This work is a contrastive investigation of numerical simulations to improve the comprehension of thermo-structural coupled phenomena of mass concrete structures during construction. The finite element (FE) analysis of thermo-structural behaviors is used to investigate the applicability of supersulfated cement (SSC) in mass concrete structures. A multi-scale framework based on a homogenization scheme is adopted in the parameter studies to describe the nonlinear concrete behaviors. Based on the experimental data of hydration heat evolution rate and quantity of SSC and fly ash Portland cement, the hydration properties of various cements are studied. Simulations are run on a concrete dam section with a conventional method and a chemo-thermo-mechanical coupled method. The results show that SSC is more suitable for mass concrete structures from the standpoint of temperature control and crack prevention. Cracking is a major concern that should be considered for mass concrete structures from the standpoints of structures and materials. Thus, for mass concrete structures in which thermal-stress-induced cracks are prone to occur, considerations should include not only rational structure types and reasonable construction sequences but also suitable cement materials. Supersulfated cement (SSC) is particularly suitable for use in the internal part of hydraulic structure mass concrete due to its slow hydration rate and very low hydration heat.2 emissions [For the cement industry, re-use binders are used for waste materials from other industries; these binders are effective for energy-saving emissions reduction to reduce energy consumption and COmissions ,2. SSC imissions . The hydmissions .For mass concrete structures, reasonable joint arrangement and stratification are significant. Concrete is poured in layers, which offers convenience in construction and improves the heat dissipation conditions. Moreover, pre-cooling techniques, such as cooling aggregates and reducing cement content, and the post-cooling of embedded cooling pipes are widely used. The first successful post-cooling technique was employed during the construction of the Hoover Dam  and is sIn most cases, proper materials may be selected to satisfy the strength requirements; reasonable construction and cooling schedules may also be established according to regular calculations. However, because regular calculations typically simplify the hydration process and difficulties arise in predicting concrete properties such as the modulus of elasticity , cracks Since the effect of the temperature and the cement hydration can promote or hinder in the concrete formation of micro-cracks which in turn influence the behavior of the material itself, the understanding of this phenomenon is very relevant. Indeed, recent works ,8 have set al. proposed a coupled thermo-chemo-mechanical model for the behavior of early-age concrete that can be implemented into the finite element method and predict the evolution of the hydration degree and heat over time [et al. found a significant linear correlation between hydration degree and the intrinsic material functions for the fracture energy [et al. proposed a model that not only took the thermal and chemical fields but also the water diffusion process coupled effects into consideration [In general, computer simulations  allow us to precisely evaluate the thermal evolution of early-age concrete by considering the hydration degree as an internal variable and other related parameters in describing the thermo-chemical-mechanical coupling process ,13,14,15ver time ,17,18. Le energy . Gawin ederation . Taking deration ,22.In this paper, one type of environment-friendly cement, SSC, is introduced, and its hydration properties are studied based on hydration heat liberation rate data. A thermo-chemical-mechanical coupled hydration model was adopted to simulate the temperature and stress field in a relatively precise way for mass concrete structures. A multi-scale framework is employed to obtain the nonlinear thermal and mechanical characteristics. The simulation model is applied in a case study on a monolith of an arch dam.t, and a and b are material parameters that can be calibrated with adiabatic test data.For mass concrete structures, the hydration heat is the main heat source, which is significant in simulating the temperature and stress field. The conventional method to calculate the hydration heat of concrete is to employ the adiabatic temperature rise model, which considers the hydration heat as a function  of hydration time. For mass concrete structures, one of the most widely used models is given by : (1)Q(t)Adiabatic tests are conducted with an initial temperature of 20–25 °C. From the viewpoint of cement hydration kinetics, concrete hydration occurs quickly under adiabatic conditions. Thus, variations in the adiabatic temperature rise are generally too low to measure after 28 days . Therefot, and α and β are material parameters.Similar to the hydration heat calculation, the elastic modulus of mass concrete is described as follows: R is a constant for ideal gases. Cervera et al. developed an analytical form of the normalized affinity based on thermodynamics [Hydration models based on the Arrhenius concept are widely accepted by researchers because they use rigorous thermodynamic theory and are consistent with experimental data . Accordidynamics . The fredynamics , a fixeddynamics : (4)Aξε˙asConsidering that the drying shrinkage behaviors usually take place on the surface of structures, and humidity diffusion coefficient is quite small ; as a consequence, for the inner part of mass concrete structures, the humidity can be treated as constant ; thus, tt is the time, The mass concrete creep can be modeled by means of the solidification theory. Bofang Zhu compared the generalized Kelvin rheological model with the Dirichlet series of creep compliance and summarized a creep model with eight parameters, which has been widely adopted in China . The forSince the creep experiments usually show the total creep strain in practice, and as has been discussed in 1 is the unit tensor.The thermal strain In a fully coupled thermo-chemical-mechanical model, the hydration degree can be used as an intermediate variable to determine the thermal or mechanical characteristics. In this work, the heat capacity and thermal conductivity of concrete are estimated using a homogenization scheme that is well-suited for heterogeneous material.A and B are material constants; in Bentz’s study [w/c varying between 0.3 and 0.5.Given the heat capacity and volume fraction values of cement, water and aggregates, the heat capacity of the matrix can be estimated using the law of mixtures : (12){cf’s study , these vThe paste matrix is made of water and cementitious powder, so a reasonable approximation of thermal conductivity can be calculated from the well-known Hashin–Shtrikman (H-S) bounds. An estimate can be determined from the measured thermal conductivities (given by : (13){λlAn estimate can then be determined by the average of the H-S bounds: According to ,33, the et al. proposed an aging degree model to link strength evolution with hydration degree and current temperature, and the aging degreemaxT represents the maximum temperature during hydration of concrete which is possibly under standard conditions (100 °C); refT is the reference temperature for the experimental calibration of the aging model; Experimental evidences show that the evolution of the concrete strength depends not only on the degree of hydration, but also on the kinetics of the hydration reaction. On the basis of this evidence, Cervera fined as ,34: (15)The Young’s modulus values vary due to the aging degree’s evolution as follows: ectively ,35.The Young’s modulus varies due to the aging degree’s evolution as follows: et al. [Based on the work of De Schutter et al. ,37, the As discussed in At present, the kinetic parameters of the hydration reaction of concrete were achieved by testing temperature or the rate of hydration heat evolution with the adiabatic tests with the following equations: Using the approach in Mechanical tests on concrete are typically conducted under isothermal conditions; therefore, the hydration process is simulated under isothermal condition in this research, so the mechanical characteristics can be linked to the hydration degree rather than to time. Young’s modulus and the Poisson’s ratio evolution for isothermal test of fly ash concrete specimen are plotted in The autologous volumetric deformation of fly ash concrete specimen are plotted in The hydration properties of SSC are different from slag Portland cement and ordinary Portland cement . SSC achThe hydration properties can be analyzed, given the kinetic parameters of the hydration reaction of the cement paste and the composition of concrete, which is listed in The compressive strength evolution for isothermal tests of the SSC specimen is plotted in 3 [The Dagangshan Hydropower Station is one of the largest hydropower projects built on the main stream of Dadu River in Southwest China. The water retaining structure of the Dagangshan water complex is a double-curvature arch dam with a maximum height of 210 m, 29 dam sections, and total concrete dosages of approximate 3,220,000 m3 . In thisFor mass concrete structures, thermal stresses are a major concern, and temperature control measures are Gordian techniques in the construction period. There are multiple factors that impact thermal stress, such as concrete material properties, boundary conditions, construction schedule and temperature control measures.The cooling effects of the cooling pipes can be estimated according to ,40,41; hThe #18 dam monolith located on the riverbed was selected as a typical structure to conduct the thermo-chemical-mechanical analysis. The 3D FE mesh model for the simulation is presented in The construction monolith #18 of the Dagangshan Dam began on 11 March 2012, and it was completed on 25 September 2014. Next, 72 layers of concrete gradually rose with the progress of construction. The initial temperature of the concrete was assumed the placing temperature , accordiIn normal conditions, the construction of the selected monolith was always behind the adjacent monoliths; the boundary condition for both sides was assumed adiabatic. The upstream and downstream surfaces of the placed concrete as well as the top surface of the concrete block and foundation were assumed directly exposed to the outside environment, where heat convection and radiation occurred.Now that the project has been completed, the distribution characters of the air temperature can be simulated based on the measured data.aT) of the measured temperature values (According to the monitoring temperature of the meteorological station at the Dagangshan Dam site, the outside air temperature (e values  can be iBased on the parameter studies in The temperature and stress evolution processes are discussed for further comparative analysis. Six typical point are selected, and points ①, ③ and ⑤ are on the downstream profile of the dam section, whereas, points ②, ④ and ⑥ are distributed over the midplane. Points ① and②, points ③ and ④, and points ⑤ and ⑥ are on the three different elevations. The position distribution of the typical points is shown in For the nodes on the downstream profile of the dam section, the temperature rises to a certain extent and then changes with air temperature. Compared with the conventional model, the temperature prediction values of the coupled model present a slower growth trend inside the mass concrete. The temperature procedure differences give rise to differentiate the effective stress development history. For points on the surface, the calculated stress values of the coupled model are relatively larger than that of conventional model. However, the opposite occurs for the inner part of dam section.The conventional heat release model of mass concrete only relates the heat liberation rate to time, which ignores the effect of real-time temperature on the hydration process. To overcome the problem, a chemical-physical coupled model is introduced.Based on the chemical-physical coupled model, the hydration properties of fly ash concrete and SSC concrete are studied.A multi-scale framework is employed to obtain the nonlinear thermal characteristics. Using a homogenization scheme that is well suited for heterogeneous materials, the hydration process of mass concrete structures can be estimated.Contrastive analysis of the coupled model and the conventional model are conducted, based on an engineering project.Temperature prediction values of the conventional model and the coupled hydration model show a difference, especially at an early age when cracking is prone to occur within mass concrete structures.The cooling effect of the cooling pipes used inside the mass concrete was neglected. Thus, the chemical-physical responses under actual working conditions should be scrutinized in future research.When using proper materials such as super sulfated slag concrete, water cooling is likely to be abrogated, which can induce cost-savings.In view of the disadvantages of the conventional temperature prediction model, this work introduces a fixed hydration model to study the non-linear thermo-chemical-mechanical responses in mass concrete structures. Focusing on the chemical-physical responses of mass concrete structures, the authors conducted numerical simulations and parameter studies. The major achievements are as follows:


R-SO), is widespread in microorganisms, but during evolution this function, conferred by the enzyme fRMsr, was lost in metazoa. We examined whether restoration of the fRMsr function in an animal can alleviate the consequences of methionine oxidation. Ectopic expression of yeast fRMsr supported the ability of Drosophila to catalyze free Met-R-SO reduction without affecting fecundity, food consumption, and response to starvation. fRMsr expression also increased resistance to oxidative stress. Moreover, it extended lifespan of flies in a methionine-dependent manner. Thus, expression of an oxidoreductase lost during evolution can enhance metabolic and redox functions and lead to an increase in lifespan in an animal model. More broadly, our study exposes the potential of a combination of genetic and nutritional strategies in lifespan control.Accumulation of oxidized amino acids, including methionine, has been implicated in aging. The ability to reduce one of the products of methionine oxidation, free methionine-R-sulfoxide (Met- Organisms are exposed to the threat of ROS during aerobic metabolism, which is associated with various reactions involving incompletely reduced species of molecular oxygen7. ROS may adversely affect biomolecules, such as DNA, lipids, and proteins, and as such have been implicated in the etiology of many disorders as well as in accelerated aging8. To prevent deleterious accumulation of oxidative damage, organisms use enzymes that participate in the removal of ROS or its consequences, often in cooperation with certain low molecular weight molecules. One such strategy is to repair oxidized biomolecules, restoring their biological functions. Many oxidoreductases have been characterized for their contributions to redox homeostasis and aging such as thioredoxin reductase, methionine sulfoxide reductase (Msr), superoxide dismutase, catalase, and other enzymes9.Molecular damage has been widely implicated in the aging process, including the damage caused by reactive oxygen species (ROS)11. Together with Cys, Met is the amino acid most susceptible to oxidation by ROS, and its oxidation results in a mixture of methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO). One of the Msrs, MsrA, can reduce both protein-based and free Met-S-SO, whereas another enzyme, MsrB, can reduce protein-based Met-R-SO, but has low efficiency with free Met-R-SO12. An additional Msr, fRMsr, was also functionally characterized that possesses high efficiency in reducing free Met-R-SO, but this enzyme was lost in animals and many plants14. In this regard, higher orgarisms, such as mammals and insects, are thought to reduce free Met-R-SO inefficiently15. Like several other antioxidant enzymes, MsrA was implicated in lifespan control in some organisms such as fruit flies and yeast, although not in mouse22, whereas MsrB and fRMsr are poorly understood with regard to their roles in lifespan control. The observed species-specific regulation of lifespan by Msrs requires further studies. In general, the effects on lifespan observed in invertebrates are more extensive than in mammals.Several such enzymes rely on the reversible Cys and Met oxidation/reduction, including Msrs which reduces oxidized Met back to Met17, expression of MsrB did not23, suggesting that this extension may be dependent on the reduction of free methionine sulfoxide (Met-S-SO in the case of MsrA). If so, the inability to reduce free Met-R-SO may be the Achilles Heel of fruit flies. To address this possibility, we generated transgenic flies that express yeast fRMsr25. These flies efficiently reduced free Met-R-SO, and we further employed this model to examine the role of this process in lifespan control and other processes.Interestingly, whereas MsrA expression extended the lifespan of fruit fliesDrosophila lines  were obtained. Among the 6 Drosophila lines carrying UAS-fRMsr in either 2nd or 3rd chromosome, 3 Drosophila lines  produced viable progeny, whose fRMsr expression was induced by GAL4 expression, and thus were chosen for further experiments. The GAL4 activator lines used in this study, da-GAL4  (stock #5460) and balancers were obtained from the Bloomington Drosophila Stock Center. A fatbody-GAL4 driver  was obtained from R.P. Kühnlein 23. Yellow-body white-eyes flies yw used as a wild type control were kindly provided by Dr. R. S. Sohal . Then, three homozygous transgenic lines  and GAL4 driver lines were backcrossed at least six times to yw to make the equivalent genetic backgrounds. After complete backcross of individual strain to yw, they were used for crosses to generate the following progeny: da-GAL4 > fRMsr2.1, da-GAL4 > fRMsr9.1, da-GAL4 > fRMsr4.2, da-GAL4 > yw, yw > fRMsr2.1, yw > fRMsr9.1, yw > fRMsr4.2.The gene coding for fRMsr  was amplified from yeast genomic DNA with 5′-GCGGCCGCTCTCAAAACTATTATTTAAAGACACATGATTTATT-3′ and 5′-GCCTCGAGATGGGCTCATCAACCGGGTT-3′ primers and then cloned into the NotI/XhoI restriction sites of pUAST vector. Germline cell co-injection with the recombinant pUAST vector containing yeast fRMsr gene and the P{2-3} plasmid carrying transposase and balancing were used to obtain transgenic flies. By using this method, 6 26. This diet was used for maintaining fly strains, counting pupa numbers, and carrying out lifespan analyses. Second, we used a regular diet supplied by the Harvard Medical School fly kitchen. Briefly, 12.65 g yeast, 7.33 g soy flour, 53.45 g cornmeal, 4.23 g agar, 4.23 g maltose, 56 ml corn syrup, 3 ml propionic acid, and 10 ml tegosept per liter of water were used27. This diet was used to examine lifespan of given fly strains. Third, we used a chemically defined diet29. Briefly, 101.07 g Diet TD.10417 (Harlan Teklad), 100 mg lecitin from soybean (Sigma), 500 mg ribonucleic acid from Torula yeast (Sigma), 100 g of dextrose, 20 g agar, 2.85 ml propionic acid, 0.255 ml of phosphoric acid (Sigma) and indicated amounts of Met (Sigma) per liter of water were used. This diet was used to examine the effect of Met levels on lifespan. All fly lines were grown on the corn meal diet mentioned above. Vials were kept with less than 50–60 eggs and then the hatched flies were transferred to one of the two types of fresh corn meal food. After mating for 1–2 days, three day old mated flies were collected using etherization, sorted by sex and used for subsequent experiments. To maintain fly strains, they were raised on corn meal food and transferred to fresh vials without anesthesia every three days. Flies were kept in a temperature-controlled chamber at 25 °C with 12 h light/dark cycle and approximately 60% humidity for all experiments.For maintanence of flies and lifespan asays, three types of diet were used. First, it was the regular corn meal diet, composed of 85.7 g corn meal (Quaker Oats Company), 50 ml golden A unsulfured molasses (Groeb Farms Inc), 71.4 g Torula yeast , 2.86 g p-hydroxybenzoic acid methyl ester (Sigma), 6.4 g agar (MoorAgar Inc) and 5.7 ml propionic acid (Sigma) per liter of water30. Fresh food (regular or defined diet) was supplied and dead flies were removed from each cage every 3 days until all flies died. Control and experimental group trials were always performed concurrently under the same conditions. Flies were maintained in a temperature-controlled incubator at 25 °C with 12-h light/dark cycle and 50–60% humidity.Newly hatched flies were sorted for sex (within 16 h) at 18  °C and then held on the regular corn meal diet with less than 40 flies per vial. The same number of male and female flies were incubated for 30 h for mating. Then, the flies were sorted for sex again and kept on the regular corn meal diet until the use. For lifespan studies, less than 50 male or female 3-day-old flies were placed in plastic cup cages, a system which we and others have routinely used for aging assays32. Briefly, fly media containing 1.3% low melting agarose, 1% sucrose, and 10 mM paraquat with or without 1 mM Met were prepared for paraquat resistance tests. 10 mM paraquat was added at 45 °C to avoid loss of activity. Male flies were prepared as described above, and then 15-day-old flies were sorted in groups of 15 flies per vial 3 days prior to the test. On the day of the test, after starvation for 6 h, flies were transferred without etherization to vials with the paraquat-containing media. The numbers of dead flies were counted every 12 h until all flies were dead. 4 replicates were performed for each control and experimental group in a temperature-controlled incubator at 25 °C with 12-h light/dark cycle and 50–60% humidity. Survival was analyzed by Log-rank test using the JMP (version 10) software (SAS Institute Inc.).Paraquat resistance tests were performed as described previously with minor modificationsMale flies were prepared as described above, and then 3-day-old flies were sorted in groups of 15 flies per vial. 4 replicates were prepared for each control and experimental group. Male flies were maintained on the regular corn meal diet for 18 days prior to the test. All flies were starved in vials with 6 ml 1% agar and kept in 12 h light/dark cycle at 25 °C. Total number of flies tested for each genotype was 60. Dead animals were counted every 12 h.Newly hatched flies were sorted for sex within 16 h at 18 °C and then 5 females were incubated with 5 males to count on pupa production during 24 h. We had 5–10 replicas of each control and experimental group. Females laid eggs for 24 h, then flies were transferred to new vials and the number of pupa was counted after enclosion.33. Then, flies were immediately collected in Eppendorf tubes by snap freezing in liquid nitrogen. Five female flies per group  were homogenized in 200 μl of phosphate-buffered saline, and the homogenate was centrifuged at 13,000 r.p.m. for 15 min. Absorbance of the supernatant was measured at 625 nm, with 675 nm used as a reference, and then compared with the absorbance among the tested strains to calculate consumed food mass.Newly hatched female flies were prepared and reared on the regular corn meal diet as described above. For the feeding assay, 0.1% (w/v) erioglaucine  was added to each diet that contains 1.3% low melting agarose and 1% sucrose. 6- and 24-day-old flies were fed this diet for 30 min after short-term starvation (12 noon–12:30 pm) as described15. Finally, fRMsr expression was visualized by using the ECL detection system (Sigma).Six-day-old flies were collected and snap frozen in liquid nitrogen. 30 flies were homogenized with disposable plastic homogenizer in Effendorf tube in PBS buffer (pH = 7.4). After centrifugation at 4 °C at 13,000 rpm for 15 min, supernant was collected for further Western blotting and methionine sulfoxide reduction activity assays. For Western blotting, 120 µg of total protein lysate was electrophoresed on NuPAGE® Novex 10% Bis-Tris gels (Invitrogen), transferred onto PVDF membranes and immunoblotted with antibodies specific to yeast fRMsrR-SO and dabsyl-Met-S-SO were prepared as previously described15, and then the reaction mixture (100 µl) containing 20 mM dithiothreitol, 200 µM dabsyl-Met-R-SO (or 200 µM dabsyl-Met-S-SO), and tissue lysate containing 200 ~ 400 µg of total protein in PBS (pH 7.4) was prepared. The reaction was carried out at 37 °C for 30 min and stopped by adding 200 µl of acetonitrile at 4 °C for 10 min. After centrifugation at 13,000 rpm at 4 °C for 30 min, supernatant was collected, that contained dabsyl-Met. Then, 50 µl of supernatant were injected onto a C18 column  to quantify dabsyl-Met at 436 nm using an HPLC instrument with a UV detector34.For protein-based Met sulfoxide reduction activity assays, dabsyl-Met-R-SO and Met-S-SO were prepared as previously described15, and the reaction mixture (100 µl) contained 20 mM dithiothreitol, 200 µM free Met-R-SO (or 200 µM free Met-S-SO), and 200–400 µg of total protein in PBS (pH 7.4). Reactions were carried out at 37 °C for 30 min and stopped by adding 200 µl acetonitrile at 4 °C for 10 min. After centrifugation at 13,000 rpm at 4 °C for 30 min, supernatant was collected that contained free Met. The supernatant (50 μl) was diluted 10 times with distilled water and prepared for OPA derivatization. OPA derivatization of amino acids and HPLC analysis were performed as described35 with minor modifications. The derivatization reagent was freshly prepared as a stock solution  at room temperature in a capped amber vial. Sample solutions (2–5 μl) were mixed with the OPA derivatization reagent to a 100 μl final volume. Following a 2 min reaction at room temperature, the mixture was injected onto a C18 column  to quantify free Met. Detection was by fluorescence of Met derivatives using a Waters 474 scanning fluorescence detector with excitation at 330 nm and emission at 445 nm.For free Met sulfoxide reduction activity assay, free Met, Met-37. 12- and 51-day-old flies were prepared and immobilized on ice for 1–2 min. After weighing, they were homogenized in 10 volumes of freshly prepared iced-cold 5% (w/v) MPA using a 1.5 ml micricentrifuge tube, a Teflon micropestle, and a handheld motor. The homogenates were incubated for 30 min on ice and centrifuged at 13,000 r.p.m. for 30 min at 4 °C. Then, the supernatants were filtered using 0.45 µm PTFE Acordisc CR 4 mm syringe filters. The filtrates (50 µl for GSH and 200 µl for GSSG) were transferred to vials and stored at – 80 °C for up to 1 month. For GSH analysis, 0, 0.5, 1, and 5 µM GSH standards or 50 µl of the supernatant were added to 1 ml of 0.1% EDTA in 0.1 M sodium hydrogenphosphate (pH 8.0). Then, 20 µl mixture was added to 300 µl of 0.1% EDTA in 0.1 M sodium hydrogenphosphate and 20 µl of 0.1% OPA in methanol. Capped tubes were incubated at 25 °C for 3 min in dark, and then the contents were filtered through 0.2 µm nylon filters and injected onto a C18 column  for further HPLC analysis. For GSSG analysis, 0, 0.5, 1, and 5 µM GSSG standards or 200 µl supernatant were incubated at 25 °C with 200 µl of 40 mM NEM for 25 min in dark and then this mixture was added to 750 µl of 0.1 M NaOH. After then, 20 µl of this mixture was added to 300 µl of 0.1 M NaOH and 20 µl of 0.1% OPA in methanol. Capped tubes were incubated at 25 °C for 3 min in dark, and then the contents were filtered through 0.2 µm nylon filters and injected onto a C18 column  for further HPLC analysis. Isocratic analysis was performed with 15% methanol in 25 mM sodium hydrogenphosphate (v/v), pH 6.0, as a mobile phase. Detection was by fluorescence using a Waters 474 scanning fluorescence detector with excitation at 350 nm and emission at 420 nm.We measured oxidized (GSSG) and reduced (GSH) glutathione levels as previously describedt test. Drosophila survival analysis on various diets and comparison of survivorship curves were carried out using the Log-rank and Wilcoxon tests.Statistical analyses were performed using the JMP (version 10) software (SAS Institute Inc.). Enzyme activities (overexpression versus control), food consumption at two different ages, and pupa production at each time point were analyzed by Student’s S-SO, whereas the activity of mouse MsrBs towards the reduction of free Met-R-SO is low, leading to the accumulation of free Met-R-SO in mouse plasma15. Drosophila contains single MsrA and MsrB genes and thus is also expected to exhibit low activity with free Met-R-SO. To restore the ability of flies to reduce free Met-R-SO reduction, which was lost during evolution of metazoa, we generated transgenic flies expressing yeast fRMsr by using the UAS-GAL4 binary system, which is commonly used to study gene expression and function in Drosophila38. Three independent homozygous UAS responder lines, fRMsr2.1, fRMsr9.1, and fRMsr4.2, were prepared that express yeast fRMsr by using this system. These responder lines and the activator lines (da-GAL4 for whole body expression and fatb-GAL4 for fat-body specific expression) were backcrossed with the control yw line at least 6 times. Then, the progeny of the cross among the three responder lines, the activator lines, and the control yw line were subjected to Western blotting . On the other hand, the da-GAL4 > fRMsr4.2 line, which expressed fRMsr at a low level, showed no lifespan extension   and the lifespan extension of males ranged from 12 to 25%  40. By reducing free Met-R-SO, fRMsr might supply additional Met which may participate in cell metabolism. Consequently, it is possible that excessive energy may promote body fat synthesis as an energy storage form. We examined starvation resistance of transgenic flies expressing fRMsr in whole body or fat body. fRMsr2.1 or fRMsr9.1 lines were crossed with the da-GAL4 or fatb-GAL4 drivers and the progeny were subjected to the analysis of starvation resistance . Moreover, when 1 mM Met was added to the same diet containing 10 mM paraquat, survivorship of da-GAL4 > fRMsr2.1 and da-GAL4 > fRMsr9.1 was further extended  . Thus, fRMsr expression in transgenic flies supports increased survivorship against paraquat-induced oxidative stress on this sugar-yeast diet. Next, we measured GSH and GSSG levels in 12- and 51-day-old flies in order to estimate oxidative stress levels in young and old flies upon fRMsr expression. In 12-day-old flies, we did not observe a difference in GSH and GSSG, whereas GSSG levels were reduced in 51-day-old flies of da-GAL4 > fRMsr2.1 and da-GAL4 > fRMsr9.1 strains, when compared with other control strains, but GSH levels were not changed . In particular, we did not observe any lifespan extension in both da-GAL4 > fRMsr2.1 and da-GAL4 > fRMsr9.1 lines on the diet without Met. Thus, cooperation between Met and fRMsr was required for lifespan extension.We hypothesized that Met may be a factor that supports lifespan extension by fRMsr by removing ROS. To test this possibility, two fRMsr-expressing strains, R-SO. However, this protein is absent in metazoa, which lost it during evolution, and this deficiency is not fully compensated for by other enzymes, leading to specific elevation of free Met-R-SO in mouse plasma15. In this study, we asked a simple question: can restoration of free Met-R-SO reduction in an animal support its physiological functions and regulate lifespan? Using fruit flies as a model, we found that ectopically expressed fRMsr increases stress resistance and extends lifespan of animals without affecting reproduction and food consumption. While lifespan extention by fRMsr was modest, we observed this effect in different transgenic lines as well as with different diets and with both virgin and mated flies. Interestingly, this effect was dependent on the levels of dietary Met: flies maintained on higher Met were better protected by fRMsr. Apparently, it is the reduction of free Met-R-SO that mediates the beneficial effects of the exogenous oxidoreductase lost during evolution and restored via ectopic expression in fruit flies.fRMsr occurs across most prokaryotes and unicellular eukaryotes, where it catalyzes the reduction of free Met-14, supporting its importance in lifespan control. What is then a possible evolutionary significance of its loss in metazoa? We hypothesize that unicellular organisms, including prokaryotes and lower eukaryotes, exhibit rapid growth if provided with necessary resources. However, feast may quickly turn to starvation due to rapid consumption. As such, unicellular organisms may face Met deficiency, in turn affecting the levels of S-adenosylmethionine, glutathione and other compounds dependent on Met supply. Many single-celled organisms are aerobic and often face oxidative stress, in contrast to multicellular organisms, which are better protected, e.g. due to cover of their skin and controlled delivery of molecular oxygen for cellular metabolism. Thus, fRMsr function may be needed by unicellular organisms more than by multicellular organisms. However, once the enzyme is lost, the ability for methionine sulfoxide reduction might be difficult to replace. Although the importance of fRMsr may have been reduced in multicellular organisms compared to the unicellular, its restoration may enhance stress resistance and support lifespan extension in at least some species.fRMsr has an important function in metabolism (by providing free methionine from methionine sulfoxide) and oxidative stress resistance (by removing ROS through cyclic oxidation and reduction of methionine). Deletion of this gene reduces lifespan of yeastR-SO and supports three major biological functions: oxidative stress resistance, protein repair, and regulation of protein function. This enzyme has been widely implicated in lifespan control. MsrB has the same protein repair and regulation functions12, but does not affect lifespan in Drosophila23. To explain this observation, one needs to focus on the differences between MsrA and MsrB. One difference is stereospecificity: Met-S-SO is a MsrA substrate, and Met-R-SO is a MsrB substrate; the other is the unequal activity of these enzymes with free Met sulfoxides. With respect to stereospecificity, the non-enzymatic oxidation of Met by ROS leads to an approximately equal mixture of Met-S-SO and Met-R-SO, and there is no evidence an isomerase is present that interconverts Met-S-SO and Met-R-SO. There is an interesting exception, Mical, a stereoselective monooxygenase that converts two conserved Met residues in actin to Met-R-SO34. However, this function points to an importance of MsrB in regulating protein function. Therefore, the distinct effects of MsrA and MsrB on lifespan control could not be explained by the biased formation of Met-S-SO or Met-R-SO.fRMsr is a relatively new addition to the group of eukaryotic Msrs, whose other members are MsrA and MsrB. MsrA is an oxidoreductase that reduces both protein-based and free Met-in vitro assays of MsrA and MsrB41. However, the actual substrates inside the cell are protein-based Met sulfoxides, and their reduction by MsrA and MsrB depends on the accessibility of specific Met residues. Aside from the effectiveness of MsrA and MsrB for the reduction of protein-based Met sulfoxides, one clear fact is that MsrB is very inefficient for the reduction of free Met-R-SO, whereas MsrA is highly active with both free and protein-based Met-S-SO. As a result, MsrA often exerts a stronger antioxidant protection.With respect to differences in catalytic efficiency for free Met sulfoxide reduction, we need to first consider the contribution of protein-based Met sulfoxide reduction to lifespan extension. Dabsylated Met sulfoxide and N-acetylmethionine sulfoxide are generally used as substrates for R-SO may have beneficial consequences on organismal physiology. The findings also point to the possibility of a combined nutritional and genetic strategy to increase lifespan, wherein a lost function, restored genetically, is supplemented with the substrate used to support this function. In addition, high Met can be toxic, and fRMsr may be viewed as an enzyme that decreases Met toxicity by repairing its oxidatively damaged form. It would be interesting to test additional functions that are lost during evolution of animals and that can be restored using a combination of genetic and nutritional approaches as well as to examine whether combinations of such functions exhibit additive effects on lifespan and stress resistance.Our findings with fRMsr transgenic flies are consistent with these differences between MsrA and MsrB and suggest that restoration of the ability to reduce free Met-Supplementary Information


In vivo, HNK markedly inhibited CNI-induced renal tumor growth; and it decreased the expression of phospho-c-Met and HO-1 and reduced blood vessel density in tumor tissues. Our results suggest a novel mechanism(s) by which HNK exerts its anti-tumor activity through the inhibition of c-Met-Ras-HO-1 axis; and it can have significant therapeutic potential to prevent post-transplantation cancer in immunosuppressed patients.Honokiol (HNK) is a small molecule with potent anti-inflammatory and anti-tumorigenic properties; yet the molecular targets of HNK are not well studied. Hyperactivation of the receptor tyrosine kinase c-Met and overexpression of the cytoprotective enzyme heme oxygenase-1 (HO-1) play a critical role in the growth and progression of renal cell carcinoma (RCC). Interestingly, the calcineurin inhibitor (CNI) cyclosporine A (CsA), an immunosuppressant used to prevent allograft rejection, can also increase the risk of RCC in transplant patients. We studied the potential role of c-Met signaling axis on CNI-induced renal tumor growth and tested the anti-tumor efficacy of HNK. Importantly, CNI treatment promoted c-Met induction and enhanced c-Met-induced Ras activation. We found that HNK treatment effectively down-regulated both c-Met phosphorylation and Ras activation in renal cancer cells. It inhibited the expression of both c-Met- and CNI-induced HO-1, and promoted cancer cell apoptosis.  Chemically, HNK is characterized as a small bi-phenolic lignan2. Recent reports have demonstrated the anti-inflammatory, anti-angiogenic, and anti-tumor properties of HNK1. It has been demonstrated that the treatment with HNK can inhibit receptor tyrosine kinase (RTK)-mediated events and induce pro-apoptotic signaling pathway(s) in tumor cells3. Thus, HNK can be a very promising agent to prevent or restrict tumor growth.Honokiol (HNK) is a natural bioactive compound originally derived from the seed and bark extract of magnolia plant species (Magnolia obovata), which has been used in traditional Chinese and Japanese medicine5. c-Met is overexpressed in both clear cell and papillary renal cell carcinoma (RCC)7. Notably, both HGF and c-Met are located on chromosome 7 and is often amplified in clear cell RCC; and germline c-Met mutations in papillary renal cancer promotes constitutive activation of c-Met9. Homodimerization of c-Met upon binding of HGF induces phosphorylation of two tyrosine residues (Y1234 and Y1235) present within the catalytic loop of the tyrosine kinase domain10. Subsequently, phosphorylation of tyrosines 1349 and 1356 in the C-terminal tail recruits signaling adaptor proteins GRb2, SHC and CRK, which mediate the activation of multiple signaling pathways11. c-Met activation promotes the expression of anti-apoptotic molecules in renal cancer cells4. However, the effect(s) of HNK on c-Met-mediated pro-tumorigenic pathway in renal cancer cells, including tumor angiogenesis, is completely unexplored.The RTK c-Met and its ligand HGF play a significant role in the growth and progression of renal cancer4. HO-1 is a catalytic enzyme involved in the degradation of potent inflammatory agent heme into carbon monoxide (CO), billiverdin and ferrous iron12. Although it plays a beneficial role in repairing tissue injury, cancer cells over-express HO-114. By the virtue of its cyto-protective effects, HO-1 plays an important role in tumor growth and the prevention of chemotherapeutic drug-induced apoptosis15. HO-1 has also been shown to induce tumor angiogenesis16. Many transcription factors, such as Nrf2 and Bach1 that regulate HO-1 expression, also play a major role in tumor progression18. However, the effect of HNK in modulating c-Met-induced HO-1 expression in RCC has not been studied.We have recently demonstrated that the induction of c-Met promotes over-expression of the cytoprotective molecule heme oxygenase-1 (HO-1) for the survival of renal cancer cells21. The CNI Cyclosporine A (CsA) inhibits T cell activation, by down-regulating the phosphatase activity of calcineurin and preventing the nuclear translocation of NFAT, a crucial factor for T cell activation, and thereby suppresses immune system22. However, independent of its immunosuppressive functions, which may lead to immune escape of cancer cells, the CNI treatment can directly induce tumor-promoting pathways26. We have shown that the CNI treatment promotes Ras activation and induces the expression of cytoprotective molecule HO-1 for the rapid growth and survival of renal cancer cells27. We have demonstrated that c-Met is also a potent inducer of the Ras-HO-1 pathway4. Interestingly, in our initial studies, we have found that HNK can inhibit CNI-induced Ras activation and HO-1 over-expression in renal cancer cells27. However, we have not yet investigated the effect of HNK treatment on c-Met-induced tumorigenic pathway(s) and it’s possible cross-talk with CNI-induced events; and if HNK can restrict renal tumor growth in vivo.Although aberrant c-Met and other RTK hyper-activation are the major drivers of renal cancer growth, recent reports clearly suggest an alarmingly high incidence of renal cancer in patients receiving calcineurin inhibitors (CNIs) as immunosuppressive therapy for the treatment of immune disorders or after having organ transplantsin vivo. Our observations suggest that HNK can act as a novel therapeutic agent for c-Met-Ras-HO-1-mediated renal cancer, with particular importance to tumor growth in immunosuppressed patients. In future, we suggest that along with anti-tumorigenic function, the anti-inflammatory property of HNK can also be utilized to sustain the immunosuppression of transplant and other patients with immune disorders.In this study, we report that HNK treatment significantly inhibits c-Met phosphorylation and c-Met-/CNI-induced Ras activation and HO-1-overexpression in renal cancer cells; and it restricts CNI-induced renal tumor growth 4. Here, we checked whether HNK treatment has any effect on c-Met-mediated signaling and Ras activation in 786-O and ACHN renal cancer cells. We observed that the c-Met ligand HGF significantly induced c-Met phosphorylation, which was markedly reduced following HNK treatment in both 786-O and ACHN (data not shown) cells  can induce Ras activation to promote the growth and progression of renal cancer; however, the role of CsA in conjunction with c-Met signaling axis has not been studiedsed Fig. ; and we sed Fig. .4. Here, we wished to study the effect of HNK on Nrf2 nuclear translocation. Analyzing the nuclear versus cytoplasmic localization of Nrf2, we found that HNK treatment markedly inhibited both c-Met and CsA-induced Nrf2 nuclear translocation ; conversely, we also found that HNK treatment retained Nrf2 in the cytoplasmic compartment, as seen with increased Nrf2 expression in the cytoplasmic fraction . Together, our results demonstrate that HNK significantly down-regulates c-Met- and CNI-induced Ras activation and Nrf2-mediated HO-1 over-expression in renal cancer cells.As discussed earlier, the expression of HO-1 plays an important role in both c-Met and CsA-mediated tumorigenic pathways in renal cancer cells. Here, we examined the effect of HNK on c-Met and CsA-induced HO-1 expression. We observed that HNK treatment significantly inhibited both HGF and CsA-induced HO-1 expression Fig. . It is e4. Here, we wished to study the effect of HNK in counteracting c-Met-induced cell survival. By MTT assay, we observed that HNK treatment significantly inhibited HGF/c-Met-induced proliferation of renal cancer cells -mediated signaling also confers tumor invasiveness by a direct effect on renal tumor cells and promotes metastasis30. Since, in this study, we also found that CsA treatment potentiates c-Met-mediated signaling, we wished to study the effect of HNK on both c-Met and CsA-induced migration of renal cancer cells in vitro by performing a wound healing assay. The ability of cells to migrate and fill the wound was quantified at 0 h and 12 h. We observed that both HGF- and CsA-induced signals increased the number of migrated cells that filled the wound area compared with control cells; whereas HNK significantly inhibited the increased migration of cancer cells following treatment with both HGF and CsA  to form tube-like structures (tube formation assay), an important step in the cascade of events that leads to new vessel formation; and if HNK can block the process. We observed that the supernatants from both HGF- or CsA-treated cancer cells increased the number of endothelial tubes formed compared with control; whereas the supernatants from cells grown in the presence of HNK significantly inhibited the tube formation ability of HUVEC cells -induced renal tumor in vivo; and HNK inhibits CsA-induced increased tumor vessel density, and it promotes apoptosis of tumor cells.Next, we examined the tumor vessel densities through CD31 staining. As expected, CsA treatment markedly increased vessel density compared with the vehicle-treated group. HNK treatment reduced tumor vessel density in both CsA- and vehicle-treated mice Fig. . To checin vitro data, CsA treatment also increased HO-1 expression (red stain) in vivo, and HNK treatment significantly inhibited CsA-induced HO-1 expression  Fig. , we obseion Fig. . These r3. c-Met-mediated Ras activation plays a major role in the accelerated growth of renal cancer, which is also a critical problem in immuno-suppressed patients24. Here, we report that HNK can effectively inhibit c-Met-mediated growth promoting pathways in RCC, involving the cytoprotective molecule HO-1. Our findings also suggest that CNI (CsA), which is used for the treatment of immune disorders, enhances c-Met activation and augments HGF/c-Met-induced Ras activation in renal cancer cells; and that HNK treatment down-regulates c-Met activation to inhibit CNI-induced renal tumor growth. Our observations show that HNK can effectively target c-Met-Ras-HO-1 pathway to restrict renal tumor growth, suggesting its use as a novel therapeutic agent in RCC.The natural product HNK, which is a small molecular weight polyphenol, has been shown to have potent antitumor activity4. Here, we show that the HNK treatment can not only inhibit HGF-induced c-Met phosphorylation but also effectively suppress HGF/c-Met-mediated Ras activation. We speculate the role of HNK-induced cellular phosphatase(s) in the down-regulation of c-Met phosphorylation, based on the reports that HNK induces SHP-1, a protein tyrosine phosphatase, to down-regulate STAT3 phosphorylation; and SHP-1 can play a role in the down-regulation of c-Met phosphorylation35. However, further studies are needed to completely understand the molecular events associated with HNK-induced down-regulation of c-Met phosphorylation.Ras mutation is not very common in renal cancer. One of the possible mechanism(s) for hyper-activation of the Ras pathway in renal cancer cells (including the cells used in this study) is due to the signaling through upstream receptor tyrosine kinases. Our recent report suggests that HGF/c-Met-mediated signaling promotes Ras activation in renal cancer cells24. Here, we found that CNI augments c-Met activation in a ligand independent manner and also enhances HGF/c-Met-induced Ras activation. However, the effector molecule(s) that mediate CNI-induced c-Met phosphorylation needs to be identified. It is known that Raf kinase inhibitory protein (RKIP) disrupts the interaction of Ras-Raf-ERK and phosphorylation of RKIP relieves the inhibitory effect of the molecule and promotes downstream signaling36. We have previously reported that HNK effectively inhibits CNI-induced phosphorylation of RKIP, and thereby blocks Ras downstream signaling27. Hence, HNK can effectively inhibit CNI-induced growth promoting signals. It has been reported that ligand independent activation of c-Met increases tumor burden and it would be of interest to know whether c-Met is activated in cancers arising in patients treated with CNI37. Nevertheless, our findings emphasize the potential cross-talk between CNI- and c-Met-induced tumorigenic pathways.In our previous studies, we have demonstrated that CNI treatment induces Ras activation and promotes renal cancer cell growth; and this effect of CNI is independent of its immunosuppressive function27. We have also demonstrated the nuclear translocation of Nrf2, the transcriptional activator of HO-1, plays critical role in promoting c-Met-mediated renal cancer cell survival4. In this study, we found that HNK can effectively inhibit the nuclear translocation of Nrf2 and down-regulates both c-Met- and CNI-induced HO-1 over-expression in renal cancer cells. In addition to HO-1 over-expression, c-Met-mediated increase in the expression of anti-apoptotic molecules, such as Bcl-2 and Bcl-XL, protects renal cancer cells from apoptosis. It has been reported that HNK exerts its apoptotic function on cancer cells possibly through cell type specific pathways, which includes p53 activation, inhibition of mTOR complex, caspase as well as PARP cleavage, and downregulation of Bcl-XL40. In renal cancer cells, our observation suggests that, HNK can promote cancer cell apoptosis through the down-regulation of c-Met-induced Bcl-2 and Bcl-XL expression, and through increased cleavage of caspase-3. Importantly, both c-Met hyper-activation and CNI treatment can promote metastasis of cancer cells30; and increased tumor cell migration is the cause for metastasis28. Here, we also report that HNK inhibits both c-Met- and CNI-induced induced renal cancer cell migration, suggesting that HNK can have therapeutic potential in restricting metastasis of RCC.Previously, we have reported that both c-Met- and CNI-mediated Ras activation leads to HO-1 over-expression in renal cancer cells42. CNI treatment also increases VEGF expression in renal cancer cells43. We have previously demonstrated that HNK can inhibit Ras-mediated over-expression of VEGF in renal cancer cells27. It has been suggested that HNK inhibits transcription factors NF-κb and STAT3, which regulate VEGF secretion in tumor microenvironment, and HNK inhibits angiogenic signals through the downregulation of VEGF receptor 2 (VEGFR2) phosphorylation46. However, the effect of HNK on renal tumor angiogenesis is not studied. In this study, we found that HNK treatment can inhibit both c-Met and CNI-induced secretion of angiogenic factors from renal cancer cells, as evident from the decrease in the extent of endothelial cell tube formation. Therefore, our findings suggest an important role of HNK to attenuate angiogenesis and vascularization that plays a crucial role in the development of renal tumors, which are highly vascular.In addition to promoting cell survival, both c-Met-mediated signaling and HO-1 can induce vascular endothelial growth factor (VEGF) expression and promote tumor angiogenesisin vivo are limited49. Also, there is an unmet need to identify novel therapeutic drugs/approaches to effectively suppress CNI-induced tumor growth19. HNK is one such promising drug with both chemopreventive and anti-inflammatory properties1; therefore, the use of HNK as a therapeutic for patients after organ transplantation can potentially have dual benefit, i.e. to restrict CNI-induced cancer and to mitigate the dose of CNI used to achieve immunosuppression in patients. In our renal tumor xenograft study, we found that CNI-induced increased tumor growth is associated with increased c-Met activation in tumor tissues. Importantly, HNK significantly inhibited CNI-induced tumor growth and is associated with decreased c-Met activation, which correlates with our in vitro findings. HNK inhibited CNI-induced HO-1 expression in tumor tissues and increased tumor cell apoptosis. We have also observed that CNI treatment increased the vessel density in tumor tissues; and it decreased with HNK combination treatment. Therefore, our in vivo findings suggest that HNK targets c-Met-HO-1 pathways in restricting renal tumor growth. c-Met inhibitors are being used in clinic for the treatment of RCC, and it will be interesting to further explore the potential benefits of using HNK in combination therapy50. Through this study, we also provide the basis for the use of HNK in preventing post-transplantation cancer.Although the chemotherapeutic effects of HNK on various malignancies have been reported, studies exploring the anti-tumorigenic effect of HNK on renal tumors In summary, for the first time our studies demonstrate that HNK inhibits c-Met-mediated tumorigenic pathways. We found that c-Met activation and HO-1 overexpression plays a critical role in CNI-induced renal tumor growth; and it is markedly inhibited by HNK treatment. Our data clearly suggest that HNK targets c-Met-Ras-HO-1 tumorigenic pathways, and a combination therapy with HNK and other target-specific agent(s) can have promising benefits for the treatment of renal tumors, with particular importance to post-transplantation cancer.Human renal cancer cell lines 786-0 and ACHN  were cultured in RPMI 1640 and EMEM medium respectively, supplemented with 10% fetal bovine serum (FBS) . All cell lines were authenticated by Short Tandem Repeat (STR) profiling from ATCC in December, 2015. The cell lines were passaged for less than three months after thawing. Human Vascular Endothelial cells (HUVEC) were obtained from Lonza and were grown in EBM-2 basal medium supplemented with EGM-2 singlequots (growth supplements) .18H18O2, Molecular Weight: 266.334) and CsA were purchased from Sellekchem  and were dissolved in DMSO. The recombinant human HGF was purchased from Peprotech .Synthesized pure Honokiol (HNK)  and transferred to a polyvinylidene difluoride membrane . The membranes were incubated with anti-phospho-MET, anti-c-MET, anti-HO-1, anti-Bcl-xL, and anti-Bcl-2, anti-cleaved-caspase-3, anti-GAPDH, , anti-Nrf2, anti-Sp1 ; or anti-β-Actin ; and subsequently incubated with peroxidase-linked secondary antibody (Santa-Cruz). Reactive bands were detected using chemiluminescent substrate . Frozen tumor tissue samples were lysed on ice with RIPA buffer; and the lysates were centrifuged and the supernatants were subsequently used for Western blot analysis. For all Western blots, 10 ug of each sample was loaded, and the primary and secondary antibodies were diluted according to the manufacturer’s recommendation.The active/GTP-bound form of Ras in the cell lysates was measured using an EZ-detect Ras activation kit .MTT cell proliferation assay  kit was used to quantify cell proliferation, following manufacturer’s protocol.Cellular apoptosis was measured using an allophycocyanin (APC)-conjugated Annexin-V and propidium iodide (PI) apoptosis detection kit . Following staining, the cells were analyzed by flow cytometry on a FACSCalibur.Nuclear extracts were prepared from the cells using a nuclear extraction kit , following manufacturer’s protocol.Cells were seeded on 6-well culture plates. When cells were grown to confluence, medium was aspirated and, using a 200 μL pipette tip, cells were scratched along the diameter of the well, to simulate the wound. Cells were washed twice with PBS and were treated in serum-free medium as indicated. At 0 h and 12 h after incubation, wound diameter in each well was photographed using phase contrast microscope to assess wound closure. Number of cells migrated to the wound area beyond the reference line were quantified using NIH ImageJ software.4 cells) on a layer of previously polymerized growth factor-reduced extracellular matrix (Geltrex) in 6-well culture plates. After 6 h, HUVEC culture well was photographed through a phase contrast microscope to assess changes in HUVEC morphology and capillary-like tube formation. The extent of tube formation was quantified by counting the number of uninhibited polygonal tube-like structures in five randomly selected microscopic fields.Conditioned medium from cancer cells was collected and utilized to culture HUVECs (2 × 106 cells/mice) were injected subcutaneously into the flanks of six-week old immunodeficient (nude) mice . Once palpable tumors were formed, mice were randomly distributed to different treatment groups as described. CsA and HNK were diluted in normal saline to reach the desired concentration before injection. Tumor volume was measured using digital caliper measurements obtained at regular intervals. The volume was estimated by following standard method, using the formula V = π/6 × a2 × b where a is the short axis and b is the long tumor axis. At the end of the study, tumors were excised and processed for further studies.Animal experiments were performed following all guidelines in accordance with Institutional Animal Care and Use Committee (IACUC) protocol approved by Boston Children’s Hospital. 786-0 cells  and tumor tissue sections were cut on a cryostat . Following standard protocols, the sections were processed for immunolabeling with anti-CD31 (1:50 dilution) , anti-phospho-c-Met (1:300 dilution), total-Met (1:300 dilution)  or anti-HO-1 (1:50 dilution) ; and subsequently labelled with a species-specific horseradish peroxidase-conjugateed secondary antibody. In tissue sections stained with anti-CD31 antibody, mean vessel density was calculated by standard grid counting method at x400 magnification.n = 5 mice per group will provide 80% statistical power  to detect significant differences of 50% or more in tumor volume between the groups. Statistical analysis was performed using SPSS software version 23 . Conservative two-tailed values of p < 0.01 were considered statistically significant in order to protect against type I errors due to multiple group comparisons. For in vitro experiments, statistical significance was determined by Student’s t test. Differences with p < 0.05 were considered statistically significant.Changes in mean tumor volume were compared by two-way repeated-measures analysis of variance (ANOVA) to test for differences in slopes between the treatment groups over time using the group-by-time interaction F-test. Fit to the data in handling tumor volume measurements from the same animal at the serial time points was accounted for using a compound symmetry covariance structure in the mixed-model longitudinal approach and this provided excellent fit to the data, as judged by the Akaike information criterion (AIC). Data are presented as mean and standard deviation (SD). Power analysis indicated that sample sizes of Supplementary information


Date of publication: 21 November 2016Mycobacterium chimaera associated with the 3T heater-cooler system used during open-heart surgeryInvasive cardiovascular infection by http://ecdc.europa.eu/en/publications/Publications/RRA-mycobacterium-chimaera-November-2016.pdfDate of publication: 18 November 2016Outbreaks of highly pathogenic avian influenza A(H5N8) in Europehttp://ecdc.europa.eu/en/publications/Publications/risk-assessment-avian-influenza-H5N8-europe.pdfDate of publication: 10 November 2016Systematic review on hepatitis B and C prevalence in the EU/EEAhttp://ecdc.europa.eu/en/publications/Publications/systematic-review-hepatitis-B-C-prevalence.pdfDate of publication: 21 November 2016HIV testing in Europehttp://ecdc.europa.eu/en/publications/Publications/HIV-testing-guidance-evaluation.pdfDate of publication: 18 November 2016Last-line antibiotics are failing: options to address this urgent threat to patients and healthcare systemshttp://ecdc.europa.eu/en/publications/Publications/antibiotic-resistance-policy-briefing.pdfDate of publication: 29 November 2016HIV/AIDS surveillance in Europe 2015http://ecdc.europa.eu/en/publications/Publications/HIV-AIDS-surveillance-Europe-2015.pdfDate of publication: 4 November 2016Influenza virus characterisation, Summary Europe, September 2016http://ecdc.europa.eu/en/publications/Publications/influenza-virus-characterisation-september-2016.pdfDate of publication: every Fridayhttp://ecdc.europa.eu/cdtrUpdated: September 2016http://ecdc.europa.eu/en/publications/peer-reviewed/Pages/index.aspx


Sport is widely recognised for the contribution it can make to international development goals. More specifically, the value of sport as a tool for development gained its impetus through the HIV/AIDS pandemic in sub-Saharan Africa. The institutionalized relationship between sport and development has mainly focussed on sport-for-development (SfD) non-governmental organisations (NGOs). This study proposed to examine the response of National Sports Associations (NSAs) towards the multisectoral approach for HIV/AIDS prevention in Zambia. The study draws on lessons learnt from how NSAs within a resource-scarce or low-income country responded to a health pandemic. While public health was previously a state and health sector preserve, the impact of HIV/AIDS pandemic influenced not only the way that a pandemic is managed but also other public health issues. A case study approach was adopted comprising of three National Sports Associations (NSAs) as units of analysis. The study utilised semi-structured interviews, documentary analysis and field observations to gain perspectives on how each NSA mainstreamed and implemented work-based health programmes. Using governance and policy network theories, the paper discusses each NSAs' role in the governance and implementation of a multisectoral approach to a health pandemic. The findings identified lack of engagement of sports agencies at strategic decision-making level, marginalisation of sport by other sectors, and variations in implementation patterns among sports agencies. Further findings indicate that lack of resources among government sport agencies or departments limited their involvement with other state or non-state actors in strategic level meetings or health policy networks. Resource-scarce conditions placed limitations on the political steer of state actors while non-state actors with foreign resources attracted collaboration from other public health policy networks. Although social change can be either positive or negative human immunodeficiency virus (HIV) which causes the acquired immune deficiency syndrome (AIDS). Coalter Other significant contributions towards the growth of the SfD sector include: the inclusion of SfD in strategic plans to achieve development outcomes by both the United Nations Despite its recognition as an agent of social change, scholars As the SfD movement is considered to be in its infancy stage, critical theorists The focus of this study is on the role played by National Sports Associations (NSAs) within public health policy strategic forums. In comparison to SfD NGOs, the ethos of NSAs is sports for sports' sake 2.When the first case of HIV was diagnosed in 1984, the response lacked high-level political commitment and strategic management NGOs gained recognition as partners in health care provision in 1993 when changes demanded by the World Bank in health provision called for the inclusion of non-state actors to promote diversity and competition one AIDS action framework; one national AIDS coordinating authority; and one country-level monitoring and evaluation system. In support of the establishments of the NAC, Morah and Ihalainen state three key reasons: (1) the need for a robust multisectoral response due to the complexity of combating the HIV/AIDS pandemic; (2) the need for a multisectoral response consisting of state and non-state actors opposed to a dominant traditional public-health paradigm which was out of tune with the challenges of combating HIV/AIDS and; (3) the lack of authority of Health Ministries to command other government departments While the state played a minimal coordinating role, the geographical limitation of NGOs Re-orienting of the response to HIV/AIDS governance meant that quasi-nongovernmental agencies like NSAs, once marginalised, were now eligible to receive donor funds under the multisectoral approach. However, since foreign donors still wield huge economic resources, the power to set agendas, priorities and preferred delivery mechanisms largely lies with them. As earlier discussed, the absence of a compelling evidence base in support of the SfD movement causes concern for potential funders to adopt sport as a development tool. Zambia's unique political, economic and social country background Most of SfD research in Zambia has focussed on micro-level analysis of SfD NGOs or cases of sport and HIV/AIDS schemes at community level. This is a meso-level analysis of NSAs The extensive use of the word “governance” in development literature over the past decade denotes a shift in focus from government to governance. Governance denotes new modes of governing that are not in the form of hierarchical control but much more of cooperative processes of governing between state and non-state actors Despite new forms of governance centring on the reduced role of the state, Bell and Hindmoor argued that governments still play a central role in making new governance a possibility or option for governing The HIV/AIDS pandemic in Zambia has significantly transformed the public health policy landscape in what was before a state actor dominated space. Today, the public health policy space is characterized as an interdisciplinary and collaborative space opposed to the dominated space found in state bureaucracies or hierarchies. Neoliberal approaches have influenced such policy spaces, enabling state and non-state actors to interact and influence policy. The days of a monolithic state controlling the policy making process are long gone. Hence, policy network theory provides analytical purchase to examine the public health landscape as “involving a diversity of actors who are mutually interdependent” Using policy network theory to examine the governance structure of HIV/AIDS under the multisectoral approach, the theory enlightens us about the following: why some actors have limited participation in decision-making; why some actors enjoy more privileges than others; why some issues get included or excluded from the agenda; and why the understanding of resource exchange by some members maximise their influence to attain their goals The use of the term “policy network” has featured a variety of models which sometimes causes confusion because the same term had previously been used to mean or refer to different interactions or interests. Marsh and Rhodes' 3.A case study approach The selected National Sports Associations (NSAs) were: Football Association of Zambia (FAZ); Netball Association of Zambia (NAZ); and the Zambia Basketball Association (ZBA) . Interviews were conducted by the principal researcher between 2010 and 2014. Interviewees were provided with information sheets via email. Verbal and written consents were obtained before proceeding with data collection. Data presented here are drawn from eleven (11) semi-structured interviews conducted with: NSA's President or the General Secretary; NSA sport development officers; government officials representing the Department of Sport Development (DSD) in the Ministry of Sports, Youth and Child Development (MSYCD); National Sports Council of Zambia (NSCZ) officials; and lastly, senior officials from the National AIDS Council (NAC). Sports Association and Sports Department senior official interviewees were purposively sampled based on their seniority and their involvement in the day to day handling of policy issues within each sport association and government department, respectively. An interview guide was used to make the process of interviewing a number of different people more systematic and comprehensive Other data collection methods included documentary analysis which focussed on public health policies, national HIV/AIDS policy documents such as the National HIV/AIDS Strategic Frameworks (NASF) 2005–2010 4.4.1.Global Health literature I think really what worked for us is to have a coordination mechanism at district, provincial and at national level. That way it's easier for us to monitor who is doing what at a micro level and at a macro level…the multisectoral approach for the Zambian context means that different sectors bring to the HIV/AIDS response their skills, their resources, and their comparative advantage.The findings revealed a wide range of stakeholders comprising of government agencies, civil society organisations, and the private sector. This reflected the new governance approaches of health pandemics, proving HIV/AIDS could not be tackled by the health sector alone The composition of the NAC was indicative of the multisectoral response at national level as it comprised of four Permanent Secretaries positioned in government ministries responsible for: Education; Health; Community Development and Social Welfare; and Sport, Youth and Child Development. The rationale for the inclusion of each of the four policy executives were as follows:Ministry of Education–education as a “social vaccine” within school settingsMinistry of Health–the NAC is under the authority of the MoH as a semi-autonomous organisationMinistry of Sport, Youth and Child Development (MSYCD)–HIV/AIDS impacts the household more severely than any other unit. Children and young people suffer the impacts of HIV/AIDS considerably more than other groups Of interest to this study among these four ministerial state actors was the MSYCD which consists of three departments, namely: Department of Sport Development (DSD); Department of Youth Development (DYD) and Department of Child Development (DCD). Notwithstanding that all three departments fall within the remit of the MSYCD Permanent Secretary (PS), only youth and child policy concerns were highlighted by a NAC official:It was deliberate that as National AIDS Council, the youths have a permanent seat on the council so that we work together with them to see how then do we address the challenges that the youths are facing and how HIV/AIDS is also affecting the progress within the youths.Further marginalisation of the value of sport in interactions between the MYSCD and NAC were evident when NAC representatives attended strategic meetings at the MSYCD office headquarters. Rather than engage all three departmental directors, a DSD Senior official reiterated that the NAC's significant focus was on the roles of the DYD and DCD in matters related to youth and child welfare:…actually, the NAC Director of Multisectoral Approach came to see the Child Department and then as we were talking, I think someone said, “no by the way we have got Sports Department [also within the MYSCD]”.The marginalisation of the Director for the DSD by the NAC was indicative of the lack of integration of sport within HIV/AIDS implementation plans. An exclusion like that above from formal strategic meetings continues to exacerbate the lack of integration of sport into development activities. The sport sector needs to integrate with planning for development outcomes at the national level and also with mainstream development agencies in order to make significant contributions towards evidence production. Policy network theory Consequently, within meso-level policy networks, the study revealed less engagement of government sport agencies (DSD), semi-governmental  and quasi-nongovernmental bodies (NSAs) in wider development activities. However, the emergence of SfD NGOs as part of the new brigade of youth services demonstrated integration of sport organisations into wider development agendas at micro level. SfD NGOs were active within NAC's subnational structures at provincial and district level, namely: Provincial AIDS Task Forces (PAFT) and District AIDS Tasks Forces (DATF) We all get invited and sometimes word gets round from NGO to NGO that there is going to be a district AIDS meeting every few months or so…all NGOs and government units get invited to talk and share about AIDS as long as they have a storyMarsh and Rhodes further stress resource dependency as a key feature of policy networks and that resources determine the level of hierarchy within networks. Despite the unrestricted access to the identified self-organising networks, lack of resources limited the involvement of most of the sports agencies from these information sharing, giving and policy shaping forums 4.2.Having noted the absence of NSAs within HIV-focused policy networks, the study sought to focus on internal organisational deliberations fulfilling the HIV/AIDS mainstreaming agenda Institutional commitment to engage in policy making networks to inform the development of in-house HIV/AIDS policy varied among the three cases. In spite of the requirements within the national sport policy We have a lot more capable doctors… they are not full time doctors of the national team, they work in hospitals so their work is very valuable now to the Football Association It [HIV/AIDS] is there in our strategic plan, …What we said is that if we keep a child active through sports, we may one way or another try to move them away from these other vices that may bring their status or the health levels down. So in one way or the other HIV/AIDS is also incorporated …as a teachers or council officers, we have programmes at work We [the ZBA] don't have a specific policy for [HIV/AIDS] but how we have gotten around the issue of not having a specific policy is to look at the [external HIV] programme itself. If it has to involve youths, we are assisted maybe through Ministry of Education guideline on behaviour change .These variations were shaped by each NSA's internal capabilities, resources and external relationships with those in health policy networks. Football, the national sport, had more resources compared to the other two cases, ZBA and NAZ. Part of its human resource were medical personnel who belong to the FAZ Medical Committee (FAZ MedCom). This internal capability was utilised for policy guidance and implementation within FAZ. The FAZ MedCom was characterised by highly restricted membership and shared basic values raising health awareness and supporting sustainable development within its strategic development plans. Netball, being a female dominated sport, placed strong emphasis on mainstreaming of gender within its strategic development plans, a key aspect of addressing the HIV/AIDS pandemic. However, despite NAZ's clear intentions for mainstreaming HIV/AIDS and gender, no formal engagement in HIV/AIDS and gender policy networks were noted within this NSA.Among all three cases, the Netball Association was the only NSA which had included the use of sport as a tool for The ZBA acknowledged its resource-poor conditions and utilised the various networks within the highly fragmented HIV/AIDS policy space to mainstream HIV/AIDS. Of interest was the acknowledgement by the association that its members possessed membership of multiple social entities. There was a sense of confidence that ZBA members had access to well-designed HIV/AIDS and gender mainstreaming programmes elsewhere such as educational institutions or places of work. Hence, developing institutional plans was seen as failing to grasp the policy rhetoric-reality gap faced by the NSA:We have not really stressed HIV and the reason is basically because…we don't have a specific budget that would look at those things .Therefore, no formal policy on HIV/AIDS mainstreaming existed. In comparison to FAZ, both ZBA and NAZ were woefully under-funded by central government and did not have the same political power as FAZ to influence HIV/AIDS policy decision-making and implementation.Within the sport and HIV/AIDS policy space in Zambia, non-state actors, the SfD NGOs, were confronted with animosity by government. The animosity developed from a perception that SfD NGOs were diverting foreign resources away from government We have worked with a lot of NGOs, with SCORE, with the Norwegians…we would bring people from the D.E.C–Drug Enforcement Commission, doctors, peer educators [who] would be able to speak to [national footballers] about HIV .We have had involvement of CHAZ and Society for Family Health at our events (Former ZBA President–Telephone Interview).We have partnerships with Sports in Action, NOWSPAR and EduSport Foundation. Through their [SfD NGOs] partnerships with other international organisations [UK sport and England Netball], they are able to relay health education through their workshops .SfD NGOs had links to a multiplicity of development sector actors enabling bilateral and multilateral collaborations involving NSAs, health sector HIV-focused NGOs and other varied civil society groups. Such collaborations were generally informal and closely matched the conceptualisation of plurilateral governance posited by Howlett, Rayner and Tollefson Of significant importance, regarding the instigation of these identified policy networks, were the restricted eligibility to foreign donor funding available only to SfD NGOs despite their geographical limitation. However, the attraction of NSAs to other actors was their countrywide geographical coverage. Hence, collaborative planning involving NSAs, the DSD and other non-state actors was essential since SfD NGOs were limited in geographical coverage during programme implementation.A new policy community The Ministry of Education, they are very capable …and we have spoken in that regard and that is why I feel once we bring the grassroots programme you know which is fully sponsored by FIFA it can bring change because, of course the Ministry of Education needs funding, we, FAZ, need funding, the Ministry of Youth and Sport needs funding .The FIFA “11 for Health” membership closely fitted Marsh and Rhodes' policy community as an “integrated community” “we have put the programme to operate at both [levels], if there is no room during PE, they [teachers] can use the programme during sports but we would have loved to have infused it into PE, that is why we are using teachers who have PE on their timetables and able to use their PE lessons for this programme” Revisiting the SfD limitation regarding the lack of an evidence base for sport as a tool for development, perhaps the composition of such policy communities as the “11 for Health” programme can enhance the gathering of evidence from different departmental/sectoral experts. Health programmes like the FIFA “11 for Health” demonstrate a shift from hierarchy (MoH) to heterarchy (multiplicity of actors in governance) 5.The paper has highlighted that public health initiatives demand an inter-organisational approach. HIV/AIDS governance as espoused in the UNAIDS “Three Ones” approach showed fragmentation and marginalisation of some sectors, particularly, the sport sector as suggested by the evidence presented. Instead, the multisectoral approach must bring different sectors together, get them involved in the complexities of inter-organisational integration and provide coordination. However, the lack of participation of sport agencies, in particular NSAs at national level has negative implications for the respective NSAs' subnational structures to emulate. Consequently, sport agencies fail to contribute to both national and subnational evidence gathering structures. This strengthens the criticism already made regarding the lack of sport's evidence base to back up the legitimisation of sport as a tool for development. Further weaknesses of sport influencing evidence gathering were revealed in how varied sport as a sector engaged in policy networks, policy communities or issue networks. These variations were influenced by each NSA's political power, internal capabilities or power of resources.The lack of engagement of NSAs in strategic decision-making tends to exacerbate the lack of influence that sport organisations and the sport sector in general may have in shaping public health policies. The challenge remains for sport policy brokers or programme designers to position themselves within development policy forums to effectively negotiate what the sector can offer to enable health-related behavioural change.While the influence of SfD NGOs on NSAs in relation to HIV/AIDS programme implementation is widely acknowledged, researching the impact of SfD NGOs on NSAs remains. Further research could look at how partnerships between NSAs and SfD NGOs coordinate evidence gathering to strengthen the legitimisation of sport in development.


In summary, our findings suggest that the BLM pump model is an attractive model to analyze the underlying mechanisms of fibrosis and test the efficacy of potential therapies. However, the choice of mouse strain, duration of BLM administration and dose must be carefully considered when using this model.The murine bleomycin (BLM)-induced fibrosis model is the most widely used in systemic sclerosis (SSc) studies. It has been reported that systemic delivery of BLM via continuous diffusion from subcutaneously implanted osmotic minipumps can cause fibrosis of the skin, lungs, and other internal organs. However, the mouse strain, dosage of BLM, administration period, and additional important features differ from one report to the next. In this study, by employing the pump model in C57BL/6J mice, we show a dose-dependent increase in lung fibrosis by day 28 and a transient increase in dermal thickness. Dermal thickness and the level of collagen in skin treated with high-dose BLM was significantly higher than in skin treated with low dose BLM or vehicle. A reduction in the thickness of the adipose layer was noted in both high and low dose groups at earlier time points suggesting that the loss of the fat layer precedes the onset of fibrosis. High-dose BLM also induced dermal fibrosis and increased expression of fibrosis-associated genes  Systemic sclerosis (SSc) is a multisystem connective tissue disease characterized by immune dysregulation, obliterative vasculopathy, and fibrosis of skin and internal organs. It has the highest disease-related mortality and morbidity with an impaired quality of life among the rheumatologic illnesses , 2. FibrThe availability of an animal model that mimics the typical features of a human disease is essential for the study of that disease. The bleomycin (BLM)-induced fibrosis model is the most widely used murine model of SSc –8. To inA few reports have shown that systemic delivery of BLM via continuous diffusion from subcutaneously implanted osmotic minipumps can cause fibrosis of the skin, lungs, and other internal organs –12. ThisIn this study, we investigated the effect of systemically administered BLM, with respect to dose-dependent changes in pulmonary fibrosis, dose-and time-dependent changes in dermal involvement, and loss of the subcutaneous adipose layer in C57BL/6J mice using the pump model. We also examined the effect of BLM on human skin maintained in organ culture.Systemic administration of BLM was done as previously described (11) with some modifications. Briefly, 8 week-old male C57BL/6J mice  were administrated BLM  using osmotic minipumps  containing either 100μl saline as vehicle or BLM for 7 days. BLM was dissolved in PBS at concentrations of 1.0 U/kg, 10 U/kg, 60 U/kg, and 110 U/kg. On day 0, osmotic minipumps were implanted under the back skin of mice slightly posterior to the scapulae under isoflurane anesthesia. Pumps were removed on day 7. Lung and skin tissues were harvested on days 7, 10, 14, 21, and 28 for histological and gene expression analysis and hydroxyproline assays. Skin samples were obtained from the dorsal area, approximately 2 cm posterior to the pump implantation site, and abdominal area. All experiments were done under a protocol approved by the IACUC of the Medical University of South Carolina (MUSC).Skin and lung specimens were fixed with 10% formalin and embedded in paraffin. Six micrometer sections of paraffin-embedded mouse skin specimens were stained with hematoxylin and eosin (H&E) or Masson’s trichrome (MT). Images were captured on a Motic BA410 Compound Microscope  using identical settings.To quantify the amount of collagen in mouse skin and lung specimens, hydroxyproline content was measured as previously described .All continuous variables were expressed as the mean ± standard deviation. All statistical analyses were done using IBM SPSS statistics 22 . Comparison among 3 or more groups was done using ANOVA with post-hoc Tukey’s test to evaluate statistical significance.® kit . Reverse transcription was performed with SuperScript Ⅳ . Gene mRNA expression levels were evaluated by quantitative PCR using the TaqMan® real-time PCR system  according to the manufacturer's protocol on a TaqMan® Gene Expression Assays . Premixed PCR primers and TaqMan probes for mouse Fn, Coll1α1, Tgfb1, Ctgf, and Gapdh and human FN, Coll1α1, Tgfβ1, Ctgf, and Gapdh were obtained from Life Technologies. Gene expression levels were normalized to Gapdh and compared with the 2−ΔΔCt method.Total RNA was extracted from mouse skin using TRIZOL Lysis Reagent  and RNeasyInjection method: Normal human skin was obtained from residual tissue following plastic surgery. All tissues were obtained according to the guidelines of the Medical University of South Carolina without any identifiers. Subcutaneous fat tissue was removed and skin tissue was cut into 1.5 cm x 1.5 cm sections. Skin tissues were injected intradermally as we previously reported , 18 withImmersion method: Normal human skin was cut with a disposable biopsy 3 mm punch, and the pieces of tissue were cultured in medium containing BLM (1 or 10 mU/ml) or 1× PBS as a vehicle control. The culture medium was replaced at 72h and consisted of DMEM supplemented with penicillin, streptomycin, and anti-mycotic agent. Skin tissues were harvested 48 hours (for RNA) or 7 days (for hydroxyproline) post-treatment.To evaluate dose-dependent changes induced by BLM, we measured hyrdoxyproline content and examined the morphology of lung and skin tissues of mice receiving BLM via the pump model. Mice treated with BLM showed a dose-dependent increase in lung fibrosis. Hydroxyproline levels were significantly greater in lungs from mice treated with high dose BLM (60 and 110 U/kg) than those treated with PBS or low dose BLM (1 and 10 U/kg)  28 days Unlike the lung, skin fibrosis did not increase in a dose-dependent manner . This isWe next compared time-dependent changes in dermal fibrosis. Mice treated with a low dose of BLM (1 U/kg) were sacrificed on days 7, 10, 14, 21, and 28. Although neither dermal thickness nor hydroxyproline levels showed any significant changes at all the time points tested , the thiTo compare the effect of different doses of BLM on dermal fibrosis, mice were treated with a high dose of BLM (60 U/kg) and sacrificed on days 7, 10, 14, 21, and 28. On day10 post-BLM treatment , dermal thickness on the back of the mice was significantly increased in BLM-treated mice compared to control mice . No signWe further analyzed fibrosis-related gene expression in back skin tissues at each time point. qRT-PCR analysis showed that the expression levels of Coll1α1, Fn, and Ctgf were significantly increased in the skin from BLM-treated mice compared with those from control mice on day 10 . The uprin vivo experiments and confirm their relevance to human tissue, we conducted ex vivo experiments using human skin maintained in organ culture. We first examined the effect of BLM intradermally injected into human skin. BLM significantly increased the levels of hydroxyproline and dermal thickness in a dose-dependent manner 7 days after injection  via minipumps for 4 weeks to C57BL/6 mice resulted in dermal fibrosis on day 28 [et al showed that dermal fibrosis in C57BL/6 mice was induced on day 28 even though administration of the BLM (150 mg/kg) was discontinued on day 7 [There are several differences that can potentially explain the discrepancy in kinetics of dermal fibrosis. One notable difference is the source of BLM. BLM purchased from different manufacturers may not have identical ingredients or identical activity levels. In fact, the same dose of BLM obtained from different manufacturers results in different mortality rates and thus toxicity in the same strain of mice (data not shown). Another important consideration that might explain the different effects of BLM is the choice of mouse strains. C57BL/6 mice are commonly used for modeling diseases and for the generation of genetically engineered mice. However, several C57BL/6 substrains exist, and these are genetically and phenotypically different. Each vendor has maintained their independent breeding stocks for years, leading to genetic differences accumulating. Furthermore, it has been reported that a chromosome 11 mutation in a strain of C57BL/6 mice was detected in stocks maintained by one commercial vendor . This unn day 28 . On the on day 7 . Lee et on day 7  showed tex vivo [We have previously shown that a single injection of TGF-β induced dermal fibrosis in human skin ex vivo . We now In conclusion, our findings show that dermal fibrosis in C57BL/6J mice using the pump model can be sustained for 10 days using a higher dose of BLM while lung fibrosis progresses in these mice even after discontinuing BLM administration. The BLM pump model is a powerful tool for the functional analysis of systemic fibrosis and the testing of potential therapies. However, the choice of mouse strains, duration of BLM administration and dose must be carefully considered when using this model.S1 FigRepresentative images of H&E (upper)–and MT (lower)–stained sections of control and BLM–treated lung tissues  and ×400 (right)).(PDF)Click here for additional data file.S2 Fig(A) Dermal thickness was measured in abdominal skin treated with BLM (1 U/kg). (B) Dermal thickness was measured in abdominal skin treated with BLM (60 U/kg).(PDF)Click here for additional data file.