Type 2 diabetes mellitus (T2DM) is regarded as one of the serious risk factors for age-related cognitive impairment; however, a causal link between these two diseases has so far not been established. It was recently discovered that, apart from high D-glucose levels, T2DM patients also display abnormally high concentrations of uric D-ribose. Here, we show for the first time that the administration of D-ribose, the most active glycator among monosaccharides, produces high levels of advanced glycation end products (AGEs) and, importantly, triggers hyperphosphorylation of Tau in the brain of C57BL/6 mouse and neuroblastoma N2a cells. However, the administration of D-glucose showed no significant changes in Tau phosphorylation under the same experimental conditions. Crucially, suppression of AGE formation using an AGEs inhibitor (aminoguanidine) effectively prevents hyperphosphorylation of Tau protein. Further study shows AGEs resulted from ribosylation activate calcium-/calmodulin-dependent protein kinase type II (CaMKII), a key kinase responsible for Tau hyperphosphorylation. These data suggest that there is indeed a mechanistic link between ribosylation and Tau hyperphosphorylation. Targeting ribosylation by inhibiting AGE formation may be a promising therapeutic strategy to prevent Alzheimer’s disease-like Tau hyperphosphorylation and diabetic encephalopathies. T2DM is characterized by high blood D-glucose and insulin resistance. Many patients with diabetes develop chronic or long-term complications, including nerve, eye, and kidney damage Nathan, . Recent et al., et al., et al., et al.  being the major phosphorylation sites  is one of the most abundant Caet al., et al., et al., High level of D-ribose in T2DM would induce more glycation, thus affected the brain, which might be a cause of cognitive impairment of patients  was observed, which occurred in a D-ribose concentration-dependent manner, even though dephosphorylated form of brain Tau protein (Tau-1) actually decreased . The addition of D-glucose, however, did not initiate any significant changes in Tau phosphorylation . This confirms that the administration of D-ribose triggers mouse brain Tau hyperphosphorylation, whereas D-glucose does not.To further demonstrate that D-ribose can trigger hyperphosphorylation of Tau, we measured the changes in Tau phosphorylation at Ser214 and Ser396 by Western blotting. As shown in Fig.Subsequent immunofluorescent experiments demonstrated the increase in phosphorylated Tau in the mouse brain Fig.. HoweverTogether, these results clearly indicate that hyperphosphorylation of brain Tau protein at Ser214 and Ser396 occurs in the presence of D-ribose, but not D-glucose.in vitro model. To this end, N2a cells were incubated for 24 h with different concentrations of D-ribose , and the yield of AGEs was measured by Western blotting  in the presence of D-ribose at the concentrations of 10 mm and higher  as an ting Fig.. The levgher Fig.′. Also, bose Fig.,b′. Thesm D-ribose for different time durations, the cell viability displayed an observable, but insignificant decrease when compared to control cells . D-glucose, in contrast, did not incur any significant changes in cell viability at the concentrations used  using a CCK-8 assay. As shown in Fig.ells Fig.. Howeverm) for 24 h. As shown in Fig.m D-ribose resulted in a significant increase in Tau phosphorylation at Thr181, Ser214, and Ser396 , while the concentration of total Tau remained unchanged. Similar to mouse brain Tau phosphorylation, pSer214 was more sensitive to D-ribose exposure (as low as 5 mm). The primary neurons showed the same result  before measuring pThr181, pSer214, and pSer396 of Tau protein. Tau phosphorylation at these three phospho-epitopes markedly increased  in direct correlation with culture time , yet simultaneously in a decrease in inactive form of CaMKII (p-Thr305) phosphorylation  was observed, together with a decrease at Thr305   Fig.. No sign= 3) Fig.,i,j,l, e= 3) Fig.. Thus, tet al., m CK59 , among which pSer214 levels displayed the largest decrease . In the absence of D-ribose, CK59 also suppressed pThr181 and pSer214 phosphorylation, but not pSer396. Again, these data verify that CaMKII plays a major role in Tau hyperphosphorylation in the presence of D-ribose.To add further proof to our assumption that CaMKII plays a major role in D-ribose-induced Tau phosphorylation, we inhibited the activation of CaMKII using a specific inhibitor CK59 , the main Tau phosphatases. Tau is dephosphorylated by PP1, PP2A, PP2B, and PP5, with PP2A showing the strongest ability to do so , to about half compared to that for the control  as observed during incubation with D-ribose , compared to the sample without inhibitor  when compared to the sample without inhibitor. AG can also suppress the activation of CaMKII under ribose treatment , an effective inhibitor for the production of AGEs to answer this question. In the presence of AG, the levels of AGEs decreased significantly  , Ser214 was the most sensitive site to D-ribose exposure. Its phosphorylation status changed in a D-ribose concentration-dependent manner. In agreement with our findings, earlier work presented evidence that the phosphorylation at Ser214 is upregulated in AD-affected brains (Sadik We choose aminoguanidine (AG) as an inhibitor of ribosylation and, as expected, observed the inhibition of accumulation of ribosylated AGE. Interestingly, it also suppressed Tau phosphorylation, in particular at the site pSer214, suggesting that AG can inhibit glycation, which would then reduce AGE levels and thus indirectly reduce the signals associated with Tau phosphorylation.As mentioned above, D-ribose produces high levels of AGEs, which in turn triggers hyperphosphorylation of Tau. Therefore, Tau phosphorylation is a direct result of ribosylated AGEs and only indirectly caused by D-ribose. This viewpoint is based on our observations as described in the results section and those of others, as cited above. Tau hyperphosphorylation is observed in the presence of D-ribose following the formation of AGEs in N2a cells. Tau phosphorylation markedly decreased upon inhibition of AGE formation in the presence of the inhibitor AG.in vitro by adding AG, resulting in the inhibition of AGE production.Figurem D-ribose, albeit only observably compared to controls, while Tau was hyperphosphorylated at the same concentration. Also, the AGEs level measured by Western blot showed to increase from 10 mm. This concentration of D-ribose (10 mm) seems to be the turning point of cell state. As Tau hyperphosphorylation is the early event in AD development, our results could be utilized to build a cellular model to simulate the initiation of Tau hyperphosphorylation by D-ribose. We not only found Tau hyperphosphorylation in D-ribose-treated cells and animals, but also linked it with glycation. These results should encourage future investigations into the details of the link between glycation and Tau modification.When looking at the correlation between cell viability and Tau phosphorylation, we found that the viability of N2a cells begin to decrease at a concentration of 10 mm D-ribose induces the increase in Tau phosphorylation, but phosphorylated Tau did not increase in the presence of 20–100 mm D-ribose. One possible explanation for this observation is that the viability of N2a cells under these conditions suffered, and the cells stopped responding to D-ribose at such high concentrations. As reported, 20 mm D-ribose induced astrocytic inflammation , pSer214 , pSer396 , and Tau-1 . Total Tau was detected using Tau-5 . Monoclonal antibodies against AGE  and β-actin  were employed.Changes in Tau kinase levels were examined using phospho-calcium-/calmodulin-dependent protein kinases II  and p25/p35 , and the following antibodies were purchased from Cell Signaling: GSK-3β (1:2000) and phospho-GSK-3β , p44/42 MAPK  and phospho-ERK/1/2 , p38 MAPK (1:1000) and phospho-p38 MAPK , SAPK/JNK (1:1000) and phospho-SAPK-JNK , and total CaMKII (1:1000) and phospho-CaMKII .Changes in Tau phosphatases were examined using PP2A-C subunit , phospho-PP2A , and PP2A . The Western blotting of dilution experiments was shown in Male C57BL/6J mice (8–10 weeks) were obtained from Vital River Laboratory Animal Technology Co. Ltd. . After 1 week of acclimatization, mice were randomly divided into five groups and, for 30 days, received either daily intraperitoneal injections of D-ribose, at doses of 0.2 or 2 g/kg, D-glucose at identical doses, or 0.9% saline (controls). There were 12 mice for each group. All mice were maintained in approved animal facilities under pathogen-free conditions. After behavioral testing, mice were sacrificed and their brains were immediately dissected out, homogenized in lysis buffer (Beyotime), and then centrifuged, to yield both supernatants for Western blots, and samples for fixation in 4% paraformaldehyde to be used for immunohistochemistry experiments. All animal experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institute of Health and were approved by the Biological Research Ethics Committee, Institute of Biophysics, Chinese Academy of Sciences .et al., 2O2 in absolute methanol for 10 min to block endogenous peroxidase, and incubated in 10% normal goat serum in PBS at room temperature for 30 min. The specimens were incubated overnight at 4 °C in anti-Tau pSer214 or pSer396 antibody solution diluted in PBS. After three washes in PBS, sections were incubated with biotin-labeled secondary antibodies . The proteins were detected using horseradish peroxidase-labeled antibodies , and staining was visualized with an AEC system .Immunohistochemistry experiments were performed as previously described  at the concentrations of 0, 5, 10, 20, 50, and 100 mm for 24 h, or 10 mm for 0, 4, 8, 24, and 48 h, or cultured with or without AG or CK59 for 24 h. Cells were then collected for cell viability testing and preparation of cellular extracts for Western blots.Mouse neuroblastoma cell line Neuro 2A (N2a) cells  were cultured in Dulbecco’s modified Eagle’s medium supplemented with 100 IU mL4 cells per well and exposed to D-ribose after 24 h, except for the control. For serial dilution tests, the final concentrations of D-ribose used were 0, 5, 10, 20, 50, and 100 mm and the CCK-8 reagent was added 24 h later. For testing of different incubation times, the final concentration of D-ribose was 10 mm and the CCK-8 reagent was added at 0, 4, 8, 24, and 48 h after adding D-ribose. Plates were incubated at 37 °C for 1 h and the absorbance recorded at 450 nm.Cell viability was assessed using a cell counting kit-8 . N2a cells were seeded into 96-well plates at a concentration of 105 cells per well. D-ribose treatment was performed 24 h later. After indicated periods of time, the growth medium was removed by aspiration, and each well was washed two times with 1 mL of PBS. The cells were harvested in ice-cold WIP buffer  containing phosphatase and protease inhibitor (1 mm PMSF). The samples were then centrifuged at 20 000 g at 4 °C for 15 min, and the supernatants were collected and analyzed for protein content using a bicinchoninate acid (BCA) assay kit . Samples were stored at −80 °C until further use for immunoblotting analysis.In the preparation of cell lysate protein extract for D-ribose exposure, N2a cells were transferred into 6-well plates at a concentration of 3 × 10Protein samples of brain tissues and cell lysate were mixed with 5 × SDS-PAGE Sample Buffer  and boiled for 10 min. Expression levels of phosphorylated Tau, total Tau, and Tau kinases and phosphatases were determined using SDS-PAGE coupled with Western blotting. Cell lysate extracts containing ∼ 30 μg protein were separated on a 12% SDS-PAGE and then transferred onto PVDF membranes (Millipore). Nonspecific binding sites of the membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween-20  for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with antibodies directed against total Tau, phosphorylated Tau, specific Tau kinase or phosphatase, AGEs, or β-actin. The following day, membranes were washed three times and then incubated for 1 h at room temperature with a horseradish peroxidase-linked secondary anti-mouse or anti-rabbit antibody . The membranes were washed again three times with PBST, and the immunoreactive bands were then visualized using enhanced chemiluminescence detection reagents . The immunoreactive bands were visualized after exposure of the membranes to Kodak X-ray film and quantified by Quantity One 1D analysis software 4.5.2 .n’ means the number of mice used. For cell experiments, ‘n’ means independent experiments. Differences between experimental groups were considered significant at a probability < 0.05 on a two-tailed test; * and ** denote P < 0.05 and P < 0.01 vs. control, respectively.All values reported represent means ± standard deviation (SE), except where otherwise indicated. Data were analyzed employing Adobe Photoshop 8.0.1, Adobe Syterm, San Jose, CA, USA, Origin 8.0, OriginLab, Northampton, MA, USA, and Microsoft Excel 2010 statistical software, Microsoft, Redmond, WA, USA. When there were multiple bands present, we used all bands for statistical analysis. For animal experiment, ‘


Macrophages are necessary in multiple processes during the immune response or inflammation. This review emphasizes the critical role of the mitogen-activated protein kinases (MAPKs) and mitogen kinase phosphatase-1 (MKP-1) in the functional activities of macrophages. While the phosphorylation of MAPKs is required for macrophage activation or proliferation, MKP-1 dephosphorylates these kinases, thus playing a balancing role in the control of macrophage behavior. MKP-1 is a nuclear-localized dual-specificity phosphatase whose expression is regulated at multiple levels, including at the transcriptional and post-transcriptional level. The regulatory role of MKP-1 in the interplay between MAPK phosphorylation/dephosphorylation makes this molecule a critical regulator of macrophage biology and inflammation. Monocytes reach all the tissues in the body via the blood and then they become macrophages  is a powerful inflammatory signal present in the membrane of Gram-negative bacteria. It regulates the expression of many genes, including TNF-α, which causes septic shock when present in large amounts. ERK-1/2 becomes phosphorylated through the interaction of LPS with LPS binding protein (a serum protein), CD14, and TLR4 , p38 is activated strongly, but ERK-1/2 and JNK-1 become phosphorylated between 2 and 5 h. Although p38 and ERK-1/2 regulate the expression of genes of the innate response, such as chemokines, TNF-α, and inducible NO synthase, the genes involved in antigen presentation are regulated by JNK-1 , is expressed ubiquitously. However, it is regulated in a cell-context manner and is able to dephosphorylate tyrosine or serine/threonine residues. MKPs dephosphorylate and consequently inactivate ERK-1/2  and cause higher susceptibility to endotoxic shock (Chi et al., Interestingly, the half-lives of mRNAs of several cytokines, including IL-6, IL-10, and TNF-α, seem to be controlled by MKP-1 through translocation of RNA binding proteins from the nucleus to the cytosol (Yu et al., In conclusion, in this review we have addressed the critical issue of the duration of ERK-1/2 kinase phosphorylation and its role in mediating macrophage proliferation or functional activation. The duration of the phosphorylation state is determined by the induction of MKP-1, thus making MKP-1 one of the critical regulators of macrophage biology.JL: review literature, discuss with the other collaborators, made part of the original data and draw the graphics. LV, JT, and TV: review literature, discuss with the other collaborators and made part of the original data. AC: review literature, discuss with the other collaborators, made the original data and 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.


Mammalian Sterile 20 (Ste20)-like kinase 3 (MST3) is a ubiquitously expressed kinase capable of enhancing axon outgrowth. Whether and how MST3 kinase signaling might regulate development of dendritic filopodia and spine synapses is unknown. Through shRNA-mediated depletion of MST3 and kinase-dead MST3 expression in developing hippocampal cultures, we found that MST3 is necessary for proper filopodia, dendritic spine, and excitatory synapse development. Knockdown of MST3 in layer 2/3 pyramidal neurons via in utero electroporation also reduced spine density in vivo. Using chemical genetics, we discovered thirteen candidate MST3 substrates and identified the phosphorylation sites. Among the identified MST3 substrates, TAO kinases regulate dendritic filopodia and spine development, similar to MST3. Furthermore, using stable isotope labeling by amino acids in culture (SILAC), we show that phosphorylated TAO1/2 associates with Myosin Va and is necessary for its dendritic localization, thus revealing a mechanism for excitatory synapse development in the mammalian CNS. •MST3 loss of function increases filopodia and reduces spine synapses in neurons•13 MST3 substrates including TAO1/2 are identified using chemical genetics•Phosphorylation of TAO kinases is critical for filopodia/spine formation•TAO1/2 binds Myosin Va at T440/475 and regulates its recruitment in dendrites Ultanir et al. show that MST3 kinase is required for normal excitatory synapse development. Using chemical genetics they identify TAO1/2 kinase as a direct substrate of MST3 that upon phosphorylation associates with Myosin Va and regulates synapse formation. Dendrite arborization and synapse formation are critical for wiring the neural circuitry . DendritProtein kinases regulate a wide range of cellular processes by phosphorylating and altering the function of their target molecules. There are more than 500 kinases in the human genome. How various kinases regulate neuronal development remains poorly understood. Previous studies by us and others have revealed that the kinase cascade of the Hippo, Wts, and Trc  kinases play important and evolutionarily conserved roles in dendrite morphogenesis .Mammalian Sterile 20 (Ste20)-like kinase 3 (MST3) belongs to the highly conserved family of Ste20-like kinases that includes Hippo as the most well studied member. MST3, also known as Serine/threonine kinase 24, is in the subfamily of the germinal centre kinase III kinases that contain an ∼275 amino acids long N-terminal kinase domain and a C-terminal regulatory domain. Originally identified as a kinase with requirement of manganese as a preferred cofactor , MST3 isMST3 is expressed ubiquitously in various tissues including the brain . MST3 siDrosophila prompted us to test whether MST3 signaling has a role in mammalian dendrite development. By inhibiting MST3 activity via expression of a kinase-dead form of MST3 or small hairpin (sh)RNA mediated knockdown of MST3 in dissociated hippocampal neuronal cultures, we show that MST3 is required for the formation and maintenance of dendritic architecture. We found that MST3 limits dendritic filopodia and facilitates the formation of spines harboring mature synapses. In utero electroporation of MST3 shRNA also confirmed that MST3 is required for dendritic spine morphogenesis in vivo. In order to investigate the molecular mechanisms by which MST3 regulates dendritic architecture, we used chemical genetics and mass spectrometry and identified 13 potential substrates of MST3. Next, by expressing phospho mutant and phospho-mimetic forms of a subset of these novel substrates, we found that expression of phospho mutant TAO1 or TAO2, or shRNA mediated knockdown of TAO1/2, is sufficient to cause increased dendritic filopodia and loss of spines, reminiscent of MST3 loss of function phenotype. We further show that downstream of TAO1/2, a Myosin Va complex specifically binds to the critical phosphorylation site found on both TAO1 and TAO2. Thus, MST3 kinase functions in the formation of dendritic spines by regulating several downstream effectors. In particular, MST3′s phosphorylation of TAO1/2, an autism spectrum disorder susceptibility gene, is critical for its regulation of Myosin Va dependent trafficking in dendrites.The homology of MST3 to the Hippo kinase in Localization of endogenous MST3 in punctate structures in the neuronal soma and dendrites was evident from immunofluorescence staining of dissociated hippocampal neurons double labeled with the dendrite marker microtubule-associated protein 2 (MAP2) A, as wel∗)  with GFP plasmid alone as control, or a plasmid with GFP and MST3 shRNA expressed via separate promoters, or mutant MST3 and GFP expressing plasmids. GFP expressing control neurons at DIV7 displayed dendritic filopodia C. Inhibi∗) C, and th∗) H that ef∗) F and S4Fol shRNA D and S1E∗ 14.5 in mice E. We fouTo identify downstream effectors of MST3, we used a chemical genetic method for kinase substrate identification  used preTo undertake covalent capture for kinase substrate identification  Figure B, we purOf 13 potential MST3 substrates identified along with their phosphorylation sites , tubulin∗− I/L/V− R for phosphorylation by MST3.This study further uncovered a phosphorylation consensus sequence for MST3: threonine (as opposed to serine) is the phosphorylated amino acid followed by Isoleucine, Leucine, or Valine. In about half of the substrates that we identified, an Arginine was found at +2, resulting in a consensus site of TTo find out whether some of the putative MST3 substrates modulate filopodia and spine morphogenesis, we tested phospho mutants and phospho-mimetic mutants of TAO1, GIPC, ARHGAP18, FMNL2, EPS8, and PAK6 by expressing HA tagged mutant substrates in hippocampal neurons at DIV3, confirming their expression by HA immunostaining A and assNext, we tested knockdown of TAO1 or EPS8 by generating three shRNAs in each case. Expressing shRNAs targeting TAO1 alone (not TAO2) or EPS8 between DIV3 and DIV7–8 did not alter filopodia of hippocampal neurons  and the ∗  against the phosphorylation site T440/T475 on TAO1/TAO2, which is almost identical in TAO1 and TAO2, cotransfected MST3-HA with wild-type TAO2 or T475A mutant TAO2 (TAO2A), and found MST3-dependent phosphorylation of the wild-type, but not mutant TAO2 A. To tesNext, we transfected hippocampal neurons at DIV13 with control shRNA or MST3 shRNA and stained these neurons at DIV16 with phosphoTAO1/2 antibody, after verifying its specificity by showing reduced immunostaining upon TAO1/2 shRNA expression A. MST3 kThousand and one amino acid1/2 (TAO1/2) kinases are Ste20-like kinases implicated in microtubule and actin regulation see . TAO1 anTo test if the phosphorylated TAO1/2 site is involved in phosphorylation–dependent protein interaction, we used a peptide pull-down method to identify binding proteins in neuronal lysates labeled by stable isotope labeling by amino acids in culture (SILAC) .We incubated iodoacetyl agarose beads conjugated with peptides corresponding to the TAO2-T475 phosphorylation site in either nonphosphorylated or phosphorylated forms A with neTo verify the phospho-specific binding between the TAO1/2 T440/T475 site and Myosin Va, we incubated lysates from HEK293 cells expressing GFP alone or GFP tagged Myosin Va-globular tail domain (GTD) with lysates from cells expressing either TAO1-HA or TAO1-T440A-HA. Immunoprecipitation using an anti-GFP antibody followed with western blot of the bound proteins with HA antibody revealed that TAO1-HA, but not TAO1-T440A-HA could bind Myosin Va C. SimilaMyosin V has been implicated in spine morphology and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptor trafficking . To testTo test if TAO1/2 knockdown affects specifically Myosin Va, we transfected cortical neurons with HA tagged Myosin Va-GTD or HA tagged Myosin Vb-GTD together with vector expressing GFP alone or GFP with TAO1/2 shRNA , and immTo find out if the T440 residue of TAO1 is important for the dendritic localization of Myosin Va-GTD-GFP, we expressed TAO1wild-type (WT) or TAO1 T440A mutant together with Myosin Va-GTD-GFP in dissociated cortical neurons at DIV13 and imaged at DIV16. TAO1WT and TAO1A colocalized with Myosin Va-GTD-GFP F, in conIn this study, we examine MST3 kinase signaling by identifying multiple potential substrates, mostly cytoskeletal regulators. In particular, the TAO1/2 kinase upon phosphorylation by MST3 on site T440/T475 associates with Myosin Va in a phosphorylation dependent manner, and this phosphorylation promotes Myosin Va recruitment in dendrites. Thus by using chemical genetic techniques, we have uncovered a kinase signaling cascade that functions in neuronal dendritic spine and synapse formation.By expressing MST3 shRNA or KD MST3 in cultured hippocampal neurons, we show MST3 is required for limiting dendritic filopodia and maintaining dendritic spine and excitatory synapse structure. Whereas the MST3 shRNAs used in our study target regions common to MST3a and MST3b, expression of the shorter MST3a isoform is sufficient for rescuing the shRNA effects. MST3b has distinct functions in axonal growth, which could be due to possible specific subcellular localization or interactions. Using pan-MST3 antibody raised against a common epitope of MST3a and MST3b, we observed a mostly dendritic and perinuclear staining for MST3 , which oNotwithstanding its broad expression pattern and its functions in apoptosis and axonal growth, little is known about the phosphorylation targets of MST3 except for NDR1 kinase . By usinSeveral of the candidate MST3 substrates identified in this study implicate MST3 in regulating the cytoskeleton . Among tN-methyl-D-aspartate (NMDA) receptors in neurons  is a PDZ domain containing protein, which regulates trafficking of membrane proteins including  neurons . Tumor sIdentification of Caspase 3 as a putative MST3 substrate is intriguing, as Caspase 3 is activated by MST3 in human trophoblasts . WhereasCystine glutamate transporter/Slc7a11, a transporter associated with glutamate toxicity in neurons, a putative substrate of MST3, is active in both neurons and glia . FinallySpine morphogenesis is regulated by NDR1/2 . NotablyExpression of phospho mutant and phospho-mimetic mutant substrates could act as dominant-negative and constitutively active forms of the effectors, thereby resulting in phenotypes similar to the loss or gain of function phenotypes of MST3. Among the tested substrates, we found that TAO1 and TAO2 phospho mutants resulted in phenotypes strikingly similar to that of MST3 loss of function. We do not rule out the involvement of any of the other putative substrates, as not all phospho mutants may act as dominant negatives. Our screen, however, was useful in identifying TAO1/2 kinases as likely downstream effectors for more in depth investigation. Using shRNA expression, we demonstrated that TAO1/2, and in particular the TAO2 activity, is required for dendritic filopodia development and maintenance of spines. We further confirmed that MST3 is necessary and sufficient to phosphorylate TAO1/2.Drosophila TAO genetically interacts with the microtubule regulator Par1 and is required for the development of the mushroom body in the Drosophila brain  via phosphorylation . MARK, ila brain . TAO1 cola brain . TAO1 mala brain . It remaTAO2 is highly similar to TAO1 in its kinase domain. TAO1 and TAO2 are expressed in the brain  and are Phosphorylation is a posttranslational modification that may alter protein function by affecting its interaction with binding partners. The observation that the TAO1A and TAO2A phospho mutant expression increases filopodia density underscores the significance of MST3 phosphorylation. We hypothesize that these mutant kinases might be binding and titrating components of TAO1/2 signaling, thus acting as a dominant negative.Using a SILAC based immunopreciptation method, we identified a protein complex including Myosin Va, Myosin light chain 6, and DrebrinE that bind peptides bearing the TAO1/2 phosphorylation site in a phosphorylation dependent manner. Unlike TAO1WT, TAO1A is unable to bind Myosin Va. We further confirmed the phosphospecific binding of Myosin Va to TAO1/2 via coimmunoprecipitation. Moreover, endogenous Myosin Va can bind endogenous TAO1 and phosphorylated TAO1/2 in neurons.Myosin Va is an abundant protein in the brain and is present in synaptic fractions . An uncoWe find that Myosin Va-GTD is localized in a punctate pattern along the dendrites and colocalizes with the recycling endosome marker Rab11, and we show that this recruitment is dependent on TAO1/2 function. Membrane transport from recycling endosomes is required for maintenance of dendritic spine structure as well as for delivery of AMPA receptors during synaptic plasticity . TAO1 coThe opposite effects of TAO1/2 shRNA knockdown on the expression of Myosin Va and Myosin Vb could reflect a homeostatic response to the reduction of Myosin Va. How TAO1/2 regulates Myosin Va remains an open question. A possibility is that TAO1/2 regulates Myosin V motor function by binding and phosphorylating components of the Myosin Va complex, and enhancing its dendritic localization.A chemical genetic screen for kinase substrate identification is an unbiased method to identify substrates in relatively intact protein lysates without the need for peptide arrays or gel electrophoresis. The combination of simple chemistry to highly enrich for peptides of substrates phosphorylated by a kinase of interest, and the ability to identify the substrates as well as the phosphorylation sites by using powerful liquid chromatography/tandem mass spectrometry renders this technique uniquely advantageous for studying downstream effectors of kinases.Phosphorylation often alters protein function by changing binding properties of substrates. We have now added a further technical advance to the kinase substrate identification screen through which we can determine the phosphorylation state dependent protein interactors that bind the substrates on the phosphorylation sites. Using a quantitative, unbiased peptide pull-down screen approach, which can be achieved by SILAC labeling of proteins in neuronal cultures, we were able to identify protein interactors of TAO1/2 that critically depend on their MST3 phosphorylation site. We believe our methodology of chemical genetic kinase substrate identification, followed by determining the functionality of the phosphorylation is a useful paradigm in delineating kinase signaling.Hippocampal neurons were cultured from E19 Long-Evans rats (Charles River Lab) in accordance with local guidelines at the University of California San Francisco (UCSF) and National Institute for Medical Research as described before . Mouse Mhttp://katahdin.cshl.org/html/scripts/main.pl. Hairpins targeting these sequences were cloned in pLentiLox 3.7, which expresses EGFP via a separate promoter in addition to expressing shRNA via a U6 promoter. Lentiviruses were generated at UCSF core facilities. All clones were verified by sequencing.All shRNA sequences were 19 base pairs long and were selected via In utero electroporation surgery was performed in accordance with local guidelines as described . EmbryosFor a detailed list of antibodies and dilutions, please see HA tagged MST3 was expressed in COS7 or HEK293T cells for 48 hr before purified via HA epitope tag using Anti-HA Affinity matrix . Kinase assays were conducted in 20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol (DTT), 100 μM ATP, 1× Phosphatase inhibitor cocktail, and 0.5 mM of an ATP analog for 30 min at 30°C. Reaction was followed by alkylation for 1 hr at room temperature by addition of 2 μl 100 mM PNBM per 30 μl of kinase reaction.Kinase substrate labeling was done in P3 and P13 mouse brain lysates obtained as described . Purifie∗IRTASLVSR) was synthesized with or without a phosphorylation at the indicated threonine and conjugated to iodoacetyl agarose. Precipitated proteins were subjected to in-bead typsin digest followed by reversed-phase liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) analysis.Cortical neuronal cultures were labeled using Multiplex SILAC , and pepHEK293T cells transfected with Myosin Va-GTD-GFP, control GFP construct, HA-TAO1, and HA-TAO1A, separately. Lysate from Myosin Va-GFP or control GFP transfected cells was incubated with Protein G beads prebound with monoclonal GFP antibody, and then lysates from either HA-TAO1 or HA-TAO1A transfected cells were added and incubated with the beads containing Myosin V-GFP or GFP alone. For immunoprecipitation from neurons precleared lysates from DIV6 or DIV9 rat hippocampal neurons were incubated with Myosin Va antibody bound Protein A beads (Sigma) overnight at 4°C.S.K.U. designed and performed spine and filopodia phenotype analysis in cultures and in vivo, MST3 substrate labeling, SILAC phosphopeptide pull-down, and wrote the manuscript with help from other authors. S.Y. designed and performed the immunoprecipitations, phosphoTAO1/2 staining, and contributed to immunostainings and biochemistry experiments on shRNA knockdown in neurons and HEK293 cells. N.T.H. designed and performed the covalent capture and MS for kinase substrate identification. J.A.O.-P. codesigned and performed MS for SILAC samples. S.C. contributed to neuronal cultures and cloned several constructs. A.L.B., K.M.S., L.Y.J., and Y.N.J. oversaw the project development.


Cell cycle ends with cytokinesis that is the physical separation of a cell into two daughter cells. For faithful cytokinesis, cells integrate multiple processes, such as actomyosin ring formation, contraction and plasma membrane closure, into coherent responses. Linear actin assembly by formins is essential for formation and maintenance of actomyosin ring. Although budding yeast’s two formins, Bni1 and Bnr1, are known to switch their subcellular localization at the division site prior to cytokinesis, the underlying mechanisms were not completely understood. Here, we provide evidence showing that Bnr1 is dephosphorylated concomitant with its release from the division site. Impaired PP1/Glc7 activity delayed Bnr1 release and dephosphorylation, Bni1 recruitment and actomyosin ring formation at the division site. These results suggest the involvement of Glc7 in this regulation. Further, we identified Ref2 as the PP1 regulatory subunit responsible for this regulation. Taken together, Glc7 and Ref2 may have a role in actomyosin ring formation by modulating the localization of formins during cytokinesis. Cytokinesis is the final stage of cell cycle, which equally distributes cellular content into two daughter cells. To achieve faithful cytokinesis, cells are equipped with elaborate cell division mechanisms. Actomyosin-dependent formation of contractile ring is critical for successful cytokinesis in diverse eukaryotes. Cdc5/Polo kinase triggers activation of Rho1/RhoA at the division site in anaphase, leading to recruitment and activation of linear actin nucleator formin The formin family proteins promote linear actin nucleation and elongation through barbed-end binding . DiaphanS. cerevisiae is a genetically tractable model organism in which regulatory mechanisms of actin nucleation by formins and their physiological significance have been elucidated. Unlike mammalian cells that contain more than fifteen formin isoforms, a budding yeast conceives only two formins, Bni1 and Bnr1 , subjected to beads beating using Fastprep  at 4°C. Cell lysates were clarified by centrifugation, incubated with c-myc antibody  for one hour at 4°C, and then incubated with protein G sepharose  for one hour at 4°C. Beads-bound Bnr1-13myc were washed three times with RIPA buffer without protease inhibitors, and subjected to the phosphatase treatment using Calf Intestinal (CIP) (NewEngland Biolabs) or rabbit Protein phosphatase 1 purified from E. coli (NewEngland Biolabs), following manufacturer’s instructions. Each phosphatase treatment was performed in the presence or absence of phosphatase inhibitors as indicated in figures (5 mM Na3VO4 and 2.5 mM ocadaic acid). Standard procedures were employed for SDS-PAGE and Western blotting. Mouse anti-c-myc antibody , rat anti-Tubαantibody (AbD serotec), goat Mpk1 antibody (SANTA CRUZ) and mouse anti-GFP antibody (Roche) were utilized to detect the western blot signals.For Bnr1-13myc western blotting, a yeast culture grown overnight Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The purpose of this study is to compare the stress and stability of plate-screw fixation and screw fixation in the treatment of Schatzker type IV medial tibial plateau fracture.A three-dimensional (3D) finite element model of the medial tibial plateau fracture (Schatzker type IV fracture) was created. An axial force of 2500 N with a distribution of 60 % to the medial compartment was applied to simulate the axial compressive load on an adult knee during single-limb stance. The equivalent von Mises stress, displacement of the model relative to the distal tibia, and displacement of the implants were used as the output measures.P < 0.001) smaller than that of the screw fixation system. The maximal value of displacement (sum) in the plate-screw fixation system was 2.46 mm, which was lower than that in the screw fixation system (3.91 mm). The peak stress value of the triangular fragment in the plate-screw fixation system model was 42.04 MPa, which was higher than that in the screw fixation model (24.18 MPa). But the mean stress of the triangular fractured fragment in the screw fixation model was significantly higher in terms of equivalent von Mises stress (EVMS), x-axis, and z-axis (P < 0.001).The mean stress value of the plate-screw fixation system was 18.78 MPa, which was significantly (This study demonstrated that the load transmission mechanism between plate-screw fixation system and screw fixation system was different and the stability provided by the plate-screw fixation system was superior to the screw fixation system. Tibial plateau fractures account for approximately 9.2 % of all tibia fractures , 2, whicTo maintain the stability of the fractured tibial plateau, various approaches have been developed. Locked plates with open reduction and internal fixation (ORIF) techniques and percutaneous lag screws with arthroscopy-assisted reduction and fixation techniques are the two most frequently used surgical approaches. The former provides stability to the fractured tibial plateau by locking head screws and the contour of the plate itself, and the lag screws maintain their fixation through compression and buttressing effect with lesser soft-tissue dissection . HoweverFinite element analysis is one of the computational methods that have received wide acceptance in the field of orthopedic research, where three-dimensional models of bone-implant construct are converted into finite elements with the application of simulated physiological loads to analyze and predict the outcome of surgery . BiomechThis study was approved by the Institutional Review Board of the Shanghai Pudong hospital. Written informed consent from the subject was obtained prior to his taking part in the study. After excluding comorbidities such as osteoarthritis, pelvic, and lower-extremity fractures, a 35-year-old healthy male with a weight of 75 kg and a height of 170 cm underwent a CT scan of his left lower limb.Initial CT data of the tibia and fibula were obtained with 1-mm cuts from the selected male subject. Three-dimensional (3D) models of the human tibia and fibula were reconstructed from the CT data in the Digital Imaging and Communications in Medicine (DICOM) format using Mimics software . Then, the previously built model was imported to Geomagic Studio Software  for further polishing and to create the fracture line. The medial plateau fracture (Schatzker type IV fracture) was simulated in the model with an oblique cut starting from 5 cm distal to the joint line of the medial tibial plateau and extending to the medial border of the tibial tubercle , as showIn this study, the properties of titanium alloy were assigned to the simulated implant models, with an elastic modulus of 110 GPa and a Poisson ratio of 0.3 , 9. It wAn axial force of 2500 N with a distribution of 60 % to the medial compartment was applied to simulate the axial compressive load on an adult knee during single-limb stance , 11. Fort test was used to compare the mean values of stress and displacement between the two models [P value less than 0.05 was regarded as statistically significant.The analysis was performed using ANSYS software. In this study, the equivalent von Mises Stress (EVMS), displacement of the model relative to the distal tibia, and displacement of the implants were used as the output measures. For statistical analysis, Student’s o models . A P valP < 0.001) smaller than that of the screw fixation system which had a value of 40.79 MPa.The stress distribution in the plate-screw system was different from that in the screw fixation system. The maximal stress value in the plate-screw system was 256.20 MPa, while that of the screw fixation system was 225.12 MPa, as shown in Fig. P < 0.001), as shown in Table It appeared that higher amounts of displacement were observed in the model with the screw fixation system, as illustrated in Fig. x-axis, and z-axis, as listed in Table The distribution of stress on the fractured surface and the small triangular fragment was compared and is illustrated in Fig. Medial tibial plateau fractures are often caused by high-energy trauma and are relatively uncommon compared with lateral tibia plateau fractures . They arThe characteristics of the stress distribution on the plate-screw and screw fixation models were analyzed through EVMS. It was observed that the stress concentration on the plate-screw system was located on the proximal part of the T-shaped plate that was in direct contact with the proximal triangular fragment and the fracture line, indicating the anti-sliding/buttress effect provided by the plate. Additionally, the mean stress on the bones (both the tibia and the fibula) was significantly smaller than that on the bones fixed with screw fixation system, as shown in Table It is interesting to note that the stress distribution on the screws of the two models shared a similar tendency. The stress concentrated on the middle section of the four proximal screws in the plate-screw system and on three screws in the screw fixation system, which just went across the fracture line and were the main part that provided the buttress and anti-sliding function. In both models, stress concentrated on the anterior distal part of the tibia Fig. . This fiExcessive displacement of implants may result in loosening and compromise the stability of fixation, as reported previously by several studies . ExcessiHowever, the screw fixation system also had its merits. The stress distributed more evenly on both implants and bones. The maximal stress on the implant and the triangular fragment was smaller than in the screw fixation system, which was attributed to the larger loading area on the percutaneous screws. In general, the screw fixation system distributed the stress more evenly and did not interrupt the loading transmission via the medial tibial plateau, which may be preferred for the fixation in young patients whose bones are biomechanically sound and do not have osteoporosis.Only a few biomechanical studies focusing on the comparison of screw-plate versus screw fixation in the treatment of tibia plateau fractures have been released. Cift et al.  comparedIn this study, with the intention to imitate the anatomical reduction of the medial tibial plateau fracture, no displacement between the fractured segments was created, which made the model the same as a simple non-displaced fracture. However, it should be noted that multiple pinning under the guidance of an image intensifier was clinically preferred to buttress plate fixation for the treatment of non-displaced tibial plateau fractures , 23. In There are a few limitations inherent in this study. First, the materials of the cortical and cancellous bone were both imitated and so may not reflect the actual conditions of bone properties. Second, cyclic loading was not simulated because the simulation of dynamic motion of the joints is time-consuming and requires substantial computer resources . Thus, tThis study demonstrated that the load transmission mechanism between plate-screw fixation system and screw fixation system was different and the stability of the plate-screw fixation system was superior to the screw fixation system. Clinically, rigid fixation with plate and screws following anatomic reduction should be preferred for Schatzker type IV fractures.


With the exception of Thera, the number of 14C determinations from other Late Bronze Age sites in Greece has been small and their contribution to chronologies minimal. The absolute dates determined for Assiros through Bayesian modelling are both consistent and unexpected, since they are systematically earlier than the conventional chronologies of southern Greece by between 70 and 100 years. They have not been skewed by reference to assumed historical dates used as priors. They support high rather than low Iron Age chronologies from Spain to Israel where the merits of each are fiercely debated but remain unresolved.Over 60 recent analyses of animal bones, plant remains, and building timbers from Assiros in northern Greece form an unique series from the 14 Even where the samples and dating techniques are more varied, as in the case of the array of absolute dates determined for the Thera eruption, these have been viewed by some archaeologists with suspicion, particularly since they are offset from the conventional chronology by around 100 years and remain the subject of lively debate 14C and historical chronologies are compatible and strengthen our conviction that the Thera 14C dates are correct 14C measurements has improved steadily and Bayesian modelling has provided a powerful tool for the analysis of the results, these results can be no better than the quality of the samples available.Until very recently the chronology of the later part of the Aegean Bronze Age was entirely based on ‘historical’ dates derived from Egypt with the aid of exported or imported objects such as Minoan or Mycenaean pottery or Egyptian scarabs. Dates based on 14C determinations have been obtained from samples of three different types: charred building timbers, charred seeds and the collagen extracted from a stratified sequence of domestic animal bones  – perhaps until c 1000 BC on the basis of the The successive buildings were of mud-brick framed with oak and suffered  regular destruction as the result of fire, earthquake or natural decay, and the immediate or almost immediate reconstruction on the debris of the preceding phases steadily increased the height of the mound and required, in consequence, the raising of the perimeter banks. Within the settlement, it was possible to distinguish between interiors which were kept more or less clean, unroofed yards where rubbish was allowed to accumulate and streets where gravel and broken pottery was regularly strewn in order to maintain a firm surface in all weathers.Excavation was directed from 1975–1989 by K.A. Wardle on behalf of the University of Birmingham and the British School at Athens. Ten separate phases of occupation . Animal bones used partially for sampling are currently held at ORAU Oxford, UK (OX1 3QY) pending eventual return to the Archaeological Museum of Thessaloniki.All archaeological materials from the excavation at Assiros are housed in the Archaeological Museum of Thessaloniki, Greece (GR 54013). Dendrochronological samples are kept for reference in the Malcolm and Carolyn Wiener Laboratory for Aegean and Near Eastern Dendrochronology at Cornell University, Ithaca (NY 14853). 14C determinations taken at a 40 year interval on a single timber with waney edge preserved gave a cutting date of 1385–1347 BC   and two  applied . Four ti14C dates but increased confidence depends on establishing Aegean oak-based sequences rather than Anatolian juniper-based ones In addition, dates obtained for the 104 year ring sequence of the samples from Phases 3 and 2, using dendrochronology cross-matching against the Anatolian master sequence, provided close agreement with the All of these dates are between 100 and 70 years earlier than anticipated in terms of the accepted chronology. The archaeological evidence, especially in the case of the Phase 3 and Phase 2 samples, makes it unlikely that these are all reused timbers. The complete dating sequence, obtained by Bayesian modelling and including the animal bone determinations undertaken in 2011, enables us to rule out this possibility.The granaries of Phase 9 yielded a rich harvest of different crop seeds – einkorn, emmer and spelt wheat, barley, vetch and millet. Given the likelihood that these had recently been harvested and probably originated from a single year's harvest, seven discrete samples of four species were analysed to clarify the cutting dates of the associated timbers. The tight series of determinations provided an earliest date for the destruction, in which they were charred, of 1378–1343 and a latest of 1370–1334 BC. We measured the span of time represented by the samples using OxCal's interval command and determined that it corresponded to a period of 0–28 years   were prepared for AMS dating at the ORAU, and 36 were successfully dated. All phases (apart from Phase 9), were represented by three or more samples. They were selected from occupation levels in every area of the site on the basis of their large size and the lack of post-discard gnawing. They had therefore been deposited while ‘fresh’ and are likely to be from animals raised for meat and under 5 years old.42 animal bones , however, were almost all consuming plants derived via a C4 rather than the normal C3 pathway  the local repertoire of fabrics and shapes is slightly different and direct comparisons between the assemblages at each of the sites are difficult. At Toumba Thessalonikis the distinctive features that make up the local Iron Age repertoire are introduced gradually from Level 3 onwards and found with Late Helladic IIIC late pottery, whilst the concentric circle decoration on wheel-made pottery, which is the hallmark of the Proto-Geometric style, does not appear until Level 2A At Assiros, as already noted, imported pottery is absent from the relatively long Phase 4 and we cannot, therefore, determine on the basis of the pottery whether this phase at Assiros should be considered contemporary with the final Bronze Age (Late Helladic IIIC late) in southern Greece or with the initial stage of the Iron Age (Early Proto-Geometric). The local EIA pottery style was already well established by the time the EPG amphora, which was shattered in the Phase 3 destruction fire, reached Assiros. The position of this phase in relation to southern Greek sequences is important in considering the impact of the absolute dates obtained on any estimation of the absolute start date of Proto-Geometric (see discussion of results below).All dated samples, from timbers, crop seeds and animal bones have been included in a Bayesian chronometric model . The timbers must therefore have been freshly cut, not reused. The assertion by Weninger and Jung In consequence, a long sequence of absolute dates has been obtained without any reference to ‘historical’ dates or any attempt to predict the length of any phase to constrain the modelling. In particular, the 36 domestic animal bone samples, formed a consistent sequence with only two outliers Phase 9, with extensive granaries and imported pottery of the Mycenaean late LH IIIA 2 period, starts between 1395–1346 BC and ends with destruction by fire 1378–1343 BC, a period of 0–19 years . Phase 7, with LH IIIC style pottery which should correspond to the period immediately after the destruction of palatial Mycenaean society in southern Greece, starts 1341–1282 BC and ends 1312–1264 BC with a span of 0–46 years . Phase 3, the second phase of the Iron Age at Assiros including a Proto-Geometric amphora  starts 114C data sets at both Assiros and Kastanas are reasonably consistent with each other, allowing for the difficulties in matching, through ceramic parallels, building levels at two sites excavated with different methods. Previous attempts to reject the self-evident offset at Kastanas from the expected historical dates rely on special pleading about the character of the Kastanas sample base rather than firm evidence Given the clear sequence of absolute dates from Assiros and their importance, our next step was to compare them with the determinations obtained from Kastanas which have been used to support the conventional chronology  and the 4 timbers used for dendrochronology and the DWM (red). Figure S9. In the Kastanas plot preferred by Weninger and Jung, the determinations are given their expected historical ages – resulting in a systematic offset from the calibration curve of around 100 years. Figure S10. In the Kastanas plot rejected by Weninger and Jung, the dates fit with reasonable conformity to the calibration curve. Figure S11. Bayesian model of Kastanas 14C data, without prior assumptions about period length or assumed ‘historical’ date. Figure S12. Kastanas: The start and end dates for stratum/Schicht 14b as derived from the Bayesian model. Figure S13. The start dates of Assiros Phase 4 and Kastanas Schicht 12 compared. Table S1. Radiocarbon AMS dates from Assiros obtained from bones with associated analytical and context data. Table S2. Oak timbers from Assiros used for dendrochronology. Table S3. Oak timbers from Assiros used for dendrochronological wiggle-matching (DWM). Table S4. Crop seeds from Assiros Phase 9 Granary Room 9, and pithos in Phase 6 Room 20. Table S5. Assiros: Results of the Bayesian modelling. Table S6. Assiros: Results of the Bayesian outlier modelling. Table S7. Kastanas: summary of date on basis of Jung's rejection of the LH IIIC Late parallels for catalogue number 91. Table S8. Kastanas: summary of date if LH IIIC Late parallels for catalogue number 91 are accepted. Table S9. Kastanas: The 14C derived date ranges for the samples from each stratum/Schicht plotted against the destruction dates for each level. Table S10. The relative chronology of key building levels at Assiros, Kastanas and Toumba Thessalonikis. Table S11. Appendix 1: Model code for the Bayesian age model of Assiros. Table S12. Appendix 2: Model code for the Bayesian age model of Kastanas.(PDF)Click here for additional data file.


To further investigate the mechanism of action of LCA, several pharmacologic agents were used. We found that the NCX inhibitor 3′,4′-Dichlorobenzamil (DCB) can significantly inhibit the relaxation of detrusor strips and a reduction of the [Ca2+]i induced by LCA, while the antagonist of muscarinic receptor and the agonist of the G protein-coupled bile acid receptor (TGR5) and the farnesoid X receptor (FXR) had no effect. In conclusion, these data suggest that the relaxation of rat detrusor induced by hydrophobic BAs is mediated by NCX. Further research is needed to carry out to demonstrate the possible pathway and provide a potential new strategy to investigation for the treatment of the low urinary tract syndromes.Hydrophobic bile acids (BAs) are thought to inhibit smooth muscle contractility in several organs. The present study was undertaken to investigate the effects of hydrophobic BAs on the detrusor contractility of rat bladder and to explore the possible mechanism. Lithocholic acid (LCA) treatment increased the micturition interval and induced a concentration-dependent relaxation of bladder detrusor strips. In addition, LCA reduced the concentration of intracellular free Ca Bile acids (BAs), main component of bile, are synthesized from cholesterol in liver microsomes, where they are conjugated with glycine or taurineBAs, in addition to their classic function in lipid digestion, act as signaling molecules with systemic endocrine functions, such as the regulation of bile acid, glucose and lipid metabolism; the immune response; and cell proliferation and differentiation22+-activated K+(BK) channel activity, resulting in a relaxation effect on resistance-size arterial smooth muscle and mesenteric artery smooth muscle102 receptor to inhibit the contraction of cultured cardiomyocytes+ channel activation, and induce the vasodilation of mouse aorta by a M3-dependent mechanismMoreover, several kinds of BAs are involved in the regulation of the motility of organs. DCA can activate TGR5 to relax the gallbladder smooth muscle and intestinal smooth muscleConsidering the important role of BAs in the smooth muscle of gallbladder and intestine, it is necessary to investigate the role of BAs on the bladder, which is a hollow organ composed of smooth muscle and to determine the distribution of BAs. In this study, we investigated the effect of hydrophobic BAs on the detrusor contractility of rat bladder.−6 M)  . Compare−6 M) . However−6 M) .−6 M) was added to the organ bath, the contraction amplitudes were significantly decreased , the contraction amplitudes showed a gradient decrease  was used to study the action of TGR5 on detrusor contraction (The expression of TGR5 mRNA (266 bp) was detected in rat bladders using reverse transcription-polymerase chain reaction RT-PCR; . The exptraction . INT-777traction  or frequtraction .+/Ca2+ exchanger, muscarinic receptor and farnesoid X receptor  for 10 min, the contraction amplitude was not significantly different when LCA was applied  and purinergic receptor antagonist  for 10 min, the contraction amplitude remained inhibited when LCA was added  was used  represents the [Ca [Ca2+]i . CompareNCX was recorded when SMCs were perfused in extracellular solution , and inhibited inward INCX by 46.07% ± 7.84% at −100 mV  could significantly inhibit bladder-voiding contraction. Bladder micturition frequency was decreased after instillation of LCA (10−6 M) into the bladder. The effects of LCA on micturition may be related to other mechanisms of the suburothelium. Strong evidence suggests that urothelial cells are involved in bladder sensory functions, which is the important part of the micturition reflexFor the first time, we demonstrated that LCA (10−9 to 10−6 M), the contraction amplitude gradually decreased, supporting a possible role of LCA on the regulation of bladder contractility. However, the detailed mechanism of action of LCA remains unclear.We investigated the effects of LCA on bladder detrusor contractility. The contraction amplitude of rat detrusor strips was significantly decreased. In addition, a concentration-response effect of LCA was also detected in the contraction experiment using detrusor strips. As the LCA concentration increased (102+ concentration ([Ca2+]i)415+ concentration2+]i was decreased when the primary isolated SMCs were treated with LCA. Calcium ions are a message transport substance, and play a key role in cell excitation and excitation-contraction coupling2+ exit mode) and the reverse mode (Ca2+ entry mode)24et al. reported that NCX plays an important role in the pathologic processes of overactive bladder syndromes (OAB)et al. reported that sodium taurocholate caused a relaxation of isolated guinea pig ileum smooth muscle strips via stimulating the NCXSome studies have shown that BAs can decrease the intracellular free CaNCX induced by LCA were recorded using the whole-cell patch clamp technique. LCA inhibited the outward INCX by 42.82% ± 17.58% at +60 mV and the inward INCX by 46.07% ± 7.84% at −100 mV. DCB is a wide-range NCX inhibitor that blocks the antiport in both the forward mode and the reverse mode. When the detrusor strips were pretreated with DCB, LCA no longer decreased the contraction amplitude of detrusor strips. In addition, when the SMCs were pretreated with DCB, the decreased concentration of [Ca2+]i induced by LCA was significantly inhibited. These results showed that the NCX is responsible for the inhibitory effects of BAs on detrusor contraction and intracellular calcium mobilization.The changes in Iet al. reported that hydrophobic BAs can act on TGR5 to activate the cAMP-PKA pathway, then opening the KATP channels to hyperpolarize the membrane and decrease gallbladder smooth muscle activityet al. reported that activation of TGR5 caused the relaxation of gastric smooth muscle. That relaxation is mediated through the inhibition of the RhoA/Rho kinase pathway via both cAMP/ePac-dependent stimulation of Rap 1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser188TGR5 is a G protein coupled receptor that has been studied in relation to BAs. It was discovered in 2002 and characterized in 2003. TGR5 is activated by multiple BAs, with LCA being the most potent natural agonist. Brigitte To explore the effects of TGR5 on the contraction of detrusor strips, INT-777 (a selective agonist of TGR5) was used. The contraction amplitude and frequency of the detrusor strips were not changed when INT-777 was added to the bath. These results suggest that the activation of TGR5 had no influence on the contraction amplitude and frequency of the detrusor strips. Our results are opposite to the results in other organs49et al. has reported that FXR is abundantly expressed in the bladder and is involved in the development of metabolic syndrome-induced OABWe used GW-4064, a selective agonist of FXR, to verify the role of BAs on the nuclear receptor. FXR is a classic nuclear receptor that is activated by BAsCholinergic and purinergic receptors are important signaling pathways regulating smooth muscle contraction, and some studies have reported that BAs might directly bind or indirectly affect muscarinic and purinergic receptor signaling1236In the present study, our results indicated that BAs is involved in the regulation of detrusor contraction, and the NCX may be the key component of downstream molecular pathway. Administration with LCA by intravesical instillation and incubation can influence the bladder micturition and contraction. Therefore intravesical instillation or injection with a purified and synthesized BAs could be developed to modulate overactive detrusor in OAB, the antagonist may be useful for increasing the bladder contractility in diabetes-induced bladder dysfunction.In conclusion, one limitation of this study is that we did not elucidate possible signal pathways of hydrophobic BAs in low urinary tract syndromes (LUTS). However, this study provides the first evidence of the relaxant effects of hydrophobic BAs on rat bladder detrusor contraction. TGR5, FXR, purinergic and muscarinic receptor have no role in the relaxant effect of hydrophobic BAs, but the activation of NCX may be responsible for the relaxant effect of hydrophobic BAs. Further studies are needed to investigate the expression and functional changes of hydrophobic BAs in LUTS.All animal handling and experimental protocols were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, and approved by the Research Council and Animal Care and Use Committee of the Third Military Medical University, China . All efforts were made to minimize animal suffering and to reduce the number of animals used. Fifty-eight adult female Sprague-Dawley rats (190–250 g) were used in our experiments. These rats which were used to measure detrusor strips contraction were killed by cervical dislocation, and the other rats were euthanized by sodium pentobarbital injection (200 mg/kg).Total RNA from rat bladder was extracted in TRIzol reagent, isolated with chloroform, precipitated with isopropyl alcohol, and dissolved in RNAse-free water. For each reaction, 2 μg of total RNA was used to synthesize cDNA using the PrimeScript RT reagent kit  and 2 × Taq MasterMix . Ileum tissue and water were used as positive and negative controls, respectively. The primers for rat TGR5 were forward 5′-GAGGGGTTCAGGAGCTTTCC-3′ and reverse 5′-CAGATTGGCAAGCAGGGAGA-3′, generating a fragment of 266 bp.SQ, Millipore, Billerica, MA, USA). The membranes were blocked with 5% bovine serum albumin (BSA), incubated with primary antibodies, rabbit anti-TGR5  and mouse anti-tubulin , and incubated with secondary antibody conjugated to horseradish peroxidase . Immunolabeled proteins were detected by the ChemiDoc XRS + Image System .The proteins were extracted for western blotting as we reported previously. Proteins (50 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes  for 1 h at 4 °C. The tissue sheets were incubated in 1% bovine serum albumin for 2 h at room temperature and then incubated with rabbit anti-TGR5 antibody (1:200) and mouse anti-α-actin antibody  for 12 h at 4 °C. The samples were washed with phosphate-buffered saline (PBS) and incubated with the secondary fluorescent antibodies conjugated with TRITC and FITC (1:500) for 1 h at room temperature. The samples were washed with PBS and incubated with 4,6-diamidino-2-phenylindole  for 10 min at room temperature for nuclear staining. Finally, the samples were imaged using a laser confocal microscope . A procedure identical to that described above was applied to the negative control group with the exception that the rabbit anti-TGR5 antibody was replaced with PBS.2, 1.2 mM MgSO4, 25 mM NaHCO3, 1.2 mM KH2PO4 and 11.0 mM glucose (pH 7.4). The bladder body was longitudinally cut into 3 × 2 × 6-mm strips and placed into a 15-ml organ bath filled with Kreb’s solution. The Kreb’s solution was bubbled with 5% CO2 and 95% O2 at 37 °C. The strip was then suspended between 2 hooks; one hook was connected to a movable stretch transducer, and the other was fixed to the bottom of the bath. After equilibrating for 30 min, the strips were stretched gradually until the stretch load was maintained at 0.75 g. The continuous curve was recorded with isometric force transducers . Gradient concentrations of LCA (10−9–10−6 M) and INT-777 , or GW-4064  were added to the bath. In addition, the M receptor blocker atropine , the purinergic antagonist, suramine hexasodium salt  and the sodium-calcium exchanger (NCX) blocker, DCB  were used to explore the possible mechanism.As previously described39−6 M) was infused at a rate of 0.2 ml/min. The continuous curves of intravesical pressure were recorded. The micturition interval and maximum bladder pressure were analyzed.Filling cystometry was performed according to previous reports413 pieces. The digestion solution contained 2.0 mg/mL type II collagenase, 2.0 mg/mL BSA, and 2.0 mg/mL trypsin inhibitor . The pieces were digested for approximately 35 min at 37 °C, and then 5 ml 10% fetal bovine serum was added to terminate the digestion. After centrifugation (1200 rpm/min for 5 min), the supernatant was discarded. Dulbecco’s modified Eagle’s media (DMEM) with 10% serum and 1% penicillin-streptomycin was added to the precipitate. The sample was beaten approximately 300 times with a dropper. The cells were filtered away from the tissue pieces with a 200-mesh (0.22 μm) cell strainer and seeded onto a glass coverslip at the bottom of a recording chamber. The SMCs were cultured in the incubator .The bladder was put into 5 ml digestion solution and cut into approximately 1-mm2+ indicator) for 30 min in an incubator. Then, the Fluo-4/AM loaded cells were washed three times and allowed to stabilize for 10 min in hank’s balanced salt solution. Real-time images of [Ca2+]i were collected at an emission wavelength of 488 nm using a Leica camera . The final data are presented as the relative fluorescent intensity (RFI): RFI = F1/F0, where F1 is the mean relative fluorescence intensity after drug administration and F0 is the mean baseline fluorescence intensity before drug administration. The experiments were carried out at room temperature.The cells were washed with PBS solution and dyed with Fluo-4/AM  using a P-97 puller . The patch electrodes had a resistance of 3.0 MΩ–5.0 MΩ when filled with the internal solution -1-piperazineethanesulfonic acid (HEPES), 8 mM D-glucose, 1 mM adenosine 5′-triphosphate disodium salt hydrate (Na2ATP), 5 mM ethylene glycol tetraacetic acid (EGTA), 4.94 mM CaCl2; pH 7.2). The extracelluar solution contained 137 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES and 10 mM D-glucose (pH 7.4). The INCX was induced by depolarization in 10 mV steps from −100 mV to +60 mV at a frequency of 1 Hz. The holding potential was set at −40 mV. Currents were recorded when SMCs were perfused in sequence with the extracellular solution, and solutions containing LCA (10−6 M) and Ni2+ (5 × 10−6 M) for 5 minutes, respectively. INCX was measured as the Ni2+-sensitive current that could be selectively inhibited by 5 × 10−6 M Ni2+. The data were recorded using EPC10 amplifiers . All of the experiments were conducted at 24 ± 2 °C.The IThe data are presented as the mean ± SD. All data were analyzed using the Statistical Package for Social Sciences, version 16.0, for Windows . Statistical comparisons were performed using Student’s t-tests. All statistical tests were two-tailed. The level of statistical significance was set at P < 0.05.How to cite this article: Zhu, J. et al. Hydrophobic bile acids relax rat detrusor contraction via inhibiting the opening of the Na+/Ca2+ exchanger. Sci. Rep.6, 21358; doi: 10.1038/srep21358 (2016).


Pygoscelis adeliae) as a bio-indicator near Dumont d’Urville, an area of the East Antarctic sector currently being considered for the establishment of a Marine Protected Area (MPA), using GPS-based tracking tags during the 2012/13 austral summer breeding season.Marine predators are ecosystem sentinels because their foraging behaviour and reproductive success reflect the variability occurring in the lower trophic levels of the ecosystem. In an era of environmental change, monitoring top predators species can provide valuable insights into the zones of ecological importance that need to be protected. In this context, we monitored the Adélie penguin (The habitat use and foraging areas of the penguins differed by breeding stage and sex and were strongly associated with patterns in bathymetry and sea-ice distribution. The first trips, undertaken during the incubation phase, were longer than those during the guard phase and were associated with the northern limit of the sea-ice extent. During the guard phase, birds strongly depended on access to a polynya, a key feature in Antarctic marine ecosystem, in the vicinity of the colony. The opening of the ice-free area was synchronous with the hatching of chicks. Moreover, a sex-specific use of foraging habitat observed only after hatching suggests sex-specific differences in the diet in response to intra-specific competition.Sea-ice features that could be affected by the climate change were important factors for the use of foraging habitat by the Adélie penguins. The extent of the foraging area observed in this study is congruent with the area of the proposed MPA. However, both penguin behavior and their environment should be monitored carefully. An average 1.5 °C increase in the air temperature is expected worldwide by the end of the 21 century . The strrn Ocean . TherefoYet monitoring a whole ecosystem is logistically difficult, if not impossible, in these remote regions of the globe. To address this issue, ecologists use sentinel species, especially meso- and top predators like seabirds and marine mammals, as they integrate and amplify effects occurring at lower levels of the food web –11. The Pygoscelis adeliae) have been well studied in Antarctica over the past four decades. Their main prey are Antarctic krill (Euphausia superba), ice krill  and Antarctic silverfish (Pleuragramma antarcticum), whose abundance is strongly dependent on primary production and sea-ice concentration, extent, and distribution [Adélie penguins , Antarctica, between November 18, 2012 and January 17, 2013. At the beginning of the breeding season, around October, males travel southwards through the pack ice to reach their colony. After the courtship, two eggs are laid. The incubation starts once the second egg is laid and lasts 32 – 34 days. Males undertake the first shift while their mate forages at sea for 11 to 14 days. After the eggs hatch, chicks are cared for by both parents for ca. 22 days during what is referred hereafter as the guard phase. Parents take turns between foraging at sea and guarding their offspring. We separated the guard phase into the early (mid-December to early January) and the late guard phases (from early January to mid-January) to account for the growing demand from the chicks and its possible influence on trip duration. When the chicks become thermally independent around mid January, they gather in crèches. At this stage, parents forage simultaneously and feed their chicks every 1 to 2 days until they fledge at the age of 50–60 days in February .http://global.mobileaction.com/download/i-gotU_download1.jsp).At the courtship, birds were marked on the breast with Nyanzol dye for identification in the colony and for the monitoring of their breeding activity throughout the season. At this occasion, observations were made and birds were sexed based on behaviour. A total of 65 birds were captured and equipped with CatLog™ GPS loggers  customized at our laboratory by the engineers of MIBE  as described in . The logOf the 65 loggers, 22 were deployed during incubation , 23 during the early guard phase , and 20 during the late guard phase  (Table 1). Although 12 loggers malfunctioned and recorded incomplete tracks, based on the time spent at sea we estimated that at least up to 70 % of the trip had been recorded in six of these twelve cases, the last 30 % corresponding to the return phase of the trip. Thus, we chose to keep these six birds in the analyses given that removing them did not modify our conclusions.www.R-project.org), using the adehabitat LT [GPS coordinates were first processed using IGOR Pro 6.12A . Duplicated coordinates, excess points before departure and after arrival at the colony were removed. Then, spatial analyses were conducted using R 3.0.1 software  is defined as the time an animal requires to cross a circle of a given radius . This meftp://ftp.ifremer.fr/ifremer/cersat/products/gridded/psi-concentration/data/antarctic/daily/netcdf/). The sea ice data do not allow fast and pack ice to be reliably distinguished, and so we did not attempt to do so here. Data collected between 63-67°S and 134-143°E were analyzed and converted to maps using the raster and fields packages in R. Depth and daily sea ice concentration values were extracted for each location on each track using bilinear interpolation from the native ice and depth grids.We used bathymetry data  and pasStatistical tests were conducted using R. The effects of gender and breeding stage on trip length (time spent at sea) and total distance travelled were tested using non-parametric Kruskal-Wallis tests, followed by Mann–Whitney rank sum tests. Statistical significance was assumed under a p-value threshold < 0.05. Normality of time spent at sea and total distance traveled was assessed with the Shapiro-Wilk test. Trips during incubation and guard stage were significantly different and, hence, were analyzed separately. Sea-ice concentration values were compared between genders and stages using Gaussian linear mixed models with random intercept by individual bird to account for repeated measurements of the same individual.2 . During incubation, the birds reached the edge of the continental shelf, the slope and eventually the open ocean of the Dumont d’Urville Sea, whereas foraging trips were confined to the shallower neritic waters of the continental shelf during the early and late guard phase. Some penguins followed the continental shelf break around 64.8°S. This trend was particularly noticeable among birds heading north-west. One penguin foraged farthermost westward along the shelf break . Trips were longer during incubation than during both early and late guard stages  but did not differ between the two guard stages . During incubation, males and females foraged for a similar duration , but during the two guard phases females spent more time at sea than males . Similarly, the mean total distance traveled by the birds also varied throughout the season . The distances were longer during incubation than during both early and late guard , but the difference was not significant between early and late guard phases . There was no difference in distances travelled during incubation  but females travelled longer distances during the early  and late guard  stages. Time spent at sea and total distance traveled were positively correlated during guard stages , and during early and late guard phases as well (p < 0.001 for both). During incubation, females and males encountered different sea-ice conditions because they foraged at different periods, females foraging in areas with extensive sea ice which had recessed when males foraged. During guard stage, males and females foraged at the same time, i.e. ice distribution in the environment was similar for females and males and, as such, greater ice concentrations along the route of the females suggest a sex-specific selection of foraging sites  colonies, [Later in the reproductive season when parents reared chicks, a coastal polynya opened in the vicinity of the colony. This polynya is a recurring feature, and results from katabatic and synoptic winds from the land, which blow the sea ice in a divergent and persistent direction, leading the sea ice away from the continent . The redolonies, ) occur iolonies, , 34–36. olonies, . If a brolonies, . This fuolonies, ). The deolonies, . The durLeptonychotes weddellii), emperor penguins and Antarctic minke whales  for the same resource [When chick-rearing Adélie penguins are foraging in the polynya we expect the intraspecific and interspecific competition to increase to some extent. In some extreme cases, this may constrained the foraging activity of the birds and lead them to search for other – potentially more distant – foraging areas. For instance, Adélie penguins from a Ross Sea colony compete with Weddell seals (resource . As a coAfter hatching, females spent longer periods at sea and traveled longer distances than males. In addition, females foraged in areas of higher sea-ice concentration than males independent of the breeding stage. This sex-specific difference in habitat use is probably linked with sex-specific differences in diet. At Béchervaise Island, females feed mainly on Antarctic krill and provide more food to their chicks than males, which feed on fish, mainly Antarctic silverfish . In our Our study shows that the foraging behaviour of Adélie penguins at Dumont d’Urville depends greatly on access to sea-ice edge, and is subsequently enhanced by the presence of a polynya in the vicinity of the colony. Both oceanic features offer concentrated and highly available sources of food. Monitoring this species over the long term would allow us to understand their capacity to adapt to environmental changes that are going to affect these two oceanic features in the future. In addition, the important intra-individual variability observed in the penguins might be driven by parameters other than environmental features and sex alone. Age, experience and physiological conditions might be related to foraging abilities and strategies, e.g. , 42, andEnvironmental change is not the only threat to Antarctic ecosystems; human impacts, such as commercial fisheries or tourism, are also likely to have an impact , 44. Our