The dynamic loads acting on concrete-filled steel tubular members under axial impacts by rigid bodies were studied herein by FEM. The whole impact process was simulated and the time history of the impact load was obtained. The effects of eight factors on the axial impact load were studied; these factors were the impact speed, mass ratio, axial pressure ratio, steel ratio, slenderness ratio, concrete strength, impact position, and boundary conditions. Besides this, the effects of concrete creep on the impact load were also considered by changing the material parameters of the concrete. The results show that axial impact load changes with time as a triangle. The peak value of impact load increases and the impact resistance improves with the growth of the axial pressure ratio, steel ratio, slenderness ratio, and concrete strength after creep occurs. As the eccentricity of the axial impact acting on a concrete-filled steel tubular member increases, the peak value of the impact load decreases. The enhancement of constraints at both ends of the member can improve the impact resistance. The creep reduction coefficients for the peak axial impact load of a concrete-filled steel tubular member under axial compression and considering the creep effect over 6 months and 30 years are 0.60 and 0.55, respectively. A calculation formula for the peak value of impact load was suggested based on the existing formula, and its accuracy was proved by finite element calculation in this study.  A concrete-filled steel tubular member is a combined member which is produced by filling a thin-walled steel tube with concrete. It is more and more widely used in actual projects as the major load-bearing member due to its high strength and good plasticity. Under the action of axial compression at the service stage, the core concrete will suffer creep as time goes on; this will cause the cooperative work of the core concrete and steel tube to be broken and cause the redistribution of their internal forces. Generally, the core concrete stress will become relaxed and the steel tube stress will increase. If the member encounters impact load at this time, the steel tube will probably enter into a plastic state and have local buckling. Concrete creep was firstly observed by Furlong  in 1967.The research on the dynamic performance of concrete-filled steel tubular members started relatively late. So far, studies on the seismic performance of concrete-filled steel tubular members are comparatively exhaustive. The existing studies on the dynamic responses of concrete-filled steel tubular members under impact load, such as the references ,12,13,14This paper explains a finite element model established in ABAQUS software to simulate the whole process when a concrete-filled steel tubular member with creep effect bears axial impact. The axial impact load is discussed by setting different values for the impact speed, mass ratio, axial pressure ratio, steel ratio, slenderness ratio, concrete strength, impact position, and boundary conditions. The peak reduction coefficient of the axial impact load due to concrete creep is concluded, and a calculation formula for the axial peak impact load of a concrete-filled steel tubular member under axial compression and considering the creep effect is suggested based on the existing formula. This will provide a theoretical basis for the impact resistance design of concrete-filled steel tubular members.The geometric model of a concrete-filled steel tubular member under impact load is as shown in D and t are the external diameter and wall thickness of the steel tube, respectively, and L is the member length. sα is the steel ratio, which can be calculated with the formula uN = 1398.2 kN for those geometric dimensions given in un = N/N set in this study was no more than 0.6, and the value of axial force N for an axial pressure ratio of 0.3 is given in x = m/M was also studied, where m and M are the impact block mass and the member mass, respectively. The member mass M is constant and M = 180 kg in this paper. x = 0.5. v is the velocity of the impact block.As shown in The effects of the eight factors of impact speed, mass ratio, axial pressure ratio, steel ratio, slenderness ratio, concrete strength, impact position, and boundary conditions on the axial impact load were analyzed. The values of the parameters given in sE = 210 GPa, the hardening modulus was t = 0.01EsE = 2.1 GPa, the ultimate tensile strength was uf = 400 MPa, and the ultimate strain was uε = 0.089. Q235 steel was used for the steel tube. A linear hardening constitutive model was used to simulate the steel tube; the elasticity modulus of the steel tube was C40 concrete was used for the core concrete. HAN Linhai’s constitutive model for core concrete in a concrete-filled steel tube , which cckf; after that, the curve becomes nonlinear. Hence, the initial point of concrete plasticity with uniaxial compression was located at 0.4 times the standard value of concrete stress strength, i.e., at 0.4fckσ = . The concrete parameters of the plastic damage constitutive model (CDP) for C40 concrete were as shown in The concrete damaged plasticity model (CDP) in ABAQUS was used to simulate the material nonlinearity of the core concrete. A damage factor was introduced in the CDP, and stiffness degeneration and unrecoverable deformation were considered when unloaded. According to the stress vs. strain curve of concrete under uniaxial compression, the stress often linearly increases with strain when the average stress on the cross section is lower than 0.4The axial impact was simulated by a rigid body with given quality and speed. At first, a concrete-filled steel tubular member bearing static axial load was calculated in ABAQUS/Standard. Then, the static calculation results in the restart document were imported into ABAQUS/Explicit to perform the calculation of a concrete-filled steel tubular member impacted with static axial load. The core concrete and steel tube were simulated using reduced integrated solid element C3D8R, and the impact block was simulated using rigid solid element R3D4. Hourglass Control was selected to analyze the grid accuracy. Sweep meshing was used to mesh for the finite element model. The meshing size of the concrete-filled steel tubular was about 0.01 and that of the impact block was about 0.04 to save calculation time.It was assumed that there is no slip between the core concrete and steel tube; therefore, concrete elements and steel elements with the same geometric location shared the same node for the finite element calculation of the concrete-filled steel tubular member. For the contact surface between the impact block and the end of the member which bears the impact, a friction contact penalty function was used in the tangential direction, and 0.1 was adopted as the friction coefficient; hard contact was used in the normal direction. One end of the member was free and the other was fixed. Using the above finite element model, the whole impact process of the concrete-filled steel tubular member with axial compression bearing an axial impact load was simulated, and energy analysis and result verification was conducted. It showed that the meshing and contact parameters in this finite element model were suitable for dynamic analysis; the hourglass effect was very small and can be ignored. “Impact load” refers to the interaction force produced on the contact surfaces where the impact block and concrete-filled steel tubular member collide with each other. In this paper, three representative concrete ages, i.e., 28 days, 6 months, and 30 years, were chosen to evaluate various factors’ effects on the impact load. The time history curve of the impact load started at 1 s and ended at 1.005 s along the horizontal axis because axial static calculation was conducted during the first 1 s.v was set to 5 m/s, 10 m/s, and 15 m/s to study its effect on impact load. The results are shown in Impact speed is one of the key factors affecting the dynamic responses of a concrete-filled steel tubular member. The speed From the time history curve of the impact load shown in From x was set to 0.5, 1, and 2 to study its effect on the impact load, and the impact velocity was kept constant at 5 m/s. The results are shown in The mass ratio From the time history curves of impact load shown in From n was set to 0.3, 0.45, and 0.6 to analyze the effect on impact load. The results are shown in The axial pressure ratio From the time history curves of impact load shown in From sα was set to 0.063, 0.108, and 0.156 to study its effect on impact load. The steel ratio was changed by changing the value of the wall thickness of the steel tube. The results are shown in The steel ratio From the time history curves of impact load shown in From The slenderness ratio λ was set to 20, 40, and 60 to study its effect on impact load. The slenderness ratio was changed by changing the value of the member length of the steel tube. The results are shown in From the time history curves of impact load shown in As shown in The concrete strength was set to C30, C40, and C50 to study its effect on impact load. The results are shown in From the time history curves of impact load shown in As shown in In order to research the impact position effect on the impact load of a concrete-filled steel tubular member under axial compression, three different impact positions were selected as shown in From the time history curves of impact load shown in As shown in In order to study the boundary condition effect on the impact dynamic responses of a concrete-filled steel tubular member, four familiar boundary conditions were chosen for the calculation, as shown in From the time history curves of impact load shown in As shown in In order to research the core concrete creep effect on the axial peak impact load of a concrete-filled steel tubular member, the above peak impact loads of the member under the variation of seven different parameters were analyzed, as shown in As shown in β. From the above discussion for the eight selected parameters, we can see that the two peak impact load curves with the seven parameters at 6 months and 30 years were basically parallel. Linear interpolation was used to calculate the reduction coefficient for the peak impact load when the concrete age is between 6 months and 30 years. According to the simplified calculation formula for peak impact load in reference [β is the creep reduction coefficient for peak impact load ; α is a correction factor for the impact load and is in the range of 1.93–2.3; m is the mass of the impact block; v is the impact speed; and T is the impact time. When the concrete age is 28 days, 6 months, or 30 years, the respective impact time calculation formulae based on the regression analysis can be obtained as follows:n is the axial pressure ratio, sα is the steel ratio, and λ is the slenderness ratio.The reduction coefficient for the peak impact load of a concrete-filled steel tubular member under axial compression considering creep was defined as the loading ratio in eference , the cal(1)The equivalent elasticity modulus was introduced to simulate the dynamic responses of a concrete-filled steel tubular member under the combined action of axial compression, creep, and axial impact. The calculation results show that the finite element model established in this paper is reasonable, accurate, and suitable for the simulation.(2)The peak impact load and contact time increased with increasing impact speed; when the impact speed was higher, the effect of concrete creep on the impact load was weakened.(3)The impact load did not change much with increasing mass ratio, while contact time increased with increasing mass ratio. For the same mass ratio, the peak impact load after core concrete creep occurring was far less than that without creep.(4)After concrete creep occurring, the peak impact load rose with the axial pressure ratio, steel ratio, slenderness ratio, and concrete strength. The axial pressure ratio, within certain limits, had a potentiation effect on the impact resistance of the member.(5)With increasing eccentricity of the axial impact, the peak impact load decreased linearly. Enhancement of the constraints at the two ends is conducive to improving the impact resistance of the member.(6)The calculation formula constructed in this paper for the peak impact load of a concrete-filled steel tubular member under axial compression considering creep is easy to use and can help in the impact resistance design of concrete-filled steel tubular members.In this paper, the whole process of a concrete-filled steel tube subjected to axial impact was calculated using the finite element method. The influences of various possible factors on the impact loads were analyzed in as much detail as possible. Concrete creep was also considered in the study. Through this research, the following conclusions can be drawn:


This cross-sectional study uses bibliometric analysis to quantify the extent and relevance of international research collaborations funded by the US National Institutes of Health. What are the trends, scope, and importance of US National Institutes of Health–funded publications with international collaboration from 2009 to 2017?In this cross-sectional bibliometric analysis, National Institutes of Health–funded publications indexed in the Web of Science database increased by 46.2%, and the percentage of those with at least 1 coauthor with a non-US affiliation increased from 28.3% to 34.8% from 2009 to 2017. Publications with US-affiliated and non–US-affiliated coauthors had a greater mean citation impact score than those with exclusively US or non-US authors.International collaboration in National Institutes of Health–funded research is a critical, growing, and high-importance component of the National Institutes of Health’s biomedical and behavioral research mission, contributing to its overall productivity. The US National Institutes of Health (NIH) is the largest funder of biomedical and behavioral research in the world. International collaborative research—a subset of NIH’s portfolio—is critical to furthering the agency’s health research mission.To quantify the extent of the NIH’s international collaborations and the relative importance of this research through the lens of publications.This cross-sectional study used bibliometric data from the Web of Science database to analyze trends in the growth of NIH-funded publications from January 1, 2009, to December 31, 2017, and examined their importance using national affiliations of all coauthors listed, h indices, and citation impact scores. All countries with coauthor affiliations in NIH-funded publications during the study period were included. Data were analyzed from October 22 through November 16, 2018.Country affiliations of coauthors’ institutions in NIH-funded publications indexed in the Web of Science database from 2009 to 2017.Trends in the number of NIH-supported publications with non-US coauthors during a 9-year period and their relative importance assessed by h index per country and category-normalized citation impact (CNCI) for groups of country affiliations in 2017.From 2009 to 2017, the annual count of NIH-funded publications increased 46.2% from 67 041 to 98 002. This increase was driven in part by an increase in publications with a non-US author alone or as a collaborator with a US author compared with those exclusively with US authors, reflected by an increase in the percentage of publications with non-US coauthors from 28.3% to 34.8%. Moreover, in 2017, publications coauthored by US-affiliated and non–US-affiliated investigators had a higher mean CNCI (1.99) than those whose authors were only US affiliated (1.54) or non–US affiliated (1.35). China became the most frequent publishing partner, with 6982 coauthored publications and the greatest increase over time among non-US countries.In a 9-year period when the NIH budget remained relatively unchanged, an increase in the number of publications occurred with a growing trend toward more international collaborations of authorship; these publications also had a higher CNCI than publications with only US or only non-US authors. The findings suggest that international collaboration is a vital and growing component of the NIH’s research output and likely reflects increased globalization of biomedical research. Supporting projects that bring together people and institutions from across the globe provides critical opportunities to advance science, identify new diseases with unique populations, and develop better diagnostics, treatments, and cures.7 Clinical trials for infectious diseases, such as human immunodeficiency virus, Ebola virus, dengue, and Zika virus, for instance, require scientists in the United States to work with investigators in affected regions. Particular scientific opportunities that benefit researchers around the world may exist in isolation, such as differences in the response to chemotherapy for breast cancer based on country of origin and genetic background; high prevalence of unique, single-gene alterations that predispose a localized population in Colombia to hereditary, early-onset Alzheimer disease; or interventions to reduce arsenic exposure and its consequences in Bangladesh.11 Rapid advancements in communication technology and the trend toward team-based research have facilitated such international collaborations, allowing scientists to freely exchange data and ideas.13The US National Institutes of Health (NIH) is the largest funder of biomedical research in the world.17 By comparing trends in the number of publications over time and a metric of scores, such as the h index, researchers can describe the relative importance of publications with coauthors affiliated with different countries. To answer larger questions about the importance of US and non-US coauthorship overall, the category-normalized citation impact 18 adds another dimension to the analysis.To assess the importance of international research at the NIH through one lens of scientific productivity—peer-reviewed publications—we evaluated the extent of NIH-funded international collaborative research and characterized trends during the past 9 years. Bibliometric analysis offers a powerful tool to better understand this output of research and provides an alternative to other traditional methods for portfolio analysis.19 We evaluated the CNCI and the number of publications overall as a function of coauthors’ country affiliations to achieve these goals. Insights into the global reach of NIH funding is critical to understand the current research landscape and inform future investments. Collaborating institutions, including those from academia, government, and the private sector, can use these findings to guide their own decision-making to build productive international research partnerships.We have conducted a bibliometric study to assess the growth of NIH-funded publications and characterize the scope of international collaboration supported by the NIH during a 9-year period when the NIH budget increased from approximately $30 billion to $35 billion but experienced an inflation-adjusted decrease.20 The WOS “analyze results” feature was used to sort records by countries or regions and download aggregate counts of NIH-funded publications by country affiliation of coauthors. Countries were categorized by income level according to World Bank country and lending groups.21 Additional search strings were used to query the WOS database for publications by institutes and centers and create reports on coauthors’ national affiliations as described above, using 2017 as the sample year. Publications with funding from multiple institutes and centers were included in each relevant report. InCites , a WOS tool, was used to calculate the CNCI for all NIH-funded publications in 2017 grouped by coauthors’ national affiliations .18 The CNCI used baseline citation rates by WOS subject area centered around a score of 1.00, with higher scores representing greater importance. All statistics were descriptive and presented counts and proportions of publications in the sample without reference to population parameters. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline was used in the reporting of this cross-sectional study. This analysis of publicly available publication data did not constitute human subjects research and did not require approval by an institutional review board or informed consent.The Web of Science  database was queried for NIH-funded publications using a comprehensive Boolean search string of the full names and acronyms of all NIH institutes and centers in the “funding text” field. We limited our analyses to the 9-year period from January 1, 2009, to December 31, 2017, because WOS only began indexing funding data in 2008, and records for 2018 were incomplete at the time of this analysis.22 For example, an author’s h index would be 20 if he or she had published at least 20 articles that had been cited at least 20 times each. This measure can also be applied to institutions or countries.24 Previous bibliometric studies have found 1-year h indices to be highly correlated with those from larger citation windows, making the statistic a valid tool for comparison.26 The data were limited to publications in 2017 to match the time frame for annual publication volume. Countries were ranked by number of publications and h index in 2017. Finally, records for all NIH-funded publications from 2009 to 2017 were sorted by citation count.Data were analyzed from October 22 through November 16, 2018. The citation report feature in WOS was used to calculate 1-year h indices for each country affiliated with NIH-funded publications in 2017. The h index is a metric that reflects the number of publications and citation counts in a single number.From 2009 to 2017, the number of NIH-funded publications increased 46.2% from 67 041 to 98 002 . The greAs a result of the differential growth rates, the proportions of each group shifted between 2009 and 2017. The fraction of studies with US and non-US coauthors increased from 24.1% (16 188 of 67 041) to 30.2% (29 580 of 98 002), whereas those exclusively with non-US coauthors increased from 4.1% (2773 of 67 041) to 4.6% (4479 of 98 002). Consequently, the overall percentage of publications with non–US-affiliated coauthors increased from 28.3% (18 961 of 67 041) to 34.8% (34 059 of 98 002). Publications with solely US coauthors accounted for 71.7% (48 080 of 67 041) in 2009 but decreased to 65.2% (63 943 of 98 002) in 2017.In 2017, the coauthors most represented in NIH-funded publications were from China, the United Kingdom, Canada, and Germany A. AlthouTo examine our most significant global partnerships in research, we identified the country affiliations most represented among NIH-funded publications in 2017. These were ranked by the total number of publications and their importance as measured by the h index . Of the The same analysis was repeated using only LMICs B. The h 21 Internationally collaborative publications tend to be most productive and have greater h indices and CNCI among European partners and Canada as well as various high-income Asian countries and China. China stood out for its most rapid growth in the same period and as the only LMIC among the top 10 represented countries. Marked increases have occurred in NIH-funded publications among LMICs, especially in Africa and Latin America, although these rates still lag behind rates of the HICs.Of the top 20 countries ranked by h index, 12 were European countries and 4 were high-income East Asian and Pacific countries as classified by the World Bank.36 Japan and the United Kingdom were the most represented country affiliations (3 publications); followed by China, Germany, Saudi Arabia, Sweden, and Switzerland (2 publications); and then France, Israel, and Singapore (1 publication). Publication topics primarily covered genomics experimental methods and computational biology.The top 10 most cited NIH-funded publications with 1 or more non–US-affiliated coauthors from 2009 to 2017 all included at least 1 US-affiliated coauthor .27,28,2946 Most publications were studies of genomics and the global burden of disease, drawing from LMICs and HICs alike. However, all first authors of the top 10 most cited NIH-funded publications in 2017 were affiliated with HICs. The percentage of LMIC-affiliated authors ranged from 2%  to 31% , with a median of 11.5% in this sample. Only 1 publication  did not include a US-affiliated coauthor.Publications with LMIC-affiliated coauthors (with or without US coauthors) had much fewer citations .37,38,39For 2017, we analyzed NIH-funded publications among the 27 institutes and centers at the NIH. Of the 98 002 publications in 2017 that reported funding from the NIH, 42 872 (43.7%) did not indicate which institute or center provided support, and 16 788 (30.5%) included funding from more than 1 institute or center and are counted multiple times. The National Cancer Institute was most often acknowledged for US-affiliated publications (7407 [62.06%]) and publications with non–US-affiliated authors (4505 [37.8%]), followed by the National Institute of Allergy and Infectious Diseases (2315 [45.7%]) and the National Heart, Lung, and Blood Institute (1840 [31.5%]). The Fogarty International Center had the highest percentage of non–US-affiliated publications in 2017 (855 [90.0%]), followed by the National Institute of Allergy and Infectious Diseases (2315 [45.7%]), the National Human Genome Research Institute (421 [44.0%]), and the National Institute of Biomedical Imaging and Bioengineering (551 [43.0%]). All of the institutes and centers at the NIH were represented among the set of publications in 2017 with at least 1 non-US coauthor.Publications with US and non-US coauthors had the greatest importance as measured by CNCI (1.99), greater than that of publications with only US coauthors (1.54) or those with only non–US co-authors (1.35). The baseline CNCI rate for all NIH-funded publications in the same year was 1.65, placing reports by US-only and non–US-only coauthors below the mean in the sample.47From 2009 to 2017, a period when the NIH budget was relatively unchanged, the number of NIH-funded publications nonetheless grew by 46.2%, reflecting a global trend in biomedical research. Much of this growth was attributable to the more rapid increase in the number and percentage of publications with non-US coauthors. Furthermore, publications with US- and non–US-affiliated coauthors had greater importance as measured by the CNCI than publications with only US- or only non–US-affiliated authors. Notably, NIH director Francis Collins, MD, PhD, has consistently voiced his support for greater international collaboration, and all 27 institutes were represented with publications having coauthors.49 However, the rankings by the CNCI in this study are important for 2 additional reasons: (1) they suggest that the trend for greater importance among internationally collaborative papers can be extended to the country level for biomedical research as a whole rather than by institute or subdiscipline within a country, as is already more thoroughly investigated53; and (2) the CNCI also follows the same broad pattern when applied to publications associated with a given funding source. Bibliometric analysis provides another element for portfolio analysis that can be used to summarize the global expansion of biomedical research.These results reinforce earlier findings that internationally collaborative publications tend to be cited more frequently.Of note, the most cited reports primarily covered genomics, methods, and the global burden of disease. The first 2 topics listed rank high among countries regardless of income, whereas burden of disease studies were most common when the scope is limited to LMIC-affiliated publications. Even among these, the proportion of LMIC-affiliated coauthors did not exceed 31%, suggesting that HICs largely drive the research. Considering the large number of collaborators needed to collect enough data for these investigations, collaboration itself was likely a mediator for the enhanced visibility and high CNCI of these publications.The predominant country affiliation for non-US authorship is China when ranked by volume, CNCI, and h index. The dramatic increase in coauthored publications may be the result of increased bilateral programs for postdoctorate trainees and visiting fellows, but a rapid survey of the data does not suggest postdoctorate trainees and visiting fellows are the sole contributors to research. Sampled articles listing Chinese-affiliated coauthors generally described equitable distribution of authorship responsibilities when such statements were present.54 However, of the various databases available, WOS captures the most publications in biomedical research with full funding information (29%) and accordingly was used for this study.54 Furthermore, publication data, including funding sources and coauthor country affiliations for each study, can be missing or inaccurate if entered into the database incorrectly.55 All authors in the database had country affiliations listed, but publications not captured by the database could nevertheless limit the internal and external validity of the study. The metrics themselves—h index in particular—must be interpreted in context and with the recognition that the country of publication can influence these statistics.24 The analysis also does not account for the balance or authorship order of US-affiliated and non–US-affiliated coauthors on a given report. Finally, coauthors’ contributions do not factor into the analysis, eliminating any potential differences among the distribution of those who write the manuscripts, for instance. The distribution of coauthors’ roles by country and country income merits further research. In addition, national affiliation does not necessarily imply different geographical locations for coauthors. International fellows working at NIH-funded facilities in the United States still qualify that publication as an international collaboration if those coauthors maintained their affiliations with non-US institutions. Future research on this topic should consider investigating the different roles and breakdown of authorship by country affiliation. Previous studies have addressed some of these concerns but often on the institutional level rather than on a global scale. Developing a more robust profile of NIH-funded work at this level may help scientists and policy makers support important biomedical research.This analysis has several potential shortcomings inherent to the methods used. Databases such as WOS do not index all publications and thus cannot provide a complete data set for bibliometric analysis.International collaborative research offers investigators new opportunities to maximize importance and interact with non-US investigators by sharing opportunities from populations having unique features and genetics, exposures, or health care delivery and bringing new technologies to emerging or existing health problems. As biomedical research continues to grow, scientists will need to work together across borders to solve increasingly complex health issues. Improving human health across the globe depends on it.


This study investigated the punching shear behavior of full-scale, two-way concrete slabs reinforced with glass fiber reinforced polymer (GFRP) bars, which are known as noncorrosive reinforcement. The relatively low modulus of elasticity of GFRP bars affects the large deflection of flexural members, however, applying these to two-way concrete slabs can compensate the weakness of the flexural stiffness due to an arching action with supporting girders. The test results demonstrated that the two-way concrete slabs with GFRP bars satisfied the allowable deflection and crack width under the service load specified by the design specification even in the state of the minimum reinforcement ratio. Previous predicting equations and design equations largely overestimated the measured punching shear strength when the slab was supported by reinforced concrete (RC) girders. The strength difference can be explained by the fact that the flexural behavior of the supporting RC beam girders reduces the punching shear strength because of the additional deflection of RC beam girders. Therefore, for more realistic estimations of the punching shear strength of two-way concrete slabs with GFRP bars, the boundary conditions of the concrete slabs should be carefully considered. This is because the stiffness degradation of supporting RC beam girders may influence the punching shear strength. Steel bar corrosion in reinforced concrete (RC) structures adversely impact on the durability and structural capacity of RC members. The consistently increasing maintenance costs of repairing and strengthening RC structures has led owners to seek more efficient and affordable solutions through the use of FRP bars . FRP barThis study investigates the structural performance of full-scale, two-way concrete slabs reinforced with glass-fiber-reinforced polymer (GFRP) bars. Slab specimens were restrained by two RC beam girders with hinged supports. The reinforcing types considered were steel bars and GFRP bars of two different diameters, which include the minimum reinforcement ratio of 0.002 recommended by the specifications of the Canadian Standards Association  . The strThe two-way concrete slabs were fabricated using normal weight and ready-mixed concrete, which was 30 MPa of the designed compressive strength. The average compressive strength of five cylindrical concrete specimens (∅100 mm × 200 mm) was obtained as 36.7 ± 1.2 MPa. The GFRP bars used in this study were manufactured by the typical pultrusion process by braiding the fiber ribs on the surface of the GFRP bar. The GFRP bars were made of polyvinyl alcohol (PVA) resin reinforced with E-glass fiber, with a fiber volume fraction of 65% by weight. The nominal diameter of D13 for the steel bar, and D16 and D19 for the FRP bars were 12.7, 16.1 and 19.1 mm, respectively.The nominal diameters were obtained by an immersion test based on density. The tensile properties of the GFRP bars were determined using the tensile test in compliance with ACI 440 3R-04 . Table 1S is the center-to-center spacing of the supports. The recommended clear cover of the concrete bridge deck was 25.4 mm [A total number of four full-scale two-way concrete slabs were fabricated with two supporting RC beam girders reinforced with steel reinforcements. At 2400 mm wide and 3000 mm long, the considered thickness of 220 mm satisfies the minimum depth calculated using the formula 1.2 (S + 3000)/30 [ 25.4 mm ; howeverρeq (=ρfrp (Efrp/Es)) along the main transverse direction at the bottom of the specimen. In order to compare with the STS specimen, the GFS1 specimens were designed using the equivalent reinforcement ratio of 0.36, similar to the reinforcement ratio of 0.39 for the STS specimen. It was expected that the flexural stiffness would be nearly the same but with different ultimate strengths. The GFS1 and GFS2 specimens were employed for the evaluation of the difference in the structural behavior near the minimum reinforcement ratio of 0.002. CSA (2012) provided a reinforcement ratio for the longitudinal reinforcement of the deck slab as a function of the effective spacing between the girders and not exceeding 67% of the transverse reinforcement. Hinges installed at the end of the two girders allowed the supporting RC beam girders to be rotated to the longitudinal direction. This design concept for the girders was intended to impose an additional flexural effect on the two-way concrete slabs so that the ultimate punching shear strength may be reduced compared to the slabs supported by the high stiffened girders, which are hardly able to deflect.Each specimen was designed employing the equivalent reinforcement ratio of The concentrated load was applied at the center of the top surface of the test specimens with a 300 mm × 500 mm loading plate on a 30-mm thick neoprene sheet, to avoid the stress concentration at the edge of the loading plate. The MTS  loading actuator was used with the capacity of 1000 kN, and the rate of loading was 1 mm/min. Two linear variable displacement transducers (LVDTs) with a measuring limit of a 100 mm stroke were also used to measure the vertical deflection at the bottom center of the slab and at the mid-span of the RC beam girders. During the loading test, the maximum crack width was measured using a crack width ruler at each designated step. The detailed test setup is shown in The specimens showed that punching shear failure occurred underneath the loading point at the ultimate state. Brittle failure occurred and the loading top and bottom surface sank, as shown in Pser, for the specimens was calculated as 1.33 × 72.5 = 96.4 kN . For the design factored load, Pf was taken as 1.33 × 1.8 × 72.5 = 173.6 kN, where the factor 1.8 was the live load combination factor specified by the ultimate level I. Compared to the design service load calculated as 96.4 kN, it was evaluated that the tested initial cracking load was higher. For the design purpose, the deflection allowance was specified as span/800 (=2.75 mm) for the vehicle load in general by AASHTO (2007), which was specified in AASHTO (2009) [O (2009)  as the aPcr), ultimate loads (Pu) and strength ratios for service (Pser) and factored loads (Pf), respectively. It was evaluated that the strength ratio of the cracking load to service load ranged from 1.4 to 1.6; and the ratio of Pu to Pf ranged from 2.1 to 2.4. This implies that the strength capacity of the two-way concrete slabs reinforced with GFRP bars was sufficient to resist the designed service and factored loads under punching shear stress, even though they were reinforced using the minimum reinforcement ratio of the GFRP bars.FRP bars are a corrosion-free reinforcing material, and the maximum allowable crack width can be within a wider range, especially when the corrosion due to reinforcement is a primary reason for the allowable crack width limitations. Other considerations with regard to acceptable crack width limits include aesthetics and shear effects. Japan Society of Civil Engineering (JSCE) (1997)  only conPser = 96.4 kN. At the factored load of 173.6 kN with a live load factor of 1.8 for ultimate level I, based on the AASHTO (2007) recommendation, the STS specimen yielded the minimum crack opening width among all the slabs, while the GFS1 specimen, which had an equivalent reinforcement ratio, exhibited a larger crack width. For the entire GFS series, the crack width at Pf increased as the reinforcement ratio decreased; however, they were within the allowable 0.5-mm limit. Accordingly, it should be noted that the GFS series equally experienced flexure in the longitudinal direction caused by the supporting RC beam girders. Thus, the boundary conditions in this study may produce greater deflection and crack widths compared to the concrete slabs on the high stiffening steel girders. Despite the additional flexure of the supporting RC beam girders, the GFS series excellently maintained the allowable crack width for both the service load and factored load.To evaluate the punching shear strength, the equations of Menétrey, ACI 440 and CSA S806 were employed and the tested and predicted punching shear strength was compared. These equations are introduced below. Menétrey’s theory (2002), which includes the structural effect of the strength ratio, punching cone angle, and structural dimensions, was adapted in this study. The equation is shown in Equation (1). (1)Fdowel) of the FRP bar is neglected due to the lower strength and stiffness of the FRP bar in the transverse direction (ACI 440 1R-15 2015).The dowel action ((2)Fsw) is replaced with stirrups crossing the punching cracks. This effect is neglected and discussed as the design safety in the analysis results.The effect of the studs ((3)Fp) is not available in this experimental test.The tendon effect ((4)The load on the two-way concrete slabs is regarded as the tire contact load of vehicle and is practically considered as a concentrated load.The assumptions for the analysis of the GFRP-reinforced concrete slabs are explained as follows.Fct is the term of the concrete tensile force introduced in Equation (2). It is a function of the loading radius, effective depth of the two-way concrete slabs, concrete tensile stress, and three influencing constants from r1 and r2. These radii are based on the specific two failure points as follows.According to the theory, the concrete tensile force is determined by integrating the vertical component of the tensile stress around the punching crack. The punching crack is assumed to be a truncated cone shape comprised between two radii, s is as follows.For the value of the radius of the column, the averaging radius of the loading plate was applied, obtained from the failure test. The angle was set for each two-way concrete slab from the experimental results for the punching shear failure surface. In this study, the average punching cone angle was used. The inclination length ρ) was replaced with the equivalent reinforcement ratio (ρeq = ρfrp (Efrp/Es))), including the modulus ratio of Efrp/Es.Influencing constants for Equation (2) are introduced in Equations (6)–(8). In the case of the applying reinforcement ratio, the reinforcement ratio . It was transferred to The Canadian Standard “Design and construction of building structures with fibre reinforced polymers”, CSA-S806-12, specifies the punching shear strength of FRP-reinforced concrete. It can be noted that these equations are the modified forms of those specified in the CSA (2004) , to accoFrom the comparison results as shown in (1)All test specimens displayed punching shear failure. The STS specimen exhibited stiffer behavior until failure than the GFS specimens that were reinforced using an equivalent reinforcement ratio. For the tested service load of the GFS specimens, the load carrying capacity was 1.4–1.6 times higher than the designed service load. Additionally, the experimental results demonstrated that the structural capacity was sufficient to resist the concentrated design load due to the vehicle load even though the amount of GFRP bars was designed using the minimum reinforcement ratio.(2)In comparison with the allowable deflection at the designed service load, the deflection of the GFS3 specimen, which was designed using the minimum reinforcement ratio, was measured as 1.42 mm. This was adequate as an allowable deflection. In comparison to the allowable crack width of 0.5 mm, the crack widths of the GFS specimens were lower than the allowable crack width . However, the allowable crack width of GFRP was larger than the allowable crack width of the RC member . The recommended allowable crack width must be well within the acceptable crack-width range for two-way concrete slabs reinforced with FRP bars, with higher strength and stronger durability under marine and ocean environments.(3)The tested punching shear strength was compared with the predicted equations. It indicated that the predicted strength largely overestimated the experimental punching shear strength as compared with previous test results, which had a similar geometry, size and structural design process but highly stiffened girders. This can be explained by the effect of flexure on the supporting RC beam girders, which may be a significant parameter in the evaluation or design of the punching shear capacity of full-scale, two-way concrete slabs reinforced with GFRP bars. During their service life, this can be accelerated due to repeated vehicle loading on slabs. Accordingly, further investigations should be conducted in relation to the correlation between the punching shear strength and the stiffness degradation of supporting RC beam girders.This study investigated the punching strength behavior of two-way concrete slabs reinforced with non-corrosive GFRP bars restrained by two RC beam girders. The punching shear strength, deflection and crack width were examined in terms of strength and serviceability performance. The punching shear strength was evaluated using the three equations, and the overestimation of the evaluated strength was discussed. The main conclusions are summarized as follows.


How these ligands influence BLM-mediated hGQ unfolding was investigated using two independent single-molecule approaches. While the frequency of dynamic hGQ unfolding events was used as the metric in the first approach, the second approach was based on quantifying the cumulative unfolding activity as a function of time. All three SM ligands inhibited BLM activity at similar levels, 2–3 fold, in both approaches. Our observations suggest that the impact of SM ligands on GQ thermal stability is not an ideal predictor for their inhibition of helicase-mediated unfolding, which is physiologically more relevant.G-quadruplex (GQ) stabilizing small molecule (SM) ligands have been used to stabilize human telomeric GQ (hGQ) to inhibit telomerase activity, or non-telomeric GQs to manipulate gene expression at transcription or translation level. GQs are known to inhibit DNA replication unless destabilized by helicases, such as Bloom helicase (BLM). Even though the impact of SM ligands on thermal stability of GQs is commonly used to characterize their efficacy, how these ligands influence helicase-mediated GQ unfolding is not well understood. Three prominent SM ligands (an oxazole telomestatin derivative, pyridostatin, and PhenDC Tm)  stabilizing small molecule (SM) ligands affect cellular biology in different ways. SM ligands that stabilize telomeric GQ structures (GQs) inhibit telomerase activity ,2 and haTm) . Pyridosmestatin ,13, and mestatin .Tm has been typically used to quantify their efficacy. To that end, thermal melting assays based on Förster resonance energy transfer (FRET), circular dichroism (CD) or ultraviolet (UV) absorption are commonly used. To illustrate, ΔTm ≈15°C was measured for human telomeric GQ (hGQ) due to 6OTD or 7OTD variants using both FRET-melting and CD melting assays at 100 mM K+  (Tm = 35°C (at 60 mM K+)   , and PDS0 mM K+)  for hGQ ectively . In thison of Tm . These l.5 range . Another pH 7.6) , in agre studies . In addi studies ,26. Therin vitro work studied the inhibition of intermolecular GQ unfolding activities of human RecQ helicases Bloom (BLM) and Werner (WRN) by acridine derivatives (N-methylmesoporphyrin IX (NMM)  on Pif1-mediated unfolding of different intramolecular GQs  easily displaces Braco19, NMM, and PhenDC3 before it repetitively unfolds the GQ  , similarIX (NMM) . More reular GQs . With ths the GQ . In thisDeciphering the underlying mechanism of helicase-mediated GQ unfolding would be an important step towards attaining a comprehensive understanding how SM ligands inhibit helicase activity. Even though SM ligands were not incorporated, two recent studies reported the structure of bacterial RecQ helicase bound to a folded GQ  and boviIn this study, we utilized BLM as a model system to investigate the impact of SM ligands on helicase-mediated hGQ unfolding. BLM is a member of the RecQ family, which is involved in various aspects of genome maintenance ,36. Bloo642–1290 that maintains both dsDNA unwinding and ssDNA translocation activities was utilized in this study. This construct consists of RecQ core of BLM containing the helicase, RecQ C-terminal (RQC) domain and helicase and RNase D C-terminal (HRDC) domain (BLM) domain .All DNA oligonucleotides  setup, equipped with an Olympus IX-71 microscope and an Olympus 60×, 1.20 NA water objective, was utilized to perform smFRET measurements. Single molecule sensitivity was achieved by an Andor Ixon EMCCD camera . A λ = 532 nm green laser (SpectraPhysics Excelsior) was used to excite the donor (Cy3) fluorophores. Emissions from donor and acceptor (Cy5) fluorophores were separated by a dichroic mirror and projected on two halves of a CCD screen.3 μm2) was targeted as the ideal density in most measurements.The quartz slides and glass coverslips were extensively cleaned, including Piranha etching, and coated with polyethylene glycol (PEG). 1% of PEG molecules were tagged with biotin to provide attachment points for biotinylated DNA molecules, which bind to biotin-PEG via neutravidin. In addition, the surface was coated with 1 mg/ml casein (overnight incubation) or 1% (v/v) Tween-20 (15-minute incubation before adding neutravidin) to reduce non-specific binding of BLM to the surface. To provide adequate statistics for the analysis, 250–350 DNA molecules per imaging area  was included in imaging buffer for measurements that included BLM. Before introducing BLM into a sample chamber, proper GQ folding was checked via smFRET histogram. We immediately started recording either short movies (15 frames) or long movies (1500–3000 frames) after introducing BLM into the sample chamber. The laser power and frame integration time  was added to the imaging buffer that contains Tris–HCl , 2 mM Trolox, 0.1 mg/ml bovine serum albumin (BSA), 150 mM KCl and 2 mM MgCl2 and pH 7.5), at which hGQ remains folded in the absence of BLM. To minimize variations in protein activity and surface quality, the measurements with SM ligands were performed immediately after those without SM ligands (will be referred to as ‘BLM-only’ case) in the same sample chamber (in the first assay) or the same slide (in the second assay).BLM activity was quantified in the presence or absence of SM ligands via two different approaches. In the first assay, we measured the rate of BLM-mediated hGQ unfolding (number of unfolding events per sec) with and without SM ligands. In the second assay, we measured the cumulative BLM activity where BLM unfolds hGQ at least once during our observation time. Both assays were performed under physiologically relevant salt conditions  is typically characterized by a higher FRET peak, we sought to increase the relative fraction of molecules that are in PC before BLM is added. As demonstrated in earlier studies, folding hGQ at room temperature, rather than at elevated annealing temperatures, increases the probability of kinetically trapping hGQ at the parallel conformation rather than the thermodynamically favored anti-parallel conformation (APC)  ,53. Ther2 and pH 7.5, under which hGQ remains folded in the absence of BLM. BLM and ATP concentrations were 300 nM and 6 mM for this assay, respectively.For the second assay Figure , a DNA c3 . After BLM dissociates from DNA, hGQ refolds and the process restarts. Therefore, multiple unfolding events can take place for a particular DNA construct within our observation time.Figure EFRET = 0.12±0.02, 0.59±0.05 and 0.70±0.03. The peak at EFRET = 0.12 is due to donor leakage into the acceptor channel and will be referred to as the donor-only (DO) peak. The standard procedure when reporting such histograms is to adjust the FRET axis such that the DO peak shifts to EFRET = 0.0. However, as the analysis of dynamic unfolding events is performed on individual smFRET traces, which are not processed with this correction, the EFRET levels corresponding to folded and unfolded states are determined without the DO correction. EFRET = 0.59 represents the anti-parallel (or hybrid) folded conformation while EFRET = 0.70 represents the parallel conformation  by dividing the total number of observed unfolding events in hundreds of smFRET traces with the total observation time. The total time is the sum of individual observation times for each molecule, as obtained from smFRET traces (from the start of recording until photobleaching or end of recording whichever comes first). To illustrate, three transitions are observed in the top trace in Figure −1. Similarly, three transitions are observed in the bottom trace in Figure −1. Table N = 544 DNA molecules independently. To form one of the bootstrapping sets, 544 rates are selected randomly with replacement from among these rates, e.g. the rate corresponding to a particular DNA molecule might be selected more than once. The average rate for this set is then found. This process is repeated 20 000 times, resulting in 20 000 different averages for this particular condition. Figure 3, respectively.We determined the average unfolding rate s− by divid3 or PDS in different chambers. The ligands were added to the chamber 15 min before BLM to allow binding to hGQ. They were also added when with BLM and ATP are introduced to maintain a high ligand concentration throughout the measurement. Figure 3, respectively. The error bars are standard deviations based on three independent measurements for each case. These results indicate that all SM ligands stabilize hGQ ∼2-fold against BLM.In this assay, unfolding of hGQ is imposed as a prerequisite for unwinding of a short duplex. Unwinding of the duplex releases a DNA strand that is labeled with Cy3, reducing the number of Cy3 molecules that remain on the surface. The more Cy3 strands are removed from the surface, the higher the BLM activity. In order to establish a reference level of activity, 300 nM BLM and 6 mM ATP were added to the sample chamber, without SM, and the number of Cy3 molecules on the surface was measured as a function of time. Similar measurements were then repeated in the presence of 1 μM L1H1-7OTD, PhenDCTm for hGQ, their impact on BLM activity showed only minor variations, which were within the uncertainties of the measurements. This raises interesting questions regarding whether ΔTm values can be taken as reference for the impact of ligands on GQ stability against destabilizing proteins. In a cellular setting, interactions with proteins and competition with Watson-Crick pairing are the primary mechanisms that destabilize GQs. Even though it is reasonable to expect that ΔTm induced by SM ligands would be a good estimator for GQ stability against Watson–Crick pairing, it is not clear whether this alone would be adequate to explain their impact against protein-mediated destabilization. Proteins may disrupt the stacking interactions between the SM ligand and GQ  reduce Pif1-mediated hGQ unfolding activity to ∼38% and ∼63% of the activity in their absence, respectively  (50) in the case of less potent ligands, while 0.25–0.75 μM ligand concentration was adequate for the more potent ligands. Our results are similar to those on the more potent acridine derivatives used in that study. It should be mentioned that the inhibition observed by Li et al. was not specific to GQ as a similar level of inhibition was also observed for dsDNA. Wu and Maizels studied inhibition of E. coli RecQ helicase by porphyrin derivative NMM and reported an inhibition constant (the concentration of NMM at which RecQ-mediated GQ unfolding activity was inhibited by 50%) of 1.7 μM  . In thisf 1.7 μM . Taking Obtaining consistent rates of unfolding in different experiments was a significant challenge. These variations are influenced by various factors including (i) the complexity of data analysis for dynamic interactions; (ii) strong dependence of unfolding rates on surface quality; (iii) variations in protein activity, for which single molecule methods are highly sensitive to; and (iv) potential variations in the folding conformations of hGQ that result in different stabilities against BLM. Given these sources of variability, we focused on comparing the relative rates observed in the presence and absence of SM ligands as measured within the same DNA chamber, with the DNA molecules from the same annealing reaction, and the same batch of proteins. We will next discuss these issues in more detail to illustrate the range of challenges associated with these measurements and the insights we acquired from our efforts.EFRET, which could be an unfolding event or just a signal fluctuation. For such cases, we adopted a conservative approach and did not consider such cases as unfolding events. Even though these criteria resulted in more consistent analysis results, we still observed about 25% variation in the obtained rates when the same data set was analyzed by different researchers independently. The consistency was not better with bias-free computational methods, such as Hidden-Markov model-based algorithms, e.g. ebFRET  or Tween-20 (1% v/v) coatings as these steps resulted in higher and more consistent BLM activity. However, we still observed significant variations in the unfolding rates among different channels and slides prepared in different days. The propensity of BLM and ligand adsorption to the surface is probably a major factor in this variability. Single molecule methods are in general highly sensitive to variations in protein activity, especially at the relatively low concentrations used in our measurements. Even though a consistent protocol was followed for how proteins are stored and thawed before measurements, there might still be variations in their activity. SM ligand adsorption to the surface has been reported to result in fluorescence quenching due to interactions of the fluorophores with the ligands that are adsorbed to the surface . These n2. In the absence of SM ligands, hGQ shows a mix of PC and APC characterized by a peak at 290 nm and a hump at 260 nm  on BLM-mediated hGQ unfolding activity was investigated using two independent single-molecule approaches. The SM ligands resulted in 2–3-fold decrease in BLM activity when the rate of dynamic unfolding events was used as the metric. Independently, we estimated the ligand-induced stabilization against BLM to be ∼2-fold when hGQ unfolding was implemented as a pre-requisite for the unwinding of a short duplex. These results provide complementary perspectives to quantifying SM ligand efficacy to commonly used thermal melting studies. A more complete understanding of the mechanisms underlying these interactions would likely require insights from both of these approaches in addition to high-resolution structure studies. This study helps this cause by introducing two independent assays that could be adapted to study other such systems. Finally, this work also provides an estimate for the level of induced stability that could be expected in a physiological setting where GQs are unfolded or destabilized by proteins.The impact of several prominent small molecule ligands (PDS, PhenDCgkz803_Supplemental_FileClick here for additional data file.


The responding property of this nanocube, reminiscent of a feature of biological molecules, arises from the fact that the GSAs in the nanocubes are connected to each other only through the hydrophobic effect and very weak intermolecular interactions such as van der Waals and cation-π interactions.Induced-fit or conformational selection is of profound significance in biological regulation. Biological receptors alter their conformation to respond to the shape and electrostatic surfaces of guest molecules. Here we report a water-soluble artificial molecular host that can sensitively respond to the size, shape, and charged state of guest molecules. The molecular host, i.e. nanocube, is an assembled structure consisting of six gear-shaped amphiphiles (GSAs). This nanocube can expand or contract its size upon the encapsulation of neutral and anionic guest molecules with a volume ranging from 74 to 535 Å Induced-fit binding, common in biological systems, is still relatively rare in artificial hosts. Here, the authors assemble a molecular cube from six gear-shaped faces, whose interdigitated design allows the cube to expand and contract in response to the size, shape, and charge of a guest molecule. As often seen in biological receptors, conformational flexibility that enables molecular hosts to respond to the shape and electrostatic surface of guest molecules is a general requirement for induced-fit binding. Most of artificial molecular hosts possessing a confined binding site are rigid structures whose conformational change upon binding of guest molecule(s) is smaller than that in biological receptors. Recently, we reported a cube-shaped molecular assembly, i.e., nanocube, from six molecules of gear-shaped amphiphiles (GSAs) such as 1Cl2  and cation-π interactions are responsible. Since the relative position of the GSAs is not much restricted, a variety of hydrophobic guest molecules with different molecular size and shape are expected to be accommodated in a nanospace (ca. 1 nm3) surrounded by hydrogen atoms on the aromatic rings of the GSAs. Furthermore, a polycationic character of the nanocube due to the pyridinium groups may facilitate encapsulation of charge-dispersed anionic species, whose binding must nevertheless overcome the energy required to release the water molecules around the anions38.Induced-fit or conformational selection is a general strategy to attain a tight binding between a molecular host and guest(s)Cl2 Fig.  in water3. The encapsulation of neutral molecules in the nanocube always causes the expansion. On the other hand, when anionic species are encapsulated, the nanocube shrinks responding to the negative charge of the guests. Thus, the response of the nanocube to guest molecules depends not only on the size and shape of the guests but also on their charged state.Here we report the expansion and contraction of a water-soluble artificial molecular host that responds to the size, shape, and charged state of guest molecules with a calculated volume ranging from 74 to 535 ÅS6 point group. All the six GSAs in the nanocube are chemically equivalent but the symmetry of each GSA in the nanocube is reduced to be the C1 point group, indicating that the three p-tolyl methyl groups  in the nanocube than the NMR timescale. , three molecules of which were cooperatively encapsulated in the nanocube, while the largest one is n-tetracosane (535 Å3)  and aromatic molecules were added in an aqueous solution of 4 and aroanti configuration  are longer than the side of the inner space of the nanocube (ca. 10 Å), long linear alkanes must be folded so as to be properly encapsulated in the cavity. However, only two kinds of 1H NMR signals for all the alkanes  and all methylenes (2.33 ppm)) were observed  (248 Å3) to two molecules of perylene (PE) (508 Å3) were also encapsulated in the nanocube. These results indicate the high adaptability of the nanocube to a wide range of molecular size of guests.Considering the fact that the molecular lengths of long alkanes with all ylenes 2.3 ppm) we1H DOSY spectroscopy. The logD value of the nanocube encapsulating two molecules of 1,3,5-tribromomesitylene (TBM), –9.96, is slightly smaller than that of the empty nanocube, –9.89 . Interestingly, a sum of the chemical shift changes of the three p-tolyl methyl signals  and that the other one (i1) is around the north or the south pole (MeP). In the cases where two molecules of anthracene and two molecules of perylene (PE) were encapsulated in the nanocube, large chemical shift changes of MeP (i1) were observed  in the nanocube is much faster than the NMR timescale.Induced-fit expansion of the nanocube by neutral guest molecules is confirmed by ved Fig. , suggest1H NMR spectroscopy indicates that the thermal stability of the nanocube became higher by binding of neutral guests and depended on the volume of the guests  is slightly smaller than the volume of decamethylferrocene (DF) (407 Å3)  (403 Å3)  is 11500 Å3, which is about half of that of TBM2@16 (24000 Å3). The longitudinal relaxation time (T1) of the p-tolyl methyl protons of PCCP2@16, 2.56 s, is longer than that of 16, 2.15 s  were not encapsulated in the nanocube  40 Å3 , which could be partly due to the chaotropic effect46. The introduction of electron-withdrawing groups in the cyclopentadienyl anion causes dispersion of the π electrons to lead to high polarizability of PCCP as seen in ClO4–. Upon the encapsulation of such chaotropic anions in the nanocube, the reformation of the water molecules that surrounded the anions restores hydrogen bonds between water molecules to make more ordered water network. Moreover, stronger electrostatic and vdW interactions between PCCP and the nanocube than the hydration of PCCP and the release of energetically destabilized water molecules in the cavity of the nanocube upon binding of PCCP also account for the large negative enthalpy change48. The entropic disadvantage is due to conformational fixation of the GSAs by the induced-fit contraction of the nanocube. The second binding is enthalpically disfavored  because of the electrostatic repulsion between the two negatively charged PCCP molecules close in vicinity in the cavity of the nanocube, so encapsulation of the second PCCP molecule was promoted by entropy . Electrostatic attractions in water are generally favored entropically due to desolvation of the structured water molecules around ionic species50. As the entropic loss arising from the induced-fit binding has already been paid in the first binding, the desolvation of water molecules around ions mentioned above would mainly contribute to the second binding. As to the encapsulation of CB, the binding constant is 1.46 × 107 M–1, which is about 20 times higher than the first binding constant for PCCP. The large negative enthalpy and entropy changes  indicate a similar binding event to the first binding of PCCP. The stronger binding for CB than for PCCP is because larger and higher polarizable CB restores more hydrogen bonds between water molecules and makes stronger vdW interactions between CB and the nanocube upon binding.As NaPCCP and CsCB are soluble in water, ITC titration experiments were carried out to determine the thermodynamic parameters of the binding of the anionic guests in the nanocube  at 130 °C is higher than that of TBM2@16 (6[TBM2@16]/[1] = 30) at the same temperature . Indeed, the formation ratio of PCCP3, has been demonstrated. Neutral guest molecules induced the expansion of the nanocube, while anionic guests the contraction of the nanocube due to electrostatic interaction between the polycationic host and the anionic guest(s) to lead to tight host-guest complexes. The nanocube can alter the size of its inner space from 14 Å (11500 Å3) to 18 Å (24000 Å3) depending on the size and the charged state of guest molecules. This high induced-fit property of the nanocube is because the GSAs in the nanocube are not connected by directional chemical bonds but only by molecular meshing, where vdW and cation-π interactions and the hydrophobic effect contribute to the adhesive force between the GSAs. In biological systems, not large but fine conformational changes precisely responding to the input (guest) are inevitable to transport signals to other molecules. Although vdW interactions are the weakest molecular interaction between atoms and has often been underestimated, one of the reasons why vdW interactions are well utilized in biological systems would be because vdW interactions endow assembled structures with a sensitive responding property.In conclusion, a fine structural change of the nanocube responding to the size, shape, and charged state of various guest molecules, whose volume ranges from 74 to 535 Å1H and other 2D NMR spectra were recorded using a Bruker AV-500 (500 MHz) spectrometer. A high pressure valved NMR tube  was used for variable temperature 1H NMR measurements over 100 °C in D2O. All reagents were obtained from commercial suppliers  and were used as received. 1 was prepared according to the literature24.2O solution of 16  in an NMR tube. The suspension was mixed by inverting the NMR tube 4 times and sonicated for 5 min. For the encapsulation of solid guests not soluble in water, 1 mg of guest was added to a D2O solution of 16  in an NMR tube. The suspension was heated at 70 °C overnight. For large hydrophobic guest molecules, heating at 90 °C was required for the completion of the encapsulation. During heating of the suspension, the hydrogen atoms neighboring the positively charged nitrogen atoms of the N-methylpyridinium rings of 1 were exchanged with deuterium atoms24. For 1,3,5-triiodomesitylene, the hydrogen atoms on the N-methyl groups of 1 were also exchanged with deuterium atoms through the very long heating. For the encapsulation of gaseous molecules, guest molecules were added through bubbling in a D2O solution of 16  for 2 min. The stoichiometries between 16 and the guest except the anionic guests, PCCP (pentacyanocyclopentadienide) and CB (CHB11Cl11–), were determined by the integrals of the 1H NMR signals of the encapsulated guest. As to PCCP and CB, the stoichiometry between 16 and the encapsulated anionic guest was determined by titration experiment using the 1H NMR signals for the nanocube encapsulating the anionic guest. 1H NMR spectra of host-guest complexes of 16 are provided in Supplementary Figs. 16 are summarized in Supplementary Table 1H DOSY NMR spectra of host-guest complexes of 16 are provided in Supplementary Figs. For the encapsulation of liquid guests not soluble in water, 1 μL of guest was added via a syringe to a Dnd CB CHBCl11–, we16 were assigned based on 1H-1H COSY and 1H-1H NOESY spectra  of the nanocubes, at which half of the nanocubes are disassembled into the monomers, was determined by variable temperature 1H NMR measurements to compare the integral values of the p-tolyl methyl signals for the host-guest complexes of 16 and for the monomer GSA. 1H NMR spectra are provided in Supplementary Figs. Disassembly temperature T1/ of the n200. Titration curves are provided in Supplementary Fig. Dilution isothermal titration calorimetry (ITC) experiments were conducted on a Malvern MicroCal iTCT1) of the monomer GSA, 1, and the 16 and PCCP2@16 nanocubes are provided in Supplementary Table Longitudinal relaxation times (Supplementary InformationPeer Review File


Immobilization of proteins has been examined to improve implant surfaces. In this study, titanium surfaces were modified with nanofunctionalized denosumab (cDMAB), a human monoclonal anti-RANKL IgG. Noncoding DNA oligonucleotides (ODN) served as linker molecules between titanium and DMAB. Binding and release experiments demonstrated a high binding capacity of cDMAB and continuous release. Human peripheral mononuclear blood cells (PBMCs) were cultured in the presence of RANKL/MCSF for 28 days and differentiated into osteoclasts. Adding soluble DMAB to the medium inhibited osteoclast differentiation. On nanofunctionalized titanium specimens, the osteoclast-specific TRAP5b protein was monitored and showed a significantly decreased amount on cDMAB-titanium in PBMCs + RANKL/MCSF. PBMCs on cDMAB-titanium also changed SEM cell morphology. In conclusion, the results indicate that cDMAB reduces osteoclast formation and has the potential to reduce osteoclastogenesis on titanium surfaces. Aseptic loosening of implants is a multifactorial process associated  with perTo mitigate this disproportional peri-implant osteoclast activity, bisphosphonates as well as recombinant OPG have been applied to implants in different studies ,10,11,12In peri-implant tissues, however, there is evidence of a biological environment that does not respond to the administration of bisphosphonates with the suppression of osteoclasts . MoreoveUtilizing the same mechanism as that of OPG, the fully human IgG monoclonal antibody denosumab (DMAB) specifically binds primate RANKL with a high degree of efficacy and has a well-established and acceptable safety profile . The safModification of titanium surfaces heralded a new direction to a more favorable interaction between the implant and the periprosthetic environment. Different methods have been developed to immobilize and release bioactive molecules from titanium specimens ,20,21,22Thus, we hypothesize that DMAB immobilized on an endoprosthetic surface could inhibit RANKL-dependent osteoclast formation and activation in the peri-implant environment at very early stages. As an oligonucleotide-mediated technique, nanofunctionalization could facilitate a targeted application of DMAB to reduce aseptic loosening with substantially lower systemic DMAB exposure, which may minimize the risk of treatment-related adverse events. As such, this study’s objective was to examine whether DMAB can be effectively nanofunctionalized on a titanium specimen in a manner that reduces osteoclast formation from stimulated human osteoclast precursor cells.Prior to testing the effect of immobilized conjugated DMAB (cDMAB), we first corroborated the ability of soluble DMAB (2 nM) to successful inhibit long-term osteoclast differentiation in vitro.Human PBMCs were used as osteoclast precursors. The cells were cultivated in the presence of the differentiation stimulators RANKL and MCSF . The lonHistochemical detection of TRAP activity revealed a strongly positive enzymatic reaction of the stimulated PBMCs B. As a rIn addition to the osteoclast differentiation medium, one group received 2 nM of soluble DMAB resulting in a substantially lower enzymatic reaction compared with that of the positive control C.The depicted groups were further examined for resorptive activity on dentine chips. After 28 days, resorption pits were visualized by staining with toluidine blue. The +RANKL/MCSF-stimulated PBMCs formed Howship′s lacunae on several sites of the chips with degraded dentine appearing darkly stained B. The -Rn = 6 donors) and resorptive activity (n = 3 donors) confirmed the eligibility of the depicted control groups for all donors. Moreover, soluble DMAB substantially reduced the accumulation of TRAP stain  and further applied at a concentration of 550 nM to the titanium with the complementary ODN anchor strands. The release of cDMAB was monitored after a standard curve for IgG/DMAB had been established. After the rinsing steps, 83% of the IgG corresponding to cDMAB remained hybridized to the ODN anchor strands. This was followed by an initially pronounced (70% of bound cDMA within 24 h) and then low continuous release, which was observed within 18 days.Evaluation of the osteoclast-specific TRAP5b activity revealed a decreased protein level (P = 0.0513) in the cDMAB group compared to the positive control +CTRL on titanium, and almost reached the level of the –CTRL group on titanium .An enzyme-linked fluorescence assay of total phosphatase activity showed that PBMCs of the +CTRL group on titanium formed large multinuclear cells B. PBMCs Scanning electron microscopy demonstrated that PBMCs after 28 days of culture on titanium had differentiated into giant well-spread cells B, showinEnhanced bony implant fixation can be achieved by increasing new bone formation onto implant surfaces, and also by inhibiting bone resorption around implant surfaces. One therapeutic approach toward those goals is to ‘functionalize’ implant surfaces via the tethering of various bioactive molecules that can stimulate osteoblasts or inhibit osteoclasts. The present study evaluated oligonucleotide-based immobilization of the anti-RANKL antibody DMAB on a titanium surface and its effect on osteoclastogenesis from PBMCs stimulated by +RANKL/MCSF.Oligonucleotide-based nanofunctionalization of titanium surfaces has been successfully applied to immobilize other bioactive molecules , for exaThe progress of implant osseointegration and loosening is a common subject of investigation. Recent studies in a New Zealand White Rabbit model reported that several weeks are required for initial implant osseointegration . The effThe current study analyzed the effects of 28 days of DMAB exposure on the ability of cultured human osteoclast precursors to differentiate into terminally differentiated bone-resorbing osteoclasts. Soluble DMAB (2 nM) affected osteoclastogenesis and abolished the terminal differentiation of osteoclasts. Although TRAP positive multinucleated cells were found, the addition of DMAB prevented the creation of resorption pits.Systemic DMAB therapy increases bone density and strength throughout the skeleton and also carries some risks for adverse events, which is acceptable for most patients with osteoporosis because denosumab substantially lowers the risk of fragility fractures. However, many patients receiving osseointegrating implants do not have low bone mass or increased fracture risk, and for those patients, systemic DMAB therapy would have a less favorable benefit–risk balance. The aim of this study was to establish proof of concept that DMAB can be nanofunctionally conjugated to titanium surfaces. This work represents a potentially important first step in establishing a therapeutic approach that leverages DMAB’s potent osteoclast-inhibiting effects while minimizing systemic DMAB exposure. DMAB has the potential to promote osseointegration even while it remains firmly tethered to implant surfaces by acting as a local ‘sink’ that sequesters RANKL, thereby depriving local osteoclast-lineage cells of an essential proresorptive factor. In contrast, the tethering of most other bioactive molecules, including BMPs, PTH receptor agonists, growth factors, and bisphosphonates, requires their timely release so they can directly bind to  their target cells. DMAB was conjugated to ODN strands and further applied to the titanium with the complementary ODN anchor strands. It was found that DMAB remained hybridized to the ODN anchor strands and was substantially released within 18 days. To test the osteoclast-specific effect of DMAB on the differentiation of PBMCs, the tartrate-resistant acid phosphatase and its osteoclast-specific isoform 5b were used, which constitutes a hallmark of active osteoclasts . As showOne limitation of the study was the impracticability of dentine assays after titanium cultivation, which does not rule out the possibility that some bone resorption may still occur in the presence of immobilized DMAB. However, the complete abolishment of terminal osteoclast differentiation is not the intended goal of implant functionalization, as bone turnover may contribute to bone regeneration and implant fixation ,32. AnotIn summary, we demonstrated that nanofunctionalized DMAB decreased the protein level of the osteoclast-specific TRAP5b and endogenous phosphatases in PBMC treated with M-CSF and RANKL for 28 days. Morphological changes in DMAB-exposed PBMCs cultured in osteoclast differentiation medium suggested an inhibitory effect of titantium-immobilized DMAB on terminal osteoclastogenesis. Collectively, these results indicate that titanium nanofunctionalized with DMAB reduces osteoclast activity by interfering with the RANKL-dependent terminal osteoclast differentiation. This in vitro study suggests that nanofunctionalization of DMAB prior to implant insertion could potentially alter the local periprosthetic milieu in a manner that favors unopposed osseointegration. Local antiresorptive treatment heralds a new direction of improving the endoprosthetic lifespan. The slow release of bioactive molecules in conjunction with improving surface osteoconductiveness may further the goals of preventing aseptic loosening.n = 8) were isolated from purchased buffy coats  to generate osteoclast-like cells. In brief, the leukocyte-rich blood portions were diluted in PBS (1:1) and layered on top of Biocoll Separating Solution . After centrifugation at 2000 rpm for 30 min, PBMCs were isolated from the gradient and purified in several rinse steps using PBS.Human PBMCs from healthy human male donors , 0.6% Penicillin/Streptomycin , and 0.01% Amphotericin B  (base medium). The PBMCs were cultured at 37 °C and 5% CO2 for 28 days and the medium was replaced three times per week with additional factors. Additional factors were added to the PBMCs in four groups: (1) no RANKL, M-CSF, or DMAB (−RANKL/MCSF −DMAB: −CTRL); (2) RANKL + M-CSF, no DMAB (+RANKL/MCSF −DMAB: +CTRL); (3) RANKL + M-CSF + DMAB (DMAB); and (4) RANKL + M-CSF + conjugated DMAB (+RANKL/MCSF + conjugated DMAB: cDMAB).To establish osteoclastogenesis, isolated PBMCs were resuspended in medium and a cell count was performed. The PBMCs were further seeded into 24-well plates as monolayer, onto dentine chips, or onto titanium specimens placed in 24-well plates at the density 6.0 × 106 cells/well. It had been shown in preliminary experiments counting the number of nonadherent cells in the supernatant after 24 h that a 2 h incubation step in the incubator at 37 °C before adding 500 μL of the medium per well was adequate to achieve sufficient adherence of the cells. All cells were supplied with alpha MEM  supplemented with 10% fetal bovine serum , 1% The +RANKL/MCSF group was treated with an osteoclast differentiation medium, which comprised the base medium described above plus 25 ng/mL of recombinant human M-CSF , on days one and three, along with 20 ng/mL of recombinant human sRANK Ligand  in all subsequent media changes. The RANKL/MCSF control group received the base medium only.To preliminarily test the effect of low-dose DMAB on osteoclast differentiation, one group of PBMCs received osteoclast differentiation medium supplemented with a single dose of 2 nM of soluble denosumab .For the subsequent examination of the effect of nanofunctionalized DMAB on osteoclast formation, 550 nM of conjugated DMAB was immobilized onto titanium discs (described below); the DMAB-conjugated titanium discs of 14.7 mm diameter were cultured in osteoclast differentiation medium.n = 6 donors). PBMCs were fixed and stained after 28 days of culture using a commercial kit , and TRAP-positivity was examined under light microscopy.Osteoclast differentiation was monitored by visual histochemistry-based assessment of TRAP staining (Osteoclast differentiation and activity were assessed by measuring the area of toluidine-blue-stained resorption pits on dentine chips. Ivory (from German Customs investigations) was cut into flat disks (9 × 9 × 1 mm) by a diamond-coated cutting band saw . Slices were placed into 24-well plates  after disinfection (70% ethanol for 48 h).n = 3 donors) in the above-mentioned groups and densities. After lysis in Tween 20 , the cells were wiped off the dentine chips with a paper towel. The chips were subsequently immersed in 1% toluidine blue , which revealed resorption lacunae that were quantified with the BZ9000 fluorescence microscope .PBMC cultures were seeded on dentin  were made of the alloy TiAl6V4 and ground with SiC paper P600 (26 μm) . The immobilization of the oligonucleotide anchor strands on the titanium specimens  was perf2O and 0.462 g potassium dihydrogen phosphate and 200 mL deionized water of total pH 6.85), 1.1 µL conjugate solution, and 43.9 µL sterile ultrapure water. The γ-sterilized titanium specimens with the immobilized oligonucleotide anchor strands were completely wetted with the hybridization solution and incubated in the dark at room temperature for one hour. Subsequently, the samples were washed four times in PBS. The last rinse took place with sterile ultrapure water. Cells were cultured for 28 days on titanium in +RANKL/MCSF and −RANKL/MCSF groups. The conjugated titanium cells were treated with osteoclast differentiation medium (+RANKL/MCSF).The hybridization of denosumab–oligonucleotide conjugates to the immobilized anchor strands was carried out directly before the cell colonization of the titanium specimens. The materials used were provided by the Max Bergmann Center for Biomaterials (TU Dresden). Titanium specimens were treated with 50 µL of a 550 nM denosumab conjugate hybridization solution consisting of 5 µL 10× PBS buffer  for quantitative human IgG2 detection. Thus, the amount of DMAB rinsed from the titanium during the hybridization process was measured using an ELISA kit  according to the manufacturer’s instructions.The staining of tartrate-resistant acid phosphatase  in monolTo depict endogenous phosphatases on the titanium test specimens, the cells were fixed analogously to the TRAP staining using a citrate and acetone solution, and after 20 min of air drying, the ELF® 97 Endogenous Phosphatase Detection Kit  was used according to the manufacturer’s instructions.Results were visualized by confocal microscopy (Zeiss LSM 710).Secretion of the tartrate-resistant acid phosphatase isoform 5b into the cell supernatant was measured using the TRAP5b ELISA kit  after 28 days of culture, following the manufacturer’s instructions.After fixation in a 4% formalin solution for 24 h, the cells cultured on the titanium were dehydrated through a graded series of ethyl alcohols (50–96%) and further processed in a Critical Point Dryer. The titanium specimens were subsequently sputter-coated with gold and observed under a scanning electron microscope (Zeiss Typ LEO 1455VP).Inferential statistical comparisons of the three groups were made using one-factorial variance analysis using the GLM procedure of the SAS 9.4 Statistical Analysis System for Windows . All values included in these analyses were averages of at least three replicate measurements. The first global test was an F-test at the alpha level of 5%. Significant failure resulted in post hoc pairwise, two-sided group comparisons with Sidak correction. Corrected p-values below 5% were considered significant.The results indicate that DMAB can be electrochemically coated onto titanium surfaces without losing its functionality. Immobilization of DMAB resulted in a reduction of terminal osteoclast differentiation from M-CSF- and RANKL-stimulated PBMCs. The inhibitory effects of DMAB appear to persist for a sufficient duration to potentially improve early implant osseointegration. The technique allows a one-time, low-dose application of DMAB, thereby minimizing systemic effects of the drug. Collectively, endoprosthetic nanofunctionalization of DMAB capitalizes on the benefits of a local therapy without compromising the efficacy of the treatment and thus has potential as a novel and clinically relevant approach to reduce the risk of aseptic loosening.