A significant part of reinforced concrete structures is subjected to intensive environmental impact during operation. This can cause local destruction and failure of buildings if obligatory measures are not taken to protect them from corrosion. This is especially true for industrial buildings, where the environment could be contaminated with aggressive products or waste. An important issue is the development of methods for calculating the load-bearing capacity and serviceability of reinforced concrete structures with corrosion damage. The main reason for this is the necessity to determine the durability and reliability of buildings and structures and the estimation of their safe operation time. As corrosion damages of concrete are a critical issue, more detailed experimental studies are needed. This paper presents experimental studies of concrete prisms under the simultaneous action of an aggressive environment and a constant level of compressive force. In total, 32 prisms under different loading conditions and in different aggressive medium were tested. Samples were divided in series, for which different load levels were chosen ( Intensive environmental impact is an important problem for concrete and reinforced concrete structures which are currently operated. This effect could be the reason for local destruction and failure of buildings ,2,3,4,5.Physical and chemical processes during corrosion are considered in a number of recent studies ,16,17,18One of the parameters of the concrete corrosion resistance is its water resistance, which can be increased, both by design measures and with special additives in the concrete . It is eAs the alternative to cementitious binder concretes, new types of acid-resistant concretes have been developed and studied. As the result of the research in , the posMany studies concern the effect of corrosion on modified concrete ,23,24,25Additives, which modify the concrete structure and properties, are of considerable interest as the means to increase its corrosion resistance. The authors of ,27,28,29An important issue is the development of methods for calculating the load-bearing capacity and serviceability of reinforced concrete structures with corrosion damages. The main reason is the necessity of determination of the durability and reliability of buildings and structures, as well as the assessment of their safe operation time. Most researchers ,44,45,46As Webster  states, Thus, in recent times, studies dedicated to qualitive and quantitative assessment of corrosion processes in concrete with the use of accelerated corrosion tests are widespread ,37. GeneElectrical measurement methods of chemical processes in concrete were also conducted by Kusak et al. ,39,40, CNamely, in ,42, fundIt could be summarized that quantitative evaluation of concrete corrosion in recent research studies is circumscribed by analysis of experimental data, based on mass or cross-section loss during the corrosion process. However, there are the number of issues of the reinforced concrete structures with corrosion damage of concrete, which are not sufficiently studied yet. Therefore, further investigation of this urgent issue and more detailed experimental studies of the effect of corrosion on concrete damage are required.3) and air-absorbing additive “Chrysoair”  were used. The dosage of the super-plasticizer was 1.2 kg for 100 kg of cement and 0.035 kg of the air-absorbing additive for 100 kg of cement.Experimental research included testing of prism samples, made of concrete with the following characteristics: Prisms were made of concrete with components ratio C:S:Gr = 1:1.01:2.28 and water-cement ratio W/C = 0.32. Additionally, the super-plasticizer “Chrysofluid” . Detailed characteristics of the concretes used during the experiment are provided in c = 2.04 and coefficient of non-uniformity Cv = 2.0. The granulometric curve for fine aggregates is presented in Fine and coarse aggregates were tested with the use of the sieve analysis procedure. As the fine aggregate, quartz sand from Slavutych quarry of the Khmelnytsky region  was used, without impurities and with a size modulus of Mmax and minimum dmin diameter of gravel grains were 20 and 3 mm, respectively. Fractional composition of coarse aggregates mostly contained 5..10 and 10..20 mm grains. The granulometric curve for coarse aggregates is presented in As the coarse aggregate, granite crushed stone from the Selishche quarry of the Rivne region  was used. Maximum DThe experimental study of concrete was performed by testing prism samples of 100 mm × 100 mm × 400 mm size according to regulations of DSTU B B.2.7–214: 2009, EN 206:2013.Samples were subjected to the action of an acid-liquid environment and axial compression in order to study the change of physical and mechanical properties of concrete under loading over time, taking into account corrosion damages of concrete.In order to study the effect of stress on the development of corrosion processes in concrete over time, 4 series of prisms were loaded with different values of initial stresses, equal to (a)Only the central compressive force without aggressive medium,(b)Simultaneous influence of compressive force and aquatic medium,(c)Simultaneous influence of compressive force and aggressive environment.Series 1–4 of prisms belonged to the main part of the research see . For eacThe following symbols were used for the marking of prisms: P—prism, and L—tested for long-term loading see . The marSeries 5 contained 4 prisms, tested in the air, subjected to long-term loading.Series 6 contained 6 prisms, tested in the air, subjected to short-term loading.Series 7 contained 6 prisms, tested on shrinkage.Series 8 contained 4 prisms, tested in aquatic medium, subjected to long-term loading.In order to take into account the whole range of various factors, which have an influence on the destruction process, series 5–8 of control samples were tested:In total, 32 prisms were tested under different conditions .Concrete prisms were tested according to the developed technique on the special stand. The constant value of compressive force was provided with the use of powerful springs.During the first 24 h after concreting, the samples were covered with plastic wrap to prevent heat loss during cement hydration. After that, the strength setting was continued at the external temperature of 20 °C and constant humidification. Concrete samples were released from the formwork on the 3rd–4th day after concreting.In order to determine the initial physical and mechanical characteristics of the studied material, control samples were investigated see . Thus, 3Micro-indicators for the determination of shrinkage deformations were fixed on 6 prisms, that were not subjected to loading (series 7).In order to assess the water resistivity of concrete, 4 samples were tested in aquatic medium under long-term loading (series 8).Simultaneously with the loading of concrete prisms, concrete cubes of 100 mm × 100 mm × 100 mm size were immersed in the aggressive environment of the same concentration in special containers of 120 mm× 120 mm× 120 mm size, in cubic form. The purpose of this experiment was observation of the corrosion depth and strength characteristics of cube samples.2SO4 with 10% concentration was used. Such concentration of the acid medium enabled the accelerated modeling of the aggressive environment impact and the assessment of the structure behavior in particular conditions. By type, these are corrosion processes that develop in concrete under the action of water containing chemicals that react with the components of cement stone and belong to highly aggressive medium. A similar environment takes place in some chemical plants, galvanic shops, chimneys of thermal power stations and reinforced concrete structures of scrubbers in emergency situations. In addition, the creation of such aggressive environment allows to simulate the effect of an aggressive environment and the simultaneous action of the load in a relatively short period of time.As the aggressive medium, the solution of sulfuric acid HPrism samples were loaded on a specially made power stand  to a givNext, special containers with aggressive medium were fixed on experimental samples of concrete prisms, and the joints were carefully sealed. Before the beginning of the experiments, the geometric dimensions, the base for concrete deformation measurement and the mass of the samples were carefully recorded using a caliper and electronic scales. During the research, these values were constantly monitored. Additionally, during the experiment, the temperature, ambient humidity and acid concentration were constantly monitored. When the acid concentration changed by more than 1%, the acid solution was replaced. It should be noted that the age of the concrete prisms from the moment of concreting until the beginning of the testing was 520 days. During this time, the main part of shrinkage deformations occurred, and the shrinkage processes of concrete were almost stabilized, which could be confirmed by the readings of devices on prisms studied for shrinkage (series 7).Measurements of longitudinal deformations of concrete were performed daily, while cross-sectional dimensions and the weight of samples were controlled every 10 days. Besides the measurement of deformations after the destruction of prisms, X-ray phase analysis of cement stone and study of the concrete microstructure were also performed in order to detect corrosion products and changes in the concrete structure. X-ray phase analysis was performed on an X-ray machine, DRON-2. For X-ray phase analysis, samples were taken from each sample from the surface and from the depth of concrete prisms. The concrete microstructure  was studied with the use of the raster electron microscope TESLA BC—300 .Samples were taken in the form of specimens with dimensions of 10 mm × 10 mm × 15 mm so that they included the outer and inner layers of the studied material. Therefore, it was possible to trace the changes in the microstructure of concrete in different areas. In order to obtain the clear idea of the concrete microstructure, the images of the samples were magnified 1000 times and fixed on photographic film. In order to analyze the microstructure of concrete, three photographs were taken: the first at the surface, the second at the distance of 4–6 mm and the third at the distance of 10–15 mm from the surface.According to the accepted research method of the concrete deformability under long-time impact of aggressive medium and compressive forces, four series of concrete prisms were tested. For series 1, the load level was equal to 0.25During the experiment, according to the accepted technique, the sizes of the cross-section and changes of deformations of concrete were measured. Destruction processes in concrete, which were observed during the test, were caused by concrete corrosion. For better understanding of deterioration processes, which are the issue of this study, it is necessary to provide a detailed explanation on the main mechanisms of concrete destruction. In general, concrete corrosion could be identified with chemical reactions of acid with components of cement stone, containing calcium.The chemical reaction could be described by Equation (1):4·2H2O in Equation (1) is gypsum. As gypsum is the salt of sulfuric acid, it simultaneously proceeds to the sulfate corrosion (see Equation (2)):CaSO2O3·CaSO4·32H2O). Under conditions of repeated wetting and drying, ettringite crystals tend to absorb additional moisture, which causes their expansion. Expanded crystals become the reason for localized crystallization pressure and internal stresses. After that, due to macroscopic expansion of the hardened material, the destruction of concrete proceeds.As the result of this reaction (2), the crystals of ettringite or “cement bacillus” are formed  and (2) describe the formation of ettringite crystals. It is important to note that the corrosion process envies different chemical processes between the components, so that the destruction of the structural element is the result of a combination of different chemical processes. 2SO4 could react with calcium components, Ca(OH)2, which results in a chemical reaction of neutralization. Therefore, C-S-H phases are destructed, and the concrete material is deteriorated, which causes the loss of mass and decrease of dimensions.Simultaneously with ettringite formation, the acid aggressive medium HAnother crucial destruction mechanism is the carbonation, which progresses in the following way: The carbon dioxide enters the concrete pores from the atmosphere and causes the carbonation reaction with interstitial water. After that, calcium hydroxide “hydrated lime” is converted. As the calcium hydroxide provides high values of pH of concrete, its destruction lowers the pH of concrete stone to the values up to 8–9 and also degrades the passivation of the reinforcement .Important issues in concrete corrosion are reactions of alkalis in concrete with aggregates. These processes are generally called alkali reactions and cause tension stresses due to components’ expansion, after which swelling and cracks appear .Among chemical causes of concrete destruction are also internal and external sulphate reactions . The proThrough the experimental research, diligent observations and a detailed analysis of the specifics of the deterioration process were made. During the whole test period, white precipitate was constantly released on the samples, which were immersed in the sulfuric acid.This substance is the product of concrete corrosion, which was formed due to chemical reactions (see Equations (1) and (2)). The white sediment was gypsum, which, as a result of reaction (2), formed the crystals of ettringite or “cement bacillus”. At the same time, the cross-sectional dimensions of the concrete samples constantly decreased see a.In the described experiments, the cross-sectional dimensions were recorded by direct measurement with the caliper with an accuracy of 0.05 mm.Experimental studies have shown that the sizes of the cross-section (width b and height h) decreased during the test, almost linearly see a. At theParameters of the aggressive medium could have a significant impact on the corrosion process, as they may act as initiators or inhibitors of the corrosion rate. On the other hand, changes in the moisture content or its temperature can be considered as indicators of the intensity of chemical reactions. Therefore, during the experiment, the temperature and environmental humidity were constantly monitored b,c. RecoThe average air humidity was 85.1% and was almost equal to its initial level see c. DuringThus, it could be assumed that temperature and humidity did not have a critical influence on the studied deterioration process.ult, applied by the power stand on the residual cross-sectional area, S . Further analysis will be more representative in coordinates of stress values, which corresponded to each loading level. Values of stresses were determined by dividing the load, Na, S see .The constant decreasing of the cross-section of prisms led to an increase of the stress level, c − tε). Additionally, graphs showing changes of stress level over time (t) and graphs of coordinates (cσ − cε) were composed. The graphs show the average values of corresponding parameters for prism-twins ):ult/S are provided in In order to represent changes in concrete strength due to corrosion, experimental and theoretical values of the ratio Nbc. For the concrete prisms tested in the air and water, no destruction occurred, and the stress level in concrete remained constant. A slight increase in deformations of concrete due to shrinkage or swelling and creep was observed. In concrete prisms immersed in water, the deformations of concrete creep were balanced by swelling deformations, and therefore total deformations remained unchanged.Analysis of the results of experimental studies shows that the destruction of concrete prisms in sulfuric acid was due to the corrosion of concrete. Corrosion processes caused the linear decrease in the cross-sectional size of prisms over time. The cross-sectional area of the experimental samples decreased according to quadratic dependency. The decrease in the cross-sectional area caused an increase in stresses in the concrete and in flexibility, which led to the destruction of concrete prisms. The destruction took place at a stress level lower than the initial strength of concrete. The reason is the decrease in the strength of concrete with corrosion damages during long-term loading, which could be taken into account by the coefficient of working conditions of concrete in an aggressive environment, γThe analysis also shows that the methodology of valid norms in all cases does not allow to determine with sufficient accuracy the strength of compressed concrete elements with corrosion damages. The difference between theoretical and experimental values was about 12.3% to 19.5% see . In geneAfter the destruction of concrete prisms subjected to the impact of sulfuric acid, the cut of the prisms in the zone adjacent to the place of concrete destruction was performed with a diamond saw . As a reThese results could also be confirmed by X-ray phase analysis . StudiesAnalysis of the results of experimental studies shows that the destruction of concrete prisms in sulfuric acid was due to concrete corrosion. As a result of concrete corrosion, a contact layer with lower physical and mechanical characteristics was formed. The thickness of the contact layer for samples of series 2 was 1.5 times smaller than for series 1. This could be explained by the use of chemical additives in samples of series 2 and more careful selection of concrete components. In the contact layer of the prism concrete, the presence of the corrosion product ettringite was detected, which causes the formation of microcracks. These microcracks become stress concentrators in concrete. This explains the decrease in the concrete strength during long-term loading, which could be taken into account by the coefficient of working conditions of concrete in an aggressive environment.c-εσ” diagrams were obtained and specifics of stress and deformation changes over time were identified. These results bring us to the conclusion that reasons for the destruction of concrete prisms are the presence of corrosion damages in the concrete and the cross-section reduction. For different series of samples, different exposure times in an aggressive environment were required for destruction: for series 1–106 days, for series 2–79 days, for series 3–56 days and for series 4–89 days. Decreases of cross-sectional sizes were also controlled and recorded. Namely, the relative reduction of the area for samples of series 1 was on average 63–65%, whereas for series 2, 3 and 4 this number reached 46–50%, 34–37% and 4–51%, respectively. Therefore, the load level has a significant influence on the residual load-bearing capacity of samples, subjected to corrosion. In addition, ultimate loads were calculated with the use of the theoretical approach and compared with experimental results. As the theoretical values were on average 15–17% higher than the obtained experimental values, it could be assumed that the stresses in the concrete at the moment of destruction were less than the value of the concrete prism strength. Therefore, exhaustion of the load-bearing capacity of concrete samples is associated not only with the cross-section decrease, but also with the reduction of material strength characteristics.Experimental research conducted in this work detailed the study of stress–strain of concrete samples, subjected to simultaneous action of aggressive impact and constant loading. As a result, changes of cross-section dimensions of samples were recorded, “The ultimate deformations of concrete with corrosion damages, which correspond to the prismatic strength of concrete in comparison with concrete without corrosion damage, were lower by 18% for material with In order to obtain reliable information about the corrosion mechanisms, which cause a reduction of the physico-mechanical characteristics of the concrete, X-ray phase analysis with the use of an electron microscope was performed. It was identified that along the section contour, a partially degraded layer of concrete was formed with a thickness of 1.5–3.7 mm, with corrosive microcracks and corrosion products. Comparison of the microstructure of intact and corrosion-damaged concrete layers enabled to indicate gypsum and ettringite crystals, which are corrosion products and are the reason for additional initial stresses in concrete.It could be concluded that according to the results of the experimental research, deterioration of concrete in aggressive environments is associated with two significant aspects: the decrease of cross-sectional dimensions and changes of physico-mechanical characteristics of the material. It was identified that theoretical algorithms for the calculation of the load-bearing capacity of damaged concrete elements overestimate strength characteristics and do not fully represent the corrosion mechanisms. It was also shown that the intensity of exhaustion of samples’ strength to a large extent depends on the level of load applied and environmental conditions.Findings of this study are important and need to be taken into consideration when the sustainability of corroded concrete structures is evaluated.


The survey comprised five sections: demographics, knowledge, usage, sources of information, and attitudes. The rate of correct answers was 64.88%; differences were detected between programs of study (p < 0.001); effect size analysis showed that these differences cannot be considered large. Individuals from applied sciences scored higher than their counterparts from social studies. Mostly, interviewees were knowledgeable about usage, but mistakenly associated antibiotics with conditions such as cold/flu or viral illnesses; also, they associated antibiotic resistance with the patient and not with bacteria. Despite these misconceptions, positive attitudes were registered overall, and students generally adhered to common practices. They cited doctors/nurses and teachers as sources of information. As a consequence, it is recommended to develop courses that address deficient knowledge regarding antibiotic resistance, especially for individuals affiliated to social disciplines.The inappropriate use of antibiotics leads to antibiotic resistance, which reduces their efficacy. The education of undergraduates is likely to influence their practices. Assessing awareness is critical in the general effort to confront the spread of antibiotic resistance. This cross-sectional investigation was carried out using the questionnaire “Antibiotic resistance: Multi-country public awareness” developed by the World Health Organization. Students from different backgrounds at the Central University participated in the study ( A major challenge for public health is the increment in resistance to common antimicrobial compounds. Consequently, antimicrobial resistance (AMR) is regarded as a priority, and the main goals of the World Health Organization (WHO) aim at containing this problem ,2. MicroLow- and middle-income countries will encounter the greatest concerns associated with public health, due mainly to the prevalence of infections, availability of antibiotics, scarcity of prescribing guidelines, and limited access to low-cost diagnostics tests ,10; AMR Previous research has highlighted that during undergraduate training, education on antibiotic use has a positive influence on the perspectives and practices of future professionals . It has In Ecuador, KAPs have been assessed in certain populations, including caretakers as well as adult and young mothers ,27. Two Consequently, the present study aimed at measuring KAPs with respect to antibiotic resistance in undergraduates associated with applied and social sciences in Quito, Ecuador. We utilized the questionnaire “Antibiotic resistance: Multi-country public awareness” developed by WHO, which has been successfully applied on different occasions ,23,31.The majority of participants were females. Individuals between 22 and 24 years of age constituted the most populous age group. Interviewees were mainly associated with programs in applied sciences . The genn = 659). Only 7% of them (n = 53) agreed that it is acceptable to use antibiotics given to someone else as long as they treat the same disease, although 12% (n = 89) chose the “Do not know” alternative (Q2). Similarly, 12% of participants (n = 89) selected the same option when asked about the appropriateness of buying previously used antibiotics when faced with sickness (Q3). The majority of them, however, recognized that this would not be correct . More than 60% of respondents associated antibiotics with UTI (urinary tract infection) and skin/wound infection; around 40% of them linked antibiotics with diarrhea, sore throat, gonorrhea, cold and flu. Around 30% of students asserted that antibacterial drugs could be employed to treat traumatic injury and fever. They also stated that head (22%) and body (21.6%) aches, malaria (14.2%), AIDS (12.7%), as well as measles (11.3%) could be treated in the same way (Q4)   . Individn = 252) of the students had used antibiotics in the previous month, and 31% (n =230) had done so in the preceding six months. Only 18% (n = 132), reported having taken antibiotics in the previous year (Q6). The majority of interviewees affirmed obtaining the antibiotics from a general practitioner  (Q7). Seventy-five percent (n = 558) admitted receiving instructions from a healthcare professional, whereas 25% (n = 187) did not or could not remember (Q8). In general, antibiotics were said to be mainly acquired in pharmacies (Q9) , although some people accepted receiving them from a relative/friend , or using stored supplies  (The outcomes showed that 34% ( n = 11) .Overall, participants affirmed that physicians and nurses were the main source of information regarding specific terms such as antibiotics and antimicrobial resistance, super bacteria, RAM, drug resistance and antibiotic resistance bacteria (Q10). Teachers and pharmacists were also mentioned, although to a lesser extent. Public campaigns and media were not cited as important .Potential actions that would help address the problem of antibiotic resistance were presented to participants (Q11) . They moAdditional statements were used to assess attitudes and perspectives regarding antibiotic resistance (Q12) . Again, The current survey aimed at studying undergraduate students’ knowledge, uses, sources of terminology, and attitudes with respect to antibiotic resistance. Knowledge was measured using multiple-choice and T/F questions; the overall correct rate was 64.88%. Previous studies have classified knowledge in terms of rate of correct responses as acceptable (≥80%), moderate (60–80%), and low (60%) ,21. AnotViral infections, fever, sore throat as well as head and body aches were mentioned as appropriate for being treated with antibiotics; these results are consistent with previous findings that have exhibited various misunderstandings concerning practices ,32,33,34Less than 40% of participants declared having used antibiotics in the preceding six to twelve months. Students mostly adhered to recommended practices such as following instructions from health care professionals, receiving professional advice, or acquiring antibiotics in pharmacies. Appropriate practices have also been observed among Ecuadorian medicine students . HoweverProper information is fundamental for refining safe practices among people, not only regarding antibiotic use , but alsThe results described herein were comparable to those documented in studies conducted in various countries ,22,28. TThis research presents some limitations. Firstly, the data is not generalizable to other regions as it was carried out in Quito at the Central University; this is important as educational standards are not consistent across the country. The aim of the study was to develop a systematic understanding of antibiotic resistance awareness among undergraduate students from different backgrounds, and also to generate quantitative data that could be used for further comparisons and evaluations. Secondly, no causal inferences could be drawn due to its cross-sectional design. Finally, the actual knowledge may be overestimated as it is based on voluntary responses; thus it is recommended to use probability sampling for further research.This cross-sectional survey was conducted from October to November 2019 amongst undergraduate students from the Central University of Ecuador in Quito, which is the oldest, largest, and most populated university in the country. Individuals were selected based on non-random criteria, as this research aimed to develop an initial understanding with respect to antibiotic awareness among university students with different backgrounds. The current study did not seek to test any hypothesis about a larger population; thus it was carried out using voluntary responses. The sample size was determined using the online calculator Raosoft , with a The questionnaire “Antibiotic Resistance, Multi-country public awareness survey”  was deveThe Ethics and Bioethics Committee for Research of the Espiritu Santo University read and approved the related protocols (Reference number: Antibio 6- 2018). All procedures involving humans were performed in accordance with the Declaration of Helsinki. The questionnaire was anonymous, voluntary and the information gathered did not put the interviewees at risk in any form. Informed consent was obtained from all subjects prior to their participation . Intervi–Wilk’s test, and homogeneity of variance was determined by Levene’s test. For non-normally distributed and homoscedastic (program of study) as well as heteroscedastic data , the Kruskal–Wallis test was employed. The mean is affected as a measure of the central tendency of distribution when this distribution is non-symmetrical. Hence, medians were employed as they depict more suitably the center of distribution in such conditions. Statistical significance was set at p < 0.05. The statistical approach based on null hypothesis significance testing does not provide information regarding the magnitude of an effect of interest , and the precision of that estimate [–Wallis test; values of 0.01, 0.06 and 0.14 denote small, medium and large differences, respectively [® version 9.9.9341360  (R2016a); figures were rendered with Python’s plotting library, Matplotlib 3.0.3 .Data description was carried out using percentages and frequencies. Normality was assessed with Shapiroestimate ,52,53. Cectively ,52,53,54This study provides a comprehensive examination of antibiotic awareness among undergraduate students in Quito Ecuador, including individuals from different academic backgrounds. The overall correct rate was 64.88%, which should be considered moderate. Students from applied sciences scored higher than those affiliated to social disciplines. In general, respondents possessed good knowledge with regard to practices, although relevant misconceptions were detected as some erroneously recognized antibiotic use for non-associated conditions. In particular, almost three-quarters of the population stated that antibiotic resistance arises when the body of the patient becomes resistant. Students quoted doctors and nurses along with teachers as sources of key information; educational campaigns were not mentioned as relevant. Respondents showed positive attitudes and mostly adhered to common practices. These outcomes provide important insights into our current understanding of antibiotic awareness in a key part of the population, which will be certainly helpful when designing not only health programs and preventive strategies, but also educational courses aimed at limiting the spread of antimicrobial resistance.


Distributed fibre optical sensing (DFOS) is increasingly used in civil engineering research. For reinforced concrete structures, almost continuous information concerning the deformations of embedded reinforcing bars can be obtained. This information enables the validation of basic and conventional assumptions in the design and modelling of reinforced concrete, particularly regarding the interaction of concrete and reinforcing bars. However, this relatively new technology conceals some difficulties, which may lead to erroneous interpretations. This paper (i) discusses the selection of sensing fibres for reinforced concrete instrumentation, accounting for strain gradients and local anomalies caused by stress concentrations due to the reinforcing bar ribs; (ii) describes suitable methods for sensor installation, strain acquisition and post-processing of the data, as well as determining and validating structurally relevant entities; and (iii) presents the results obtained by applying DFOS with these methods in a variety of experiments. The analysed experiments comprise a reinforced concrete tie, a pull-out test under cyclic load, and a flexural member in which the following mechanical relevant quantities are assessed: the initial strain state in reinforcing bars, normal and bond shear stresses, deflections as well as forces. These applications confirm the benefit of DFOS to better understand the bond behaviour, but also demonstrate that its application is intricate and the results may lead to erroneous conclusions unless evaluated meticulously. The response of structural concrete members is governed by the interaction between the reinforcement and the surrounding concrete. Since the first use of reinforced concrete structures, researchers have been concerned with the understanding and proper modelling of this interaction. However, it was hardly possible to investigate the stress transfer between concrete and reinforcement experimentally due to limitations of the measurement technology available and the fact that the interface remains difficult to access without influencing its structural behaviour. The latest developments in measurement technology, especially in fibre optical sensing, bear the potential to greatly improve the understanding of this interaction.Traditionally, the investigation of the concrete-to-steel interaction was carried out on pull-out tests with small bonded lengths. Under the assumption of nominal bond shear stresses of constant magnitude over the bonded area, these simple tests allow the deriving of bond stress–slip relationships, which can then be used to model the stress transfer between concrete and reinforcement. Despite being widely used, the traditional experimental setup remains controversial, as it does not represent the stress conditions in an actual structure: for instance, compression fields are generated by the bearing plate used in the tests, and the concrete cover exceeds practical values. Hence, many efforts were undertaken to study the steel strain variation directly in structural concrete elements. By using embedded electrical resistance strain gauges, researchers succeeded in experimentally demonstrating the contribution of the concrete between the cracks e.g., ,3,4,5,6),4,5,63,42 groove carved in the bars by planing. Thereby, they obtained the steel strain distribution along the bar at 10.4 mm spacing, reaching the plastic steel range [With the development of fibre optical sensors, the measurement of strain profiles using a series of strain gauges became less common. Initially, optical fibres were also designed as discrete sensors, typically using fibre Bragg gratings (FBG), in which sensors are written into an optical fibre. Temperature or strain changes over the grating length alter the properties of the reflected light, which is used to quantify the strain or temperature difference. Fundamentals on the functionality of FBG can be found in ,8. Such el range . In a reel range . In theiAlthough FBG is applicable over longer sensing distances and requires a significantly smaller intrusion of the host material, it only provides information at the locations of the sensors, i.e., critical locations might be missed unless a large number of gratings at small spacings is employed as by Kenel et al. ,11. HoweThe c-OFDR based strain measurement has recently found broad application in civil engineering research , where iDespite the versatility of DFOS, most current applications are limited to small deformations and monotonic loading. The limitation to small deformations is provided by the measurement ranges of the spectrometers currently in use . Galkovski et al. (2021) reported the possibility to extend the measurement range of the spectrometer by setting intermediate reference states and superposing the data afterwards . Their mOnly a few applications of DFOS under repeated and high cyclic loading are known to the authors. Broth and Hoult (2020) used nylon-coated fibres to assess the deformation of slender and deep reinforced concrete beams subjected to dynamic loads (3600 cycles of three and four-point bending applied at 1 Hz). The DFOS revealed a localised increase in strains with the number of cycles, attributed to the loss of tension stiffening. The dynamic loads and subsequent loading to failure did not cause an impairment in the DFOS performance . FernandThe present paper discusses the particularities of using DFOS to instrument reinforcing bars in RC. This comprises practical recommendations for new and experienced users. Procedures are proposed for instrumenting embedded reinforcing bars with DFOS, including methods to avoid damaging the fibres while casting. Furthermore, methods for the determination of steel stresses in the reinforcing bars, nominal bond shear stresses between concrete and reinforcing bars, bond-slip and crack widths are presented, discussing potential problems and limitations. These comprise (i) the interpretation of local phenomena typically unaccounted for by conventional models, but captured by high-resolution DFOS, and the quantification of their impact on the above-mentioned values, and the global response of the elements, (ii) the difficulties of dealing with signal anomalies  and establishing transparent post-processing methods to eliminate them, and (iii) accounting for the initial strain states that inevitably exist in structural concrete. Furthermore, the challenges arising in long-term and high cyclic tests are addressed.The unprecedented high spatial resolution of DFOS sheds light on local and global phenomena, some of which might not be part of the intended investigations nor anticipated so that this advantage might turn into a drawback if the analyser is no longer able to interpret or trust the obtained strain measurements. The development of measurement anomalies, such as noise and outliers, renders proper interpretation of the results more challenging. Thus, it is not surprising that questions concerning measurement reliability arise with the increasing use of this technology. The present paper discusses the accuracy and determines the challenges and limitations on simple elements in which the deformation response is largely understood and verifiable. Once the challenges regarding application methods and limitations are mastered, DFOS can be incorporated into complex experiments and structures with due care, providing valuable insights into their internal strain distributions and thus an understanding of their mechanical response.The following section investigates the ability of various single-mode fibres to accurately measure some particular effects observed in reinforcing bars embedded in concrete, such as strain discontinuities at concrete cracks and strain gradients caused by the contribution of concrete in bearing tensile forces between cracks. Furthermore, the performance of these fibres under repeated loading is studied. Lastly, the strain profiles obtained with the fibres on reinforcing bars embedded in concrete will be analysed. These issues are discussed based on experimental results but without claiming generality or completeness.All the DFOS data presented in this paper were acquired with the optical distributed sensor interrogator ODiSI-6104 supplied by Luna Innovations . The optMuch research has been carried out to investigate the suitability of various coating types, e.g., ,23,25. TFor this purpose, a steel plate with changing cross-sectional geometry was tested under direct tension. The specimen was cut from a 10 mm thick S355 J2 steel plate. Its total length measured 1345 mm and the cross-section width varied between 30 and 120 mm. The cross-sectional area and hence, the resulting strains were chosen such that they represent typical strain conditions in a structural concrete element under tension. Discontinuities of the cross-section were used to simulate cracks, and a tapered cross-section simulated the tension stiffening effect. The advantage of using a steel plate is that the gradients and discontinuities do not change with the load cycles (provided that no fatigue cracks appear), whereas in an embedded reinforcing bar, new cracks may appear, and tension stiffening may also be affected by the deterioration of bond with repeated loading ,35.The instrumented steel plate was tested under repeated tensile loading. −1). In contrast, regarding the dynamic cycles, loads of the same intensity were applied in a sinusoidal form at a frequency of 5 Hz under force control to ensure a constant load amplitude. The applied force, the machine displacement and the gauge strains were recorded continuously, at a rate of 100 Hz in the dynamic cycles and 5 Hz in the monotonic cycles. The DFOS measurements (gauge spacing of 0.65 mm and a sampling frequency of 4.2 Hz) were only carried out during the monotonic cycles. A total of one million cycles were executed over seven days. The ambient temperature was recorded throughout the test duration.The test was carried out in a universal testing machine of the type Schenck 480 kN. The upper and lower load were set to 60.5 kN and 20 kN, respectively. The loading type altered between sequences of monotonic and dynamic loading. The monotonic loading was applied under displacement control with a constant rate (0.01 mm.sF = 60.5 kN). The DFOS data shown here are raw data that were down-sampled to 1 Hz as were the other measurements. The difference between the DFOS strain profiles and the one obtained from the simulation is provided in x > 700 mm) and increased gradually toward the lower end. To investigate the reason behind this small but non-negligible change, the development of the strain difference at around x = 967 mm over the load cycles was studied. −6 K−1 based on the ODiSI-A user guide [N = 106, as well as with the variation over the cycles. The reason for the gradient in er guide ), whereaThis example illustrates the ability of DFOS to measure strain gradients and discontinuities, which is essential in order to obtain meaningful measurements in structural concrete elements. The polyimide-coated fibres are recommended as they demonstrate a higher accuracy, as reported in ,28. The As outlined in the previous section, polyimide-coated fibres are better suited to measure strain gradients than acrylate-coated fibres and show a higher accuracy. This confirms the findings of Mata-Falcon et al., who also studied the performance of polyimide-coated and acrylate-coated fibre optical sensors by investigating their ability to measure strain gradients on a steel plate with variable cross-sections , similarTo address these contradicting conclusions, the authors investigated both types of fibre coatings on bare reinforcing bars and on reinforcing bars embedded in concrete. Based on the investigations on bare reinforcing bars, which are presented in a companion paper , it was L × b × h = 300 × 150 × 150 mm3. Self-compacting concrete with a maximum aggregate size of 16 mm was used, with an average compressive strength of 38 MPa after 28 days . The stsilicone d. Finall−1 loading rate in a universal testing machine of type Schenck 480 kN. A total of 11 loading cycles in the elastic range of the reinforcing bar, with an upper load of F = 46 kN ≈ 0.6 yF were applied, followed by two cycles in the plastic range before the specimen was loaded to failure. Only data from the first elastic cycle are presented and discussed here.The specimen was tested in displacement control at 0.04 mm.sThe effect of the local disturbance by a rib is investigated in The wavelength of the undulations coincides with the rib spacing, and local peaks occur systematically at the inter-rib areas, as clearly demonstrated by the polyimide-coated fibre in this test, and thus cannot be considered as random noise. On the other hand, as these peaks also appear in the bare parts with ribs—though with a smaller amplitude–, attributing them to bond action alone  may be mThis test demonstrates that the use of conventional strain gauges to measure reinforcing steel strains inside concrete produces significant disturbances in the deformation behaviour of the reinforcement as, locally, bond is lost over the instrumented length. This is caused by the milled surfaces to accommodate strain gauges and the silicon employed to seal them d.Furthermore, the suitability of polyimide-coated fibre optical sensors for applications in reinforced concrete members is demonstrated by the test. The mechanical origin of the jagged strain distribution can be attributed to the presence of ribs. Therefore, measurements with polyimide-coated fibres can be processed with a suitable filter to obtain mean strains over a rib spacing, which are representative for structural concrete applications while maintaining a short activation length and a high sensitivity . Using fSpecific procedures for the instrumentation of bare reinforcing bars, data acquisition, some general aspects for validations, and the determination of steel stresses are published in the companion paper . These aFor the measurement of strains in embedded reinforcing bars, it is recommended to use polyimide-coated single-mode fibres with a thin coating. Their small size produces a negligible alteration of the interface between concrete and steel. The fibres can either be placed in a groove or attached along the longitudinal ridge. The authors prefer the first configuration as (i) the fragile fibre is protected from damage during casting and compaction, and (ii) it was observed in some cases, when glued directly on the bar, the adhesive cleaved to the concrete and delaminated from the bar. In other studies, a Teflon layer  or a silThe bars should be instrumented with at least two fibres arranged on opposite sides to capture potential bending effects. If the specimen and reinforcement layout permits, it is recommended to lead the fibre ends out of the concrete at a suitable location, i.e., away from areas with high stress concentration, such as supports or load introduction regions. The parts of the fibre located outside the formwork during casting should be protected from mechanical damage . In anchorage investigations, it would affect the structural behaviour and the measurement quality if the fibre was led out of the concrete at the embedded bar end, where large relative displacements between the reinforcing bar and the surrounding concrete occur. In such cases, it is preferable to loop the fibre around the bar end in a protective plastic tube that runs inside an indentation connecting the grooves on either side of the reinforcing bar at its end e.Once the concrete is cast, parts of the fibre are no longer accessible. The user should register the coordinates of all relevant points that lay inside concrete beforehand . The local coordinates within the bar are important, in addition to the position of each bar in the specimen, i.e., in the global coordinate system. Precise positioning and documentation are essential for reproducibility and reliable data post-processing.Furthermore, from the moment the concrete is poured into the formwork, the reinforcement deforms due to the concrete hardening process (hydration heat) and shrinkage. A reference measurement needs to be set before casting to quantify the resulting initial strain state of the composite element. Such shrinkage strain measurements with DFOS are presented and discussed in The fibre optical sensors on cast-in bars usually display more anomalous readings and require more post-processing than on bare steel. The user will usually have to identify and cancel out outliers and mitigate peaks resulting from local phenomena. Based on the analysis in An interesting value that can be obtained directly from the quasi-continuous fibre measurements is the relative displacement between concrete and steel, denoted as slip δx a. By intIn a reinforcing bar embedded in concrete and monotonically loaded in tension, the slip vanishes at the locations of the global strain minimum between two adjacent cracks. Note that due to its low tensile strength, the deformations of the uncracked concrete are very small compared to those of the steel and can usually be neglected.Adding up the slip from the adjacent crack elements on either side of a crack  crack openings in such cases: with the decrease of the applied load, slip reversal occurs starting from the crack, along with a potential deterioration of the bond shear stresses ,42. ThesOnce the acquired strain data have been post-processed, it can be employed to determine the stresses in the reinforcing bars, provided that the stress–strain characteristics of the latter are known. Commonly, such constitutive laws for reinforcing bars are defined using the elongation of a base length containing a multitude of ribs. DFOS, on the other hand, distinguishes between strains at and between the ribs, but as observed by Galkovski et al. , mean loFor a realistic translation of strains into stresses, accurate knowledge of the stress–strain characteristics of the instrumented reinforcing bar is essential. Overly simplified constitutive models lead to biased results. If the constitutive law of the reinforcing bar has been determined by material testing, the stresses can essentially be obtained from the strains directly using the experimentally observed stress–strain relationship, suitably smoothed where appropriate and fitted to the average behaviour if several material tests have been carried out. However, this requires knowledge of the experimental stress–strain curve of each reinforcing bar and cannot be used for comparison with model predictions, which are typically based on few parameters, i.e., Young’s modulus Alternatively, the model proposed by Ramberg and Osgood  Figure b can be The parameters to-Pinto , may be The deformation behaviour of steel reinforcing bars exhibiting a yield plateau, nowadays typically quenched and self-tempered (QST) straight bars, is accurately reproduced by the following expressions :(4)σs=εsThe parameters lus) see d. This dThe parameters defining the material models should be determined in material characterisation tests, ideally on bars prepared identically as those used in the actual experiments . Still, the material parameters are subject to scatter and additionally depend on the methods used in the material characterisation. For instance, the yield limit and the tensile strength depend on the applied loading (displacement) rate, with relevant variations even within the strain rates allowed in standards for uniaxial tension tests on reinforcing bars . DependiOnce the steel stresses have been obtained from the strain data, they can be used to determine the forces acting in instrumented parts of the tested specimens. To this end, the geometry and the loading type must be known. For instance, in elements (such as reinforcing bars) subjected to normal forces and uniaxial bending, which are instrumented with at least two fibres, following the common hypothesis of Euler–Bernoulli, a plane can be fitted to the measured strains and subsequently, the stresses in the entire cross-section can be determined based on the procedures outlined above. Integration of the stresses then provides the normal force and bending moment acting on the cross-section see b. FinallThis allows, e.g., determining the contribution of reinforcing bars to shear force transfer in beams, also known as dowel action . Furtherx  based strain data or average strains determined using linear variable displacement transformers (LVDT). In the latter case, mean DFOS strains over the same section as covered by the LVDT must be compared.By integrating the quasi-continuous strains, deformations can be calculated (similar to the determination of bond-slip) and, if at least two fibre optical sensors are installed, inclinations or curvatures can also be obtained. These values again can be compared to measurements obtained using other instrumentation, such as LVDTs, inclinometers, DIC, or actuator strokes of testing machines.Finally, the steel stresses obtained from the measured strains can be compared with the stresses determined from applied loads and geometrical information, particularly in cracked cross-sections of reinforced concrete specimens. Precise knowledge of the materials’ constitutive laws is a prerequisite for reliable comparisons.The following section presents examples of the application of DFOS in experiments on structural concrete elements. In the first example, the initial strain state in an embedded reinforcing bar (ERH) and its implications for DFOS measurements are discussed. Subsequently, the derivation of normal and bond shear stresses as outlined in Shrinkage designates the decrease in the volume of stress-free concrete. It consists of several contributions, with autogenous shrinkage and drying shrinkage being predominant . The forand 0.7‰ .In an unreinforced, perfectly unrestrained concrete specimen, shrinkage deformations would not cause any stresses. In reinforced concrete specimens, however, the deformations are internally restrained by the reinforcement even in the absence of external restraint. This internal restraintcauses tensile stresses in the concrete When studying the global response of structural concrete elements, this self-equilibrated initial stress state is often neglected, and the response of the element is assumed to start with the loading. However, the shrinkage-induced stresses can have a pronounced influence on the crack formation and stiffness: the initial tensile stresses in the concrete cause cracking at reduced loads than expected in initially stress-free elements. The corresponding apparent tensile strength must not be confused with the effective concrete tensile strength. Moreover, neglecting the initial strains and stresses caused by shrinkage leads to an underestimation of the concrete tensile contribution between the cracks  and an oTaking shrinkage stresses and strains into account is challenging, and without measurements on a specimen, only rough estimates are possible. Even if strain measurements are available, determining the initial stresses is not straightforward owing to the complex nature of the time-dependent processes and their dependence on the actual exposure and dimensions of a specimen. Standards provide empirical formulas to approximate free shrinkage, which though is subject to significant scatter. A better approximation is obtained by shrinkage measurements on plain concrete specimens, which are cast using the same concrete and stored in the same conditions as the specimens under investigation.For structural concrete elements instrumented with DFOS, the initial strains can easily be obtained. Sensors installed prior to casting indicate strain and temperature changes with respect to this reference state . In contrast to other strain measuring methods, no additional work steps are required.2 cross-section and 1000 mm length  suggests a physical cause behind the observed strain fluctuations. The elapsed time between the reference state (before casting) and the shrinkage measurement can be excluded as a reason since the loose sensor sections outside the concrete did not reveal any anomalous readings. The most plausible explanations are (i) local strain variations between the shrinking cement paste and the aggregates that do not shrink and (ii) internal micro-cracking caused by the resulting restraint stresses. Indeed, non-shrinking particles, such as aggregates and unhydrated cement, restrain the shrinkage of the cement paste, which explains why shrinkage strains of concrete are much smaller than those of cement paste and decrease with the relative aggregate volume . As theIn the example given, a change in the ambient temperature was also measured by DFOS. This is recognisable on the vertical shift concrete b, and waFor subsequent measurements, it is recommended to set a new reference state at the beginning of testing and superpose the smoothed shrinkage strains in the post-processing. −1 and increased ten-fold after the onset of yielding. The obtained properties are provided in The derivation of normal and bond shear stresses as outlined in In addition to the DFOS, one concrete surface of each RC tie was instrumented with a 3D-DIC system to track the global deformation of the specimen. A speckle pattern consisting of black circular points with a diameter of 1.2 mm was applied using a speckle roller. The images were captured at 1 Hz with two cameras (FLIR 12.3 MP) using 28 mm focal length Zeiss lenses with a baseline of 942.6 mm and a resulting resolution of 3.34 px/mm. The correlation was performed with the software VIC-3D  using a−1. During the experiment, the DFOS measurements were tracked continuously. Once the measurement became unstable  was measured before clamping the specimen in the universal testing machine. The load was applied to the reinforcing bar ends deformation controlled at a loading rate of 0.01 mm.sF = 175 kN. The locations where yielding started are indicated on the bar also shown in F = 150 kN and 178 kN). The impact of the material law employed to translate strains into stresses and further into bond shear stresses is discussed in the following, using half of the crack element between YS 3 and YS 5 at the two load steps marked in yielding c. Finallyielding c. The stx = 65 to 105 mm, which results in zero bond shear stresses in this part as well. The vanishing bond shear stresses, contradicting the commonly postulated bond shear stress–slip relationships, are the result of the measured gradual strain increase from the yield limit to the onset of hardening, whereas steel stresses are presumed to remain constant in this range (yield plateau).As expected, the different models hardly have any influence on the results in the elastic range, except when the strain exceeds the strain radients a, middleradients a, bottomstresses b, middlestresses b, bottomIn a seminal paper, Shima et al. (1987) used strain gauges inside the reinforcing bar (as described in The disturbed regions with strains beyond the yield strain but inferior to the strain at the onset of hardening result in zero bond shear stress when applying the constitutive law presented in F and the relative slip This section describes the results of a pull-out test (PO) according to the RILEM standard . Figure To assess the plausibility of the results obtained with DFOS, the bond shear stress values were averaged over the bonded length and compared to the nominal values obtained by dividing the applied load by the perimeter of the bonded length of the reinforcing bar. With the use of DFOS, it could be shown that the bond shear stresses were far from being constant over the bonded length of a pull-out test as often presumed given the short embedment length. Furthermore, their profiles vary with increasing number of load cycles, redistributing toward the unloaded end. The quality of DFOS in this test was not affected by the repeated loading action.In PGz≈16 mm is the inner lever arm between sensors PG1 and PG2; and (iii) the reinforcing bar deflectionzPG.Note that while the deflections of the reinforcing bar determined from Equation (10) corresponded well with the measured vertical displacements of the beam, determining the deflections of a beam from the curvatures of a reinforcing bar is not recommended in practice, as the goodness of the results is highly dependent on the accurate knowledge of the small distance between the sensors x = 1.65 m changes from about 2.3 kN on the left side of the crack to −1.9 kN on its right side, corresponding to a shear force of 4.2 kN carried by the reinforcing bar at the crack; note that the global shear force at this loading stage amounted to 73 kN.The instrumentation proved to be suitable to determine all derived parameters and revealed that the curvature of the reinforcing bar was localised at the cracks, rather than being uniform over longer distances as commonly assumed. The bending moment and the shear forces along the bar also depend on the location of cracks: at cracked sections, the local bending (and curvature) of the bar is highest. In the shear zones, the curvature and hence, the bending moment change signs between cracks, and the shear forces exhibit discontinuities at the cracks, indicating that part of the applied shear force is carried by the reinforcing bar, which is known as dowel action. For instance, for the highest shown load (light grey curve), the shear force at the crack at As expected, integral values, such as the bar deflection and the bending moment have a much smoother distribution than derivatives, such as the shear force in the reinforcing bar.Distributed fibre optical sensing (DFOS) bears the potential to achieve a long-standing ambition of many researchers: to measure the internal strain distribution of RC structures with adequate resolution and effort, yet without biasing the behaviour. This paper, together with the companion paper , summariFor instrumenting reinforcing bars embedded in concrete, the use of fibres with a chemically bonded coating, placed inside a groove and covered with a two-component epoxy, has proven to lead to accurate results. However, the high spatial and temporal resolution makes it important to distinguish between global and local phenomena when analysing the acquired data and to select the size of the virtual sensor according to the target measurement. An example for measuring local deformations are the jagged strain distributions. As outlined in The strain state of the reinforcement can be measured from the first contact with fresh concrete—provided that the fibre optical sensor is installed  before casting, such that subsequent measurements refer to the bare state. As discussed in The strains induced in the fibres represent a combination of mechanically and thermally induced strains, as observed even in laboratory conditions . In longIn reinforced concrete elements, DFOS captures the variation of steel strains along an embedded reinforcing bar. The steel stresses can be obtained with knowledge of the stress–strain relationship, and the bond shear stresses follow from equilibrium. The slip, i.e., the relative displacement between concrete and reinforcing bar, is obtained by integrating the strains along the bar and neglecting or approximating the minor concrete displacements. A local bond stress–slip relationship can thus be obtained. However, the magnitude of the bond shear stresses remains sensitive to the post-processing methods applied and depends strongly on the material law used to convert the measured strains to stresses. While the derived values in the elastic and fully plastic range are reliable and could be shown to correspond on average to the nominal bond stress obtained in pull-out tests , the conRepeated loading  did not


The role of tyrosine kinase inhibitors (TKI) in the neoadjuvant setting and the optimal duration of therapy remains poorly defined. As such, we aim to evaluate the impact of neoadjuvant TKI on oncological and functional outcomes in our cohort of patients with rectal GISTs.A retrospective analysis of 36 consecutive patients who underwent treatment for rectal GIST at the National Cancer Centre Singapore from February 1996 to October 2017 was analysed. Surgical, recurrence and survival outcomes between the groups who underwent neoadjuvant therapy and those who underwent upfront surgery were compared.p = 0.04) and lower mitotic count  when compared with the non-neoadjuvant group. With TKI pre-treatment (median duration 8.8 months), majority of patients (82%) achieved at least partial response to the therapy coupled with a significant downsizing effect of up to 39% (median size of 7.1–3.6 cm), resulting in similar rates of sphincter-sparing surgery  when compared with the non-neoadjuvant group. In general, neoadjuvant group had lower rates of local recurrence  and higher overall survival  as compared to the non-neoadjuvant group.Patients who received neoadjuvant treatment had significantly larger tumours (median size 7.1 vs. 6.0 cm, Neoadjuvant TKI has the benefit of downsizing unresectable rectal GIST to benefit from sphincter-sparing procedure and also confers protection against local recurrence and improves overall survival. Gastrointestinal stromal tumours (GISTs) belong to a spectrum of mesenchymal tumours that range from indolent tumours to malignant sarcomas. After acquiring a mutation in the tyrosine kinase receptor c-KIT CD117), the interstitial cells of Cajal (ICC), which are located in the myenteric plexus in the gastrointestinal wall and serve as a pacemaker of the gut, lose normal growth control and result in tumour formation [17, the iApproximately half of the patients at first presentation are not eligible for curative surgical resection due to the presence of metastatic disease . Even wiUnlike its gastric counterpart, rectal GISTs have the worst clinical outcomes and poorest prognosis . AlthougThis study aims to find out the clinical profile of patients with rectal GIST in our local population and determine if neoadjuvant TKI prior to surgery improves the outcomes for this rare disease.The data were retrospectively collected from a prospective database of patients who underwent treatment for rectal GIST at the National Cancer Centre Singapore from February 1996 to October 2017. The conduct of the study was approved by the SingHealth Centralized Institutional Review Board (CIRB 2018/3065). A total of 36 consecutive patients were accrued and analysed. Preoperative parameters such as patients’ age, gender, race and ECOG status were analyzed.All patients had a histological diagnosis of GIST and underwent preoperative imaging with a Computed Tomography (CT) scan of the chest, abdomen and pelvis or a Positron Emission Tomography (PET)-CT scan. Patients with distal metastatic disease were excluded from the analysis. Primary tumour size, mitotic count, stage according to NIH-Fletcher risk classification, imaging and endoscopic findings were reviewed and discussed at a multidisciplinary tumour board meeting .Reasons for the recommendation of neoadjuvant therapy were collated. Patients recommended for neoadjuvant therapy had unresectable tumours  or borderline resectable tumours in close proximity to vital structures such as the anal sphincter. In addition, they had to be of Eastern Cooperative Oncology Group (ECOG) status 0 or 1 prior to receiving neoadjuvant therapy. The dose, duration and tumour response to neoadjuvant therapy were evaluated with the Response Evaluation Criteria in Solid Tumours (RECIST) criteria based on the CT scan performed after completion of neoadjuvant therapy . PatientPatients who underwent upfront surgery without neoadjuvant therapy were labelled as the non-neoadjuvant group. There were patients from the non-neoadjuvant group who were offered but did not undergo surgery, due to lack of consent or defaulted surgery. The type of surgery, residual tumour (R) classification and surgical complications were tabulated. Duration and response to adjuvant imatinib, together with the DFS and OS, were also compared between the neoadjuvant and non-neoadjuvant group. DFS is defined as all GISTs cancer events including local, regional and distant relapse; second malignancies and deaths without recurrence were censored. OS is defined as all deaths including those without recurrence.t test was used for normally distributed continuous variables while the Mann–Whitney U test was used for non-parametric continuous variable. p < 0.05 was considered statistically significant. Statistical analysis was performed on SPSS version 19.0.Categorical variables were analyzed using the chi-square test or Fisher’s exact test when cell sizes are small (< 5). Haldane’s approximation was utilized by adding a constant of 0.5 to each cell if any observed frequencies were 0. The two-sample p = 0.04) and lower mitotic count  as compared to patients who did not receive neoadjuvant therapy.There were 11 patients in the neoadjuvant group and 25 in the non-neoadjuvaunt group. The demographics between the two groups are generally comparable (Table The top reasons for neoadjuvant therapy were due to an unresectable tumour (55%) or high morbidity surgery (36%) Table . Most paSurgical Outcomes after Neoadjuvant Gleevec.p = 0.26) and stoma creation , that was sphincter preserving , via a minimally surgical approach  (Table p = 0.47) and had fewer surgical complications . Importantly, the non-neoadjuvant group experienced intraoperative complications (16%) consisting of ruptured or perforated tumour resulting in local and distal recurrence  while the neoadjuvant group had no intraoperative complications and only developed one case of rectosigmoid fistula that was managed with a transanal excision. All patients in the non-neoadjuvant group who did not undergo surgery only had biopsies to confirm the diagnosis and subsequently defaulted follow-up. In terms of CKIT exon 9 mutation, the neoadjuvant group was able to achieve better rates of R0 resection (100 vs. 50%), with lower rates of overall recurrence (100 vs. 50%) as compared to the non-neoadjuvant group. There was no difference in terms of the use of sphincter-sparing approach (100 vs. 100%), surgical complications (0 vs. 0%) and 1-year overall survival (100 vs. 100%) between both groups. Keeping in mind the inherent limitation of running subgroup analysis in such a small population (2 in the non-neoadjuvant group and 1 in the neoadjuvant group), pooled data from various institutions of this rare tumour may better facilitate subgroup analysis to find out if the type of mutation affects the prognosis in the setting of neoadjuvant therapy.Between the neoadjuvant and non-neoadjuvant groups, both groups underwent similar rates of resection , the neoadjuvant group underwent a shorter duration of adjuvant therapy  and achieved greater rates of complete response  based on RECIST, though it did not achieve statistical significance (Table p = 0.40).Although both the neoadjuvant and non-neoadjuvant therapy groups underwent adjuvant therapy , with the former having a lower rate of local recurrence  (Table p = 0.63). For both groups, all recurrence cases did not undergo surgical treatment. Instead, adjuvant chemotherapy was restarted in 75% (12/16) and 100% (2/2) in the neoadjuvant group. Unfortunately, 7 patients are still currently undergoing therapy and we are unable to run further analysis to evaluate the efficacy of the treatment. There were 2 neoadjuvant cases that experienced tumour recurrence, specifically distal recurrence involving the liver and peritoneum. Both cases had CKIT exon 11 mutation and experienced recurrence at 3.4 and 3.3 year after the initial surgery, respectively. Both cases are still undergoing imatinib therapy. The neoadjuvant group had a longer OS  as compared to the non-neoadjuvant group  and OS (100 vs. 100%) and 3-year DFS  and OS  between the neoadjuvant and non-neoadjuvant groups were similarly high. At the 5-year mark, the neoadjuvant group had poorer DFS , but had better OS  as compared to the non-neoadjuvant group.The recurrence rate for the neoadjuvant group was statistically lower than the non-neoadjuvant group  with lower mitotic count  as compared to those who had no neoadjuvant therapy. A duration of 8.8 months of neoadjuvant therapy was useful in downsizing tumour to a median size of 3.6 cm after a 2.7 cm or 39% size reduction to improve resectability to the extent of achieving R0 resection, with low complications and improvements in sphincter-sparing rates. Neoadjuvant TKI improved the eligibility of sphincter-sparing procedures to the level that was comparable to the non-neoadjuvant group for patients who were considered to be surgically inoperable or challenging. Notably, neoadjuvant therapy generally conferred protection from local recurrence and improved OS regardless of tumour size and tumour risk stratification.Our study’s findings may provide further insights insofar as to aid the future development of a neoadjuvant therapy guidelines. We found that patients who received neoadjuvant therapy had larger tumours .In reported literature, the mean size of GIST ranges from 10 to 13 cm with those greater than 5 cm more likely to be symptomatic  . On the Surgical resection with negative margins represents the most effective chance of long-term survival in patients with non-metastatic, low-risk GISTs . LikewisAdjuvant imatinib following surgery is well established for high risk GISTs. According to two independent high-impact studies conducted by Joensuu et al. and Mietinen et al., the initiation of adjuvant imatinib for high-risk rectal GIST to decrease the risk of recurrence and metastasis is recommended , 31. In In this study, we found that neoadjuvant therapy enabled a modification in tumour size and/or density to permit the utility of minimally invasive sphincter-sparing surgery in 36% of our patients. It is important to note that resistance to relevant secondary mutations may occur after neoadjuvant therapy and hence the duration of treatment should be as short as possible to facilitate timely complete resection . NeoadjuDifferent imaging modalities are used to monitor the response after systemic therapy. The most commonly used is RECIST . HoweverGiven the complexity and rarity of rectal GIST, a multidisciplinary approach with a team of surgical oncologists, medical oncologists, radiologists and pathologists with expertise in sarcomas is imperative to provide tailored management for the patient. Recurrence can develop up to 10–15 years after surgical therapy and therefore requires long-term follow-up.Inferences drawn from our study should be tempered by the fact that it is based on a single-centre retrospective study. The retrospective nature of this study renders it susceptible to selection bias. Given that this is a single centre study looking at a rare condition, it is compounded by the low incidence of rectal GIST resulting in our study sample to be small and hence prone to type 2 errors. Also, there are no optimal cut-off values for initiation and termination for neoadjuvant therapy. Future studies in the area of inflammatory indices may look into optimal cut-offs for patients. In addition, patients selected for neoadjuvant imatinib and the selection of surgical technique were also not properly defined. The lack of these selection criteria muddles the conclusion of our study. Furthermore, in Tables Although this study is small in number and respective in designs, it adds to the increasing evidence to support the use of neoadjuvant imatinib therapy for the treatment of rectal GIST. In selected cases that are not amenable with surgical resection at the onset, administration of neoadjuvant imatinib of approximately 9 months has the benefit of reducing the tumour size by 39% to improve resectability to achieve R0 resection with low complication rates using sphincter-sparing techniques. In addition, it confers protection from local recurrence and improved OS regardless of tumour size and tumour risk stratification. The benefits of neoadjuvant imatinib also extend to GIST of other sites. Neoadjuvant imatinib has been shown to downsize tumour to achieve complete surgical resection in gastroesophageal GIST –41. The In conclusion, our study found that a therapeutic strategy that combines neoadjuvant imatinib therapy with surgery should be considered for patients with rectal GISTs that are large, marginally resectable or unresectable or have a close relationship with vital pelvic structures, such as anal sphincters or nerves. Neoadjuvant imatinib may potentially increase the proportion of patients able to undergo curative conservative surgery rather than the more morbid extended resection.


PIK3CA occur in a subset of human HCC. Alpelisib, a selective PIK3CA inhibitor, has been approved by the FDA to treat PIK3CA mutant breast cancers. In this manuscript, we evaluated the therapeutic efficacy of alpelisib, either alone or in combination, for the treatment of HCC. We tested alpelisib in mouse HCC induced by hydrodynamic injection of c-Met/PIK3CA(H1047R) (c-Met/H1047R), c-Met/PIK3CA(E545K) (c-Met/E545K), and c-Met/sgPten gene combinations. Alpelisib slowed down the growth of c-Met/H1047R and c-Met/E545K HCC but was ineffective in c-Met/sgPten HCC. Mechanistically, alpelisib inhibited p-ERK and p-AKT in c-Met/H1047R and c-Met/E545K HCC progression but did not affect the mTOR pathway or genes involved in cell proliferation. In human HCC cell lines transfected with PIK3CA(H1047R), alpelisib synergized with the mTOR inhibitor MLN0128 or the CDK4/6 inhibitor palbociclib to suppress HCC cell growth. In c-Met/H1047R mice, alpelisib/MLN0128 or alpelisib/palbociclib combination therapy caused tumor regression. Our study demonstrates that alpelisib is effective for treating PIK3CA-mutated HCC by inhibiting MAPK and AKT cascades. Furthermore, combining alpelisib with mTOR or CDK4/6 inhibitors has a synergistic efficacy against PIK3CA-mutated HCC, providing novel opportunities for precision medicine against HCC.Hepatocellular carcinoma (HCC) is the sixth most common primary cancer with an unsatisfactory long-term survival. Gain of function mutations of  Hepatocellular carcinoma (HCC) is currently ranked as the sixth most common primary cancer and the fourth leading cause of cancer-related deaths worldwide . SorafenTP53, CTNNB1, AXIN1, ARID1A/B, and ARID2, have been detected in human HCC subsets [PIK3CA account for ~3% and rank among the top 20 mutations in human HCC based on the COSMIC database  p110α, which is critical in cancer development [PIK3CA mutations deregulate the PI3K/AKT/mTOR cascade and ultimately lead to cancer formation. PIK3CA mutations map to three sites, E542 and E545, at the helical domain , and H1047, in the kinase domain  [PIK3CA mutations [elopment , 9. In pPIK3CA mutations. Both preclinical and clinical studies have reported significant efficiency of alpelisib against tumors with GOF PIK3CA mutations [PIK3CA-mutated HCC has never been investigated. In the present study, we explored the therapeutic efficacy of alpelisib, either alone or in combination with other drugs, such as the mTOR inhibitor MLN0128 or the CDK4/6 inhibitor palbociclib against GOF PIK3CA-mutated HCC both in vitro and in vivo.Alpelisib, an orally administered PIK3CA inhibitor, has been approved by the FDA to treat breast cancers with utations –16. HowePIK3CA in liver tumors, we analyzed the PIK3CA genomic alterations in human HCCs based on the COSMIC database [PIK3CA mutations occur in 0.9–3.6% HCC samples  and PIK3CAE545K (E545K), to test this hypothesis. Successful transfection of H1047R was confirmed by western blot analysis, showing an increased expression of PI3K-p110α level . Overexpression of E545K also increased the sensitivity of HCC cells to alpelisib, although the increased sensitivity was much milder than that observed in H1047R transfected cells  .First, we tested alpelisib in the c-Met/H1047R model. As we and others previously published, total liver weight was used to estimate liver tumor burden –21. In bWe repeated alpelisib treatment studies using the c-Met/E545K HCC model , and p-AKT(Thr308)  and YAP in tumor lesions  Fig. . However08) Fig. , alpelis08) Fig.  and S11.(Ser473) levels, although the inhibition was milder than observed in the c-Met/H1047R model  and YAP expression in c-Met/E545K HCC  levels in both c-Met/H1047R and c-Met/E545K HCCs. In contrast, alpelisib inhibited p-STAT3(Tyr705) and YAP expression in c-Met/H1047R HCC, but not in c-Met/E545K corresponding lesions  and YAP levels in H1047R cells, but not E545K cells  and YAP inhibition might be involved in alpelisib dependent antitumor activity against HCC. Additional studies are required to address this critical issue.One intriguing observation of the present data is that H1047R, the kinase domain ice Fig. . At the ons Fig.  and S17aAnother important conclusion of the study is that alpelisib suppressed HCC angiogenesis in all the mouse models tested. Notably, even in c-Met/sgPten HCC lesions, where alpelisib demonstrated no efficacy, the treatment led to a decreased microvascular density. Thus, the results indicate that PI3K signaling is necessary for efficient angiogenesis in HCC. On the other hand, the data suggest that suppressing angiogenesis alone is insufficient to cause tumor regression. This observation is consistent with previous reports . NeverthAt the molecular level, alpelisib treatment effectively inhibited AKT and the downstream mTOR pathway in vitro. However, in mouse HCC models, alpelisib treatment could not suppress the mTOR cascade despite the effective inhibition of p-AKT. These findings imply that additional pathways modulate mTOR activation independent of AKT in vivo. Previous studies demonstrated that amino acid levels and the LKB/AMPK pathway could regulate the mTOR cascade , 26. AddPIK3CA mutations. We chose these molecules based on the pathway analysis using alpelisib monotherapy: alpelisib cannot inhibit the mTOR pathway and has a limited impact on cell cycle regulators. The combination with MLN0128 induced a further inhibition of the PI3K/AKT/mTOR signaling, as evidenced by the significant downregulation of p-mTOR, P-RPS6, and p-4EBP1 levels. Combination with palbociclib led to a pronounced suppression of cell cycle targets, demonstrated by the significantly decreased expression of p-RB, PCNA, CYCLIN A, and CYCLIN D1  before administration to mice. All these drugs were dissolved in DMSO for in vitro studies.The constructs used in this study, including pT3-EF1α-c-Met, pT3-EF1α-HA-PIK3CAH1047R, pT3-EF1α-HA-PIK3CAE545K, pX330-sgPten, and pCMV/sleeping beauty transposase (SB), have been described previously , 22, 29.2 atmosphere. For IC50 values detection, all HCC cells were seeded in 24-well plates and administrated with increasing concentration of drugs in triplicate for 48 h. Subsequently, tests were performed by crystal violet staining. After incubating in lysis solution for 20 min, the diluted solution was added in 96-well plates to measure OD values at 590 nm by the BioTek ELX808 Absorbance Microplate Reader (ThermoFisher Scientific). All experiments were repeated at least three times.The human HLE, SNU449, Huh7, SNU-475, SNU387, MHCC97-H, SNU-182, and HepG2 HCC cell lines were used in this study. HLE, Huh7, MHCC97-H, and HepG2 cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS (Gibco) and penicillin (100 U/mL)/streptomycin (100 μg/mL) (Gibco), while SNU449, SNU-475, SNU387, SNU-182 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium supplemented with 10% FBS (Gibco) and penicillin (100 U/mL)/streptomycin (100 μg/mL) (Gibco). Cell lines were maintained at 37 °C and the 5% COFemale wild-type FVB/N mice were obtained from Charles River Laboratories . The hydrodynamic injection was performed as described previously in detail . SpecifiLiver specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were trimmed at 5 μm in thickness. For immunohistochemistry (IHC), slides were microwaved in 10 mmol/L citrate buffer (pH 6.0) for 10 min for antigen unmasking, followed by a 30 min cool down at room temperature. Subsequently, the slides were incubated in 10% goat serum for 30 min and in Avidin/Biotin solution  for 10 min, respectively. Next, primary antibodies  together with the Halt™ Protease Inhibitor Cocktail (ThermoFisher Scientific), and concentrations were determined with the Pierce™ Microplate BCA Protein Assay Kit (ThermoFisher Scientific). Proteins were then denatured by boiling in Tris-Glycine SDS Sample Buffer (Bio-Rad). Western blot analysis was performed in SDS-PAGE, and then gels were transferred onto nitrocellulose membranes (Bio-Rad) by electroblotting. After incubation in 10% nonfat milk for 1 h at room temperature, membranes were incubated with primary antibodies (Supplementary Table The combination index (CI) was determined according to the Chou–Talalay method . HCC celt-test and Tukey–Kramer test were carried where appropriate. Probability (P) values < 0.050 were considered statistically significant.Statistical significance was assessed using the GraphPad Prism 6.0 software (GraphPad Software Inc.). Student’s Supplementary FiguresSupplementary Table S1Related Manuscript File


Candida Bombicola. The fermentation products predominantly consist of the diacetylated lactonic form that coexists with the open-chain acidic form. A systematic series of modified SLs were prepared by ring opening of natural lactonic SL with n-alkanols of varying chain length under alkaline conditions and lipase-selective acetylation of sophorose primary hydroxyl groups. The antimicrobial activity of modified SLs against Gram-positive human pathogens was a function of the n-alkanol length, as well as the degree of sophorose acetylation at the primary hydroxyl sites. Modified SLs were identified with promising antimicrobial activities against Gram-positive human pathogens with moderate selectivity . SL-butyl ester exhibited the best antimicrobial activity (MIC = 12 μM) and selectivity (TI = 33) among all SLs tested. Kinetic studies revealed that SL-ester derivatives kill B. cereus in a time-dependent manner resulting in greater than a 3-log reduction in cell number within 1 h at 2×MIC. In contrast, lactonic SL required 3 h to achieve the same efficiency.Sophorolipids (SLs) are glycolipids that consist of a hydrophilic sophorose head group covalently linked to a hydrophobic fatty acid tail. They are produced by fermentation of non-pathogenic yeasts such as  Bacterial resistance has spread worldwide and is now causing a global health crisis, which requires a global action plan . For exaCandida Bombicola [Sophorolipids (SLs) are a family of glycolipids produced by fermentation of non-pathogenic yeasts such as ombicola ,7. They ombicola ,9,10. Moombicola ,12 and wombicola , antivirombicola , anticanombicola ,16, and ombicola  propertiombicola ; and modombicola . Despiteombicola ,20 and gombicola . Howeverombicola ,23,24,25ombicola . Selectiombicola ,12,23. TIn this study, a family of SL esters b with inE. coli and several Gram-positive bacteria. The MICs of SL-methyl ester against B. cereus, B. subtilis, S. aureus, and L. innocua were estimated to be 50, 50, 200, 100 μM, respectively . The SL-butyl ester exhibited superior or equivalent activity relative to natural LSL for B. cereus, B. subtilis, and L. innocua, and to the control antibiotic, streptomycin. SL-pentyl ester was only active against B. cereus and L. innocua. Further increase in the ester chain length  did not show any antibacterial activity up to 200 μM. This loss of activity likely indicated that longer carbon chain esters failed to confer sufficient hydrophilicity to maintain a good hydrophilic–hydrophobic balance. Furthermore, LSL and all the SL-ester derivatives exhibited poor activity against E. coli (MIC > 200 μM). The lack of activity against E. coli is likely due to the presence of an outer membrane that acts as an additional barrier, as well as other defense pathways that are not present in Gram-positive bacteria [To assess the structure–activity relationship (SAR) of SLs, a family of SL-esters was synthesized from natural lactonic SL by transesterification using sodium alkoxides of different lengths b. The anectively . As the bacteria . LSL selbacteria .B. subtilis, S. aureus, and L. innocua. However, monoacetylation decreased the MIC against B. subtilis, S. aureus, and L. innocua from 50 to 25 μM. While increasing the degree of acetylation so that both the 6′- and 6″-positions were acetylated decreased the MIC to 12 μM for B. Cereus, it did not exhibit any antibacterial activity against B. subtilis, S. aureus, or L. innocua. These results indicate that SL-ester chain length, sophorose unit degree of acetylation, and cell morphology all play an important role in antibacterial activity. These results are consistent with a previous study on the effect of acetylation degree on the antibacterial activity of LSL [The degree of acetylation was reported to substantially affect several SL properties, including oil phase emulsification as well as antibacterial, spermicidal, and anti-HIV activities ,23,28. TB. cereus. B. cereus inocula (107 CFU/mL) were exposed to SL derivatives at 1×MIC, 2×MIC, and 4×MIC in PBS buffer at pH 7.4. At 15 min, 45 min, 1 h, and 3 h, aliquots were spread onto agar plates for cell counting. As shown in B. cereus, natural lactonic SL is less effective in killing this bacterium than the modified SL-ester derivatives, thus showing the benefit of molecular editing in enhancing the killing efficiency of sophorolipids.To further investigate if the growth inhibition was associated with cell death, a killing kinetic study was conducted on B. cereus cells at 2 min, a rapid decay in fluorescence intensity was observed due to self-quenching caused by migration of the probe into membranes. Subsequent addition of the SL-esters at 4 min led to a sharp increase in the fluorescence signal, thus indicating enhanced permeability of the cell membrane driven by the presence of the SL derivative. This is consistent with the time–kill studies that showed rapid bactericidal activities of SLs on B. cereus. Endpoint fluorescence data were recorded at t = 60 min and are displayed in To probe the role of LSL and the SL-esters as membrane-active agents, dye leakage experiments were carried out. Polymyxin B, a well-known membrane-active agent, was used as a positive control. Polymyxin B is a cyclic peptide with long, hydrophobic tails that can insert into cell membranes causing cell lysis. First, time-course membrane-depolarizing studies were conducted A. At timB. cereus, B. subtilis, and L. innocua). The SL-octyl ester was the most toxic of the SL-ester series, possibly due to its high hydrophobicity and affinity to cell membranes. Similarly, previous work with antimicrobial peptides (AMPs) showed that increasing their hydrophobicity enhanced their binding affinity to mammalian cell membranes [50/MICB. cereus, where EC50 is the concentration that corresponds to 50% cell death after 16 h incubations. A potential antimicrobial candidate must selectively kill pathogen cells without harming the host mammalian cells. To test the ability of SLs to effectively discriminate between bacteria and mammalian cells, a cytotoxicity assay was previously reported by our team . The SL-embranes ,30. In c3), methanol (CH3OH) and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific . All bacteria culture media and agars were purchased from Sigma-Aldrich . All other chemicals and solvents were purchased in the highest available purity from Sigma-Aldrich and were used without further purification.Novozym 435 was obtained from Novozymes . Lipase PS-C was purchased from Sigma-Aldrich . Sodium metal (large pieces in kerosene) was purchased from Fisher Scientific . 3,3′-Dipropylthiadicarbocyanine iodide (diS-C3(5)) and polymyxin B were purchased from Sigma-Aldrich. Tetrahydrofuran (THF), chloroform ). Upon completion, the reaction mixture was cooled to room temperature, acidified with 1 M HCl or glacial acetic acid to pH 4–5, and concentrated by rotoevaporation. Precipitation in ice-cold water (0.5 L), followed by filtration and dryness under vacuum, yielded the desired product as an off-white solid (80–85%). Longer alkyl chain SL esters (C4–C8) were further purified by flash chromatography. The SL esters were characterized by 1H NMR and LC-MS.Lactonic SL and SL esters were prepared with good purity (>95%) following the experimental procedures previously reported by us ,22,23,242 atmosphere, and the reaction progress was monitored by TLC. Upon completion, the enzyme was filtered out, followed by washing with THF (twice). After removal of solvent, the residue was subjected to silica gel column chromatography using chloroform/methanol as eluent (gradient: from 95/5 to 80/20) to afford the final products as off-white solids (75–85%).Selective acetylation of the sophorose primary hydroxyls was achieved using two different immobilized lipases (lipase PS-C and Novozym 435). To a solution of SL-ethyl ester  and vinyl acetate (5 or 10 eq) in dry THF (50 mL), either lipase PS-C (1.23 g) for 6′′-acetylation or Novozym 435 (1.5 g) for 6′- and 6′,6″-acetylation was added. The resulting mixture was stirred at 40 °C under an NStaphylococcus aureus (ATCC 33807), Bacillus cereus (ATCC 4342), Bacillus subtilis (ATCC 21332), Listeria innocua (ATCC 33090), and Escherichia coli (ATCC 53323) were provided by Ravi Kane’s lab at Rensselaer Polytechnic Institute. Mueller–Hinton broth (MHB) was used for the Staphylococcus aureus culture, while tryptic soy broth (TSB) was used for the other strains. All strains were grown to the stationary phase for 24 h at 37 °C with agitation at 220 rpm. After overnight incubation, cultures were diluted to 5 × 106 CFU/mL prior to use.Bacteria strains 6 CFU/mL) were added to each well, and the final DMSO concentration was 5%. Wells containing the same volumes of DMSO/PBS buffer and culture medium, with and without bacteria, were used as positive and negative controls, respectively. Streptomycin was used for comparison. The plate was then incubated with shaking for 16–30 h at 37 °C to ensure that cultures had reached stationary phase growth and the absorbance readings were recorded at a wavelength of 600 nm using an absorbance microplate reader. Absorbance data from 16–30 h were used to calculate the percent inhibition for each test condition. MIC was recorded as the lowest concentration of drug that inhibited more than 90% of bacterial growth. All assays were carried out in triplicate.The minimum inhibitory concentration (MIC) was determined using the microdilution technique. Modified sophorolipid (SL) derivatives were dissolved in DMSO to make 2 M stock solutions. A dilution series was made by diluting the stock solution to a range of concentrations from 200 µM to 4 µM and a final volume of 100 μL in 10% DMSO/phosphate-buffered saline (PBS) in each well of a 96-well plate. Bacterial inocula  and SLs  in 5% DMSO/PBS buffer at room temperature with constant shaking at 200 rpm. Aliquots of 20 µL were withdrawn periodically at t = 0, 0.25, 0.75, 1, and 3 h, and a series of 10× dilutions were made before spreading onto nutrient agar plates. The agar plates were then incubated at 37 °C for 12 h. The killing rate was measured by comparing colonies grown on each agar plate with colony counts from the untreated control.The bactericidal activity of modified SLs against The activity of SL derivatives against M1 activated macrophages (AMOs) obtained after the stimulation of Raw 254.7 (ATCC) murine macrophages with LPS  for 4 days was assessed following the procedure reported in the literature by us .B. Cereus grown to mid-log phase was adjusted to 2.5 × 106 CFU/mL in fresh MHB. Subsequently, 100 μL of cell suspensions were mixed with 50 μL of LSL and SL-ester solutions  and 50 μL of diS-C3(5) , followed by incubation at 37 °C for 1 h. Polymyxin B , a well-characterized membrane-depolarized peptide, and PBS were used as positive and negative controls, respectively. Fluorescence data were recorded at 670 nm with excitation at 622 nm on Molecular Devices SPECTRAmax Plus 384 UV-VIS High Throughput Microplate Spectrophotometer and normalized against the negative control.The disruption of bacterial membranes after treatment with LSL and SL-ester derivatives was tested using a fluorescence probe, diS-C3(5), which can be quenched by the electrically polarized membrane. B. cereus in a time- and concentration-dependent manner resulting in greater than a 3-log reduction in cell number within 1 h at 2×MIC. In contrast, LSL required 3 h to cause a 3-log reduction in B. cereus. The results of a depolarization assay performed using diS-C3-(5) as a fluorescent probe revealed that, except for LSL, all structurally edited SLs show a strong ability to enhance membrane permeability. Longer carbon chain SL esters exhibit a strong depolarizing effect despite their inability to kill bacterial cells. In contrast, LSL failed to enhance membrane permeability despite its good antimicrobial activity. These results strongly suggest that, while membrane disruption may be involved in LSL and SL-ester antimicrobial activity, additional pathways are involved.In summary, we designed and synthesized a library of SLs that exhibit promising antimicrobial activities against Gram-positive human pathogens with moderate selectivity (TI = 6–33). SL-butyl ester exhibited the best selectivity among all the SLs tested. Kinetic studies revealed that SL-ester derivatives kill