To compare the risks of hospitalization for heart failure (HHF) associated with sulfonylurea (SU), dipeptidyl peptidase-4 inhibitor (DPP-4i), and thiazolidinedione (TZD) as add-on medications to metformin (MET) therapy using the data of Korean adults with type-2 diabetes from the Korean National Health Insurance database.We identified 98,383 people who received SU , DPP-4i , or TZD  added to initial treatment of MET monotherapy in patients with type-2 diabetes. The main outcome was the hospitalization for HHF. Hazard ratios for HHF by type of second-line glucose-lowering medication were estimated by Cox-proportional hazard models. Sex, age, duration of MET monotherapy, Charlson Comorbidity Index and additional comorbidities, and calendar year were controlled as potential confounders.The observed numbers  of HHF events were 1,129 (658) for MET+SU users, 710 (455) for MET+DPP-4i users, and 110 (570) for MET+TZD users. Compared to that for MET+SU users (reference group), the adjusted hazard ratios for HHF events were 0.76  for MET+DPP-4i users and 0.96  for MET+TZD users.DPP-4i as an add-on therapy to MET may lower the risks of HHF compared with SU. Heart failure (HF) is one of the main health burdens of cardiovascular disease (CVD) in people with type-2 diabetes mellitus (T2D) . PathogeMetformin (MET) is the most commonly used starting drug for patients with T2D on the basis of international guidelines . In KoreDPP-4i is commonly used because it is associated with a relatively low risk of hypoglycemia and weight gain . HoweverOur previous study revealed that MET+DPP-4i for T2D was associated with a lower CVD risk than that of MET+SU . HF is aThis study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System (IRB number 4-2015-1023). All datasets were anonymous and de-identified. As such, the need for informed consent was waived.The National Health Insurance Service (NHIS) covers approximately 50 million people in Korea. In the NHIS claims database, diagnoses are coded using the International Classification of Disease version 10 (ICD-10). Insured individuals are required to undergo general health screenings every 2 years . The heaIn Korea, MET is the most commonly prescribed drug in Korea . MET canComorbidities were adjusted using the Charlson Comorbidity Index and calculated by individual conditions, such as hypertension (ICD-10 codes I10–I15 and/or ATC codes C02–C03 and C07–C09), dyslipidemia (ICD-10 code E78 and/or ATC code C10), atrial fibrillation (ICD-10 code I48), chronic kidney disease (ICD-10 code N18), and microvascular complications of diabetes, including diabetic retinopathy , diabetic neuropathy , and diabetic nephropathy . CVD included myocardial infarction (ICD-10 codes I21–I23), cerebrovascular diseases , unstable angina (ICD-10 code I20.0), HF (ICD-10 code I50), and transient cerebral ischemic attack (ICD-10 code G45).A subgroup analysis was performed to account for additional covariates, such as body mass index (BMI), waist circumference, systolic blood pressure, total cholesterol level, high-density lipoprotein (HDL) cholesterol level, low-density lipoprotein (LDL) cholesterol level, triglyceride level, fasting glucose level, serum creatinine level, smoking status , and family history of stroke and heart disease. These variables were measured via health screening within 1 year before the index date. We used available data from patients who had undergone health screening .Baseline characteristics were compared between patients treated with the different second-line glucose-lowering drugs. Cox proportional hazard regression models were used to calculate the hazard ratios (HRs) and 95% confidence intervals (CIs) for the incidence of HHF. We tested the proportional hazards assumption on the basis of Schoenfeld residuals . The METThe patients were divided into the following three groups: MET+SU , MET+DPP-4i , and MET+TZD . The mean index age of the study population was 58.3 ± 11.1 years, and 53.4% were men. Compared to MET+SU users, MET+DPP-4i users and MET+TZD users were younger, had a higher prevalence of microvascular complications such as diabetic retinopathy, neuropathy, and nephropathy, had a lower prevalence of hypertension, and had a higher prevalence of dyslipidemia . Over 34To assess whether a history of CVD affected the relationship between the types of add-on medication and the risk of HHF, we repeated the analysis separately for people with and those without a history of CVD . CompareThe NHIS claims data included all physician-diagnosed disorders coded by ICD-10 but did not include objective data on exposure levels to CV risk factors. Therefore, we performed a subgroup analysis with available health screening data. In total, 35,297 people (35.9%) remained with available screening data and 498 per 100,000 person-years experienced HHF. Using MET+SU as a reference group, the HRs for HHF adjusted by age and sex were 0.74 (95% CI 0.61–0.89) for MET+DPP-4i and 1.11 (95% CI 0.77–1.59) for MET+TZD, respectively . MoreoveThe present study of nationwide real-world data demonstrated that people with T2D who were prescribed DPP-4i as a second-line drug alongside MET therapy had relatively lower risks of HHF than did those who were prescribed SU regardless of their CV history. These findings were directionally consistent across the subgroups that were available for further analysis using health screening data. Additionally, people who were prescribed TZD tended to have lower risks without statistical significance. This result could be explained by the fact that TZD was not prescribed to people at high risk of HHF because of doctors’ concerns about the high risks of HHF associated with TZD reported in previous studies , 15.et al. revealed that individuals treated with DPP-4i had significantly lower risks of HHF among patients with no baseline CVD. The risk of HHF was lower in those treated with DPP-4i monotherapy than SU, pioglitazone, or insulin therapy alone , or numbers (percentages) unless otherwise stated. CVD, cardiovascular disease; MET, metformin; SU, sulfonylurea; DPP-4i, dipeptidyl peptidase-4 inhibitor; TZD, thiazolidinedione; HDL, high-density lipoprotein; LDL, low-density lipoprotein.(DOC)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


This paper intends to evaluate the influence of content of rice husk fiber and cementitious materials on mechanical properties and thermal conductivity of thermal insulation wallboards. Thermal insulation wallboard contained different mass of rice husk fiber was prepared when the weight of cement, fly ash, cellulose ether, naphthalene superplasticizer, and recycled concrete aggregates was equal. Scanning electron microscopy (SEM) shows the internal structure of the insulation wallboards is very dense. Compared to thermal conductivity of blank group (0.9600 W/m·°C), B2 (0.1997 W/m·°C) and C2 (0.1810 W/m·°C) measured by the DRCD-3030 intelligent thermal conductivity tester can meet certain engineering requirements. Average compressive strength, flexural strength, and thermal conductivity of wallboards decreases with content of rice husk fiber increasing when other materials mass are the same. Under the same conditions of curing time and rice husk content, average compressive and flexural strength increase with the increase of the amount of cementitious material. Concrete materials in human social and economic development are indispensable. With the progress of society and people’s living standards continues to improve. The per capita demand for concrete has increased year by year, indirectly lead to ecological damage, environmental pollution, and other issues become increasingly prominent. Gravel and sand as non-renewable resources have shown a shrink tendency under the huge consumption. To ensure high-quality aggregate supply more difficult, which will inevitably affect the construction and national economic development. With the acceleration of civil engineering construction, huge amounts of construction waste are generated, of which nearly half are waste concrete. The utilization rate of China’s construction waste is less than Europe and the United States. Waste concrete make into recycled aggregates, replacing normal natural aggregates has become an important issue, and researched by scientists around the world –23.The use of recycled concrete aggregate as a building material is now gaining popularity in China’s construction materials. High-performance concrete, stub columns, and beams can be cast by recycled concrete aggregates, and have good mechanical properties according to literature reported –27. Howe(1) Cement: The cement used in this experiment was ordinary Portland cement 42.5 , as shown in The cement, fly ash, concrete powder, cellulose ether, naphthalene superplasticizer, and rice husk fiber were mixed in different proportions to make different kinds of cement-based materials, as shown in The contents of the experimental materials were calculated and measured according to the requirements for mix proportion. Recycled concrete aggregates, cement, fly ash, cellulose ether, naphthalene superplasticizer, and rice husk fiber were sequencely added into a small mortar mixer. The mixer was covered and mixed for 2 min to achieve uniform mixing. A certain amount of running water was added to the mixture, and continued stirring for 3 min. The consistency of all cement-based slurries was adjusted to about 8CM. The cement-based slurry was then poured into a particular mold . Specimens were demolded after 3d, as shown in The phase composition of products was examined by X-ray diffraction . Scanning electron microscope  was used to conduct morphological studies of the samples. The compressive strength and flexural strength of specimens was tested through the ordinary compression/bending test machine. The thermal conductivity of the insulation panels was measured by the DRCD-3030 intelligent thermal conductivity tester , as shown in The composition of concrete powder was analyzed through XRD, as shown in SEM images of concrete powder are shown in Compressive strength and flexural strength of the specimens in the same age decreasing with the increase of the amount for rice husk fiber when the content of cement, fly ash, concrete powder, cellulose ether, and naphthalene superplasticizer are same, as shown Figs 7, 14, and 28d of average compressive strength for groups B1 ~ B4 were 6.5, 9.4, and 10.4Mpa, 3.7, 4.6, and 4.9Mpa, 4.0, 3.7, and 4.0Mpa, 1.9, 2.3, and 2.3Mpa, respectively . AverageFor groups C1 ~ C4, average compressive strength at 7, 14, and 28d were 8.0, 13.0, 13.4Mpa, 5.5, 6.7, and 6.6Mpa, 3.5, 4.2, and 3.1Mpa, 2.4, 2.8, and 2.8Mpa, respectively . The aveFor the group numbers A1 ~ A4, 7, 14, and 28d of average flexural strength were 1.7, 3.3, and 3.6Mpa, 1.4, 1.4, and 1.5Mpa, 1.0, 1.0, and 1.1Mpa, 0.8, 0.8, and 0.9Mpa, respectively, as shown in Average flexural strength of groups C1 ~ C4 was 4.1, 4.4, and 4.2Mpa, 2.2, 2.6, and 2.6Mpa, 1.9, 2.1, and 2.0Mpa, 1.5, 1.5, and 1.5Mpa, respectively, as shown in In conclusion, average compressive strength and flexural strength of cement-based materials are greatly reduced with the increase of the content of rice husk, and the intensity of 7 ~ 28d is slow or negative when mass of cementitious materials was equal, as shown in 3. When the content of rice hull is the largest, the value of thermal conductivity λ is minimum, indicating the better the insulation performance. The average compressive strength of the specimens should be greater than 4.0 MPa according to the actual engineering needs. The thermal conductivity of thermal insulation wallboards was tested and the results are shown in Thermal insulation wallboards can well be cast by the mixed slurry of cement, fly ash, cellulose ether, naphthalene superplasticizer, rice hull, and recycled concrete aggregates when slurry consistency was about 8CM. B2 and C2 specimens of compressive strength is bigger than 4MPa, and can be applied to actual engineering. The content of cementitious materials and rice hull has an important influence on the mechanical properties of the resulting thermal insulation wallboards, affecting their application. Average compressive strength, flexural strength, and thermal conductivity of the specimens decreased as the content of rice hull increased. Thermal conductivity of C2 was 0.1810 W/m·°C less than B2 (0.1997 W/m·°C) and blank group (0.9600 W/m·°C) when curing time was 28days. Therefore, C10 will be chosen and used in certain engineering.S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Antimicrobial resistance (AMR) driven by antibiotic consumption is a growing global health threat. However, data on antimicrobial consumption patterns in low- and middle-income countries (LMICs) is sparse. Here, we investigate the patterns of antibiotic sales in humans and livestock in urban Nairobi, Kenya, and evaluate the level of awareness and common behaviours related to antibiotic use and AMR amongst human and veterinary pharmacists.A total of 40 human and 19 veterinary drug store pharmacists were interviewed in Nairobi in 2018 using a standard questionnaire. Data recorded included demographic variables, types of antibiotics sold, antibiotic customers, antibiotic prescribing practices and knowledge of antibiotic use and AMR.Our study shows that at the retail level, there is a considerable overlap between antibiotic classes (10/15) sold for use in both human and veterinary medicine. Whilst in our study, clinical training significantly influenced knowledge on issues related to antibiotic use and AMR and respondents had a relatively adequate level of knowledge about AMR, several inappropriate prescribing practices were identified. For example, we found that most veterinary and human drug stores (100% and 52% respectively) sold antibiotics without a prescription and noted that customer preference was an important factor when prescribing antibiotics in half of the drug stores.Although more research is needed to understand the drivers of antibiotic consumption in both human and animal populations, these findings highlight the need for immediate strategies to improve prescribing practices across the pharmacists in Nairobi and by extension other low- and middle-income country settings. Antibiotic resistance (AMR) has been described as one of the most serious public health threats of this century -3. AntimAs in most cities in low and middle income countries (LMICs), in urban Nairobi the high incidence of bacterial diseases and antimicrobial resistance in clinical medicine is a major public health challenge . In bothPharmacists (both human and veterinary) play a pivotal role in enhancing antimicrobial stewardship initiatives, not just by highlighting the AMR problem, but also by influencing crucial prescribing decisions ,9. To fuA cross-sectional study targeting human and veterinary drug stores in urban Nairobi, Kenya was carried out in January 2018 as part of the UrbanZoo project . BrieflyA draft of the questionnaire was pre-tested with five drug stores (three human and two veterinary), and refined on basis of the feedback from the pre-testing sessions before final dissemination. In each drug store a detailed questionnaire was used to collect data on socio-demographic variables, training on antibiotic use, types of antibiotics sold (by class), the four antibiotic classes most commonly sold, variation in antibiotic sales, antibiotic sources, antibiotic customer characteristics, and antibiotic prescribing practices. A summary of the collected data are presented in Table S1 in 2 or Fisher exact test using R package stats [Descriptive statistics were prepared for all data including frequencies and percentages for categorical variables  and means, medians, standard deviations (SDs), quartiles, and ranges for quantitative variables  depending on the distribution of the data. We used a χge stats  to descrP < 0.05 to be statistically significant.Next, we aimed to describe practices and evaluate the factors associated with drug prescribing amongst human and veterinary pharmacists. To achieve this, we collected data on information provided to customers after purchasing antibiotics as an indicator of good prescribing practices. This included: whether pharmacists provided customers with information on dosage, directions for use , storage instructions, side effects, expiry date and contra-indications . The datIn order to assess the internal consistency of the ten statements evaluating the level of knowledge on AMR, Cronbach’s alpha coefficients were calculated for each statement. Internal consistency is a measure of item-total correlations and reliability of the scale, thus describing the extent to which all items in a test measure the same concept or construct . An unstPrincipal Component Analysis (PCA) using polychoric correlation  was usedThe scores of the first PCA component were used as measure of knowledge of AMR, and the higher the knowledge score, the higher the implied knowledge of AMR of that respondent. A generalised linear model was used to investigate the possible influence of type of drug store (human or veterinary), clinical training (present or absent), education level (high or low), and range of antibiotics sold in the drug store (proxy for store size) on the level of knowledge of AMR.Ethical approval for this study was obtained from the International Livestock Research Institute (ILRI) Institutional Research Ethics Committee (ILRI IREC) (project reference: ILRI-IREC2017-35).P = 0.01, Fisher exact test). Additionally, for participants who underwent clinical training, professional development programmes/trainings aimed at continuing education in AMR were an important source of information on antibiotic stewardship .A total of 59 participants were interviewed – 40 from human drug stores and 19 from veterinary drug stores   were reported as the main provider of antibiotics to human drug stores (78%). On the other hand, distribution companies  were reported as the main provider of antibiotics to veterinary drug stores (58%).P = 0.20; Mann-Whitney U test) between human drug stores  and veterinary drug stores .The average daily number of customers purchasing antibiotics was not significantly different  believed that AMR occurs when their body becomes resistant to antibiotics rather than the bacteria themselves that develop resistance. Similarly, 40% and 53% of human and veterinary respondents respectively suggested that AMR is only a problem for regular consumers of antibiotics. More than half (52%) of the respondents interviewed responded neutrally or disagreed with the statement that antibiotic resistant bacteria could be spread from person to person , meaning respondents with clinical training had a higher knowledge about AMR than those who had not undergone such training. The level of knowledge about AMR did not differ by store type, education level or range of antibiotics available in the store (P > 0.05)  (Table S5 in the Fifty-two per-cent (21/40) of the human drug stores reported that they sold antibiotics without a prescription while all veterinary drug stores sold antibiotics without a prescription. Multivariable logistic regression analysis revealed prescribing practices did not vary significantly by clinical training, store type, range of antibiotics sold and/or education level , followed by price of the antibiotic (in >50% of the stores). Of note, 28% and 31% of human and veterinary pharmacists respectively considered customer preference as an important factor when prescribing an antibiotic.In this study, we aimed to investigate the patterns of antibiotic sales in humans and animals in a large and rapidly developing city in a LMIC: Nairobi, Kenya. We also evaluated the level of awareness and common behaviours related to antibiotic use and AMR amongst human and veterinary pharmacists. Our study was based on gathering antibiotic sales data from human and veterinary drug stores across the city, where sales data were interpreted as representing antibiotic usage.Our study shows considerable overlap in the antibiotic classes (10/15) sold for human and animal use in urban Nairobi, with marked variations in the sale of some antibiotic classes such as cephalosporins and fluoroquinolones – mostly found in human drug stores. This overlap in antibiotic classes, including of critically important antimicrobials , highligThe most common symptoms prompting antibiotic purchase in humans were similar to those reported in other studies, respiratory tract infections and diarrhoeal disease ,26,27. BIn the current study, tetracyclines, sulphonamides, penicillins, and macrolides were the most commonly purchased veterinary antibiotics and poultry farmers were the major consumers of antibiotics. Further, our findings indicate that colistin – a drug considered of last resort in human medicine  – was anKnowledge about antimicrobial resistance among pharmacists has only been studied to a limited extent in LMICs . ConsistWhilst the majority of the pharmacists we interviewed have an understanding of the threat posed by AMR to public health, our data highlight the poor quality of community pharmacy practice, most notably the dispensing of antibiotics without prescriptions and the inclusion of customer preference as an important factor when selling antibiotics. Antibiotics were dispensed without prescription in 53% and 100% of the human and veterinary drug stores respectively; a finding consistent with similar studies in Tanzania (92.3%) , Serbia Restating the particular relevance of training to antibiotic stewardship measures, the role of enhanced training in antimicrobial prescribing and AMR has been identified in surveys of both medical personnel and the public, both in Kenya and globally . ResultsSimilar to other studies ,52, our This is the first study designed to capture the overlapping patterns of antibiotic sales in humans and livestock in a developing city via an epidemiologically-structured approach. A variety of approaches are available for assessing patterns of antibiotic use in humans and animals . ConsideMonitoring and surveillance of antibiotic use in LMICs is challenging, but vital, as it provides valuable information for public health policy. Our study shows that at the retail level in urban Nairobi, there is a considerable overlap between antibiotic classes available for use in both human and veterinary medicine. Whilst the majority of human and veterinary pharmacists showed high knowledge about antibiotic use and antimicrobial resistance, inappropriate prescribing practices were noted, highlighting the need for continued education to the pharmacists and the public, about prudent antibiotic prescribing and use. Although further research is necessary to understand the drivers of antibiotic consumption in both populations, it is clear that interventions are urgently required to improve prescribing practices across the pharmacists.


However, simultaneous realization of acoustic logic devices with subwavelength size, broad bandwidth and passive structure still poses a great challenge. To overcome it, we propose a subwavelength acoustic logic gate which consists of binary-phase passive unit cells placed into a multi-port waveguide. Based on the phase manipulations of the unit cells, we experimentally and numerically realize three basic logic gates OR, NOT and AND, and a composite logic gate XOR with a uniform threshold of 0.4 Pa based on linear acoustic interferences. More importantly, We also design a composite logic gate XNOR by a four-port waveguide, and composite logic gates NOR and NAND and a logic operation A⊙(B+C) based on two logic gates. We demonstrate a 0.6λ-length, 0.3 By using photonic crystals2, nanophotonic plasmon networks4, and metal slot waveguide networks6, optical logic gates have been realized based on linear interferences of input and control signals with a certain phase difference9.As major critical components in modern electronic integrated circuits, Boolean logic gates have attracted more and more attentions owing to its capability of implementing Boolean functions. Motivated by great potentials in next-generation photonic integrated circuits, the Boolean logic gates have been introduced in the field of optics17, such as acoustic computing, acoustic information processing and integrated acoustics. Based on a nonlinear dynamical effect of a driven granular chain composed of spherical particles, Li et al.13 have experimentally realized an acoustic switch and logic gates AND and OR. Beyond that, the rapid development of sonic crystals (SCs)22 and acoustic metamaterials (AMMs)29 has provided feasibility for realizing linear acoustic logic devices. By using the SCs, Bringuier et al.30 have designed logic gates NAND, XOR and NOT based on the linear interferences of input and control signals. Zhang et al.31 have theoretically proposed and experimentally demonstrated basic Boolean logic gates XOR, OR, AND and NOT by using self-collimated beams in the SCs with line-defects. However, the acoustic logic devices fabricated with the SCs inevitably involve large structure size at low frequency. By introducing density-near-zero AMMs34, a series of Boolean logic gates AND, OR, XOR and NOT and a logic operation have been realized based on two input signals with a certain phase difference35. Additionally, as a category of AMMs, acoustic metasurfaces with the characteristics of ultrathin planar structure and wholly controlled phase delay40 have been introduced to design acoustic analog computing systems for nth-order ordinary differential equations41. Moreover, the recent emerging acoustic topological insulators47 immune to backscattering has great significance for the development of acoustic logic devices. As an example, Xia et al.48 have proposed acoustic topological insulators with reconfigurable and programmable functionalities realize two types of acoustic switches and tunable logic gates OR and XOR. Furthermore, to realize acoustic logic gates with broad bandwidth, Zuo et al.49 have designed an acoustic logic gate based on multi-port circular waveguides, in which its working bandwidth is larger than 5 kHz and all logic functions and a complex logic operation can be realized by modulating signal phases. The aforementioned works have realized acoustic logic gates and operations with high efficiency. However, most of them have been designed based on active manipulations of the phases of input and control signals, and their structure sizes are still large, which severely limits their integration and practical applications. Simultaneous realization of acoustic logic devices with subwavelength size, broad bandwidth and passive structure still poses a great challenge.Inspired by the optical logic gates, the design of acoustic logic devices has also become a research hotspot owing to a variety of important potential applicationsIn this work, we propose a subwavelength acoustic logic gate which consists of binary-phase passive unit cells placed into a multi-port waveguide. Based on the phase manipulations of the unit cells, we design the basic logic gates OR, NOT and AND, and two composite logic gates XOR and XNOR with a uniform threshold of 0.4 Pa based on the linear acoustic interferences, and the fractional bandwidth (the ratio of the bandwidth to the center frequency) can reach about 0.2. The measured results agree well with the simulated ones. Finally, we also present the realizations of two composite logic gates NOR and NAND and a logic operation A⊙(B+C) based on two logic gates.l = λ/2 and width w = λ/10) consists of two symmetric Helmholtz resonator arrays and a straight channel50 with a tunable width w1, where λ is the wavelength. The cavity width w2 is determined by w1, and the other parameters l1 = 0.03 l, l2 = 0.225 l, w3 = 0.05w and w2 = w − w1 − 4w3. The unit cell filled with air is fabricated with epoxy resin to satisfy the sound hard boundary condition. The wavelength λ of input acoustic signals is selected as 10 cm .As shown in Fig. w1/w). Note that, in the range 0.12–0.70, the phase delay of the unit cell can cover the whole 2π range, and the transmission is larger than 0.85. Here, the two selected unit cells I and II (black hollow dots) with the parameter w1/w of 0.22 and 0.69, which correspond to the transmissions TI = 0.94 and TII = 0.99 and the difference of the phase delays φI − φII = π, are used to design acoustic logic gates.Figure d = 0.1λ and d1 = 0.055λ. Note that the width and length of the logic gate structure are only about 0.3λ and 0.6λ, respectively. When the unit cells in the channels A and B are different  or not (encode “0”) acoustic signal from the respective input port. In addition, to determine the output states, we introduce a uniform threshold At = 0.4 Pa. When the output amplitude is larger than At, the output state is 1/TRUE, while the output is 0/FALSE with the output amplitude smaller than At.Figure 49. Figure As shown in Fig. We also realize the logic gate NOT with the same structure as the logic gate XOR, in which A is the input port, and B is the control port with a constant signal. As shown in Fig. l2), in which the parameter w1/w is a constant of 0.12. Here, we select the unit cell III (the parameters w1/w = 0.12 and l2 = 6.5 mm) with the transmission TIII = 0.71 and the phase delay φIII = φI + π/6, which is utilized to realize the output amplitude less than 0.4 Pa for the input state {0, 0} and the interference cancellation between the input and control ports.In addition to the logical gates constructed by the three-port waveguide structure, we design the logic gate AND based on a four-port waveguide structure, in which the rectangular solid at the center of the three-port waveguide Fig.  is replaAs shown in Fig. The proposed logic gates have broadband characteristic. As shown in Fig. To further demonstrate the aforementioned logic gates, we experimentally measure output time-domain signals for these logic gates. The measurement setup and the photograph of the sample are shown in the Fig. t = 0–3 ms, 3–6 ms, 6–9 ms and 9–12 ms, respectively. Figure Figure t = 0–3 ms and 3–6 ms, respectively, and the constant signal placed at the port B acts as a control port  based on the logic gates OR and XNOR , which is shown in Fig. In conclusion, we have demonstrated the binary-phase acoustic logic gate based on the multi-port waveguide structure, in which the width and length of the logic gate are only about 0.3λ and 0.6λ. The results show that the basic logic gates OR, NOT and AND, and the composite logic gate XOR with a uniform threshold of 0.4 Pa are realized based on the linear acoustic interferences, which arises from the phase manipulations of the binary-phase unit cells. The measured results agree well with the simulated ones, and the fractional bandwidth can reach about 0.2. Moreover, we design the composite logic gate XNOR by using the four-port waveguide, and realize the composite logic gates NOR and NAND and the logic operation A⊙(B+C) based on two different logic gates, corresponding to the logic gates NOT and OR, the logic gates NOT and AND, and the logic gates OR and XNOR, respectively. The proposed acoustic logic gates have advantages of the subwavelength size, broad bandwidth, and passive structure, which have important potential applications in acoustic computing, acoustic information processing and integrated acoustics.The designed acoustic logic gates are fabricated with epoxy resin by three-dimensional printing technology. To obtain the same input signals, two same cylindrical acoustic sources are placed on the right side of the input ports, and the distance between the unit cell and the cylindrical source is 5 mm. The input signals are generated from the cylindrical sources driven by a power amplifier. On the left side of the output port, a 1/4 inch microphone (Brüel & Kjær type-4961) is adopted to measure output signals. The measured data is recorded by the Brüel&Kjær 3160-A-022 module, and is analyzed by the software PULSE Labshop.ρair = 1.21 kg/m3 and sound speed cair = 343 m/s. The mass density, the longitudinal wave velocity and the transversal wave velocity for epoxy resin are 1180 kg/m3, 2720 m/s and 1460 m/s, respectively.The finite element method (COMSOL Multiphysics software 5.2a) is utilized to numerically simulate the characteristics of the acoustic logic gates. The pressure field is calculated in the Acoustic-Solid interaction module, and the Helmholtz resonator arrays filled with air are fabricated with epoxy resin to satisfy the acoustic-structure boundary conditions. In addition, the boundaries of the waveguides are set as the sound hard boundary, and those of the input and output ports are the plane wave radiation boundary. The parameters used for air under an ambient pressure of 1 atm at 20 °C are mass density


Results obtained indicate that C-3ʹ methylation reduces the transcription regulating activity, as shown by the more pronounced suppression of the SL biosynthesis genes DWARF27 (D27) and CAROTENOID CLEAVAGE DIOXYGENASES (CCD7 and CCD8) and higher induction of the SL signaling repressor gene DWARF53 (D53) in MP13 treated seedlings. These results are consistent with a recent study on the biological activities of MP13 and AR8.Strigolactones (SLs) are a well-known class of plant hormones, which are involved in a number of developmental and adaptation processes and mediate different interspecific interactions. In spite of the growing knowledge on SL biosynthesis and signal transduction, effects of structural modifications on the activity and efficiency of SLs and their analogs remain largely elusive. SLs are characterized by the presence of a lactone ring (D-ring) that is connected by an enol ether bridge to a second moiety. In this study, we investigated the effect of additional D-ring methylation of SL analogs on their transcription regulating activity. For this purpose, we compared the SL analogs MP13 and AR8, which differ only by the presence of a methyl group at the C-3ʹ atom in the latter. Transcription regulating activity was determined by quantitative real-time PCR measurement of transcript levels of SL-dependent, feed-back regulated genes in treated wild type and  However, it is largely unknown whether structural changes in SLs have an impact on their activity in determining the expression of their own biosynthetic genes and in regulating gene expression in general. In this study, we examined the effect of methylation at the C-3ʹ in the D-ring of SL analogs on their activity in determining the transcript levels of genes involved in SL biosynthesis and perception, using roots of rice seedlings treated with AR821 MP1322 two analogs that differ in C-3ʹ methylation, and, as a control, GR24, the common SL analog (hl-2 (high-tillering dwarf1-like), a ccd7 mutant.23Previous studies have shown that an iron-containing carotenoid isomerase DWARF27 (D27) and two CAROTENOID CLEAVAGE DIOXYGENASES (CCD7 and CCD8) are sequentially required to produce the SL precursor carlactone (CL) from all-plastids . SeveralL analog . Assuminhl-2 (high-tillering dwarf1-like (hl)) seedlings were grown hydroponically under normal Hoagland’s nutrient solution for 2 weeks with 12-h photoperiod (200 μM m−2 s−1). Then, rice seedlings were subjected to phosphorus deficiency for another 1 week and treated with 2.5 µM of each SL analog for 6 hours. Total RNA was extracted from rice roots using TRI-Reagent with Direct-zol RNA MiniPrep Kit (Zymo Research). Reverse transcription reaction was performed with the Bio-Rad iScript cDNA Synthesis Kit using 1 µg of total extracted RNA following the manufacturer’s instructions. Primers used for quantitative real-time PCR (qRT-PCR) analysis are listed in (Supplementary Table S1). qRT-PCR was performed in a StepOne™ Real-Time PCR Systems (Applied Biosystems) using SYBR Green Supermix to monitor double-stranded DNA (dsDNA) synthesis following the manufacturer’s instructions. Relative expression levels of genes were determined using a comparative Ct method as previously described24 and rice Ubiquitin (ubi) gene (Supplementary Table S1) was used as the internal control to normalize target gene expression.For this purpose, rice Nipponbare (wild type) and D27, D17 and D10 were significantly suppressed, and expression of an SL signaling repressor gene D53 was strongly induced by MP13 and GR24 treatment in both wild type and hl-2 (ccd7) mutant, as compared with mock and methylated AR8  reduces the capability to regulate the expression of SL-dependent genes, which is in line with our recent study that shows that this modification leads to a significant decrease in different biological activities.22The results obtained reveal that transcript levels of SL biosynthesis genes ated AR8 . Indeed,7 mutant . These r26 elucidating the impact of structural diversity on gene expression regulating activity of SLs could be very helpful in developing analogs that can be employed to optimize some important crop traits and resilience to biotic and abiotic stresses. In addition, such studies are expected to help in understanding why plants produce and release structurally diverse SLs.Considering the important role of SLs determining plant architecture and interaction with beneficial and detrimental organisms,


Prestressed concrete structures are used in various fields as they can reduce the cross-sectional area of members compared with reinforced concrete structures. In addition, the use of high-performance and strength concrete can help reduce weight and achieve excellent durability. Recently, structures have increasingly been constructed using high-performance and strength concrete, and therefore, structural verification is required. Thus, this study experimentally evaluated the structural performance of a long-span bridge deck slab joint, regarded as the weak point of structures. The specimens were designed with a 4 m span for application to cable-stayed bridges. To ensure the required load resistance and serviceability, the specimens comprised of 120 MPa high-performance fiber-reinforced concrete and were prestressed. The deck slabs satisfied all static and fatigue performance as well as serviceability requirements, although they were thinner than typical concrete bridge deck slabs. The study also verified whether the deck slabs were suitable to help implement precast segmental construction methods. Finally, the results confirmed that the structural performance of the developed prestressed concrete (PSC) deck slab was sufficient for the intended bridge application as it achieved a sufficiently large safety factor in the static and fatigue performance tests, relative to the design requirement. High-performance concrete not only improves the durability of structures by ensuring high quality, but also enables weight reduction owing to its excellent structural performance. Recently, high-performance materials such as ultra-high-performance concrete, whose strength has been significantly increased compared with normal concrete, have been attracting attention as they help realize innovative performance improvements and light-weight structures ,2. Bridge deck slabs are frequently damaged members because they directly support live loads through contact with the wheel load of vehicles. Therefore, continuous efforts have been invested to improve the quality and structural performance of these slabs by utilizing high-performance materials. In particular, many research and construction examples that utilize a precast method for accelerated bridge construction can be found in the literature . The preThese previous studies were focused on RC–type–joints because of the high adhesion performance of UHPC. However, research on prestressed concrete (PSC)–type–joints suitable for a long-span structure is relatively insufficient.In this study, the behavior of joints in thin long-span bridge deck slabs was evaluated experimentally. The specimens were fabricated with a thickness of 150 mm and a span of 4 m. The high-performance concrete of 120 MPa was applied to resist the flexural moment caused by this relatively thin long span. A method to apply prestress using post-tension was adopted for this type of joint. This was because it could address problems such as deflection and cracks that could be disadvantageous owing to the relatively thin thickness of the slabs.To verify the structural performance of the 120 MPa prestressed concrete (PSC) deck slab with reinforced steel fibers, various specimens were designed and fabricated, as summarized in The cross-section of the deck slab specimens was designed in accordance with the Korean Highway Bridge Design Code (Limit State Design) (2015)  and the The tendon material was SWPC 7BL, which is a low-relaxation steel with seven strands and a 15.2 mm nominal diameter. The yield and tensile strengths of the strands were 1600 and 1881 MPa, respectively. Full prestressing was applied using the post-tension method to prevent tensile stress being applied to the overall section, while the tendons were placed in a straight line in groups of five at 1.0 m intervals. Moreover, the eccentricity of the tendons was 20 mm, and flat-type anchors, typically used for prestressed deck slabs, were used.2 content larger than 90%, were applied to SC120f. Coarse aggregates were not used. The steel fiber used exhibited tensile strength higher than 2000 MPa and a diameter of 0.2 mm. Further, polycarboxylate superplasticizer  was used. A combination of calcium sulfoaluminate (CSA) type expansion admixture and glycol-based shrinkage reducing agent was used as the shrinkage reducing agent.High-strength concrete with a compressive strength of about 120 MPa, sustained tensile strength through internal fiber reinforcement, and exceptional durability as compared with conventional concretes was applied to the test specimen. The properties of the materials used in SC120f are listed in All the specimens, except for SC120f-PSC-NJ with no joint, were fabricated by match casting. After installing a formwork, the rebar and tendons were placed, and then the concrete was first poured into one side of the joint. The poured concrete was allowed to cure for approximately 2–3 days, and then the concrete was poured into the other side using match casting. Subsequently, high-temperature steam curing (at about 90 °C) was conducted. After curing, epoxy was applied to the interface of the joint where the segments abutted, and prestressing was applied using a hydraulic jack. At this time, the jacking force was determined to maintain the effective prestress considering the immediate losses such as elastic shortening of the concrete, anchorage losses, and frictional losses. After jacking, grouting was conducted to complete the specimens .In the static loading tests, the deflection, strain, and joint widening of each deck slab specimen were measured, and the behavior of the specimens was examined using a 3500-kN static actuator , with the load being applied until failure. The load was applied using a displacement-controlled method at a rate of 3 mm/min. Furthermore, the load was applied using a third-point loading method by ASTM C78/C78M  such thaThe results of the static load test revealed that the SC120f-PSC-NJ specimen without the joint exhibited typical flexural behavior similar to general beam members. As the load increased, many tensile cracks appeared and grew at the bottom of the deck, while the concrete was crushed at the top of the deck slab between the load points during the final failure. At the moment of failure, the maximum load was 435.65 kN, and the deflection of the center of the deck slab under the maximum load was 88.06 mm.For the SC120f-PSC-J1 and J2 specimens with joints, joint widening occurred from the start of loading, and then cracking occurred in the concrete adjacent to the joint. The joint widened as the load increase, and failure occurred as the concrete at the top of the joint was crushed. The SC120f-PSC-J1 and J2 specimens with joints had relatively few cracks compared with the SC120f-PSC-NJ specimen without a joint, given that the cracks form around the initial cracks initiated by the separation of the joints. The measured maximum loads were 322.07 and 289.04 kN for the SC120f-PSC-J1 and J2 specimens, respectively, while the deflections of the center of the deck slab under the maximum load were 58.6 and 60.18 mm, respectively .To evaluate the level of safety in terms of strength, the experimental results were compared with the ultimate limit state load required by the design code. The ultimate limit load was calculated by considering the third-point load test conditions from the ultimate moments induced by the ultimate limit state load combination specified in the design code. As a result of verifying the structural performance of the PSC deck slabs with joints, the maximum loads of the SC120f-PSC-J1 and J2 specimens were 74% and 66% of that of SC120f-PSC-NJ, respectively. Although the presence of the joints caused the maximum load to decrease, the values were 3.67 and 3.29 times higher than the ultimate limit load specified in the design code, as shown in The Korean Highway Bridge Design Code (Limit State Design) specifies that deflection of a bridge concrete deck slab caused by live loads and their dynamic effects must not exceed the following limits, where L is the distance between the centers of the deck slab supports: deck slab with no passage of people, L/800; deck slab with passage of a limited number of people, L/1000; deck slab with passage of many people, L/1200. Furthermore, the design code specifies a maximum crack width of 0.2 mm for PSC members and 0.3 mm for reinforced concrete members. For the SC120f-PSC-NJ specimen, both the deflection and crack width at the center of the deck slab, generated at the serviceability limit state, satisfied the values specified in the design code . For the2, 103, 104, 105, 106, and 2 × 106 cycles. The fatigue loads were applied at a rate of 2–3 times/s using a fixed-point fatigue–loading method. The fatigue load (110 kN), obtained by multiplying the standard truck (KL-510) rear wheel load (96 kN) of the Korean Highway Bridge Design Code (Limit State Design) (2015) by the impact load factor of 15%, was applied to verify the performance in a more severe situation than the fatigue level required by design (80% of the standard truck rear wheel load was considered for fatigue examination). Moreover, the performance of the joint was verified in the harshest environment by applying the fatigue load to the joint interface of the specimen over a wheel–ground contact area of 231 mm × 577 mm. After 2 × 106 fatigue–loading cycles, the residual strength test was conducted in the same manner as the static loading test to enable a comparison under the same conditions. Therefore, the boundary conditions were adjusted again in the same way as in the static loading test.In the fatigue loading test (SC120f-PSC-JF), a total of two million fatigue load cycles were applied to the specimen using a 1000 kN dynamic actuator . When simple supports are applied as the boundary conditions for the fatigue loading test, a load imbalance can arise due to the impact of the load or the movement of the specimen during fatigue loading. Therefore, fatigue loads were applied after the specimen was fixed to the structural test frame with steel rods through the holes in the fixed part of the specimen . In addi6 fatigue loading cycles, although no cracking was observed in the concrete deck slab body. In the residual strength test, a failure mode similar to those used for static loading tests (SC120f-PSC-J1 and J2) was observed, as shown in As a result of applying fatigue loads to the SC120f-PSC-JF specimen, the joint slightly widened up to 2 × 10The load–deflection curve of the fatigue loading test specimen (SC120f-PSC-JF) obtained from the residual strength test is compared with those of the static loading test specimens (SC120f-PSC-J1 and J2) in 6 fatigue cycles have occurred. Similarly, in the residual strength measurement test conducted after the occurrence of 2 × 106 fatigue cycles, the deflection and cracking were within the limits specified in the design code (The deflection of the SC120f-PSC-JF fatigue loading test specimen shown in ign code .(1)In the static loading test, the flexural strengths of the specimens with joints were 74% and 66% of that of the specimen without a joint. However, they were about 3.7 and 3.3 times higher than the levels required by the design code.(2)In addition, the deflections and crack widths of the specimens with joints satisfied the limit of the design code at the serviceability limit state.(3)After two million fatigue load cycles were applied, the residual strength of the specimen with a joint was similar  to the flexural strengths of the specimens with joints in the static loading test.(4)In the fatigue loading test, the deflection at the serviceability limit according to the number of loads, was about 28.2–37.2% of the allowable deflection specified in the design code, and the joint widening of the specimen was 19.2–24.8% of the limit.This study verifies the structural performance of joints in thin long-span bridge deck slabs that used 120 MPa UHPC reinforced with steel fibers. The effect of the PSC–type–joint by post-tensioning methods on static and fatigue performance were verified experimentally. The following conclusions were derived:


The purpose of the work was to propose analytical model considering double confinements (provided by both transverse reinforcements and a wide flange steel section), which was verified by the nonlinear finite element analysis considering concrete-damaged plasticity. The scope of the effort and the procedures to achieve the aim of this study included the identification of the concrete confinements provided by both transverse reinforcements and a wide flange steel section based on the elasto-plastic model in tension for both rebar/steel sections and elasto-buckling for rebars in compression. The influence of rebar buckling in the compression zone on flexural moment strength was also investigated with and without considering confining effects offered by steel sections. The analytical approach predicted a post-yield behavior of composite beams based on the confining effect offered by both the shear reinforcement and wide steel flange sections. However, for beams without axial loads, the compressive zones with high and partial confinements for concrete sections at the yield and maximum load limit state were limited when compressive buckling failure was not considered, preventing the confining factors from significantly influencing the flexural load resisting capacity. An accurate flexural capacity of composite beams can be obtained when rebar was modeled with buckling in the compression zone. The use of concrete-encased steel beams requires an understanding of the individual merits of the two materials. Many tests have been performed to explore the post-yield behavior of the steel columns encased in structural concrete ,3,4,5,6.One of the studies of concrete-encased steel beams was carried out by , who preThe main goal of the present study was to analytically predict the post-yield moment–curvature relationships based on the double confining effects, which were subsequently compared with experimental results and nonlinear finite element analysis. This study focused on the strain compatibility-based simplified method. The material properties and stress–strain relationships included elasto-hardening and elasto-buckling of rebar steels to predict the post-yield behavior of the concrete-encased steel beams. h and Kp, which were then validated by the test data and numerical results using non-linear finite element analysis considering concrete plasticity. In this model, rebars were assumed to buckle and lose their strength due to local buckling in the compression zone when the concrete cover reaches the peak strength. Buckling of rebar in the compression zone contributes to the formation of a hinge length and has an influence on the post-yield behavior. Simplified analytical equations based on strain compatibility were then proposed to estimate the equilibrium neutral axis depth and flexural moment strength. Mathematical expressions derived for the rapid evaluation of the flexural strength of the composite beams were implemented in Matlab for automated estimation of the post-yield behavior of the composite beams. The flexural load-carrying capacity and post-yield behavior were finally examined at the yield limit (the limit of elastic behavior and the beginning of plasticity), the maximum load limit state (the limit state at which the maximum loads are supported), and ultimate load limit state (the limit state at which the strength of the structures is terminated). The confining factors did not significantly influence the flexural load resisting capacity calculated based on strain compatibility. This was because the compressive zones at the yield and maximum load limit state were small, as shown in highly confined zones with concrete stress–strain profiles. However, a decrease in moment strengths was observed when rebars buckled during compression. Rebar buckling in the compression zone is not frequently considered in the flexural analysis of beams. Confinement factors for steel and concrete materials were identified by exploring the influence of wide flange of steel sections on the concrete confinement. The concrete zone confined by the steel flange was simplified to model confinement factors, KThis study considered the confinement effect on concrete caused by lateral reinforcement and structural steel. It was assumed that parabolic arching of the area of the effectively confined concrete core occurred between the reinforcing bars in the cross section ,15. Simicf = Kpcof and ccf = Khcof in Equations (1) and (2), respectively, where Kp and Kh were defined as confinement factors for partially and highly confined concrete, respectively. Concrete stress–strain relationships representing the four zones, shown in In this study, arching similar to that suggested by ,5 was adc,maxε), corresponding to the maximum confined concrete compressive strength (ccf), while maintaining a constant value after the longitudinal rebars under compression reached the concrete strain (cuε). It was assumed that the spalling of the concrete cover caused the rebars to buckle and lose their strength. Researchers  suggesteThe steel beam encased in structural concrete considered with nomenclature describing the section is shown in c1 is the neutral axis of the section at the maximum limit state. The mean stress factors α and centroid factors γ are derived in Equations (A1)–(A7) and (A8)–(A14) in c2, c3, and c4, representing the zones confined by the steel section shown in c1) are also established in Equations (3)–(11). The Mander confining curve was used in these equations. For the unconfined concrete based on the Mander curve , confined concrete (cyan region), partially confined concrete (yellow region), and highly confined concrete (orange region), are shown in Equations (3)–(11). Here, For confined concrete based on the Mander curves  and (13), where the neutral axis, Equation (15) can be rewritten as below,Equation (19) can be rewritten as below,Equation (13) can be expressed as follows:The nominal moment strength at the yield limit state was then obtained using Equation (22)Equation (22) can be rewritten as follows:h and Kp.The steel section (H-250 × 250 × 9 × 14) with a yield strength of 350 MPa and reinforcing steels (4-HD25) with yield strengths of 550 MPa were used in the analysis. Both have a Young’s modulus of 200,000 MPa. The concrete encasing steel section and rebars had compressive and tensile strengths of 21 MPa and 2.1 MPa, respectively, both with a Young’s modulus of 21,538 MPa. The yield strengths of the H-steel, reinforcing bars, and the concrete compressive strength were obtained from test samples. In In the FEA model, concrete damaged plasticity model for the behavior of concrete and the elasto-plasticity model for rebar and steel sections were considered. The stress–strain relationships for these materials are shown in Researchers  suggestebo/fco, Kf, and viscosity, which were defined to be 1.16, 0.6667, and 0.001, respectively.Here, The elements of type C3D8R were chosen to represent the structural behavior of the steel–concrete composite beams, as shown in The definition of interactions between the reinforcing bars, steel sections, and the concrete is an important issue for modeling steel–concrete composite beams. Previous studies adopted the embedded method to model the bond behavior between the concrete and reinforcements ,21,22,23A rigid body object (JIG) was used with the dimensions 300 mm × 500 mm to transfer the concentrated load to the specimen, preventing the local failure of a single node in the FEA model, and relieving stress concentration. The FEA results were compared with test data to verify the reliability of the proposed tie modeling technique. It was found that the FE models constructed using the proposed modeling technique accurately predicted the post-yield behavior of the tested steel–concrete composite beam.The stress status maximum and ultimate limit state were demonstrated in the In The authors obtained test data of h and Kp) was also shown with moment–strain relationships represented by Legends 2, 3, and 4 in h with 2.0 and Kp with 1.5 were compared with FEA data (refer to Legend 1a in h (2.0) and Kp (1.5) (refer to Legend 4 in h and Kp was implemented as 1.The post-yield behavior of the precast composite beams was obtained based on the iterated strain compatibility approach using Equations (3)–(27). The analytically calculated flexural strengths  Significant implications of the information presented in the body of the studyUnderstanding the post-yield structural behavior of composite beams composed of structural steel and concrete is quite a complicated issue. One of the most fundamental requirements in predicting an accurate post-yield behavior of composite beams was to understand the constitutive relationships of concrete with all the confinements provided by the surrounding structural elements such as stirrups and the wide flanges of steel sections. Simplified but accurate analytical equations based on iterated strain compatibility were proposed to estimate the equilibrium neutral axis and flexural moment strength reflecting the buckling of reinforcing rebar in the compression zone. The post-yield behavior of composite beams was accurately estimated, considering confining effects on the concrete by shear reinforcement and the steel flange. A numerical investigation of the composite beams using finite element analysis (FEA) based on damaged concrete plasticity was also presented with nonlinear parameters to verify the analytically predicted post-yield behavior with the two confining effects. In this study, the moment–strain relationships for different confining factors were obtained by an analytical model based on strain compatibility. The influence of confinement in concrete caused by rebar and steel on the post-yield behavior of composite beams was explored. (2) Research impact both within and beyond academiaFor deep beams with an L/d ratio of 3.9, inconsistencies at the yield limit state were demonstrated between the flexural strengths calculated based on strain compatibility and those obtained by the nonlinear finite element investigation, indicating that the flexural strength of deep beams was not accurately estimated when only the curvature of the sections based on strain compatibility was considered. However, the flexural capacities obtained by a nonlinear FEA based on the 9 m shallow beam with a L/d ratio of 20 were relatively well compared with the analytical estimates based on strain compatibility both at yield and maximum load limit states, respectively, when the effects of deep beam and concrete plasticity were minimized. The deep beam effect was one of the reasons for the difference in the prediction of the post-yield deformations and the flexural capacities of composite beams at the maximum load limit state because the analytical estimation based on strain compatibility cannot consider the plastic rotation due to inelastic energy dissipation.(3) Influence of the steel sections on the flexural capacities of the concrete-encased steel beamsStudies that focused on the consideration of double confinements (provided by both transverse reinforcements and a wide flange steel section) are absent from the literature to some extent in conventional composite beam analysis, ignoring the contribution of confined concrete by steel sections. The highly and partially confined zones for compressive concrete sections at the yield and maximum load limit state were limited, being unable to fully activate the confining effects on the flexural load resisting capacity. However, the changes of flexural load resisting capacity were observed due to the confining effects by the steel section when compressive buckling failure was considered. When considering confining effects by the steel section, a decrease in moment strengths was observed when rebars buckled during compression. However, the retarded decrease in moment strengths was observed from the beam when compressive buckling failure was not considered. Rebar buckling in the compression zone is not frequently considered for the flexural analysis of beams. However, it was shown that an accurate flexural capacity of composite beams can be obtained when rebar was modeled with buckling in the compression zone. The analytical algorithm based on strain compatibility developed in this study can be extended to understand the post-yield behavior of columns with axial loads.


This study compares the antibiotic consumption rates over a period of five years in two hospitals in Eritrea, Orotta National Referral and Teaching Hospital (ONRTH) and Hazhaz Zonal Referral Hospital (HZRH). Antibiotic consumption is an important parameter in the study of antibiotic use. However, no published data on consumption rates exist for these two hospitals, thus the aim of the study is to measure and compare a five year antibiotic consumption trends of the two hospitals’ medical wards using Defined Daily Dose per 100 bed-days (DDD/100-BD). Antibiotics dispensed from January 2014 to December 2018 were considered. Non-parametric Pearson’s correlation coefficient was used for comparison of consumption, while non-parametric Friedman’s test measured annual rates. The total antibiotic consumption in the HZRH was almost double that of ONRTH. The analysis showed that antibiotic consumption in ONRTH was significantly decreased from 2014 to 2018 while there was no significant difference in consumption in HZRH. Benzyl penicillin was the most consumed antibiotic in HZRH and ONRTH throughout the study period at 87.8DDD/100-BD and 35.4 DDD/100-BD respectively. Ceftriaxone and ciprofloxacin were among the most commonly consumed antibiotics in both hospitals. Establishment of Antibiotic stewardship program would benefit both hospitals greatly, and further studies need to be done to establish the national antibiotic consumption baseline. Overuse and misuse of antibiotics are among the leading factors in the complex web of causation in antimicrobial resistance (AMR). The wide use of antibiotics, whether appropriate or not, exerts a selective pressure by reducing the reproductive success of some microorganisms thereby accelerating the development of AMR –4. The aThe increasing resistance of bacteria to antibiotics has been pointed out as one of the main public health concerns globally, as treatment for growing list of infections becomes less effective and previously relatively safe medical procedures like surgery and organ transplants would become increasingly risky . AMR incAntibiotic consumption is an important parameter in the study of antibiotic use and there are several metrics of measuring antibiotic consumption. The most commonly used method is the one recommended and updated by the WHO Collaborating Centre for Drug Statistics Methodology World Health Organization (WHO) . It is bExpressing antibiotic consumption in DDD per 100 patient-days (DDD/100-BD) allows hospitals to compare their consumption with other hospitals regardless of differences in quality and quantity of antibiotics , 18. No A five-year retrospective study which focused on the antibiotics dispensed to the internal medicine wards of two hospitals in Asmara was conducted, i.e. in Orotta National Referral and Teaching Hospital (ONRTH) and Hazhaz Zonal Referral Hospital (HZRH). Eritrea has six administrative regions and the central region is the main hub of the country. HZRH is a regional referral hospital for the central region. These two hospitals are found in Asmara, the capital and the most populated city in the country, located in the central region. Eritrea has two national referral hospitals and currently no privately owned hospitals are operational in the country. ONRTH is one of the two national referral hospitals in the country with a functional Medicine and Therapeutics Committee (MTC) while HZRH is a secondary level zonal referral hospital which has established an MTC in 2019. ONRTH is a multi-specialty national referral hospital with medical, surgical, pediatric, emergency, intensive care unit (ICU), gynecology and obstetrics wards while HZRH is a secondary level zonal referral hospital with medical, pediatric and emergency wards.Antibiotics which were classified as J01 category (antibiotics for systemic use) under the ATC classification system and antibiotics available in the latest edition of the Eritrean National List of Medicines (ENLM) 2015 were included in this study . Amount The DDD is a globally accepted unit of measuring drug consumption of different strengths, pack sizes or combinations. It can be used to compare rates between regions, countries, hospitals and wards –24. NumbAntibiotic consumption data were aggregated at the fifth  level of the ATC classification and expressed in DDD and DDD/100-BD. Data were entered and analyzed using Microsoft Excel ® 2010 and after data cleaning, it was exported to IBM SPSS ® version 23 for descriptive and analytical studies. Non-parametric Pearson’s correlation coefficient was used to test for comparison of rates between the two hospitals. While annual change in antibiotic consumption was tested using nonparametric Friedman’s test. All statistical tests were considered significant at p-value of < 0.01.Total antibiotic consumption differed between the two hospital wards. Antibiotic consumption was consistently higher in Hazhaz Medical Ward (HMW) than in Orotta Medical Ward (OMW). During the five years, the consumption in HMW showed considerable variation, while there was relatively small variation in OMW .The mean antibiotic consumption rate in DDD/100-BD versus year graph shows that antibiotic consumption rate was much higher in HMW than OMW throughout the five years. The consumption rates peaked in HMW in 2015 and fell down in 2016 and started to slowly increase thereafter, the rate of increase was higher in 2017 but it never approached the peak seen in 2015. In OMW, on the other hand, the line graph of antibiotic consumption shows less fluctuations and a small peak was observed in 2016 after which the consumption rate started to decrease consistently exhibiting the lowest rate in 2018 .r = 0.151; p = 0.93). Differences in annual antibiotic consumption rates were significant in OMW for the five years under study χ2  = 24.487, p<0.0001; while the annual variations in HMW were not significant χ2  = 2.86, p = 5.82. Follow-up pairwise comparisons were conducted using Wilcoxon test and controlling for Type I errors across these comparisons at the 0.005 level using Bonferroni procedure. The mean antibiotic consumption difference between 2014 and 2018 and between 2016 and 2018 were significant at p-values of 0.001 and 0.002 respectively. The remaining eight different comparisons were not significant. Consumption of antibiotics was not correlated between the two hospitals .Consumption of antibiotics was not correlated between the two hospitals  was higher than the 117.6 DDD/100-BD reported from a similar secondary level hospital study in New Zealand . The meaAntibiotic consumption showed opposite trends, decreasing in OMW and increasing in HMW . It decrThe three most commonly prescribed antibiotics in the world are penicillins, third-generation cephalosporins, and fluoroquinolones . Our stuCiprofloxacin was the most commonly prescribed antibiotics in OMW with a five year mean 7.6 DDD/100-BD , this isAfter benzyl penicillin G, gentamycin and amoxicillin with 17.7 and 16.9 DDD/100-BD respectively were the most commonly prescribed antibiotics. In HMW gentamycin is often used as an empirical treatment owing to its broad spectrum activity, alongside benzyl penicillin G or ampicillin; especially for the treatment of lower respiratory tract infections and infective endocarditis. It is also used as add-on antibiotic in case of pneumonia management, when patients don’t respond to benzyl penicillin G treatment alone. The consumption of amoxicillin in OMW is 4.3 DDD/100-BD, much lower than the 16.9 DDD/100-BD in HMW. This variation in consumption of oral amoxicillin may be due to the difference in the setting, one being tertiary hospital which have complicated and seriously ill patients’ resulting in its low utilization, physicians opting for its parenteral forms for inpatient use. Oral amoxicillin is a preferred discharge medicine, i.e. given to patients at discharge time to continue to take it at home.Third generation cephalosporins are thought to be medicines that are frequently consumed in hospitals , 34 and A number of fluctuations in antibiotic consumption were seen over the five year period and this could be due to the availability of the medicines in the country, and might not reflect a true picture of how they would have been consumed had their availability been consistent. Yet, one objective of medicine consumption studies is to identify inconsistencies in medicine supply, and demonstrate how consumption of medicines are drastically affected by the availability of medicines on the market, which in turn leads to overuse of readily available broad spectrum antibiotics –37.The findings of this study can be used as a starting point in the implementation and strengthening of Antimicrobial Stewardship Programs (ASPs) and encourage other hospitals throughout the country to measure their antibiotic consumption rates. Antibiotic consumption measurement and reporting are an important part of ASP. Many studies , 38, 39 This study used the amount of antibiotics dispensed to medical wards to estimate the consumption rates of antibiotics and this might not always reflect the actual amount “consumed” by patients. However, even with this inherent limitation of the study, we believe the methodology adopted would be more than adequate to give a clue to the antibiotic consumption rate using a standardized metric. Future studies should focus on computing antibiotic consumption from all wards and use days-of-therapy to calculate antibiotic consumptions.This study measured antibiotic consumption trends in medical wards of two hospitals using DDD/100-BD methodology. The total antibiotic consumption in the HMW was almost double of that OMW. The trend analysis showed that antibiotic consumption in OMW was significantly decreased from 2014 to 2018 while there was no significant difference in consumption in HMW. Benzyl penicillin was the most consumed antibiotic in HMW and OMW throughout the study period at 87.8DDD/100-BD and 35.4 DDD/100-BD respectively. Ceftriaxone and ciprofloxacin were among the most commonly consumed antibiotics in both wards, given these two antibiotics are under the Watch Group they must closely be observed by establishing an ASP in their respective hospitals. Further studies which focus on all the wards and across different regions need to be conducted to generate a complete picture of antibiotic consumption patterns across the country, which would be instrumental in setting up a national antibiotic consumption baseline.S1 File(XLSX)Click here for additional data file. 4 May 2020Antibiotic consumption study in two hospitals in Asmara from 2014 to 2018 using WHO’s Defined Daily Dose (DDD) methodologyPONE-D-20-06944Dear Dr. Amaha,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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The specific roles of these authors are articulated in the ‘author contributions’ section.”If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.b)  Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.  http://journals.plos.org/plosone/s/competing-interests).  If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” :Though data is limited, this could form baseline information to develop antibiotic usage policy.Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: While this is very relevant topic, drug utilization data presented in DDDs only give a rough estimate of consumption and not an exact picture of actual use. Although it enables the researchers to assess trends of antibiotic comsumption and to perform comparisons. Despite these limitations, this study can povide baseline information on the recent status of antibiotic overuse or misuse in two hospitals in Asmara. I hope the authors will succeed in developing monitoring system for antibiotic use.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: No


To the Editor—In the absence of effective treatments for coronavirus disease 2019 (COVID-19), many hospitalized COVID-19 patients receive antibiotics.2 Thus far, the literature does not indicate that antibiotics are effective in treating COVID-19, and the incidence of bacterial coinfections appears low.2 One analysis reported that while 8% of COVID-19 patients experienced a bacterial or fungal coinfection, 72% of COVID-19 patients received antibiotics.2Clostridioides difficile infection (CDI) is associated with broad-spectrum antibiotics frequently used for COVID-19; CDI is thus a significant concern for COVID-19 patients.3 Patients at higher risk of severe COVID-19 frequently also have risk factors for CDI such as advanced age and weakened immune systems.3 COVID-19 treatments themselves, which often involve extended hospital stays, can also increase a patient’s risk of developing healthcare-associated CDI.3 CDI has been identified in patients who received antibiotics as part of their COVID-19 treatment.5Given the patient safety risks posed by CDI, effective antibiotic stewardship remains critical throughout the COVID-19 pandemic. However, pandemic-related changes to healthcare delivery  have made antibiotic stewardship interventions even more challenging. We present a systems engineering approach to evaluate and modify antibiotic stewardship programs within the constraints of COVID-19 responses, with the overall goal of reducing CDI.Antibiotic stewardship initiatives can involve persuasive  and restrictive  approaches; these initiatives are used together with appropriate diagnostic and infection prevention measures. These strategies require complex behavioral changes and can involve significant resources such as real-time access to antibiotic stewardship staff for consultation.6 Prescribers and antibiotic stewardship team members are facing higher and more complex patient loads. The wide-ranging symptoms of COVID-19 may mimic other infections, and a worsening of symptoms is frequently seen 1–2 weeks into the disease that can make it difficult to identify potential coinfections. The length of hospitalization for many patients increases risks of developing healthcare-acquired infections such as ventilator-associated pneumonia that may require additional antibiotic treatment.7 These factors, combined with a lack of effective treatment options for severe COVID-19, have resulted in high levels of antibiotic use among COVID-19 inpatients.2The COVID-19 pandemic response has further increased the complexity of antibiotic stewardship.8The structure and effectiveness of an antibiotic stewardship program is dependent on the individual work-system context, including characteristics of the patient population, organizational culture toward antibiotic stewardship, availability of infectious disease and pharmacy personnel, accessibility of clinical decision support tools, and existing policies to support antibiotic stewardship. A systems engineering approach can be used (1) to fully evaluate the roles that work-system elements play in complex antibiotic stewardship interventions and (2) to develop modifications to these elements to support the implementation of interventions. The Systems Engineering Initiative for Patient Safety (SEIPS) provides a framework for this approach. SEIPS defines work-system elements: person(s), technology and tools, environment, tasks, and organization. The interaction of these elements influences care processes and outcomes.9 Teams can then develop strategies to support antibiotic stewardship within the individual work system to optimize patient and organizational outcomes.Given the urgent needs to ensure appropriate antibiotic use and reduce CDI risk in COVID-19 patients, a systems engineering approach such as SEIPS can be used to understand the various work-system factors that are involved in antibiotic stewardship and CDI prevention. The interaction of these elements drives antibiotic stewardship, COVID-19 treatment, and CDI prevention processes in each work system; thus, they influence critical patient and organizational outcomes. This SEIPS-based approach provides flexibility for teams to evaluate their own work-system–specific barriers and facilitators to antibiotic stewardship practices within their COVID-19 response.Table Research is needed to understand whether, when, and how antibiotics should be used to treat COVID-19 patients while minimizing adverse effects such as CDI. CDI in COVID-19 patients should be investigated to identify risk factors such as use of specific antibiotics, previous CDI, and/or presentation of COVID-19 gastrointestinal symptoms. Research findings should be incorporated into comprehensive evidence-based antibiotic stewardship programs. In the meantime, the urgency of preventing CDI in COVID-19 patients requires adjusting antibiotic stewardship interventions to fit within current COVID-19 protocols. The SEIPS-based approach presented here can help local antibiotic stewardship teams and decision makers adjust existing plans and develop new approaches to support antibiotic stewardship and reduce CDI during the COVID-19 pandemic.


The percentages of immunopositive cells for PGR and for KLF9 in GE and for PTEN in ST were higher while those for ERα in GE but not ST were lower, in tumors of MET vs. CON patients. The numbers of Ki67- and TUNEL-positive cells in tumor GE and ST did not differ between groups. In human Ishikawa endometrial cancer cells, MET treatment (60 μM) decreased cell numbers and elicited distinct temporal changes in ESR1, KLF9, PGR, PGR-B, KLF4, DKK1, and other tumor biomarker mRNA levels. In the context of reduced KLF9 expression (by siRNA targeting), MET rapidly amplified PGR, PGR-B, and KLF4 transcript levels. Our findings suggest that MET acts directly in EC cells to modify steroid receptor expression and signaling network and may constitute a preventative strategy against EC in high-risk non-diabetic women.Metformin (MET) is increasingly implicated in reducing the incidence of multiple cancer types in patients with diabetes. However, similar effects of MET in non-diabetic women with endometrial cancer (EC) remain unknown. In a pilot study, obese non-diabetic women diagnosed with type 1, grade 1/2 EC, and consenting to participate were randomly assigned to receive MET or no MET (control (CON)) during the pre-surgical window between diagnosis and hysterectomy. Endometrial tumors obtained at surgery  contains supplementary material, which is available to authorized users. Obesity is also associated with insulin resistance and an increased risk (1.5–2-fold) in developing type 2 diabetes (heretofore referred as diabetes) [Endometrial cancer (EC) is the most common gynecologic malignancy in the USA, with approximately 60,000 new diagnosed cases per year . Obesityiabetes) , 5. GiveMetformin  is an oral hypoglycemic agent used in the management of diabetes. MET lowers blood glucose levels by inhibiting gluconeogenesis and improves insulin sensitivity by increasing peripheral glucose uptake and utilization , 8. The The present study examined the short-term effects of MET on tissue proliferation, apoptosis, and expression of estrogen receptor-α (ERα), progesterone receptor (PGR), specificity protein (Sp)–related transcription factor Krüppel-like factor 9 (KLF9) and tumor suppressor phosphatase and tensin homolog (PTEN) in endometrial tumors of non-diabetic obese women with EC. The tumor biomarkers identified to be MET-responsive in vivo were further evaluated for MET effects in vitro using the human endometrial cancer cell line Ishikawa. The collective results identify novel networks related to direct MET effects on endometrial carcinoma cells.Human subject protocols were approved by the University of Arkansas for Medical Sciences Institutional Review Board. Patient recruitment was listed under Clinical Trial No. NCTO1877564. Eligibility criteria were non-diabetic obese women (BMI ≥ 30) with histologically confirmed EC and no diagnosis of other cancers, who show normal liver and renal function, and who consented to the study. Exclusion criteria were smoking, previous metformin use, or regular alcohol consumption. A total of 13 patients were enrolled within a period of 1 year (2013–2014) and were randomized to receive either oral MET or no MET for 28–32 days during the pre-surgical window between diagnosis and hysterectomy  and their working dilutions used at incubation conditions of 4 °C for 16–24 h. Immunoreactivity was detected using the VECTASTAIN Elite ABC Kit (Vector Laboratories) and biotinylated anti-rabbit secondary antibodies (Vector Laboratories), and slides were counterstained with hematoxylin. The stained sections were digitized using the Leica Digital Pathology Whole Slide Scanner (Aperio ImageScope). The number of nuclear-stained and non-stained glandular epithelial (GE) and stromal (ST) cells in 3–4 random visual fields (~ 100 cells per field) for each tissue section was separately counted manually. Cells were scored as non-staining  based on sections that were processed in parallel with the omission of the primary antibody. Data are expressed as a percentage of the number of nuclear-stained cells, relative to the total number of cells (stained + non-stained) counted.Tumor samples were formalin-fixed and paraffin-embedded; sections (5 μm) were processed by heat-induced epitope retrieval (citrate buffer) and subsequent incubation with designated antibodies as previously described . Table 12/95% air) at 37 °C. Metformin  was dissolved in phosphate-buffered saline  and used at a final concentration of 60 μM for treatments. This dose approximates the plasma concentration of MET in diabetic patients treated with the drug [3 cells in MEM-FBS -phenol red. Cells were synchronized by serum starvation in MEM-phenol red containing 2% FBS (v/v) for 24 h and then transferred to MEM-FBS (10%)-phenol red for treatment with MET or vehicle control (PBS). Cellular apoptosis was measured using cells initially plated in 12-cm dishes, and 24 h later, cells were treated with MET. For gene expression analysis, cells were seeded at a density of 1 × 105 per well in 12-well plates and cultured to 50–60% confluence in MEM-FBS-phenol red prior to MET treatments. For the 48-h incubation period, cells were replenished with fresh media containing MET or no MET at 24 h.The human endometrial epithelial carcinoma cell line Ishikawa  was previously authenticated and determined to express functional estrogen and progesterone receptors . Cells wthe drug . Cell viTBP) mRNA was used as the normalizing control.Treated cells were collected at select time points for analyses. Cellular viability was evaluated at 48 h post-initial treatment (MET or PBS) by trypan blue exclusion using the Vi-Cell cell viability analyzer (Beckman Coulter Inc.). MET- or PBS-treated cells were stained with annexin V and propidium iodide (Trevigen) at 48 h post-initial treatment and quantified for percent of apoptotic cells in the early (annexin V–positive/propidium iodide–negative) and late (positive for both annexin V and propidium iodide) stages using the Becton Dickinson LSRFortessa Flow Cytometer, as described previously . RNA isoKLF9 (50 nM concentration) (Dharmacon) using Lipofectamine 2000 (Invitrogen), as previously described [KLF9 siRNAs were processed for Western blot analyses using rabbit anti-rat KLF9 antibody  small interfering RNAs (siRNAs) and siRNAs directed against escribed . Cells wdy Table , followiP ≤ 0.05) by one-way ANOVA followed by Tukey’s post hoc test (for three or greater groups) or by Student’s t test (for two groups).Statistical analyses were performed using SigmaStat . Data (mean ± SEM) were analyzed for statistical significance , were lost to follow-up, and/or did not complete the MET treatment regimen. Four patients from each group who successfully completed the study provided the tumor samples used in the analyses. The two groups were comparable in age and BMI and showed normal fasting blood glucose levels, the latter confirming their non-diabetes status Table . Other mImmunohistochemistry was used on patient tumor samples to assess the relative expression of proteins that are known to be positively ) and negatively (progesterone receptor (PGR); Krüppel-like factor 9 (KLF9); phosphatase and tensin homolog (PTEN)) associated with EC. A representative H&E-stained section of tumor tissue Fig.  and reprCCND1) mRNA levels , ERα mRNA levels were reduced early (at 2 h) by MET treatment, but this suppression was not sustained at 24 h post-treatment , PGR-B isoform (1.25-fold), and PTEN (1.5-fold) transcript levels by 24 h but not earlier at 2 h  and p53 (at 24 h) transcript levels were elevated, while those of tumor promoters c-FOS (at 24 h) and telomerase  were reduced (relative to t = 0), with MET treatment , a well-known progesterone-regulated gene [The expression of other genes that are implicated in EC, owing to their tumor-promoting or tumor-repressive functions, was evaluated in MET-treated Ishikawa cells at the same time points (2 h and 24 h post-MET treatment). The analyses for the same genes were not performed in EC tumors due to limited tissue amounts. Tumor suppressors Krüppel-like factor 4  and PGR-B mRNA levels were numerically higher with MET, but the increase did not reach the level of significance (P = 0.06).The transcript levels of a select number of genes were evaluated for response to MET after an extended treatment period  Fig. . RelativKLF4 is a PGR-regulated gene [KLF9 siRNAs or scr siRNAs were analyzed for KLF9 protein (by Western blot of nuclear lysates) and KLF9 transcript levels (QPCR). The reduction in nuclear KLF9 protein  in siKLF9-transfected cells was accompanied by rapid and concurrent increases (within 2 h) of total PGR, PGR-B isoform, and KLF4 transcript levels. The specificity of these effects was displayed by the absence of comparable changes elicited on p53 mRNA levels  study involved a short-term MET treatment  of non-diabetic obese women with EC, while the in vitro experiments analyzed the direct effects of MET on proliferation, apoptosis, and gene expression in the human endometrial carcinoma cell line Ishikawa. While the in vivo results were limited by the small patient sample size due in part to the prevalence of type 2 diabetes in obese women with EC , the nonPTEN mRNA levels in human colon cancer cells [KLF4, cFOS, p53, and TERT were altered by MET in the direction consistent with their respective anti- (increased for KLF4 and p53) and pro- (decreased for cFOS and TERT) tumorigenic activities. ERα is known to directly bind to p53 tumor suppressor to repress its function [TERT expression is transcriptionally enhanced by the binding of ERα to TERT promoter [Our primary endpoints for MET outcomes in vivo were proliferation (Ki67 immunostaining) and apoptotic (TUNEL assay) status and changes in nuclear levels of tumor biomarker proteins ERα, PGR, KLF9, and PTEN. Reported outcomes of MET on proliferation and apoptosis markers in tumors of patients with EC have been inconsistent, with some showing no er cells . Moreovefunction , and TERpromoter . Our resERα transcript levels comparable with that noted for ERα protein with MET intake in vivo preclude the exclusive participation of systemic factors and implicate direct mechanisms in MET-elicited responses. Consistent with our results, cell lines established from endometrial tumor tissues obtained from women undergoing surgery for EC showed reduced ERα transcript levels with MET [ERα may constitute a key mediator of MET in targeting EC, but the underlying mechanism for this remains unknown. Similar to our findings reported here in non-diabetic obese women with EC, MET caused a reduction of ERα expression in endometrial tumors of diabetic women relative to non-treated women . In diabwith MET .KLF4, DKK1) may suggest a simple linear pathway by which MET’s elevation of PGR expression can orchestrate anti-tumorigenic outcomes, this may not necessarily be the case, given the temporal course in gene expression changes. For example, the relative increases in KLF9, KLF4, and DKK1 transcript levels (by 2 h) preceded those of PGR and PGR-B, which were observed at 24 h. Further, while the early increase in KLF9 transcript levels was maintained through 24 h and 48 h post-MET, DKK1 and KLF4 transcript levels were reduced to basal levels by 24 h and needed additional MET treatment to rise to levels above controls , but this reduction was not observed by 24 h and similarly required additional MET treatment to be maintained below those of control cells. Taken together, our results suggest that MET may elicit early  and late  responses on PGR/ERα gene networks and that maintenance of some of these MET effects on gene expression  may require additional/continuous exposure to MET, possibly involving the activation of other signaling pathways. In this regard, the recent report that the development of EC tumors in a xenograft mouse model was significantly suppressed by the combined administration of MET and medroxyprogesterone acetate, when compared to individual treatments [Ligand-activated PGR has been implicated as a tumor suppressor in EC . We showeatments , is consKLF9 transcript levels with MET was temporally and closely aligned with, respectively, the reduction of ERα and TERT and the induction of KLF4 and DKK1 transcript levels and preceded the expression changes noted for PGR, PGR-B, PTEN, cFOS, and p53 transcripts. Given that MET induction of KLF9 transcript levels occurred early and was maintained at all time points examined , we suggest that KLF9 is a major component of MET signaling. By siRNA targeting of KLF9 in Ishikawa cells to mimic the low levels found in EC tumors [KLF9 in tumor cells by rapidly increasing transcript levels for PGR, PGR-B, and KLF4, all of which exhibit anti-tumor properties. The lack of a similar effect of KLF9 siRNA targeting on p53 transcript levels in the background of MET indicates the specificity of MET effects on KLF9-associated signaling.We previously reported the loss of KLF9 expression in EC tumors . AlthougC tumors , we founERα expression and estrogen-mediated proliferation of Ishikawa cells through AMPK activation and subsequent inhibition of mTOR signaling [The present study did not address how MET may directly act on EC cells to influence the expression of genes with pro- and anti-tumor effects. A well-established mechanism of MET action involves the inhibition of mammalian target of rapamycin (mTOR) signaling via increased phosphorylation and hence activation of AMPK. A recent study using high MET doses (5 mM) reported the inhibition by MET of ignaling . Reductiignaling , 56, andignaling , 58. Basignaling , 48, 54,Limitations of this study include the small patient sample size, the short duration of MET treatment, and the lack of pre-treatment patient samples  that would allow for a more accurate evaluation of MET effects. Moreover, our in vivo findings did not mimic the in vitro effects of MET on cell proliferation and apoptosis, as might be expected from their distinct contexts and treatment duration. Nevertheless, our findings provide support to the emerging body of evidence on the potential preventative use of MET for non-diabetic women who are at high risk for EC and offer mechanistic insights on steroid receptor signaling as a key pathway mediated by MET. The promising findings presented herein warrant further studies with a larger non-diabetic patient population to expand current understanding of novel networks to exploit for prevention of EC.ESM 1(PDF 43 kb)ESM 2(DOCX 17 kb)