Polymethylmethacrylate (PMMA) has been applied clinically and biomechanically repair loose pedicle screws. Controversies have arisen over data due to uncontrolled cement properties, various locations and sizes of fenestrated holes in repair screws, irregular holes and different bone densities of specimens. In this study, the pullout strength was compared for two techniques, the modified technique to use PMMA to augment a threaded hole and the traditional technique with retrograde injection of a PMMA filling, for standard loose screws in porcine vertebrae. Both techniques provided statistically significant results for sufficiently randomized specimens and experimental procedures. The difference in the pullout strength between conical and cylindrical screws for the aforementioned cement augmentation techniques was also investigated. Twenty-four single-level fresh-frozen lumbar vertebrae from L1 to L6 were harvested from four mature pigs. A total of 0.8 ml of PMMA was retrograde injected into screw holes with a 5.5 mm diameter, followed by insertion of a 5.0 mm diameter repair screw in the traditional group (n = 12). A stiff threaded PMMA hole was created with a 4.5 mm tapping screw before insertion of repair screws in the modified group (n = 12). Two screw geometries were randomly assigned as cylindrical (n = 6) and conical (n = 6) in each group. The correlations between filling techniques, screw geometries and axial pullout strength were analyzed. An appropriate screw trajectory and insertion depth were confirmed using X-ray imaging prior to pullout testing in both groups. For a given screw geometry , the pullout force of the modified group was significantly higher than that of the traditional group. There was no significant difference in the pullout force between the screw geometries for a given filling technique. The cement augmentation technique is far more influential than the screw outer geometry. The modified PMMA technique created a greater anchor force than the traditional method and could be an alternative for revision of pedicle screw loosening. Increased screw size and augmentation of loose screws with polymethylmethacrylate (PMMA) cement are commonly used techniques in revision surgery5. However, screws with PMMA-augmented cement are preferable because screw stability cannot be achieved only by increasing the screw diameter in a halo screw hole10.Pedicle screw fixation is a common and useful procedure for treating various spinal disorders, including fracture, deformity and instability. Pedicle screw loosening is one of most frequently reported complications associated with the necessity of revision surgery, with a reported rate ranging from 0.8 to 50% and possibly, even higher in patients with osteoporosis12. However, cement leakage along the broken pedicle hole can cause devastating results12. Other studies have included the use of perforated cannulated screws, which improved the screw anchoring strength by allowing the injection of cement through the perforation13. A study showed that solid screws with retrograde cement prefilling exhibited notably higher pullout strength than cannulated screws with cement injection through perforation14. Both perforated cannulated screws and solid screws with cement filling techniques had better pullout performance than screws without cement augmentation.Various PMMA augmentation techniques have been used in previous studies, including direct injection of PMMA into the hole of the vertebral body prior to screw insertion15; however, the results were unreliable because there were too few specimens, the specimens had mixed bone density, irregular loosening holes were formed and the traditional filling method was not explained.A modified transpedicular screw augmentation method was compared to the traditional filling method and used to repair loose pedicle screwsIn this study, the pullout strength was compared for two techniques, the modified technique to use PMMA to augment a threaded hole and the traditional technique with retrograde injection of a PMMA filling, for standard loose screws in porcine vertebrae. Both techniques provided statistically significant results for sufficiently randomized specimens and experimental procedures. The difference in pullout strength between conical and cylindrical screws based on the aforementioned cement augmentation techniques was also investigated.An appropriate screw trajectory and insertional depth were confirmed using axial and sagittal X-ray imaging prior to pullout testing Figs. , 2. In tIn the modified group, a 5.5 mm diameter enlarged screw hole was formed Fig. A, and a In the traditional group, cylindrical and conical repair screws had mean pullout strength values of 3386.90 ± 370.94 N and 3338.80 ± 548.37 N, respectively; these values were 5095.63 ± 859.67 N and 4755.99 ± 819.60 N in the modified group, respectively  of PMMA cement was examined; further investigations with various cement volumes and larger sample sizes might be necessary.Our study shows the pullout strength was higher in the modified group than in the traditional group, and the cement augmentation was far more influential than the screw outer geometry. We believe that this modified filling technique could be an important alternative for revision of pedicle screw loosening.32. The track was followed with a standard straight pedicle probe to a depth of 48 mm.This study was performed using twenty-four single-level fresh frozen lumbar vertebrae from L1 to L6 that were harvested from four mature pigs (weight 100–120 kg). All animals were healthy before harvesting and never exposed to any drugs or procedures that could affect bone density. All the specimens were separated into individual vertebrae after stripping away the surrounding musculature, ligaments, and periosteum. All the specimens were embedded in 10% formalin solution  for 24 h before the biomechanical and imaging studies. An enlarged screw hole was prepared in one side of each vertebra, which was drilled using a 5.5 mm “twist” metric drill bit attached to a Dremel 4000 rotary tool mounted on a Dremel WorkStation Model 220–01. This trajectory was selected based on previously reported morphometric characteristic data33, the screw was carefully removed, leaving a stiff threaded PMMA hole. A cylindrical or conical repair screw sized according to that used in the traditional technique was implanted . Failure was defined as the maximum load or the load peak prior to a decrease in load associated with increasing displacement36. After the pullout test was completed, the specimen and screws were closely examined for signs of fracture, and any damage was carefully recorded.Each of the 24 specimens was potted in a metal box using a specific acrylic mixture  to prepare a cubic specimen with dimension of 8 cm × 8 cm × 6 cm Fig. A. Judicip < 0.05.Statistical software  was used to analyze the pullout force of all the specimens. All of the measurements were collected for 24 vertebrae and the results were expressed as the mean ± standard deviation (SD). An ANOVA test with post hoc analyses was performed to evaluate the differences between groups. Differences were considered statistically significant at


We develop a generalized interpolation material point method (GIMPM) for the shallow shelf approximation (SSA) of ice flow. The GIMPM, which can be viewed as a particle version of the finite element method, is used here to solve the shallow shelf approximations of the momentum balance and ice thickness evolution equations. We introduce novel numerical schemes for particle splitting and integration at domain boundaries to accurately simulate the spreading of an ice shelf. The advantages of the proposed GIMPM‐SSA framework include efficient advection of history or internal state variables without diffusion errors, automated tracking of the ice front and grounding line at sub‐element scales, and a weak formulation based on well‐established conventions of the finite element method with minimal additional computational cost. We demonstrate the numerical accuracy and stability of the GIMPM using 1‐D and 2‐D benchmark examples. We also compare the accuracy of the GIMPM with the standard material point method (sMPM) and a reweighted form of the sMPM. We find that the grid‐crossing error is very severe for SSA simulations with the sMPM, whereas the GIMPM successfully mitigates this error. While the grid‐crossing error can be reasonably reduced in the sMPM by implementing a simple material point reweighting scheme, this approach it not as accurate as the GIMPM. Thus, we illustrate that the GIMPM‐SSA framework is viable for the simulation of ice sheet‐shelf evolution and enables boundary tracking and error‐free advection of history or state variables, such as ice thickness or damage. Our material point method for ice shelf flow enables error‐free advection of history variables, such as damage, and ice front trackingThe method can be readily implemented into existing finite element software , and is suitable for large‐scale applicationThe method is verified against analytical solutions for steady‐state flow and front evolution, and tested on an idealized marine ice sheet Over 80% of the Antarctic Ice Sheet drains into the ocean through floating ice shelves , fracture or thermomechanical weakening , are not well‐suited to the Eulerian approach due to the artificial diffusion or dispersion inherent to Eulerian advection schemes. For example, this artificial or numerical diffusion smears sharp edges and therefore compromises the accuracy of damage advection and evolution. Furthermore, Eulerian approaches require separate schemes to approximate ice front evolution, such as level‐set  was originally introduced by Sulsky et al. , 1995 foMany variants of the sMPM have been formulated that retain the basic procedure, but exhibit higher accuracy. These variants are largely motivated by the need to mitigate the well‐known “cell‐crossing error” in sMPM. This error arises from mapping between the material point and the background grid using linear shape functions, which have discontinuous gradients between grid cells so that abrupt transfers of stiffness occur as material points cross cell boundaries or become unevenly distributed between neighboring cells. The first sMPM variant to mitigate this error was the generalized interpolation material point method (GIMPM) developed by Bardenhagen and Kober , which cHere, we develop an implementation of the GIMPM for simulating shallow‐shelf ice flow. To our knowledge, this is the first ever implementation of MPMs for shallow ice flow. Our GIMPM formulation solves the momentum and mass balance equations for ice flow and thickness evolution, and enables natural tracking of the ice front and grounding line. In Part II , followed by the numerical discretization using the finite element and generalized material point methods. We will use indicial notation for vectors and tensors to describe the strong and weak forms of the governing equations and use matrix notation to present the corresponding discretized linear system. We will use Einstein's summation convention only for spatial indices, where repeated indices imply summation. For brevity, we occasionally avoid indicial notation, and use bold face letters to denote vectors, tensors and matrices.2.1i,j∈1,2 correspond to the horizontal plane, x=xie^i denotes the in‐plane spatial coordinates, e^i are the basis vectors for the Cartesian coordinate system, ρ is the ice density, g is the acceleration due to gravity, H is the ice thickness, s is the top surface elevation, η¯ is the depth‐averaged effective viscosity, and τb is the basal traction described by a friction law. For simplicity, here we assume the friction law asβ^ is a friction parameter and vi are the horizontal velocities for which the SSA is solved. Note that the above equation can used to specify several friction laws currently available in Elmer/Ice  asIce shelves and ice streams can be modeled under the assumption of plug flow, where horizontal velocities and strain rates are constant over depth. Consequently, the incompressible Stokes equations are modified to exclude vertical shear and vertically integrated to derive the SSA that describes the horizontal force or momentum balance as∂Tij∂xj+η=12n is the flow law exponent, ε˙e is the scalar second invariant or effective strain rate ε˙e=ε˙ijε˙ji/2, and B is a flow rate factor dependent on temperature and ice fabric. The depth‐averaged effective viscosity used in the SSA takes the same form as , where b is the vertical coordinate of the ice basal surface. We take z as positive in the upward direction, where z=0 corresponds to sea level.In the above equation, Equation , but useσij=σijD−pδij is the Cauchy stress, p is the hydrostatic pressure, δij is the Kronecker's delta, and n^ is the unit (outward) normal to the ice front. Equation A boundary condition at the ice front is set according to the seawater pressure at the ice terminus opposing ice flowσijn^j=ρwAppropriate Dirichlet conditions for velocity are set at all other boundaries.2.2w(x) and integrating over the domain. After applying the divergence theorem and introducing the boundary conditions, we obtain:Ω and Γcf represent the area of the ice domain and calving front boundary, respectively. Dirichlet conditions for velocity are applied along the final boundary, represented by Γk. Here, wi is set to zero in accordance with the fixed velocities, and the integral over Γk is eliminated  is the nodal shape function associated with node I, and Nn is the number of nodes of the chosen finite element . Substituting the continuous representations for v and w from Equation v. The element tangent stiffness matrix K and residual force vector f can be split into components and expressed as follows:v1 and v2 are the vectors of nodal velocity components, the vectors f1 and f2 contain the gravitational forcing, and the element submatrices of the tangent matrix are given byI and J denote the nodal indices of the element and ΩE is the domain of the element, as indicated by the superscript, ‘E’. The right‐hand side of Equation All variables in Equation fined asv=∑Ifollows:[K11K12K2At boundary elements for the calving front,H, ρ, b, B, and ∇v must be mapped to the Gauss points from the nodes, where B and ∇v are used to calculate the depth‐averaged effective viscosity η¯.Following standard finite element procedure, the integrals in Equations 2.3w and the trial function v belong to different function spaces. In the GIMPM, each material point or particle is assigned with a particle characteristic function, χp, that must satisfy partition of unity in the reference or undeformed configurationThe original formulation of the GIMPM  is chosen as a Dirac delta function, then the sMPM is retrieved. We assign a rectangular domain to each material point over which χp is defined, which we will refer to as the GIMPM domain of a material point. To satisfy Equations Ω into GIMPM domains Ωp by introducing a specified number of material points for each active background grid cell. In this formulation, we will update the lengths of the GIMPM domains due to deformation  rather than area (Ap), but we use Ap here because the SSA is inherently 2‐D.Note that if hp0, may be expressed as an area‐averaged form of the initial continuum field h0(x) as0’ indicates the initial time step. The validity of Equation m, the particle characteristic functions may be used as a basis to represent the material property throughout the computational domainNp is the number of material points in the domain. We use Equation Tij), gravitational driving force (ρgH(∂s/∂xi)), and basal traction (τb)i, which we substitute into Equation 0 continuous representations for w using Equation SIp are the GIMPM weighting functions corresponding to node I (of the fixed background Eulerian mesh) evaluated at a material point location asThe values of material point variables may be initialized by integrating properties of the continuum body against the particle characteristic functions. For example, the initial value of material point property, h0(x) ashp0=1Ap0∫l domainhm(x)=∑p= domain1hm(x)=∑p=ϕI to be element‐wise linear Lagrange interpolants. These shape functions at the two nodes of the linear element can be defined asξx is the local (or isoparametric) coordinate (between −1 and +1) of any material or integration point within the parent element. For illustration, we plot both the linear and GIMPM shape functions in 1‐D in supporting information Figure 1 continuous by smoothing the discontinuous gradient observed in the linear shape functions, which mitigates cell‐crossing error. Material points influence all elements that their GIMPM domains overlap. Note that in practice, we evaluate the convolutions in Equations χp and the linear shape functions within each element rather than for each node individually, which allows us to assemble stiffness matrices is a similar manner to the FEM , as defined for the GIMPM functions in Equation We take the nodal shape functions Comparing Equations SJp and ∂SJp/∂xi with ϕJp and ∂ϕJp/∂xi, respectively, in Equations Wp, is a function of both the element area, AE, and material point areas, Ap, as given byBy replacing Wp replaces Ap as the integration weights in Equations Ap becomes the area of overlap between a GIMPM domain and the element. However, reweighted GIMPM is mostly unnecessary because material point weight is already smoothly distributed between neighboring elements unless severe overlaps or gaps develop between neighboring GIMPM domains. We largely avoid these errors in our simulation studies, and therefore do not apply the reweighting to our GIMPM simulations here.Thus, in the reweighted sMPM, 3t=0, the ice domain is discretized into a specified number of material points per grid cell as described in Section B and density ρ for all material points. However, these quantities can be treated as spatially varying and history‐dependent. In this section, we detail the numerical procedure for a typical computational cycle, according to the simplified representation given in Figure At time 3.1mp is the material point massTo allow a solution of the SSA, the velocity field and thickness are initialized on the grid by mapping from the material points Figure , where tbI, is computed as the maximum value of the bedrock elevation (zbed) or the ice base elevation according to hydrostatic equilibrium aszsea=0 is the sea level.Ice thickness is mapped to the grid from material points as3.2p by mapping from the nodes. The friction parameter (β^p), bedrock elevation (zbedp), and rate factor (Bp) must also be defined at the material point level, which may require mapping from the nodes as well. Any scalar grid property, hI, may be interpolated to the material points asThe second half of the SSA initialization procedure is focused on updating material point variables Figure . SurfaceSimilarly, for gradients, the mapping isI has bI=(zbed)I, it is marked as grounded; otherwise, it is floating. If a material point belongs to an element whose surrounding elements have a mix of grounded and floating nodes, then that it is clear that material point is near the grounding line, and its grounding status is determined using the same procedure used for the nodes. Otherwise, it inherits the grounding status of its surrounding nodes.Lastly, material points are marked as grounded or floating. Defining grounding status at material points (or at Gauss points in the FEM) rather than at nodes during the SSA solution has been shown to provide a more accurate estimate of grounding line dynamics during the SSA solution , an algorithm that can remove FLIP noise using a set of k additional projections  can accumulate a small amount of error in conjunction with our boundary treatment at the ice front  and reference (undeformed) spatial coordinates. The material point deformation gradients (Fp) are tracked over time, and are updated asI is the second‐order identity tensor. In the sMPM, the determinant of Fp is used to update the material point area asIn the GIMPM, material point area is calculated as the product of the lengths defining the rectangular GIMPM domain.x^p of the GIMPM domain edges asOur implementation currently includes two schemes to update GIMPM domain lengths. The lengths should be updated carefully in order to minimize overlap or separation of GIMPM domains over time, and thus maintain partition of unity as precisely as possible throughout the domain. The first scheme updates the lengths of a GIMPM domain such that the resulting rectangular domain approximates the quadrilateral domain that would be obtained if the position of each corner of the GIMPM domain was updated individually . More detailed derivations of the above scheme can be found in Coombs et al. ((lp0)i are the original domain lengths and Uij=FkiTFkj is the symmetric material stretch tensor, which is equivalent to the deformation gradient rotated into the original Cartesian reference frame , which is updated explicitly according to the Lagrangian description of surface mass conservation for a column of ice at time step m+1 asa˙pm (m a−1) is the sum of the basal and surface accumulation rates. We add damage as a history variable in Part II  within the element, which allows us to enforce the boundary condition at the element edge that divides active and inactive elements. An illustration of this treatment is shown in Figure 4.2lp exceeds a given threshold. We implement a similar procedure for the sMPM, where a pseudo‐domain length is tracked using the accumulated strain of a material point in Cartesian directions i and initial (lp0)i domain length corresponding to the splitting direction i, from the parent material point being split. For a unidirectional split, the non‐split current and initial domain lengths are inherited from the parent material point without modification. The deformation gradient and velocities of the parent material point are transferred directly to the new material points, but direct transfer of thickness may cause visible thickness oscillations in areas of steep thickness gradients. We propose to mitigate these oscillations by instead reassigning thickness to each split material point as∂Hp∂xi, must be interpolated from the grid. Figure Each new material point is then assigned half the current point ashsp=Hp+∂Hpoint ashsp=Hp+∂H5In this section, we consider several examples using the GIMPM and sMPM for SSA simulations to validate and test the methods. We quantify error in modeled stress and front propagation versus analytical solutions in 1‐D, and further demonstrate front propagation in 2‐D. We then test the methods on an idealized marine ice sheet to show that they can maintain steady state grounding line positions over time and can advect passive scalar fields without artificial diffusion.5.1(v2)p=0). This experiment was previously used to verify a finite‐difference front‐tracking scheme , seawater density (ρw=1028 kg m−3), and the flow rate factor (B=1.9×108 Pa s1/3). The flux at the upstream boundary is given as Q0=v0H0, where we take the velocity, v0=300 m a−1 and the thickness, H0=600 m. The solution for the spreading rate is given asx1‐axis =(4C/Qox1+1/H04)1/4 and v1(x1)=Q0/H(x1), respectively =0, but including an ice front at any location on the domain will not change the steady‐state upstream provided the ice front boundary conditions . Figure Figure x1<∼40 km, as ice shelf surface slopes are high and therefore finer mesh resolution is needed for improved accuracy. In general, the GIMPM is more accurate than the reweighted sMPM, as the latter still does not alleviate cell crossing errors as fully. This is evident in Figure The stress response using the reweighted sMPM and the GIMPM are given in Figure (xI)1≤5 km. For each grid and method, three material point resolutions are tested: 4, 9, and 16 material points per cell. Each time step, we compute the normalized errors 1≥5 km. Figure We further investigate the performance of our methods by conducting a mesh convergence study. We run the steady‐state flow‐band model again using the GIMPM, sMPM, and reweighted sMPM, but now we test each method using four different grid resolutions: 5, 2.5, 1.25, and 0.625 km. We use a time step increment of 5 days and run each simulation for 150 years. For consistency between all grid resolutions, we also extend the inflow thickness and velocity correction scheme, applied previously to the first element of the primary domain, to include all elements in the primary domain with a downstream nodal coordinate of For large deformation that occurs over 150 years, Figure In Figure 5.2xc, at time t can be found from the relation Q0t=∫0xcH(x′)dx′ . Thus, this study demonstrates that the GIMPM can accurately simulate the stresses, geometry, and ice front position of an evolving ice shelf.For all grid and material point resolutions from the mesh convergence exercise, and using the GIMPM and reweighted sMPM, we track the ice front over 300 years using one month time steps, where the initial position of the ice front is set to 5.3χp for the particle characteristic function requires the use of perfect rectilinear elements. However, the reweighted sMPM can be classified as a special case of the GIMPM that uses the Dirac delta function instead of the hat function for the particle characteristic function. The upstream boundary (red) corresponds to an arc extracted from a circle with 70 km radius with a central angle of 10°. Flow is axisymmetric with respect to the vertical axis defined at the center of the circle, and we set free slip conditions at the lateral boundaries by enforcing that the normal component of velocity is zero. At the upstream boundary, a constant thickness of 400 m and an inflow velocity of 500 m a−1 is enforced. We evenly initialize 9 material points per cell on an inflow domain beyond the upstream boundary (not shown), and allow the system to evolve until the ice front reaches the downstream edge of the computational grid, which occurs after 86.6 years.To test our front propagation scheme in 2‐D, we simulate the radial spread of an unconstrained floating ice tongue. This benchmark example was considered under steady‐state conditions in previous studies  method  quickly diminish from 1.0 to ∼0.8 over 5 years. By 100 years, the diffusion increases in severity, and these furthest‐downstream, non‐zero values diminish to ∼0.5. Consistently, the results from the DG method show the spatial spread of the damage region to be about twice that observed from the GIMPM results  methods assemble shape functions according to the shapes of the material point domains, and alleviate cell‐crossing error. Unlike the GIMPM, CPDI methods are not restricted to tracking rectangular domains, and instead may track parallelograms  method (Zhang et al., Although not illustrated in this paper, an additional advantage of our GIMPM‐SSA model is that complex 3‐D multiphysics can be represented while still being efficient enough to couple with Earth system models. Because horizontal velocities are vertically invariant within the SSA framework, 3‐D processes can be approximated locally with each material point using a series of vertical layers, and subsequently vertically integrated if needed for implementation into the next SSA solution. While the same can be applied to mesh‐based Eulerian methods, the associated advection schemes are not only dissipative, but involve solving a matrix equation for each 3‐D field that scales in computational expense with the number of layers used. However, as for 2‐D fields, horizontal advection of 3‐D fields using the MPM‐SSA framework avoids artificial diffusion and only requires explicitly updating 2‐D material point locations, rather than solving a matrix equation as in Eulerian schemes. We employ this 3‐D approach to model orthotropic damage evolution in ice shelves in Part II (Huth et al., 7Error‐free Lagrangian advection or transport without numerical diffusion or dispersionComputationally inexpensive, explicit time updates that do not require solving a matrix equation for ice thickness and history variables, such as damage. History variables may be tracked in 3‐D on a series of vertical layers associated with each material point. Horizontal advection of 3‐D fields is naturally accounted for during the 2‐D material point position update, because horizontal velocities are vertically invariant.Natural tracking of the ice front and grounding line at sub‐element scaleAccurate schemes for boundary treatment and redistribution of thickness during particle splitting that facilitate simulations over long timescales.A framework consistent with the well‐established conventions of the finite element method for shallow shelf ice flowWe presented the generalized interpolation material point method for shallow shelf ice flow, and verified that this formulation can reproduce and maintain analytical solutions for steady state ice flow and ice front advection. The advantages of this formulation include:By choosing the particle characteristic functions to be either the Dirac delta or the “hat” functions, the present formulation can reproduce the existing implementations of sMPM and the GIMPM. We demonstrated that the sMPM shape functions are very sensitive to cell‐crossing errors and uneven distributions of material points, likely due to the quasi‐static and highly nonlocal stress regime of the SSA. By simply modifying the shape functions with a reweighting scheme in the sMPM, we can significantly decrease this sensitivity to cell‐crossing errors. This numerical error is almost entirely alleviated using the GIMPM without the reweighting scheme, so it is more appropriate for many applications on timescales of decades to centuries. However, a major advantage of the reweighted sMPM over the GIMPM is that it is applicable with adaptive and non‐uniform quadrilateral and triangular mesh discretization, which is ideal for accurately resolving grounding line dynamics. Future work is necessary to mitigate errors in the GIMPM associated with the intense distortion and gaps in the material point distribution observed in high shear regimes over long timescales. Potential solutions for this error involve developing material point reinitialization schemes, improving GIMPM domain updating schemes, and/or implementing different shape functions. In addition, future developments should focus on implementing additional physics to fully take advantage of the GIMPM‐SSA treatment of history variables; this could be particularly beneficial when parameterizing complex 3‐D processes using a series of vertical layers assigned to each material point. Thus, the GIMPM‐SSA model can potentially develop into a powerful tool for studying large‐scale, coupled ice sheet processes simultaneously, thus enabling the accurate prediction of ice sheet response to climate change and eventually global sea level rise.Figure S1Click here for additional data file.


Hepatic mitochondrial metabolism was profiled under fed and fasted states by the incubation of isolated mitochondria with [13C3]pyruvate, targeted metabolomics of tricarboxylic acid (TCA) cycle intermediates, estimates of oxidative phosphorylation (OXPHOS), and hepatic gene and protein expression. De novo lipogenesis was higher in the HFr/HF mice compared to their HF counterparts. Contrary to our expectations, hepatic oxidative function after fasting was induced in the HFr/HF group. This differential induction of mitochondrial oxidative function by the high fructose-driven ‘lipogenic’ environment could influence the progressive severity of hepatic insulin resistance.Diets rich in fats and carbohydrates aggravate non-alcoholic fatty liver disease (NAFLD), of which mitochondrial dysfunction is a central feature. It is not clear whether a high-carbohydrate driven ‘lipogenic’ diet differentially affects mitochondrial oxidative remodeling compared to a high-fat driven ‘oxidative’ environment. We hypothesized that the high-fat driven ‘oxidative’ environment will chronically sustain mitochondrial oxidative function, hastening metabolic dysfunction during NAFLD. Mice (C57BL/6NJ) were reared on a low-fat , high-fat , or high-fructose/high-fat  diet for 10 weeks. De novo lipogenesis was determined by measuring the incorporation of deuterium from D Non-alcoholic fatty liver disease (NAFLD) is a common co-morbidity of obesity and is prevalent in over 70% of patients with type-2 diabetes mellitus (T2DM) ,2. LipidUnder normal physiology, the metabolic plasticity of the liver allows it to efficiently switch from lipid storage mechanisms, during fed conditions, to lipid oxidation during fasting. Approximately 60% of the triglycerides in a human liver arise from the adipose tissue derived plasma non-esterified fatty acid (NEFA) pool, with new lipid synthesis from carbohydrates (de novo lipogenesis) and free fatty acids from the diets contributing around 25% and 15% respectively . While dChronic exposure to diets rich in fat/lipid calories or refined carbohydrate calories  are common strategies to induce insulin resistance and NAFLD in rodent models ,17,18. Tp ≤ 0.05). Furthermore, the body weights of the HF mice were significantly higher than the HFr/HF mice . Liver weights were higher in the HF  and HFr/HF groups  relative to the LF group  under fed conditions, with the weights remaining similar between HF and HFr/HF groups. No significant differences in liver weights were observed after overnight fast between any of the three groups. While fed plasma glucose remained similar between groups, fasted plasma glucose was higher in the mice reared on a HF diet compared to those reared on a LF diet . The HFr/HF group had the highest hepatic glycogen levels under fed conditions . There was a non-significant increase in fed plasma insulin following HF diet  and HFr/HF diet  feeding when compared to the LF diets (1.24 ± 0.40). Fasted plasma insulin remained similar between groups. Plasma non-esterified free fatty acids (NEFA) under fed conditions were significantly higher in the HF group  compared to both LF  and HFr/HF groups . However, following an overnight fast, NEFA levels were slightly higher (non-significant) in the LF group , compared to both HF  and HFr/HF group . The fed to fasted change in NEFA levels illustrate the blunted induction of lipolysis in both the HF and HFr/HF mice. Under fed conditions, liver triglycerides were higher in the high-fat fed mice  compared to both LF  and HFr/HF groups . Following fasting, liver triglycerides remained similar between groups. Both HF and HFr/HF mice had bigger deposits of inguinal adipose tissue under fed and fasted conditions. The adipose tissue weight in the HF mice were significantly higher than the HFr/HF group under fed conditions . Gene expression profiles of common markers of inflammation, profiled under fed conditions,  or fructose calories are commonly used to induce insulin resistance and NAFLD in rodent models. Our HFr/HF diet was customized to contain higher calories from fructose along with 2.5 time more lipids than the LF diet. We rationalized that the HFr/HF macronutrient environment is more reflective of the Western diets consumed by humans relative to the HF diet. The metabolic characteristics in rodents following the consumption of these diets for 10 weeks are presented in ACC, FASN, SCD1, and ELOVL6, where their expression levels were higher in the liver of HFr/HF mice compared to their HF counterparts (We determined whether the two macronutrient compositions (HF vs. HFr/HF), which were both predicted to induce insulin resistance and NAFLD, differ in their ability to induce hepatic de novo lipogenesis. Based on the deuterium incorporation into total lipids, the representative snap shots of which are provided in terparts D. OveralThe phosphorylation of AKT was higher in the mice on an HF diet under fed conditions relative to the HFr/HF group A,B. Undep ≤ 0.05) in the HFr/HF mitochondria compared to the HF group (p ≤ 0.05) of COX IV compared to the HF mice  both pyruvate and lactate levels in all the three dietary treatment groups  under fed and fasted conditions. This allowed us to determine whether HF vs. HFr/HF feeding altered the efficiency to switch from utilizing carbohydrates during the fed state to utilizing free fatty acids during fasted states. Fasting significantly decreased , suggesting that the mice reared on the HF diet were less efficient in switching from carbohydrate utilization during fed conditions to free fatty acid utilization during fasting conditions.Furthermore, fasting significantly induced plasma β-hydroxybutyrate levels in all three groups C. Howevep ≤ 0.05  in the HFr/HF group vs. the HF group at 5 min of mitochondrial incubations  achieved statistical significance at p ≤ 0.05 A. A simip ≤ 0.05 . In the ubations B. Overal13C3]pyruvate, and the incorporation of 13C was followed into the TCA cycle intermediates using GC-MS  was observed only for fumarate and malate  compared to their HF counterparts (Schematic representation of oxygen consumption rates (OCR) under basal, oligomycin, FCCP, and rotenone/antimycin under fed and overnight fasted states are presented in terparts H–J. No sterparts K–L. Overp ≤ 0.05) in all the three groups of mice (p ≤ 0.05), compared to their HF counterparts  diet. It is also important to highlight that the rates of de novo lipogenesis in the liver of the HF mice were significantly lower than their LF  counterparts. These results highlight the robust suppression of hepatic de novo lipogenesis by the high-fat dietary environment, which is commonly used in rodent models to induce obesity and NAFLD . Thus, tWe rationalized that normal feeding-to-fasting metabolic transition will induce hepatic mitochondrial function , amplifyThe remodeling of mitochondrial oxidative function is a dynamic process, especially considering the fact that the oxidative machinery comprises of multiple biochemical networks including β-oxidation, TCA cycle, ketogenesis, OXPHOS, and ATP synthesis. These biochemical networks do not remodel in the same direction or at the same rate during the progression of NAFLD ,39,40. DIn summary, our results suggest that the variations in dietary macronutrient environments can differentially influence the rate of mitochondrial oxidative remodeling and lipogenesis. The impact of these results is in the fact that these differences in mitochondrial remodeling can influence the rate of progression of NAFLD and the severity of hepatic insulin resistance, which are modulated by various dietary environments. The dietary treatments (HFr/HF vs. HF), which were applied for 10 weeks in this study, only resulted in mild hepatic insulin resistance; thus, they are reflective of the early stages of NAFLD onset, when the initial remodeling of mitochondrial oxidative metabolism is active. Thus, the observed induction of mitochondrial oxidative function associated with the high-fructose/high-fat environment could be an early compensatory response to the macronutrient overload. How the prolonged induction of mitochondrial oxidative function, fueled by the HFr/HF diet, impacts the progressive severity of NAFLD needs to be further explored.This study was approved by the Institutional Animal Care and Use Committee at the University of Maryland, College Park, MD. Male C57BL/6NJ mice were procured from Jackson Laboratory  at 4–6 weeks of age. Once mice were around 8 weeks old, the standard chow was replaced with either a low-fat control diet , high-fat diet , or high fructose/high-fat . The majority of lipid calories in these custom diets were derived from animal fat (lard), the free fatty acid composition of which is well known . All theg. The supernatant was collected in a fresh ice-cold 15 mL falcon tube after passing through a double-layered cheesecloth pre-soaked in ice-cold MSHE buffer. The supernatant was centrifuged at 8000× g for 10 min at 4 °C. The supernatant was discarded; then, the pellet was re-suspended in 3 mL of ice-cold MSHE and centrifuged at 8000× g for 10 min at 4 °C. The process is repeated with 2 mL of MSHE buffer and the remaining pellet, containing approximately 2.5 to 3.5 mg of mitochondrial protein re-suspended in 100 µL MSHE buffer without BSA. The protein concentration was measured using the Pierce protein assay kit .Mice were anesthetized using isoflurane and blood collected from the posterior venacava, which was then spun at 2500 g for 10 min to separate the serum, which was stored at minus 80 °C for later analysis. Fresh liver was collected in ice-cold phosphate-buffered saline  and washed with ice-cold PBS. Around 0.6–1.0 g fresh liver was minced in 3 mL of MSHE buffer ; pH 7.2). The minced liver was transferred to a 30 mL Dounce homogenizer and homogenized with four strokes; the first having 10 turns, the second and third having 5 turns, and the fourth having 2 turns. The homogenate was transferred to an ice-cold 15 mL falcon tube. The Dounce homogenizer was rinsed with an additional 4 mL of MSHE, which was mixed with the homogenate. Then, the liver homogenate was centrifuged for 10 min at 4 °C at 800× 2PO4, 2 mM MgCl2, 3 mM HEPES, 1 mM EGTA and 0.2% fat free BSA; pH 7.2) containing either (1) 5 mM glutamate, 2.5 mM malate, and 1 mM uniformly labeled [13C3] pyruvate or (2) 5 mM glutamate, 2.5 mM malate, and 1 mM unlabeled pyruvate. Mitochondrial protein aliquots from each liver were incubated for 0, 5, and 10 min at 37 °C. These time points were selected to stimulate mitochondrial respiration and the subsequent changes in mitochondrial TCA cycle intermediates, as a reflection of mitochondrial activity. Mitochondrial respiratory activity was stopped by placing samples on ice for 1 min before centrifugation at 3000× g for 5 min at 4 °C. The supernatant was collected in a separate Eppendorf tube, and the pellet was washed with 250 µL of ice-cold PBS. The pellet was centrifuged again at 3000× g for 2 min at 4 °C, and the supernatant was discarded. The pellet was covered with 100 µL cold methanol and stored for the determination of 13C enrichments in the TCA cycle intermediates by GC-MS. The second aliquot of mitochondria incubated with unlabeled pyruvate was prepared similarly to determine the concentrations of TCA cycle intermediates by GC-MS.Mitochondria (500 µg based on protein measurements) was incubated in 1 mL MAS-3 buffer  by microwaving for 90 s, which is followed by conversion to their respective TBDMS (Tert-butyldimethylsilyl) derivatives [The TCA cycle intermediates in the liver tissue (≈15 mg) and isolated mitochondrial protein (500 µg) were extracted with 750 µL of chloroform: methanol (2:1) and 250 µL of water. The aqueous phase was dried under nitrogen, and the TCA cycle intermediates were converted to their oximes with 20 µL of 2% methoxamine hydrochloride in pyridine  with helium as the carrier gas, followed by selective ion monitoring on MS  under electrical ionization conditions .g for 15 min. The isolated glycogen pellet was washed two times with ice-cold ethanol and the free glucose units were liberated by incubation (1 h at 55 °C) in 250 μL of buffer  containing 0.5 mg of amyloglucosidase (31.2 units/mg of solid). The incubation mixture was lyophilized to dryness, and the di-O isopropylidene acetate derivative of glucose was formed before GC-MS analysis as described above. Liver glycogen was estimated as free glucose units liberated from the amyloglucosidase activity relative to a known amount of stable isotope labeled glucose internal standard spiked into the sample.Liver glycogen pellets were isolated as previously described . BrieflySerum NEFA concentrations were determined using the NEFA-HR2 analytical kit . Liver triglycerides from approximately 20 mg of tissue were Folch extracted in 750 µL of chloroform:methanol (2:1). The extracted triglycerides were dried down before determining the triglyceride content using the triglyceride determination kit  with triolein as the standard.2O2 production in mitochondria was measured using the oxidation of the fluorogenic indicator amplex red  in the presence of horseradish peroxidase (HRP). Mitochondria (64 µg) were incubated at 37 °C with HRP (0.2 U/mL) and amplex red reagent (100 µM) prepared in MAS-3 buffer with 5 mM glutamate and 2.5 mM malate. Fluorescence was recorded using the Cytation 5 spectrophotometer  with 530 nm excitation and 590 nm emission wavelengths. Standard curves were constructed by adding known amounts of H2O2 to the assay medium in the presence of the reactants.The rate of Hp-(trifluoromethoxy), phenylhydrazone , antimycin A, and rotenone (2.53 μM) were utilized for this test. Isolated mitochondria (10 µg/well) were added to the chilled test cell plate containing the media  and centrifuged at 2000× g for 20 min at 4 °C. Then, the plate was incubated at 37 °C for 10 min before starting the Cell Mito Stress Test. The OCR were measured using Seahorse xFP analyzer  as follows: no equilibration, 5 basal readings, 5 readings with oligomycin, 3 readings with FCCP, and 3 readings with rotenone and antimycin. Indices of mitochondrial respiratory function were determined from the OCR values utilizing the calculations provided by the manufacturer.Changes in the respiratory function of the isolated hepatic mitochondria were determined using the Seahorse XF Cell Mito Stress Test Kit . Modulators of mitochondrial oxidative phosphorylation, namely Oligomycin (5 μM) F0-F1ATPase inhibitor, carbonylcyanide Total RNA was extracted from 20 mg snap-frozen liver tissue using 500 µL Trizol reagent  and mRNA mini-prep kit . The cDNA was reverse transcribed from 1 μg mRNA using cDNA synthesis kit . Quantitative real-time PCR was performed by amplifying 25 ng of cDNA, 150 nM of forward and reverse primers , and 5 μS473, COX IV, and GAPDH . A Total OXPHOS Rodent WB Antibody Cocktail  was used to profile mitochondrial complex proteins involved in oxidative phosphorylation. Images were collected using a Gel Doc XR+ system .Liver (around 20 mg) and mitochondrial homogenates were lysed with protease and phosphatase inhibitors. Protein content was measured using a Pierce BCA protein assay kit . Liver and mitochondrial OXPHOS proteins were separated using Bolt 8% Bis-tris Plus gels , transferred to a nitrocellulose membrane, and blocked with 5% milk in tris-buffered saline with Tween 20. Then, the membranes were probed with the primary antibodies of interest including Akt, pAktp ≤ 0.05. All statistical analyses were conducted and the graphs were plotted utilizing Prism 7 .Based on our previous experience, we expected the standard deviation for our measurements to range between 20 and 30%. With the statistical power (1-β) set at 80% and an alpha error of 5%, we calculated a minimum sample size requirement of five mice per group. All the data reported are presented as means ± standard error of means (SEM). All results (except the mitochondrial respiration data in


The RAF kinase inhibitor protein, RKIP, is a dual inhibitor of the RAF1 kinase and the G protein-coupled receptor kinase 2, GRK2. By inhibition of the RAF1-MAPK (mitogen-activated protein kinase) pathway, RKIP acts as a beneficial tumour suppressor. By inhibition of GRK2, RKIP counteracts GRK2-mediated desensitisation of G protein-coupled receptor (GPCR) signalling. GRK2 inhibition is considered to be cardioprotective under conditions of exaggerated GRK2 activity such as heart failure. However, cardioprotective GRK2 inhibition and pro-survival RAF1-MAPK pathway inhibition counteract each other, because inhibition of the pro-survival RAF1-MAPK cascade is detrimental for the heart. Therefore, the question arises, what is the net effect of these apparently divergent functions of RKIP in vivo? The available data show that, on one hand, GRK2 inhibition promotes cardioprotective signalling in isolated cardiomyocytes. On the other hand, inhibition of the pro-survival RAF1-MAPK pathway by RKIP deteriorates cardiomyocyte viability. In agreement with cardiotoxic effects, endogenous RKIP promotes cardiac fibrosis under conditions of cardiac stress, and transgenic RKIP induces heart dysfunction. Supported by next-generation sequencing (NGS) data of the RKIP-induced cardiac transcriptome, this review provides an overview of different RKIP functions and explains how beneficial GRK2 inhibition can go awry by RAF1-MAPK pathway inhibition. Based on RKIP studies, requirements for the development of a cardioprotective GRK2 inhibitor are deduced. PEBP1) inhibits tumour metastasis initiation and acts as an endogenous tumour suppressor [The phosphatidylethanolamine-binding protein 1  and MEK2 (MAP2K2) are the MAPKK. Upon activation by RAF1, MEK1 and MEK2 phosphorylate the MAPKs, ERK1 (MAPK3) and ERK2 (MAPK1) [The first RAF kinase, the v-Raf, was discovered in 1983 . The gen (MAPK1) .Raf1 kinase is indispensable for embryonic growth and differentiation, and knockout mice are not viable [Raf1-deficient (Raf1−/−) mice die around mid-gestation due to enhanced apoptosis, which causes major defects of the placenta and the liver [The t viable . Raf1-dehe liver ,40. The he liver , (ii) thhe liver , and -mediated phosphorylation of RKIP on serine-153 ,58. ThisRKIP is a ubiquitously expressed protein with multiple functions. As inhibitor of the pro-survival RAF-MAPK pathway, RKIP acts as an endogenous tumour suppressor. A major RKIP-mediated tumour suppressive function is based on its enhancement of tumour cell death and apoptosis. Proapoptotic activities of RKIP involve the inhibition of RAF1 together with other mechanisms. By the inhibition of RAF1, RKIP prevents the translocation of RAF1 to mitochondria . MitochoIn addition to RAF1 kinase inhibition, RKIP interferes with other growth-promoting pathways and enhances proapoptotic stimuli. Among several important signalling pathways, RKIP interferes with NF-kappa B signalling by the inhibition of upstream kinases TAK1 , NIK  and IKKA and IKKB  . The netAnother apoptosis-enhancing function is attributed to the interaction of RKIP with GSK3B (glycogen synthase kinase 3 beta), which is a suppressor of many oncogenic signalling cascades. RKIP enhances the suppressive functions of GSK3B by interaction with GSK3B and reduction of inhibitory GSK3B phosphorylation on T390 by P38 MAP kinase (MAPK14) . By thisFinally, RKIP was also reported to de-repress the expression of death receptors (DR) such as DR5 . By thisOne of the main functions of RKIP is metastasis suppression, which is mediated by multiple mechanisms. A major pathway of RKIP-dependent metastasis suppression involves the inhibition of MYC. Since MYC is a direct target of the RAF1-MEK1/2-ERK1/2 cascade, RKIP-mediated metastasis suppression by MYC inhibition is a direct consequence of RAF1 inhibition. By inhibition of the RAF1-MAPK pathway, RKIP decreases ERK1/2-mediated MYC phosphorylation and activation. As a consequence, MYC-mediated LIN28 expression is repressed by RKIP. In a next step, downregulation of LIN28 by RKIP de-represses let-7 . Within By inhibition of the NF-kappa B signalling pathway, RKIP also interferes with early metastasis and the induction of epithelial–mesenchymal transition, EMT . EMT is Another metastasis-suppressing activity of RKIP involves inhibition of the STAT3  signalling pathway ,78. RKIPH. pylori infection [Based on cellular and mechanistic data, many researchers have investigated whether RKIP could also act as a tumour suppressor in vivo. On one hand, experimental data document that overexpression of RKIP blocks proliferation and/or metastatic progression of various experimental tumour models in vivo ,82,83,84nfection .In sum, RKIP is an endogenous tumour metastasis suppressor that is widely expressed in non-tumour cells. However, as an endogenous tumour suppressor, this protein is regularly downregulated within the tumour environment. The major functions of RKIP as a tumour suppressor are mediated by its main activity as an inhibitor of the proto-oncogenic and pro-survival RAF1-MAPK pathway .RKIP not only inhibits the RAF1 kinase but also the G protein-coupled receptor kinase, GRK2 -MAPK pathway ,118,119.Does RKIP act as RAF1 inhibitor in vivo? This question was addressed in Tg-RKIP mice with B6 and FVB background. Data showed that RKIP caused myocardial dysfunction and cardiolipotoxicity ,22. The As documented by published studies, all of these RKIP-induced Pparg target genes promote symptoms of heart failure and/or cardiac lipid overload, i.e., Adipoq , Cidec , Retn 1 and Ucp1As detailed above, inhibition of the pro-survival RAF1-MAPK pathway by RKIP enhances the cardiotoxic lipid overload and cardiomyocyte apoptosis. However, inhibition of GRK2 by RKIP promotes cardioprotective signalling in cells and cardiomyocytes ,20. Why Agtr1a transcript levels , which is elevated on biopsy specimens of failing human hearts [The overstimulation of Agtr1 in Tg-RKIP mice with heart failure is reflected by the downregulation of Agtr1-specific binding sites  and cardt levels b. Decrea of RKIP . During n hearts ,130 and TGFB1-Tgfb1 [Col1a1 and Col1a2 , Col3a1 , Ccn2  and Fn1 (fibronectin 1)  b. All ofNfe2l2; Nrf2) [Complementary studies with RKIP-deficient mice found that endogenous RKIP levels are sufficient to enhance myocardial fibrosis under conditions of enhanced myocardial stress imposed, e.g., by chronic pressure overload induced by transverse aortic constriction or carbon tetrachloride treatment . Fibrosi2; Nrf2) ,138,139.2; Nrf2) .Nnt (nicotinamide nucleotide transhydrogenase) gene locus in the B6 C57BL/6J background whereas in the B6 C57BL/6N background, intact Nnt promotes mitochondrial ROS generation under chronic pressure overload [The profibrotic activities of RKIP could be neutralised by a defective overload ,140. Comoverload ,23. Taken together, different studies with RKIP-transgenic mice and RKIP knockout mice demonstrate that RKIP accelerates and/or promotes symptoms of cardiac dysfunction, heart failure and cardiac fibrosis in vivo ,21,22,23GRK2-Grk2, which accounts for the impaired contractile reserve of failing hearts by desensitisation and downregulation of cardiac β-adrenoceptors [Grk2 deficiency (Adrbk1+/−) in failing hearts restores the contractile response and contributes to the cardioprotective profile of GRK2-Grk2 inhibitors [GRK2-Grk2 levels are normalised [Grk2 levels of heart failure rats [During the pathogenesis of heart failure, there is an upregulation of oceptors ,17,141. hibitors ,16,17. Urmalised . Likewisure rats . Grk2 transcript levels in vivo, i.e., the Grk2 transcript levels of Tg-RKIP hearts were 1.50 ± 0.06-fold higher than those of non-transgenic FVB controls  and enhances the β-adrenoceptor-mediated contractile activity of cardiomyocytes  is downregulated in human heart failure [ADRB1 is considered to involve the exaggerated GRK2 activity of failing hearts [Adrb1 transcript levels  desensitisation and downregulation.While  failure ,149,150.g hearts ,149,150.t levels . InsteadP hearts . The absAdrb1) is the predominant β-adrenoceptor in the heart and mediates catecholamine-induced inotropic and chronotropic responses in the mouse [Adrb1−/− mouse hearts, which lack the β1-adrenoceptor [Adrb2 deficiency in Adrb2−/− hearts are only detected during exercise stress [Adrb2 were significantly increased 1.80 ± 0.27-fold compared to those of non-transgenic FVB hearts  is mainly expressed in adipose tissue [Adrb3 were low in Tg-RKIP and FVB hearts  levels could account for a minor role of the β2-adrenoceptor in heart failure development. The β2-adrenoceptor not only activates Gs proteins but also stimulates Gi-mediated signalling [Adrb2) induce a heart failure phenotype with progressive fibrotic dilated cardiomyopathy in mice [The β1-adrenoceptor is causally involved in the development of heart failure ,156. Vicgnalling ,158. By gnalling ,160. In  in mice . Nnt locus, because in the presence of an intact Nnt locus and unrestrained mitochondrial ROS generation under cardiac stress such as pressure overload, endogenously expressed RKIP levels are sufficient to promote detrimental cardiac effects such as myocardial fibrosis [Ace and Grk2 upregulation as indicators of an overactivated RAAS  and sympathetic nervous system. Therefore, the net effect of the dual RAF1 and GRK2 inhibition by RKIP in vivo is a detrimental cardiac phenotype, which predisposes to cardiac fibrosis, cardiac dilation and heart failure.Acting as a GRK2 inhibitor, RKIP promotes β2-adrenoceptor sensitisation and enhances cardioprotective Gi-stimulated signalling . Howeverfibrosis ,140. In NPPA and NPPB [Grk2, which reflects sensitised β-adrenoceptor-stimulated cAMP signalling [This review aims to delineate how the multifunctional protein and GRK2 inhibitor RKIP accounts for a heart failure phenotype despite cardioprotective signalling stimulated by GRK2 inhibition. The cardiotoxic effects of the tumour suppressor RKIP can be attributed to inhibition of the pro-survival RAF1-MAPK pathway. By its RAF1-inhibitory function, RKIP could promote cardiomyocyte death and cardiotoxic lipid overload . RKIP-meand NPPB ,163. In gnalling . Beneficgnalling ,23. By the inhibition of GRK2 and sensitisation of β-adrenoceptor-dependent Gs/Gi-mediated signalling, RKIP exerts an inotropic effect in mice . In patiTaken together, RKIP exerts cardiotoxic effects by multiple pathways (i) as a tumour metastasis suppressor and inhibitor of the RAF1-MAPK axis and (ii) as a GRK2 inhibitor .Studies with the multifunctional GRK2 inhibitor RKIP have revealed much information on the requirements for a cardioprotective GRK2 inhibitor. Experiments with the RAF1-inhibitory RKIP documented and confirmed that an intact pro-survival RAF1-MAPK pathway is an important feature of cardioprotective GRK2 inhibition ,21,22. BGrk2 knockout causes the lethal phenotype of Grk2−/− mice [Despite well-documented beneficial activities, GRK2 inhibition—like all therapeutic approaches—can have adverse effects. Adverse effects of complete GRK2 inhibition are caused by the fact that GRK2 is an indispensable kinase. The homozygous −/− mice . Therefo−/− mice , could bPparg-induced Adipoq and Ucp1 levels [Adipoq and Ucp1 levels contribute to heart failure pathogenesis [Adipoq and Ucp1 levels is a desired, positive therapeutic effect and not an adverse effect. Moreover, by this mechanism, GRK2 inhibitors are expected to counteract symptoms of cardiac cachexia as a potentially life-threatening condition in severe heart failure [Adverse effects of Grk2 inhibition are also detectable in adult mice. Inhibition of Grk2 in adult mice leads to weight gain under conditions of a high-fat diet intake . This ob1 levels ,174,175,1 levels . Since iogenesis ,122, the failure . The Grk failure ,177, whi failure . As deta failure . To circ failure  and coul failure . Based o failure . As an a failure ,170 and Taken together, studies with the multifunctional protein, RKIP, delineated major requirements for a cardioprotective GRK2 inhibitor. RKIP is an endogenous tumour metastasis suppressor with detrimental cardiac effects due to RAF1-MAPK pathway inhibition and its specific mode of GRK2 inhibition. Based on detrimental RKIP outcomes, future approaches of cardioprotective GRK2 inhibitor development can avoid cardiotoxic side effects and design compounds, which do not inhibit pro-survival kinases such as RAF1 but neutralise pathological GRK2 activities without disturbing indispensable physiological GRK2 functions.What about the ongoing translational efforts of RKIP-mediated tumour metastasis suppression? The inhibition of RAF(1) kinase(s) has detrimental cardiac side effects. In this respect, RKIP resembles many approved anticancer treatment modalities, which are similarly associated with cardiovascular adverse effects . However


RING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC. Colorectal cancer ranks as the fourth most deadly cancer in the world, resulting in approximately 900,000 deaths annually, and accounts for almost 10% of all annually diagnosed cancers globally . PathogeThe RING  finger (RNF) proteins, owing to RING finger domains based on structural similitude, are involved in a variety of biological processes , 14. ItsHere, we report for the first time that RNF141 was upregulated in CRC tissues, and was positively correlated with T stage. Moreover, both in vivo and in vitro data showed that RNF141 induced CRC cell proliferation, migration, invasion, and metastasis, but inhibited apoptosis through increasing the level of activated KRAS. These findings strongly indicated that RNF141 promoted CRC tumorigenesis and was a potential therapeutic target.P = 0.625). However, both Western blot and IHC analyses showed that, compared with adjacent normal tissues, RNF141 protein level was significantly elevated in CRC tissues . Together, these results indicated that RNF141 upregulation might play an important role in colorectal tumorigenesis.To identify whether RNF141 was involved in colorectal tumorigenesis, we first examined the mRNA and protein levels of RNF141 in 64 paired CRC and corresponding adjacent normal tissues. There was no significant difference in RNF141 mRNA level between CRC and peritumor tissues  in HCT116 (16.92 ± 0.37% vs 9.56 ± 0.43%), SW480 (14.55 ± 0.85% vs 8.80 ± 0.52%), DLD-1 cells (24.95 ± 0.66% vs 16.37 ± 0.27%) and HT29 (20.87 ± 0.54% vs 12.93 ± 0.51%) Fig. . In addiSubsequently, wound healing assay and transwell assay were employed to evaluate the effect of RNF141 on cell migration and invasion. RNF141 knockdown significantly impaired the migratory capacity of HCT116, SW480, DLD-1, and HT29 cells, resulting in an evident decrease of wound closure Fig. . SimilarMeanwhile, to determine whether RNF141 promoted angiogenesis, we carried out the tube formation assay in vitro. As shown in Fig. The above studies demonstrated that RNF141 promoted the tumorigenic potential of CRC cell in vitro. We next established a subcutaneous xenograft model to explore tumor-forming capacity of RNF141 in vivo. Nude mice injected with LV-sh-RNF141-infected HCT116 cells were found to have smaller volume and lower weight tumor than those injected with LV-sh-NC-infected cells at the assigned day Fig. . MeanwhiE.coli system. According to the RNF domain, RNF141 was divided into two peptides of 1H-RNF141 (1-144aa) and 2H-RNF141 . Because the 1H-RNF141 peptide formed inclusion body, only 2H-RNF141 and RNF141 proteins were made the purification. The purified proteins were identified by SDS-PAGE and Western blotting analysis   Fig. s. Taken tHaving established the promotive effect of RNF141 on KRAS activity, we next analyzed the role of RNF141 on KRAS signal pathway, MEK-ERK signaling. As shown in Fig. To explore how RNF141 facilitated KRAS membrane enrichment, we attempted to analyze the interaction between RNF141 and traffic proteins related to KRAS. Interestingly, we also found the traffic protein LYPLA1  also known as APT1  in proteins database from LC–MS/MS , colorectal cancer 45%) and lung adenocarcinoma 35%), and has been found to play pivotal role in oncogenesis , 35. In %, and ha% and lunIn conclusion, our study uncovered that the expression of RNF141 in CRC tissue was significantly upregulated, and closely associated with high T stage of CRC patient. Our systematic analysis revealed the in vivo and in vitro positive impacts of RNF141 on CRC. Mechanistically, RNF141 interacted with KRAS and promoted KRAS activity via increasing its enrichment on plasma membrane. Owing to the target proteins screened out by LC–MS/MS including more than 30 cancer-related proteins, further works are required to elaborate the interaction network of RNF141 that involves in the CRC tumorigenesis.Fresh surgically resected tissue samples, including CRC tissues and matched adjacent normal tissues from 64 CRC patients who underwent complete surgical excision between January 2014 and December 2016, were randomly obtained from the Second Hospital of Hebei Medical University, Shijiazhuang, China. The diagnosis of CRC was confirmed independently by two pathologists. After excision, 64 pairs of CRC tissues and matched adjacent normal tissues were randomly selected, and every pair was divided into three portions: one portion was directly snap-frozen in liquid nitrogen for Western blot; another portion was immediately immersed in RNAstore reagent from Tiangen Biotech Co., Ltd  and stored in −80 °C freezer for quantitative real-time polymerase chain reaction (RT-PCR); another one portion was fixed in 4% paraformaldehyde (PFA) for further paraffin embedding. This study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University, and written informed consent for collecting and preserving samples and details was obtained from each patient before the study  staining and immunohistochemistry (IHC) according to the standard procedures . The IHCTM RT reagent kit from Takara Biomedical Technology (Beijing) Co., Ltd . The cDNA was used for RT-PCR using the above PrimeScriptTM RT reagent kit on GeneAmp7300 RT-PCR System . GAPDH was used as an internal control. The specific primer sequences were designed as following: RNF141: Forward primer 5′-GTTGGTTCGAGAGAGTGGCT-3′, Reverse primer 5′-TGGTACAGACCACCCG TACA-3′; KRAS: Forward primer 5′-CGGCGGCGGAGGCAGCA-3′; Reverse primer 5′-CCCGCCGCCGCCTTCAGT-3′; GAPDH: Forward primer 5′-CAGGGGGGAGCCAAAAGGGTCA-3′, Reverse primer 5′-TGGGTGGCAGTGATGGCATGGA-3′.Total RNA was extracted from tissues using TRIzol reagent (Tiangen). Complementary DNA (cDNA) was synthesized from total RNA through reverse transcription using PrimeScript2. HCT116, SW480, DLD-1, and HT29 cells were authenticated by Viva Cell Biosciences Ltd , and the reports of Cell Line Identification were presented in Supplementary data The human CRC cell lines HCT116, SW480, DLD-1, and HT29 were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences . The HCT116 and HT29 cells were cultured in McCoy’s 5A Medium  and SW480 cells and DLD-1 cells were cultured in RPMI 1640 medium , all of which are supplemented with 10% FBS (Gibco BRL), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator with 5% COSupplementary methodsSupplementary figure legendsSupplementary table 1Fig. s1Fig. s2Fig. s3Fig. s4Fig. s5Fig. s6Supplementary data 1supplementary data 2Supplementary data 3Supplementary data 4


Afterwards, the surface morphology was observed again and the surface topography of the specimen was investigated by measuring selected 3D parameters of the ST structure, Sz and Sa. The results of investigations into the influence of selected HPWIJ treatment parameters on the surface QF removal efficiency obtained are also presented. Univariate regression functions were developed for the mean stripping efficiency based on the following: dry ice mass flow rate 2 and spray angle κ. Based on these functions, the values of optimal parameters were determined that allow the maximum efficiency of the process to be obtained. A 95% confidence region for the regression function was also developed. The results demonstrated that HPWIJ treatment does not interfere with the geometric structure of the base material, and they confirmed the possibility of using this treatment as an efficient method of removing a worn paint layer from bases made of various metal and plastic materials, and preparing it for applying a new layer during renovation.The paper presents the results of investigations into the possibility of using ahigh-pressure water-ice jet as a new method for removing a worn-out paint coating from the surface of metal parts (including those found in means of transportation) and for preparing the base surface for the application of renovation paint coating. Experimental investigations were carried out in four stages, on flat specimens, sized S × H = 75 × 115 mm, cut from sheet metal made of various materials such as steel X5CrNi18-10, PA2 aluminium alloy and PMMA polymethyl methacrylate (plastic). In the first stage, the surfaces of the samples were subjected to observation of surface morphology under a scanning electron microscope, and surface topography (ST) measurements were made on a profilographometer. Two ST parameters were analysed in detail: the maximum height of surface roughness Sz and the arithmetic mean surface roughness Sa. Next, paint coatings were applied to the specimens as a base. In the third stage, the paint coating applied was removed by means of a high-pressure water-ice jet (HPWIJ) by changing the values of the technological parameters, i.e., water jet pressure p The continuous dynamic development of the means of transport requires the use of various materials in their manufacture. One of the main elements of vehicles of various types is their body, which is usually protected against corrosion with a paint layer. However, in order for the paint coating to fulfil its function, it is necessary, among other things, to properly prepare the surface of the base for the coatings to be applied. This refers both to factory and refinish coatings. During vehicle transport operations, factory paint coating may be damaged for various reasons; therefore, the preparation of the base for the application of the refinish paint coating becomes of particular importance.Most frequently, damage to the factory paintwork in the vehicle body occurs as a result of natural wear and tear, such as erosion or ultraviolet radiation . Other fPrior to regeneration of paintwork, a thorough assessment of the condition of the factory paintwork is carried out. The size and amount of visible damage as well as the type and origin of discoloration occurring on the car body are assessed. It is only then that a decision is made as to the method of paint stripping and preparation of the base for renovation. Old paintwork is usually removed using mechanical methods. Their advantage is high efficiency and the possibility to remove traces of corrosion occurring under the paint coating. The disadvantage of these methods is interference in the geometric structure of the base material prepared by the manufacturer. Therefore, when there is no need to interfere with the topography of the base material, chemical methods  or thermal methods (pyrolysis furnaces) are used for paint removal. However, it is not always possible to use one of these methods, for example due to the necessity to disassemble the car body or the proximity of components that are sensitive to temperature or chemical agents. In such cases, sandblasting in hermetic chambers can be used. However, in this case, the topography of the base material is damaged after sandblasting ,6,7. In In the search for surface treatment methods that do not damage the base, more and more attention is being paid to air-ice ,9 or watVery few publications have investigated the use of a water-ice jet where carbon dioxide ice is used as an abrasive. The effectiveness of the surface treatment in removing paint layers using a high-pressure water-ice jet, using different nozzle variants, is presented in . Frozen Processing can also involve a cryogenic jet of liquid nitrogen . The posThe vast majority of tests are conducted on prototype test benches. The difference between the high-pressure water-ice jet (HPWIJ) presented in this paper and the ice jet technology, known as ice abrasive water jet (IAWJ), is the pressure and the abrasive used. The IAWJ method uses liquid nitrogen at the appropriate pressure and the abrasive is water ice. In the presented paper, the carrier jet is water and crushed carbon dioxide ice is used as the abrasive. A significant advantage of treatment with a high-pressure water-ice jet is the removal of surface layers without introducing surface tension . In the The possibility of using a high-pressure water jet without any additives seems to be safe for metal substrate materials and plastic parts. High-pressure water jet has previously been used to process such delicate materials as fish muscle and skin ,32. The 2 dry ice particles. After treatment, the ice particles sublimate into the atmosphere and filtered water is reused. The hardness of dry ice particles is estimated at 2 on the Mohs scale, making them comparable to rock salt or calcite. These properties of dry ice increase the erosivity of the water jet, but there is no negative impact on the topography of the base material.High-pressure water jet treatment possesses an unquestionable advantage: after treatment, water is filtered and reused, thus protecting natural resources . In ordew, dry ice mass flow rate 2i and spray angle) on the surface removal efficiency QF was also investigated. Regression functions were developed for the average paint removal efficiency as a function of: dry ice mass flow rate 2 and spray angle κ. Based on these functions, the values of the optimal parameters to obtain the maximum process efficiency were determined.An experimental study was conducted to see if the application of high-pressure water-ice jet (HPWIJ) causes changes in the substrate material after paint removal. The influence of selected processing parameters . Following the guidelines from the Nobikor manufacturer, the surfaces painted were seasoned for 24 h. Once the primer had dried, the specimens were additionally coated with a non-metallic base paint: phthalic carbamide enamel with the trade name of Autorenolak F manufactured by Polifarb Cieszyn . The coat obtained was dried at ambient temperature (ca. 20 °C) for 10 days. Then, one layer of Spectral colourless paint was applied and seasoned for 7 days.Experimental investigations were carried out to determine the impact of high-pressure water-ice jet treatment on the surface morphology and the surface topography structure (ST). It was also important to obtain reliable information on the selection of appropriate hydraulic and technological parameters of the cleaning process to ensure maximum surface paint removal efficiency while not interfering with the substrate material. Several substrate materials were selected for testing and paint coating were applied to them. The main consideration was their application in various means of transportation vehicle . The mechanical properties of the materials used were also taken into account, as they determine the degree of damage to their geometric surface structure in the process of removing paint coatings with a water-ice jet . The firk = 200 mm. Water jet pressure pw = 20 MPa and pw = 35 MPa was used during the tests. The dry ice flow rate was altered in the range −1 with a step of 26 kg·h−1. The distance between the sprinkler head outlet and the surface treated  was l2 = 150 ÷ 400 mm and it was altered every 50 mm. The spray angle was changed in the range κ = 60 ÷ 90° with a step of 15°. Each test was repeated three times. Variants of the tests performed for X5CrNi18-10 steel substrate specimens are shown in Paint stripping was carried out using a four-hole helical nozzle with a water hole diameter of φ = 1.2 mm (TS = 4 × 1.2 mm). The appropriate shaping of the water-ice jet was carried out in the sprinkler head with an experimentally pre-determined length Lw = 25 MPa and pw = 30 MPa.An analogous study was conducted for samples with PA2 aluminum alloy substrates and polymethyl methacrylate (PMMA). After determining the central values  for the dry ice output and the working length of the stream and the angle of spray, tests were carried out for the stream with pressure pExperimental investigations were carried out to determine the impact of the water-ice jet on the base material. For this reason, the macro- and microstructure of the surfaces of both untreated samples and samples after removal of the varnish coating with a high-pressure water-ice jet were evaluated. A JOEL JSM-5500LV scanning electronmicroscope was used to evaluate the surface morphology of the specimens examined. It was equipped with a computer system for recording and measurements that allows archiving the results obtained. The surface topography (ST) of the specimens was assessed on the basis of measurements made by means of a Talysurf CLI 2000 spatial profilometer manufactured by Taylor-Hobson. The measurement results recorded were processed and analysed using TalyMap software. The samples for measurement were 3 × 3 mm in size. Two hundred and one profiles were recorded during the measurement. The distance between the profiles was 15 µm. On one profile 1501 points were registered. The distance between the points of the profile was 2 µm. Each measurement was performed in single-pass mode. Measurements were made at the same sample location, i.e., the center of the sample. The maximum height of surface roughness (Sz) and the arithmetic mean surface roughness (Sa) were recorded during testing. The definitions and notation of spatial parameters are well known ,40. For w = 20 MPa. w = 35.A summary of the measurements of the arithmetic mean surface roughness Sa and the maximum surface roughness height Sz of the X5CrNi18-10 steel samples before paint coating is presented in w = 20 MPa. w = 35 MPa.A summary of the measurements of the arithmetic mean surface roughness Sa and the maximum surface roughness height Sz of the PA2 aluminium alloysamples before paint coating is presented in w = 20 MPa. w = 35 MPa.A summary of the measurements of the arithmetic mean surface roughness Sa and the maximum surface roughness height Sz of the PMMA polymethyl methacrylate samples before paint coating is presented in Fm2·h−1 was determined during testing. The mean value of the surface treatment efficiency 2·h−1 and the standard deviation and spread were calculated for three samples treated with identical parameters.Experimental tests were carried out in accordance with the static factor-selection programme shown in The mean values of the object’s outputs −1] or {b} of the unknown coefficients in Equation (5) was calculated from the matrix formula:Using matrix calculus, the column vector  results .The boundaries of the confidence region for Regression Function (1) were determined from the following formula:N—number of measurement points in the experimental design N = 5, 6, 7 or 9,L—number of unknown coefficients in Regression Equation (1); here, L = 4, results .The test results after statistical processing according to the algorithm presented in the study  were useThe regression equations developed take the following form.2·h−1] of the removal of the paint coating from the surface of the X5CrNi18-10 steel specimen from the dry ice mass flow rate −1]:Dependence of the average surface efficiency 2·h−1], of the removal of paint from the surface of the PA2aluminiumalloy specimen from the dry ice mass flow rate −1]:Dependence of the average surface efficiency 2·h−1], of the removal of the paint coating from the surface of the X5CrNi18-10 steel specimen from the working jet length Dependence of the average surface efficiency 2·h−1], of the removal of the paint coating from the surface of the PA2 aluminium alloy specimen from the working jet length Dependence of the average surface efficiency 2·h−1], of the removal of the paint coating from the surface of the PA2 aluminium alloy specimen from the spray angle κ [°]:Dependence of the average surface efficiency 2·h−1] of the removal of the paint coating from the surface specimen from the water-ice jet pressure pw [MPa]:X5CrNi18-10 steelDependence of the average surface efficiency PA2 aluminium alloypolymethyl methacrylate PMMAThe surface quality of the base after treatment plays an important role in the paint stripping process. In order to verify whether the High-Pressure Water Ice Jet (HPWIJ) treatment does not damage the base, the results of 3D surface topography (ST) and morphology of the surface of the specimens before and after paint stripping were compared.An example of a scanning electron microscope SEM image of the surface morphology of an X5CrNi18-10 steel specimen before paint coating is presented in w = 20 MPa and pw = 35 MPa), scanning electron microscope images of the surface were observed, an example of which is shown in After removal of the paint coating using a high-pressure water-ice jet, carried out with different treatment parameters .On the basis of the results obtained from the research conducted, it was established that the removal of paint coatings from various base materials is possible without disturbing the structure of the base material. However, in order to accomplish this, it is necessary to know the values of the technological parameters which enable the cleaning process to be carried out correctly. From the perspective of practical applications, for operators that employ a high-pressure water-ice jet for removing paint coatings, it is not only the effect of such technology on the base material that is of great importance but, above all, this is the impact on the surface treatment efficiency achieved during work. First, it should be noted that paint stripping with a high-pressure water jet cannot be compared to conventional abrasive blasting methods ,45,46,472 dry ice particle output on the surface paint coat stripping efficiency of X5CrNi18-10 steel for jet pressures pw = 20 MPa and pw = 35 MPa. As the pressure of the water jet increases, its velocity increases, and so does the velocity of CO2 dry ice particles that are accelerated by it. The higher kinetic energy of dry ice particles results in a stronger mechanical (impact) effect on the surface being treated. The sublimation of CO2 particles in the treatment region is also more intense. In addition, a higher-pressure water jet exerts a greater force on the paint coating to be removed. This is why the microcracks in the top layers of the paint film are separated and torn apart more quickly. Consequently, a higher pressure water-ice jet treatment leads to an increase in the surface paint coat stripping efficiency. At the same time, it should be noted that a higher pressure water jet, with its higher kinetic energy, is able to accelerate a larger mass of dry ice to the maximum speed than a lower pressure jet.2·h−1 is obtained with dry ice mass flow rate −1 and water jet pressure pw = 20 MPa. An increase in dry ice flow rate results in a higher number of CO2 particles hitting the surface being cleaned. Weakened by the impact of the dry ice particles and of the water jet, the outer layer of the paint coatis removed more quickly. Doubling the CO2 dry ice output (−1) (2·h−1). The optimum dry ice particle output to be used for stripping with water-ice jet pressure pw = 20 MPa is −1. This being the case, surface paint coat stripping efficiency reaches the value (2·h−1). With an increasing CO2 output, the decreasing number of dry ice particles to be used to remove the paint coating on the “unit surface” indicates that the “supercooled” water jet fully accelerating dry ice particles added to it is a tool with maximum erosivity. A flow rate of CO2 particles that is too high causes the nozzle pass to be “choked” with excess ice. The water jet is then unable to accelerate dry ice particles so that they could achieve maximum velocity. Therefore, with a flow rate being too high, dry ice particles have less kinetic energy compared to particles that are fully accelerated. The end result is a decrease in surface paint coat removal efficiency. In addition, using CO2 mass flow rate that is too high results in increased treatment costs, which is undesirable from a practical point of view. For example, with dry ice mass flow rate of −1, surface paint coat stripping efficiency decreased to QF = 0.116 m2·h−1. Under these conditions, more than 75 CO2 dry ice particles have to be used to obtain 1 mm2 of cleaned surface.In the case of X5CrNi18-10 steel specimens, a minimum surface paint coat stripping efficiency of  kg·h−1) a leads tw = 35 MPa  (−1 (for pw = 20 MPa)  b and m˙L 20 MPa) a is exce2  and the surface being treated on the maximum surface efficiency QF of paint coat stripping. It should be noted that when using a working jet length that is too small, high erosivity is achieved but not the highest treatment efficiency is obtained. In the case of the base made of X5CrNi18-10 steel, an increase in the working length of the water-ice jet from l2 = 150 mm to l2 = 200 mm is accompanied by an increase in the surface paint coat stripping efficiency by 18 % on average when using a water-ice jet with pressure pw = 20 MPa (2 = 150 mm to l2 = 250 mm. Any further increase in the working jet length from l2 = 150 mm to l2 = 300 mm only results in a 9% increase in stripping efficiency. The use of length l2 = 350 mm leads to a 13% reduction (compared to that obtained with l2 = 150 mm) in the surface paint coat stripping efficiency. For the greatest spray length used of l2 = 400 mm, the reduction in treatment efficiency is just over 41%. The results from the use of a water-ice jet with pressure pw = 35 MPa (w = 20 MPa).= 20 MPa a. The ma= 35 MPa b are simk = 200 mm is presented in 2 = 150 mm to l2 = 200 mm is accompanied by an increase in the surface paint coat stripping efficiency caused by an increase in the impact area of the water-ice jet.The surface paint coat stripping efficiency as a function of the working jet length for PA2 aluminium alloy specimens using a water-ice jet created in a sprinkler head with length Lw = 20 MPa (2 = 150 mm to l2 = 250 mm. Further increase of the working length from l2 = 150 mm to l2 = 300 mm only results in a 9% increase in surface paint coat stripping efficiency. The use of l2 = 350 mm leads to a 15% reduction (compared to that obtained with l2 = 150 mm) in surface paint coat stripping efficiency. For the largest working jet length used l2 = 400 mm, the decrease in treatment efficiency is just over 41% (w = 35 MPa (w = 20 MPa).For a water-ice jet with pressure p= 20 MPa a, this iover 41% a. The re= 35 MPa b are sim2 dry ice particles with the temperature of 194.6 K, a local spot depression of its temperature occurs, which leads to the formation of thermal stress that causes microcracks, which initiate the erosion process [w = 20 MPa and pw = 35 MPa is presented in In the paint stripping process, apart from dry ice output and the distance between the sprinkler head outlet, the spray angle is also of great importance a,b. This process . The effWith an increase of the jet spray angle κ = 30 ÷ 60°, there is an almost linear increase in surface paint coat stripping efficiency. Further increase of the spray angle up to κ = 90° causes an increase of the treatment efficiency yet that is not as sharp as observed with smaller spray angles. It can therefore be accepted that the best machining results are obtained with a spray angle of κ = 75 ÷ 90°.w = 20 MPa to pw = 25 MPa increased the treatment efficiency to ∆2·h−1 for the PA2 aluminum alloy. Further increase of the jet pressure to pw = 30 MPa, with unchanged other processing parameters, resulted in paint removal efficiency of 2·h−1. This is an outgrowth of the treatment efficiency by ∆2·h−1 compared to that obtained for a jet with pressure pw = 25 MPa.The effect of water jet pressure on paint removal efficiency is shown in w = 35 MPa does not cause any changes in the geometric structure of the surface of X5CrNi18-10 steel and PA2 aluminium alloy.The use of a high-pressure water-ice jet with maximum pressure pw = 20 MPa and dry ice flow rate of −1. The use of a water jet with pressure pw = 35 MPa and dry ice flow rate −1 causes traces of jet impact in the form of small chipping of the base material (PMMA polymethyl methacrylate).In the case of brittle materials, such as polymethyl methacrylate PMMA, which form bases for lacquer coatings, it is necessary to use a water-ice jet with the maximum working pressure of pw = 20 MPa, the best results are obtained when dry ice flow rate is −1, while for water jet pressure pw = 35 MPa, this output should not exceed −1.The highest surface treatment efficiency is obtained with a higher water jet pressure and an appropriately increased dry ice particle flow rate. For water jet pressure p2 = 250 mm and the spray angle needs to be κ = 75 ÷ 90°.In order to obtain the maximum paint coat stripping efficiency irrespective of the water jet pressure and the dry ice particle flow rate, the working length of the jet needs to be lw = 20 ÷ 35 MPa.Increasing the pressure of the water jet results in higher efficiency of paint removal. The increase of processing efficiency is proportional in the tested range of jet pressure pThe above research proves that one of the effective methods of removing paint coatings is the use of a high-pressure stream of water with ice. This method of processing allows for effective preparation of the substrate for the application of a renovation paint coating, while simultaneously maintaining appropriate processing parameters in that it does not change the geometric structure of the substrate.The optical microscope by Kestler—Vision Engineering Dynascope Ltd. with the ND 1300 Quadra-Chek measuring system and the sensitivity of ±0.001 was used to measure the surface quality of the processed materials. Based on the research and analysis of the results, it was found that: