At the upstream site, which had a lower vegetation cover and highest soil temperatures, the poised electrodes inhibited CH4 emissions by ∼45% . CH4 emissions were not significantly impacted at the downstream site. N2/N2O emissions were generally low at both sites and were not impacted by poised electrodes. We did not find a direct link between bioelectrochemical treatment and microbial community membership; however, we did find a correspondence between environment/function and microbial community dynamics.Dissimilatory metal-reducing bacteria are widespread in terrestrial ecosystems, especially in anaerobic soils and sediments. Thermodynamically, dissimilatory metal reduction is more favorable than sulfate reduction and methanogenesis but less favorable than denitrification and aerobic respiration. It is critical to understand the complex relationships, including the absence or presence of terminal electron acceptors, that govern microbial competition and coexistence in anaerobic soils and sediments, because subsurface microbial processes can effect greenhouse gas emissions from soils, possibly resulting in impacts at the global scale. Here, we elucidated the effect of an inexhaustible, ferrous-iron and humic-substance mimicking terminal electron acceptor by deploying potentiostatically poised electrodes in the sediment of a very specific stream riparian zone in Upstate New York state. At two sites within the same stream riparian zone during the course of 6 weeks in the spring of 2013, we measured CH Riparian zones, which are the areas of land adjacent to streams, are often hotspots for biogeochemical transformations . These ein situ experimentation. Here, we utilized a BES to study the interactions between microbial communities and the presence of iron(III) and humic substances as potential terminal electron acceptors under anaerobic conditions in a riparian zone.Bioelectrochemical systems (BESs) are engineered systems that utilize a biocatalyst, such as bacteria or enzymes, at electrodes. Microbial BESs capitalize on the ability of, for example, dissimilatory metal-reducing bacteria to respire with solid-state electrodes via extracellular electron transfer . These Bin situ. For example, our recent work in Arctic peat soils has shown that bioelectrochemical manipulation designed to enrich for dissimilatory metal reduction actually increased methane emissions from soils. In this case, there was likely a bottleneck in the degradation of plant organic matter, which was widened by bioelectrochemical manipulation and stimulated the production of fermentation-like products , environmental parameters , and ecosystem function (rates of CH4 and N2/N2O emissions). The objective of this work is to provide a foundation for elucidating the complex relationships between microbial community structure, community function, and ecosystem function ; ultimately, the goal is to better inform modeling efforts through a more complete understanding of these complex relationships. One of our main objectives was to find correlations between changes of the environment, the community structure, and microbial function during the experimental period for several different experimental systems at two sites, rather than to describe the microbial community (α diversity) as a snapshot measurement, which does not inform about its function  or unpoised electrodes (control) during a sampling period of 6 weeks in the spring of 2013. We also gathered environmental data during the experiment, including: soil temperature, pH, dissolved oxygen concentration, conductivity, iron concentrations and speciation, and anion concentrations. Once per week, bacterial biofilms were harvested from both poised and unpoised electrodes for 16S rRNA gene sequencing to determine microbial community structure. Here, we demonstrated the capability of small alterations to redox conditions to impact carbon release to the atmosphere, and provide a foundation for future examinations of biogeochemical cycling using in situ bioelectrochemical manipulations. Our hypothesis was that placement of iron(III)/humic substance-mimicking electrodes would reduce the emissions of methane in a very specific riparian zone of Fall Creek in Freeville, NY, USA.At two distinct sites adjacent to an agricultural field within the riparian zone of Fall Creek in Freeville, NY, we measured CHThis experiment was conducted within Fall Creek, which is a third order stream in Central New York that passes through the Homer C. Thompson Vegetable Research Farm and a Cornell University facility in Freeville, NY . To create the base of the chamber, the bottom 2-cm of the bucket was removed leaving a 17-cm-long cylinder; when placed in the soil, 11-cm of the soil chamber was below the surface and 6-cm extended above the soil surface , while each working electrode (WE) consisted of six 6-cm × 6-cm × 0.635-cm blocks connected in parallel . Having multiple electrodes in parallel for the WEs allowed for the harvesting of biofilms during different stages of the experiment. The WE and reference electrode (RE) were placed inside the chamber, while the CE was placed outside the chamber on the opposite side of the membrane to maintain an electrical connection while preventing cathodic hydrogen or methane production inside the soil chamber. Electrodes were connected to microcontroller-based potentiostats (SHE using a microcontroller-based potentiostat (The working and counter electrodes (CE) were manufactured from medium-extruded graphite plates . The CE consisted of a 6-cm × 6-cm of 0.635-cm thick block , we indicate this in the text as N2/N2O emissions. We prepared and used beeswax-coated calcium carbide tablets, which react with water to form acetylene gas, as described by 2O reduction . N2O separation was performed using a Supel-QTM PLOT capillary column  with ultra-pure helium carrier gas (2.6 mL min-1) and 95:5 Ar:CH4 make-up gas (8.2 mL min-1) and an electron capture detector set to 250°C. CH4 and C2H2 separation was performed using a Carboxen 1006 PLOT capillary column  and a flame ionization detector set to 200°C with H2 gas (30 mL min-1), air (400 mL min-1), and N2 makeup gas (25 mL min-1). The oven temperature was initially set to -22°C for 4.7 min, then increased to 30°C for 0.85 min and finally increased to 80°C for 2.5 min to allow for elution of all three gasses of interest. Calibration curves were made using serial dilutions of 1 ppm N2O, 20 ppm CH4, and 2.5% C2H2 (Airgas Inc.). Emissions were calculated as the slopes of the linear regression (concentration vs. time) curves (R2 > 0.9) for each measurement period (flux).Measurements were taken every Monday and Thursday during the 6-week experimental period. We measured denitrification rates using the acetylene inhibition method, where acetylene is added to the soil to inhibit reduction of N2O to N2 , allowineduction . Gas emi2+ and total Fe using the ferrozine assay  and probes . Soil water was collected from within each soil chamber (2–7 cm below the soil surface) using porous soil moisture samplers , vacutainers , and 21G needles . These samples were analyzed for nitrate, nitrite, chloride, and sulfate concentrations using a Dionex ICS-2000 ion chromatograph with IonPac AS-18 analytical column and 25-μL sample loop. Immediately following the collection of soil water, 0.5 mL of each sample was transferred to another vacutainer containing 0.5 mL of 0.5 N HCl; these samples were analyzed for Fene assay .® DNA Isolation Kit . Bacterial extraction product was then amplified in duplicate 50-μL polymerase chain reactions (PCRs) according to ® E-Z Pure Kit . Cleaned product was again confirmed with gel electrophoresis and barcoded amplicons for each sample were pooled at equimolar ratios to a final concentration of 8 ng DNA μL-1. The single, pooled amplicon mixture was sequenced at the Cornell University Biotechnology Resource Center using an Illumina MiSeq . Data were quality filtered with the quantitative insights into microbial ecology (QIIME) platform  using a sterile blade. Samples were placed on wet ice immediately in the field, and then stored at -20°C until the completion of the experiment. Following biofilm harvesting, the bare electrodes were returned to the soil to keep the WE surface area constant throughout the experiment. Genomic DNA was extracted using a PowerSoilm v 1.6; . Forwardm v 1.6; , and appm v 1.6; .4 emissions bi-weekly in each of 12 chambers split between two riparian zone sites over the 6-weeks experimental period and calculated the absolute average CH4 emission (Table 1). We observed differences between the two riparian zone sites, which were 50 m apart, in regards to CH4 emissions, showing that this specific riparian zone was heterogeneous. The absolute average CH4 emissions at the upstream site were considerably higher compared to the downstream site; absolute average CH4 emissions with the unpoised electrode (control) chambers were 1.96 ± 1.42 mg CH4∗m-2∗h-1 at the upstream site compared to 0.79 ± 0.65 mg CH4∗m-2∗h-1 at the downstream site (Table 1). On average, the absolute CH4 emissions from the poised electrode chambers at the upstream site were ∼50% lower than those from the unpoised electrode chambers, but due to the large temporal error not significantly different . At, the downstream site the absolute average CH4 emissions from the poised electrode chambers were similar compared to those from the unpoised electrode chambers . However, there were large temporal trends throughout the experimental period. The CH4 emissions for each chamber were highest at the beginning of the experiment (earlier in the spring) and decreased throughout the 6-week experimental period (data not shown).We measured CH4 emissions (mg CH4∗m-2∗h-1) from chambers with poised electrodes against the CH4 emissions from chambers with unpoised electrodes at each site for each measurement day, resulting in an average percentage change in CH4 emission ratios of poised vs. unpoised electrode chambers . At the upstream site, we observed a 44% reduction in normalized CH4 emission  for the poised vs. unpoised electrode chambers . This is in agreement with our hypothesis: placement of iron(III)/humic substance-mimicking electrodes will reduce the emission rate methane compared to the control in a riparian zone, since it is thermodynamically more attractive to sustain iron/humic substance reduction /humic substances as the terminal electron acceptor) than methanogenesis . However, for the downstream site for which we observed lower soil temperatures . Noteworthy is that the temperature difference between the upstream and downstream sites may have caused for a small error in our data (∼1%). Based on our hypotheses, we anticipated lower CH4 emissions for the poised electrode chambers. It remains, however, unclear why the normalized CH4 emissions were indeed lower at the upstream site and not at the downstream site with poised electrodes. This could be due to environmental differences between sites , but further work is required to determine the precise reason(s).To more precisely determine the effects of potentiostatic manipulation, we normalized average CH2/N2O emissions (μg N2O-N∗m-2∗h-1) between sites or treatments (Table 1). These data were also compared using percent change based on paired sample analysis in the same manner as the normalized CH4 emissions, and there were no significant changes between groups at either site . Regardless, the discrepancies with our CH4 emission results from adjacent field sites show the heterogeneous character of field research, and the importance to test results in the field rather than only at the bench.There were no differences in the absolute average Np < 0.001, two-tailed t-test). The pH values across both sites were close to each other and near neutral, with the upstream site averaging at 7.3 ± 0.2 and the downstream site at 7.2 ± 0.2 , and ranged between 6.8 and 19.1°C  across sites and treatments, and were below detection limit (0.1 ppm) for most samples (consistent with the relatively low N2/N2O emissions rates). We did not observe a difference between sites and treatments for total iron concentrations from soil pore water, which averaged 10.97 ± 9.0 μM. Approximately 95% of this iron was in the reduced form (Fe2+), and there were no differences between sites or treatments in iron speciation. At the upstream site, SO42- concentrations were lower in chambers with poised electrodes than those with unpoised electrodes, however, there was not a significant difference in absolute SO42- concentrations at the downstream site (Table 1). These values were corrected in the same manner as the normalized CH4 emission rates, visualizing an average reduction in normalized SO42- concentration of 39% with poised vs. unpoised electrodes . It is unclear whether the difference in SO42- concentrations at the upstream site was related to the reduction in CH4 emissions, although sulfate reduction is more energetically favorable than methanogenesis, but less favorable than iron reduction  and decreased normalized SO42- concentration (via increased sulfate reduction) at the upstream site, while neither decrease occurred at the downstream site.Dissolved oxygen was low across both sites due to water tables at or near the soil surface , and both sites showed average concentrations of less than 1 mg Leduction . Based o- concentrations were lower in pore water collected from chambers containing poised electrodes at both sampling sites (Table 1); these values were corrected in the same manner as normalized CH4 emission rates. At the upstream sites, chambers with poised electrodes had normalized Cl- concentrations that were 17% lower than those with unpoised electrodes ; at the downstream site, normalized Cl- concentrations were, on average, 55% lower than in chambers with unpoised electrodes . These lower Cl- concentrations were a result of Cl- transport from the inside to the outside chamber through the anion exchange membrane because of ion imbalances due to the electrochemical activity  did not reveal any clustering of samples by site or treatment . However, there was a clear clustering of samples with time, which was highlighted by UniFrac distances between the first 2 weeks (weeks 1–2) and last 2 weeks (weeks 5–6) of the experimental period . This result likely suggests that most of the broad differences in the microbial community phylogenetic structure were based simply on changing environmental parameters and community function with time, rather than experimental treatment.We obtained a total of 72 samples  from the 12 chambers during 6 weeks and achieved an average sequencing coverage of 29,782 sequences (16S rRNA gene) per sample with an average length of 251 bp. We first analyzed the beta diversity, which quantifies the differentiation between sample communities, using UniFrac distances . Based oFigure 4). Similar to the beta diversity data analysis, we grouped the taxonomy data for weeks 1–2 and weeks 5–6 with the objective to identify phyla that changed during the experimental period of 6 weeks. At the upstream site, the community composition at the poised electrodes between weeks 1–2 and 5–6 showed a 10% increase in Proteobacteria  and a 35% decrease in Bacteroidetes  during the experimental period. Proteobacteria and Bacteroidetes populations were not different at the unpoised electrodes between weeks 1–2 and 5–6 . At the downstream site, both poised and unpoised electrodes enriched for Proteobacteria between weeks 1–2 and 5–6, which was accompanied by a decrease in relative abundance of Bacteroidetes . Specifically, the poised electrodes resulted in a 14% increase in Proteobacteria and a 30% decrease in Bacteroidetes  while the unpoised electrodes caused a 20% increase in Proteobacteria and a 50% decrease in Bacteroidetes  between weeks 1–2 and 5–6. The increase in Proteobacteria in both poised and unpoised treatments could be due to the presence of the graphite electrodes, which provide a conductive surface that promotes both biofilm growth and long-range electron transport through soil microenvironments . We applied this to OTU abundance profiles from all samples  as well as data from only the upstream site samples . Upon cross-validation, the machine-learning algorithm returned a relatively high error rate , indicating that there were no indicator OTUs that could differentiate poised from unpoised electrode conditions. This machine learning approach confirmed our other findings from the principal coordinate analysis of UniFrac-based beta diversity  and the other taxonomic classification analysis  that the bioelectrochemical manipulation did not cause systematic, or reproducible, changes in the microbial membership.We used a machine learning approach to find OTUs that are predictive of samples taken from either poised or unpoised electrodes . First, we performed principal component analysis with our OTU tables, and second we performed correspondence analysis to map environmental metadata onto the principal component results as vectors. The resulting direction of the vector (arrow) indicates that the specific variable corresponded strongly with community structure, while the resulting length of the vector indicates the magnitude of the association . When we used data from across all sites and treatments, the variables of strongest correspondence with the OTU tables were: N2/N2O emissions, pH, and Cl- concentrations . When limiting the analysis to the upstream site (where unpoised electrode chambers had higher CH4 emissions), however, the CH4 emissions were the most strongly weighted variable, with N2/N2O emissions, pH, Cl- concentrations, and temperature being other variables . Noteworthy is that temperature and time as a vector cannot be distinguished in our work because of slowly rising soil temperatures during our experimental period  or environmental parameters were associated with microbial community membership. We performed correspondence analysis to compare OTU abundance profiles with gas emission and environmental metadata , which we anticipated because the presence of a terminal electron acceptor with the redox potential of iron(III) for iron reduction is thermodynamically more favorable compared to carbon dioxide for methanogenesis. Furthermore, manipulation resulted in a decrease in SO42- and Cl- concentrations in soil porewater . However, at the downstream site, which had a lower magnitude of emissions (nearly 50%) from the chambers with unpoised electrodes , poised electrodes had no effect on CH4 emissions. This is likely due to the lower overall CH4 emission rates at this site, which would preclude inhibition via bioelectrochemical manipulation since methanogenic activity was already low.When we manipulated the soil redox environment using poised electrodes mimicking iron(III), there was a significant decrease in CH4 emission from the upstream site, an increase in the relative abundance of Proteobacteria and a decrease in Bacteroidetes . However, we found no systematic link between community structure and experimental treatment . Further quantitative analysis showed that these microbial community changes corresponded with differences in the environmental parameters (pH and Cl- concentrations) and CH4 and N2/N2O emissions .We also observed, together with the reduction in CH4 emissions at the upstream site in conjunction with bioelectrochemical manipulation (using soil-based electrodes) demonstrates the fragile balance that governs biogeochemical cycles in these soils, and highlights the measurable impact that microbial competition has on ecosystem-scale processes. It is clear that microbial community structure and function is subject to influence from a wide array of biological, chemical, and physical factors, which in turn can have a measurable impact on the landscape level. Accurate modeling of biogeochemical processes is important for predicting responses to climate change, determining regulatory limits for anthropogenic pollutants, and designing effective best management practices  at one riparian zone site. Poised electrodes provided an inexhaustible source of electron acceptor for iron reducers, and, at our upstream riparian zone site, resulted in reduced CH4 emissions that could be a result of iron- or humic substance-reducers outcompeting methanogens for carbon sources and nutrients. However, we did not see such changes in CH4 emissions at the downstream site. Furthermore, we showed that bioelectrochemical manipulation had minimal effects on the microbial community structure. This was confirmed by machine learning analysis, which was unable to develop an algorithm to predict sample grouping with an error rate below 30%. We did, however, find a correspondence between the community composition and the function of the microbiota (CH4 emissions). Despite the lack of direct changes in microbial community structure at the upstream site with poised electrodes, the reduction (∼45%) of CH4 emissions together with its correspondence to the composition of microbiota suggests that the balance between competing anaerobic microbial processes can have a major impact on landscape-level processes.In summary, we manipulated the sediment redox environment by poising electrodes capable of being used as electron acceptors for iron(III)-reducing microbes and observed a response in ecosystem function (in this case, CHKey Points:•Potentiostatic manipulation was used to change redox dynamics in riparian soils.•4 emissions but did not change the microbial community.Manipulation inhibited CH•Microbial community dynamics corresponded to environment and ecosystem function.EF, LM, MW, and LA designed the study; EF and LM performed the field research; EF, LM, and AP performed the sample analysis; EF, LM, JW, AP, RL, MW, and LA analyzed the data, EF, LM, JW, AP, RL, MW, and LA wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Salix nigra had the highest Importance Value at every site. Species with minor Importance Values were Celtis laevigata, Acer rubrum, and Plantanus occidentalis. Productivity increased more following the introduction of river water in affected sites compared to the reference. Aboveground net primary productivity was highest at the reference site (2926 ± 458.1 g·m−2·year−1), the intact site; however, there were greater increase at the sites in the Loosahatchie Chute, where measurements ranged from 1197.7 ± 160.0 g m−2·year−1·to 2874.2 ± 794.0 g·m−2·year−1. The site furthest from the notching was the most affected. Pulsed inputs into these wetlands may enhance forested wetland productivity. Continued monitoring will quantify impacts of restored channel hydrology along the Mississippi River.This research presents the initial results of the effects of hydrological restoration on forested wetlands in the Mississippi alluvial plain near Memphis, Tennessee. Measurements were carried out in a secondary channel, the Loosahatchie Chute, in which rock dikes were constructed in the 1960s to keep most flow in the main navigation channel. In 2008–2009, the dikes were notched to allow more flow into the secondary channel. Study sites were established based on relative distance downstream of the notched dikes. Additionally, a reference site was established north of the Loosahatchie Chute where the dikes remained unnotched. We compared various components of vegetation composition and productivity at sites in the riparian wetlands for two years.  The Lower Mississippi River sub-basin is characterized by low, flat topography, alluvial soils in a wide flood plain, relatively high rainfall, and high water tables [The Mississippi River is the fourth longest and tenth largest river in the world. The Mississippi River Basin (MRB) is the fourth largest watershed in the world, draining more than 3,220,000 kmr tables . The Misr tables . Dams anr tables . This har tables . Prior tr tables .Zea mays L.) and soybeans (Glycine max (L.) Merr.) [By the 1980s, an estimated 2.8 million ha of bottomland hardwood forest remained of the original 10 million ha, and much of the habitat that has remained is highly fragmented and hydrologically altered ,8. Appro) Merr.) ,12.3−), and subsequent transport of N via the Mississippi River to the Gulf of Mexico causing hypoxia in the nearshore waters off Louisiana [2 during midsummer, currently the second largest in the world in area [The major driver of degradation for the Gulf of Mexico is upstream water management and agricultural practices in the MRB . As landouisiana ,15,16. Aouisiana , such asouisiana . The higouisiana . These c in area ,20,21,223− by more than 80% [Wetlands provide a vital ecosystem service of improving water quality by treating and removing a variety of waste products with reported reductions of NOthan 80% . Undistuthan 80% ,24,25, pthan 80% ,27. The than 80% ,29,30. Hthan 80% ,31,32. MConceptually, our understanding river ecosystems includes both the River Continuum Concept  where tScaphirhynchus albus) and the Interior Least Tern , as well as other recreationally and commercially valuable wildlife. In the fall of 2008, twelve notches were excavated in nine existing dikes at Loosahatchie Chute to restore flow to more than 17 km of secondary channel , as well as other wildlife.Loosahatchie Bar is located on the west bank of the Mississippi River, opposite Memphis, Tennessee, between river miles 736.5 and 742.8 on the border of Shelby County, Tennessee, and Crittenden County, Arkansas . Dikes mSalix nigra Marsh.; Study site selection and data collection began February 2009. Sites were selected in relation to the Redman Point-Loosahatchie Bar Environmental Restoration Project ; Table 1The river stage for the Mississippi River at Memphis, Tennessee, was measured at the Weather Bureau Gage, where the National Weather Stage Gage height of 0 m is measured at an elevation of 56.1 m; this is based on older USGS topographic maps and NGVD29 benchmarks. Flood stage is at gage height 10.4 m. Initial measurements began during winter 2009 and continued until autumn 2011 . Sites w−1 with a pH of 5.6 to 7.3; at a depth from 12.7 to 43.2 cm, the CEC was 22–40 meq 100 g−1 with a pH of 5.6 to 7.3; at a depth from 43.2 to 106.7 cm, the CEC was 7.0 to 18 meq 100 g−1 with a pH of 6.1 to 8.4; and at a depth from 106.7 to 152.4 cm, the CEC is 5.0 to 18 meq 100 g−1 with a pH of 6.1 to 8.4. The soil at the Mid site was classified as Crevasse fine sand from data collected 23 September 2008. At a depth from 0 to 20.3 cm, the CEC was 1.8 to 6.8 meq 100 g−1 with a pH of 5.6 to 8.4; and at a depth from 20.3 to 152.4 cm, the CEC was 1.4 to 6.1 meq 100 g−1 with a pH of 5.6–8.4. Soil at the Near and Far sites have not been surveyed.Soil was classified according to the United States Department of Agriculture (USDA) National Resources Conservation Service Web Soil Survey. The Ref site soil data were collected 17 November 2008. The soil was classified as Bowdre silty clay and was frequently flooded. At a depth from 0 to 12.7 cm, the cation-exchange capacity (CEC) was 25 to 45 meq 100 g2) resulting in the biomass per unit area (kg·m−2) for each plots for both years.Total aboveground NPP was calculated as the sum of stem growth and litterfall. Woody stem growth productivity was calculated using diameter at breast height (dbh) measurements of all trees with dbh greater than 10.0 cm. Trees were tagged with an aluminum identification tag positioned ~1.3 m from the base of the tree . The collection traps were constructed from 1.91 cm diameter polyvinyl chloride (PVC) pipe, measuring 0.25 × 0.25 m, with a screened net bottom. Each trap was elevated (~3 m) above the ground to prevent inundation during high water periods , right. et al. (1987) [i.e., cm2·m−2). Importance Values equal the sum of relative density and relative dominance for a maximum value of 2.Tree species composition analysis used equations 1–3 modified from Barbour . (1987) . Basal at-test. Differences were considered significant at α < 0.05.To determine if restoring hydrology had increased productivity, NPP among sites within each sampling year was tested with general linear model for analysis of variance . SignifS. nigra, which also had the highest Importance Value for every site (Celtis laevigata Willd.), red maple (Acer rubrum L.), American elm tree (Ulmus americana L.), and sycamore Trees at the Near site were more exposed to wind compared to the other sites, which were protected by surrounding forests, contributing to stem breaks and canopy debris on the forest floor. Tree density at all sites was dominated by ery site . Species−1, however, these were the largest trees increasing in basal area from 3.26 cm2·m−2 in 2009 to 3.47 cm2·m−2 in 2010 and 3.67 cm2·m−2 in 2011  with C. laevigata being of minor importance (0.04) for 2009–2011  with P. occidentalis being of minor importance (0.02) for 2009–2011  with A. rubrum (0.18–0.19) and U. americana (0.02) being of minor importance for 2009–2011 . For all sites combined, there was no significant difference in stem growth increase in 2011 compared to 2010 .Stem growth in 2010 ranged from 595.2 ± 237.6 g m−2·year−1 at the Near site to 734.5 ± 107.3 g·m−2·year−1 at the Mid site, and was not significantly different between sites , with both the Far  and the Mid sites  being significantly more productive than the Ref site (542.7 ± 26.5 g m−2·year−1). The Near site (672.2 ± 47.6 g·m−2·year−1) was not significantly different from the other sites. For all sites combined, litterfall increased in 2011 compared to 2010 . The largest change in litterfall was the increased productivity at the Far site  followed by the Near site  or the Ref site .Litterfall collected in 2010 ranged from 433.0 ± 60.5 g m3,8 = 4.064, p = 0.050; −2·year−1) and lowest at the Near site . Both the Mid (2446.1 ± 303.1 g m−2·year−1) and Far (1907.7 ± 564.8 g m−2·year−1) sites did not differ from each other or the other sites in NPP. In 2011 aboveground NPP ranged from 1197.7 ± 160.0 to 2926.1 ± 458.1 g m−2·year−1, however, there was no significant difference between sites  in litterfall, contributing 56% of total NPP, compared to 42% contribution the previous year. Total NPP increased at the Near site by 169.5 g m−2·year−1 despite the loss of 69.70 g m−2·year−1 in stem productivity. The Mid site had increased production in both litterfall (51.5 g m−2·year−1) and stem growth (197.7 g m−2·year−1), and thus in total NPP (249.2 g m−2·year−1). The Far site had the greatest changes in both litterfall (354.6 g m−2·year−1) and stem growth (611.9 g m−2·year−1), which is reflected in the 50% increase in the total NPP (966.5 g m−2·year−1). The first year the Far site had 766.7 g m−2·year−1 less total NPP than the Ref site, and following hydrologic restoration it was within 51.9 g m−2·year−1 of total NPP compared to the Ref site. Total NPP at the Ref site increased the least , having an increase in stem growth of 278.3 g m−2·year−1 and a decrease in litterfall of 26.6 g m−2·year−1.NPP increased in 2011 compared to 2010  and total NPP (1467 g m−2·year−1) than a control site , and stem growth at all sites ranged between . In anoMeasurements of the soil structure at the sites revealed that the restored sites had a higher proportion of silt than sand and clay particles . The resThe effectiveness of functions of restored hydrology that maintains restored wetland flora and soil carbon resources may take centuries to return to a healthy state . As ecosThe functioning of the LC and the effectiveness of the restoration can be understood within the context of the River Continuum Concept  and theThe initial results of the effects of hydrological restoration on forested wetlands in the Mississippi alluvial plain near Memphis, Tennessee showed that enhancing the connectivity of the river by notching rock weirs led to greater increases in productivity of the study sites as a result of improved exchange between the river and its floodplain. The site furthest from the notching was the most affected. Pulsed inputs into these wetlands appeared to have enhanced forested wetland net primary productivity. However, continued monitoring is needed to quantify the long-term impacts of restored channel hydrology on forest productivity.


During mitosis, Bub1 kinase phosphorylates histone H2A-T120 to promote centromere sister chromatid cohesion through recruitment of shugoshin (Sgo) proteins. The regulation and dynamics of H2A-T120 phosphorylation are poorly understood. Using quantitative phosphoproteomics we show that Bub1 is autophosphorylated at numerous sites. We confirm mitosis-specific autophosphorylation of a several residues and show that Bub1 activation is primed in interphase but fully achieved only in mitosis. Mutation of a single autophosphorylation site T589 alters kinetochore turnover of Bub1 and results in uniform H2A-T120 phosphorylation and Sgo recruitment along chromosome arms. Consequently, improper sister chromatid resolution and chromosome segregation errors are observed. Kinetochore tethering of Bub1-T589A refocuses H2A-T120 phosphorylation and Sgo1 to centromeres. Recruitment of the Bub1-Bub3-BubR1 axis to kinetochores has recently been extensively studied. Our data provide novel insight into the regulation and kinetochore residency of Bub1 and indicate that its localization is dynamic and tightly controlled through feedback autophosphorylation. Bub1 kinase phosphorylates histone H2A-T120 at the centromere to recruit shugoshin proteins and promote sister chromatid cohesion during mitosis. Here the authors show that Bub1 autophosphorylation on T589 influences Bub1 dynamics at the kinetochore and restricts H2A-T120 phosphorylation to centromeres. The accurate traverse through mitosis results in equal allocation of duplicated sister chromosomes and is critical for cellular and organism health. To ensure this, eukaryotes have evolved a safeguard mechanism known as the spindle assembly checkpoint (SAC), which functions during both meiosis and mitosis1234659611in vitro target of Bub1 and this phosphorylation may directly contribute to APC/Cdc20 inhibition151617Bub1 also has kinase-dependent functions during mitosis. Cdc20 is an Here we use a quantitative proteomics approach to identify Bub1-specific autophosphorylation sites. We show that Bub1 is significantly autophosphorylated outside the activation segment and kinase domain, including at the conserved threonine 589 (T589). We show the Bub1 activity is primed in interphase but does not fully mature until mitosis. Immunofluorescence with a phosphospecific antibody indicates that autophosphorylation at T589 is prevalent during early mitosis. Alanine substitution of this residue (T589A) results in chromosome missegregation and incomplete sister chromatid arm resolution as a result of non-localized H2A-T120 phosphorylation and ectopic Sgo1 recruitment. Fluorescence recovery after photobleaching (FRAP) experiments reveal that Bub1-T589A and Bub1-kinase dead  exhibit more rapid kinetochore turnover than wild-type (WT) protein. Forced localization of Bub1-T589A to the kinetochore (KT) refocuses H2A-T120 phosphorylation and Sgo1 localization to the kinetochore. We propose that spatially constrained H2A-T120 phosphorylation, and thus sister chromatid cohesion, are promoted by a positive feedback mechanism formed by autophosphorylation of Bub1 at T589 that regulates the dynamics of Bub1 kinetochore docking.13C6,15N4]arginine and lysine (Arg10/Lys8). Immunoprecipitated, mitotic, Bub1-WT and Bub1-KD expressed in differentially labelled cells were separately subjected to a non-radioactive in-vitro kinase assay. This autophosphorylation amplification step was introduced to increase the occupancy of phosphorylation sites within Bub1 and thus increase phosphopeptide detection and, importantly, to allow distinction between genuine autophosphorylation sites and phosphorylation incurred by co-precipitating kinases. We also considered this approach superior to an in-vitro assay of recombinant proteins as Bub1 mitotic modifications, localization and binding partners may all contribute to genuine and physiologically relevant Bub1 autophosphorylation. The experiment was performed in triplicate with minor changes: the amino acid labelling was reversed in one replicate  and small non-polar (at positions +2 and +3) residues relative to the phosphoacceptor, as well as an exclusion of acidic residues surrounding the phosphosites. Of the phosphosites that were not considered to be Bub1 dependent (phosphopeptide:peptide ratio <3), 50%  were followed by a proline, suggesting that they may be targets of a proline-directed kinase such as CDK1 or mitogen-activated protein kinase, in agreement with previous observations222325Twenty phosphosites were significantly upregulated in Bub1-WT compared with Bub1-KD and thus considered potential Bub1 autophosphorylation sites , red. Thin vivo autophosphorylation sites. Anti-pT589 staining of fixed cells clearly decorated kinetochores and overlapped the Bub1 signal in prophase and prometaphase  domain in regulating kinase activity with conflicting results1929rylation . KinetocLac repressor (LacI)-fused Bub1 WT and Bub1 KD in cells stably expressing a 256 copy array of the lac operator sequence (LacO) in an arm of chromosome 1 , the cytoplasmic:kinetochore GFP ratios in these cells were found to be significantly higher than the cells expressing Bub1-WT ; The parallels in the phenotype observed in Bub3-depleted cells and Bub1-T589A cells were surprising, considering that Bub1-T589A localized efficiently to the kinetochore, as measured by indirect immunofluorescence, and exhibited normal activity towards H2A in vitro . To detein vitro . As an ain vitro . Althoug2 cellsP<0.001, one-way ANOVA). In contrast, we found no major difference in cytoplasmic diffusion  and Cdk1 phosphorylation of a number of consensus sites in Sli15/INCENP restricted premature chromosomal passenger complex localization to the spindle5457Considerable evidence from the literature supports this model of Bub1 function. First, all conditions in which proper Bub1 kinetochore targeting is impaired result in the spread of the H2A-pT120 signal and/or Sgo1 displacement along chromosome arms. Our data here show that depletion of Bub3 or loss of the Bub1–Bub3 interaction result in unchecked H2A-T120 phosphorylation and Sgo recruitment. Similarly, depletion of KNL-1 or ectopic localization of the Bub1 kinase domain to chromosome arms led to uniform H2A-T120 phosphorylation on chromatin14474849503552Crystal structures suggest that Bub1 may be a constitutively active kinase19The H2A-pT120-Sgo1 pathway serves as an adaptor to facilitate Aurora B inner centromeric accumulation6364How T589 phosphorylation changes the dynamics of Bub1 shuttling remains unclear. One possibility is that phosphorylation may induce conformational changes that alter affinity of Bub1 to kinetochores. Binding of Bub3 as measured by immunoprecipiation revealed no difference between this mutant and Bub1-WT however, and any change caused by this mutation might be small and restricted to the shuttling pool of Bub1 (∼ 50%) and thus not easily detected by such steady-state assays. Alternatively, it is possible the pT589 motif forms a docking site for a protein interaction motif, which would allow for the dynamic exchange of Bub1 between the cytoplasm and kinetochores. For example, 14–3–3 binding to pThreonine motifs has been shown to control the nucleocytoplasmic exchange of a number of proteins2 in DMEM (Hyclone) supplemented with 10% fetal bovine serum or supplemented bovine growth serum (PAA). HeLa cells for the generation of stable isogenic cell lines were a generous gift of Patrick Meraldi−1). U20S cells expressing a 256 copy array of the Tet operator sequences were a kind gift of David Spector , MG132  and nocodazole . Transient plasmid transfections  and subcloned into the KpnI site of pcDNA3.1–3xMYCBub1 WT was cloned into the pcDNA5/FRT/TO expression vector modified to include an N-terminal triple MYC tag (HindIII–Kpn1) followed directly in-frame by EYFP (KpnI–BamHI), generated by PCR amplification from pEGFP-N1 (Clontech). For artificial kinetochore targeting of Bub1-T589A and Bub1-Δ229–256, the enhanced GFP sequence was substituted for hMis12. For construction of the LacI-expressing plasmid, 2, 10 mM EGTA and 4% formaldehype)−1. Other antibodies were used at 1 μg ml−1, unless otherwise indicated, as follows: anti-MYC , anti-Bub1 (ref. Cells were grown on cover slips and were arrested in mitosis either by nocodazole (330 nM) or by 10–12 h release from thymidine block. Fixation was performed by incubation with PTEMF . For isolation of the cytoplasmic and cytoskeletal fraction of Bub1, mitotic cells stably expressing Bub1-WT, KD or T589A were harvested by shake-off after thymidine release, washed twice in PBS and lysed for 10 min on ice in cytoskeletal buffer  and 1 mM ATP). The lysate was spun down for 4 min at 3,200 r.p.m. and the resulting supernatant (S1) constituted the cytoplasmic fraction. The original, non-cropped blots for all western blottings in this study are shown in Supplementary Fig. 4For immunoblotting and immunoprecipitations, cells were lysed with RIPA buffer containing 150 mM Tris-HCL pH 7.5, 150 mM NaCl, 10 mM NaF, 1% NP-40, 0.1% Na deoxycholate and a protease and phosphatase inhibitor cocktail that included 20 mM B-glycerophosphate, 0.1 mM sodium vanadate, 10 mM sodium pyrophosphate, 1 μg mlrsb.info.nih.gov). Image processing was performed in Image J or Photoshop and images shown in the same figure have been identically scaled.Cells were imaged by confocal microscopy on an inverted Olympus IX80 microscope equipped with a WaveFX-Borealin-SC Yokagawa spinning disc (Quorum Technologies) and an Orca Flash4.0 camera (Hamamatsu). Image acquisition was performed using Metamorph software (Molecular Devices). Optical sections were acquired with identical exposure times for each channel within an experiment and then projected into a single picture using ImageJ (F(t)=A(1−eτt−), where A=fraction recovery. Half-time recovery was determined using the formula T1/2=ln0.5/−τ.For FRAP experiments, the cells were grown in glass-bottom lab-tek chambered slides (Thermo Scientific). FRAP analysis was performed on Leica DMI600B equipped with a heated chamber (37 °C) and a Mosaic active illumination system , which allowed for simultaneous bleaching and acquisition, and an ImageEM (512 × 512) camera (Hamamatsu). The microscope and Mosaic were operated by Metamorph. The GFP-tagged Bub1 protein at both kinetochores and the cytoplasm was bleached using a 405-nm laser (diode 475 mW power at 100%) and excited at 491 nm (detection filters 536/40 nm). Individual kinetochores or cytoplasmic regions were bleached by a 400-ms laser pulse. Image acquisition (every 150 ms) began 15 frames before bleaching and continued for an additional 750 frames post bleaching. The bleached region in each case was a circular region of 15 pixel diameter and only kinetochores that remained visible within this region for the length of the experiment were included in the analysis. Quantification of fluorescence recovery was obtained using the FRAP profiler plugin of ImageJ, which accounts for correction of overall bleaching. Recovery rates for cytoplasmic and kinetochore Bub1 WT, KD and T589A were determined after fitting a single exponential curve (which showed the best fit) using the formula 2 heated to 37 °C. Bub1 stable cell lines were subjected to depletion of endogenous Bub1 for 48 h, then synchronized in mitosis after a further 16-h block with thymidine. Image acquisition was started 12 h after release. Only cells visibly expressing the GFP-tagged Bub1 were included in subsequent analysis.Live-cell imaging was performed on the above indicated microscopy system that is also equipped with a motorized stage (ASI) and an incubator with atmospheric CO2. The immunoprecipitates were then subjected to a cold in-vitro kinase assay  at 30 °C for 30 min. The reaction was stopped by the addition of SDS–PAGE sample buffer. The Bub1-WT and Bub1 KD immunoprecipitates were then mixed, resolved by SDS–PAGE and visualized by Coomassie brilliant blue staining. The band corresponding to the size of 3 × MYC-Bub1 was excised and processed for MS analysis−1 of trypsin or was added in an enzyme/substrate ratio of 1:50 of each Lys-C, GluC and elastase.293T cells were cultured in heavy or light amino acid-containing medium for five generations before transfection with 3 × MYC-tagged Bub1-WT or Bub1-KD. Cells were incubated for 36 h, after which nocodazole was added for an additional 16 h. Cells were harvested, lysed in RIPA lysis buffer and 3 × MYC-tagged Bub1 WT or KD were immunoprecipitated with anti-MYC antibodies for 2 h. The immunoprecipitated Bub1 was washed 3 × with RIPA lysis buffer, 1 × with RIPA buffer including 300 mM NaCl and a final buffer exchange with kinase reaction buffer lacking ATP and MgClm/z 300 to 2,000) were acquired in the FT-Orbitrap with a resolution of 60,000 at m/z 400. A maximum of five peptides were sequentially isolated for fragmentation in the linear ion trap using collision-induced dissociation. The Orbitrap lock mass feature was applied to improve mass accuracy. To improve phosphopeptide analysis, the multistage activation option in the software was enabled and the neutral loss species at 97.97, 48.99 or 32.66 m/z below the precursor ion were activated for 30 ms during fragmentation (pseudo-MS3).All peptide samples were separated by online reverse-phase nano liquid chromatography and analysed by electrospray tandem MS (MS/MS). Using a nanoACQUITY ultra-performance liquid chromatography system (Waters), samples were injected onto a 14-cm fused silica capillary column with an inner diameter of 75 μm and a tip of 8 μm (New Objective) packed in-house with 3-μm ReproSil-Pur C18-AQ (Dr Maisch GmbH). The LC setup was connected to an LTQ-Orbitrap MS (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source (Proxeon Biosystems). Peptides were separated and eluted by a stepwise 180 min gradient of 0%−100% between buffer A (0.2% formic acid in water) and buffer B (0.2% formic acid in acetonitrile). Data-dependent acquisition was performed on the LTQ-Orbitrap using Xcalibur 2.0 software in the positive ion mode. Survey full-scan MS spectra (from Raw data files including SILAC quantitation were processed using the MaxQuant software suite (version 1.0.12.5). Generation of peak lists was performed with the following MaxQuant parameters: top 12 MS/MS peaks for 100 Da, 3 data points for centroid, Gaussian centroid determination and slice peaks at local minima. During the peak list generation, MaxQuant identified potential SILAC pairs based on mass differences of specified labelled amino acids, intensity correlation over elution time and so on. Mascot  was used for peptide identifications. The initial precursor mass tolerance was set to ±7 p.p.m., whereas an accuracy of ±0.5 Da was used for MS/MS fragmentation spectra. Carbamidomethylation was set as fixed modification and methionine oxidation, protein N-terminal acetylation and phosphorylation (STY) were considered as variable modifications. Putative SILAC pairs were searched with their respective labelled amino acids as fixed modification, whereas peaks that were not assigned to any of the SILAC pairs were searched using R10 and K8 as variable modifications. Enzyme specificity was set to Trypsin/P, that is, allowing cleavage N-terminal to proline in the context of [KR]P. Up to two missed cleavages were allowed. The minimum required peptide length was set to six amino acids. Searches were performed against IPI human  that was concatenated with reverse database sequences . Further, MaxQuant filtered Mascot results using additional parameters such as the number of labelled amino acids (maximum of three) in the identified peptide sequence and the measured mass accuracy as a function of intensity. As an additional quality measure to increase identification stringencies, we only accepted phosphorylation site identifications with Mascot scores of at least 12 or PTM scores of at least 30. Quantitation of SILAC pairs was performed with the following parameters: re-quantify; for protein quantification, discard unmodified counterpart peptides, except for oxidation and acetyl protein N-terminal; use razor and unique peptides; minimum ratio count of 1; minimum score 0; and minimum peptides 1. The initial maximum false discovery rates were set to 0.02 and 0.05 for peptides and proteins, respectively, and further reduced by Mascot score filtering as described above. False discovery rates were calculated as (number of hits in the reversed database/number of hits in the forward database) × 100%. Whenever the set of identified peptides in one protein was equal to or completely contained in the set of identified peptides of another protein, these two proteins were joined in a single protein group. In cases where the peptides have more than one phosphorylation site, some of these phosphorylation sites are identified as multiply phosphorylated peptides, whereas others are identified on multiple singly phosphorylated peptides.A summary of all quantifiable SILAC pairs identified on Bub1 from each of the three independent experiments is shown in Unless otherwise stated, all experiments were performed in triplicate. Image quantification was performed using Image J. For quantification of signal intensities at kinetochores, the CREST/MYC signal was used to generate a binary mask to include kinetochore and centromere signals. Integrated signal intensity was measured in all relevant channels and intensities indicated are values relative to CREST or MYC. Unless otherwise indicated, a minimum of ten cells were quantified per condition for all experiments involving kinetochore quantification. For H2A-pT120 and Sgo1 signal intensity at chromosome arms, the following formula was used to measure the corrected total cellular fluorescence. CTCF=integrated density−(area of selected cell × mean fluorescence of background readings). For t-tests (two-tailed) for determination of significance of the differences or as otherwise indicated. A P-value of <0.05 was considered to be statistically significant.For the statistical analysis for significance of FRAP data, the relationship between Bub1 WT, KD and T589A were compared among treatments by testing the equality of a set of parameters using F-tests, derived from the error sum of squares of the null model and the full model in SAS. All other statistical analysis was performed with Sigmaplot. Data are expressed as means±s.e.m. The data were analysed by ANOVA or How to cite this article: Asghar, A. et al. Bub1 autophosphorylation feeds back to regulate kinetochore docking and promote localized substrate phosphorylation. Nat. Commun. 6:8364 doi: 10.1038/ncomms9364 (2015).Supplementary Figures 1-4Bub1 phosphosites and autophosphorylation sites identified by SILAC and Mass spectrometry.HeLa stable cell lines expressing 3xMYC-GFP-tagged Bub1 WT were treated with Bub1 siRNA, transfected with an mRFP-H2B expressing plasmid, and synchronized in mitosis by release from a thymidine release 72 hours after transfection. The movie shows chromosome movement of a representative cell. The time stamp is in min:secs.HeLa stable cell lines expressing 3xMYC-GFP-tagged Bub1 KD were treated with Bub1 siRNA, transfected with an mRFP-H2B expressing plasmid, and synchronized in mitosis by release from a thymidine release 72 hours after transfection. The movie shows chromosome movement of a representative cell. The time stamp is in min:secs.HeLa stable cell lines expressing 3xMYC-GFP-tagged Bub1 T589A were treated with Bub1 siRNA, transfected with an mRFP-H2B expressing plasmid, and synchronized in mitosis by release from a thymidine release 72 hours after transfection. The movie shows chromosome movement of a representative cell. The time stamp is in min:secs.


HTLV-1 causes proliferation of clonal populations of infected T cells in vivo, each clone defined by a unique proviral integration site in the host genome. The proviral load is strongly correlated with odds of the inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is evidence that asymptomatic HTLV-1 carriers (ACs) have a more effective CD8 + T cell response, including a higher frequency of HLA class I alleles able to present peptides from a regulatory protein of HTLV-1, HBZ. We have previously shown that specific features of the host genome flanking the proviral integration site favour clone survival and spontaneous expression of the viral transactivator protein Tax in naturally infected PBMCs ex vivo. However, the previous studies were not designed or powered to detect differences in integration site characteristics between ACs and HAM/TSP patients. Here, we tested the hypothesis that the genomic environment of the provirus differs systematically between ACs and HAM/TSP patients, and between individuals with strong or weak HBZ presentation.We used our recently described high-throughput protocol to map and quantify integration sites in 95 HAM/TSP patients and 68 ACs from Kagoshima, Japan, and 75 ACs from Kumamoto, Japan. Individuals with 2 or more HLA class I alleles predicted to bind HBZ peptides were classified ‘strong’ HBZ binders; the remainder were classified ‘weak binders’.The abundance of HTLV-1-infected T cell clones in vivo was correlated with proviral integration in genes and in areas with epigenetic marks associated with active regulatory elements. In clones of equivalent abundance, integration sites in genes and active regions were significantly more frequent in ACs than patients with HAM/TSP, irrespective of HBZ binding and proviral load. Integration sites in genes were also more frequent in strong HBZ binders than weak HBZ binders.Clonal abundance is correlated with integration in a transcriptionally active genomic region, and these regions may promote cell proliferation. A clone that reaches a given abundance in vivo is more likely to be integrated in a transcriptionally active region in individuals with a more effective anti-HTLV-1 immune response, such those who can present HBZ peptides or those who remain asymptomatic.The online version of this article (doi:10.1186/1743-422X-11-172) contains supplementary material, which is available to authorized users. The absolute abundance ; LM; LMSi isTo measure the diversity of clone abundance in the infected cell population from each individual, we used the oligoclonality index which isEstimation of total numbers of clones in the blood (observed and unobserved) was carried out using a computational diversity estimation approach (DivE), as described previously. Brieflyhttp://ftp.ncbi.nih.gov/gene/), CpG island data from UCSC tables).C tables, and hishttp://www.R-project.org/). A multivariable linear regression model was used to compare log PVL with the number of HBZ-binding HLA alleles per individual, taking into account disease status, as well as age and sex in the Kagoshima cohort-only analysis as these have been suggested to vary with PVL. Differences in OCI, PVL and total number of sisters between cohorts were analysed by non-parametric Mann-Whitney U tests as distributions were non-normal. Spearman correlation was used to determine the correlation in each cohort between log PVL and either log estimated number of clones in the blood or OCI. A likelihood ratio test was used to compare a null multivariable linear regression model associating log clone number with log PVL, disease status and HBZ binding capacity to an alternative model which added an interaction term between PVL and HBZ binding capacity. This allowed a test of whether the association between total clones in the blood and PVL differed with HBZ binding capacity. A second likelihood ratio test compared the same null model with a different alternative model including an interaction term between log PVL and disease status. This method was repeated for the association of OCI with log PVL.Statistical tests were performed using R version 2.15  divided by the mean number of that mark within 10 Kb of in silico random sites (N random). Mann-Whitney U tests were used to compare the numbers of specified epigenetic marks near integration sites from ACs versus HAM/TSP patients. Spearman correlation was used to test the association between log absolute abundance of an integration site clone and the frequency of a specified epigenetic mark within 10 Kb of the integration site.Correction for multiple comparisons was made using a Bonferroni-Holm correction to control the family-wise error rate for each set of tests  carried out across all analysed epigenetic marks and integration site subsets. Two epigenetic marks  were analysed but not reported as their results were very similar to reported ones; the p values from their analyses were included in the calculation of the Bonferroni-Holm correction. Correction for multiple comparisons was also made across subsets in the analyses  of integration sites in genes.A multivariable logistic regression model was used to test whether disease status was independently associated with integration site in a gene, active genomic region and inhibitory genomic region at the integration site level  or patient level (characteristics of integration sites averaged per patient). Host HBZ binding status and PVL, as well as log absolute clone abundance were controlled as additional factors in the model as these may vary with disease status and integration site environment. A multivariable logistic regression model was also used to test the association of integration in the same orientation as a gene or TSS with disease status . A further multivariable logistic regression tested the association of host HBZ binding status with integration in a gene (controlling for disease status and clone abundance) at the patient and integration site level.Figure S2. Integration in a gene does not vary by viral load or AC cohort and there is no association of disease status of orientation with respect to flanking gene. Figure S3. Integration in transcriptionally active regions is more frequent in asymptomatic controls than HAM/TSP patients. Figure S4. Transcriptionally active integration sites are more frequent in AC than HAM/TSP patients. (PDF 137 KB)Additional file 1: Figure S1: Clone sister numbers, PVL and abundance classification in sequenced samples.


AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase that is activated by cellular perturbations associated with ATP depletion or stress. While AMPK modulates the activity of a variety of targets containing a specific phosphorylation consensus sequence, the number of AMPK targets and their influence over cellular processes is currently thought to be limited.In vitro kinase assays were conducted for preliminary validation of 12 novel AMPK targets across a variety of cellular functional categories, including transcription, translation, cell migration, protein transport, and energy homeostasis. Following initial validation, pathways that include NAD synthetase 1 (NADSYN1) and protein kinase B (AKT2) were hypothesized and experimentally tested to provide a mechanistic basis for AMPK regulation of cell migration and maintenance of cellular NAD+ concentrations during catabolic processes.We queried the human and the mouse proteomes for proteins containing AMPK phosphorylation consensus sequences. Integration of this database into Gaggle software facilitated the construction of probable AMPK-regulated networks based on known and predicted molecular associations. in silico procedures and in vitro experiments to produce testable hypotheses for AMPK regulation of cellular processes.This study delineates an approach that encompasses both The online version of this article (doi:10.1186/s12918-015-0156-0) contains supplementary material, which is available to authorized users. AMP-activated protein kinase (AMPK) has emerged as a major regulator of cellular metabolism through activation of signaling cascades that are protective against stress. AMPK is composed of three subunits , and its activation occurs via AMP-dependent and -independent mechanisms leading to phosphorylation of the α subunit at Thr172. While elevation of the AMP: ATP ratio promotes AMP binding to the γ subunit, which permits activation by LKB1, CaTP ratio . In addiTP ratio .Once activated, AMPK initiates an array of signaling cascades by phosphorylating proteins with a βϕβXXXS/TXXXϕ consensus sequence  ,6. AMPK in silico search of the AMPK phosphorylation consensus sequence to the human and mouse proteomes. The most stringent AMPK consensus phosphorylation sequence  was qet al. reported that AMPK activation increases cellular NAD+ levels independent of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD+ synthesis [+ biogenesis, glutamine-dependent NAD+-synthetase (NADSYN1) also contributes to maintaining cellular NAD+ by catalyzing the final step in its biosynthesis. NADSYN1 and NAD+ are precursors for many signaling molecules involved in a variety of cellular processes, particularly for redox balance [In vitro kinase assays of recombinant NADSYN1 showed that AMPK can phosphorylate NADSYN1  and a mutant peptide with Ser to Ala substitution. In vitro kinase assay in the presence of AMPK showed increased 32P incorporation to the S641 peptide and SAMS peptide, a 13-residue peptide with a sequence around the AMPK target on acetyl CoA carboxylase and the standard positive control for AMPK kinase activity [32P incorporated into the S641A peptide was much lower, which was comparable to background or reaction without peptide. Similar results were obtained with the use of full length recombinant NADSYN1   or a peptide with an alanine substituted for the serine to support AMPK phosphorylation of AKT2 at Ser268  such as cell migration and angiogenesis, both of which require prolonged metabolic flux to achieve energy homeostasis. For example, AMPK positively regulates EC migration and differentiation facilitating angiogenesis under hypoxia ,13. With8 Figure C. Next, 8 Figure A. Furthe8 Figure B. Additi8 Figure C. Immunos Figure D. Next,  Figures F, 5G. TaAfter assembling a list of proteins containing the AMPK phosphorylation consensus sequence, the resulting database was integrated into Gaggle software to gain an understanding of the subcellular systems or functional categories that the identified targets represent. We also utilized this software to determine the number of connections each target has with other proteins. Central node substrates were identified as proteins with a high level interconnectivity within a functional category, which indicates a greater influence a protein has on a phenotypic response. Highly connected nodes were chosen for further validation as AMPK targets using kinase assays. Predicted phosphorylation sequences of the identified proteins chosen are illustrated in Figure Beta Arrestin 1 (ARRB1) regulates many signaling cascades through steric hindrance of GPCRs and serves as a scaffold for several AMPK regulated signaling molecules, including extracellular-signal-regulated kinases 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) ,19. AddiDDX56, also known as nucleolar helicase of 61 kDa (NOH61), is a putative RNA helicase implicated in a number of cellular processes involving alteration of RNA secondary structure . DDX56 i2+ signaling and Ca2+-sensitive chaperone functions in the ER [2+ efflux to cause ER stress [2+ homeostasis [2+ and SERCA activity could be through phosphorylation of CALNX Ser-247 and/or Thr-93, functioning as a protective mechanism  is a type I integral ER transmembrane chaperone that transiently binds to the newly synthesized glycoproteins to assist in their folding and transport . Becausen the ER . This phR stress . Althougeostasis , the mecHigh-mobility group AT-hook 1 (HMGA) is a transcriptional regulator that preferentially binds to the minor groove of A/T-rich regions in double-stranded DNA to form transcriptionally active multiprotein-DNA complexes that regulate the expression of target genes such as the insulin receptor (IR) . HMGA1, Klf2 genetic knockout results in defective recruitment of pericytes and vascular SMCs, impaired vasculogenesis, and lethality [LPA4 promoter contains a KLF2 binding site [Krüppel-like Factor 2 (KLF2) is a transcription factor that regulates diverse biological processes by direct DNA binding or association with transcription co-regulators such as acetyltranferases, cAMP response element binding protein (CREBP), and p300 -37. KLF2ethality . The mecing site . Given ting site , AMPK ma1 and α2 null embryos are lethal ~10.5 days post-conception because of poor expression of genes important for growth and survival [Treacle, commonly referred to as TCOF, is a nucleolar protein involved in ribosomal gene transcription. TCOF interacts with upstream binding factor (UBF) and promoter selectivity factor SL1 to promote RNA polymerase I activity ,43. TCOFsurvival . If AMPKCharged multivesicular body protein 1B (CHMP1b) also plays a role in gene regulation through chromatin structural maintenance . CHMP1b Cleavage and polyadenylation specificity factor 2 (CPSF2) is the 100-kDa subunit of the CPSF that recognizes the AAUAAA consensus sequence and interacts with proteins such as the poly (A) polymerase to prompt cleavage of template mRNA and initiation of poly (A) addition in pre-mRNA 3'-end formation . This prNuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) is a substrate for casein kinase and cyclin-dependent kinase (CDK) and its phosphorylation is important for cell-cycle regulation . Upon miZuotin-related factor 1 (ZRF1) executes both cytosolic and nuclear functions. In the nucleus, ZRF1 binds to the DNA sequence: 5’-GTCAAGC-3’ and facilitates histone 2A K119 ubiquitination leading to chromatin remodeling and transcription activation . In the in silico database together with biological validations suggests that AMPK is a master regulator of a broader range of cellular processes at more levels than previously recognized. Identifying AMPK targets by phosphorylation consensus sequence mapping is a simplification of the biochemistry that occurs at the substrate-enzyme interface because tertiary structure and binding domain chemistry is important for kinase substrate specificity and accessibility. However, arguably, the constraints of the active site require a minimum signature sequence within the catalytic pocket that mirrors a chemical signature flanking the phosphorylation site for catalysis to occur [in silico screening of the phosphorylation consensus sequence for candidate substrates of a kinase is a useful tool, the presence of such a phosphorylation consensus sequence in a given protein does not necessitate its phosphorylation in the cell. Further, some predicted AMPK targets offer slight variation to the exact consensus sequence, which presents a limitation of in silico analysis. Clearly, additional validation of individual targets and networks is necessary. Several biochemical tools are available and essential for such validation including in vitro kinase assays using recombinant protein substrates and phospho-antibodies for cellular and in vivo studies. Additional levels of AMPK phosphorylation specificity can be achieved with AMPK α1 and α2 knockout MEF cells and conditional knockout mice following treatment with AMPK agonists/activators such as exercise, caloric restriction, metformin, and AICAR. This database provides investigators with an AMPK-related network development tool that should stimulate experimental validation and offers new insights into AMPK involvement in health and disease.AMPK has emerged as a master regulator of metabolism by serving as an energy sensor. However, the multiple modes of AMPK activation, many of which remain elusive, indicate that AMPK’s function may extend beyond a mere energy sensor. Here, our to occur . Althoughttp://www.ncbi.nlm.nih.gov/sutils/blink.cgi?mode=query). Using the less stringent consensus phosphorylation sequence, in which φ and β were not specified to be a specific amino acid, a Scansite search was also performed to compare with the ENSEMBL search to the SWISS-PROT protein database [Using an R script, the AMPK phosphorylation consensus sequence, βϕβXXXS/TXXXϕ  was mapped to human and mouse proteomes imported from ENSEMBL proteome database -7. To fidatabase -7. The gdatabase .32P) ATP, 5 mM MgCl2, 1 pM AMPK (Sigma Aldrich), and the putative target recombinant protein (1 nM) in a 50 μl reaction volume at 37°C for 1 h. Proteins were then resolved by using SDS-PAGE, stained with Coomassie blue, and subjected to autoradiographic analysis. Peptide assays were performed under the same conditions but with 3 nM peptide, (γ-32P) ATP 1 mM, and 1 pM AMPK. SAMS peptide (3 nM) was used as positive control. Peptide reactions were terminated by blotting samples onto Whatman grade P81 ion-exchange chromatography paper, rinsed in 1% phosphoric acid, washed in acetone, and allowed to air dry prior to submersion in 1 ml scintillation fluid. The radioactivity was measured with a Beckman LS 6500 scintillation counter.AMPK kinase assays were performed using full-length recombinant proteins in 50 mM HEPES (pH 7.4), 1 mM AMP, 1 mM . DNA was purified using Qiagen PCR purification kit prior to qPCR analysis. The following primers were used for qPCR analysis of immunoprecipitated DNA: NADSYN1 promoter forward, ATTCCTTGGCTTCCTACTGC; NADSYN1 promoter reverse, GTGTCTTGATAGATGGGCTACAG; MMP-2 promoter forward, GCAGAAGGAAAGAGGTAAGGAAG; MMP-2 promoter reverse, GAAGGAATGGTCAGAAACAGATG.Cells were incubated in 0.75% paraformaldehyde and harvested in FA lysis buffer  prior to sonication with a Bioruptor 200. Protein was immunoprecipitated overnight at 4°C, washed with wash buffer  and eluted with 100 μl elution buffer , 0.1% SDS, 0.5% Sodium Deoxycholate, 1% Triton X-100, 10% Glycerol, 1 mM PMSF and protease inhibitors. The obtained cell lysates were then resolved by 10% SDS-PAGE, blocked with 5% milk, rinsed with TBST, probed with NADSYN, FOXO3a, β-actin, AMPK, MMP2, ATF2 primary antibodies from ABcam cat# ab139561, ab12162, ab8227, ab110036, ab86607, and ab47476 respectively, P-FOXO3a and P-ATF2 primary antibodies from Cell Signaling (Cat # 8174 and 9221), and secondary antibodies (Cat# 7074S and 7076S). The blotted proteins bands were made detectable by Emerson chemiluminescence substrate (ECL) and image captured on HyBlot CL autoradiography film.Cells were lysed in 10 mM Tris (pH 7.4), 0.1 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM NaMMP-2 activity was determined using Amersham Matrix Metalloproteinase-2 (MMP-2) Biotrak Activity Assay System RPN2631 protocol.+ was measured in C2C12 cell lysates using EnzyChromTM NAD+/NADH Assay Kit (E2ND-100) and protocol from BioAssay Systems.The cellular concentration of NADData are expressed as means ± SEM of at least three independent experiments. Comparisons of mean values between two groups were evaluated using a two-tailed Student’s t-test, Wilcoxon signed-rank test or Mann–Whitney U test. Unless otherwise indicated, *p < 0.05 was considered statistically significant.


With support from USAID-funded projects, the Dominican Republic (DR) started organizing a unified pharmaceutical system (SUGEMI) in 2010. Implementing SUGEMI included developing standard operating procedures (SOPs) for all system components, appointing personnel for national and regional pharmaceutical units, and training on-site staff responsible for pharmaceutical supply management. Strengthening and sustaining SUGEMI is now dependent on in-depth training of public health system staff responsible for supply management functions.SIAPS involved key personnel early in the elaboration of SOPs and training activities and helped design and implement a 12-week on-site/off-site certificate course on pharmaceutical supply. Each of the six program modules includes preparatory activities ; on-site sessions (discussing readings and instructions for on-the-job practice), and on-the-job site practice .Rapid capacity building has contributed to a nationwide implementation of SUGEMI in less than three years. Major outcomes were: National and Regional Pharmaceutical Unit staff members were trained to replicate trainings for SUGEMI implementation in 1,105 primary care facilities and 143 hospitals, and two on-site/off-site site courses have been completed . All students successfully fulfilled the academic requirements to obtain their certificates. Half of the graduates in the first course and all in the second were employed in a public health facility.Basic training in operational procedures is a necessary first step when implementing a national pharmaceutical system. However, consolidation and sustainability demands professionals with in-depth knowledge of concepts and tools commonly used in supply management. A hybrid on-site/off-site approach directed toward health workers in the public sector assures: reinforcement of the theory through practical experience, implementation of a national pharmaceutical system, and immediate introduction of good pharmaceutical management practices in their particular labour sites. All trainings had an immediate operative purpose–the implementation of particular SUGEMI component—fixing knowledge, through practice.Implementing a national pharmaceutical system offers a unique opportunity to consolidate theoretical concepts with practical on-the-job experiences. Involving personnel in the elaboration and training in implementing SOPs, and an on-site/off-site course simultaneously strengthens SUGEMI and builds capacity of personnel in pharmaceutical supply management.


Mono, di, and tri-acetoxy resveratrol were synthesized, a cell line of human intestine origin and microsomes from rat liver served to determine their in vitro inhibition of CYP3A4, and compared to that of resveratrol. Docking simulation served to predict the affinity of the synthetic model compounds to the enzyme. Modelling of the enzyme’s binding site revealed three types of interaction: hydrophobic, electrostatic and H-bonding. The simulation revealed that each of the examined acetylations of resveratrol led to the loss of important interactions of all types. Tri-acetoxy resveratrol was the weakest inhibitor in vitro despite being the more lipophilic and having the highest affinity for the binding site. The simulation demonstrated exclusion of all interactions between tri-acetoxy resveratrol and the heme due to distal binding, highlighting the complexity of the CYP3A4 binding site, which may allow simultaneous accommodation of two molecules. Finally, the use of computational modelling may serve as a quick predictive tool to identify potential harmful interactions between dietary compounds and prescribed drugs.Many dietary compounds, including resveratrol, are potent inhibitors of CYP3A4. Here we examined the potential to predict inhibition capacity of dietary polyphenolics using an  Cytochrome P450 enzymes (P450s) are very important for the metabolism of a wide range of endogenous compounds , as well as xenobiotics including drugs, environmental pollutants and dietary products1236481111213t-Resveratrol  is a polyphenol found in grape skin and red wine. The ability of resveratrol to inhibit CYP3A4 both in vitro and in vivo has been well established and it has been suggested that resveratrol may act as an irreversible, mechanism-based inactivator of this enzyme1516171819152124in vitro and in vivo, allowing resveratrol sulfates to act as an intracellular reservoir for resveratrol181950 values and greater affinity for CYP3A4 than t-resveratrol or its glucosides282914313233t-Resveratrol contains three hydroxyl group (OH), which may act simultaneously as both H-bond donors and H-bond acceptors35in vitro models presenting different isozymes of CYP3A, i.e., human CYP3A4 in Caco2/TC7 cell culture, and CYP3A in rat liver microsomes (RLM), and an in silico docking model allowed us to define structural components required for a compound to potently inhibit CYP3A4.A study of the effect of lipophilicity on the interactions of stilbenes with CYP3A4 revealed that methoxy-stilbenes have lower ICThe oxidation of testosterone by CYP3A4 was followed by the measurement of the residual testosterone in the reaction medium during incubation with Caco-2/TC7 cells . Under tThe effects of MAR, DAR and TAR on the viability of Caco-2/TC7 cells were measured as described. None of the compounds caused a reduction of Caco-2/TC7 cell viability when applied up to a level of 100 μM, except for 100 μM of TAR, which caused a minor reduction in the cells proliferation . The conThe metabolism of testosterone by Caco-2/TC7 cells in culture was used to study the interaction of acetoxy stilbenes with CYP3A4. All of the examined acetoxy stilbenes inhibited the metabolism of testosterone in a dose-dependent manner, significantly different from control, and maximal inhibition was achieved by application of 50 μM . However3H(N)]  were similar to that of resveratrol ] . Inhibitveratrol .The orientation and residue bonding of ketoconazole inferred from the X-ray structure of human CYP3A4 (PDB code 2V0M) served as a model for computing the interactions of resveratrol and the molecules synthesized here. To validate the suitability of the selected docking model, we first docked ketoconazole and ensured that its bonding to the CYP3A4 binding site was restored to the initial state, as in the original 2V0M structure. According to the ketoconazole-docking simulation, the binding site can be described in terms of three main features: a solid hydrophobic region, H-bond donors/acceptors and electrostatic interactions. The hydrophobic cluster includes the alkyl and alkyl-pi interactions between CYP3A4 residues Leu210, Phe241, Ile301, Ala305 and Leu482, and the chlorinated aromatic ring and imidazole ring of the ketoconazole molecule. The main H-bond donor residue is CYP3A4 Arg372, which interacts with the ketonic oxygen atom of ketoconazole; whereas the oxygen atoms of Ala370 serve as H-bond acceptors. The nitrogen atom of the heme group provides further anchoring to the molecule through its electrostatic interactions (anion-pi) with the imidazole ring of the ketoconazole , as compared to resveratrol  and 10. t-resveratrol on CYP3A4, both in vitro and in vivo, are well documented in the literature151617181920282931In recent studies, evidence has accumulated to indicate that there are potent interactions of clinical importance between CYP3A4 and dietary polyphenols, including flavonoids, phenolic acids, phenolic alcohols, stilbenoids and lignans50 values and greater affinity for CYP3A4, as compared to the parent resveratrol and its glucosidesin vitro and in silico models. This finding highlights the importance of the electrostatic interactions of the molecule at the active site of the enzyme, in addition to hydrophobic and H-bondsA previous study described the effect of a molecule’s lipophilicity on its interactions with CYP3A4 using synthetic modifications of resveratrol. That study found that methoxy-stilbenes have lower IC50 values in agreement with those presented in previous works1640414250 values for the acetoxy stilbenes since we could not achieve sufficient inhibition in the TC7 cells or in the RLM assay. The above results clearly demonstrate the importance of structural properties in determining the inhibition capacity of closely related polyphenolics against CYP3A4.The CYP3A4-mediated metabolism of testosterone using Caco-2/TC7 cells was designed to last 8 h, to optimally represent passage through the intestine. A linear curve was observed over a 2 to 6 h incubation period, leading to the selection of this time interval for the study the inhibition of human intestinal CYP3A4. We monitored the substrate consumption and, in this context, the treatments were significantly different from the control throughout all of the examined incubation periods. Resveratrol inhibited CYP3A4 in a manner similar to that of ketoconazole, with calculated ICIn a recent study, examination of the docking of ketoconazole, resveratrol and resveratrol-aldehyde at the binding site demonstrated the role that hydrophobic and electrostatic interactions play in the interactions of polyphenolics with CYP3A417454647Docking of the acetoxy analogues at the enzyme cavity was shown to produce a new array of bonds, i.e. two hydrogen bonds with Arg372 and the B ring of TAR, resulting also in an increased distance from the heme prosthetic group  and 10. in vitro inhibition of the enzyme by this molecule. Further and following its insertion into the active-site cavity, TAR may introduce a new set of available functional groups for secondary substrates to interact with, rather than inhibit enzyme activity via direct interaction with the catalytic site of CYP3A4. The latter suggestion could be examined by simultaneous insertion of two ligands into the cavity, but one should note that even the simultaneous insertion of two identical molecules dramatically decreases the degrees of freedom allowed in by nature, e.g. in the sense of order and orientation of approach, and the presence of other substrates or strongly bound water molecules. However, and unfortunately, that option is not available in the present simulation software.The results of the computational analysis suggest that TAR is inserted in an opposite orientation, in which, unlike previously studied stilbenes, the A ring faces the hydrophobic region of the binding site and is distally above the heme group E,F. In a454647in vitro and highlights the importance of the ligand’s proximity to the heme group of CYP3A4.A simulation of the docking of acetoxylated stilbenes, resveratrol and ketoconazole at the active site of CYP3A4 provided a mechanistic explanation of the lower inhibition caused by the more hydrophobic compounds, showing that the replacement of a hydroxyl group on the aromatic rings leads to a large loss of hydrophobic, H-bond and electrostatic interactions. Interestingly, the CDOCKER energy values of the acetoxy stilbenes were found to be relatively low, probably due to their low conformational energies. The molecules synthesized here, together with the computer simulation, allowed us to reveal a unique behavior of CYP3A4, which may explain its documented behavior involving a very wide spectrum of substrates. Our findings suggest the option of a simultaneous insertion of two molecules into the active site, which adds to reports that some molecules with a strong affinity for the binding site change the properties of that binding site and actually “open” it for additional moleculesIn conclusion, this work demonstrates for the first time that a substrate-induced modification of the active site of CYP3A4 can be predicted using simulation models. These predictions are in agreement with results achieved using crystallography for an abundant drug, e.g. erythromycin, and its interactions with CYP3A4t-Resveratrol , testosterone (99%) and ketoconazole (98%) standards were purchased from Sigma Aldrich, Israel. Testosterone  was purchased from . All other chemicals and reagents were purchased from Sigma Aldrich, unless otherwise noted.1H NMR and 13C NMR spectra of the products were obtained with a 200-MHz Bruker NMR spectrometer .The synthesis of acetoxylated analogues of resveratrol was performed using acetylation reactions, as described by1H NMR : δ 2.26 , 6.21 , 6.49 , 6.99–7.08 , 7.49–7.54 . 13C NMR : δ 20.9 (CH3), 103.2, 106.1, 116.5, 122.9, 128.3, 130.2 (CH), 135.1, 136.6, 148.0, 151.6, 159.7, 171.2, 178.2 (C).: 1H NMR : δ 2.27 , 6.45 , 6.78 , 6.86 , 7.04–7.15 , 7.51–7.57 . 13C NMR : δ 20.9 (CH3), 109.3, 111.8, 111.9, 124.8, 127.6, 129.2, 129.5 (CH), 136.9, 140.8, 151.7, 153.4, 157.1, 159.6, 171.1 (C).: 1H NMR : δ 2.30 , 6.83 , 7.07–7.26 , 7.56–7.61 . 13C NMR : δ 20.9 (CH3), 115.7, 118.1, 120.4, 123.1, 128.1, 128.7 (CH), 130.7, 137.7, 151.9, 152.9, 170.7, 171.1, 174.8 (C).: 2 and 95% air. The DMEM was supplemented with 20% fetal bovine serum, 100 units/mL penicillin , 100 μg/mL streptomycin , 1% non-essential amino acids solution  and 1% L-glutamine solution . The medium was pre-warmed to 37 C prior to contact with the cells. The Caco-2/TC7 cells were used between passages 25 and 35 and grown in 75-cm2 flasks. New flasks were seeded at a concentration of 3–4 × 104 cells/cm2.Caco-2/TC7, a subclone of Caco-2, expresses a high level of CYP3A4 enzyme, which is quite common in the human intestine515254554 cells/cm2 in 12-well plates. The medium was changed every second day and the cells were allowed to grow and differentiate for up to 14 days after confluence was reached. On the day of the experiment, testosterone, ketoconazole, resveratrol, MAR, DAR and TAR were dissolved in methanol, sonicated for 10 min  and then added to the treatment medium (maximum methanol concentration in the medium was 0.1%). Testosterone, used as a substrate of CYP3A4 enzyme, was added to each cell well in all of the treatments at a final concentration of 200 μM. The actual concentration of testosterone in the reaction medium at time 0 was 31 μM, as determined using the below described HPLC method. The treatments in this study were as follows: Control 1 included only testosterone incubated with Caco-2/TC7 cells and Control 2 included a specific inhibitor of CYP3A4, ketoconazole (10 μM). The stilbene treatments included resveratrol (10 μM) and the acetoxy analogues MAR, DAR and TAR at different concentrations . All treatments were carried out for three incubation periods, 2, 4 and 6 hours (in addition to time 0), and there were three replicates of each treatment/incubation time combination. At the end of incubation, the culture medium was transferred to Eppendorf vials, the cell were harvested by scraping after addition of HPLC-grade methanol (200 μl) and then transferred to the Eppendorf vials. Then, 100 μl of HPLC-grade acetonitrile was added and the Eppendorf vials were shaken vigorously and then, centrifuged at 14,000 RPM for 5 min . The supernatant was then filtered through 0.22 μm filter into HPLC vials.Caco-2/TC7 cells were seeded at 2–3 × 10High pressure liquid chromatography (HPLC) analysis was carried out on a Surveyor HPLC system  using a RP-C18 Luna column 250 mm × 4.6 mm × 5 μm . Elution was performed with water (Solvent A) and acetonitrile (Solvent B) at a flow rate of 0.7 mL/min. The mobile phase gradient was modified as follow: B in A (v/v), from 5–25% for 5 min, from 25–75% for 10 min, from 75–95% for 5 min, followed by holding for 2 min, dropped again to 5% for 1 min and held for 4 min. Samples were examined with a photodiode array detector at 244 nm for testosterone and its metabolite, and at 280 nm and 306 nm for resveratrol and its derivatives. Peaks were scanned between 196 and 450 nm.4 cells per well and incubated for 24 h. Then, the cells were treated with DMEM (control) and the acetoxy stilbene analogues MAR, DAR and TAR at different concentrations  for an additional 8 h. Cells were incubated with 3--2,5-diphenyl tetrazolium bromide  for 1 h and there was a follow-up incubation with DMSO for 20 min. The formation of the colored formazan dye was assessed colorimetrically at 550 nm in an ELX 808 Ultra microplate reader  using KCJunior software .The effects of the acetoxy stilbene analogues on the proliferation of Caco-2/TC7 cells were assessed by the MTT assay. Cells were seeded onto 96-well plates at a density of 10Male rat liver microsomes (RLM) were prepared from adult male Sprague-Dawley rats (175–199 g), according to published protocol with minor modifications2HPO4, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.4) containing 8 nM testosterone  and 1.5 mM NADPH. Resveratrol, acetoxy stilbene analogues and ketoconazole were first dissolved in 30% methanol and sonicated, and then stock solutions were prepared in reaction buffer (maximum methanol concentration in each well was 0.95%). The different treatments were added to the reaction wells to achieve final concentrations of 10, 25, 50 and 100 μM. All treatments were assayed in triplicate with the following controls: a buffer control containing the reaction buffer instead of the inhibitor (no treatment); a methanol control containing 0.95% methanol instead of the inhibitor, to account for possible effects of this solvent; and a RLM control that did not contain any microsomes, to provide a background value. Reactions were then incubated for 4 h with continuous shaking at the optimal temperature of 37 °C. To stop the reaction, 1 mL of ethyl ether was added, followed by vortex-mixing. Ether and aqueous layers were allowed to separate for 10 min and then transferred to −80 °C for 15 min, to completely freeze the aqueous phase. The aqueous phase was left in the chemical hood for 1 h, to allow the complete evaporation of ether traces. The aqueous phase was shaken with 2 volumes (400 μl) of dextran coated (0.5%) charcoal slurry  for 1 min and centrifuged at 3000 g at 4 °C for 5 min. The supernatant (300 μl) was collected into scintillation vials and mixed with 5 mL scintillation fluid. 3H was measured as disintegration per 5 min using a liquid scintillation β counter .The interaction of CYP3A with testosterone was examined using a tritiated water assay adapted fromThe crystal structure of human CYP3A4 determined with ketoconazoleHow to cite this article: Basheer, L. et al. Inhibition of cytochrome P450 3A by acetoxylated analogues of resveratrol in in vitro and in silico models. Sci. Rep.6, 31557; doi: 10.1038/srep31557 (2016).