Here, we investigated the expression of enzymes in H2S synthesis and the mechanism regulating colonic smooth muscle function by H2S. Expression of cystathionine‐γ‐lyase (CSE), but not cystathionine‐β‐synthase (CBS), was identified in the colonic smooth muscle of rabbit, mouse, and human. Carbachol (CCh)‐induced contraction in rabbit muscle strips and isolated muscle cells was inhibited by l‐cysteine (substrate of CSE) and NaHS (an exogenous H2S donor) in a concentration‐dependent fashion. H2S induced S‐sulfhydration of RhoA that was associated with inhibition of RhoA activity. CCh‐induced Rho kinase activity also was inhibited by l‐cysteine and NaHS in a concentration‐dependent fashion. Inhibition of CCh‐induced contraction by l‐cysteine was blocked by the CSE inhibitor, dl‐propargylglycine (DL‐PPG) in dispersed muscle cells. Inhibition of CCh‐induced Rho kinase activity by l‐cysteine was blocked by CSE siRNA in cultured cells and DL‐PPG in dispersed muscle cells. Stimulation of Rho kinase activity and muscle contraction in response to CCh was also inhibited by l‐cysteine or NaHS in colonic muscle cells from mouse and human. Collectively, our studies identified the expression of CSE in colonic smooth muscle and determined that sulfhydration of RhoA by H2S leads to inhibition of RhoA and Rho kinase activities and muscle contraction. The mechanism identified may provide novel therapeutic approaches to mitigate gastrointestinal motility disorders.Hydrogen sulfide (H CBScystathionine‐β‐synthaseCChcarbacholCSEcystathionine‐γ‐lyaseDL‐PPGdl‐propargylglycineMLCKmyosin light‐chain kinaseMLCPmyosin light‐chain phosphataseNaHSsodium hydrosulfide2S) is produced via both enzymatic and nonenzymatic pathways in mammalian cells, but most of the H2S levels in tissues are attributed to enzymatic synthesis  and cystathionine‐γ‐lyase (CSE), are expressed in several tissues, but the pattern of expression has been reported to be tissue‐specific. Expression of CBS is mainly found in the central and peripheral nervous system, whereas expression of CSE is mainly found in the vascular system and liver  system include two sources: a luminal sulfate‐reducing bacterial source in the large intestine and an endogenous generation by different cells within the wall of GI tract , and epithelial cells  is an essential step in smooth muscle contraction , which initiates phosphorylation of MLC20 and MLC phosphatase (MLCP), which dephosphorylates MLC20. MLCP is a heterotrimer with an 110‐ to 130‐kDa regulatory subunit (myosin phosphatase target subunit 1 (MYPT1), a 37‐kDa catalytic subunit of type 1 phosphatase (PP1cδ), and a 20‐kDa subunit of unknown function  and cystathionine‐γ‐lyase (CSE) antibodies were purchased from Proteintech ; [32P]ATP was purchased from PerkinElmer ; HRP‐conjugated secondary antibodies were obtained from Cell Signaling Technology ; PVDF membranes were obtained from Millipore ; Effectene Transfection Reagent, QIAEX®II was from Qiagen ; culture medium (Dulbecco's modified Eagle's medium) was from Fisher Scientific ; l‐Cysteine and dl‐propargylglycine (PPG) were from Sigma . All other reagents were from Sigma .Rabbits  weighing 4–5 lbs were purchased from RSI Biotechnology , and mice  were purchased from Jackson Laboratories . Rabbits and mice were acclimatized at the facility administered by the Division of Animal Resources, Virginia Commonwealth University. The Institutional Animal Care and Use Committee of Virginia Commonwealth University approved all the procedures conducted. Colons from normal human subjects were obtained from a nonprofit organization known as National Disease Research Interchange  that provides human organs and tissue. The studies involving human tissues are approved as exempt from VCU Institutional Review Board.2PO4, 1.2 mmol/L MgSO4, 2.54 mmol/L CaCl2, 25 mmol/L NaHCO3, 11 mmol/L glucose at 37°C, and pH 7.4. Small colonic segments were threaded onto a glass rod. The longitudinal muscle with adherent myenteric plexus was removed by radial abrasion with Kim wipes. Circular muscle layer of the colon was used to isolate smooth muscle cells as described previously  Eagle's essential amino acid mixture, 0.1% collagenase [type II], and 0.1% soybean trypsin inhibitor). After the 30‐min digestion period, tissues were washed with collagenase‐free medium (50 mL) and muscle cells were allowed to disperse spontaneously. Cells were collected by filtration through 500 μm Nitex followed by centrifugation twice at 350g for 10 min. Smooth muscle cells were cultured in DMEM containing 10% fetal bovine serum and cells passaged once after attaining confluence. For experiments, muscle cells were used in the first passage.The colon from rabbit, mouse, and human were dissected out and after emptying the contents was placed in oxygenated Kreb's solution composed of 118 mmol/L NaCl, 4.75 mmol/L KCl, 1.19 mmol/L KHEcoRI). Smooth muscle cells in culture were transiently transfected for 48 h with recombinant plasmid cDNAs. To monitor transfection efficiency, muscle cells were cotransfected with 2 μg pcDNA3 vector and 1 μg of pGreen Lantern‐1 DNA  were resolved on 10% SDS‐PAGE and the proteins were transferred to PVDF membranes. The membranes were incubated overnight with CSE or CBS antibodies followed by incubation with appropriate secondary antibody conjugated with horseradish peroxidase. Enhanced chemiluminescence was used to visualize protein bands on the membrane.Expression of CSE and CBS was measured by western blot as described previously  or NaHS (1 mmol/L) for 10 min. In some cases, the cells were preincubated with CSE inhibitor, DL‐PPG (1 mmol/L) for 10 min before the addition of CCh in the presence or absence of l‐cysteine or NaHS. Cells were homogenized in the lysis buffer and GTP‐bound RhoA was immunoprecipitated using monoclonal antibody  that specifically recognizes RhoA‐GTP. RhoA‐GTP bound antibody were pulled down by protein A/G, washed with lysis buffer, and processed for separation by SDS‐PAGE. The level of activated RhoA was evaluated by western blot analysis.RhoA activation was evaluated in pull‐down assays using specific anti‐RhoA‐GTP antibody and protein A/G beads , 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P‐40, 150 mmol/L NaCl, 1 mmol/L PMSF, 10 μg/mL aprotinin, 10 μg/mL pepstatin A, and 10 μg/mL leupeptin. Aliquots containing an equal amount of protein (50 μg) were incubated overnight at 4°C with Rho kinase‐2 antibody and protein A/G agarose. The immunoprecipitates collected by centrifugation were washed twice with a medium containing 10 mmol/L MgCl2 and 40 mmol/L HEPES (pH 7.4) followed by incubation for 5 min at 4°C with myelin basic protein (MBP) (1 mg/mL). The reaction was initiated by the addition of 10 μCi of [32P]ATP (3000 Ci/mmol) and 20 μmol/L ATP at 37°C and terminated after 10 min by spotting the reaction mixture onto phosphocellulose disks to capture phosphorylated MBP. The disks were washed with 75 mmol/L phosphoric acid to remove free radioactivity. Phosphorylation of MBP was determined from the radioactivity on disks by liquid scintillation.Rho kinase activity was measured by immunokinase assay as previously described  Kreb's medium (pH of 7.4) at 37°C. The isometric force generated by circular muscle was measured by mounting the tissue between a glass rod and isometric transducer  connected to a computer recording system. A resting tension of 1 g was given and the muscle strips were allowed to equilibrate. Muscle strips were contracted with 10 μmol/L CCh in the presence or absence of different concentrations of l‐cysteine or NaHS pretreatment for 10 min. In some experiments, muscle strips were pretreated with 10 μmol/L glibenclamide (a KATP channel blocker) before the addition of NaHS or l‐cysteine. Tissue weight was recorded after the experiment and contraction was calculated as area under the curve in response to CCh alone and compared with CCh with l‐cysteine or NaHS  was preincubated with a different concentration of l‐cysteine or NaHS for 10 min and then with CCh for another for 10 min. A final concentration of 1% acrolein was used to terminate the reaction and a drop of cell suspension was placed on a slide under a cover slip. In some experiments, muscle cells were pretreated with 10 μmol/L glibenclamide (a KATP channel blocker) before the addition of NaHS or l‐cysteine. Cell length was measured by scanning micrometry. Cell length in the absence of any treatment was taken as resting cell length. Contraction was expressed as a decrease in mean cell length from control cell length.Contraction in dispersed muscle cells was determined by scanning micrometry as described previously .Data are expressed as mean ± SEM. “Expression studies by western blot demonstrated the presence of CSE protein (66 kDa) in cultured muscle cells of the colon of mouse, rabbit, and human Fig. . Under sl‐cysteine (0.1 μmol/L to 10 mmol/L) or NaHS (0.01 μmol/L to 1 mmol/L) for 10 min before the addition of CCh and measurements of AUC were recorded again to estimate the amount of inhibition of contraction in response to l‐cysteine or NaHS.Isometric contraction was measured in muscle strips isolated from the colon of rabbit. Muscle strips were equilibrated to a passive tension of 1 g for 1 h before experiments were conducted. Area under curve (AUC) measurement representing force versus time in response to CCh (10 μmol/L) was defined as muscle contraction above basal. AUC was recorded for the first 2 min in response to CCh. The strips were incubated with various concentrations of n = 4). CCh‐induced contraction was inhibited by l‐cysteine and NaHS in a concentration‐dependent fashion  (65 ± 4% inhibition vs. 61 ± 3% inhibition in the presence of glibenclamide) or NaHS (1 mmol/L) (82 ± 6% inhibition vs. 79 ± 7% inhibition in the presence of glibenclamide). Glibenclamide had no effect on CCh‐induced contraction , suggesting that inhibition of CCh‐induced contraction by l‐cysteine was not due to activation of KATP channels.As shown previously  or NaHS (1 mmol/L) and then treated with different concentrations of CCh (0.01 nmol/L to 1 μmol/L) for 10 min. CCh caused a concentration‐dependent contraction in muscle cells with EC50 of 10−9 ± 3 × 10−10 mol/L. Maximal contraction  was obtained with 1 μmol/L of CCh. Addition of l‐cysteine or NaHS shifted the contractile response to CCh to the right with EC50 values of 3.5 × 10−8 ± 5 × 10−9 mol/L and 9 × 10−8 ± 7 × 10−9 mol/L, respectively, suggesting inhibition of CCh‐induced contraction by H2S  induce biphasic contraction in muscle cells: a rapid contraction within 30 sec followed by a sustained contraction for 20 min  for 10 min did not affect the inhibition of contraction by l‐cysteine (10 mmol/L)  or NaHS (1 mmol/L) . Glibenclamide had no effect on CCh‐induced contraction . These results demonstrate that H2S inhibits muscle contraction and the effect of H2S does not involve KATP channel activation. Control studies showed that inhibition of contraction by levcromakalim (10 μmol/L), a potassium channel activator, was reversed by glibenclamide .Contraction in response to maximal dose of CCh (1 μmol/L) was inhibited by ion Fig. C. Glibenl‐cysteine was blocked by treatment of cells with the CSE inhibitor, dl‐propargylglycine (DL‐PPG) (1 mmol/L) for 10 min  for 10 min caused significant inhibition (59 ± 3% inhibition) of contraction in response to CCh. The inhibitory effect of l‐cysteine was attenuated (15 ± 8% inhibition) in the presence of DL‐PPG (1 mmol/L). In contrast, inhibition of sustained contraction by NaHS (1 mmol/L) was not affected by DL‐PPG (59 ± 3% inhibition vs. 60 ± 9% inhibition with DL‐PPG), suggesting that the inhibition of muscle contraction by l‐cysteine was mediated by the activation of CSE and generation of H2S.Inhibition of CCh‐induced contraction by min Fig. . Treatmel‐cysteine (10 mmol/L) or NaHS (1 mmol/L) for 10 min also inhibited sustained contraction in mouse and human. In mouse colonic muscle cells, contraction (31 ± 3% decrease in cell length) in response to 1 μmol/L CCh was inhibited by l‐cysteine (45 ± 4% inhibition) and NaHS (57 ± 6% inhibition)  and NaHS (47 ± 5% inhibition)  or NaHS (1 mmol/L) for 10 min caused inhibition of RhoA activity in response to CCh  for 10 min reversed the effect of l‐cysteine on RhoA activity in response to CCh  for 10 min caused an increase in the RhoA activity, measured as an increase in the incorporation of GTP into RhoA using an antibody that specifically recognizes RhoA‐GTP. Pretreatment of cells with CCh Fig. A. These CCh Fig. A. DL‐PPGCCh Fig. A.2S signals. H2S alters the function of KATP channels via S‐sulfhydration  was analyzed using a biotin switch assay. Basal sulfhydration of RhoA was not detected in control cells  or NaHS (1 mmol/L) for 10 min, suggesting inhibition of Rho kinase activity by H2S . These results suggest that endogenous and exogenous H2S inhibits Rho kinase activity in colonic muscle cells. The kinase assay was specific for Rho kinase. CCh (1 μmol/L)‐stimulated Rho kinase activity was blocked by the addition of Rho kinase inhibitor Y27632 (10 μmol/L) .Pretreatment of cells with increasing concentrations of 2S generation by l‐cysteine to inhibit Rho kinase activity was tested by two methods: (1) by transfection with CSE‐specific siRNA in cultured muscle cells, and (2) by treatment with a selective CSE inhibitor, DL‐PPG in dispersed muscle cells. CCh induced a significant increase in Rho kinase activity in cultured muscle cells  that is similar to the increase in dispersed muscle cells. Pretreatment of cells with l‐cysteine (10 mmol/L) for 10 min significantly inhibited CCh‐stimulated Rho kinase activity (63 ± 8% inhibition) in cells transfected with control siRNA and the inhibition was blocked in cells transfected with CSE‐specific siRNA   for 10 min also inhibited CCh‐stimulated Rho kinase activity in cells transfected with control siRNA (66 ± 7% inhibition) and the inhibition, however, was not affected in cells transfected with CSE‐specific siRNA (60 ± 7% inhibition).The involvement of endogenous Hl‐cysteine or NaHS in dispersed muscle cells. Pretreatment of cells with l‐cysteine (10 mmol/L) for 10 min significantly inhibited CCh‐stimulated Rho kinase activity (53 ± 7% inhibition) and the inhibition was blocked by DL‐PPG (1 mmol/L)   for 10 min before treatment with l‐cysteine (10 mmol/L) or NaHS (1 mmol/L) for 10 min also inhibited Rho kinase activity in response to CCh in mouse and human. In mouse colonic muscle cells, inhibition by l‐cysteine was 43 ± 6% and inhibition by NaHS was 58 ± 5%  and carbon monoxide (CO), is justified to be defined as a physiological transmitter  or l‐cysteine (substrate of CSE), pharmacological inhibitors of enzymes that generate H2S  and molecular approaches , several functions of H2S have been demonstrated in the cardiovascular system, central nervous system, GI tract, and energy metabolism  selective expression of CSE in smooth muscle cells of rabbit, mouse, and human, where it is responsible for H2S production; (2) inhibition of CCh‐induced contraction by l‐cysteine and NaHS in muscle strips and dispersed smooth muscle cells of rabbit and blockade of inhibition by a selective inhibitor of CSE (DL‐PPG), but not by a selective inhibitor of KATP channels (glibenclamide); (3) S‐sulfhydration of RhoA by NaHS and attenuation of CCh‐induced RhoA and Rho kinase activities and muscle contraction by both l‐cysteine and NaHS; (4) blockade of l‐cysteine‐induced inhibition by DL‐PPG or CSE siRNA; and (5) inhibition of Rho kinase activity and muscle contraction by l‐cysteine and NaHS in colonic smooth muscle cells from mouse and human.In the present study, we investigated the expression of H2S synthesizing enzymes also varies with the different regions of the GI tract. Expression of CSE is more abundant in the proximal regions , compared to distal regions (ileum and colon) of rat GI tract. In contrast, expression of CBS was low in proximal regions (duodenum and jejunum) and high in the distal regions (ileum and colon)  or mmol/L  (Yang et al. 2S concentrations in GI smooth muscle and the significance of H2S at low μM concentration as a messenger molecule are yet to be determined. Our studies also showed that the effect of H2S is not dependent on the activation of KATP channels, but dependent on the S‐sulfhydration of RhoA.2S in mediating the inhibitory effect on muscle contraction. Like S‐nitrosylation by nitric oxide, S‐sulfhydration by H2S appears to be the common post‐translational mechanism to alter the function of proteins (Mustafa et al. 2S (Dhaese and Lefebvre 20 phosphorylation and muscle contraction (Murthy et al. 2S exerts an inhibitory effect on RhoA activity causing attenuation of Rho kinase activity and disinhibition of Rho kinase‐mediated MLCP activity, augmentation of MLC20 dephosphorylation, and inhibition of sustained contraction. Blockade of l‐cysteine effect by DL‐PPG in dispersed muscle cells and CSE siRNA in cultured muscle cells provides evidence for the involvement of CSE activation.In the present study, we have identified RhoA as S‐sulfhydration target of H2S inhibits muscle contraction via S‐sulfhydration of RhoA and inhibition of RhoA and Rho kinase activities.In summary, our studies demonstrated the selective expression of CSE in colonic smooth muscle cells and identified the molecular mechanism by which HK. S. M., A. N., and H. W. participated in research design; A. N., H. W., S. B., B. B., and K. S. M. conducted experiments; K. S. M. and A. N. performed data analysis; K. S. M., A. N., and S. B. wrote or contributed to the writing of the manuscript. None contributed to new reagents or analytical tools.None declared.


The threshold of sea ice concentration (SIC) is the basis for accurately calculating sea ice extent based on passive microwave (PM) remote sensing data. However, the PM SIC threshold at the sea ice edge used in previous studies and released sea ice products has not always been consistent. To explore the representable value of the PM SIC threshold corresponding on average to the position of the Arctic sea ice edge during summer in recent years, we extracted sea ice edge boundaries from the Moderate-resolution Imaging Spectroradiometer (MODIS) sea ice product , MODIS images (250 m), and sea ice ship-based observation points (1 km) during the fifth (CHINARE-2012) and sixth (CHINARE-2014) Chinese National Arctic Research Expeditions, and made an overlay and comparison analysis with PM SIC derived from Special Sensor Microwave Imager Sounder  in the summer of 2012 and 2014. Results showed that the average SSMIS SIC threshold at the Arctic sea ice edge based on ice-water boundary lines extracted from MOD29 was 33%, which was higher than that of the commonly used 15% discriminant threshold. The average SIC threshold at sea ice edge based on ice-water boundary lines extracted by visual interpretation from four scenes of the MODIS image was 35% when compared to the average value of 36% from the MOD29 extracted ice edge pixels for the same days. The average SIC of 31% at the sea ice edge points extracted from ship-based observations also confirmed that choosing around 30% as the SIC threshold during summer is recommended for sea ice extent calculations based on SSMIS PM data. These results can provide a reference for further studying the variation of sea ice under the rapidly changing Arctic. In recent years, global climate change has drawn increasing attention . AccelerRemote sensing is an important tool to monitor sea ice over a wide region. Passive microwave (PM) remote sensing can distinguish between sea ice and sea water, which are widely used to study the variation of sea ice extent at the Arctic scale ,4,5,6. SA typical SIC threshold is 15%, which was first used by Parkinson et al. to define the location of the sea ice edge for measuring the Arctic sea ice extent . AccordiIn addition, the SIC threshold at the sea ice edge used in the released sea ice products is not always consistent. A threshold of 15% is used in the product of “Sea Ice Index” , and theIn these contexts, this paper focused on exploring a representative ice-water discrimination threshold for the SSMIS  in the Arctic in recent years. obs is the observed brightness temperature; and TBow, TBFY, TBMY are the typical brightness temperatures from open water, first-year, and multi-year ice, respectively. Cow, CFY, CMY are the concentrations of the three surfaces, respectively. In practice, the left hand side of Equation (1) are usually uses the polarization ratio (PR) and spectral gradient ratio (GR) both derived from brightness temperature, Equations (2) and (3) for the reason that they are independent of the physical temperature [The sea ice concentration data  used in perature :PR = [TBased on the PR and GR, the first-year ice concentration (CF) and the multiyear ice concentration (CM) are calculated from the following equations:The total sea ice concentration (CT) is the sum of the first-year and multiyear concentrations:i, bi, and ci  are functions of a set of brightness temperatures. These brightness temperatures, referred to as algorithm tie points, are the observed PM radiances over areas of known ice-free ocean, First Year (FY) sea ice, and Multi Year (MY) ice for each channel. The coefficients in Equations (3)–(5), are given in The coefficients a4 and Ref6 are the reflectance from MODIS bands 4 and 6, respectively. Then, the sea ice can be discriminated using the following satisfied criteria [2 is the reflectance from MODIS band 2. With the MODIS land mask and cloud mask defined areas input, MOD29 data provide sea ice, ocean, land, and cloud information in the tile grid. The MOD29 sea ice product is the MODIS daily sea ice product, which can be used to extract sea ice edges for a comparing with the SSMIS SIC. MOD29 data  used in criteria : are shown in When the sea ice edge lines and points were extracted from relatively high resolution data such as MOD29 sea ice products, MODIS images, and ship-based observations, they were then overlaid to coincide with SSMIS SIC , in order to calculate the mean SIC at sea ice edges, which was used as the ice-water discrimination threshold for PM data. These comparisons were to explore the representative ice-water discrimination threshold for SSMIS in the Arctic in recent years.We calculated the SSMIS SIC corresponding to the extracted MOD29 sea ice edges in four different marginal seas. The results are shown in From the statistical results of the different years b, we canThe results of the SSMIS SIC at the Arctic sea ice edges corresponding to the visual interpretation from 4 senses MODIS images are shown in In addition, by comparing the results from the MODIS images and from the corresponding MOD29 product, we found that the threshold values from MOD29 (36% on average) were generally higher than those from the MODIS images (35% on average).It can be seen from Currently, much attention has been paid to the variation and the trend of Arctic sea ice using passive microwave remotely sensed data ,29, but Overall, the thresholds obtained from the comparisons were rather similar; different comparison results indicated that the representable threshold value of Arctic sea ice edge during the summer was higher than the well-known 15% . The meaAlthough the threshold values from the MOD29 and MODIS images were both higher than 15%, MOD29 (36% on average) was generally higher than that from the corresponding MODIS images (35% on average). The correlation between the SIC of the ship-observed sea ice edge points and the corresponding SSMIS was moderate, which may be due to the following factors. First, there were some errors in the SSMIS SIC due to the SIC retrieval algorithm, as well as the existing errors for the sea ice ship-based observations due to the influence of in-situ human observations. Second, the spatial resolution between the SSMIS and the ship-based observation SIC was different as the resolution of the SSMIS data was 25 km and the ship observational data were 1 km.It should be noted that different SIC algorithms, with the spatial resolution of each channel that enters into these SIC algorithms may influence the threshold results. Although Comiso and Parkinson , HeygsteSince a representable SIC threshold of 15% or 30% would influence the exact Arctic sea ice extent, we compared the difference of the calculated Arctic sea ice extent based on a 15% and 30% SIC threshold for both the NT SIC algorithm and Bootstrap SIC algorithm with a spatial resolution of 25 km for SSM/I and SSMIS data, and the ASI SIC algorithm with a spatial resolution of 6.25 km for AMSR-E and AMSR2 PM data. 5 km2 and 6.24 × 105 km2 for the NT and Bootstrap SIC algorithms, and 3.99 × 105 km2 for the ASI SIC algorithm with a spatial resolution of 6.25 km. The averaged relative differences at the minimum and maximum extents from 2002 to 2016 based on 15% and 30% SIC thresholds were 5.63 × 105 km2 and 1.24 × 105 km2 for the NT and Bootstrap SIC algorithms, and 1.48 × 105 km2 for the ASI SIC algorithm. We deduce that the improvement of the SIC algorithms and the satellite sensors’ spatial resolution will reduce the impact of choosing different SIC thresholds on the calculated sea ice extent. However, these influences should not be ignored when using different SIC products for the long-term trend of Arctic sea ice extent.The maximum differences of the calculated sea ice extent based on 15% and 30% SIC thresholds were 10.33 × 10This paper explored the representable value of the SSMIS sea ice concentration threshold at the Arctic sea ice edge during summer in recent years. The MOD29 sea ice product, MODIS images, and ship-based sea ice observations from CHINARE-2012 and CHINARE-2014 were used to extract Arctic sea ice edges. Based on these extracted ice edge lines and points, we made a comparison and statistical analysis of the SSMIS sea ice concentration at the Arctic sea ice edge in the summer of 2012 and 2014. Overlay analysis of the sea ice edges extracted from the MOD29 sea ice product and SSMIS SIC showed that the average SIC threshold of the four Arctic marginal seas was 33%. The average SIC threshold (42%) of the Chukchi Sea was higher than that in other marginal seas, 34% for the Laptev Sea, 29% for the East Siberian Sea, and 29% for the Greenland Sea. The average SIC thresholds in 2012 and 2014 were 32% and 34%, respectively, with an average value of 33%. Therefore, the commonly used 15% SIC threshold at the Arctic sea ice edge could be adjusted to 30% in the recently rapid changing Arctic.A comparison of four scenes of MODIS visual interpretation sea ice edges and the corresponding SSMIS SIC indicated that the average SIC threshold value was 33% on 9 June 2012 for the Chukchi Sea, 37% for the Greenland Sea ice on 15 July 2012, 34% for the Laptev Sea on 30 June 2014, and 34% for the East Siberia Sea on 10 July 2014. All these values further confirmed that using 30% as the sea ice edge threshold with respect to the 15% was more reasonable.The threshold SIC values from overlying the MOD29 and MODIS visual interpretation of the sea ice edge lines on the same date and the same sea region were approximately similar. MOD29 was slightly higher than that of the MODIS visual interpretation as the MOD29 sea ice product misjudged sea water as sea ice in some areas, resulting in a higher SIC at the sea ice edge.The average SIC at the Arctic sea ice edge points extracted from ship-based observations during CHINARE-2012 and CHINARE-2014 was 31%, which also showed that choosing around 30% as the SIC threshold is recommended for sea ice extent calculation based on SSMIS SIC data. These results and conclusions can provide a reference for further development and improvement of PM SIC products and studying the variation of sea ice under the rapidly changing Arctic.


Leptodictyum riparium in bags at 3 representative sites of the river. Biological effects were assessed by metal bioaccumulation, ultrastructural changes, oxidative stress, as Reactive Oxygen Species (ROS) production and Glutathione S-transferase (GST) activity, as well as Heat Shock Proteins 70 (HSP70s) induction. The results showed that L. riparium is a valuable bioindicator for toxic metal pollution of water ecosystem, accumulating different amounts of toxic metals from the aquatic environment. Toxic metal pollution caused severe ultrastructural damage, as well as increased ROS production and induction of GST and HSP70s, in the samples exposed at the polluted sites. To assess the role and the effect of toxic metals on L. riparium, were also cultured in vitro with Cd, Cr, Cu, Fe, Ni, Pb, Zn at the same concentrations as measured at the 3 sites. Ultrastructure, ROS, GST, and HSP70s resulted severely affected by toxic metals. Based on our findings, we confirm L. riparium as a model organism in freshwater biomonitoring surveys, and GST and HSP70s as promising biomarkers of metal toxicity.This study evaluates the effects of toxic metal pollution in the highly contaminated Sarno River , by using the aquatic moss  The contamination of rivers is of special concern, since they may transport contaminants, among which toxic metal, to areas far removed from any local pollution source, thus posing at risk even pristine ecosystems.Leptodictyum riparium (Hedw.) Warnst (Bryophyta). Bioaccumulation ability, tissue localization, toxic effects of metals and antioxidant activity in this species were investigated in previous studies [L. riparium resulted the most effective among 3 freshwater plant species in bioaccumulating metals in vitro [Toxic metal pollution is a major concern worldwide, and the use of living organisms for monitoring these pollutants is widely accepted . Whitton studies –5, confiin vitro .Among the damages induced by environmental stress, and namely by toxic metal pollution, the disruption of main metabolic pathways, primarily due to the unfolding of enzymes and proteins, represents one of the first, and most devastating, effects on living organisms. As a consequence, cells react by activating a number of defense mechanisms: the induction, synthesis and activation of chaperons represent the most evident and widespread of them . Among tVarious abiotic stressors lead to the overproduction of Reactive Oxygen Species (ROS) in plants. These molecular species are highly reactive and can play a dual role in plants. In fact, they can either act as toxic agents or work as signals, which then trigger and regulate biological processes, such as cell death or adaptation responses to environmental stress, or even main physiological processes, such as cell proliferation and differentiation –10. As tThe Sarno River , is known as one of the most polluted rivers in Europe: the whole basin  has been declared as “area with high risk of environmental crisis” . The heain vitro results. To this purpose, we evaluated the biological effects of water pollution by toxic metals in one of the most polluted river in Europe using the aquatic cosmopolitan moss L. riparium, a species able to strongly accumulate toxic metals [in vitro, considering metal bioaccumulation, oxidative stress (content of ROS and GST antioxidant enzyme activity), ultrastructural damage and HSP70s induction.This study aimed at proposing an experimental protocol that allows determining contribution of the different pollutants to the overall biological effect, by carefully comparing in field and c metals . BiologiL. riparium was widely present in the Mediterranean area, and in the Sarno river basin from springs to the river mouth; in order to use similar specimens for the experimental design, samples were collected at the Botanical Gardens of the University of Naples “Federico II,” Italy. Moss grew in a basin at a depth of 20–25 cm and temperature of 17–20°C. Homogeneous samples of about 2 g fresh weight (fw) were washed with deionized water and placed into 1 mm2 meshed nylon bags, as recommended by Kelly et al. [y et al. . Six bagy et al. , and feaA summary of the three selected sites are provided below:“Rio Foce” (site A) near the spring, before pollution sources, as control site; 40°49’56.269” N, 14°35’27.103° E. Sarno River spring is located at 30 m asl, at the slope of the Saro Mount. This represents the Western extremity of Picentini Mounts, characterized by a forest extension of over 40,000 hectares, rich in beech, maple, alder, and chestnut, and numerous streams that make the area the richest potable drinking water tank in southern Italy.“San Marzano sul Sarno” (site B), before the confluence with the Alveo Comune Nocerino; this site is affected mainly by urban pollution. The Alveo Comune Nocerino is originated by the confluence of Solofrana and Cavaiola, and is the main Sarno tributary; 40°46’47.971” N, 14°34’23.296” E.“Scafati” (site C), the heavily polluted site, after the confluence of Alveo Comune Nocerino collecting Cavaiola e Solofrana streams, affected by heavy industrial  and agricultural  wastewaters. 40°44’48.812” N, 14°31’37.653” E.No specific permissions were required for these locations/activities because they were not necessary and we confirm that the field studies did not involve endangered or protected species.in vitro the moss to the same metal concentrations water solution and in the same condition measured in the experimental 3 sites. In addition, to assess the effects of the single metals, the moss was exposed in vitro to single toxic metal concentrations as measured at the most polluted environment (site C). Treatments consisted in the addition to the growth medium of the metals as soluble salts: CdCl2, Cr(Cl)3, CuSO4, FeCl2, NiCl2, Pb(CH3COO)2, ZnCl2. In this way the studied metals were readily available to the moss.In parallel with in-situ toxic metal exposition, a twin experiment was performed to further evaluate the effects on ultrastructure and biochemistry of metals using manipulative laboratory infrastructure. For this purpose we exposed In vitro cultures were done as previously reported [reported ; after creported  and in tExperiments on gametophyte cultures were run in triplicate and repeated three times.3, 0.2 mL of 60% HF and 1 mL of 30% H2O2 (ultra-pure reagent grade). Digestion of samples was carried out in a microwave digestion system (Milestone Ethos 900) for a total time of 30 min. Concentrations of selected toxic metals , expressed on a dry weight basis, were determined by ICP-MS (Perkin-Elmer Sciex 6100) on three subsamples for each site. Analytical quality was checked by analysing the Certified Reference Material BCR 61 “aquatic moss”. Precision of analysis was estimated by the coefficient of variation of 3 replicates and was found to be <10% for all elements.After exposure along the River Sarno, moss samples were air-dried at 40°C to constant weight and then frozen in liquid nitrogen, pulverized and homogenized with a ceramic mortar and pestle. About 300 mg of moss powder was mineralized with a mixture of 6 mL of 70% HNO4 for 1.5 h at room temperature, dehydrated with ethanol and propylene oxide and embedded in Spurr’s epoxy medium [Leaflets on the moss stem ca. 5 mm below the apex were used for TEM observations. Samples were fixed in 3% glutaraldehyde in phosphate buffer (pH 7.2–7.4) for 2 h at room temperature and post-fixed with buffered 1% OsOy medium . Ultra-ty medium .L. riparium samples of 1–5 g fw were frozen and powdered in liquid nitrogen using a mortar and pestle. HSP70s were extracted in 50 mM phosphate buffer at pH 7.5 with 10% glycerol; the homogenate was then filtered through four layers of muslin and centrifuged for 20 min at 20,000g at 4°C. The supernatant fraction was designated as the crude extract and used for SDS-PAGE analysis and Western blots. SDS-PAGE was performed using 10% acrylamide resolving gel with a 4% stacking gel. Before loading, samples were boiled for 10 min, in the presence of BBF to ensure protein denaturation. Proteins were subjected to electrophoresis under a constant voltage of 180 V, 40 mA for 90 min. For Western blot analysis, the separated polypeptides were transferred from gels to a nitrocellulose membrane  soon after the SDS-PAGE run, then incubated for 2 h at room temperature with antibodies raised against bovine heart HSP70 (Sigma). Western blotting on the same extracts using anti-tubulin antisera (Sigma) were made to check the equal loading of the sample lanes, as previously described [escribed . The resescribed . As contA fluorescent technique using 2’,7’-dichlorofluorescin diacetate (DCFH-DA) has been used for quantitative measurement of ROS production. DCFH-DA is de-esterified intracellularly and turns to nonfluorescent 2’,7’-dichlorofluorescin (DCFH). DCFH is then oxidized by ROS to highly fluorescent 2′,7′-dichlorofluorescein (DCF) . BrieflyGlutathione S-transferase  activity was measured using a commercial kit . The conjugation of GSH to 1-chloro-2,4-dinitrobenzene (CDNB) catalyzed by GST was monitored at 340 nm for 4 min (SyneryTM HTX Multi-Mode). The reaction mixture contained 4 μL of extract and 196 μL of reaction solution (200 mM GSH and 100 mM CDNB in Dulbecco’s buffer at pH 7). The activity was calculated with ε = 9.6 mM−1 cm−1 . A GST uOne-way ANOVA was used to analyse the differences in metals concentration between sites (in-field experiment), and differences in ROS content and GST activity between treatments in-vitro experiment. The one-way ANOVA was followed by Student-Neuman-Keuls test for post hoc comparisons. Prior to analysis, data not matching a normal distribution  were log-transformed to correct for skewed distributions. Results were reported as mean ± standard deviation. For the in-field experiment, the relationships between toxic metals content, ROS and GST were assessed using Pearson correlation analysis. Data from all sites  were analysed together.Metal concentrations measured in moss bags exposed at site A  are signMoss samples exposed at site A showed an ultrastructure comparable with control, unexposed samples, collected at the Botanical Gardens (data not shown) . h site A . Treatmeh site A .L. riparium exposed in bags along the Sarno River for 7 d showed strong differences in the amount of proteins reacting vs HSP70 antisera , and aquatic mosses are known to be suitable bioaccumulators of trace elements . Mosses in vitro and exposed to the toxic metal mixture, confirmed that low toxic metal concentrations as measured at the site A did not induce ultrastructure damage [in vitro data we can conclude that the toxic metal concentrations measured at sites B and C cause severe and increasing alterations; this is consistent with our previous studies [in vitro test of fully soluble salts could also explain the more visible plasmolysis in the in vitro-treated samples compared with the in field-exposed ones. Intriguingly, samples treated with the metal mixture similar to the water of site B are all plasmolysed, but samples exposed at site B were not: this could be possibly caused by a different forms and bioavailability of pollutants. This hypothesis has been supported by Sassmann et al. [The Sarno River, one of the most polluted in Europe, is known for having been subjected to illegal disposal of waste from the leather industry . Toxic me damage . On the  studies ,7,19,22. studies ,31. The n et al. , who fouin vitro with single toxic metals induced harmful effects on the moss, resulting in plasmolysis, swollen chloroplasts, and thick membrane appearance; moreover, empty cells appeared along the leaflet section. Toxic metals induced harmful effects, possibly in relation to their toxicity, concentration and/or availability.Culturing L. riparium cells showed that the most damaged organelles were the chloroplasts. In our samples these organelles became heavily swollen and developed large plastoglobules in the stroma. Chloroplasts are a common target of metal toxicity in different plant taxa, as comparable damage was already reported for toxic metal-treated L. riparium [Funaria hygrometrica (Hedw.) [Scorpiurum circinatum (Brid) [Pellia neesiana (Gottsche) Limpr. [Lunularia cruciata L. Dum [L. minor and Elodea canadensis Michx. [The ultrastructural analyses of riparium , and oth (Hedw.)  and Scorm (Brid)  the live) Limpr.  and Lunua L. Dum , as wells Michx. , 37.Toxic metal exposure is known to induce oxidative stress  and lipiDamaged membranes could be the source for the lipid droplets and plastoglobules –40, whicPlant vacuoles, which are also increased in some of our treated samples, are also known to be involved in autophagy phenomena and are reported to be a major site for the degradation of macromolecules , 45.L. riparium samples exposed along the Sarno river path showed strong GST activity, increasing from site A to site C. This can enable the cells to better scavenge the pollution-induced ROS increase. Therefore, it can be suggested that plant adaptive response(s) to pollution-induced oxidative stress may involve antioxidants like GST. The correlation between GST activity and ROS levels implies that the induction of activity of this enzyme by pollution is attributable to enhanced ROS. Therefore, activation of glutathione-S-transferases could enhance ROS quenching.Curbita maxima) subjected to metal stress [Heavy metals stimulated the GST activity. Notably this effect seems not being species-specific, since it has been found in several studies, also on pumpkin .L. riparium increased the activity of HSP70s in parallel with a higher accumulation of toxic metals. This result is consistent with the increased HSP70s and antioxidant activity found in experiments with the same moss exposed in vitro to toxic metals [Increased levels of HSP70s are usually resulting from the toxic action of pollutants taken up by living organisms, which have not been scavenged by detoxification systems, and negatively influence the correct folding of native proteins . The mosc metals . It is aLemna minor L. [L minor, L. riparium, although presenting a simpler anatomical organization, typical of mosses, presents a surprising more preserved ultrastructure. In particular, the thylakoid system still appears recognizable even in the samples exposed in the most polluted site and /or at the highest metal concentrations. In addition, the measured bioaccumulation confirms that the moss is not able to avoid the presence of metals, but rather it accumulates them. The relative "resistance" of the moss is explained by the biochemical responses measured. In particular both HSPs and antioxidant response could protect the moss from proteotoxic and oxidative stresses respectively. The observed strong responses of L riparium could explain its hard resistance to pollutants and suggest it as an excellent bioindicator of pollution in aquatic systems.A previous experiment was carried out in Sarno River using the aquatic macrophyta minor L.  and a coin situ and carefully measuring pollution in river waters. Always for the first time, these alterations have been validated by comparing the response in field with the parameters measured in the laboratory under controlled conditions. Overall, our results suggest that L. riparium is able to respond to pollution by modifying biological parameters, such as HSP70s, ultrastructural organization, enzyme and antioxidant activity, suggesting that this species may be used as an effective bioindicator and bioaccumulator of water toxic metal pollution.In conclusion, for the first time to our knowledge, a comprehensive study has described the effects of pollution in moss in a river, by comparing both ultrastructural damage with biochemical and physiological parameters observed S1 Table(TIFF)Click here for additional data file.


WDR5 is a highly conserved WD40 repeat-containing protein that is essential for proper regulation of multiple cellular processes. WDR5 is best characterized as a core scaffolding component of histone methyltransferase complexes, but emerging evidence demonstrates that it does much more, ranging from expanded functions in the nucleus through to controlling the integrity of cell division. The purpose of this review is to describe the current molecular understandings of WDR5, discuss how it participates in diverse cellular processes, and highlight drug discovery efforts around WDR5 that may form the basis of new anti-cancer therapies. Increased understanding of the complexity of eukaryotic life has led to growing awareness of the phenomenon of ‘moonlighting’, in which a protein characterized in one context is found to play roles in other, often quite diverse, cellular processes . That prBIG-3, for BMP-2 Induced Gene 3 kb, and accurately predicted to encode a WD40 repeat-containing protein that folds into a seven-bladed β-propeller structure , the homolog of the mammalian SET1 and MLL (mixed lineage leukemia) protein complexes that catalyze histone H3 lysine 4 (H3K4) di- and tri-methylation [HOX genes, and that whole organism depletion of WDR5 in Xenopus embryos causes not only a decrease in H3K4 methylation, but also severe developmental defects [Studies of skeletal development in mice showed that Wdr5 promotes cellular differentiation and proper bone formation ,4. Overeelopment . At abouhylation ,8. It wahylation . They sh defects . Further defects , and tha defects . Recogni defects ,12.Trichoplax have a WDR5 homologue that is ~90% identical to human WDR5 within the WD40 repeat region  are marks of transcriptionally active chromatin, laid down by the SET1/MLL family of histone methyltransferases (HMT), also called trithorax group (TrxG), MLL-like, or COMPASS complexes . The cenThere are six non-redundant mammalian SET1/MLL HMT complexes, each with a distinct regulatory role ,19,20,21Although the actions of WDR5 in scaffolding the assembly of SET1/MLL HMT complexes are perhaps its best understood function, there is ample evidence that WDR5 functions in a multitude of processes, both in and out of the nucleus. Indeed, WDR5 associates (directly or indirectly) with nearly 200 different proteins in HeLa cell nuclear extracts and is roughly 10 times more abundant than other WRAD proteins , suggestStudies of chromatin gradually revealed a complicated interplay between the genetic code, regulatory proteins, and post translational modifications on histones, observations that were unified by the “Histone Code” hypothesis. Put forward by Strahl and Allis, the histone code hypothesis proposes that by being “written,” “read”, and “erased” by various regulatory proteins, histone post-translational modifications enable regulation of the transcriptional state of a piece of chromatin ,36. HistOne of the earliest studies in this regard, by the Allis group, led to the notion that WDR5 itself is an H3K4-methylation reader ,37. By ‘Although the methylation status of H3K4 may not directly influence its binding to WDR5, the methylation status of R2 in H3 does A. ArginiThree important points are raised by this discussion. First, the ability of WDR5 to respond—positively and negatively—to modifications of the H3 tail strongly implies that one of its main functions in the cell is as an epigenetic reader. Second, the recurring theme of recognition of both the H3 tail and the SET1/MLL proteins via the Win site seems unlikely to be coincidental, implying an important (and yet to be discovered) functional consequence. Perhaps shared usage of the Win site allows stepwise changes in the functional state of chromatin, marking a poised gene via H3R2me2-dependent recruitment of WDR5, which can then transition at chromatin into a functional SET1/MLL complex. Or perhaps interactions at the Win site are highly dynamic, allowing WDR5 to constantly monitor the modification status of a particular segment of chromatin and respond to the marks it reads by changes in its recruitment or binding partners. Finally, the recurring re-use of the Win site by SET1/MLL proteins, the H3 tail, other WDR5 complex members (below)—and possibly by proteins yet to be discovered—issues a strong caution to interpreting the results of experimental perturbation of WDR5  in terms of one selective function or another. Clearly, as studies of WDR5 continue, more emphasis needs to be placed on development of approaches and reagents that can tease apart the significance of the manifold interactions mediated by the Win site.Drosophila, this KANSL1-WDR5 interaction appears to be required for efficient recruitment of the NSL complex to chromatin, as mutations that disrupt this interaction reduce NSL chromatin binding [MOF  is a histone H4 acetyltransferase that assembles into two distinct functional complexes, the MSL  complex and the non-specific lethal (NSL) complex . WDR5 as binding . Because binding ,52. But Another intriguing appearance by WDR5 is within the nucleosome remodeling and deacetylase (NuRD) complex, a chromatin-associated protein complex that performs the dual-roles of chromatin remodeling and histone deacetylation. The NuRD complex originally surfaced within the context of transcriptional repression, but it is now clear that it functions in a multitude of chromatin-centric events, including gene activation and DNA repair . SeveralGiven that WDR5 assembles into so many different multi-protein complexes, it is somewhat surprising to learn that WDR5 can be directly contacted by sequence-specific transcription factors. Yet several such direct interactions have been described—with Oct4 , Twist1 HOXA genes [Besides the well-described moonlighting activities mentioned above, there is a growing body of evidence, less mechanistically developed, suggesting that these functions may just be the tip of the WDR5 iceberg. For example, WDR5 associates with chromatin remodelers through its interactions with CHD8 ,62 and IXA genes ,73. WDR5XA genes , GClnc1 XA genes , and HOXXA genes  lncRNAs,Early on in the study of chromatin-modifying complexes, a curious parallel was noticed between the histone monomers of chromatin and tubulin monomers of microtubules: both assemble into polymers that are post-translationally modified in specific ways. In the case of tubulin, this phenomenon is particularly important during cytokinesis, where post-translational modifications of microtubules play a key role in orchestrating events at the mitotic spindle and the midbody . Strahl WDR5 localizes to the mitotic spindle and to the midbody in dividing human cells ,81. ThisOn the surface, WDR5 is nothing special. Its architecture—WD40 repeats forming seven β-propellers—is shared by many scaffolding proteins of multisubunit complexes . Indeed,It is possible that the moonlighting of WDR5 in multiple complexes is a mechanism that cells use to coordinate broad changes in the activity of different epigenetic regulatory complexes. This coordination, for example, could allow multiple WDR5-containing complexes to be regulated simultaneously, such as in ESC differentiation where down-regulation of WDR5  would beAs to how WDR5 manages its diverse extracurricular activities, a clue may come from the repeated reuse of the WBM and Win sites. It is intriguing to note that for all direct WDR5 interaction partners that have been studied in detail the interactions occur through either the Win or the WBM sites. Despite extraordinary conservation of residues in WDR5 —conservaIn the last decade, it has become apparent that epigenetic regulatory proteins can be targeted by small molecule inhibitors for therapeutic benefit. There are currently dozens of small molecule epigenetic inhibitors in various stages of clinical trial in the United States , targetidK ~ 1 nM) and inhibits the HMT function of MLL1 complexes in vitro, consistent with the requirement of the MLL1–WDR5 interaction for robust methyltransferase activity [MLL1 gene (MLLr), a common occurrence in acute myelogenous leukemia (AML). This concept was, in turn, based on the idea that MLLr cancers almost always retain one wild-type copy of MLL1, and are uniquely dependent on the HMT activity of wild-type MLL1-complexes for survival [HOXA genes, driving cellular differentiation and apoptosis. Subsequently, however, it became clear that MLLr cancers are not dependent on MLL1, but instead rely on MLL2 [For the Win site, however, two types of inhibitors have been discovered. One Win site inhibitor, MM-401 , is a maactivity . Dou andsurvival . And indsurvival , where i on MLL2 —notable  on MLL2 . It seemdK ~ 100 nM), inhibits MLL1-HMT activity in vitro and shows inhibition of cancer cell lines in culture. Interestingly, the impact of OICR-9429 on MLLr cancer cells has not been reported. But it has been shown to inhibit AML cells that carry the p30 isoform of the transcription factor C/EBPα [TP53 mutations in human cancer—are uniquely sensitive to OICR-9429. Here, there is no direct contact between oncogenic p53 variants and WDR5, but rather sensitivity to compound appears to result from selective induction of MLL1 expression by the oncogenic p53 variants, the impact of which is mitigated by Win site blockade and inhibition of MLL1-HMT activity. Given the magnitude of involvement of p53 gain of function mutants in cancer, these findings open the door to a potentially huge therapeutic impact of a drug-like Win site inhibitor.The Structural Genomics Consortium has generated a set of more traditional small molecule inhibitors of the Win site ,95,96,97r C/EBPα , who shoThe examples of MM-401 and OICR-9429 raise two important issues. First, it is not always clear why or how a Win site inhibitor will block cancer cells. The premise on which MM-401 was established has been challenged, and yet it clearly results in selective inhibition of MLLr cells in culture. We do not know how WDR5 makes contact with p30 C/EBPα, yet targeting the Win site of WDR5 can inhibit cells bearing this oncogenic C/EBPα variant. And there is no direct contact between p53 gain-of-function mutants and WDR5, yet OICR-9429 appears highly effective in that setting. These examples suggest that, despite all that is known about WDR5, the application of Win site inhibitors may best be determined empirically. Perhaps this is not surprising, given the extreme multi-tasking that WDR5 can perform. Potent Win site inhibitors would likely displace all proteins that engage this site on WDR5, and it may very well be that the particular Win binding protein that predominates in any given context, or other perturbations in a cell that render it sensitive to loss of a particular Win interactor, determine whether a cell lives or dies in response to Win site blockade. Second, it is possible to achieve selective inhibition of some cell types, but not others, with Win site inhibitors. Given that WDR5 itself is essential in most cancer cells , and thaWDR5 is a fascinating example of a protein that sits squarely at the crossroads of development, epigenetic control, transcription, and cell division. Its multitude of interactions, with both proteins and RNAs, endows it with a broad range of molecular functions, and it appears to manage the volume of traffic at its surface by creative reuse of two main binding sites. WDR5 has emerged as a prime target for anti-cancer drug discovery efforts, which have already been shown to selectively inhibit cancer cells carrying specific genetic lesions. But what makes WDR5 such an intriguing and important protein also makes it especially challenging to study, and it is very likely that it will take many more years to fully appreciate which activities of this moonlighting master are most relevant to normal and diseased cellular states.


E. coli (ETEC) is one of the most frequently encountered in impoverished regions where it is estimated to kill between 300,000 and 700,000 children and infants annually. Critical ETEC virulence factors include pili which mediate the attachment of the pathogen to receptors in the intestinal lumen. In this study we show that the ETEC virulence regulator Rns positively regulates the expression of CS14 pili. Three Rns binding sites were identified upstream of the CS14 pilus promoter centered at −34.5, −80.5, and −155.5 relative to the Rns-dependent transcription start site. Mutagenesis of the promoter proximal site significantly decreased expression from the CS14 promoter. In contrast, the contribution of Rns bound at the promoter distal site was negligible and largely masked by occupancy of the promoter proximal site. Unexpectedly, Rns bound at the site centered at −80.5 had a slight but statistically significant inhibitory effect upon the pilin promoter. Nevertheless, this weak inhibitory effect was not sufficient to overcome the substantial promoter activation from Rns bound to the promoter proximal site. Thus, CS14 pili belong to a group of pili that depend upon Rns for their expression.Although many viral and bacterial pathogens cause diarrhea, enterotoxigenic  Escherichia coli (ETEC) remains a leading cause of diarrheal disease [First identified nearly 50 years ago as the causative agent of non-vibrio cholera, enterotoxigenic  disease ,2,3,4,5. disease ,7,8. Tra disease . High in disease . Sporadi disease ,12. Alth disease ,14. Two types of enterotoxins have been extensively characterized, either of which is capable of causing diarrhea . One is cooBp [ETEC virulence is also dependent upon afimbrial adhesins and pili that mediate attachment to host cell receptors in the lumen of the intestine ,21,22,23cooBp . cooBp, one centered at −40.5 and the other −110.5. Mutagenesis of either site decreased Rns-dependent expression from cooBp; however, mutagenesis of the promoter proximal site produced a larger defect than mutagenesis of the promoter distal site [DNase I footprinting revealed two Rns binding sites upstream of the Rns-dependent transcription start site of tal site . A similtal site . As withtal site ,34,35.Although Rns binding sites were also identified upstream of the CFA/I pilus promoter, to date CS1, CS17, CS19, and PCF071 remain the most extensively characterized Rns-activated pilus promoters ,28,34. Ipir-dependent origin of replication that has previously been described [attPHK022 for IntHK022-mediated integration at attBHK022 in pir deficient hosts and aadA for selection with spectinomycin and streptomycin [csuB1o, csuB2o, and csuB3o were mutagenized by inverse PCR of intermediate cloning plasmids with primers 566/567, 564/565, and 769/770 to generate restriction sites for SpeI, KpnI, and AgeI, respectively. The mutagenized promoter fragments were then cloned into pHKLac1 as per above. All promoter fragments were sequenced to verify the absence of PCR unintended mutations. Each reporter plasmid was then integrated into the chromosome of MC4100 and single integrants were verified by colony PCR as previously described [WS3294A, provided by S. Savarino , is a clinical isolate of ETEC that infects humans . Bacteriescribed . It carrptomycin . Rns binescribed . Plasmidescribed ,38.rns+). Reporter strains were transformed with pGPMRns<Tn>2 (rns::kan) a plasmid that carries the rns gene disrupted by a kanamycin (kan) resistance cassette to determine the level of Rns-independent transcription. Transformed reporter strains were cultured aerobically at 37 °C in Lysogeny broth (LB) medium with 100 µg/mL ampicillin and harvested in late log phase. Cells were lysed and assayed for β-galactosidase activity as previously described [To determine the level of Rns-dependent transcription from the CS14 promoter, Lac reporter strains were transformed with pGPMRns  at 4 °C. Bacteria were lysed in a French press  MBP-Rns was collected from the soluble fraction by amylose affinity column chromatography and eluted with elution buffer . The eluent was dialyzed against 10 mM TrisCl, pH 7.6, 50 mM KCl, 1 mM β-mercaptoethanol, 30% vol/vol glycerol and stored at −80 °C. Protein concentration was determined by the Bradford method relative to a standard curve of bovine serum albumin.MBP-Rns was purified as previously described ,28. In b32P by PCR with one primer labeled at its 5’ terminus and one unlabeled primer. Labeled PCR products were purified by recovery from non-denaturing acrylamide gels and equilibrated with MBP-Rns in 10 mM TrisCl, pH 7.6, 50 mM KCl, 1 mM DTT, 0.4 mM MgCl2, 0.2 mM CaCl2, 2 ng/µL polydI-dC, 10 µg/mL bovine serum albumin [4OAc, 50 µg/mL tRNA, 80% vol/vol ethanol. Precipitated DNA was washed with 70% vol/vol cold ethanol, dried, then suspended in 4 µL 80% vol/vol formamide, 50 mM Tris-Borate, pH 8.3, 1 mM EDTA, 0.1% wt/vol xylene cyanol and bromophenol blue. Denatured DNA fragments were separated on sequencing gels and imaged by exposure to phosphorimager plates . Sequence ladders were generated by the Maxam-Gilbert method [CS14 promoter fragments were labeled with  albumin . The solt method . 32P end-labeled oligonucleotide 346 was combined with 63–65 μg of total RNA and 0.8 mM deoxynucleotides. The oligonucleotide was extended with SuperScript™ III Reverse Transcriptase according to the supplier’s protocol . Primer extension products were separated on DNA sequencing gels alongside Maxam-Gilbert sequencing ladders and imaged by exposure to phosphorimager plates.Rns-dependent and -independent transcription start sites were mapped as previously described . In briecsuB, from ETEC strain WS3294A into a promoterless Lac reporter to construct pCS14Lac2. The reporter plasmid was then integrated into the attBHK022 site of Lac− K-12 strain MC4100 to construct reporter strain GPM1132. This strain produced only 7 ± 0.5 (n = 3) Miller units of β-galactosidase when transformed with pGPMRns<Tn>2 (rns::kan). However, 2050 ± 207 (n = 3) Miller units were produced when the GPM1132 was transformed with the Rns expression plasmid pGPMRns. The 292-fold increase in β-galactosidase expression from the CS14 promoter csuBp was statistically significant . Thus, these results clearly demonstrate that Rns activates the CS14 promoter and places CS14 pili into the Rns-dependent subgroup of class 5 pili. In terms of phylogeny, CS14 pili belong to the class 5 group—more recently termed the α-clade—of adhesive pili ,36. The csuBp, we next used primer extension to map the promoter’s Rns-dependent transcription start site. Our analysis revealed one primer extension product that mapped to a deoxyguanosine 21 bp upstream of the start codon for csuB , the generation of which facilitated screening for mutagenized binding sites. The resulting constructs were cloned into Lac reporter plasmids which were subsequently integrated into the attBHK022 site of MC4100 so that the function of each binding site could be evaluated in vivo  reduced, but did not abolish, Rns-dependent transcription from csuBp . When strain GPM1132 was transformed with an Rns expression plasmid, the expression from the wild-type promoter increased to 2050 Miller units, a ca. 300-fold increase above the basal level of expression . MutagencsuBp increased 1.3-fold when csuB2o was mutagenized  appeared to be a low-affinity binding site and mutagenesis of it had no effect on promoter activity as long as the promoter proximal site (csuB1o) was intact. Only in the absence of csuB1o did csuB3o contribute towards activation of csuBp. The distance of csuB3o centered at −155.5 from csuBp is near the upper limit for activator interactions with the carboxy-terminal domain of the α subunit of RNA polymerase [csuB3o and RNA polymerase. Despite these remaining mechanistic questions, we have definitively shown that Rns activates the CS14 pilin promoter. CS14 pili belong to class 5 or the α-clade of ETEC pili. Thus, members of this clade are related by both pilin phylogeny and similar regulatory control, as CS1, CS2, CS4, CS17, CS19, and CFA/I pili are all positively regulated by Rns [The presence of a third Rns binding site also differentiates the CS14 promoter from CS1, CS17, CS19, and PCFO71 pilus promoters which have only two Rns binding sites ,28. Howelymerase . Howeverd by Rns ,27,36,40


SCN1A mutations account for ∼80% of cases. Animal studies have implicated mutation-related dysregulated cortical inhibitory networks in its pathophysiology. We investigated such networks in people with the condition.Dravet syndrome is a rare neurodevelopmental disorder characterized by seizures and other neurologic problems. Transcranial magnetic stimulation using single and paired pulse paradigms was applied to people with Dravet syndrome and to 2 control groups to study motor cortex excitability.Short interval intracortical inhibition (SICI), which measures GABAergic inhibitory network behavior, was undetectable in Dravet syndrome, but detectable in all controls. Other paradigms, including those testing excitatory networks, showed no difference between Dravet and control groups.There were marked differences in inhibitory networks, detected using SICI paradigms, while other inhibitory and excitatory paradigms yielded normal results. These human data showing reduced GABAergic inhibition in vivo in people with Dravet syndrome support established animal models. Substantial developmental delay is a common feature; further problems, such as gait disorder, feeding or appetite difficulties, behavioral problems, and sleep disturbance, also occur. Development is normal prior to seizure onset.SCN1A, encoding the α subunit of a voltage-gated sodium channel (Nav1.1), have been found in >80% of affected individuals; the majority of these mutations arise de novo,5 and the SCN1A variants include both missense and truncating mutations in addition to whole gene deletion, suggesting a loss of function mechanism.6Mutations in SCN1A mutations cause the features of DS have been investigated using knockout mouse models (Scn1a+/− and Scn1a−/−). Homozygous Scn1a−/− mice had seizures and ataxia, leading to death by day 15, while heterozygous Scn1a+/− mice showed spontaneous seizures, but some survived past 15 weeks.7 Whole-cell sodium current recordings from mouse neurons suggest that DS-causing mutations do not affect sodium currents in excitatory pyramidal cells, a finding that may be explained by upregulation of other sodium channels, such as Nav1.3. Recordings from GABAergic inhibitory interneurons using the same mouse models show reduced sodium currents.8 This leads to an increased threshold for action potential generation in affected inhibitory cells.6 It has been suggested that DS-causing SCN1A mutations reduce the activity of cortical inhibitory networks, with the resultant imbalance of neuronal activity leading to seizures. Apart from seizures, Scn1a+/− mice exhibit behavioral problems such as hyperactivity, cognitive difficulties, and impaired social interaction. These may be attributable to reduced GABAergic inhibition in frontal cortical networks.9 A recent in vivo study of inhibitory interneurons in the same mouse model of DS, however, demonstrated normal spontaneous firing patterns, suggesting that the picture may be more complex.10The mechanisms by which 11While GABAergic inhibition has been well-studied in mouse models of DS, little has been done to understand how inhibitory networks are affected in humans with DS. An EEG-fMRI study of 10 people with DS could not identify a common network underlying epileptiform discharges.In the current study, transcranial magnetic stimulation (TMS) was used to probe cortical excitability in people with DS. TMS can be used to stimulate motor cortex noninvasively, triggering a motor evoked potential (MEP) in a target muscle. The resting motor threshold (rMT) measures the ease of activation of motor cortex via excitatory networks, while paired pulse paradigms with variable interstimulus intervals test inhibitory and excitatory intracortical circuitry.12 We also hypothesized that rMT and intracortical facilitation (ICF), which are less influenced by GABAergic inhibition, would be normal in DS.We hypothesized that GABAergic inhibition would be reduced in people with DS compared to control groups. GABAergic inhibition can be measured using inhibitory paired pulse paradigms such as short interval intracortical inhibition (SICI) and long interval intracortical inhibition (LICI).13 A safety questionnaire was completed for each participant. Participants gave written informed consent. In those lacking capacity, a relative was asked to give assent.The project was approved by the local ethical review board ; TMS parameters were within current safety guidelines.14) and the presence of an SCN1A mutation confirmed in a clinically accredited laboratory.People with DS were identified using an existing clinical registry. Inclusion criteria for people with DS were as follows: clinical criteria for diagnosing DS  muscle.15 The following interstimulus intervals were used: for SICI, 2 and 5 ms; for ICF, 10 and 15 ms; for LICI, 100, 150, 200, and 250 ms.The following paradigms were tested: rMT, SICI, ICF, and LICI. Exact definitions and experimental details of the paradigms used have been published.12 SICI is mediated by GABAAergic intracortical circuits16 while ICF is mediated by glutamatergic intracortical circuits, possibly alongside a reduction in GABAergic inhibition.12 LICI is mediated by GABABergic intracortical circuits.12rMT is a measure of motor cortex excitability, with high rMT implying low excitability.p Values of 0.05 or lower were interpreted as statistically significant. Post hoc analysis was performed using Tukey honest significant difference (HSD) test. All analysis was performed using IBM  SPSS Statistics, version 22.0.rMT for cases and the 2 control groups was compared using univariate analysis of variance (ANOVA). SICI, ICF, and LICI results for cases and controls were compared using multivariate ANOVA for each paradigm. SCN1A ; 5 completed all TMS paradigms, while the 6th subject was unwilling to continue after initial measurement of rMT. All participants with DS had a known mutation in SCN1A . Controlp = 0.61; sex: Fisher exact test p = 0.68).t test, p = 0.14). Drugs used in the 2 groups do not match exactly; 3/6 with DS were taking stiripentol, which is rarely used outside of DS, while some epilepsy controls were taking drugs such as carbamazepine and lamotrigine, which are typically avoided in DS. Mean reported seizure frequency in the DS group was 1.3 seizures per day, while epilepsy controls reported a mean of 1.0 seizure per day; there was no significant difference . Healthy controls were taking no medication, and reported no seizures.Those with DS were taking a mean of 3.2 medications each, while the epilepsy controls were taking a mean of 2.5 medications each ; there wt test p = 0.90), implying similar baseline cortical excitability in the groups. Meaningful comparison between epilepsy groups and healthy controls is difficult, since antiepileptic medication is known to affect rMT. A univariate ANOVA comparing rMT in all 3 groups showed significant differences (p = 0.03); post hoc analysis confirmed a significant difference only between epilepsy controls and healthy controls .rMT was measured in 6 people with DS. Average rMT was 74.7% of maximum machine output, SD 10.8%, range 55%–85% . This avp < 0.0001). Post hoc analysis with adjustment for multiple comparisons (Tukey HSD test) confirmed a significant difference between DS and each control group at both ISIs . There was no significant difference between non-DS epilepsy controls and healthy controls at either ISI .SICI paradigms using ISI of 2 and 5 ms did not cause inhibition in our DS group. Instead, there was significant facilitation, with mean MEP responses of 2.18 (SD 0.47) at 2 ms and 1.74 (SD 1.03) at 5 ms normalized to unconditioned responses. By contrast, all 20 controls, including those with and without epilepsy, showed inhibition at an ISI of 2 ms , and most also showed inhibition at an ISI of 5 ms . Data for SICI across DS and control groups are shown in p = 0.58, LICI p = 0.65).There was no significant difference in ICF or LICI between any group   while the excitatory effects were dominant at longer ISIs . In our DS group, the excitatory effects appeared to be dominant at shorter ISIs. This may be because the subthreshold conditioning stimulus failed to activate a sufficient number of inhibitory interneurons to trigger detectable inhibition.LICI, which is believed to be mediated by the same GABAergic interneurons, was robustly detectable in all groups, with no difference detected between DS and controls.Aside from a different ISI, an obvious difference between SICI and LICI paradigms is that the conditioning pulse in SICI is given at subthreshold intensity, while that in LICI is given at suprathreshold intensity.Scn1a+/− mouse inhibitory interneurons showed a reduction in sodium currents, leading to an increased threshold for action potential generation in these cells6–8; subthreshold conditioning stimuli in our SICI paradigm failed to generate any detectable inhibition on subsequent MEPs in people with DS. Suprathreshold stimuli were still able to generate action potentials in Scn1a+/− mouse inhibitory interneurons; the suprathreshold conditioning stimuli in LICI triggered an inhibitory effect on the MEP similar to that seen in controls.There is a striking parallel between our findings in people with DS and the data from mouse models of the condition. Scn1a+/− mouse.10 The lack of SICI, however, suggests that these inhibitory networks may show reduced sensitivity.The presence of normal LICI suggests that inhibitory cortical networks are functionally intact in people with DS, as indeed they are in the 10It is worth noting that a TMS impulse activates axons directly, while physiologic activation of the same neurons would occur synaptically. The spreading depolarization ahead of a synaptically triggered action potential is much larger than needed to discharge the membrane and therefore might be relatively insensitive to small changes in membrane threshold. Thus, the relative insensitivity of inhibitory neurons to subthreshold magnetic stimuli in DS may not imply an equally significant reduction in the physiologic firing of these neurons. A pattern of reduced sodium channel function but relatively preserved spontaneous firing patterns has been shown in animal models.SCN1A mutation. This is consistent with their clinical presentation; none of our DS group members was seizure-free.The fact that our patients with DS were all on antiepileptic drug (AED) treatment implies that the therapies used did not fully neutralize the pathologic effects of the A receptors on corticospinal neurons, while LICI is mediated via GABAB receptors.16 It is possible that expression or activity of these receptors might be affected unequally in DS, such that GABAB-mediated inhibition functions normally and GABAA-mediated inhibition does not. Another possibility is that the longer ISIs in LICI paradigms are important; for example, the SCN1A mutation might delay release of GABA from the inhibitory cortical neurons, thus preventing inhibition at the shorter ISIs of SICI. A further explanation would be that the mechanisms controlling SICI are normal in people with DS, but the inhibition is not detectable as the SCN1A mutation leads to extra facilitation not found in controls. Facilitation can be found at short ISIs in healthy people, a process termed short ICF, but this is typically triggered by a suprathreshold conditioning stimulus.17 ICF uses a subthreshold stimulus, and was similar in DS and controls in our study. None of these alternative explanations is supported by the animal data.There are other potential explanations for the lack of SICI and the presence of LICI in DS. SICI is believed to be mediated via GABArMT was lower in healthy controls compared to either the DS group or epilepsy controls, although the comparison between DS and healthy controls did not reach statistical significance. The difference is most likely due to effects of the AEDs used by our DS and epilepsy populations. There was no difference in rMT between the DS group and epilepsy controls, who were on a similar number of medications. Likewise, ICF was similar in DS and controls. These findings are in keeping with our hypothesis that DS would not affect rMT or ICF.18 We consider that disparities between drug types cannot explain the unusual pattern of SICI in our DS group.AEDs are known to modify TMS results, with different drugs affecting different measures. Our inclusion of a control group with epilepsy is helpful in this regard, as it demonstrates that SICI is robustly detectable in people with epilepsy of various types despite long-term AED treatment, which aids a meaningful comparison between cases and controls. It was not possible to match exactly the AED exposure of our patients with DS and epilepsy controls. Sodium channel-blocking AEDs, including commonly used AEDs like lamotrigine and carbamazepine, may exacerbate DS; they are typically avoided in this population. Conversely, stiripentol is in common use in DS, but is rarely used to treat other types of epilepsy. Given the impossibility of matching drugs exactly, we instead compared the number of AEDs. Moreover, although sodium channel blocking AEDs are widely recognized to increase rMT, they do not affect SICI.15 supporting the specificity of our results for DS.People with DS commonly have substantial learning difficulties and may have other behavioral problems; performing TMS can be challenging in this population. However, this seems an unlikely explanation for the robust differences seen in SICI between our DS and control groups, since other TMS paired pulse measurements (ICF and LICI) were no different vs controls. We have previously reported TMS results in people with alternating hemiplegia of childhood, a neurodevelopmental disorder associated with learning and behavioral difficulties of comparable severity to DS; this group had normal SICI,Neurology.org). All our participants are adults, in whom the electrophysiologic profile may differ from that in children.19 Only small numbers of people with DS could be recruited. The differences in SICI between the DS and control groups are statistically significant, but need cautious interpretation and replication.Our DS group is smaller than our control groups. DS is a rare condition, and many people with DS are not able to undergo TMS testing, due to learning and behavioral difficulties. People with a more severe phenotype were not approached for this study, since it was believed unlikely that they would tolerate testing; this is a potential bias (table e-1 at We used TMS to study motor cortex excitatory and inhibitory networks in people with DS. There were clear abnormalities in inhibitory networks, detected using SICI paradigms, while other inhibitory and excitatory paradigms yielded normal results. These human data show reduced GABAergic inhibition in vivo in DS, supporting established animal models. The specific absence of SICI, in the presence of normal LICI, suggests that inhibitory networks in people with DS are functionally intact, but have a reduced sensitivity to low-intensity stimuli. TMS measures may provide rapid-readout biomarkers for cortical excitability and its modulation by treatment in DS.


Streptomyces griseus. Here, we identify AdpAch, an ortholog of AdpA, as a “higher level” pleiotropic regulator of natamycin biosynthesis with bidirectional regulatory ability in Streptomyces chattanoogensis L10. DNase I footprinting revealed six AdpAch-binding sites in the scnRI–scnRII intergenic region. Further analysis using the xylE reporter gene fused to the scnRI–scnRII intergenic region of mutated binding sites demonstrated that the expression of scnRI and scnRII was under the control of AdpAch. AdpAch showed a bi-stable regulatory ability where it firstly binds to the Site C and Site D to activate the transcription of the two pathway-specific genes, scnRI and scnRII, and then binds to other sites where it acts as an inhibitor. When Site A and Site F were mutated in vivo, the production of natamycin was increased by 21% and 25%, respectively. These findings indicated an autoregulatory mechanism where AdpAch serves as a master switch with bidirectional regulation for natamycin biosynthesis.AdpA, an AraC/XylS family protein, had been proved as a key regulator for secondary metabolism and morphological differentiation in  Streptomyces is regulated by a complex regulatory network involving pathway-specific, pleiotropic, and global regulators which respond to a variety of physiological and environmental condition alterations   , Streptoomogenes , Streptormitilis , Streptocus 5008 , and Struligerus .strR . These data clearly demonstrate that AdpAch could specifically bind to the scnRI–scnRII intergenic region and could control the expression of these two pathway-specific genes.To test this hypothesis, electrophoretic mobility shift assays (EMSAs) were applied. As shown in ch protected in the scnRI–scnRII intergenic region, DNase I footprinting assays, in absence or presence of purified recombinant AdpAch, were performed. In our previous studies, we had determined the transcription start site (TSS) of the two pathway-specific genes, scnRI and scnRII  were observed. With respect to the scnRI TSS, the AdpAch-binding Site A locates at positions +8 to +54, Site B at positions -20 to +2, Site E at positions -161 to -114, and Site F at positions -283 to -259 . The six AdpAch-binding sites were spread over the scnRI–scnRII intergenic region. Notably, Site A was located downstream of the scnRI TSS, while Site B overlapped the -10 region of the scnRI promoter. Site F was located downstream of the scnRII TSS, and Site E overlapped the -35 region of the scnRII promoter. This data suggest that AdpAch might have a negative regulatory ability for the expression of these two pathway-specific genes. Additionally, the results from the DNase I footprinting assay also reveal that AdpAch may have higher affinity to Site C and Site D than to the others.To identify the exact DNA sequences that AdpAd scnRII . As seench identified in S. griseus and S. coelicolor have been reported to have the consensus binding sequence, 5-TGGCSNGWWY-3  . To further study the roles of these consensus sequences in the AdpAch-binding ability, EMSAs were carried out using the probes containing either the sequences of wild-type (wt) binding sites or the mutated sites . As shown in Figure 3B, no binding shift was detected for the mutated sites A–F when compared with their corresponding wt targets. Taken together, these data demonstrated that AdpAch indeed has six binding sites in the scnRI–scnRII intergenic region and the consensus sequence is essential for the binding activity of AdpAch.The orthologs of AdpAleotide) . After ach than the other sites. This suggests that there may be affinity differences for AdpAch between the six binding sites. To test this possibility, competitive EMSAs with 50- to 100-fold excess of unlabeled fragments of six AdpAch-binding sites were used to compete with each labeled fragment. As shown in Figure 4A, 100-fold excess of unlabeled SB′ (Site B) and SF′ (Site F) could not completely abolish AdpAch complex formation with the labeled probe SA (Site A). However, the same amount of unlabeled SC′ (Site C), SD′ (Site D), and SE′ (Site E) outcompeted the labeled probe SA. This result indicated that AdpAch binds to Site A more tightly than Site B and Site F, but less tightly than Site C, Site D, and Site E. Following this way, we could conclude that Site B has less affinity for AdpAch than others, except for Site F , which was the weakest affinity among the six binding sites , and Site D was the strongest affinity of these six sites . The affinity of Site E for AdpAch was between that of Site C and Site A . Therefore, we determined the affinity of AdpAch to different binding sites in the following order: Site D > Site C > Site E > Site A > Site B > Site F.In the DNase I footprinting analysis, Site C and Site D were occupied with a lower concentration of AdpAch in the scnRI–scnRII intergenic region were adjacent to either the scnRI or the scnRII start codon. This raised the possibility that this intergenic region might harbor a bidirectional promoter allowing AdpAch to regulate transcriptions of the divergently transcribed flanking genes, scnRI and scnRII . To investigate the promoter activities of the two pathway-specific genes with each of the AdpAch-binding sites, we used the promoter-probe plasmid pIJ8601 carrying the xylE gene, encoding catechol 2,3-dioxygenase, as the reporter. As shown in Figure 5A, the transcriptional profiles of scnRI were severely decreased when the AdpAch-binding Site C and Site D were mutated. Conversely, its transcriptional activity was increased when Site A and Site B were mutated and remained almost unchanged when Site E and Site F were mutated. For the promoter activity of scnRII, we did not detect any consistent differences when Sites A, B, and C were mutated, but mutation in the Sites D and E resulted in a large decreases of up to 70 and 40%, respectively, compared to those of the wt. The mutation in Site F resulted in a statistically significant increase . These findings indicated that expressions of scnRI and scnRII are both under the control of AdpAch, which has a completely different regulatory ability (activation or inhibition) when binding to different binding sites.The binding sites of AdpAin vitro that failed to be exhibited in vivo. In order to test this possibility and reveal the function of the six AdpAch-binding sites in natamycin biosynthesis in vivo, a series of mutants were constructed as described in Experimental procedures. As shown in Figure 6A, compared to the WT strain, the level of natamycin production in the R-mA (mutation in Site A) and R-mF (mutation in Site F) had increased by 21 and 25%, respectively. However, the constructed strains of R-mC (mutation in Site C), R-mD (mutation in Site D), and R-mE (mutation in Site E) showed up to 31, 42 and 15% reductions, respectively. The natamycin production of R-mB (mutation in Site B) mutant exhibited almost no change. This finding indicated that the AdpAch-binding Sites A and F play negative roles for natamycin biosynthesis, while the functions of the Sites C, D, and E were positive. Quantitative real-time PCR (qRT-PCR) analysis showed that the promoting effect of site mutation on natamycin production was due to alteration of the pathway-specific genes at the transcriptional level .There have been some reports where effects upon DNA-binding sites were found Streptomyces spp. have developed complicated mechanisms to adapt to altered circumstances  serve as quorum-sensing signaling molecules for activating natamycin production in S. chattanoogensis L10 . This is quite different from the function of PimR in S. natalensis where the deletion of pimR almost completely destroys the transcription of pimM . This is why we did not detect the shifted band with low concentration AdpAch (∼1 pM) in the binding reaction of our previous study . Based on the short distances between the AdpAch-binding sites which are spread over the scnRI–scnRII intergenic region, there may be complicated interactions between different AdpAch dimmers to explain this.In most cases, AdpA acts as an activator for the target genes, except for itself where it exhibits an autorepression . In this mutated . Howeverch binds to Sites A–F with the following affinities: Site D > Site C > Site E > Site A > Site B > Site F . These data are consistent with the footprinting assay where the regions of Site C and Site D were previously protected at a lower AdpAch protein concentration . This gives a hint that the regulatory ability of AdpAch may occur in a growth phase-dependent manner. In the early stage, AdpAch firstly binds to the Site C and D to recruit RNA polymerase to the promoter and initiates the transcription of scnRI and scnRII. This in turn triggers natamycin production . When AdpAch is accumulated to a certain critical level, it will bind to other binding sites located near the TSS. A DNA loop may be formed via the interaction between different AdpAch dimers, thus preventing RNA polymerase from access to the promoter of the pathway-specific genes . Reduced transcription of the pathway-specific genes will result in a low rate of natamycin production.With further analysis using competitive gel shift assays, we could conclude that AdpAch in the control of natamycin biosynthesis reveals an artful adaptive mechanism in microbial cells. Microorganisms produce molecules with antibiotic activity and expel them into the environment, presumably enhancing their ability to compete with their neighbors  for natamycin production.All plasmids and bacterial strains used in this study are listed in escribed . Escherich, histidine-tagged protein was purified from the soluble fractions of E. coli BL21 (DE3) harboring the plasmids pET32a-chadpA, as previously described (scnRI–scnRII intergenic region was amplified by PCR using primer pair RI–RII-F and RI–RII-R. The PCR product was then cloned into a pTA2-vector (TOYOBO) to generate the plasmid pT-RI–RII. The biotin-labeled probe RI–RII was made with 5′-biotin-labeled M13 universal primer pair using pT-RI–RII as a template by PCR amplification. The probes A (295 bp), B (281 bp), C (294 bp), D (282 bp), E (288 bp), F (284 bp), mA (295 bp), mB (281 bp), mC (294 bp), mD (282 bp), mE (288 bp), and mF (284 bp) were prepared following the above-mentioned method. In the EMSAs assay, 1 ng of the probe was incubated with varying quantities of AdpAch, at 25°C for 30 min in the buffer . For the competition assay, 100 times of excessive un-labeled probes and non-specific DNA were added to the reaction buffer, respectively. Reactions were displayed on 5% acrylamide gels for separation in 0.5× TBE buffer. EMSA gels were then electro-blotted onto the nylon membrane and UV-fixed by UV crosslinker. Labeled DNA was detected with streptavidin-HRP and BeyoECL plus  as described by the manufacturer.His-AdpAescribed . The Brach protein was ultra-filtered with YM-10 (Millipore) for 10 kD cut-off and eluted in 20 mM Tris buffer, pH 7.5. Then, FAM-labeled probe was amplified using 5′-(6-FAM)-labeled M13 universal primers from plasmid pT-RI-RII, followed by gel recovery. About 50 ng of fluorescently labeled probe was added to the reaction mixture to a final volume of 50 μl. After binding of the AdpAch protein to 5′-(6-FAM)-labeled probe , 0.01 U of DNase I (Promega) was added for 1 min at 30°C, followed with equal volume of 100 mM EDTA to stop the reactions and extracted by phenol/chloroform. After precipitation with 40 μg of glycogen, 0.75 M ammonium acetate (NH4Ac), and ethanol, the digested DNA mixture was loaded into ABI 3130 DNA sequencer with Liz-500 DNA marker (MCLAB). DNA sequencing ladder was prepared according to Thermo Sequenase Dye Primer Manual Cycle Sequencing Kit (USB).DNase I footprinting assay was performed as previously described . Firstlych-binding sites A–F was replaced by the sequence of EcoRV restriction sequence sites using overlapping primers  was used with UV detector set at 303 nm. Mobile phase and gradient elution process were as described previously (Natamycin production was confirmed by HPLC analysis with the Agilent 1100 HPLC system. HC-Ceviously .PY, Q-TB, and Y-LT performed the experiments. X-MM assisted with the primary data analysis. Y-QL supervised the project and revised the manuscript. All authors reviewed 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.