An integrated multi-physics simulation capability for the design and analysis of current and future nuclear reactor models is being investigated, to tightly couple neutron transport and thermal-hydraulics physics under the SHARP framework. Over several years, high-fidelity, validated mono-physics solvers with proven scalability on petascale architectures have been developed independently. Based on a unified component-based architecture, these existing codes can be coupled with a mesh-data backplane and a flexible coupling-strategy-based driver suite to produce a viable tool for analysts. The goal of the SHARP framework is to perform fully resolved coupled physics analysis of a reactor on heterogeneous geometry, in order to reduce the overall numerical uncertainty while leveraging available computational resources. The coupling methodology and software interfaces of the framework are presented, along with verification studies on two representative fast sodium-cooled reactor demonstration problems to prove the usability of the SHARP framework. Many examples of nonlinear multi-physics phenomena occur in a spectrum of scientific fields, raising the need to develop and validate accurate and stable numerical modelling and solution procedures. Such robust coupling methods are often used in radiation hydrodynamics, nuclear reactor analysis, fluid–structure interaction and climate model problems because of the need to accurately resolve the fine-scale effects in physics evolution .Traditional solution techniques for coupled multi-physics phenomena have often relied on operator-split (OS) coupling strategies, which can introduce several sources of numerical errors in the solution fields as a result of inconsistent spatio-temporal treatment of the nonlinear terms. It is imperative to verify the accuracy preservation in these methods for problems of interest since effective resolution of the disparate characteristic physical scales is non-trivial. In this paper, we introduce an integrated multi-physics coupling capability with a multi-resolution hierarchy that is designed to ultimately span the full range of length and time scales present in relevant nuclear reactor design and safety analyses.In order to produce a flexible multi-physics simulation capability, two obvious approaches can be pursued. In one approach, pieces of existing single-physics codes can be assembled into an overall coupled simulation code with appropriate interfaces to communicate between the components. This is generally referred to as a ‘bottom-up’ framework approach . The The implementation of a verified multi-physics solver code also imposes a number of requirements on the overall design aspects of the framework. Hence we need flexible interfaces and robust solver options that encompass variations in a hierarchy of coupling algorithms affecting the frequency and degree of coupling between the physics. Since the choice of a coupling method is both physics and problem-specific, SHARP includes a spectrum of numerical techniques to tackle the relevant scales in physical phenomena that is relevant to nuclear reactor design. The necessary background on the methods is provided in the following sections.(a)y is the solution vector that is dependent on both space and time, respectively, and F is the nonlinear operator representing the coupled system and n is the total number of unknowns. For ease of comprehension, we can write F as in the second equality of equation , and its mth component is the nonlinear residual stemming from the mth physics component and may depend effectively on all other fields, for example, Fm(y)=Fm.For illustration, let the nonlinear vector-valued function representing a coupled partial differential equation (PDE) system be written in a general form as(b)O(Δt), even though high-order time integration might have been used for the individual physics components  with P being the total number of coupled physics components, Ni,j represent the nonlinear block corresponding to physics i coupled to solution from physics j and yi is the ith physics component solution.An example discrete coupled system of equations to be solved is of the following form:Ni,i(yi)∀i∈ require only the inversion of the single physics operator, this fixed point iteration procedure can be continued to generate a sequence of solutions that converge to the true coupled physics fields. Equation  coupling strategies. The organization of the paper is as follows. In §2, a brief description of the components of the SHARP framework is provided along with some implementation details for the interfaces. Then we show results in §3 on verification studies performed on a fully heterogeneous assembly-scale problem in order to gauge efficiency and accuracy metrics that give the necessary foundation for simulating a large-scale, realistic nuclear engineering benchmark validation problem.2.One can construct a multi-physics reactor core modelling code in many ways, and numerous efforts have attempted to do so by providing a stepping stone for future efforts . What diAs stated in §1, the ‘bottom-up’ approach lends itself naturally to leverage existing well verified libraries such as the Mesh-Oriented datABase (MOAB ), for haIn practice, the coupled system may be solved on multiple physics meshes, each of which models part or the entire physical domain of the problem but resolving relevant spatial scales pertaining to a single physics. In order to perform efficient coupled calculations, the results must be transferred from the physics/mesh on which they are generated (source) to the physics/mesh for which they provide initial or boundary conditions (target) due to nonlinearity introduced by physics coupling. ‘Two-way’ transfer is required in cases where the physics depend on each other's solution fields, for example in reactor analysis where neutronics computes total fission heat generation based on material properties that are temperature dependent, which are computed by thermal-hydraulics component using the heat generation source term computed by neutronics.L2 projection) each dependent physics component solution from the source to the target, with appropriate conservation prescriptions [riptions . The Cou(a)MOAB is a library for query and modification of structured and unstructured mesh and field data associated with the mesh . MOAB caEntity: basic entity in the discrete model, e.g. vertex, quadrilateral and tetrahedron.— Entity set: arbitrary collection of entities and other sets.— Interface: database object or instance from an implementation point of view.— Tag: set of data annotated on objects of any of the other three data types.— The data model in MOAB consists of four fundamental data types:This data model, while simple, is flexible enough to represent the necessary data to run coupled physics simulations. In particular, data tags can be used to store both fine-grained solution data on individual vertices and elements and coarse-grained annotation of sets to identify them as boundary conditions, material types or processor partitions. With such a uniform mechanism to push and pull data through the MOAB mesh layer, a clear physics abstraction can be created to transfer the solution from a source physics mesh to a target physics mesh, to be used as a source term or to evaluate dependent parameters.One of the critical aspects in assembling a multi-physics modelling code is mapping the results from one physics domain to another. We use MOAB as a ‘data backplane’ to link physics through their spatial domains; and we use MBCoupler to project coupled physics fields between those domains. MBCoupler has been demonstrated to be accurate and was recently shown to have more than 65% strong scaling up to 262 000 processors on the BG/Q (Mira at Argonne). This tool allows the source and target meshes to be partitioned and distributed across processors in a way that is performance optimized for the individual physics associated with the mesh. Target-to-source mesh point location is performed in parallel, with bounding box-based acceleration used to determine possible source mesh processors containing every point and with KD-tree  decomposition used locally on each processor. MBCoupler can transfer solutions using piecewise linear and quadratic FE shape functions along with options to use spectral element (SE) shape functions depending on physics discretizations. In this paper, MBCoupler is used to map the results computed by one physics module onto the discrete mesh used by another, in order to evaluate the nonlinear terms in the tightly coupled, successive iterative splitting scheme.In the SHARP framework, we have implemented MOAB interfaces to several different physics components that are relevant to fast reactor physics analysis.(b)CouPE aims to enable scalable and extensible coupling of different physics components that are nonlinearly dependent on each other. The SHARP multi-physics framework for solving reactor analysis problems employs validated and verified efficient mono-physics codes with MPI-level parallelism to implement several coupling strategies including OS and tightly coupled, iterated methods. The current design of CouPE is intended to satisfy the need for a loosely coupled software framework even when the physical phenomena are strongly coupled to each other.(i) Use existing libraries and physics codes in order to minimize development time and leverage man-years invested in these tested codes.(ii) Enable a flexible and accurate data exchange framework between codes in a mesh-, numerics- and physics-aware fashion; in other words, data exchange while maintaining consistency, accuracy and conservation of key fields that contribute to inaccuracy and stability issues in coupling.(iii) Provide flexible data containers and physics objects that facilitate and simplify the evaluation of the nonlinear residuals representing the fully discrete PDE for different physics components.(iv) Provide the ability to use different kinds of multi-physics coupling strategies within the same architecture with runtime object polymorphism, avoiding recompilation for each problem.The motivations in designing CouPE are the following:−4 – 10−8 [CouPE aims to integrate all the physics components under a unified framework in order to exchange the solution from one physics to another (interfaces to MBCoupler) and converge the coupled physics solution fields to user-specified tolerances  without4 – 10−8 .Similar to the PETSc library, CouPE is designed to allow the user to specify command-line arguments in order to control the dynamic behaviour of the coupled solver. The parameter specifications include the input for individual physics components, input mesh parameters, and type of solver along with ability to enable dynamically choosing the fidelity of the physics being coupled. This is made possible by abstracting out behaviour of the core object until runtime. The advantage of such a method is that the implementation of the coupled physics driver and the accompanying physics components need to be compiled, linked and verified only once and then can be re-used in a variety of different coupling methods (e.g. loose versus tight coupling). The coupled fields are iterated until convergence to user-required accuracy for any problem of interest. Results obtained by using this coupled physics driver based on the CouPE library are shown in §3.(c)ψg and ϕg(r) are the angular and scalar flux for energy group g, respectively, Σt,g is the total material cross section in energy group g at position r, r in energy group g′ travelling in the direction Ω′ is scattered into energy group g with direction dΩ about Ω, and PROTEUS is a high-fidelity deterministic neutron transport code  (formerlExpressing the angular flux in its even (keff and its corresponding eigenmode ϕ(r), provides critical information for the design of nuclear reactors. Since the scalar flux solution is obtained as a solution of the eigenproblem, only the shape of the flux can be ascertained and the magnitude is determined based on the total power load chosen during operation.The final form of the even-parity equation is obtained by substituting the definitions equation  and equaRp interpolation, where p is the total number of parameters used. In this paper, we use the effective cross-section generation capability provided by MC2-3 code [A nuclear reactor core is typically composed of hundreds of different materials and isotopes, each with different cross sections. The cross section of the material in equation  is great2-3 code  that canPROTEUS has a hierarchical multilevel solver based on space–angle–energy parallelism which internally uses PETSc's Krylov methods and SSOR preconditioners. This solver is capable of using several existing petascale parallel machines with demonstrated scalability of over 70%  at over 250 000 processors (BlueGene/P ). Recent(d)M<0.3). Then, the conservation of mass, momentum and energy equations are written asu is the velocity field, r are the spatial coordinates, t is the time, ν is the kinematic viscosity, Cp is the heat capacity, T is the temperature, λ is the conductivity, p is the pressure, ρ is the density and q′′′s and The Nek5000 computational fluid dynamics solver is based on the SE method developed by Patera . For thePN−PN−2 method [PN−PN formulation [PN−PN methodology tends to yield improved solution estimates, and this is consequently the formulation used for the calculations performed here.The open-source Nek5000 code supports two different formulations for spatial and temporal discretization of the incompressible Navier–Stokes equations: the 2 method –37 and Pmulation ,39. Both−3 s to 10−6 s. The Poisson equation for the pressure, the most computationally expensive stage, is solved using an AMG preconditioner. Nek5000 does not rely on external linear algebra packages. Nek5000 is massively parallel and employs the MPI standard for parallelism.Time integration is performed using a backward differentiation/extrapolation scheme of the second order (BDF2/EXT2) using variable time stepping. The Courant number is maintained below 0.5, with time steps in the range 10Typically, the solution of thermal-hydraulic modelling of reactor cores involves the solution of a modified conjugate heat transfer problem in rod bundles (fuel assemblies). The heat generated in cylindrical pins containing the nuclear fuels is removed by an external coolant flowing in parallel to the pins. In addition to the equation described by , additioqs′′′ estimated by equation  is the temperature- and density-dependent fission cross section of the material, Wg is the group-dependent power production per fission reaction and ψg is the angular flux for energy group g.With a solution to equation , the volequation  is used — The inlet fluid surface has uniform prescribed velocity and fixed temperature.— The inlet and outlet solid surfaces of the duct have an adiabatic temperature boundary condition.— Standard outflow boundary conditions are specified at the outlet fluid surface.— The wall surfaces of the pins and the inner surfaces of the duct have a non-slip velocity boundary condition.— The outer surfaces of the duct have an adiabatic temperature boundary condition.The boundary conditions for the described conjugate heat transfer problem in rod bundles are standard and are applied at the inflow, outflow and wall surfaces as follows:(e)At the most basic level of capability, integration of a given physics code into this system requires reading the mesh along with the associated data and writing solution fields back onto the mesh after their computation. This enables the interfaces in CouPE to transparently interface with the physics solvers and to pull/push the necessary data to drive the global nonlinear solver to convergence.L2 global error of the numerical solution Unum computed on the corresponding physics mesh using the following definition:Ω is the spatial domain and Uref is the reference solution computed on a refined/resolved spatio-temporal grid.The convergence criteria for the solvers are determined based on the actual 3.Verification of single-physics codes is a daunting task, and implementing the process for a complex multi-physics simulation requires quantifying errors at many stages. The spatial projection errors that occur when the solution field is transferred from one physics to another dominate the coupling errors along with the treatment of the nonlinear terms. In this paper, we present two demonstration problems that will help determine the accuracy of the solvers. First, a simplified hexagonal assembly (SAHEX) is taken and we test the data transfer mechanism for optimal accuracy and consistency as the source and target meshes are refined. Then, a realistic assembly that was employed in the EBR II reactor (the instrumented XX09 assembly) is solved for various SS and quasi-static transient problems, in order to investigate the advantage of using tightly coupled solvers to numerically resolve the relevant scales in the problem. The boundary conditions for the problems described are summarized in §2.(a)The neutronics solution without any coupled feedback is essentially a linear generalized eigenvalue problem that computes the fundamental eigenvalue, eigenmode pair. The neutronics solver assumes constant material-dependent temperature/density values to initiate computation of cross sections which are nominal values obtained with the initial conditions (ICs) from thermal-hydaulic solver. Owing to the nonlinear nature of the coupled physics, the hydraulics solver assumes an axially cosine shape as the heat source to compute the thermodynamically equilibrium solution to a user specified tolerance.— compute coupled initial SS solutions at rated conditions and— perform a quasi-static transient where the power, temperature and density evolve based on the change in total reactivity.Each problem was tested in a two stage process:t0 is the transient initiation time, T0 is the initial converged temperature solution, δt is the duration of the transient at the inlet and α is the damping parameter to control the magnitude of the perturbation .The type of transient examined in the paper is a simplified loss-of-heat-sink, where the temperature of the fluid at the inlet boundary is specified as a function of time and the evolution in the coupled fields is computed. This simulates an accident scenario when the heat exchangers fail to remove excess heat from the coolant, thereby increasing the inlet temperatures steadily, causing feedback effects from different sources to interact nonlinearly between the physics:(b)a priori using the MC2 to obtain parametrized 9-group cross sections as a function of temperature and density. Since this is an isolated assembly model, a vacuum boundary (non-reentrant) condition is applied on the top and bottom and reflective boundary condition on all other outer surfaces of the neutronics model. The velocity and temperature boundary conditions are applied at the bottom surface of the model  and outlet boundary conditions are applied at the top surface. In terms of the mesh resolution, the hydraulics solver uses a comparatively coarser mesh than neutronics, consistent with the use of quadratic elements and nature of spectral discretization.The SAHEX problem geometry was designed to an outer duct wall that encloses six fuel pins and a control pin in the centre. Meshes with varying resolution were used for neutronics and thermal-hydraulics solvers to resolve the spatial scales in the model and the geometry consisting of fuel pins, cladding, control rod, steel can and sodium coolant. This model is carefully chosen to create a verification test case for solving realistic assemblies to be addressed later. The SAHEX geometry and a representative resolved physics mesh are shown in In this section, the coupled results for the simplified single assembly are shown first and then the verification studies performed on this problem will be discussed.(i)a, the SS solution fields for the individual physics were computed. The profile of the integral power based on the angular flux computed from solving equation (Following the procedure detailed in §3equation  and the (ii)rtolNE=10−7 and rtolTH=10−4, respectively, in order to capture relative variations in the fine-scale scalar flux and temperature profiles. CouPE computes the rate of convergence and determines the stopping criteria for the coupled physics iteration. Every call to the thermal-hydraulic solver is preceded by a conservative solution projection of the power solution field computed in neutronics, which is used to recompute the volumetric power source in equation (The convergence criteria for the neutronics and hydraulics solver were specified to be equation .keff are tabulated in O(Δx2) as expected, since the neutronics discretization is based on linear Lagrange basis functions. It is also imperative to note that the parallel performance of the solvers (on 32 processors) measured using the computational cost per Picard iteration  increases nearly linearly with the number of degrees of freedom.It is essential to rigorously verify spatial accuracy constraints in order to conserve the total energy specified for the problem configuration. We performed several successive refinements of the neutronics and hydraulics meshes, and quantified the error convergence in the solution projection and parallel data transfer algorithms. The results based on a reference mesh solution for the convergence of a,b for a coarse and fine spatial resolution. It is evident from the scales and the integral of the profile distribution that the global energy is conserved during field transfers.The projected power solution at the end of the SS iteration from the source neutronics mesh to the target thermal-fluid mesh, after applying conservation prescriptions, is shown in figure 6(iii)t=t0, by updating the inlet boundary conditions as shown in equation , and the overall time steps are reduced consistently to resolve the spatial and temporal scales in successive simulations starting with Δtcoarse=0.02 s. The criticality constant keff rapidly converges as the number of time steps is halved while approaching the reference solution as O(Δt). Note that feedbacks based on both temperature and density are necessary to show the complex nonlinear coupling between the neutronics and thermal-hydraulics physics for this test problem since the case where only Doppler feedback is considered shows larger sensitivity to the inlet temperature change. In other words, the density and Doppler expansion feedback are competing effects as validated from theory and experimental observations.In all the cases, the number of subcycling steps performed in thermal-hydraulics was specified to resolve the transient change in temperature. Several transients have been performed to test for sensitivity of the coupled field solutions to different feedback effects. a. The corresponding evolution of the temperature profiles is shown in figure 8b. Note that the significant change in power profile corresponding to only a minor penetration of the high temperature front within the domain indicates a very fast response (high sensitivity) to the boundary condition in the system.The total power decrease in the assembly as the transient progresses can be observed in figure 8(i) the time-step size necessary for this transient to maintain accuracy and(ii) the importance of the inclusion of all types of feedback effects.The coupled physics simulation capability with the SHARP framework was tested on the SAHEX problem for a loss-of-heat-sink transient and the results obtained have been verified by spatial and temporal solution convergence studies. The sensitivity tests lead to important conclusions on:(c)a. The coupled loss-of-heat transient problem is simulated with this problem to observe spatio-temporal sensitivity and nonlinear feedback effects.Detailed specifications for the XX09 assembly can be found in ,41. Owin(i)t=6×10−6). This step size was chosen to resolve the temporal evolution of the flow and temperature fields to approach an SS solution. A perturbation of the inlet temperature (with α=0.2 in equation (keff reduces as the transient proceeds, owing to the negative feedback effect from the increase in fluid temperature flowing through the domain. The Doppler temperature increase is evident by observing the change in the profile at the axial centre of the assembly as shown in The coupled physics results for the XX09 assembly problem use sub-cycled thermal-hydraulic solvers (5000 time steps, with a step size of Δequation  equivale4.The SHARP framework has been designed to tackle explicitly heterogeneous reactor assembly and full core geometry configurations and has been demonstrated to be convergent, and accurate in the implementation of the algorithms and the solver. The capability of the framework to flexibly choose different coupling schemes also makes it an important tool to determine the optimal solver method to resolve relevant scales for problems of interest. Using existing physics components expands the scope of SHARP to encompass different physics models with varying fidelity in equations, discretizations and requirements through a uniform abstraction layer. A structural mechanics solver, Diablo , is curr


Neuropilin 1 (NRP1) is expressed by neurons, blood vessels, immune cells and many other cell types in the mammalian body and binds a range of structurally and functionally diverse extracellular ligands to modulate organ development and function. In recent years, several types of mouse knockout models have been developed that have provided useful tools for experimental investigation of NRP1 function, and a multitude of therapeutics targeting NRP1 have been designed, mostly with the view to explore them for cancer treatment. This review provides a general overview of current knowledge of the signalling pathways that are modulated by NRP1, with particular focus on neuronal and vascular roles in the brain and retina. This review will also discuss the potential of NRP1 inhibitors for the treatment for neovascular eye diseases. Neuropilin 1 (NRP1) is expressed in many cell types, including neurons and blood vessels, and constitutive NRP1 knockout mice are embryonically lethal with both neural and vascular defects . Since i1Both NRP1 and its homolog NRP2 are glycoproteins encoded by genes that are alternatively spliced into full-length transmembrane receptors and shorter soluble forms  Fig. 1)Fig. 1). NRP2 was identified based on its homology to NRP1 . The ami11NRP1 and s12NRP1 are encoded by the first 11 and 12 exons of the NRP1 gene, respectively, and s9NRP2 is encoded by the first 9 exons of the NRP2 gene  clone library  . NRP1 alThe b1 and b2 domains are also required for NRP1-mediated cell adhesion activity . The cel1.2www.phosphosite.org), an excess of 485 high throughput proteomic discovery-mode mass spectrometry analyses identified post-translational phosphorylation of human NRP1 at Y920, which is located next to the SEA domain. Post-translational modification of the NRP1 cytoplasmic tail may enable modulation of NRP1 signalling, but their existence has yet to be validated through conventional biochemical analyses.The 44 amino acid NRP1 cytoplasmic domain has no known catalytic activity, but contains a SEA motif that is able to recruit the PDZ domain-containing adaptor protein synectin , also kn1.3The c domain of NRP1 mediates homodimerisation and heterodimerisation with NRP2 . ExperimNRP1 and NRP2 interact with transmembrane proteins of the plexin (PLXN) family to convey semaphorin signals, with NRPs serving as the ligand binding and plexins serving as signal transducing subunits  e.g. . VEGF165in vitro .in vitro  and the TGFβ receptor 2 (TGFBR2), independently of TGFβ1 binding . BiochemIn human mesenchymal stem cells, NRP1 also interacts with PDGFRα in the presence of PDGFA, PDGFB or VEGF165, and with PDFGRβ in the presence of PDGFB or VEGF165 . It ther2The functional significance of SEMA3 and VEGF signalling through NRP1 has been studied in several different contexts, including in neuronal and vascular development, tumourigenesis, ocular neovascular disease and immune system function . This re2.1The mouse embryo hindbrain and retina are widely used models for the qualitative and quantitative description of molecular and cellular mechanisms regulating neovascularisation. The hindbrain is vascularised early in development to support the growth of rapidly proliferating neural progenitors and the function of their progeny, and vascularisation follows a stereotypical process  are the second most common type of retinal vascular disorder after diabetic retinal disease, can occur at almost any age, but mostly in patients aged over 50, and they cause sudden unilateral loss of vision . While cNRP1 to treatment response in patients with neovascular AMD  axons and dendrites  in the extracellular matrix (ECM) and on the cell surface. In agreement with this idea, VEGF189 has a high affinity for ECM and is therefore poorly diffusible, whilst VEGF121 is the most diffusible VEGF-A isoform and VEGF165 has intermediate properties , most notably 951, 1054, 1175 and 1214, and these residues then bind intracellular adaptor proteins to initiate signal transduction . For exaIn contrast to VEGFR2, VEGFR1 is thought to function mainly as a decoy receptor in angiogenesis by trapping VEGF to prevent VEGF binding to VEGFR2 and therefore inhibiting VEGFR2 signalling . In suppIn vivo, the interaction between NRP2 and VEGFR2 is important for developmental lymphangiogenesis  neurons to ensure their neuroprotection during their migration from the nasal placode into the brain, and RGC neurons in the retina to enable their axon guidance  cells; thus, overexpressing NRP1 alongside VEGFR2 increases VEGF165-induced endothelial cell chemotaxis compared to overexpressing VEGFR2 alone . Moreove4.4.1Nrp1cyto/cyto mice)  . Accordiia model . Mechaniia model . Togetheia model .Nrp1Y297A/Y297A and Nrp1D320K/D320K mice in vivo, ip cells  and NRP1ip cells . The impip cells , the mouip cells  and the ip cells . Even thip cells . Supportip cells .Nrp1 knockout mice; tamoxifen-inducible, endothelial NRP1 knockout mice (Nrp1-null (floxed) alleles (Nrp1fl/fl) were crossed to mice expressing a tamoxifen-inducible Cre transgene under the control of the endothelial Pdgfb promoter  of PAE cells co-transfected with NRP1 and VEGFR2 decreased after VEGF165 stimulation, whereas it was unaltered in PAE cells transfected with NRP1 or VEGFR2 alone in vitro mulation  and paximulation , this stmulation . It is lmulation , and intmulation .A subsequent study from our lab showed that NRP1-mediated ABL1 signalling is also required for fibronectin-induced activation of CDC42 . CDC42 iECM-driven, NRP1-dependent actin remodelling promotes both physiological and pathological angiogenesis in the mouse . Thus, tImatinib-treatment additionally causes a small decrease in vascular extension of the primary plexus across the developing retina . This phDespite ample evidence that NRP1 helps to convey extracellular matrix signals by modulating integrin function, the molecular mechanism by which NRP1 interacts with integrins is not yet understood. In particular, it remains to be examined whether NRP1 interacts with integrin β1 directly, or if their interaction is only indirect as part of a multiprotein complex. Moreover, it is not yet clear how the VEGF- and ECM-driven NRP1 pathways described above intersect with NRP1-mediated TGFβ signalling, discussed in the next section.5.2TGFB1, TGFB2 and TGFB3 genes. Each TGFB gene encodes a pro-protein of 50 kDa, which is cleaved by furin enzymes and then dimerises; the cleaved dimer binds to the dimeric pro-protein, also referred to as latency-associated peptide (LAP), through non-covalent interaction. Upon secretion, the LAP/TGFβ complex is covalently linked to the latent TGFβ binding protein (LTBP) via a disulfide bond to maintain TGFβ in an inactive state Tgfb1-nulopoiesis . The defopoiesis . Also agopoiesis . It rema5.3in vivo studies are required to establish whether lack of PDGF signalling is responsible for the defective function of visceral smooth muscle lacking NRP1. It also remains to be investigated whether PDGF enables bridge formation between NRP1 and PDGF receptors to increase signal transduction in a mechanism akin to that induced by VEGF165 during NRP1-VEGFR2 complex formation and signalling (see above).NRP1 can modulate signalling induced by the platelet-derived growth factor PDGFA and PDGFB. As mentioned above, NRP1 interacts with PDGFRs, and NRP1 downregulation reduces PDGF-induced PDGFR phosphorylation and therefore cell migration and proliferation of human mesenchymal stem cells . NRP1 al6We recently reported that NRP1 is expressed in the endothelial cells of neovascular lesions in the OIR model B Raimon. MoreoveNRP1 (rs2070296) is associated with decreased response to ranibizumab (Lucentis), as assessed by treatment-induced improvement of visual acuity . Moreover, this SNP does not appear to be in linkage-disequilibrium with any other known coding SNPs (linkage-disequilibrium data from the 1000 Genomes Project). It has not yet been examined whether this SNP is associated with regulatory elements that control gene expression.NRP1 is expressed by both endothelial cells and RPE cells in choroidal neovascular membranes of AMD patients . Agreeinl acuity . The cauin vitro assays and/or examined their ability to inhibit tumour growth and angiogenesis in mouse models. Many of the principle findings obtained through these studies will likely be relevant for ocular disease. The search for NRP1 blocking agents has so far focussed mainly on identifying compounds that inhibit VEGF164 binding to NRP1, but recent studies raised the possibility that VEGF-independent NRP1 functions may also be possible therapeutic targets, such as manipulating SEMA3A signalling through NRP1 or NRP1-mediated ECM signalling via integrins.The importance of NRP1 in animal models of vascular pathology and tumourigenesis, combined with multiple descriptions of NRP1 expression in human disease, suggests that NRP1 may be a useful therapeutic target for several vessel-associated diseases. NRP1 function may be inhibited with function blocking antibodies, peptides, small molecules or micro RNA (miRNA) mimics. Most studies seeking to target NRP1 function have assessed a candidate agent's ability to modulate endothelial cell phenotypes in commonly used 6.16.1.1A, which targets the NRP1 a1-a2 domains to inhibit SEMA3A binding, and anti-NRP1B, which targets the b1-b2 domain to inhibit VEGF164 binding (d) of anti-NRP1A and anti-NRP1B are 0.9 nM and 0.4 nM for human NRP1, respectively, and both antibodies also bind mouse NRP1  peptides . The Cenrisation . Aside frisation .6.1.2.2in vitro. In vivo, A7R administration reduces vascular density and tumour size in a breast cancer cell xenograft model, but has no direct effect on cancer cell proliferation (The heptapeptide ATWLPPR (A7R) was originally thought to antagonise the VEGF interaction with VEGFR2 , but is feration . The vasferation , and, wiferation .6.1.2.350 of 3 μM, but has no antagonistic effect on VEGF binding to VEGFR1- or VEGFR2-expressing PAECs , even though it was less potent in blocking VEGF binding to NRP1 (IC50 = 34 μM compared to IC50 = 16 μM for EG00229)  . Furtherft model . However6.26.2.1Based on work in the OIR mouse model, it has been proposed that exogenous SEMA3A may be administered to redirect neovascularisation from the vitreous into the retina in retinopathies  see Sec. In effein vitro  and examined whether anti-SEMA3A therapy would be well tolerated. Whether anti-NRP16.2.2www.fda.gov) as a drug to treat neovascular eye disease. When administered systemically, as in cancer treatment, Imatinib can cause bone marrow suppression due to its potent inhibition of the hematopoietic stem cell regulator KIT (NRP1 also promotes OIR-associated neovascularisation by mediating ECM-driven ABL1 activation . Should ator KIT . However6.312NRP1 has antitumor activity when overexpressed in prostate cancer cells injected into rats (The intravitreal administration of a NRP1-derived ligand trap, effective for both SEMA3A and VEGF164, reduced myeloid infiltration and pathological neovascularisation in the OIR model . Howevernto rats . Moreovento rats . It has 6.4in vitro and in tumour xenograft models (The discovery of miRNAs that regulate NRP1 expression  is a relt models . Althoug7VEGF blockade is now first line therapy for the treatment of macular oedema in ischemic retinopathies. In the case of exudative AMD, anti-VEGF therapy stabilises sight in over 90% of patients over several years of treatment , wherebyInefficacy of anti-VEGF therapy may result from the treatment's inability to target established vascular lesions and/or prevent the emergence of new lesions driven by VEGF-independent angiogenesis pathways, such as ECM-induced angiogenesis. In addition, several rodent studies suggest that blockade of all VEGF isoforms in mouse models increases neuronal death in the mouse retina . As mostAs long-term anti-VEGF monotherapy for exudative AMD has limited efficacy, does not work in all patients and has potential neuronal side effects, there is a need for alternative treatments for this and possibly other ischemic eye diseases. An alternative strategy to treat neovascular disease may be to preferentially target VEGF165 over other VEGF isoforms in circumstances where preserving VEGF120 signalling through VEGFR2 may be beneficial, especially for the long-term treatment of chronic diseases such as exudative AMD. Here, wherein blockade of all VEGF isoforms would likely increase neuronal death in the retina, preservation of VEGF120 may be sufficient to convey neuroprotection through signalling to VEGFR2-expressing neurons . In thisAs NRP1 regulates angiogenesis and permeability induced by multiple signals, including VEGF, ECM and semaphorins, targeting NRP1 pathways may offer numerous windows for intervention in eye disease. Yet, it is the characteristics of the individual pathologies that should govern considerations on which type of NRP1-based targeting approach might be explored. For example, a total blockade of NRP1 signalling might be advisable when there is a need to block VEGF-dependent, VEGF-independent and SEMA3-dependent signalling through NRP1. Alternatively, it may be preferable to exclusively target VEGF-dependent and SEMA3-dependent NRP1 signalling, whilst leaving ECM/NRP1-driven angiogenesis untouched to harness NRP1's positive roles in facilitating vascular tissue integration and thereby restoring blood flow to hypoxic tissues. More specifically, it may be desirable to promote NRP1-mediated signalling to guide vessels along ECM remnants of degenerated vasculature whilst blocking harmful NRP1 functions, such as its ability to promote vascular hyperpermeability. With NRP1 emerging as a promising therapeutic target for eye disease and with encouraging therapeutic agents being developed, further studies are clearly needed to define the specific mechanisms by which NRP1 activates different disease-related pathways and to hone in on pathology-specific applications.


The need for the development of new cancer therapies and push for the design of new targeting techniques is on the rise, and would be useful for cancers that are resistant to current drug treatments. The understanding of the genome has significantly advanced cancer therapy, as well as prevention and earlier detection. This research highlight discusses a potential new type of cancer-targeting molecule, Sweet-P, which is the first of its kind. Sweet-P specifically targets the microRNA-144 binding site in the 3′ untranslated region (3′ UTR) of the human glucocorticoid receptor β (GRβ), which has been demonstrated to increase expression. GRβ has been shown to be highly expressed in cells from solid tumors of uroepithelial carcinomas, gliomas, osteosarcomas, and hepatocellular carcinomas, as well as in liquid tumor cells from leukemia patients. In non-cancerous diseases, GRβ has been shown to be highly expressed in glucocorticoid-resistant asthma. These maladies brought the need for the development of the Sweet-P anti-GRβ molecule. Sweet-P was shown to repress the migration of bladder cancer cells, and may serve as a new therapeutic for GRβ-related diseases. Oncotarget, we have discovered that the glucocorticoid receptor β (GRβ) causes migration (movement) of human bladder cancer cells . Bladder cancer is the 4th most common cancer in men, and the 5th most overall . Almost three-quarters of bladder cancer patients may have a recurrence, and one-third experience progression, causing the need for constant lifelong surveillance and treatment . The long-term therapy results in bladder cancer being the most costly cancer for lifetime regimen , which brings the need for new and better treatments. Because we showed that GRβ plays a role in bladder cancer migration, we set out to construct the first anti-GRβ molecule, which we termed Sweet-P, with the goal of providing a potential new therapy. Sweet-P was designed as a peptide nucleic acid (PNA), conjugated to the Trans-Activator of Transcription (TAT) protein from HIV (for cellular delivery) to specifically target the 3′ untranslated region (3′ UTR) of human GRβ. Sweet-P functions by specifically blocking the microRNA-144 (miR-144) binding site in the 3′UTR of human GRβ , which increased Akt1 guided proliferation . We also showed that Sweet-P inhibition of human GRβ increased PTEN expression in bladder cancer cells  /coregulators, coactivator squelching through the transactivation domain, and through inactive α/β dimers that bind in the nucleus r cells  . There m. The GRβ interaction with β-catenin and transcription factor-4 (TCF-4) was shown to positively regulate astrocyte activity, leading to increased proliferation , 24. This observation further supports our previous finding of GRβ stimulation of growth , albeit via Akt1 activation and PTEN inhibition. Also, GRβ was shown to increase migration of glioblastoma cells . However, the interaction of miR-144 with the GRβ 3′UTR in glioblastoma or ALL is unknown. In LNCaP-ARA70β prostate cancer cells, which express increased levels of GRβ, Ligr et al. reported increased cellular growth and proliferation . Furthermore, treatment with methotrexate in peripheral mononuclear and lymphocyte cells resulted in decreased GRβ expression, thus increasing GC sensitivity . Additionally, Piotrowska et al. demonstrated in Hut-78 and Raji B-lymphoma cells, MCF-7 breast cancer cells, and HT-29 colon carcinoma cells that known growth inhibitors trichostatin, sodium butyrate, and 5-Aza-20-deoxycytidine treatment suppressed GRβ and enhanced GRα with an increase in GC sensitivity , 29. However, miR-144 levels, proliferation, or migration were not assessed in these studies. Nevertheless, these observations indicate the necessity of developing an anti-GRβ therapy to specifically target GRβ-related cancers.Sweet-P may have several clinical applications as GRβ has been shown to be involved in other cancer types. For example, treatment with GCs as a first line therapy in acute lymphoblastic leukemia (ALL) is effective due to its ability to arrest cell growth and trigger apoptosis. Unfortunately, resistance to therapeutic GCs is common, which has been attributed to increased levels of GRβ or decreased GRα . In mice, increased GC levels induce apoptosis in thymocytes . Because of the anti-inflammatory effects of GCs, they are commonly prescribed to asthma patients. Many studies have demonstrated an elevated expression of GRβ and GC-insensitivity in the airways of asthma patients –34. Christodoulopoulos et al. showed that approximately 8% of cells in large and 2% of cells in small airways of patients were GRβ positive. However, mild asthma patients had an increase of 14% (7 fold) in GRβ positive cells in the small airways, but no change in expression in large airways. Alarmingly, in fatal asthmatic patients, the airways showed a dramatic increase in GRβ positive cells to 21% (2.5 fold) in large and 35% (17 fold) in small airways . Hamid et al. reported an increased number of GRβ immunoreactive inflammatory cells in the airway T-cells of GC-resistance patients when compared to GC-sensitive or healthy patients . In tuberculin-driven cutaneous inflammatory lesions of patients with GC-resistance asthma, increased number of cells expressing GRβ was also reported . Furthermore, Goleva et al. demonstrated in bronchoalveolar lavage macrophages that GC-insensitive asthmatics have elevated GRβ mRNA and protein levels in comparison to GC-sensitive patients . Of most interest, the authors reported enhancement of dexamethasone-induced GRα transactivation in GC-insensitive asthmatics after RNAi silencing of GRβ. As such, Sweet-P suppression of GRβ may serve useful for GC-insensitive asthmatic patients.In non-cancerous diseases, the resistance to GCs due to high levels of GRβ have been reported, and Sweet-P can potentially be used as a novel therapy. Most clinically relevant is the anti-inflammatory and immunosuppressant effects of GCs, which have been shown to decrease levels of cytokines, chemokines, and vasoactive agents. GCs reduce the movement of leukocytes to inflamed areas, and the function of immunocompetent cells  but is essential for proper erythropoiesis by downregulating RAB14 . Also, miR-144 has been shown to promote nasopharyngeal carcinoma through the downregulation of PTEN, a regulator of the PI3K/AKT pathway , and induce breast cancer and hepatocarcinoma cell proliferation through the downregulation of Runx1, a tumor-suppressor gene . Interestingly, estrogen treatment (E2) in SkBr3 breast cancer and HepG2 hepatocarcinoma cells increased the expression of miR-144 through the PI3K/ERK/Elk1 transduction pathway , which may serve as a positive activator of GRβ. Solakidi et al. showed in HepG2 and SaOS-2 cells that GRβ and ERα were localized mainly in the nucleus, particularly concentrated in nuclear structures which suggest a direct involvement of GRβ and ERα in nucleolar-related processes . However, the interaction of ERα and miR-144 signaling to increase GRβ activity has not been studied. In contrast to oncogenic properties of miR-144, the loss of miR-144 expression has been shown to be related to the progression of colorectal cancer through the derepression of mTOR, a cell growth and metabolism regulator . However, because the decrease in miR-144 expression leads to colorectal cancer progression, GRβ may not have an involvement. Also, miR-144 has been shown to inhibit the migration, invasion, and proliferation of carcinomas such as rectal cancer  and osteosarcoma , which was attributed to the downregulation of ROCK1 . High levels of GRβ was shown in SaOS-2 osteosarcoma cells, which suggest that miR-144 and GRβ signaling may be differentially regulated in bone cancer. Similarly, miR-144 inhibited migration and proliferation of hepatocarcinoma cells by the downregulation of AKT3  and non-squamous cell lung carcinoma through the downregulation of ZFX . Due to the diverse targeting of many different genes, inhibiting the global function of miR-144 during cancer therapy could be detrimental through off-target effects, and result in the de-repression of oncogenes. The specificity of Sweet-P blocking only the interaction of miR-144 with the 3′UTR of GRβ (Our work highlights miR-144’s role in inducing migration of bladder cancer cells via GRβ; however, miR-144 has been demonstrated to play roles, both positive and negative, in many other forms of cancers and diseases. For example, miR-144 has been shown to contribute to the pathogenesis of Alzheimer’s disease through the downregulation of ADAM10 R of GRβ  to supprIn conclusion, our discovery of Sweet-P targeting GRβ in bladder cancer sheds light on a novel drug therapy that specifically targets a gene known to cause growth, proliferation, migration, and GC hormonal therapy resistance. At this point, we have shown that the Sweet-P molecule suppresses GRβ in bladder cancer. In addition, we have shown that Sweet-P only targets GRβ and not other miR-144 regulated genes. More importantly, Sweet-P inhibits the ability of cancer cells to migrate. We will also be testing the effect of the Sweet-P molecule on other types of cancer. Essentially, Sweet-P may be used as a treatment option for several different carcinomas where GRβ is highly expressed including bladder, prostate, lung, or glioblastoma, as well as for liquid tumors such as in leukemia. Sweet-P can be beneficial for non-cancerous diseases also, such as asthma and GC-insensitive disease states caused by increased GRβ. Thus, Sweet-P serves as the first anti-GRβ molecule that may provide a new therapy.


AR) gene polymorphism in humans is linked to aggression and may also be linked to reproduction. Here we report associations between AR gene polymorphism and aggression and reproduction in two small-scale societies in northern Tanzania (Africa)—the Hadza (monogamous foragers) and the Datoga . We secured self-reports of aggression and assessed genetic polymorphism of the number of CAG repeats for the AR gene for 210 Hadza men and 229 Datoga men (aged 17–70 years). We conducted structural equation modeling to identify links between AR gene polymorphism, aggression, and number of children born, and included age and ethnicity as covariates. Fewer AR CAG repeats predicted greater aggression, and Datoga men reported more aggression than did Hadza men. In addition, aggression mediated the identified negative relationship between CAG repeats and number of children born.The androgen receptor ( Aggression in traditional and modern societies is sometimes deployed to acquire resources and, therefore, social status and reproductive opportunities –6. Even AR) gene contains a polymorphic and functional locus in exon 1, comprising two triplets (CAG and GGN). This locus supports a regulatory function that responds to T, with fewer CAG repeat clusters being more effective in transmitting the T signal [AR gene polymorphism with personality traits and behavioral correlates, including antisocial behavior and aggression [AR polymorphism and personality traits [AR CAG repeats with aggression, anger and hostility. Men with fewer AR CAG repeats reported more aggression and sired more children. A previous study of these relationships among Hadza men did not find these associations [AR gene polymorphism and aggression.The effect of androgens, such as T, operates through stimulation of androgen receptors –23. The T signal . Moreovegression . The resgression ,26 and vgression , whereasy traits . Butovsky traits  investigciations , with thciations  assessedAR gene polymorphism and aggression and reproduction in men living in one of two small-scale societies, the Hadza and the Datoga of Tanzania. The Hadza are hunter-gatherers that are egalitarian and monogamous and have nominal leadership with no clear division into age classes [Here we report associations between  classes ,31–34. C classes . Hadza m classes ,36. Cons classes . The Dat classes , polygyn classes ,40. As i classes –43, the  classes  that inc classes .AR gene, due to differences in mating systems and related cultural differences in attitudes towards aggression. Manning [We predicted differences between Hadza men and Datoga men in aggression and in CAG repeats of the  Manning  suggests Manning , and men Manning . FinallyWe studied 210 Hadza men and 229 Datoga men (aged 17–70 years) from northern Tanzania, Africa. Self-reports of aggression and buccal epithelium samples (for genotyping) were collected between 2007 and 2013 as part of a larger study. We secured age by using a calendar of well-dated and memorable events in local history, as the majority of Hadza and Datoga are not literate and do not keep birth records. Each participant was assigned to a 10-year age group  following previous protocols .extremely uncharacteristic of me) and 5 (extremely characteristic of me). The translation of the BPAQ into Swahili (n = 204) and Datoga (n = 190).Participants were interviewed in Swahili by the first author or a trained local assistant. They were asked to provide information including their age, sex, marital status, number of children, ethnicity and aggression history . All questions were read aloud in one-to-one dialogues and further explanations were provided, if necessary. Self-reported aggression was assessed with the Buss-Perry Aggression Questionnaire . The BP Swahili  was donen = 166) and Datoga (n = 114) was isolated using the Diatom DNA Prep 200 extraction kit . The AR PCR was performed with the primers, 5′–(FAM)tccagagcgtgcgcgaagtgat–3′ and 5′–(FAM)cgactgcggctgtgaaggttg–3′. The reaction mixture was as described previously [Buccal epithelium samples were collected for DNA analysis. Genomic DNA of Hadza (eviously . The folhttp://kimura.univ-montp2.fr/~rousset/Genepop.htm) was used to test the Hardy–Weinberg equilibrium. Kolmogorov–Smirnov tests did not indicate deviation from normality for total aggression scores in either the Hadza or the Datoga sample (ps > .10). t-tests were used for group comparisons of reports of aggression, age, and number of children. A Mann-Whitney U-test was used in the case of the number of AR CAG repeats due to significant deviations from normality in both ethnic groups. We used the AMOS extension to IBM SPSS (v. 21.0) for structural equation modeling in order to identify associations of AR CAG repeat number, ethnicity and age with the number of children, mediated through BPAQ aggression scores. A test for mediation was carried out in accordance with Baron and Kenny [The Genepop software  and local governmental agencies , were obtained prior to conducting this study. All subjects gave their informed, verbal consent prior to participation. Verbal consent was deemed appropriate given the low literacy rates in traditional Hadza and Datoga, and was approved by the MSUEC and the COSTECH.AR CAG repeats for the Hadza and Datoga samples. There were significant differences between Hadza and Datoga for the BPAQ total score and sub-scale aggression scores, with the Hadza reporting less aggression. The median number of AR CAG repeats was greater in the Hadza than in the Datoga  in the 60+ group. Older men had more children than younger men. The Hadza men had more children than Datoga men between 20–29 years, whereas a tendency to have more children after the age of 40 in Datoga men was demonstrated  than in Datoga men (15–31), consistent with previous findings [Polymorphism of the findings ,30. Thisfindings . The Datfindings , with nofindings , as wellfindings .AR gene and total aggression. Our second hypothesis was partly confirmed: Datoga men were more aggressive than Hadza men, but no age effect on aggression was found. Perhaps ethnic differences reveal an effect of social environment, rooted in different population histories and cultural factors.The structural equation modeling analyses produced several notable results. Our first hypothesis was confirmed: We documented a negative association between the number of CAG repeats in the AR gene may affect the development and maintenance of masculine traits and male reproduction. That is, along with the association between the number of AR CAG repeats and aggression, we may expect the negative correlation of AR CAG repeats with general threat potential . These qualities may be beneficial in different ways in hunter-gatherers, pastoralists, and horticulturalists, and the modulating role of AR in energy allocation under conditions of energy limitation might be addressed in future research. For example, AR CAG repeat number may modulate the impact of T on body size and body composition for Ariaal men suffering nutritional deficit [The current research suggests that the  deficit .AR polymorphism was mediated by aggression.Our findings provide support of our third hypothesis: We identified a positive association between total aggression and the number of children. The negative association of CAG Our research indicates a difference in the number of children in Hadza and Datoga men achieved after the age of 50. This may be interpreted as differences attributable to different life trajectories and marriage patterns. Beginning in early childhood, boys in the two societies are subjected to different social and environmental pressures . Hadza men start reproducing in the early 20s, but their reproductive success later in life is associated with their hunting skills . In the The current research includes several limitations. We used data on self-reported number of children, but Hadza and Datoga men sometimes have extramarital sexual affairs and, thus, the actual number of children might be higher. Conversely, it is possible that some children who are related to a particular man were fathered by his wife’s extramarital partner (in the Hadza) or by his brothers or other relatives, as is culturally permissible in the Datoga. Our findings are in concordance with other research, demonstrating that even among the relatively egalitarian Hadza there is selection pressure in favor of more masculine men –62. At tAR gene may be relevant to aggression and reproduction in men in traditional small-scale societies, where modern methods of contraception are not practiced. We show that aggression, ethnicity, and age predict the number of children sired by men. The number of children sired by men among the Hadza hunter-gatherers and the Datoga semi-nomadic pastoralists differed significantly only for ages 50 older–an effect that may be attributable to polygyny among the Datoga.In summary, we suggest that the expression of the S1 File(DOC)Click here for additional data file.


The effects of electrical stimulation of median nerve with a continuous theta burst pattern (EcTBS) on the spinal H-reflex were studied. Different intensities and durations of EcTBS were given to the median nerve to 11 healthy individuals. The amplitude ratio of the H-reflex to maximum M wave (H/M ratio), corticospinal excitability and inhibition measured using motor evoked potentials (MEPs), short-interval intracortical inhibition and facilitation (SICI/ICF), spinal reciprocal inhibition (RI), and postactivation depression (PAD) were measured before and after EcTBS. In result, the H/M ratio was reduced followed by EcTBS at 90% H-reflex threshold, and the effect lasted longer after 1200 pulses than after 600 pulses of EcTBS. In contrast, EcTBS at 110% threshold facilitated the H/M ratio, while at 80% threshold it had no effect. Maximum M wave, MEPs, SICI/ICF, RI, and PAD all remained unchanged after EcTBS. In conclusion, EcTBS produced lasting effects purely on the H-reflex, probably, through effects on postsynaptic plasticity. The effect of EcTBS depends on the intensity and duration of stimulation. EcTBS is beneficial to research on mechanisms of human plasticity. Moreover, its ability to modulate spinal excitability is expected to have therapeutic benefits on neurological disorders involving spinal cord dysfunction. Plasticity is an intrinsic reaction to adapt to environmental pressures, physiologic changes, and experiences . There aSpinal plasticity is important in the functional recovery of several neurological disorders. For instance, synaptic plasticity is known to occur simultaneously with axonal sprouting and cellular proliferation after spinal cord injuries (SCIs) and may contribute to improve motor function –8. AnothIn contrast, spinal plasticity may also cause neurological disorders. Spasticity, which is one of the most common sequelae of SCIs and strokes, may be the best example. Although spasticity involves complex changes in the spinal cord and muscles –15, it iSo far, only a few noninvasive protocols, including transcutaneous electrical nerve stimulation (TENS) , patternIn the present study, we developed a protocol for producing an inhibitory LTD-like effect in the spinal cord. Continuous theta burst stimulation (cTBS) based on repetitive TMS over M1 has shown its efficiency and efficacy in the suppression of cortical excitability through a LTD-like mechanism –31. We tEleven healthy subjects without history of neurological disorders were recruited. Eight of the subjects  underwent the complete experiments and 10 subjects participated in the experiment of spinal reciprocal inhibition. One subject, who did not have a consistent H-reflex in the forearm, was excluded. All participants signed their informed consent prior to the participation. The project protocol was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan.Each subject was seated in a comfortable chair. EMGs were performed with 1 cm-diameter Ag/AgCl-plated surface electrodes placed 2 cm apart over the tested muscles in the dominant hand as instructed separated below in each experiment. The EMG of the flexor carpi radialis muscle , extensor digitorum communis muscle , and abductor pollicis brevis muscle  were recorded. The EMG was amplified and band-pass filtered (3 Hz to 2 kHz) by Digitimer D360 amplifiers . Signals were recorded at a sampling rate of 5 kHz and stored on the computer for later analysis by Signal software  through a power 1401 data acquisition interface . If the target muscle was not relaxed (as monitored with an EMG gain of ×5000) during the test, the data would be scrapped and the test redone.EcTBS was adapted from the theta burst protocol (TBS) of repetitive transcranial magnetic stimulation (rTMS)  and consμV, in a random order. A total of 6 trials given every 6–8 sec were recorded in each condition. The intensities that produced the 2 maximum H-reflexes were used for testing the effect of EcTBS afterwards. Mmax was also recorded from the same FCR muscle by using supramaximal stimulation. Then EcTBS was applied on the median nerve. Two maximum H-reflexes  and Mmax were recorded again at 0, 15, 30, 45, and 60 min after the end of cTBS EcTBS. The H/M ratio was calculated by dividing the peak-to-peak amplitude of H-reflex by the peak-to-peak amplitude of Mmax. Two H/M ratios were then averaged for analysis.The effects of EcTBS were firstly evaluated with the maximum H-reflex and Mmax. H-reflexes were recorded from FCR by stimulation of the median nerve. Electrical median nerve stimulation was performed through surface electrodes to stimulate the median nerve in the antecubital fossa with the duration of 1 ms. In the baseline condition, the H-reflex recruitment curve was recorded with the stimulus intensities at 90%, 100%, 120%, 140%, 160%, 180%, and 200% of H-reflex threshold, which was defined as the minimum stimulus current to the evoked-potential with an amplitude of 50 We started the experiments with EcTBS600 at 90% and 110% H-reflex threshold intensity and discovered a better inhibitory effect at an intensity of 90% (see below for the results). Since we aimed to find an optimal inhibitory protocol, we doubled the stimulus duration to have EcTBS1200 at 90% and 80% H-reflex threshold for the following study. We expected to achieve a better result by doubling the length of stimulation because studies of rTMS usually find that the effect lasts longer after longer periods of stimulation. In addition, 80% H-reflex threshold may have produced better inhibition than 90%, since there was better inhibition at 90% than at 110% threshold using EcTBS600.μV in more than five out of ten trials from the contralateral APB while the subject was maintaining a voluntary contraction of about 20% of the maximum using visual feedback. Subjects received the stimulation in a random order either of the test stimulus alone or of conditioning-test stimuli for a total of 12 trials per condition. The intertrial interval was 4.5–5.5 s. SICI/ICF was calculated as the ratio of the mean conditioned MEP over the mean unconditioned test MEP. The unconditioned test MEPs in SICI/ICF assessments were used to evaluate any change in the size of MEP.In this session, TMS was used to evaluate the effect of EcTBS on excitatory and inhibitory circuits in the motor cortex. TMS was given using a hand-held figure of eight coils with a mean loop diameter of 70 mm  connected to a Magstim Bistim2 machine . The coil was placed tangentially to the scalp over the “motor hot-spot” with the handle pointing backwards. The “motor hot-spot” was marked on the scalp and defined as the location where magnetic stimulation produced the largest MEP in the APB muscle. APB was selected as the target muscle because like FCR it is innervated by the median nerve but has a clearer MEP than FCR. Short-interval intracortical inhibition and intracortical facilitation (SICI/ICF) were assessed in the baseline condition and at 30 min after the EcTBS1200 at 90% H-reflex threshold. SICI/ICF were tested using a paired-pulse technique with a subthreshold conditioning stimulus followed by a suprathreshold test stimulus of TMS at interstimulus intervals (ISI) of 3, 7, and 10 ms . The conμs, and the intensity was adjusted to produce an M wave around 50 μV from EDC [In a separate experiment, RI in the same forearm was assessed. RI assesses the interaction between stimulation of the radial nerve and the H-reflex produced by stimulation of the median nerve . Electrifrom EDC –36. We rPAD was tested before and 30 min after spinal EcTBS1200 at 90% H-reflex threshold in the same forearm to understand the presynaptic mechanism of the spinal reflex in EcTBS. Electrical stimulation was performed via surface electrodes. Stimulation duration lasted for 1 ms, and the intensity of the stimuli was adjusted to evoke maximal H-reflexes from FCR in the baseline condition. Four conditions composed of two electrical median nerve stimuli at ISIs of 1, 3, 5, and 7 sec were given every 30 ± 10% sec in a random order for a total of six trials in each condition. PAD was calculated as the ratio of the conditioned (second) H-reflexes to the unconditioned (first) H-reflex at each trial and was averaged for each condition.Experiments were done in different days at least two weeks apart from each other in a random order.t-test (baseline and 30 min after EcTBS1200). For PAD, a two-way ANOVA with factors of TIME (before and 30 min after) and ISI  was performed to compare PAD before and after EcTBS. Furthermore, a one-way ANOVA on unconditioned baseline H-reflex and conditioned H-reflexes at ISI of 1, 3, 5, and 7 sec followed by post hoc tests was performed to test the effect of ISI on PAD. Differences were considered significant at a level of 5% or below.A two-way repeated measure ANOVA on values normalized to the baseline with factors of TIME  and PROTOCOL were applied to compare the effects on the H/M ratio between different EcTBS protocols. A one-way ANOVA analysis on nonnormalized values followed to test the effect of TIME  for the effect of the individual protocol. The results of MEP, SICI/ICF, and RI were analyzed by the paired F = 2.129, P = 0.127). The effect of EcTBS600 at different intensities was then compared on values normalized to the baseline  = 14.515, P = 0.007), for EcTBS600 at 110% H-reflex threshold facilitated the H/M ratio  = 6.394, P < 0.001), while EcTBS600 at 90% H-reflex threshold inhibited the H/M ratio  = 2.580, P = 0.043), mainly at 30 min after EcTBS. We then compared the effects of EcTBS1200 at 90% and 80% threshold and revealed an INTENSITY × TIME interaction  = 10.148, P = 0.015)  = 3.173, P = 0.018), while EcTBS1200 at 80% threshold failed to produce any effect  = 1.953, P = 0.110). Furthermore, we compared the effects of EcTBS600 and EcTBS1200 at the same intensity of 90% threshold. Two-way ANOVA showed a significant interaction between TIME × DURATION (EcTBS600 and EcTBS1200)  = 3.187, P = 0.028), indicating that EcTBS1200 produced a more profound inhibitory effect than EcTBS600. We further tested the amplitude of Mmax and found that none of the protocols of EcTBS changed Mmax .There was no significant difference between the baseline H/M ratio in all conditions of experiment 1 (baseline . A two-w= 0.015) . A follot-tests revealed that EcTBS1200 at 90% threshold did not alter the amplitudes of unconditioned MEP  , ICF at ISI of 10 ms , and SICI/ICF at intermediate ISI (7 ms)  (Paired = 0.917) , SICI at= 0.975) .t-tests revealed no significant difference at all phases after EcTBS1200 .The results of RI (before and 30 min after EcTBS1200) at all ISIs are shown in F = 6.976, P = 0.002) but no significant effect of TIME  = 0.668, P = 0.441) and no TIME × ISI interaction  = 1.307, P = 0.298). The results indicate that PAD was not changed by EcTBS. We therefore put pre- and post-EcTBS data together to evaluate the effect of ISI on PAD. A one-way ANOVA showed a significant effect of ISI indicating that the second H-reflex was significantly suppressed by the first H-reflex. The post hoc test confirmed that the second H-reflex was significantly suppressed at ISI of 1, 3, and 5 second(s) but not at 7 seconds .The results of PAD (before and 30 min after EcTBS1200) at all ISIs are shown in In our experiments, the H/M ratio was suppressed following EcTBS at 90% H-reflex threshold. Eighty seconds of EcTBS containing 1200 pulses suppressed the H/M ratio for 45 min or more, while 600 pulses of EcTBS produced a shorter-lasting inhibitory effect. Interestingly, increasing the stimulus intensity to 110% threshold did not enhance the suppressive effect but produced a facilitation instead. By contrast, 80% threshold produced no detectable effect on the H/M ratio. None of the protocols changed the size of Mmax. Moreover, spinal reciprocal inhibition, the size of MEP, and SICI/ICF remained unchanged at 30 min after EcTBS1200 at 90% threshold, when the H-reflex was most suppressed.EcTBS modifies the H/M ratio and leaves the amplitude of Mmax unchanged, suggesting that EcTBS mainly affects the circuit of the H-reflex. The H-reflex involves a monosynaptic loop, in which the afferent consists of Ia fibers from the muscle spindles and the efferent consists of alpha motor axons, within the spinal cord . The lacTo further clarify the mechanism of EcTBS at the spinal level we examined spinal reciprocal inhibition in the same nerve. There are three known phases of RI in the forearm. The first phase that occurs at ISI of 0 ms or so in the radial-medial stimulation is regulated by the glycinergic disynaptic inhibitory pathway, while the second phase that occurs at ISI of 10–20 ms includes the presynaptic Ia inhibitory pathway , 41. TheMoreover, we tested PAD to identify the location of the plasticity within the synapse of the H-reflex. Postactivation depression was thought to be due to the presynaptic mechanism involving a decreased probability of neurotransmitter release from previously active Ia-afferent terminal –46. No c2+ influx that tends to induce LTP [At first glance, we were surprised to see that EcTBS600 at 110% threshold facilitated the H-reflex. cTBS600 using rTMS generally shows an inhibitory effect over the cerebral cortex , althougduce LTP , 53. AltDifferent from EcTBS over M1, EcTBS600 at 90% threshold only produced a relatively short-lasting inhibitory effect around 30 min after the end of EcTBS, indicating that spinal circuits are less modifiable than those in the brain. It is reasonable since the brain needs to be more flexible than the spinal cord for, say, learning and memorizing. After doubling the length of stimulation, EcTBS1200 is capable of producing a more profound and consistent inhibitory effect for 45 min or more. This result implies that, at least within a certain range, the longer the stimulation lasts, the stronger and longer-lasting after-effect on the H-reflex it may produce. This is consistent with a previous report of TBS using rTMS . NeverthEcTBS modulates the H-reflex likely through a mechanism involving synaptic plasticity in the human spinal cord. The effect of EcTBS depends on its stimulus intensity. Intensities higher than the H-reflex threshold tend to facilitate the H-reflex, while intensities slightly below the threshold suppress H-reflexes. Moreover, a longer-lasting EcTBS induces a more consistent and longer-lasting after-effect than a shorter EcTBS. Hence, EcTBS1200 at 90% threshold is an efficient protocol to produce an inhibitory LTD-like effect in the human spinal cord and it can be used for exploring mechanisms of human plasticity, understanding the pathophysiology of diseases relevant to spinal plasticity and, hopefully, providing therapeutic benefits on spinal disorders in the future.


Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets. Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer and the second-leading cause of cancer-related death in men in the Unites States . The andThe best-characterized variant is AR-V7 , which contains exons 1-3 followed by 16 unique amino acids from a cryptic exon 3b , 9. PrevOne known action of AR is to alter metabolism. Several studies have shown that AR signaling stimulates aerobic glycolysis, lipid metabolism, and several anabolic processes in PCa –17. HoweA majority of metabolic cancer research to date has focused on the role of glycolysis. Increased glycolysis yields more metabolic intermediates to fuel several anabolic processes to produce more building blocks  for the cells to proliferate rapidly . HoweverPrevious studies have also integrated metabolic profiling with genomic studies in LNCaP cells to identify transcriptional networks with AR serving as a critical regulator of metabolism , 29, 30.To characterize the functions of AR-V7 in PCa, we generated an LNCaP cell line with doxycycline (Dox) inducible expression of AR-V7 (LNCaP-AR-V7-pHage). AR-V7 lacks the hinge region and LBD found in full-length AR while retaining the NTD and DBD followed by 16 unique amino acids from a cryptic exon 3b Figure . We induPrevious studies have shown that activation of AR with R1881 increased glycolysis , 16. To in vitro citrate levels is consistent with patient tumor derived metabolomics data. Citrate levels are high in the normal prostate and in androgen-dependent PCa but are low in CRPC tumors, which frequently express AR-V7 , fumarate, malate, and oxaloacetate (OAA) were increased by AR, only AKG and OAA were increased by AR-V7 Figure  and 4B. 2  and the 2  revealedWe next used [U-13C]-glucose metabolic flux analysis to examine AR isoform-specific progression through the canonical TCA cycle. In this labeling scheme depicted in Figure 2 during its conversion to succinyl CoA). In contrast, if the cells utilized reductive carboxylation, then the mass spectrometer would detect m+1 isotopomers for the TCA intermediates. Interestingly, after 3 hours of [1-13C]-glutamine labeling, AR and AR-V7 significantly increased m+1 isotopomers of citrate and malate compared to vehicle control  were consistent with these findings, but regulation may also occur at additional levels. This study suggests that AR-V7 does not simply substitute for AR, but exhibits gains of function that may include the ability to grow more efficiently in an oxygen poor environment. Lastly, this study yields potential pathways to target in combination with current CRPC therapies to better treat patients whose tumors express AR-V7. For example, AR-V7's reliance on glutamine suggests the use of a glutaminase inhibitor could be beneficial although additional pre-clinical studies are needed to determine whether AR-V7 tumors are strongly dependent on glutamine. Calithera is currently testing a glutaminase inhibitor, CB-839, in a phase I clinical trial in solid tumors that does not include PCa. If AR-V7 tumors are dependent on glutamine, then CRPC should be considered in subsequent trials , 46.14C) and oleic acid (1-14C) were purchased from Elmer LNCaP cells were purchased from the American Type Culture Collection . The LNCaP-AR-V7-pHage cell line is an LNCaP cell line with TET (doxycycline) regulated AR-V7 expression generated in our lab . ParentaLNCaP and LNCaP-AR-V7-pHage cells were lysed by four rounds of freeze/thaw treatment using 1X Reporter Lysis Buffer (Promega) containing 0.4 M NaCl. A 7.5% SDS gel was used to resolve 20 μg of protein. Proteins were then detected using the previously described AR441 antibody  at a 1:1TRIzol reagent (Invitrogen) was used to prepare RNA from the treated LNCaP and LNCaP-AR-V7-pHage cells. cDNA Synthesis Master Mix (GenDEPOT) was used to reverse transcribe the total RNA (1 μg) to make cDNA to measure target genes. Target gene expression was analyzed using SYBR green PCR Master mix and an ABI 7500 Fast sequence detection system. Previously described PSA, FKBP5, and 18S primer sets were used , 49. TheLNCaP and LNCaP-AR-V7-pHage cells  were changed to RPMI 10% charcoal stripped medium and treated with vehicle (EtOH), 1 nM R1881, or 20 ng/mL of doxycycline (Dox) for six days. Medium and treatments were replenished on day 3. Cells were harvested and counted utilizing a Coulter Counter as previously described -glucose (11 mM), [1-13C]-glutamine (2 mM), or [5-13C]-glutamine (2 mM) for 0 and 3 hours. After the incubation period, medium was removed and the cells were gently washed with PBS, flash-frozen with liquid nitrogen, harvested with a mixture of water and methanol (1:4), and stored at −80°C.For processing, the frozen samples were thawed and then mixed with 600 μl of ice-cold chloroform. The resulting homogenate was then mixed with 260 μl of ice-cold water and vortexed again for 2 min. The homogenate was incubated at −20°C for 20 min and centrifuged at 4°C for 10 min to partition the aqueous and organic layers and remove the protein at the interface. The aqueous and organic layers were combined and dried at 37°C for 45 min in an automatic Environmental Speed Vac® system . The extract was reconstituted in 500 μl of ice-cold methanol: water (1:1) and filtered through a 3 KDa molecular filter  at 4°C for 90 min to remove proteins. The filtrate was dried at 37°C for 45 min in a speed vacuum and stored at −80°C until mass spectrometry analysis. Prior to mass spectrometry analysis, the dried extract was re-suspended in 50 μL of methanol:water (1:1) containing 0.1% formic acid and then analyzed using SRM (selected reaction monitoring). Ten microliters were injected and analyzed using a 6490 QQQ triple quadruple mass spectrometer (Agilent technologies) coupled to a 1290 series HPLC system via SRM. Metabolites were targeted in both positive and negative ion mode, ESI voltage was +4000 V in positive ion mode and −3500 V in negative ion mode. Approximately 9-12 data points were acquired per detected metabolite. Samples were delivered to the MS via normal phase chromatography using a Luna Amino column  at 400 μl/min gradient spanning 80 % B to 2 % B over a 20 minute period followed by 2% B to 80% B for a 5 min period and followed by 80% B for 8 minute time period re-equilibrate the column. Buffer A was comprised of 5 mM ammonium acetate (pH = 9.9) in water and Buffer B was 100% acetonitrile. To assess the validity of our method for calculating isotopomers, we determined the complete isotopomer distributions for each metabolite.13C/13C+12C) X100] and was corrected for isotopomeric distribution and natural abundance.Data analysis was performed in Quantitative Analysis ) and estimated the % of isotopomer incorporation using the formula [% of Incorporation = 2 incubator at 37°C. The next day, the medium was changed to stripped serum and the cells were treated with either EtOH, 10 nM R1881 or 20 ng/mL Dox for 72 hours. After this initial incubation, the media and treatment were changed out and replaced with a fresh set of stripped media and treatment for another 72 hours for a total treatment time of six days. When harvesting the cells, the medium was aspirated, and the cells were gently washed with PBS. Next the cells were stained with Oil Red O for 15 minutes at room temperature. After the stain was removed, the cells were washed with water and then imaged under a Nikon Phase Contrast 2 ELWD 0.3 microscope at 20X magnification.LNCaP and LNCaP-AR-V7-pHage cells were plated at 50,000 cells per well in a 6-well plate and allowed to adhere overnight in a humidified 5% CO14CO2 was collected in CO2 traps and counted using a scintillation counter [LNCaP and LNCaP-AR-V7-pHage cells were plated in 24-well CellBIND  plates at a density of 60,000 cells per well in RPMI 10% FBS medium. The next day the cells were changed to 10% charcoal stripped medium and treated with vehicle (EtOH), 10 nM R1881, or 20 ng/mL of Dox for 24 hours. After 24 hours, radiolabelled palmitate or oleate was added for 3 hours. Then 50 μL perchloric acid was added and allowed to incubate for 1.5 hours. Lastly,  counter .All data for the experiments were presented as mean ± SEM. The mean displayed was the average of at least triplicate biological samples in each experiment. Each experiment was repeated at least three independent times with the exception of the metabolomics and metabolic flux assays. Both student's t-test and one-way ANOVA followed by a Tukey post-hoc statistical analysis were used to analyze the results using GraphPad PRISM. Differences were considered to be statistically significant at p < 0.05.