). There was no significant change in the number of GCs per square millimeter seen in the fish fed the 8CM diet compared to the control diet. The counts of EGCs that had infiltrated into the LP showed a significant increase seen in the fish fed the 24CM diet compared to all other diet groups (p = 0.004); however, there was no significant change in the number of EGCs that had infiltrated into the LP seen in the fish fed the 8CM or 16CM diet compared to the control group. Images showing increased EGCs and GCs can be seen in Fig. p = 0.001). This is illustrated with representative images in Fig. p = 0.046), although there was no notable change in the fish fed the other CM-containing diets compared to the control  correlation between many of the histological parameters , tmprss9 correlated with LP width and SNV, GILT correlated with LP width and SNV, and mmp13 correlated with GCs and SNV.Pearson’s correlation analyses revealed significant  are discussed herein to elucidate the transcriptional effect of chronic exposure to dietary CM on inflammatory processes in the DI of Atlantic salmon.The microarray experiment carried out in the current study indicated that the dietary inclusion of 24 % CM resulted in the modulation of the expression of a relatively small number of transcripts related to inflammatory responses, and all of the BLASTX-annotated genes identified by both SAM and RP in the microarray experiment were qPCR validated. The three microarray-identified and qPCR validated genes that were positively correlated with physical signs of inflammation  system, which is heavily involved in remodeling of the extracellular matrix (ECM) for the promotion of tumor progression and metastasis  and negative correlation of mmp13 transcript expression with GCs and SNV, were not expected. In Sahlmann et al.  at day 5 in a 20 % soybean meal (SBM) feeding trial, but was not differentially expressed at any other time points in the 1-week trial. Collectively, these results suggest that mmp13 expression response to PMs may be dynamic and time-dependent. The potential roles of mmp13 in PM-associated acute versus chronic inflammation of salmon DI warrant further investigation.Two of the qPCR analyzed genes that were not identified by the microarray were also shown to be responsive to CM inclusion. The first gene, n et al. , mmp13 wGILT transcript expression displayed significant positive correlation with LP width and SNV but did not show any differential expression between the fish fed the different diets. GILT has been shown to be heavily involved in many aspects of immunity; it is constitutively expressed in professional antigen presenting cells and can be induced in many other cells by inflammatory cytokines , tumor necrosis factor alpha induced protein 8-like protein 2 (2.4-fold upregulated), b-cell lymphoma 6 protein homolog (1.5-fold upregulated), and c-c motif chemokine 19, which was represented by two upregulated transcripts (both 2.4-fold upregulated).The RP analysis revealed four genes with GO terms directly related to “inflammatory response” that were not identified by SAM analysis  chain transcripts (ranging from 1.5-fold to 3.3-fold downregulated). Igs are present in many mucosal surfaces and are necessary for adaptive mucosal immunity . It is likely that other cellular processes were altered in the CM fed fish, and that these genes may play roles in the modulation of these processes. Those genes are briefly discussed herein to elucidate possible further effects of CM outside of an inflammatory response.There were some transcripts that were identified by both SAM and RP to be responsive to CM and were qPCR validated, but did not correlate with any of the histological parameters . As shown in the aforementioned studies, the response to dietary PMs is very dynamic and changes significantly over time. In the current study, a high level of variability was also seen between fish, which may have limited our ability to identify DEGs. As a result, it is possible that other cellular processes were altered in response to long-term exposure to dietary CM that we were not able to detect in our analyses.The gene expression analyses carried out in this study present a snapshot of the Atlantic salmon DI transcriptome response to long-term exposure to dietary CM. The dynamic transcriptome response of salmon intestine to PM-containing diets has been documented in various studies, including Sahlmann et al. , which sTranscriptional modulation was paired with histopathological changes in the DI of salmon fed the CM-containing diets that are evidence of the manifestation of an inflammatory response. Quantitative measurements were taken in place of a conventional semi-quantitative scoring system for most histological parameters analyzed to increase the accuracy and sensitivity of the analyses carried out in detecting pathophysiological changes. Notable changes detected include an increase in the population of GCs in the epithelium of fish fed the 16CM and 24CM diets. GCs are responsible for the production and maintenance of the mucus layer that protects the epithelium of the intestine . Both of these papers reported histology results that are congruous with what is reported here. Other studies have also reported differential transcript expression and decreased growth performance in Atlantic salmon fed SBM  that are responsive to long-term dietary CM exposure. While high levels of biological variability may have limited out ability to detect all of the genes that were responding to the CM-containing diets, we believe that this study provides valuable new information for understanding the long-term effects of dietary CM exposure in the DI of Atlantic salmon.The data presented in the current study demonstrate an inflammatory reaction in response to long-term dietary CM exposure in the DI of Atlantic salmon post-smolts. This conclusion was established based on decreased overall growth performance in response to dietary CM inclusion, which was paired with the modulation of the expression of some novel inflammation-relevant genes and histopathological changes associated with inflammation . Some of the identified genes correlated significantly with histopathological parameters and allowed for the identification of novel molecular biomarkers of inflammation Supplemental Table 2(DOCX 20 kb)Below is the link to the electronic supplementary material.


In light of the wide systemic influence of ER signaling in regulating a host of biological functions in multiple tissues, it is difficult to infer the direct role of ER on the heart. Therefore, we developed a mouse model with a cardiomyocyte-specific deletion of the ERα allele (cs-ERα−/−). Male and female cs-ERα−/− mice with age/sex-matched wild type controls were examined for differences in cardiac structure and function by echocardiogram and differential gene expression microarray analysis. Our study revealed sex-differences in structural parameters in the hearts of cs-ERα−/− mice, with minimal functional differences. Analysis of microarray data revealed differential variations in the expression of 208 genes affecting multiple transcriptional networks. Furthermore, we report sex-specific differences in the expression of 56 genes. Overall, we developed a mouse model with cardiac-specific deletion of ERα to characterize the role of ERα in the heart independent of systemic effects. Our results suggest that ERα is involved in controlling the expression of diverse genes and networks in the cardiomyocyte in a sex-dependent manner.Estrogen exerts diverse biological effects in multiple tissues in both animals and humans. Much of the accumulated knowledge on the role of estrogen receptor (ER) in the heart has been obtained from studies using ovariectomized mice, whole body ER gene knock-out animal models,  Estrogen receptors (ER) are involved in multiple biological processes in a variety of tissues including the cardiovascular system, adipose tissue, and sex organs ERs have been implicated in several pathophysiological conditions including diabetes and obesity with implications for cardiovascular disease −/−. The availability of mice with cs-ERα−/− will enable, for the first time, to investigate the role of ERα in cardiac tissue independent of peripheral effects. In addition, since ERs are important therapeutic targets, development of in vivo models of selective modulation (such as cardiac specific deletion) will enable better understanding of ER effects in specific tissue −/− manifests significant variations in the expression profile of genes involved in metabolism, cell growth and differentiation, muscle architecture, and relaxation. Finally, we delineate sex differences associated with the absence of ERα in the heart and identify key transcriptional/receptor hubs that are involved in ERα mediated regulation/signaling in cardiac tissue.As a first step in characterizing the role of ERs in the heart, independent of systemic effects of ERs, we have generated a mouse model with a cardiomyocyte-specific deletion of ERα (cs- ERαhttp://grants1.nih.gov/grants/olaw/references/phspol.htm). Animals were housed in AAALAC-Accredited animal facility overseen by experienced veterinary personnel and animal care staff. Animals were euthanized via carbon dioxide inhalation, and all efforts were made to minimize suffering.All experiments were conducted according to a protocol approved by the animal experiment committee at Washington University School of Medicine in Saint Louis  and in accordance to ‘Principles of laboratory animal care’ . The sequences of the forward and reverse primer used are ATGGTCATGGTAAGTGGCA and CCTCTGCCATTGTCTAGCTT, respectively. Both male and female Knockout animals showed reduced expression of the ERα gene in the cardiac tissue as shown in Mice with cardiomyocyte-specific ERα deficiency were generated by crossing mice with Exon 3-floxed ERα alleles obtained from NIEHS flox/flox) and cardiomyocyte-specific ERα knockout (cs-ERα−/−) male and female mice of C57BL/6 phenotype were bred and maintained at Washington University School of Medicine animal facility. Mice had free access to regular diet and water. At approximately 18–20 weeks of age, four mice from each group  were sacrificed after drawing blood for biochemical analysis, and their hearts were isolated and flash frozen for further analysis. Serum was separated by centrifugation from the collected blood samples for measurement of non-esterified fatty acids (NEFA), triglycerides (TG) and cholesterol concentrations. All experiments were performed in compliance with the guidelines for the care and use of research animals established by Animal Studies Committee of Washington University in St. Louis School of Medicine.Wild type , TG , and cholesterol . These assays were performed by the Diabetes Research Center (DRC) at Washington University School of Medicine. Blood glucose concentrations were measured by using commercial blood test strips  by using a drop of tail blood.Echocardiograms were performed using non-invasive ultrasound imaging with the Vevo2100 Ultrasound System  at 18–20 weeks of age as described previously Total RNA was extracted from ∼50 mg of pulverized cardiac tissues using Qiagen Universal RNA isolation kit  following the manufacturer’s recommended protocol. On-column DNase removal was performed and total RNA concentration and purity were measured by absorbance ratio at 260 nm and 280 nm. Total RNA quality was then determined by Agilent 2100 bio analyzer (Agilent Technologies) according to the manufacturer’s recommendations. All samples used in the study had a RNA integrity number (RIN) number of 7 or above. RNA amplification, hybridization, and detection were performed at the Genome Technology Access Center, Washington University in Saint Louis. RNA transcripts were amplified by T7 linear amplification . For reverse transcription, 400 ng of total cellular RNA sample (11 µl) was mixed with an oligo-dT T7 primer (1 µl), reaction buffer , dNTP mix (4 µl), RNase Inhibitor (1 µl), and Arrayscript RT enzyme (1 µl) and then incubated at 42°C for 2 h. After a three minute incubation on ice, the cDNA underwent second strand synthesis after addition of water (63 µl), 10x second strand buffer (10 µl), dNTP mix (4 µl), DNA polymerase (2 µl), and RNase H (1 µl). This cocktail was incubated at 16°C for two hours. Following a column cleanup using Zymo DNA Clean and Concentrator 5s (Zymo Research) according to the manufacturer’s protocol, in vitro-transcription (IVT) was carried out by adding 10x T7 reaction buffer (2.5 µl), T7 biotin-NTP mix (2.5 µl), and T7 RNA polymerase enzyme mix (2.5 µl) and then incubated at 37°C. The IVT reaction was carried out for 14 hours. Following reaction termination with water (75 µl), the amplified RNAs (aRNA) were cleaned with RNA columns provided in the MessageAmp TotalPrep kit. The aRNAs were then quantitated on a spectrophotometer, and quality determined by the Agilent 2100 bio analyzer (Agilent Technologies) according to the manufacturer’s recommendations. 750 µg of each aRNA in water (5 µl) was suspended in Illumina “HYB” buffer (10 µl), heated to 65°C for five minutes, and allowed to cool to room temperature. The samples were applied to Illumina Mouse Ref-8v2 Expression BeadChips and hybridized at 58°C for 16–20 hours at high humidity. Arrays were washed according to Illumina standard protocol. Immobilized, biotinylated aRNAs were then detected by staining with cy3 streptavidin (1 µg cy3-SA per 1 ml of Illumina “Block E1”) for 10 minutes at room temperature. Arrays were washed and dried according to Illumina standard protocol. Arrays were scanned on an Illumina BeadArray Reader. Laser power and PMT voltage were kept constant for Cy3 scans. Images were quantitated by Illumina Beadscan, v3.2 metric. Raw and normalized data sets for all samples involved have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (GEO) repository under the accession number GSE55936.Structural parameters from ECHO were normalized to body weight prior to statistical analysis. Echo and substrate data were analyzed using a 2-Way ANOVA model . Microarray data were imported into Illumina Genome Studio software. On-array spot replicates were averaged by Genome Studio and individual spot probe was reported. The bead chip data were normalized using the cubic spline method in the Illumina Genome Studio software package and exported in log2 factorial design was used to investigate the patterns of differentially expressed genes between sexes, genotypes, and any interactions between these two factors . The 16 samples were divided between two Illumina Mouse Ref-8v2 Expression Bead Chips in a pattern sufficient to identify and correct for batch effects, if any, between chips. Probe sets were filtered from the data set if fewer than 3 replicates in any group had detection p-values greater than 0.05. The filtered data were imported into the statistical program Partek Genomics Suite v6.6 for analyses. Initially, a three-way ANOVA model with interaction  was applied to identify possible batch effects and if necessary to adjust the data accordingly. The 3-way ANOVA model was reapplied to the batch adjusted data to determine statistical differences, and this analysis was followed by contrast comparisons using Fisher Least Squares difference to determine the statistical significance. Fold changes between groups were calculated as the antilog of the log ratio of the two groups of interest . It was expected that the statistical noise  would be greater at low fold changes. Therefore, to confirm the sensitivity of the analysis with respect to fold change, the analysis was repeated on 20 different random combinations of the 16 Bead Chips. The distribution of fold changes for probe sets with a p-value <0.05 from the real combinations are plotted in A replicated (n = 4) 2For exploratory analysis, Hierarchical clustering (HCL) of the significant genes was carried out using Partek genomics suite. HCL analysis was performed on intensity data derived from the ANOVA analysis yielding 254 and 74 probe sets that are differentially expressed between genotype and sex, respectively. The fold changes were subject to normalization, shifting genes to mean of zero and standard deviation of one prior to clustering. Clustering was performed with complete linkage where the distance between two clusters is equal to the distance between the two furthest members of those clusters. The results from clustering analysis are provided in the The significant gene lists, both for genotype and sex variations, were analyzed using Metacore. Analysis of the significant probe sets revealed that there were 208 and 56 identifiable unique genes that were different between genotype and sex, respectively. The gene set thus identified at the FDR 0.05 level was used to identify GO pathways and processes. GO was primarily used to probe the cellular localization of genes, the processes in which they are involved, the metabolic networks, and their potential pathways.To investigate the biological functions of the differentially expressed genes, pathway analysis was conducted using MetaCore. Analysis was performed using the 208 genes that were different between the knockout and wild type mice and for the 56 genes that were different between sexes. The dataset containing gene identifiers and corresponding expression values were uploaded on to the web portal and the identifiers were mapped to its corresponding object in MetaCore’s knowledgebase. Networks for the identified and mapped molecules were then algorithmically generated based on their connectivity. As our interests were primarily on the modulation of genomic actions by ERα, we conducted enrichment analysis for pathways and built network for transcription factors and receptors for the gene list. The transcription factor network analysis generates a list of transcription factors that have targets among the uploaded gene list, and the receptor network generates a list for which ligands are present in the uploaded data set. It then draws the shortest paths between these lists. The networks were generated and scored.FDR threshold has been argued to be too restrictive in conducting pathway analysis −/− mice). In contrast to whole-body ERα knockout mice, which are obese −/− mice were viable, overtly normal, and did not display significant variations in serum substrate concentrations nor weight , there were no significant genotypic variations in the remaining functional parameters. However, sex differences were observed in multiple structural parameters, such as, left ventricle posterior wall diameter, the left ventricle internal dimension, and the inter-ventricular septum  networks and pathways from this analysis are shown in −/−mice . The net−/−mice  and 3.−/−and WT mice. Though estrogen activated expression and translocation of GLUT4 has been shown to be vital for glucose disposal, our microarray analysis failed to reveal any significant difference in Glut4 expression between knockout and wild type animals. However, we observe up regulation of Rab31, a member of the Ras oncogene family, which is known to modulate glucose homeostasis by alterations in GLUT4 partitioning between the cell membrane and intracellular vesicles , Estrogen Receptor Alpha (ERα), and C-Myc are at the hub of transcriptional modulation connecting most of the significant differentially expressed genes  type receptor; interleukin 13 receptor alpha 1 (Il13ra1); Notch 1 precursor; toll-like receptor 2 (Tlr2); and interleukin 28 receptor alpha (Il28ra), several of which have known roles in cardiac metabolism.Because ERα is known to have significant regulatory roles in transcriptional modulation and receptor mediated signaling, network analysis was performed to identify differentially expressed networks between cs-ERαed genes . More th−/− mice . All of The GO analysis based on sex is distinct from that of genotype. The primary sites of localization for sex significant gene-products are in the extracellular space and matrix . The lisNetwork analysis based on sex difference presents a smaller network of receptor genes compared to genotypic differences. Only one receptor coding gene, Ly96 (MD-2), is down-regulated in females. However, there are 4 receptor ligand encoding genes that are differentially expressed between female and male mice. In females, Pf4 (Cxcl4) and Ccl11 (Eotaxin) are down-regulated whereas Mdk (Midkine) and Ptn (Pleiotrophin) are up-regulated . As obse−/− mice (i.e. both sex and genotype differences are observed) (bserved)  A, B. Si−/− mice raised under normal diet in absence of external stress were viable and overtly normal. Our data suggests that ERα modulates numerous genes in the heart that are involved in transcriptional regulation, metabolic control, and oxidative stress in a sex-specific manner. This study has identified potential networks through which ERα may affect cardiac biology. Our study also identified novel genes that are potentially under direct regulation by ERα and whose role in the heart is yet unclear, and we have shown how their biological relevance can be identified through Wnt signaling. It is likely that alterations in the dietary pattern, aging, or induction of metabolic stress could reveal additional phenotypic and metabolic differences for which further studies could lead to a better understanding of cardiac biology and potentially, improved treatment in a sex-dependent manner.In conclusion, cs-ERαFigure S1Hierarchical Cluster analysis of genotype significant genes from cardiac mRNA microarray results. mRNA expression patterns were established for cardiac tissue from both male and female, ERα −/− and wild type mice. HCL analysis was performed on intensity data using partek genomic suites for the significant gene list. FDR (0.05) corrected genes with a p value of 0.05 or less were considered significant.(DOCX)Click here for additional data file.Figure S2Hierarchical Cluster analysis of sex significant genes from cardiac mRNA microarray results. mRNA expression patterns were established for cardiac tissue from both male and female, ERα −/− and wild type mice. HCL analysis was performed on intensity data using partek genomics suite for the significant gene list. FDR (0.05) corrected genes with a p value of 0.05 or less were considered significant.(DOCX)Click here for additional data file.


Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings.Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean ( The amplification and detection of signal from nucleic acid targets to test for the presence of specific genetic markers in sample material are of ever increasing importance in a vast array of application areas. These include trait detection applications in the agricultural sector, as well as clinical diagnostics, testing for food-borne pathogens, environmental testing, and many others –3.Collectively, the field of nucleic acid based testing may be termed “molecular diagnostics,” and its central step most often consists of nucleic acid amplification techniques (NAATs). NAATs owe their increasing popularity to their extremely high sensitivity, specificity, speed, and operational simplicity. NAATs, in fact, increasingly complement or may replace traditional methods such as culturing techniques –6 and anSince its inception in the late 1980s, the polymerase chain reaction (PCR) has been the mainstay technique for the amplification of nucleic acids. Although PCR-based testing in laboratories is well established and extremely successful, the reliance of PCR on precise thermal control  resulting in formation of filaments of protein coated oligonucleotide complexes. Binding of GP32 to the oligos is followed by UvsY and finally UvsX, the recombinase. The UvsX/oligo complex searches the template DNA for homologous sequences and subsequently extends the primers. Target amplification is detected with a dual labeled oligonucleotide probe, containing a fluorophore (either TAMRA or FAM) and quencher separated by an abasic site , of compRPA has been successfully employed in a number of reports for both human IVD –27 and vIn order to constitute a fully integrated diagnostic system for the detection of genetic traits, the chosen sample processing procedure and the RPA biochemistry have to be augmented by a compatible read-out approach that is capable of monitoring a signal generated by the target amplification event and thus delivering the diagnostic information. Moreover, this detection method has to maintain the overall operational simplicity and portability of the complete POC system. An extremely useful tool to achieve these requirements is the use of a novel type of fluorogenic oligonucleotide probe specifically designed for RPA, in combination with appropriate fluorescence detection equipment. This approach is described in greater detail elsewhere , 16.Zea mays L.), soybean (Glycine max), and cotton (Gossypium sp.). Currently, GMO crops are grown in ~30 countries worldwide over an accumulated area of ~1.5 billion hectares [One potential application for the use of NAAT-based diagnostic devices is to develop field detection methods for genetically modified (GMO) crops. Since the first commercialization in the USA in 1996, millions of farmers demonstrate confidence in the benefits of GMO crops as evident by high adoption rates , especiahttps://www.bio.org/sites/default/files/Product-Launch-Stewardship-11272012.pdf) [As part of the stewardship efforts supporting the commercialization of GM crops, international organizations such as BIO (Biotechnology Industry Organization) developed policy guidance documents (012.pdf)  which maProtein-based lateral-flow devices (LFD) are the current gold standard for the rapid, on-site test format for GMO sample screening , 9, 32. Monsanto's Genuity Roundup Ready 2 Yield (RR2Y) and its predecessor Roundup Ready® soybean present a unique case study for the application of RPA technology to distinguish the two products at the molecular level. The unique protein expressed in RR1 and RR2Y soybean is the same CP4 EPSPS, preventing the use of protein-based LFD method to differentiate the two products.The current publication reports the use of new NAAT-based technology platform to develop a field detection method, using RR2Y-specific assay as an example. This represents the development of a rapid and inexpensive field-deployable detection method that can effectively differentiate between samples from different GMO events, in this case RR1 and RR2Y soybean that express the same CP4 EPSPS protein. The report also demonstrates the ease and reliability of the method starting from a laboratory sample such as purified DNA template or whole seed sample encountered in a field.Typically, 100 seeds were collected and crushed in Oster Blender  with ice crushing blade (#4961 USA). One scoop (~90 mg) of the seed powder was transferred to a tube containing 4 mL of lysis buffer (0.2 M NaOH). Each tube was shaken for approximately 5 seconds and incubated at room temperature (RT) for 1 minute to let the particulates settle down.™ kit (Zymo Research) using a modified method of the manufacturer's instructions. Briefly, 1.3 mL of DNA Binding Buffer (with β-mercaptoethanol (BME)) was added to each 2 mL screwcap tube, followed by addition of ~90 mg of ground seed sample. Seed solution was mixed in a Disruptor Genie for 2 minutes and centrifuged at 10,000 ×g for 2 minutes. Supernatant (900 µL) was transferred to a Zymo-Spin IIC Column in a collection tube and centrifuged at 10,000 ×g for 2 minutes. Zymo-Spin IIC Column was transferred to a new collection tube and 200 µL of DNA Pre-Wash Buffer was added, followed by centrifugation at 10,000 ×g for 1 minute. Plant/Seed DNA Wash Buffer (500 µL) was added to the Zymo-Spin IIC Column and centrifuged at 10,000 ×g for 1 minute. DNA was eluted from the Zymo-Spin IIC Column by incubating in presence of 30 µL of elution buffer at RT for 2 minutes, followed by centrifugation at 10,000 ×g for 30 sec.DNA was extracted from ground seed using the ZE Plant/Seed DNA miniprepOligonucleotide primers and probes used to develop the RPA assays were purchased from Biosearch Technologies . The RPA reactions were typically carried out at 39°C, unless otherwise noted, using a device that maintains constant temperature combined with fluorescence detection . Reaction contents were mixed prior to amplification and again at five minutes during incubation. The output of the reaction was monitored in real time using the Twista Studio Software  with fluorescence measurements taken every 20 seconds for a total of 15 (or 20) minutes .µL gp32, 120 ng/µL uvsX, and 30 ng/µL uvsY, 0.5 U of ExoIII, and 0.25 U of POL. Assay pellets were generated by lyophilizing reaction mixtures containing RR2Y primers at 420 nM, lec primers at 240 nM, RR2Y and lec specific probes at 120 nM each, 100 ng/µL creatine kinase, 5.5% PEG 35 K, 6% Trehalose, 2.5 mM ATP, 50 mM phosphocreatine, and 240 µM of each dNTP. To initiate an amplification reaction, the lyophilized pellets were rehydrated with 46.5 µL of rehydration buffer . Tubes were spun down after thorough mixing by vortex. One µL of purified DNA or a loop (~1 µL) of seed extract or buffer alone for no template control (NTC) was added to the rehydrated pellets. Pellets were mixed by vortex and spun down and 2.5 µL magnesium acetate was added  to initiate the reaction. Samples were mixed by vortex and spun down prior to transferring the tubes to the Twista fluorescent reader.RPA assays with purified DNA as the template were performed as described below. Lyophilized RR2Y RPA Exo pellets were obtained from TwistDx. The RR2Y RPA Exo pellets comprised all essential components including the critical recombinase and polymerase proteins, such as 900 ng/Preassigned cutoff values are determined based on statistical analysis of a large set of data. These cutoff values are used to generate an algorithm that can translate the fluorescent amplification curves into positive/negative/invalid calls by the Twista fluorescent reader. The algorithm considers background fluorescence values, onset of amplification, and the strength of the signal between two defined time points (5–8 minutes) to determine the results of the assay. Using this algorithm, Twista can report the assay results without any manual intervention.PCR amplification was carried out in a thermocycler  using the following primer pairs.The oligonucleotide primers were purchased from Integrated DNA Technologies . lec was carried out using the Qiagen Multiplex PCR Mastermix (2x) and 100 ng of genomic DNA in a final reaction volume of 40 µL. PCR amplification for RR2Y was carried out using the HotStart-it™ Taq Mastermix (2x) and 100 ng of genomic DNA in a final reaction volume of 50 µL. RR1 and RR2Y reactions were performed using 200 nM forward and reverse primers, whereas the lec reactions were performed using 100 nM primers. The following cycling conditions were used for RR1 and lec reactions: 95°C for 10 minutes, 44 cycles of 94°C for 15 seconds, 62°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The following cycling conditions were used for RR2Y reactions: 95°C for 2 minutes, 34 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes.PCR amplification of RR1 and™ Gel Loading Dye (Invitrogen) and run in 8-well 1% precast agarose gel containing 1% ethidium bromide. Pictures of agarose gels were captured using the Molecular Imager® Gel Doc™ XR.The PCR products were mixed with 10x BlueJuice™ Kit for Roundup Ready Soybean  according to the manufacturer's protocol. Briefly, 100 soybean seeds were counted and ground on high speed for 20 seconds. Seed powder and water were mixed in a ratio of 1 : 5 (w/v) and manually shaken for 30 seconds. About 12 mL of the extract was transferred carefully in order not to collect any solids into a three-ounce sample cup. The bottom portion of the test strip was dipped in the extract for 5 minutes. Strips were photographed at the end of five minutes for documentation. A pink band corresponding to the Control band developed in all strips indicating that the strips have functioned properly. Presence of the desired product is indicated by the appearance of an additional pink band above the Control band.For lateral-flow strip experiments, conventional, RR1, and RR2Y soybean were tested in triplicate with the QuickStixTo determine long-term stability of RPA reaction pellets at different temperatures, all experimental parameters such as template, buffers, analyst, protocol, and detection devices were kept constant. Reaction pellets were stored at four different temperatures: ≤−15°C (reference), 2–8°C, 22–28°C (ambient), or 35–40°C. Reaction pellets were produced as a single batch, packaged in a dry-room facility as strips of 8 PCR tubes, vacuum-sealed, and shipped on dry ice. The ≤−15°C, 2–8°C, and 22–28°C (ambient) storage units were tracked via an automated system to ensure accurate temperatures. The 35–40°C storage unit was tracked on a weekly basis by manual recording. The reaction pellets from all four temperature conditions were tested at 18 defined intervals over a year.yijk is the observed response of the jth well of the ith temperature at the kth time; μ is the overall mean, τi is the effect of the ith temperature; θi(j) is the random effect of the jth well in the ith temperature; δk is the effect of the kth time; τδik is the effect of the interaction between the ith temperature and the kth time; and εijk is the residual error.At each time point, eight reactions were tested per storage condition including six reactions using 0.1% RR2Y genomic DNA as positive control template and two reactions using genomic DNA from conventional soybean. Positive, negative, or invalid calls were made based on a slope cutoff of 90 mV/min for FAM and 600 mV/min for TAMRA. Statistical analysis was used to determine overall results of stability experiments using the following analysis of variance (ANOVA) model:n would be followed by a nonsignificant difference at time n + 1. As a result a biologically meaningful difference was also used to evaluate the data. When the average response reached half of the mean at time zero, the pellets were considered unstable.Pairwise comparisons of each time point to time zero were defined within the ANOVA model and tested using Dunnett's test at the 5% level. There were multiple cases where statistical significance did not indicate a lack of stability. For example, a significantly lower response at time This report describes an assay designed to detect a DNA sequence of a genetic element in the soybean genome coding for CP4 EPSPS that confers tolerance to the herbicide Roundup®.lec), as an internal control. Amplification of an endogenous gene such as lec confirms (1) the general activity of the RPA amplification/detection “machinery” of the reaction; (2) the effectiveness of the sample preparation procedure; and (3) the input of the correct sample type (soybean in this case).The assay described here is designed in a duplex format to allow simultaneous amplification and detection of the RR2Y insertion and the endogenous soybean gene, lectin . Purified genomic DNA isolated from conventional, RR1, or RR2Y soybean seeds were used as templates to determine the specificity of the assay, as observed by detectable signal in 5–15 minutes  of RR2Y material in a mixture with 99.5% of non-RR2Y  soybean seed material. Due to the nature of the sample preparation procedure, in practice about 10–30 ng of total soybean DNA was analyzed per RPA reaction which corresponds to about 4000–12000 copies of genomic DNA. In a typical reaction at the specified discrimination level of 0.5% (w/w), 20–60 copies of the RR2Y target may be present to act as template for RPA. The copy numbers were calculated based on the genome size of soybean. In order to meet this requirement, first, the sensitivity of the RR2Y analyte detection must be high enough to be able to detect as low as 20 copies per reaction and, second, the assay must perform without significant loss of sensitivity, even in the presence of a comparatively large amount of total soybean DNA. lec control reaction  are to some degree in competition with each other . Additionally, at the discrimination level of 0.5%, a large imbalance of starting amount of template exists between the two component reactions, with the lec target being up to 200 times more numerous than the RR2Y analyte. These two factors need to be accounted for in the relative performance of the two assays. A duplex assay challenged with a low number of RR2Y target copies in the background of conventional soybean DNA would reflect the performance of the assay in a POU setting, compared with that described above  on the performance of the assay . All the lec target were stable for one year in a freezer or refrigerator or at RT  are potentially unstable, some of them even under refrigerated conditions, and are, therefore, provided in a lyophilized format as preformulated reaction pellets in microcentrifuge PCR strip tubes. Each strip of lyophilized RPA pellets was vacuum-sealed in a foil pouch. Sealed strips of lyophilized RPA reaction pellets were stored at different temperatures: in a freezer (<−15°C), in a refrigerator (2–8°C), at room temperature , and in an incubator (35–40°C). Eight reaction tubes from the respective storage temperature were tested for assay performance at regular intervals over a period of one year. RPA assays for theor at RT . However20 weeks . The RR2x months . IncreasTo test the feasibility of RPA in POU setting, we performed a set of experiments. The current benchmark for field testing involves protein-based LFD that detects the presence of the target protein in crude seed extracts . The uni lec target , − (negative), or ?  at the end of the reaction (data not shown). Results from the screen display matched the amplification curves (data not shown), as expected. lec gene as a control with soybean samples, irrespective of the specific trait(s) of interest for a given RPA assay.In summary, the present studies demonstrate that RPA-based field detection assays can be fast and simple and performed by a relatively untrained user. The ability of RPA assays to detect DNA sequences unique to GMO crops provides an advantage over LFD for GMO crops that share the same protein. RPA assays also have intrinsic advantages over PCR assays. RPA assays are simpler, easier, and faster than PCR assays for an end-user. Instrumentation used for the RPA-based assays is relatively cheaper than a PCR thermocycler, largely owing to the isothermal nature of the RPA reaction. In particular, the current report presents an RPA-based field detection method that can specifically detect RR2Y soybean. The assay was developed in a duplex format allowing simultaneous detection of the endogenousDetection of a number of novel traits could benefit from employing RPA, including RNA interference-based traits where the inserted DNA in the transgenic event does not encode a protein (Vistive® Gold Soybean). RPA technology provides benefit to products with multiple stacked traits by delivering insertion-specific detection of the individual traits. The technology offers a significant breakthrough for the development of detection methods at the elevators and ports and in the field to ensure regulatory and product stewardship compliance and validate product claims.


Salmonella Typhi from blood culture is the standard diagnostic for confirming typhoid fever but it is unavailable in many developing countries. We previously described a Microwave Accelerated Metal Enhanced Fluorescence (MAMEF)-based assay to detect Salmonella in medium. Attempts to detect Salmonella in blood were unsuccessful, presumably due to the interference of erythrocytes. The objective of this study was to evaluate various blood treatment methods that could be used prior to PCR, real-time PCR or MAMEF to increase sensitivity of detection of Salmonella.Isolation of Salmonella in spiked blood was improved when whole blood was first lysed using erythrocyte lysis buffer prior to DNA extraction. Removal of erythrocytes and clotting factors also enabled reproducible lysis of Salmonella and fragmentation of DNA, which are necessary for MAMEF sensing.We tested ammonium chloride and erythrocyte lysis buffer, water, Lymphocyte Separation Medium, BD Vacutainer® CPT™ Tubes and dextran. Erythrocyte lysis buffer was the best isolation method as it is fast, inexpensive and works with either fresh or stored blood. The sensitivity of PCR- and real-time PCR detection of Salmonella detection by PCR and real-time PCR and has allowed lysis and fragmentation of Salmonella using microwave radiation (for future detection by MAMEF).Use of the erythrocyte lysis procedure prior to DNA extraction has enabled improved sensitivity of Salmonella in blood.Adaptation of the blood lysis method represents a fundamental breakthrough that improves the sensitivity of DNA-based detection of  Salmonella enterica serovars Typhi and Paratyphi A and B cause enteric fever, febrile illnesses that cannot be clinically distinguished from one another and which are difficult to differentiate from other causes of febrile illness  serovars, in particular, S. Typhimurium and S. Enteritidis, are cardinal causes of invasive bacterial disease  in young children and HIV-infected adults and diagnostic tests are needed to identify these infections and to differentiate them from enteric fever   with gel-based identification of amplicons is potentially more sensitive and rapid than blood culture and reportedly detects as few as 10 CFU ml. et al. .Salmonella  locus that is conserved among all Salmonella serovars. Following microwave-accelerated lysis, released and fragmented bacterial DNA is hybridized to a molecular probe, allowing detection of as low as 1 CFU of Salmonella in 1 ml of bacteriologic medium in <30 s. The MAMEF Salmonella assay is specific and does not detect other bacterial species commonly isolated from blood  is an ultra-fast, highly sensitive DNA hybridization-based methodology that has been adapted to allow more sensitive detection of . et al.  to detecSalmonella in culture medium, initial attempts to detect bacteria in diluted whole blood following lysis were not reproducible due to congealing of erythrocytes and clotting factors during microwave heating. Recognizing that about 60% of Salmonella in the blood of an infected individual reside in the mononuclear cells and that direct plating of buffy coat yielded sensitivity comparable to blood culture   . Salmonella spp., E. coli, P. aeruginosa and K. pneumoniae were grown in HS bacteriological medium , 5 g Hy Yest 412  in 1 l distilled water) at 37°C. Media were supplemented with guanine (0·001% w/v) and 50 μg ml−1 carbenicillin for growth of CVD 1940(pGEN206); culture medium for CVD 909 was supplemented with 2, 3-dihydroxybenzoate (DHB) (0·0001% w/v). Whole human blood with sodium heparin anticoagulant was purchased from Innovative Research . For the buffy coat experiments, fresh blood was obtained from volunteers under the approval of the University of Maryland, Baltimore Institutional Review Board and used within 8 h. All blood donors provided written informed consent.Attenuated 3 CFU of S. Enteritidis strain CVD 1940 (pGEN206) were inoculated into 2 ml of whole blood and the buffy coat was separated by mixing the blood with 2 ml of 1× PBS, layering carefully onto lymphocyte separation medium  and centrifuging at 400 g for 10 min. The buffy coat was removed and lysed with a final concentration of 1% Triton X-100 . Viable counts were performed by mixing the lysed buffy coat with 20 ml of molten HS-agar supplemented with guanine (0·001% w/v) and 50 μg ml−1 carbenicillin. Colonies were counted after 24-h incubation at 37°C and the percentage of intracellular bacteria recovered was determined in relation to the initial inoculum.7 × 10−1 of NaCl), as necessary.Six techniques to separate white blood cells from erythrocytes were evaluated, including erythrocyte lysis buffer, ammonium chloride lysis buffer, distilled water, BD Vacutainer® CPT™ tubes, dextran, and lymphocyte separation medium, as described below. Following erythrocyte lysis, white blood cell pellets were suspended in various diluents  and 1× TE buffer plus 5 g lg using a swing bucket rotor followed by removal of the supernatant, thus removing the erythrocyte debris and leaving behind a pellet consisting of white blood cells and a small amount of erythrocytes. The pellet was resuspended in 20 ml of fresh erythrocyte lysis buffer , inverted five times and centrifuged again to ensure complete lysis of erythrocytes. The supernatant was removed leaving behind a white blood cell pellet.Two ml of whole human blood were mixed with 18 ml of 1× erythrocyte lysis buffer diluted from a 10× stock  or ammonium chloride buffer alone (8·26 g ammonium chloride in 1 l distilled water). Each sample was inverted 10 times, incubated at room temperature for 2 min and centrifuged for 5 min at 400 g, thus removing the erythrocyte cell debris and leaving behind a white blood cell pellet. The pellet was resuspended in 20 ml water, inverted five times and centrifuged again to ensure complete erythrocyte lysis. The supernatant was removed leaving behind a white blood cell pellet.Two millilitres of whole blood were mixed with 18 ml of distilled water. The sample was inverted 10 times, incubated at room temperature for 2 min and centrifuged for 5 min at 400 g for 15 min. The buffy coat layer was transferred to a fresh tube, resuspended and washed in 1× PBS and centrifuged at 16 000 g for 3 min to remove the PBS.Buffy coat was obtained by drawing 8 ml of whole blood into a BD Vacutainer® CPT™ cell preparation tube containing sodium heparin and centrifuged in a swing bucket centrifuge at 1500 −1 sodium citrate and incubated for 30 min at room temperature (Boyum g for 3 min to remove the erythrocytes and pellet the white blood cells.Buffy coat was obtained by mixing 2 ml of fresh whole blood with 2 ml of 2% dextran (Sigma Aldrich) in 0·9% normal saline containing 25 mmol lre Boyum . The buf®). The mixture was centrifuged at 400 g using a swing bucket rotor for 5 min to separate the buffy coat from the erythrocytes and plasma.Buffy coat was obtained by mixing 2 ml of fresh whole blood with 2 ml of 1× PBS and layering onto 2 ml lymphocyte separation medium  according to the manufacturer's instructions. The purified DNA was eluted in 300 μl of nuclease-free water.DNA was extracted from 2 ml of whole blood spiked with 4·4 × 10μl PBS and subsequently spiked with 4·4 × 103 CFU, 4·4 × 104 CFU, 4·4 × 105 CFU or 4·4 × 106 CFU of CVD 909 and DNA extraction was performed using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer's instructions. The purified DNA was eluted in 100 μl of nuclease-free water.Two millilitres of whole blood was lysed using erythrocyte lysis buffer as described above. The white blood cell pellets were resuspended in 200 −1 MgCl2, 2·5 U Green Taq DNA polymerase , 0·2 mmol l−1 dNTP and primers oriCF (5′- TTATTAGGATCGCGCCAGGC-3′) and oriCR (5′- AAAGAATAACCGTTGTTCAC-3′) which detect all Salmonella at a final concentration of 0·5 μmol l−1 and combined with 5 μl of extracted DNA in a reaction volume of 50 μl in a T100™ Thermal Cycler   according to the manufacturer's instructions. Concentrations of CVD 909 were obtained by serial dilution in HS bacteriological medium. The purified DNA was eluted in 300 μl PBS and subsequently spiked with three ten-fold dilution series of CVD 909 (ranging from 0·3 CFU to 6 × 105 CFU). DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer's instructions. Viable counts of CVD 909 were obtained by serial dilution on HS-agar plates. The purified DNA was eluted in 50 μl of nuclease-free water or AE buffer from the QIAamp kit.Two millilitres of whole blood was lysed using erythrocyte lysis buffer as described above. The white blood cell pellets were resuspended in 200 S. Typhi putative fimbrial-like adhesion protein and were taken from Nga et al. . Cycling parameters were as follows: initial denaturation of 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 3 s, annealing and extension at 60°C for 30 s.Primers used for real-time PCR targeted STY0201, a et al. 9 CFU ml−1) were diluted to the desired concentration in PBS and 10 μl of bacteria were added to the white blood cell suspension. The bacteria-spiked white blood cell samples were then heated in a Frigidaire microwave  for 25 s on 30% power. In order to determine the optimal media for microwave lysis, lysis in 1 ml of water was compared to the same volume of 1× TE buffer, 1× PBS, 0·05× PBS, APF-LB and 1× TE buffer plus 5 g l−1 of NaCl. A cracking scale for the gold lysis bow-tie structures after exposure to microwave irradiation was determined on a scale of 1–5, with 1 representing small cracks and 5 representing complete disintegration of the gold bow ties. For visualization of DNA fragments on a gel, 1 ml overnight bacterial culture containing 109 CFU Salmonella was pelleted, resuspended in 1 ml TE plus 5 g l−1 NaCl and added to white blood cells separated from 2 ml whole blood by erythrocyte blood cell lysis buffer. The suspension was microwave lysed and DNA was ethanol precipitated with 0·1× volume of 3 mol l−1 sodium acetate pH 5·2 and 2× volume of prechilled molecular grade ethanol. DNA was pelleted, washed with 70% ethanol, air-dried and resuspended in 30 μl of TE and the entire volume was electrophoresed on a 2% agarose gel. DNA fragments were visualized by staining with ethidium bromide to identify double stranded DNA or SYBR® Gold  to identify single stranded DNA. DNA fragment size was verified with the Agilent 2100 Bioanalyzer .In order to focus microwaves, gold bowtie lysis triangles were deposited on glass slides as described previously  was found to be taken up by the mononuclear cells, results which are corroborated in previous studies by Rubin et al. , 1× erythrocyte lysis buffer removed erythrocytes most effectively, and yielded the purest sample of white blood cells Fig.. When thtran Fig.. HoweverSalmonella in blood could be improved by targeting white blood cells instead of using whole blood, we used conventional gel-based PCR. We found that when the QIAamp Mini blood kit was used to extract DNA from spiked white blood cells (separated from 2 ml whole blood using 1× erythrocyte buffer), we could detect 4·4 × 105 and 4·4 × 106 CFU of S. Typhi CVD 909 by PCR. In contrast, when the QIAamp Midi blood kit was used to extract DNA from 2 ml spiked whole blood, only the 4·4 × 106 CFU sample was detected  values.Following the promising results using gel-based PCR, we determined whether combining RBC lysis with a mini QIAamp kit could also lead to improved detection by real-time PCR. First, we spiked whole blood with various concentrations of a et al.  who showa et al. . We founa et al. . As expeSalmonella in whole blood by MAMEF were unsuccessful due to congealing of erythrocytes and clotting factors under microwave heating (results not shown). The role of clotting factors in congealing was supported by the fact that when plasma and serum were microwave lysed, samples also congealed under exposure to microwave heat (results not shown).Previous attempts to detect Salmonella are intracellular and focused our attempts on harvesting and lysing white blood cells. We tested microwave lysis of white blood cells spiked with Salmonella and suspended in various diluents. When approx. 1000 CFU of bacteria were serially diluted in PBS down to 0·1 CFU, added to purified white blood cells, resuspended in 1 ml TE plus 5 g l−1 NaCl and microwave lysed, 100% lysis was observed reproducibly in spiked samples containing 100 CFU or more as determined by pre- and postlysis viable counts . As a result of complete removal of erythrocytes using 1× erythrocyte buffer, we were able to avoid congealing under microwave heat.We took advantage of the fact that approx. 60% of −1 NaCl), the peak fragment size was approx. 100 bp. Similarly, when the white blood cells were resuspended in 0·05× PBS, which contains approx. 6 mmol l−1 sodium chloride, DNA fragments of about 100 bp were also observed (Table −1 sodium chloride) boiled over in the microwave due to overheating at these high salt concentrations and sample was lost in the microwave cavity. Optimal conditions for microwave lysis were ultimately identified as white blood cells suspended in APF-LB . Eventually, we were able to estimate the effectiveness of microwave lysing and DNA release by visually inspecting the cracking of the gold bow tie after microwave lysis with more cracking signalling maximal DNA release and fragmentation  was the same as was previously optimized for broth samples as described by Tennant et al. . On the other hand, when too much salt was present, the sample reached boiling temperature too rapidly and the sample boiled over in the microwave Table . When thed Table . In contt et al. ) or 1× Tt et al. , side bySalmonella suspended in bacteriological medium can be successfully lysed using microwaves, allowing highly sensitive detection of Salmonella DNA by MAMEF  makes it too expensive for routine use in many resource poor areas. Among the buffy coat extraction methods, dextran is the most promising because it is easy to perform, inexpensive, easy to handle and requires only an inexpensive, small microcentrifuge. However, it is highly effective only when used with fresh blood.Salmonella after erythrocyte lysis, it is conceivable that if 1000 CFU of Salmonella were present in a sample of blood, 600 CFU (60%) would be recovered from the intracellular portion along with 160 CFU (16%) of the 400 extracellular Salmonella, totalling 760 CFU, i.e. 76% of the CFU present in the entire blood sample.For utilization in field situations where blood may be collected from a remote site and transported for some hours to a blood processing laboratory, erythrocyte lysis buffer is the preferred method for isolation of white blood cells, as it does not require fresh blood for optimal separation. Additionally, as we showed that we could recover an additional 40% of extracellular Salmonella infection in young and malnourished subjects from whom only small volumes of blood are typically collected to make a laboratory diagnosis.Despite the limitations inherent in each separation method, one major advantage common to all is that any volume of blood may be processed and the white blood cells resuspended in any volume necessary for detection by molecular methods such as MAMEF or PCR or quantitative real-time PCR. This is particularly important in et al. . Likewise, when real-time PCR was used, we were able to detect as low as 0·3 CFU S. Typhi mixed with white blood cells from 2 ml of blood. In contrast, detection of DNA extracted from whole blood using a midi kit shows a detection limit of more than 40 CFU S. Typhi. The increased sensitivity is most likely due to the fact that we could elute the DNA in a smaller volume using the QIAamp DNA Blood Mini kit than the QIAamp DNA Blood Midi kit. As it is not possible to have <1 CFU, we hypothesize that we are also detecting dead bacteria which is an advantage of using molecular methods.It is important to stress that testing white blood cells alone from typhoid fever patients does not decrease sensitivity, as has already been demonstrated by Wain et al. , where cSalmonella suspended in a buffy coat fraction in preparation for detection by MAMEF. Work is currently underway to determine the level of sensitivity of MAMEF.We have also shown that we can effectively lyse Salmonella using microwave radiation (for future detection by MAMEF). We are currently evaluating whether erythrocyte lysis and subsequent detection by real-time PCR is faster and more sensitive than blood culture in a typhoid endemic setting. These blood treatment methodologies may also be applicable for the typhoid/paratyphoid diagnostic assay (TPTest) that detects IgA antibody to Salmonella antigens elaborated by mononuclear cells (Khanam et al. Salmonella, but which are capable of performing molecular biology procedures.We conclude that adaptation of the erythrocyte lysis procedure has enabled improved sensitivity of detection by PCR and real-time PCR following DNA extraction and has allowed lysis and fragmentation of