Most adolescent girls in the UK do not meet government physical activity recommendations and effective interventions are needed. This study reports the results of a feasibility trial of PLAN-A, a novel school-based peer-led physical activity intervention for adolescent girls.A two-arm cluster randomised controlled feasibility study was conducted in six English secondary schools (4 intervention & 2 control). Year 8 (age 12-13) girls were eligible and randomisation was at school-level. The intervention involved training Year 8 girls , who were identified by their peers as influential, to provide informal support to their friends to increase their physical activity. Feasibility of the intervention and the research was examined, including: recruitment, training attendance and data provision rates, evidence of promise of the intervention to affect weekday moderate-to-vigorous physical activity (MVPA), intervention cost and estimation of the sample size for a definitive trial. Accelerometer and questionnaire data were collected at the beginning of Year 8 (Time 0), the end of Year 8 (10-weeks after peer-supporter training) and the beginning of Year 9 (Time 2).Four hundred twenty-seven girls were recruited (95% recruitment rate). 55 girls consented to be a peer-supporter and 53 peer-supporters were trained (97% of those invited). Accelerometer return rates exceeded 85% at each time point and wear time criteria was met by 83%, 71% and 62% participants at Time 0, 1 and 2 respectively. Questionnaire data were provided by >91% of participants at each time point. Complete-case adjusted linear regression analysis showed evidence of a 6.09 minute  between-arms difference in weekday MVPA at Time 2 in favour of the intervention arm. On average PLAN-A cost £2685 per school to deliver (£37 per Year 8 girl). There were no adverse events. A trial involving 20 schools would be adequately powered to detect a between-arms difference in weekday MVPA of at least six minutes.The PLAN-A intervention adopts a novel peer-led approach, is feasible, and shows evidence of promise to positively affect girls’ physical activity levels. A definitive trial is warranted.http://www.isrctn.com/ISRCTN12543546ISCTRN, ISRCTN12543546, Registered on 28/7/2015, URL of registry record: The online version of this article (10.1186/s12966-018-0682-4) contains supplementary material, which is available to authorized users. Physical activity is associated with benefits to young people’s physical and psychological health –3. The URecent evidence syntheses have identified that previous interventions have had small positive effects on the physical activity of adolescent girls , 11, witCorrelates of girls’ physical activity include physical self-perceptions, self-efficacy, perceived competence and enjoyment . Yet, giDespite the potentially important role of peers in adolescent girls’ physical activity, there is a lack of robust trials  that targeted adolescent girls’ physical activity could hold promise as a means of promoting physical activity. We therefore developed the PLAN-A intervention  and in line with the UK Medical Research Council (MRC) framework on the development and evaluation of complex interventions , tested The study was a two-arm cluster randomised controlled feasibility study in six secondary schools to compare the PLAN-A intervention against a usual-practice control with embedded process and health economics evaluations. The study protocol was published in 2016 , 26 and Schools were eligible and invited to participate via letter and email to the Head Teacher if they were; state-maintained mainstream secondary schools in Wiltshire and South Gloucestershire (South West England), had girls in Year 8 (aged 12-13 years), were above the median of the local Pupil Premium Indicator  , were noFollowing the completion of baseline data collection, all schools were randomly allocated (school being the unit of randomisation), at an intervention:control ratio of 2:1 stratified within Local Authority area (Wiltshire & South Gloucestershire). Four schools were allocated to the intervention arm and two schools to the control arm by a member of the Bristol Randomised Trials Collaboration who was blind to the school identity and independent of the study. The statisticians, and all team members except the Project Manager, Research Associate and Fieldworkers were blind to allocation.The intervention was adapted from the ASSIST intervention through detailed formative research and its refinement and piloting is described in detail elsewhere . BrieflyCommensurate with ASSIST, the PLAN-A intervention was primarily grounded in Diffusion of Innovations theory (DOI)  in whichPeer-supporter training was delivered by female trainers who had attended a three-day (≈ 15 hours) education programme delivered by the study team . Trainers were recruited as freelance individuals  or through physical activity/sport promotion teams to have a range of experiences in working with young people and physical activity promotion. Training covered gender inequalities in physical activity, the PLAN-A concept  and theory in addition to teaching trainers about the sessions, activities and resources and giving time to practice delivery. Peer-supporter training was delivered by trainers working in pairs and trainers had time to work in their pairs and establish their delivery plan. Trainers were given a manual containing all resources needed.Who do you respect? Who are good leaders in sport or other group activities? Who do you trust? and Who do you look up to?). Girls could list up to five girls for each question. The 18% of girls with the most nominations were invited by letter to be a peer-supporter and attended a peer-supporter briefing where the role and training were explained and written information and consent forms were distributed. 18% were invited with the aim of recruiting 15% of the year group as peer-supporters in line with DOI.Prior to randomisation, Year 8 girls in all schools completed a four-item peer-nomination questionnaire to identify the influential girls in their school . Peer-supporters were provided with return transport from school to the training by coach  and were chaperoned by a member of school staff. Training for one school was held on the school site due to the school being unable to release staff to chaperone. The interactive training blended education about the importance of physical activity, inspiration and empowerment of the girls to be active and development of the interpersonal skills needed to be a peer-supporter , immediately post-intervention  and follow up .At baseline (Time 0) participants reported their date of birth and ethnicity. Socio-economic position was estimated by: (1) an index of multiple deprivation (IMD) using home postcode and the English Indices of Deprivation  for eachRecruitment of schools and Year 8 girls (opt-out consent rate) was recorded at Time 0 and retention was recorded at Time 1 and 2. Recruitment of peer-supporters (consent rate) and peer-supporter trainers was recorded by the field team as well as attendance of peer-supporters at the briefing meeting and each peer-supporter training day.Participants wore a waist-worn ActiGraph accelerometer  during waking hours which recorded data at 30Hz for seven days. Participants were instructed to remove the accelerometer while sleeping, showering/bathing and participating in water sports. Scores derived from ActiGraph accelerometers have been shown to provide reproducible and valid estimates of physical activity amongst adolescents . PeriodsFull details of the psychosocial questionnaires will be published in a separate process evaluation paper. For the purposes of reporting data provision rates in this paper, pupils completed a survey  using Samsung Galaxy Table The economic analysis aimed to assess the feasibility of collecting the data required to cost the intervention and conduct a cost-effectiveness analysis in a future definitive trial. The affordability and potential cost-effectiveness of the intervention was also explored. A public-sector perspective was taken assuming that if scaled up the intervention would be funded by schools or local government. Data were collected using expense claim and data collection forms, completed by the research team, students and school contacts. Students’ quality of life was assessed using the EQ-5D-Y . ResponsDescriptive summary statistics were calculated by trial arm at baseline. Continuous data were analysed using means and standard deviations or median and inter-quartile range, where the data were markedly skewed. School and student recruitment and retention were presented as a CONSORT flow chart for schools by trial arm. Recruitment, retention and data provision were also summarised descriptively.Summary statistics for the  primary and secondary outcomes are presented, by intervention and control group according to the allocation of the student’s school  analysis). Mixed effects linear regression was used to estimate the adjusted differences in means between intervention and control groups. School-level variance in the outcome was accommodated by inclusion of school as a normal distribution random effect, and any remaining differences in the baseline assessment of the outcome measure, and local authority area (by which the randomisation was stratified), was accommodated by their inclusion as covariates. Each difference in means is presented with its 95% confidence interval. As this study was a feasibility study and not powered to detect differences between arms, p-values are not presented.Two pre-specified sensitivity analyses were conducted assessing the impact of missing data. Firstly, where there were 5% or more missing MVPA measures, missing MVPA data were imputed for Time 1 and Time 2, in turn using Multiple Imputation by Chained Equations . Fifty iThe school-level intra-class correlation (ICC) coefficient for weekday MVPA was estimated in a variance component random effects analysis. As this gives an imprecise estimate in a study of this size, the data were compared with ICC estimates from other studies , 41, 42.School and participant recruitment and retention are shown in Fig. At each time point, accelerometer return rates exceeded 85% Table . At TimeThe trial arms were well balanced at baseline Table . IMD i.. Peer-suAlmost all ) peer-supporters attended all of the training (Table There was no evidence of a difference in weekday or weekend MVPA between intervention and control groups at Time 1 Table . There wThe intra-class correlation (ICC) on weekday minutes of MVPA  from the PLAN-A sample at each time point was; Time 0: <0.00 , Time 1: 0.02  and Time 2: <0.0001 . Based on these findings, and ICCs from other studies involving adolescent girls, sample size calculations for a definitive trial were based on an ICC of 0.01 . To detect a 10-minute between arms difference in weekday MVPA with 90% power, an alpha of 5%, and with a design effect of 1.7 (based on 70 students per school), 560 students are required (280 per arm)  , to allow peer-supporters to work together and focus on a shared goal with their (non-school teacher) trainers, and to inspire peer-supporters with a special or “grown up” experience. As such, it would be ideal to deliver peer-supporter training off the school site. In future implementations of PLAN-A it would therefore be helpful to develop a persuasive pitch to school head teachers explaining the relatively low burden placed on school staff by PLAN-A, compared to other interventions , 44, 45 We showed that the research required to evaluate the effectiveness and cost-effectiveness of the intervention in a larger trial is feasible. Questionnaire data provision was very high (>90%) at all time points. Accelerometer return rates exceeded >85% at each time point, however compliance with the wear time criteria decreased to 62% at Time 2 and was 5-7% lower in the control versus intervention group. Despite this, the level of completely missing accelerometer data  was low. This suggests that whilst participants were willing to wear the accelerometers, strategies are needed to ensure that they are worn for long enough on measurement days, especially amongst control group participants, in a future definitive trial. Such strategies could include incentivising sufficient wear time rather than, or in addition to, monitor return , 47, ameThe study showed that the PLAN-A intervention has the potential to positively affect adolescent girls’ MVPA (≈ +6 minutes per weekday) and reduce their sedentary time (≈ -23 minutes per weekday) compared to controls 4-5 months post-intervention. The estimates in this study suggest that the between-arm difference represents a stemming of the decline in MVPA that was seen in the control group. Previous school-based physical activity interventions for adolescent girls show very small effects  and someThe evidence presented above suggests that the conduct of a larger definitive trial to determine the effectiveness and cost-effectiveness of the PLAN-A intervention is warranted. Sample size calculations suggested that this study should include 20 schools (ten intervention & ten control) and 1400 Year 8 girls to have sufficient statistical power to detect a 10-minute between-arms difference in MVPA. The details of minor amendments to the intervention content that were identified in the process evaluation will be described in a separate paper. In comparison to other studies PLAN-A has the potential to be good value. PLAN-A cost £37 per Year 8 girl and £6 per additional minute of weekday MVPA (based on the between-arms comparison at Time 2) which compares favourably, for example, to the multicomponent Physical Activity 4 Everyone trial  which haA main strength of this study was its methodology. The PLAN-A intervention was carefully adapted from an effective adolescent smoking cessation intervention model through formative work with key stakeholders . As suchIt is feasible to implement a secondary school-based peer-led physical activity intervention for adolescent girls based on trained influential students providing informal support to their year group peers. It is also feasible to study this intervention using a robust cluster-randomised trial design. The intervention shows promise to stem the age-related decline in adolescent girls’ physical activity, and overall the evidence presented suggest that a definitive trial is warranted to investigate the effectiveness and cost-effectiveness of the intervention.Additional file 1:Consort 2010 extension for pilot and feasibility trials checklist. Completed CONSORT checklist (version adapted for reporting pilot and feasibility studies). (DOC 225 kb)Additional file 2:Template for Intervention Description and Replication (TIDieR) checklist for PLAN-A.Completed TIDieR Checklist describing the PLAN-A intervention. (DOCX 16 kb)Additional file 3:Baseline descriptive statistics of peer-supporter and non-peer-supporter students. Descriptive data for peer-supporters and non-peer-supporters at baseline. (DOCX 15 kb)Additional file 4:EQ-5D-Y and QALYs results, by treatment arm. Baseline, Time 1 and Time 2 EQ-5D-Y results and corresponding QALY estimates, by treatment arm (DOCX 14 kb)Additional file 5:Sensitivity analysis of evidence of promise for intervention effects on weekday MVPA at Time 1 and Time 2 using the imputed data set and complete data where accelerometer wear was one or more valid day. Sensitivity analysis of evidence of promise for intervention effects on weekday MVPA at Time 1 and Time 2 using the imputed data set and complete data where accelerometer wear was one or more valid day (DOCX 13 kb)Additional file 6:Exploratory interaction of the intervention effect on weekday MVPA at Time 1 and Time 2 and peer supporter status. Exploratory interaction of the intervention effect on weekday MVPA at Time 1 and Time 2 and peer supporter status. (DOCX 13 kb)


The interface delamination as well as the composite-phase damage is the most serious failure dominating the bi-material machining. In this paper, an original finite element (FE) model was developed to inspect the key mechanisms governing the induced damage formation when cutting this multi-phase material. The hybrid composite model was constructed by establishing three disparate physical constituents, i.e., the Ti phase, the interface, and the CFRP phase. Different constitutive laws and damage criteria were implemented to build up the entire cutting behavior of the bi-material system. The developed orthogonal cutting (OC) model aims to characterize the dynamic mechanisms of interface delamination formation and the affected interface zone (AIZ). Special focus was made on the quantitative analyses of the parametric effects on the interface delamination and composite-phase damage. The numerical results highlighted the pivotal role of AIZ in affecting the formation of interface delamination, and the significant impacts of feed rate and cutting speed on delamination extent and fiber/matrix failure.In hybrid carbon fiber reinforced polymer (CFRP)/Ti machining, the bi-material interface is the weakest region vulnerable to severe damage formation when the tool cutting from one phase to another phase and  The enhanced mechanical properties and improved structural functions have given the material a high demand for manufacturing key aircraft structures subjected to high thermo-mechanical stresses. A typical application is the use of wing-fuselage connections in the new-generation Boeing 787 Dreamliner. The CFRP-to-Ti coupling typically provides the best combination of metallurgical and physical properties including high strength-to-weight ratio, low density, and superior corrosion/erosion resistance that favor energy saving in industrial applications ,2,3. Gen3 .Prior to their post applications, structural components made of hybrid CFRP/Ti composite are mostly manufactured in near-net-shape in order to achieve dimensional tolerance and assembly requirement. However, due to the disparate machinability behaviors of each stacked constituent, the manufacturing hybrid composite exhibits the most challenging task in industrial sectors. For instance, the titanium phase exhibits high mechanical properties, low thermal conductivity, and strong chemical affinity to tool materials, which commonly results in high force/heat generation, serious tool wear (abrasive wear and adhesion wear), and short tool life ,6,7. In i.e., the Ti-phase cutting, interface cutting, and CFRP-phase cutting. Among them, the interface region  represents the most difficult-to-cut zone vulnerable to severe damage formation when the tool edges are cutting from one phase (Ti phase) to another phase (CFRP phase) and vice versa. The interface region is usually characterized as a physically intermediate transition zone that really exists in the bi-material machining process. During interface cutting, the interface area usually suffers changeable chip-separation modes and experiences severe mechanical/physical phenomena transition exerted at the bi-material contact boundary. In such circumstances, the interface region becomes the most challenging cutting zone as compared to absolute Ti-phase cutting and absolute CFRP-phase cutting while machining the hybrid composite. The discontinuity of the tool-work interaction governing interface cutting commonly makes the machining behavior more complicated and interrelated. Inspections of interface damage in CFRP/Ti cutting via the experimental method have been shown to be very challenging and highly difficult [i) the key mechanisms and physical phenomena controlling the CFRP/Ti interface cutting, (ii) the parametric effects on the interface machining and subsequently induced damage extent, and (iii) the machinability classification of hybrid CFRP/Ti machining, i.e., which region of cutting actually reflects the machinability of pure Ti-phase cutting (MTi), stacked material cutting (MCFRP/Ti), and pure CFRP-phase cutting (MCFRP), respectively. In addition, the conventional experimental method is cost-prohibitive and time-consuming. In contrast, the numerical approach should be a qualified tool helpful to enable feasible inspections of the damage formation when cutting this bi-material. Furthermore, although a considerable amount of scientific work has dealt with single Ti-cutting modeling and single CFRP-cutting modeling, comprehensive numerical studies concerning hybrid CFRP/Ti machining have still been only rarely reported.In hybrid CFRP/Ti machining, typically three cutting stages are involved, ifficult . Despiteifficult ,2,12 havetc. The key objective of this investigation aims to establish an FE model for damage predictions and failure analyses when orthogonally cutting hybrid CFRP/Ti composite. The established numerical model incorporated three physical constituents, i.e., the Ti phase, CFRP phase, and interface layer. The CFRP/Ti model was rigorously validated prior to its post-application. The multiple aspects of machining responses including cutting process, interface delamination, and subsurface damage formation were precisely investigated via finite element (FE) analyses. A particular concentration was made to characterize the dynamic process of delamination formation and affected interface zone (AIZ). The numerical results highlighted the significant role of AIZ and bi-material interface consumption (BIC) in controlling the induced interface damage formation.These are the key incentives that motivated the current work to develop an original finite element (FE) model to address the mentioned topics. To inspect the fundamental mechanisms controlling the bi-material machining, the simplified orthogonal cutting configuration (OCC) was adopted. The OCC represents a convenient way to reveal the most fundamental machining physics governing the various actual manufacturing operations of hybrid CFRP/Ti composite, e.g., drilling, grinding, i.e., the tool part, Ti part, interface part, and CFRP part with total dimensions of 2 mm × 1 mm (L × H) for the workpiece material.In the current work, a 2D orthogonal cutting model was developed by using the commercial software Abaqus/Explicit code . To simulate the hybrid cutting operation, the machining process was specified as shown schematically in i.e., negative X-axis) to finalize the cutting simulation. The tool was configured by defined geometries of rake and clearance angles (α = 12° and γ = 7°) as depicted in X direction), as shown in The cutting tool was modeled as a rigid body and imposed by a cutting velocity on its reference node toward the horizontal direction (i) Zone A denoted the separated chip layer, (ii) Zone B signified the predefined separation path (joint layer) and (iii) Zone C represented the machined Ti surface, where mesh generations exhibited different characteristics. Both Zones A and B were defined by very fine mesh density, whereas Zone C was constructed by coarse mesh element density far away from the vicinity of the tool-work contact region. The surface-to-surface contact algorithm was used to model the interaction between the cutting tool, Zone A and Zone B. The kinematic contact algorithm was assigned to the contact pairs in order to avoid element penetrations. Friction in the orthogonal cutting commonly occurs at the contact surfaces of tool and workpiece, rake face and chip surface. The frictional shearing stress is the average of the shearing stress at the tool and chip interface. In the present work, the Coulomb’s friction law was utilized to describe the contact behavior.The Ti phase was modeled as a fully isotropic and homogeneous material. A four-node plane-strain thermally coupled quadrilateral element type CPE4RT, which has better convergence properties was utilized for a coupled temperature-displacement analysis and enhanced hourglass control was selected for the whole set of the Ti elements. The entire Ti phase was divided into three physical zones:  and globally exhibits anisotropic properties. However, for simulations in a macro-mechanical model, the reinforced laminate has commonly been assumed as an equivalent homogeneous material (EHM) by most numerical study cases ,14,15,16To link the Ti phase and CFRP phase together, an interface layer was introduced in the FE model and simulated as a quick transition zone by using a cohesive element. It should be stressed that the use of the interface layer here serves as a technical control for the “Ti-to-CFRP” contact management during simulation. A triangular traction-separation cohesive formulation with linear softening was used to represent its mechanical response. The assembly of the interface layer with both the Ti phase and CFRP phase was carried out by setting a constraint type join (tie constraint). Furthermore, two contact pairs (Int-A and Int-B) with specifications of penalty contact algorithm and rough friction formulation were established in the interface zone, Ti phase and CFRP phase as shown in E), thermal expansion coefficient (αT), thermal conductivity (λ) and specific heat (pc) were considered to be temperature-dependent in order to accurately represent the property variation of titanium phase versus thermal influence during the cutting process.The material behavior of the Ti phase was assumed to be isotropic and elastic-plastic with thermal softening by using the isotropic plasticity model available in the Abaqus/Explicit code. The material properties for the Ti phase (Ti6Al4V) are summarized in T is the cutting temperature of Ti6Al4V alloy during the machining process, mT is the material melting temperature, and rT is the room temperature. A, B, C, m and n are material constants, which are usually determined by fitting the strain-stress curves obtained by split-Hopkinson bar. In this simulation, the JC material constants are selected carefully from the open literature [In FE modeling, accurate material flow stress models are very much required to capture the constitutive behavior of the work material under high strain/strain rate/temperature encountered in machining. The constitutive model proposed by Johnson-Cook (JC) ,20 was aterature ,22 as shi.e., failure initiation law and failure evolution law, to describe the failure responses of ductile material as shown in In order to simulate the chip separation process, an energy-based ductile failure criterion was applied in the FE computation. The failure damage criteria consist of two-stage laws, Step 1: Damage Initiation. The JC failure model was used as a damage initiation criterion, which contains five failure parameters that need to be determined (D1–D5) as presented in Equation (2). In the JC failure model, damage initiation is assumed to happen when a scalar damage parameter (ω) reaches 1. The ω parameter is defined based on a cumulative law as described in Equation (3).P is the hydrostatic pressure, 1D–5D are JC damage parameters, ω is the scalar damage parameter and 1D–5D in the cutting simulation.In the above equations, Step 2: Damage Evolution. When the ductile material damage is initiated, the strain-stress relationship no longer accurately represents the real material behavior. Based on this, Hillerborg’s fracture energy proposal [fG) as a material parameter, and the fracture energy is represented as follows:proposal  was usedL denotes the characteristic length, In the equation, L) was defined as a half typical length of a line across a second order element. As the direction in which fracture occurs was not known in advance, so the definition of characteristic length was used.The FE model applied the planar quadrilateral continuum element (CPE4RT), and then characteristic length (lD) was used for the joint layer according to the following equation:A linear damage parameter  was used for the chip layer according to the following equation:In contrast, an exponential damage parameter (D represents the damage parameter (lD or eD).At any given time during the FE calculation, the equivalent flow stress in the material is given by the following equation:fG is provided as an input parameter and theoretically is a function of Poisson’s ratio (υ), Young’s modulus (E), and fracture toughness (CK) as shown in Equation (9). Considering the different fracture mechanics [fG)I for the joint layer (Zone B) and (fG)II for the chip layer (Zone A). The (fG)I denotes the fracture energy of mode I which is a tensile mode  whereas (fG)II signifies the fracture energy of mode II which is a shearing one .In this study, echanics  occurrini.e., fiber-tensile failure, fiber-compression failure, matrix-tensile failure, and matrix-compression failure, as summarized in 11 signifies the stress in the fiber direction, σ22 denotes the stress in the transverse direction, and σ12 represents the in-plane shear stress.For CFRP phase, the simulated material was unidirectional carbon/epoxy T300/914 laminate  and its mechanical/physical properties are summarized in i.e., when one type of the fiber/matrix failure occurs, the relative material properties will be degraded as shown in i) the first variable noted HSNFTCRT represents the fiber-tensile failure mode; (ii) the second HSNFCCRT represents the fiber-compression failure mode; (iii) the third HSNMTCRT represents the matrix-tensile failure made and (iv) the fourth HSNMCCRT represents the matrix-compression failure mode.The element erosion of the CFRP phase is conducted through the concept of stiffness degradation., During the FE computation, the material properties at each integration point were evaluated and degraded depending on which set of failure mode was used. If any failure index reached unity, the relevant material properties were automatically reduced to zero according to the implemented stiffness degradation scheme. The procedure was repeated until the occurrence of complete chip formation.etc. The surface-based traction-separation law with linear softening was adopted to produce the mechanical responses of the cohesive interaction. The failure initiation law required to motivate damage among the interface layer is based on the quadratic stress criterion as illustrated in the following equation.The interface model used here aims to serve as a technical control for the “Ti-to-CFRP” contact management and to facilitate the characterization of interface damage formation during the simulation. It should be noted that in real CFRP/Ti configurations, some of them do not exist with such a third layer and only combine together for machining. The interface layer was modeled by using cohesive interaction allowing interfacial-damage propagation between the two joint phases as a fracture mechanics phenomenon with a very small thickness (probably 5 μm). Note that the use of a small interface thickness aims to minimize its influence on some other machining responses such as Ti/CFRP chip separation modes, force generation, 33, σ13 and σ23 represent the normal traction stress, and shear traction stresses in two directions, respectively; In which, σnG, sG and tG).Once the damage onset was satisfied, the Benzeggagh-Kenane (BK) damage criteria  and potenG, sG and tG are the released rate energies in the normal and two shear directions respectively; In which, nnK, ssK and ttK), the interface resistance in each direction  could be ignored. Besides, the input parameters for interface zone were also selected carefully from the literature, in which it had already been validated with experimental results and indicated good suitability for multi-material modeling. Therefore, the validation work was performed solely focused on Ti phase verification and CFRP phase verification by referring to the open literature. Each model was improved and refined carefully until it was capable to replicate consistent results with the experimental observations. For validation purposes, all the numerical simulations were run under the same cutting conditions as used in the literature.i.e., valley, peak, and pitch, as compared with the experimental results from the literature [The Ti-phase model was validated rigorously by means of force generation and chip morphology with experimental data from the literature ,35,36, wversus fiber orientation (θ). It was noticeable that the predicted force magnitudes of both cutting force and thrust force yielded a strong correlation and a consistent variation trend with the experimental results gained by Iliescu et al. [Moreover, the CFRP-phase model was validated through the simplest manner of force generation (cutting force and thrust force) comparison. u et al. , which ccv and f), the cutting conditions used in the FE simulations were selected based on the compromise selection of the optimal parametric range for both the two phases of machining. Special focus was made on the analyses of the cutting process, interface damage formation, and parametric effects on fiber/matrix damage extent.In this Section, numerical analyses concerning hybrid CFRP/Ti cutting are presented with the aim of better machining comprehension. Since Ti phase cutting and CFRP phase cutting require different optimal cutting parameters  and the thrust-force component (tF), which signify the tribological interactions between tool rake and chip surfaces, together with tool flank and machined surfaces, respectively. It was noticeable that typically three cutting stages referring to the Ti-phase cutting, interface cutting, and CFRP-phase cutting could be seen from the force signal variation and the chip-morphology evolution.The cutting process of hybrid CFRP/Ti exhibits quite differently from the single-composite and single-metal cutting cases due to the multi-tool-work interaction domains. The disparate natures of each constituent make the chip separation modes more interrelated and coupled governing the bi-material interface consumption (BIC) ,38. The t) approximately exceeded 1.0 ms, the cutting-force component was observed to suffer a gradual reduction. The physical phenomenon could be explained by the decreased uncut Ti-chip thickness contributing to the reduction of force resistance when the tool tended to finalize the Ti-phase cutting. With tool advancement, especially when the cutting edges cut into the interface region, the previously-resected Ti chip adhered on the tool rake face and replaced the tool edges for further chip separation. Due to the transition from tool-Ti interaction to tool-CFRP interaction, the cutting force magnitudes underwent quick drop throughout the interface cutting. In such circumstances, the chip separation mode shifted gradually from plastic-defamation into brittle-fracture, which inevitably resulted in the serious transfer of mechanical/physical loads exerted on the tool-work system. The harsh cutting conditions dominating the CFRP/Ti interface cutting was the key contributor promoting the severe damage formation in the bi-material interface.As depicted in With further cutting progression, the tool tip cut across the interface region completely and induced a large extent of delamination damage focused on the interface area as shown in the magnified view of Moreover, when the cutting tool approached the finish of the cutting process, a significant increase of cutting force generation became pronounced as portrayed in cv = 40 m/min, f = 0.2 mm/rev and θ = 0°. It was noticeable that the key characteristic of the interface damage was a “V-shape” like notch damage focused on the CFRP-Ti contact boundary. A large extent of delamination damage became pronounced in both tool-cutting direction (X-direction) and through-thickness direction (Y-direction) as depicted in In CFRP/Ti cutting, the bi-material interface consumption (BIC) signifies the tremendous mechanical/thermal energy transfer arising from disparate phase cutting, the interrelated chip separation modes, and mixed machining responses controlling the hybrid cutting process. BIC plays a significant role in affecting the final surface quality and subsurface damage formation. The interface cutting can be identified as the weakest region vulnerable to serious damage formation. X-direction displacement versus cutting time (t) as shown in To specify the dynamic process of delamination formation and also the affected interface zone (AIZ), nine interface nodes beneath the trimmed plane were selected to characterize their X displacement evolution of the selected interface nodes with respect to the cutting time (t) under the fixed cutting conditions of cv = 40 m/min, f = 0.2 mm/rev, and θ = 0°. It was apparent that the X displacements of the selected interface nodes generally underwent three variation stages, i.e., the initial variation stage, rapid variation stage, and steady variation stage during the total cutting duration (t = 3.0 ms). At the beginning of cutting, all the selected interface nodes were predicted to suffer slow-rate displacement variation, which indicated that the chip removal process initially exhibited slight/minor effects on the interface region. However, with the cutting progression, the machining operation gradually exerted significant influences on the output responses of the interface region with evidence that all the selected interface nodes began to experience high-extent displacement fluctuations and quickly enter into the rapid variation stage. The physical phenomena were predicted to take place in the Ti-phase cutting period approximately at a cutting time of 1.0 ms. Such evidence signified the advent of the affected interface zone (AIZ) prior to the interface-cutting period as illustrated in X displacement compared to their counterparts far away from the trim plane. The reason might be due to the different levels of influences arising from tool-work interactions. When the cutting time approximately reached 2.0 ms, the X displacements of the selected interface nodes gradually achieved the steady variation state, indicating the diminishing trend of the cutting influences on the interface region. For instance, as shown in X and Y directions reached their stable values. In such a case, the AIZ could be defined probably at a duration of tX direction except the interface node 1. The dramatic displacement increase of interface node 1 demonstrated that the node had been deleted completely from the interface zone.i.e., XD, YD, and DS were introduced for evaluation in this study, where XD signified the delamination length in the tool-cutting direction (X-direction), YD denoted the delamination length in the through-thickness direction (Y-direction) and DS implied the delamination area of the damage zone as shown schematically in DS) was defined as illustrated in Equation (13).To quantify the delamination extent occurring in the interface zone, three indicators, versus cutting time (t). The results confirmed that all the delamination indicators  exhibited similar evolution trends as the X displacements of the selected interface nodes versus cutting time (t) in i) Ti-phase cutting, (ii) interface cutting, and (iii) CFRP-phase cutting in terms of their respective cutting lengths. However, such a category actually couldn’t reflect the real machining behavior of the bi-material system due to ignorance of the interactive cutting influences arising from each-phase cutting. In reality, the entire hybrid cutting process could be divided into three basic zones based on the interactive influences of each phase machining.i) Pure Ti-phase cutting tMTi).(ii) AIZ cutting tMCFRP/Ti).(iii) Pure CFRP-phase cutting tMCFRP)., indicating that no delamination occurred in the interface zone. With the continuation of the cutting process, especially when t reached the AIZ, the three delamination indicators totally suffered a dramatic increase with elevated cutting time. Such a phenomenon signified that the delamination damage was principally formed at the AIZ cutting period. Besides, the subsequent reduction of the delamination indicators in the AIZ might be caused by the bouncing-back effects from adjacent CFRP elements affected by the cutting operation. Moreover, with further increased t, especially in the pure CFRP-phase cutting zone, XD, YD and DS gradually reached their steady values, which meant that the later cutting process (post CFRP-phase cutting) generated less effect on the interface delamination formation.During the pure Ti-phase cutting zone, all I” indicates the cutting duration of AIZ and the interface delamination extent (D) was measured based on the YD length (delamination length in the through-thickness direction (Y-direction)). As can be seen in cv and f were found to have significant effects on the AIZ cutting time. Specifically, cv exhibited a negative impact on the AIZ cutting duration while the impact of f was positive. A parametric combination of high cv and low f commonly resulted in the lowest AIZ cutting duration. Moreover, as is evident in cv was found to be negative, i.e., an increase of cv resulted in the reduction of D, while the impact of f was positive. The mechanisms controlling the variation phenomena could be associated with their specific influences on AIZ. This was because when cv was elevated, typically a reduced cutting time of AIZ was achieved as illustrated in f usually led to an increased AIZ  under the fixed cutting conditions of cv = 40 m/min and f = 0.2 mm/rev as depicted in θ exhibited significant influence on the evolution of the various types of fiber/matrix damage during the chip removal process. The subsurface damage extent increased with elevated θ, especially the elevation became pronounced when θ was above 60° as depicted in et al. [Apart from the severe interface imperfection, the subsurface damage occurring in the composite phase is also a particular concern when machining hybrid CFRP/Ti composite. As discussed above, the composite-phase damage takes place following four types of fiber/matrix failure, n et al. , and Wann et al.  when cutcv and f on the cutting-induced composite-phase damage. Note that the composite-phase damage (DCFRP) was evaluated based on the type of fiber/matrix failure that caused the largest extent of damage  might benefit the reduction of composite-phase damage formation when machining hybrid CFRP/Ti composite. Therefore, based on the above analyses, the optimum cutting parameters for composite damage minimization should consist of small fiber orientation, high cutting speed, and low feed rate when machining this bi-material system.In addition, In this paper, a finite element model was developed to simulate the chip formation process with orthogonal cutting of hybrid CFRP/Ti composite. The proposed OC model attempted to address the fundamental mechanisms dominating hybrid composite machining. The numerical studies provided a better comprehension of various cutting phenomena induced in the hybrid cutting process. Based on the results acquired, key conclusions can be drawn as follows.i.e., Ti-phase cutting, interface cutting, and CFRP-phase cutting. The chip separation modes controlling the bi-material cutting comprise both elastic-plastic deformation and brittle fracture. The changeable chip removal mechanisms typically resulted in extremely harsh conditions dominating the interface cutting. Severe force fluctuation was observed as the main characteristic governing the hybrid CFRP/Ti cutting. Serious chip adhesion on the tool rake face was found to be the key factor contributing to the dramatic increase of cutting force generation in post CFRP-phase machining.(1) The disparate natures of each stacked constituent resulted in the hybrid cutting process exhibiting three distinct cutting stages, (2) Numerical results highlighted the occurrence of severe delamination damage concerning the CFRP/Ti interface. FE analyses revealed that the initiation and evolution of interface delamination primarily took place at the cutting period of AIZ. The formation of CFRP/Ti interface damage involved a series of detaching and tearing variations governing the AIZ cutting time. The key mechanisms governing the interface damage formation could be attributed to the coupling effects of fracture modes I and II arising from the unique mechanical loads exerted on the bi-material interface region. The key morphological characteristic of the interface delamination was a “V-shape” like notch focused on the CFRP-Ti contact boundary.cv and f) indeed exhibited pronounced effects on the interface delamination extent via the manner of their influences on the AIZ. For minimizing severe interface damage formation, a parametric combination of high cutting speed and low feed rate is strongly preferred.(3) Parametric studies confirmed the significant role of the cutting speed and feed rate in affecting the AIZ cutting duration. The input variables  The fiber/matrix damage promoted in hybrid CFRP/Ti cutting was carefully studied i) pure Ti-phase cutting, (ii) AIZ cutting and (iii) pure CFRP-phase cutting. Also, among them, the AIZ cutting in reality reflects the real machinability of the stacked CFRP/Ti material (MCFRP/Ti), which can be considered as the most difficult-to-cut zone due to the occurrence of interrelated cutting influences and the existence of the weakest interface zone. In such a case, to fundamentally improve the machinability of the hybrid CFRP/Ti composite, special focus should be made concerning the in-depth mechanism investigation and cutting-parameter optimization of the AIZ cutting zone. In the future, more systematic studies should be performed to address precisely the aforementioned issues.(5) Another key finding revealed in this paper showed that the entire hybrid cutting could be physically divided into three cutting zones from the viewpoint of the interrelated influences arising from each-phase machining: (


Early-life adversity is associated with accelerated cellular ageing during development and increased inflammation during adulthood. However, human studies can only establish correlation, not causation, and existing experimental animal approaches alter multiple components of early-life adversity simultaneously. We developed a novel hand-rearing paradigm in European starling nestlings (Sturnus vulgaris), in which we separately manipulated nutritional shortfall and begging effort for a period of 10 days. The experimental treatments accelerated erythrocyte telomere attrition and increased DNA damage measured in the juvenile period. For telomere attrition, amount of food and begging effort exerted additive effects. Only the combination of low food amount and high begging effort increased DNA damage. We then measured two markers of inflammation, high-sensitivity C-reactive protein and interleukin-6, when the birds were adults. The experimental treatments affected both inflammatory markers, though the patterns were complex and different for each marker. The effect of the experimental treatments on adult interleukin-6 was partially mediated by increased juvenile DNA damage. Our results show that both nutritional input and begging effort in the nestling period affect cellular ageing and adult inflammation in the starling. However, the pattern of effects is different for different biomarkers measured at different time points. Experiencing adverse conditions during early life is associated with reduced survival and an increase in morbidity from many different sources, even if the adult environment is benign. For example, in humans, childhood poverty and maltreatment are associated with premature mortality and an increased risk of major non-communicable diseases such as coronary heart disease124579101113The impact of early-life adversity may be measurable at the cellular level long before the onset of adult disease and senescence. Specifically, studies from multiple taxa have shown that exposed individuals show accelerated telomere attrition in proliferating tissues151617181920221824272829Evidence from humans for links between early-life experience, telomere attrition and adult health is necessarily correlational. Thus, the possibility can never be ruled out that the individuals experiencing early-life adversity are a non-random component of the population, and thus that associations with early adversity are non-causal by-products of common genetic or environmental third factors18191933Amount; either Plenty or Lean), and the begging effort required to obtain it . Amount was manipulated by restricting nestlings in the Lean groups to approximately 73% of the food given to the Plenty groups, who were fed to satiety, on their most recent feedEffort was manipulated by stimulating approximately twice the daily duration of begging from the Hard groups as from the Easy groups. Amount and Effort were combined factorially to give four experimental groups: Lean-Hard, Lean-Easy, Plenty-Hard, and Plenty-Easy. Hence, our experimental treatments involved types of adverse experience that occur naturally in our study species, but allowed the causal separation of factors that are usually confounded in the wild and in brood-size manipulation experiments. We measured the attrition of erythrocyte telomeres over the course of the manipulation, and in the juvenile period immediately following it. We also simultaneously measured a marker of oxidative damage to DNA, 8-hydroxy-2′-deoxyguanosine (8-OHdG) at the same time points. The motivation for measuring oxidative damage to DNA is that an increase in such damage is one of the pathways through which organism-level stress may accelerate the shortening of telomeresWe hand-reared nestlings from days 5 to 15 post-hatching, independently varying two types of adverse experience, the amount of food provided , and in the period after the manipulation . None of the correlations between the 8-OHdG measures and the telomere length change measures was significant . There were no significant treatment effects on 8-OHdG on days 5 or 15 . HoweverThe two markers of adult inflammation, HS-CRP and IL-6, were not significantly correlated with one another . There was a significant interaction between Amount and Effort in predicting both markers . For HS-Our experimental paradigm allowed us to separate the effects of different types of early-life adverse experience, namely nutritional inadequacy (Lean Amount) and high begging effort (Hard Effort), on development and cellular ageing. Only Lean Amount, not Hard Effort, affected final skeletal size, suggesting that the Effort treatments had not substantively impaired energy availability for growth. . The extent of variation in post-manipulation telomere attrition did not appear to be explained by the extent of post-manipulation weight gain. We confirmed previous observations that longer telomeres shorten faster, even when regression to the mean has been corrected forFor DNA damage, there was no evidence of an increase due to experimental treatment during the manipulation itself, but the consequences of the treatments became manifest in the period after the manipulation had ended. Only the combination of Hard Effort and Lean Amount increased DNA damage; in isolation, neither adversity type showed significant evidence of impact. Our measures of DNA damage did not significantly predict the rate of telomere attrition. We had expected they might, since oxidative damage to DNA is one of the processes affecting the rate at which telomeres are erodedWe measured inflammation in adulthood using two plasma markers, HS-CRP and IL-6. Though both are widely used as indicators of chronic inflammation, IL-6 is considered the less general marker of overall inflammatory activity, and its associations with subsequent morbidity tend to be weaker42310The complexity of the relationship between early adversity and adult inflammation is further highlighted by the fact that, although juvenile DNA damage levels predicted adult IL-6, developmental telomere attrition did not predict any aspect of adult inflammation. We predicted that it would do so, since developmental telomere attrition has been proposed as an integrative marker of the impact of adversity during development2728291425Our results produced the incidental observation that the Lean-Easy group were heavy for their size as juveniles. The developmental induction of high adult body weight by early-life adversity has been observed in many species, including humans47484950Our experimental paradigm had a number of limitations. All nestlings experienced early parental separation, and therefore no group represents an un-manipulated control. However, altricial passerines can be very readily hand-reared if taken prior to filial imprinting, and patterns of growth and development were in line with our previous studies of parent-reared nestlings19201825Although we found evidence that both nutritional inadequacy and begging effort contribute to the effects of early-life adversity on cellular ageing and adult inflammation, we found several cases where their effects combined non-additively. Moreover, the patterns of effects of the two treatments were rather different across telomere attrition, DNA damage, and the two markers of adult inflammation. In observational studies of early-life adversity and ageing, different types of adversity are often summed together into a single adversity score115346Sturnus vulgaris, taken from 8 wild nests in monitored nesting boxes in Northumberland under Natural England permit 20121066. The study was approved by the Animal Welfare and Ethics Board at Newcastle University and the UK Home Office (licence PPL 70/8089). The study was carried out in accordance with the Home Office licence and the Association for the Study of Animal Behaviour (ASAB) guidelines for the use of animals in research. All birds fledged successfully, but two  died prior to 20 months. The European starling is a gregarious, medium-sized passerine with a maximum longevity of 22.9 years given in the AnAge databaseSubjects were 32 European starlings, From the day after arrival, experimental groups received nine feeds a day. In the Plenty groups, each feed was to satiety, whilst for the Lean groups the amount fed was approximately 73% that fed to the corresponding Plenty group on the most recent feed. The Hard groups additionally received nine ‘sham’ feeds each day. During these sham feeds, which had approximately the same duration as a genuine feed (2 minutes), nestlings were stimulated to beg, but no food was delivered.At regular intervals between approximately 07:00 and 21:00 each day, the nest lid was removed and all nestlings delivered standardized 0.5 ml aliquots of food via a 25 ml Eppendorf repeater pipette. Food consisted of a blended mixture of high-meat cat food, apple sauce and vitamins, a mixture previously shown to be appropriate for hand-rearing starlingsFor the Hard groups, at nine points each day evenly interspersed between the feeds, we removed the nest lid and used the feeding pipette to stimulate the nestlings to beg for two minutes (the approximate duration of a feed) without delivering any food. All nestlings begged readily for at least part of the time.th feed), when nestlings were regrouped by natal family rather than experimental group. From day 15 until fledging (around day 21), all nests were fed to satiety on a constant rotation. Once birds had fledged, they were kept in mixed-treatment cages until they had been observed eating for themselves, and then moved to permanent large mixed-treatment aviaries  in flocks of 16.The experimental manipulations were continued until day 15  were taken by puncture of the alar or metatarsal vein on days 5, 15, 56, and again as young adults at 20 months, using a 25-gauge needle and capillary tube . Blood samples were immediately centrifuged to separate cells from plasma, and frozen to −80 °C for later analysis.2 calculated from the reference curves were 0.98–0.99 both for the telomere and the control gene assays. Intra-plate mean coefficients of variation (CVs) for Ct values were 1.7% (telomere assay) and 0.4% (control gene assay). Inter-plate CVs for Ct values based on repeated samples were 2.7% (telomere assay) and 0.7% for (control gene assay). T/S ratios were calculated using the ΔΔCt methodr > 0.99). Plate accounted for no variation in T/S ratios once effects of individual and natal family were accounted for. Mean CV for the relative T/S ratios was 18%. For 5 birds , the GAPDH assay failed and they were removed from the analysis. We have encountered this issue in previous studies of the same population1920Relative erythrocyte telomere length was assessed from DNA extracted from day 5, 15 and 56 blood samples using the quantitative real-time PCR amplification approachWe determined oxidative damage to DNA from day 5, 15 and 56 samples by measuring 8-hydroxy-2′-deoxyguanosine (8-OHdG), using the colorimetric assay EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit . When the DNA base guanine is damaged by oxidation, it is excised by repair mechanisms; excised bases from all tissues go into the circulation before being excreted. The assay measures plasma abundance of these excised oxidized bases. Whilst there is some debate on the interpretation of the assayPlasma samples recovered from EDTA blood taken at age 20 months were assessed for the two inflammation markers HS-CRP and IL-6 by solid phase sandwich enzyme-linked immunosorbent assay (ELISA) protocols, using a pigeon HS-CRP ELISA Kit , and a chicken IL-6 ELISA Kit . Both are widely used as plasma markers of chronic inflammation: IL-6 is an inflammatory cytokine, whilst CRP is an acute phase protein involved in the inflammatory response. HS-CRP concentration was expressed in μg/ml and IL-6 in pg/ml. The respective inter-assay CVs were 3.1% and 8.3%. Two birds had died by 20 months, and for a further four samples , plasma volume was insufficient to obtain an IL-6 value.Sex of starling nestlings is not phenotypically obvious and so experimental groups could not be balanced for sex. Instead, birds were sexed genetically to control statistically for sex differences. Molecular sexing followed the standard approachet al.For a single-number summary of telomere shortening between sampling points, we used the D measure suggested by Verhulst Data were analysed using linear mixed models using package lme4How to cite this article: Nettle, D. et al. Early-life adversity accelerates cellular ageing and affects adult inflammation: Experimental evidence from the European starling. Sci. Rep.7, 40794; doi: 10.1038/srep40794 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This study aimed to investigate the chronic effects of caudal artery denervation on morphometric parameters of the tail vascular smooth muscle and on physical exercise-induced thermoregulatory and cardiovascular adjustments in rats. Male Wistar rats were subjected to caudal artery denervation or the sham procedure. Approximately 26–28 days after these procedures, their thermoregulatory and cardiovascular parameters were evaluated at rest and during or following a fatiguing treadmill run. At the end of the experiments, the rats were euthanized, and samples of their tails were removed to evaluate morphometric parameters of the vascular smooth muscle surrounding the caudal artery. Denervated rats showed morphological adaptations, including increased arterial wall thickness and wall-to-lumen ratios. In resting rats and following the fatiguing exercise, caudal artery denervation barely affected the thermoregulatory and cardiovascular parameters evaluated. By contrast, caudal artery denervation attenuated the increase in tail skin temperature, decreased the spontaneous baroreflex sensitivity, and exacerbated the increases in mean arterial pressure in exercising rats. The increased wall-to-lumen ratio of denervated rats correlated negatively with the maximum tail skin temperature attained or cutaneous heat loss sensitivity but correlated positively with the maximum diastolic blood pressure attained during exercise. In conclusion, cutaneous denervation induces vascular remodeling characterized by morphological adaptations of the tail vascular smooth muscle. This vascular remodeling likely underlies the impaired tail heat loss and blood pressure adjustments in denervated rats subjected to physical exercise. Therefore, we have highlighted the importance of cutaneous vascular innervation integrity in thermal and cardiovascular control in stress-challenged rats. In this sense, our findings advance the understanding of thermoregulatory and cardiovascular system reactions after a sustained cutaneous vascular innervation injury, which is essential for the treatment of some diseases, such as Parkinson's disease and type 1 and type 2 diabetes mellitus. The regulation of cutaneous blood flow depends on the thermal and pressure demands of the body, and sympathetic constrictor tone is one of the main regulators of skin vascular resistance  homeostasis. Nevertheless, a few days after vascular sympathetic dysfunction, the cutaneous blood flow and heat loss are restored to basal levels  artery induces immediate increases in cutaneous blood flow and heat loss  upon passive exposure to heat on the 26–28th days after denervation  and were conducted in accordance with the regulations provided by the Brazilian National Council for the Control of Animal Experimentation. Efforts were made to minimize the number of rats used and to minimize their suffering.Wistar rats  were used in all experiments. Initially, four animals were housed in each standard polypropylene cage. After CAD or sham denervation (Sham-CAD), the rats were housed individually until the end of the experiments. The rats were housed under controlled light (lights on from 05:00 to 19:00 h) and ambient temperature (24.0 ± 1.0°C) conditions. Standard rat chow and tap water were provided ad libitum.Adult male Different groups of rats were subjected to three different sets of experiments, as described below and in Table n = 9) and rats subjected to Sham-CAD (n = 9). Twenty-six days after these procedures, the rats were once again subjected to an incremental-speed exercise.Prior to denervation (or sham denervation), the rats were subjected to an incremental-speed exercise to measure their intrinsic aerobic capacity, as determined by the time to fatigue and the maximum speed attained. By matching their body mass and intrinsic aerobic capacity, these rats were divided into two groups, rats subjected to CAD (n = 10) or Sham-CAD (n = 8) procedure. Nineteen days after these procedures , the rats received an abdominal temperature sensor implant to measure their TCORE. Twenty-six days after the CAD or Sham-CAD procedure, the rats were subjected to the first of two experimental trials, which were separated by a 48-h interval: resting on the treadmill or running at a constant speed until volitional fatigue. The order of the trials was balanced and crossed-over. During the experimental trials, TCORE, TSKIN , and time to fatigue were measured.The rats were subjected to the CAD (n = 8) or Sham-CAD (n = 6) procedure. The experimental procedures and measurements were the same as those in the second set of experiments with the exceptions that the pulsatile arterial pressure was measured and physiological responses were recorded for 30 min post-exercise. Twenty-four days after the CAD or Sham-CAD procedure, rats received abdominal temperature sensor implants and arterial catheter implants to measure their TCORE and cardiovascular parameters, respectively. Two days later, the rats were subjected to the first of two experimental trials, which were separated by a 48-h interval.Rats were subjected to the CAD  analyses of the caudal artery. Alternatively, the caudal artery sections from the other rats were used to confirm the effectiveness of denervation.For all surgical procedures, rats were anesthetized with an intraperitoneal injection of a cocktail containing ketamine and xylazine . After anesthesia induction, the rats were treated with prophylactic doses of different antibiotics (including 48.000 IU/kg intramuscular benzylpenicillin). A post-operative analgesic (1.1 mg/kg subcutaneous flunixin meglumine) was also administered.The procedure used for caudal artery denervation was described previously ; thus, the distal portion is largely poikilothermic and mimics the environmental temperature  was inserted into the abdominal cavity via two small (~2 cm in length) incisions in the abdominal skin and in the The left common carotid artery was identified through a ventral incision in the neck, and its proximal portion was occluded. A small cut was made in the artery, thereby allowing for the insertion of a polyethylene catheter  filled with heparin diluted in isotonic saline. The free end (PE-50) of the polyethylene tubing was tunneled subcutaneously and exteriorized at the cervical dorsal area  positioned next to the treadmill. TSKIN was measured using a thermocouple fixed to the right lateral surface  located 1 cm from the base of the tail. The close proximity to the base of the tail enabled more sensitive measurements of changes in TSKIN that occurred as a function of changes in local blood flow  was measured by a thermocouple taped to the ceiling of the treadmill chamber located close to the stimulating grid at the rear end of the treadmill belt.TCORE or TSKIN) from the average value obtained throughout the resting experiments to analyze the variability in TCORE and TSKIN. Next, all deviation values were averaged for each animal and then averaged for each group.We determined the deviation of each temperature measurement  was used to determine the heat loss threshold (HLTHR), which represents TCORE at the initiation of the rapid increase in TSKIN. Heat loss sensitivity (HLSEN) was defined as the regression slope of the five time points that followed the threshold and corresponded to the steepest part of the rising curve. This analysis has been used in recent studies with exercising rats to determine whether mechanisms underlying modified cutaneous heat loss have a central and/or peripheral origin , systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP) were determined from pulsatile arterial pressure recordings using AcqKnowledge 3.7.0 software .The tape-recorded arterial pressure signal was sampled at 2 kHz. The SAP values were identified beat by beat, and the pulse interval was calculated as the interval between two consecutive systolic peaks using a customized routine . Time- and frequency-domain analyses were performed during resting, constant-speed exercise and after exercise using an 8-min period selected from continuous recordings after stabilization of the cardiovascular parameters. In exercising rats, the middle point of the 8-min recording corresponded to half of the time to fatigue. The power spectral density was obtained with a fast Fourier transformation, and the size of the evaluated segment was established using 512 points with 50% overlap. The spectral power for very low frequency ([VLF] < 0.2 Hz), low frequency ([LF] = 0.2–0.75 Hz), and high frequency ([HF] > 0.75 Hz) bands was evaluated. The LF-to-HF ratio of pulse interval variability was calculated to assess the sympatho-vagal balance in the heart  using light electrical stimulation (0.5 mA). In each session, the rats ran at a constant speed of 18 m·min−1, and this speed was increased by 1 m·min−1 every 2 min until the rats were fatigued. Fatigue was defined as the moment when the rats could no longer maintain the running pace and exposed themselves to the light electrical stimulation for 10 s  attained by the rats. During constant-speed running, performance was evaluated by determining the time to fatigue (min). The SMAX attained during the incremental exercise sessions was calculated by modifying the following equation proposed by Kuipers et al. (MAX = S1 + [S2(t/120)], in which S1 is the speed reached in the last completed stage (m.min−1), S2 is the increment in treadmill speed at each stage (m.min−1), and t is the time spent in the uncompleted stage (s).Performance was evaluated during incremental-speed running by determining the time to fatigue (min) and the maximum speed . The constant exercise bout was interrupted when rats were fatigued . In the third set of experiments, the thermoregulatory and cardiovascular parameters continued to be recorded for an additional 30 min after the end of the exercise to evaluate whether CAD impaired physiological responses during the post-exercise period.The treadmill speed was maintained at a constant value of 18 m·minWhen the cardiovascular and thermoregulatory parameters were stable for at least 20 min , the resting values for these parameters were recorded over the next 45 min.Before removing tail samples, rats were anesthetized and successively transcardially perfused with a saline solution and paraformaldehyde . The tail samples were stored in 70% ethanol prior to starting the decalcification procedure. After decalcification, tissue samples were dehydrated in a graded series of ethanol , embedded in resin (2-hydroxymethacrylate), and polymerized at 56°C. The paraffin blocks were cut (10 μm), and tail slices were placed on slides. The slides were stained with hematoxylin and eosin and then coverslipped with entellan.Photomicrographs of transverse sections of the caudal artery were acquired (10-fold increase), digitized and analyzed off-line using ImageJ software. The vascular and lumen perimeters, which correspond to the contour of the vascular and lumen areas, respectively, were determined by the software after initial calibration. The vascular and lumen diameters were also determined by the software; in this case, four different measurements were collected for each rat and averaged. The arterial wall thickness was calculated by subtracting the inner diameter from the outer diameter and then dividing the result by two. Furthermore, the wall-to-lumen ratio was calculated to characterize the presence of vascular remodeling  in a 2% glyoxylic acid solution (diluted in 0.1 M phosphate buffer adjusted to pH 7.4 with 10 M NaOH). The slices were dried (for 20 min in a stream of cold air) and covered with mineral oil before being heated at 60°C for 30 min. Next, the slices were coverslipped and visualized under a fluorescence microscope. Catecholaminergic innervation of the caudal arteries was visualized and photographed using a microscope equipped with an HBO 100-W mercury light source . The effectiveness of the caudal artery denervation procedure was confirmed by the absence of catecholamine fluorescence in caudal artery slices  and across time points using two-way ANOVAs with repeated measures analysis for only the time factor. When applicable, Tukey's post-hoc test was used. Inter-group differences in body temperature variability, HLTHR, HLSEN, HR and SAP variability, spontaneous baroreflex sensitivity, body mass, time to fatigue, maximum speed attained, and morphometric parameters were evaluated using unpaired Student's t-tests. The correlations presented in the results were assessed using the Pearson coefficient. α < 0.05 was considered statistically significant.Data normality was assessed using the Shapiro-Wilk test, and all normally distributed data are expressed as the means ± standard errors of the means (SE). The TFluorescence induced by glyoxylic acid was evaluated 26–28 days after the CAD or Sham-CAD surgery Figure  and was CORE, TSKIN, MAP, and HR  and higher TCORE variability  than the control rats. Regarding the cardiovascular responses  and HR values than the Sham-CAD rats . Next, we evaluated the autonomic control of the cardiovascular system by performing a spectral analysis of the HR and SAP recordings . During the 26-day period between surgery and the second test, the body masses of rats from both groups increased by ~80 g with no inter-group differences .We initially subjected the rats to an incremental-speed running exercise that was performed before (intact rats) and 26 days after the denervation or sham procedure to investigate whether CAD changed the rats' physical performances. We selected this exercise protocol because a previous study showed that the variation coefficient of the time to fatigue was lower during incremental-speed running than during constant-speed running  .The exercise intensity used during constant-speed running corresponded to ~70% of the maximum speed attained by the rats  Figure . As expeCORE and TSKIN of the control rats by 1.5 and 5.0°C, respectively. Regarding the TCORE response, two-way ANOVA revealed an interaction between the groups and exercise time points ; however, post-hoc analysis did not identify any differences in means between CAD and Sham-CAD rats during treadmill running . Additionally, CAD attenuated the exercise-induced increase in TSKIN, which was ~2°C lower in CAD rats than in Sham-CAD rats at fatigue , than Sham-CAD rats  without affecting the increase in HR , TCORE was still higher than the resting values of both groups  or HLSEN  during exercise and the wall-to-lumen ratio. We also observed a positive correlation between the maximum DAP attained during treadmill running and the wall-to-lumen ratio .Pearson's correlation coefficients were used to evaluate the strength of the association between CAD-mediated physiological changes during exercise and the CAD-mediated increase in the wall-to-lumen ratio. As shown in Figure SKIN attained or HLSEN and the wall-to-lumen ratio and by the positive correlation between the maximum DAP attained and the wall-to-lumen ratio  on thermoregulatory and cardiovascular adjustments in rats subjected to treadmill running. The denervated rats exhibited attenuated exercise-induced increases in cutaneous heat loss, an exacerbated increase in MAP Figure  and reduo Figure .SKIN and TCORE variability, thereby confirming our previous results  are possible compensatory pathways. Supporting the first of these hypotheses, a previous human study demonstrated that individuals who showed lower skin vasodilation during exercise exhibited greater hyperthermia-induced hyperventilation (Hayashi et al., Tail skin vasodilation is the main route for heat loss in exercising rats (Wilson et al., In exercising CAD rats, cutaneous heat loss dysfunction was accompanied by an exacerbation of the increase in MAP Figure . ConsistSKIN. Two non-mutually exclusive mechanisms may explain this observation. First, as increased sensitivity to catecholamine is a classic adaptation developed after tail artery denervation, we postulate that the accelerated increase in blood pressure is induced by greater tail sensitivity to catecholamines of neural and humoral origins (Kamikihara et al., One of our hypotheses was that impaired vasodilation in CAD rats would increase peripheral resistance and then place a burden on the cardiovascular system. However, our data do not support this hypothesis, as the increase in blood pressure in the CAD rats compared to that in the Sham-CAD rats occurred before the CAD-induced changes in TDespite the exaggerated increase in MAP, HR was not affected by CAD, suggesting an impaired baroreflex sensitivity in these rats. Indeed, the evaluation of spontaneous baroreflex sensitivity confirmed our hypothesis; CAD impaired sensitivity during exercise. Supporting the lack of inter-group differences in HR during exercise, the spectral analysis of the pulse interval did not differ between CAD and Sham-CAD rats. Interestingly, the lack of CAD-mediated changes in running-induced tachycardia is unexpected because resting CAD rats displayed a trend toward higher HR values.SKIN during the initial 10-min post-exercise period. Notably, CAD rats were more reactive to vasoconstrictor agents and likely displayed greater vasodilation than Sham-CAD rats in response to the suppression of these agents, as expected with exercise termination. Another explanation is that CAD rats require a longer period to achieve maximum TSKIN values during exercise. This hypothesis corroborates our previous findings that CAD delays the increase in TSKIN but does not change the maximum TSKIN value attained in rats that were passively exposed to heat (Lima et al., SKIN of CAD rats at the beginning of the post-exercise period may have reduced peripheral resistance, thereby preventing the observation of inter-group differences in cardiovascular parameters.CAD rats exhibited a transient increase in TSEN was reduced in CAD rats, indicating that changes in peripheral control of the vasomotor tone occurred.The hypothesis that morphological adaptations to the cutaneous denervated vasculature are associated with altered cardiovascular and thermoregulatory adjustments during exercise was confirmed in the present study. However, the adaptations observed following sympathetic denervation of the tail occurred in the opposite direction from those observed in the denervated ear arteries of “young adult” rabbits, in whom sympathetic denervation reduced the total wall thickness and the cross-sectional area of the tunica media Bevan, . These iAerobic performance was reduced during the second incremental exercise relative to the first one irrespective of the experimental group Figure . This reIn conclusion, CAD attenuated and enhanced exercise-induced increases in cutaneous heat loss and blood pressure, respectively. Moreover, the denervation procedure promoted vascular remodeling in the caudal artery, as evidenced by the increased arterial wall thickness and the wall-to-lumen ratio. Notably, CAD-induced vascular remodeling was associated with altered physiological responses during exercise, suggesting that vascular remodeling at least partially contributes to the attenuated cutaneous heat loss and enhanced blood pressure observed in denervated rats during exercise.CORE during either hot or cold thermal stress (Kenny et al., Understanding thermoregulatory and cardiovascular system reactions after an abrupt and sustained cutaneous vascular innervation injury (Lepori et al., Conceived and designed the research: MM-L, WP, IF, CC, NL, and SW; Performed the experiments: MM-L, WP, IF, and JJ-S; Analyzed the data: MM-L, WP, IF, JJ-S, AF, CC, NL, and SW; Interpreted the experimental results: MM-L, WP, IF, AF, CC, NL, and SW; Prepared the figures: MM-L and SW; Drafted the manuscript: MM-L and SW; Edited and revised the manuscript: MM-L, WP, CC, and SW; Approved the final version of the manuscript: MM-L, WP, IF, JJ-S, AF, CC, NL, and SW.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A growing body of evidence suggests that sodium-glucose cotransporter 2 (SGLT2) inhibitors appear to be a powerful option to improve the cardiovascular (CV) prognosis in high CV-risk patients with type 2 diabetes. Despite a significant reduction in major adverse CV events with SGLT2 inhibitor treatment, however, an unexpected increased risk of amputation was observed in the CANVAS program and the subsequent pharmacovigilance analysis. Although the underlying mechanisms are currently unknown, because amputation has a large negative impact on patient clinical course, clinicians want to know the exact reason for the increased amputation in the canagliflozin treatment. We herein discuss a need to elucidate the actual reasons with more appropriate statistical consideration, taking into account individual clinical course potentially involved in the diabetes-related amputation. Decreases in the hardendpoints by canagliflozin might result in an alternate increase in the other diabetes-related complications, including amputation. In addition, if amputation occurred after stopping canagliflozin, the incidence might be caused by worsened glycemic control and a decrease in hematocrit, accompanied by a subsequent worsening of diabetic foot disease. More detailed approach considering individual clinical course potentially involved in the amputation, would help to further unravel the cause for suspected risk of amputation with canagliflozin. A great deal of clinical interest was generated by the EMPA-REG OUTCOME trial, which evaluated the cardiovascular (CV) safety of empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, in patients with type 2 diabetes (T2D) and high CV risk . Based oAccumulating evidence has demonstrated that canagliflozin treatment can achieve favorable long-term improvement in glycemic and metabolic parameters and is well tolerated as either mono-therapy or in combination with other anti-hyperglycemic agents, including insulin –8. FurthConversely, due to their unique mode of action, SGLT2 inhibitors have several signature adverse effects, such as genital/urinary tract infection, and such adverse effects were observed in both CV safety trials. In contrast, in the CANVAS program an unexpected increase (approximately twofold) in the risk of lower-extremity amputation  was observed in the canagliflozin-treated group . Most reAlthough the risk of lower-extremity amputation in patients with T2D has declined substantially during the past couple of decades , 12, gloAlthough it may not be appropriate to compare the results of separate studies, we are left to question why the risk of amputation was increased in the CANVAS program, but not in the EMPA-REG OUTCOME trial . FocusinWe next noticed that adverse events related to volume depletion occurred more often in the canagliflozin-treated group than in the placebo group, despite a similar incidence in the EMPA-REG OUTCOME trial, which might accordingly contribute to the circulatory failure in the distal peripheral arterial beds. However, diuresis and subsequent volume depletion would be more evident at the early phase of SGLT2 inhibitor treatment, inconsistent with the phase of increased incidence of amputation.With regard to adherence to the study drugs, a total of 25% of the participants in the EMPA-REG OUTCOME trial and 29% in the CANVAS program discontinued their assigned treatment. In the EMPA-REG OUTCOME trial, the incidence of stroke showed a higher tendency in the analysis of the intent-to-treat population than in the on-treatment analysis. When administration of an SGLT2 inhibitor is stopped, several SGLT2 inhibitor-mediated favorable effects on metabolic and hemodynamic parameters might be eliminated , followed by progression of distal peripheral arteriosclerosis prone to ischemia and foot ulcer, leading to possible increases in amputation. Although it is still not easy to determine or explain the specific reason for an increased frequency of amputation with canagliflozin treatment, more detailed statistical approaches considering the individual clinical course potentially involved in diabetes-related amputation should further help us to understand why and how its risk increased in the canagliflozin-treated population.For clinicians, especially in the cardiovascular field, the emerging SGLT2 inhibitors should be a promising option to improve CV outcomes in the care of patients with T2D at high CV risk. If possible reason(s) for the increased risk of amputation with canagliflozin treatment are uncovered behind the large CV benefit, our understanding of SGLT2 inhibitors would be much better for providing effective and safe CV-diabetes care. The ultimate goal is to exploit the CV benefit of SGLT2 inhibitors, while avoiding adverse effects, for appropriate patients who need SGLT2 inhibitor-mediated CV benefits, because SGLT2 inhibitor treatment in the recent landmark studies markedly reduced the incidence of MACE paramount to the clinical outcomes.


Continuously operating Global Navigation Satellite Systems (cGNSS) can be used to convert relative values of vertical land motion (VLM) derived from Interferometric Synthetic Aperture Radar (InSAR) to absolute values in a global or regional reference frame. Artificial trihedral corner reflectors (CRs) provide high-intensity and temporally stable reflections in SAR time series imagery, more so than naturally occurring permanent scatterers. Therefore, it is logical to co-locate CRs with cGNSS as ground-based geodetic infrastructure for the integrated monitoring of VLM. We describe the practical considerations for such co-locations using four case-study examples from Perth, Australia. After basic initial considerations such as land access, sky visibility and security, temporary test deployments of co-located CRs with cGNSS should be analysed together to determine site suitability. Signal to clutter ratios from SAR imagery are used to determine potential sites for placement of the CR. A significant concern is whether the co-location of a deliberately designed reflecting object generates unwanted multipath  in the cGNSS data. To mitigate against this, we located CRs >30 m from the cGNSS with no inter-visibility. Daily RMS values of the zero-difference ionosphere-free carrier-phase residuals, and ellipsoidal heights from static precise point positioning GNSS processing at each co-located site were then used to ascertain that the CR did not generate unwanted cGNSS multipath. These steps form a set of recommendations for the installation of such geodetic ground-infrastructure, which may be of use to others wishing to establish integrated InSAR-cGNSS monitoring of VLM elsewhere. Vertical land motion (VLM), i.e., subsidence or uplift, arising from natural  and/or anthropogenic  phenomena affects geodetic benchmark heights and complicates the sea level record at tide gauges ,2. EarthThe first key difference is that GNSS provides estimates of VLM in terms of ellipsoidal heights at discrete points, whereas InSAR provides estimates of range change in the satellite’s slanted line of sight (LoS) on regional scales (10 s to 100 s of km) at spatial resolutions reaching <10 m. The LoS of a SAR satellite is most sensitive to VLM, due to the usually steep incidence angle of the sensor, but the near-polar orbital plane of SAR satellites means that it also contains any motion in the east-west and, to a much lesser extent, north-south directions. Therefore, to directly compare/integrate the two measurement types, either the 3D GNSS coordinates should be mapped into the 1D InSAR LoS, or the vertical component of motion should be extracted from the LoS InSAR measurement. The latter may be achieved by transforming all LoS displacements into the vertical direction under the assumption that no relative horizontal motion is occurring ; throughThe second key difference is that GNSS provides VLM in an absolute sense with respect to some defined reference frame ,12,13,14In order to make precise comparisons between these two measurement types and thus fully integrate InSAR and GNSS, a ground network of artificial InSAR corner reflectors (CRs) can be deliberately co-located with continuous GNSS (cGNSS). The CRs provide a high-quality, temporally stable phase response in SAR imagery, which is required for reliable InSAR time series of VLM . The del2) CR-GNSS networks include 40 CRs co-located (within 50 m) with episodic-occupation GNSS stations throughout the Surat Basin, Australia [There is limited published literature on monitoring VLM using deliberately co-located cGNSS and CRs. Studies that do combine InSAR and cGNSS to measure VLM tend to do so without the use of CRs ,18,24,25ustralia , and natustralia ,37,38.Here we use the example of Perth, Australia, to describe a series of tests conducted before new permanent installations of co-located ground infrastructure to support integrated cGNSS and InSAR monitoring of VLM. In documenting these efforts, we aim to provide some framework for efficiently establishing operational integrated satellite-based VLM monitoring that may be applicable elsewhere. In Perth, InSAR and cGNSS, coupled with repeat levelling over many 10 s of km (not described in this study), are currently being used to measure, map and monitor subsidence caused principally by groundwater extraction ,40,41. OThe Perth region hosts a mixture of existing cGNSS infrastructure . The cGNThis includes two cGNSS (PERT and STLG) that are on deep-seated (~1.5 m) pillars, a new cGNSS co-located with a tide gauge at Fremantle (FMTL), and HIL1, that is also located on a tide gauge.  have unobstructed sky visibility down to 10 degrees for cGNSS and in the satellite LoS for CRs, (ii) are secure against theft and/or vandalism, (iii) have the landowner’s permission, and (iv) can host ground monuments that, ideally, are connected to bedrock. In sedimentary basins such as in Perth, however, bedrock is not always close to the ground surface at sites that satisfy items (i–iii), so deep-seated foundations have had to be used instead.Mature, tried and tested standards and recommended practices (SARPs) for the installation of cGNSS have been compiled by numerous national government or international agencies . In AustOur particular concern was the potential of multipath  caused by the co-located CR, which is a deliberately designed reflecting object. Characterisation of carrier-phase multipath at cGNSS sites is not only non-trivial, but also can adversely affect VLM rates determined from the daily ellipsoidal height time series ,50. MultWe desire to avoid multipath from the CR, but achieve a close enough co-location to allow for regular short-range repeat levelling (following standards outlined in ) to moni√hr and 0.3 mm/√hr, respectively, and ambiguities were fixed to integers, with the coordinates represented in the IGS08 reference frame after transforming the fiducial-free outputs using JPL ’repro 2.1’ daily Helmert (seven) transformation parameters.We chose the PPP technique over a network solution (which outputs double difference (DD) carrier-phase residuals) because in the latter, it not possible to discriminate which site is generating the multipath. We used the NASA JPL GIPSY software version 6.4 for the daily processing, with JPL fiducial-free ’repro 2.1’ orbits and satellite clocks held fixed, a 10 degree elevation angle cut-off, correcting for satellite and receiver phase centre variations using the IGS08 models, modelling tidal ground displacements according to the IERS Conventions 2010 , and estThis GIPSY-based PPP GPS analysis procedure has been used for monitoring small geophysical movements of a few mm per year ,57,58. TIn addition to the general considerations  is directed in the SAR satellite’s LoS. This can be achieved using the freely available, physics-based Systems Tool Kit modelling environment , and theFollowing the test CR installations, intensity images were analysed to assess the suitability of the CR deployment. For each site and satellite, we calculated a time series of the SCR at the location of the CR using a set of co-registered intensity images. For each image, the SCR is calculated by comparing the backscatter intensity in a window containing the CR response  relative to the backscatter intensity from surrounding scatterers in a 15 × 15 pixel window . A test The brightness of the CRs is greater in TSX than Sentinel-1B imagery  were made permanent by bolting the reflector to a specially constructed concrete foundation. Each foundation is one-metre square with a 0.6 m central core attached to metal reinforcing rods driven into the ground until refusal (~2 m) for added stability. Local levelling ties between the CR and cGNSS at both PERT and STLG were completed in October 2016 to first-order levelling standards, where the two-way levelling disclosure must be <2nchmarks .We would expect any strong generator of multipath to manifest in the ellipsoidal heights and LC carrier-phase residuals within 24 h of CR installation, and certainly within the ~20-day CR test periods used in The long-recording (since 1897) Fremantle tide gauge was not previously co-located with a cGNSS , and assFinding a suitable site proximal to the tide gauge proved problematic in the developed town of Fremantle. Numerous sites were rejected for one, more or all of the reasons covered in . Following the successful GNSS test deployment, TSX backscatter intensity imagery was used to identify prospective sites in the school grounds with low background clutter for the temporary CR deployment  co-located cGNSS and CR installation is soon to be constructed at the Beenyup wastewater treatment plant BEEN in , which iWe repeated our series of site-suitability experiments, carrying out a simultaneous GNSS and CR test deployment. A temporary GNSS receiver was installed 12–15 December 2016. The average of the four days of PPP GPS LC carrier-phase residuals was 10.8 mm, commensurate with the values for PERT and STLG , but noiPrior to the test deployment period at Beenyup, the Sentinel-1B satellite began to acquire and release SAR imagery over Perth. Therefore, backscatter intensity imagery from both TSX and Sentinel-1B could be analysed simultaneously to select a location for the CR test installation. The CR was deployed for four overpasses of both satellites . Like PEThe test experiments and procedure described here may be of use to others wishing to install co-located cGNSS and InSAR CR ground infrastructure for operational integrated monitoring of VLM elsewhere. In short, test co-locations of GNSS and CRs, followed by analysis of GNSS LC carrier-phase residuals (from PPP processing) and SCRs (from backscatter intensity imagery) are used to efficiently identify suitable sites before the installation of permanent ground infrastructure. Our primary concern was GNSS multipath caused by the co-located CR. We managed this by placing the CR at least ~30 m from the GNSS and not in visible line of sight. This strategy was vindicated by the test installations, and later confirmed after the permanent installations. On average, installation of a CR at the sites in this study resulted in SCRs of ~29 dB for TSX and ~13 dB for Sentinel-1B. These values are commensurate with SCRs observed for CRs installed in natural environments with X- and C-band SAR satellites , and sugThe advantages and disadvantages of the two techniques discussed in this paper are described elsewhere in the literature ,65, inclOne consideration for cGNSS-derived VLM demonstrated by  is that The accuracy of the VLM estimate from InSAR is primarily a function of the frequency of acquisitions, the length of the observation period, and the magnitude of changes in atmospheric refraction ,73, the In order to combine the cGNSS and InSAR measurements, the simplest approach will be to use a 1D polynomial ,75, refeThe co-located installations of ground infrastructure in Perth described here have been dictated by the existing locations of cGNSS on deep-seated pillars (PERT and STLG), the desire to determine VLM at tide gauges to correct the tide gauge record  (FMTL), Finally, when active SAR transponders ,81 becomUsing the example of Perth, Australia, we have described the practical considerations necessary for installing ground-based infrastructure to monitor VLM using cGNSS and SAR satellites. We present a series of recommendations that readers may adopt during the installation process, including the use of SCRs calculated from SAR intensity imagery to install CRs in regions of low background clutter, and tests on the spatial separation of the CR from the cGNSS to reduce multipath effects. In Perth, these installations will facilitate the integration of InSAR and cGNSS measurements to measure sub-cm scale VLM in an absolute reference frame. This will aid in our efforts to (1) constrain VLM in response to groundwater extraction and managed wastewater recharge, and (2) measure the impact of VLM upon the sea level record from local tide gauges.