Human HepG2 cells and male Wistar rats were exposed to ethanol and simultaneously treated with probiotic V or Met alone as well as in combination. Endoplasmic reticulum (ER) stress markers, inflammatory markers, lipid metabolism, reactive oxygen species (ROS) production, and oxidative stress were evaluated, using qRT-PCR, Oil red O staining, fluorimetry, and HPLC. In vitro, probiotic V and Met in combination prevented ethanol-induced cellular injury, ER stress, oxidative stress, and regulated lipid metabolism as well as inflammatory response in HepG2 cells. Probiotic V and Met also promoted macrophage polarization towards the M2 phenotype in ethanol-exposed RAW 264.7 macrophage cells. In vivo, combined administration of probiotic V and Met ameliorated the histopathological changes, inflammatory response, hepatic markers (liver enzymes), and lipid metabolism induced by ethanol. It also improved the antioxidant markers (HO-1 and Nrf-2), as seen by their protein levels in both HepG2 cells as well as liver tissue using ELISA. Hence, probiotic V may act, in addition to the Met, as an effective preventive treatment against ethanol-induced hepatic injury.Alcohol-induced liver injury implicates inflammation and oxidative stress as important mediators. Despite rigorous research, there is still no Food and Drug Administration (FDA) approved therapies for any stage of alcoholic liver disease (ALD). Interestingly, metformin (Met) and several probiotic strains possess the potential of inhibiting alcoholic liver injury. Therefore, we investigated the effectiveness of combination therapy using a mixture of eight strains of lactic acid-producing bacteria, commercialized as Visbiome® (V) and Met in preventing the ethanol-induced hepatic injury using  Alcohol-induced hepatic injury is the main root of morbidity and mortality universally among individuals who misuse alcohol . ChronicMetformin (Met) is a commonly approved antihyperglycemic drug of the biguanide family commonly used to control type 2 diabetes. In humans, there is a resilient connection between hepatic insulin resistance and steatohepatitis as well as fatty liver. In many animal and human studies, Met has shown protection in NASH and nonalcoholic fatty liver disease (NAFLD). In NAFLD patients, Met treatment shows decreased liver dysfunction . The accLactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium longum, and Lactobacillus plantarum, had shown to possess the antioxidant potential and be able to scavenge the superoxide radicals and hydroxyl radicals. These are the radicals that would lead to oxidative stress and therefore upregulation of pro-inflammatory cytokines. The in vitro mechanism of action of probiotics could partially be explained by its antioxidant potential, which reduced the oxidative stress and further damage associated with it  and allowed to swell on ice for 20 min with sporadic mixing. Samples were vortexed and centrifuged at 10,000 × g at 4°C for 10 min. The supernatant obtained was used as a cytoplasmic extract. Later, the pellet was washed with the lysis buffer and resuspended in the nuclear extraction buffer  and incubated in ice for 30 min. Nuclear extract was collected by centrifugation at 12,000 × g for 15 min at 4°C. Proteins levels were checked using Bradford reagent To analyze HO-1 levels in the ethanol-induced hepatic injury model, 100 mg of liver tissue was mixed with 1 ml PBS and homogenized. The homogenate was centrifuged at 5000 rpm for 10 min at 4°C, and the resultant supernatant was used as the liver tissue homogenate. Proteins levels were checked using Bradford reagent.Collected cell-free supernatant, nuclear extract, and liver tissue homogenate were measured through enzyme-linked immunosorbent assay (ELISA) according to the protocol given by the kit manufacturer .p values less than 0.05.All respective experiments were performed at least thrice. Data of the replicates were calculated as mean ± SD. The GraphPad Prism 7 software  was used to analyze the results. The differences between all groups were analyzed using a one-way analysis of variance (ANOVA). Variations between groups were considered to be significant at μl/ml) and Met  individually on HepG2 cell was assessed in the presence and absence of 100 mM ethanol  also was assessed as a measure of cell viability on HepG2 cells treated in the presence and absence of 100 mM ethanol  shows a dose-dependent increase in cell viability  when compared to ethanol-exposed HepG2 cells at 48 h. The effect of Met at various concentrations in the presence of 100 mM ethanol also was assessed as a measure of cell viability of HepG2 cells. The viability of cells exposed to 100 mM ethanol in the presence of Met alone showed a dose-dependent decrease in cell viability  when compared to ethanol-exposed HepG2 cells at 48 h, which is suggestive of the toxicity of Met at higher concentration.Neither the individual treatment with probiotic V nor with Met at different concentrations showed significant cell death of HepG2 cells in the absence of 100 mM ethanol. However, there was a slight reduction observed in the viability of the cells treated with 3 mM Met alone. Treatment with 100 mM ethanol on HepG2 cells induced cell death, and therefore, only 64.6% cell viability was observed after 48 h of 100 mM ethanol treatment as compared to the untreated cells . To see if probiotic V could prevent the ethanol-induced toxicity, HepG2 cells were treated with probiotic V in the presence of 100 mM ethanol. Our results indicate that probiotic V treatment at different concentrations , but this difference was not significantly different when compared to the cells treated with only 100 μl/ml probiotic V in the presence of ethanol (85.5%). As our study is aimed at seeing if probiotic V and Met could work in synergy to prevent the ethanol-induced toxicity of HepG2 cells, we chose the combination of 10 μl/ml probiotic V and 1 mM Met, because this combination significantly improved the cell viability of ethanol-exposed HepG2 cells, compared to their respective individual treatments.As the treatment with probiotic V alone showed a dose-dependent increase, and the treatment with Met alone showed a dose-dependent decrease in the viability, we checked if the combination of probiotic V and Met could work in synergy to improve the cell viability of HepG2 when treated in the presence of 100 mM ethanol. The combinatorial doses of probiotic V and Met did not significantly affect the viability of HepG2 cells treated in the absence of 100 mM ethanol across all the different combinations. However, we observed that probiotic V treatment at various concentrations rescued the toxicity of 3 mM Met and improved the cell viability. This suggests that probiotic V can improve the efficacy of metformin by reducing its toxicity at a higher concentration. Also, the viability of the cells treated with 100 mM ethanol in the presence of different combinations of probiotic V and Met was improved as compared to ethanol-treated HepG2 cells in the absence of probiotic V and Met, which suggests the beneficial role of probiotic V and Met as combinatorial treatment. The most significant difference in the cell viability was observed with a combination of 100 The HepG2 cells when treated with ethanol showed a change in their morphology as seen by the constricted cell membrane and distorted shape of the cells  as compa8 CFU/day) demonstrated normal hepatocytes and central vein with full restoration. Moreover, feeding probiotic V and Met in combination could significantly prevent hepatic lipid accumulation and safeguard the hepatic cells as compared to probiotic V or Met alone . The ethanol-fed rats demonstrated a dramatic increase in fat droplets with cellular inflammatory infiltrations and disorderly arrangements of hepatocytes as the representative pathological change compared to the control group. Compared with this, Met (75 mg/kg) depicted congested central vein and disorderly arranged hepatocytes. On the other hand, probiotic V  when compared with control cells (1.54 ± 0.28 nM/ml). Co-administration with probiotic V and Met in combination reduced the levels of MDA more efficiently (4.76 ± 0.8 nM/ml) as compared to ethanol-exposed HepG2 cells. However, there was a significant difference in the levels of MDA in either probiotic V (8.04 ± 0.53 nM/ml) or Met (8.04 ± 0.55 nM/ml) treated groups as compared to the combinatorial dose of probiotic V and Met in ethanol-exposed HepG2 cells . TherefoμM at 37°C for 1 hour in the dark. Cotreatment of ethanol-exposed cells with probiotic V and Met in combination was associated with reduced ROS production in comparison with HepG2 cells exposed to ethanol alone, as well as compared to the individual treatment of probiotic V or Met  . ExpressAccumulation of lipids was analyzed through Oil red O staining of cells microscopically and quantified using a spectrophotometer at 570 nm by isopropanol extraction method . To deteThe total lipid accumulation was found to be elevated in ethanol-exposed HepG2 cells compared to control cells. Upon administration of probiotic V and Met alone as well as in combination with ethanol-exposed HepG2 cells, the amount of lipid accumulated was decreased at 48 h. The results demonstrated that complete lipid accumulation was observed in ethanol-exposed HepG2 cells, which was restored by probiotic V and Met in combination, with better efficacy as compared to the individual treatment of either probiotic V or Met .ALT, AST, and ALP are fundamental biochemical markers indicative of liver injury. The present study showed a significant increase in serum transaminases (ALT and AST) and ALP levels in the ethanol-fed rats compared to the control group. The elevated serum levels can be due to the outflow of cellular enzymes into the bloodstream. Treatment with either probiotic V or Met significantly reduced the serum levels of ALT, AST, and ALP as compared to the ethanol-fed group, which was further downregulated by the two in the combination when compared with the ethanol-fed group as well as individual treatment of probiotic V or Met.Also, serum levels, as well as hepatic contents of total cholesterol (TC) and triglyceride (TG) in the ethanol-fed rats, were significantly higher compared to those in the control group, which were dramatically reduced in the combinatorial treatment of probiotic V and Met in comparison to the ethanol-fed group as well as individual treatment of probiotic V or Met . These rChronic alcohol consumption leads to abnormal lipid metabolism in the liver by decreased adenosine monophosphate-activated protein kinase (AMPK) activation which leads to increased sterol regulatory element binding protein 1c (SREBP-1c) expression, further leading to increased lipogenesis through activation of downstream lipogenic genes like acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in ethanol-exposed HepG2 cells as well as ethanol-fed rats. Individual treatment of either probiotic V or Met administration activates adenosine monophosphate-activated protein kinase (AMPK), a critical regulator of lipid metabolism, which is otherwise inhibited in the presence of ethanol-exposed HepG2 cells and ethanol-fed rats. Activation of AMPK by either treatment of Met or probiotic V inhibits the expression of transcription factor, i.e., sterol regulatory element binding protein 1c (SREBP-1c) , thereby inhibiting the ethanol-induced lipogenesis. Consistent with the altered expression of SREBP-1c, it also decreases the expression of downstream lipogenic enzymes like acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) as compared to ethanol-exposed HepG2 cells as well as ethanol-fed rats, which was more significantly reduced as compared to either individual treatment of probiotic V or Met.γ and HSL are present at basal levels; however, treatment with ethanol upregulates the hepatic expression of PPAR-γ and HSL in comparison to the control group, leading to increased lipogenesis. The treatment with either probiotic V or Met downregulated the expression levels of PPAR-γ and HSL which resulted in reduced lipogenesis as compared to the ethanol-exposed HepG2 cells as well as ethanol-fed rats. This was further diminished more significantly by the two in combination when compared with the ethanol group as well as probiotic V- and Met-unaided groups , and receptor mediating inflammatory response, i.e., TLR-4, was found to be upregulated, and mRNA expression levels of anti-inflammatory cytokine like IL-10 and oxidative stress markers like heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf-2) were found to be downregulated in ethanol-exposed HepG2 cells and ethanol-fed rats. We observed that mRNA expression levels of CYP2E1 and TLR-4 as wells pro-inflammatory markers were dramatically diminished, and anti-inflammatory cytokine IL-10 and oxidative stress markers (HO-1 and Nrf-2) were largely upregulated in ethanol-induced HepG2 cells and ethanol-fed rats treated with probiotic V and Met alone. However, probiotic V or Met in combination could additionally diminish the expression of pro-inflammatory markers and more significantly elevated the IL-10, HO-1, and Nrf-2 expression levels as compared to probiotic V or Met-unaided groups.Ethanol-induced hepatic injury can also cause chronic inflammation. Thus, we evaluated the effects of probiotic V and Met alone, as well as in combination on inflammatory response and oxidative stress markers Figures  and 11. in vitro and in vivo models of ethanol-induced hepatic injury.As shown in α, TLR-4, iNOS, and NADPH oxidase 1 (NOX-1) and increased expression levels of M2 phenotypes, such as IL-10 and Arginase-1 as compared to ethanol-induced RAW 264.7 cells  found that metformin at a dose of 200 mg/kg/day attenuated the liver injury caused by chronic ethanol exposure . Also, ivo model . TherefoL. acidophilus can ameliorate ER stress in HepG2 cells [in vivo [Ethanol-induced ROS production plays a key role in the progression of ALD and limits the cytoprotective gene expression . TherefoG2 cells  and HT-2[in vivo  by decreγ play a crucial role in lipid metabolism. In the liver, increased lipogenesis was observed with the hepatic overexpression of PPAR-γ [γ [L. plantarum administration reduced the hepatic weight and the mRNA expression levels of lipogenic genes PPAR-γ and HSL [Lactobacillus rhamnosus GG activated AMPK and thereby downregulated the expression levels of SREBP-1c, ACC, and FAS in ethanol-induced liver injury model [Chronic alcohol consumption exerts a detrimental effect on lipid metabolism in the liver resulting in hepatic steatosis . During f PPAR-γ . HSL traPAR-γ [γ . Our rep and HSL . Also, cry model , 55.Another important marker of alcohol-induced liver injury is demonstrated by the increased levels of TG, which is indicative of hepatic steatosis. Fatty acid oxidation and tricarboxylic acid (TCA) circulation get disturbed due to excessive alcohol intake which affects the lipid metabolism, causing increased accumulation of TG in the liver and therefore elevates the serum levels of TG . FeedingLactobacillus [Ethanol-induced liver injury is linked to an increased level of nitrative stress. In a mouse model, it was observed that the ethanol-fed group was having an increased mRNA expression of ROS-generating enzymes like iNOS by 3.6 fold . iNOS exbacillus  can prevAlterations in the development of ROS/RNS (reactive nitrogen species) result in the onset of oxidative stress. Acetaldehyde and MDA are produced due to oxidation of the alcohol during its metabolism, and an increased level of MDA is an oxidative stress biomarker . Met aloPersistent ROS results in inflammation which leads to a major number of illnesses in humans. Cells that are impaired due to oxidative stress induce inflammation upon alcohol ingestion . Chronicα cytotoxicity and IL-1β secretion are known to be increased by ethanol exposure in the HepG2 cells and in rat primary hepatocytes [α in colorectal cancer (CRC) and type 2 diabetes [α and IL-1β, which were downregulated more significantly by cotreating probiotic V and Met in combination as compared to the individual treatment of either agent. TNF-α also downregulated by the production of IL-10, which otherwise is decreased in HepG2 cells challenged with 100 mM ethanol [TNF-atocytes , 58. Metdiabetes . Our res ethanol  consisteL. reuteri) and antibiotic metronidazole revealed modest improvement of the inflammatory pathway: LPS/TLR-4/NF-κB/TNF-α [TLR-4 plays an important role in the development of ALD. In HepG2 cells, increased TLR-4 expression was induced by 100 mM ethanol for 20 h . In the α and IL-6 in RAW 264.7 cells stimulated by 100 mM ethanol for 24 h and that Met-induced RAW 264.7 macrophage towards the M2 phenotype [α, and NOX-1. Our study observed the combined treatment of probiotic V and Met also upregulated the expression levels of M2 phenotypic markers like Arginase-1 and IL-10 compared to ethanol-treated RAW 264.7 cells. This study additionally concludes that combinatorial treatment of probiotic V and Met may serve as a preventive target for ALD progression as it helps in translating the pro-inflammatory M1 phenotype macrophages (tissue-damaging infiltrative macrophages) to M2 phenotype macrophages (anti-inflammatory tissue restorative cells) more significantly compared to the probiotic V or Met-unaided groups.In the liver, macrophages also play an acute role in the progression of alcohol-induced inflammation and the accumulation of infiltrating macrophages . Excessihenotype . Given thenotype , it was in vivo hepatic injury by CYP2E1 inhibition. The reduced expression levels of CYP2E1 induced by probiotic V and Met are associated with the increased Nrf-2 translocation as the Nrf-2 translocation and HO-1 activation accelerate the heme decomposition, resulting in a reduced CYP2E1 protein level [κB or MAPK, triggering the production of inflammatory mediators like TNF-α, IL-1β, and IL-6, thereby preventing the ethanol-induced hepatic injury.In the current study, the combination of probiotic V and Met helped Met in potentiating its antioxidative and anti-inflammatory effects. Our findings indicate that probiotic V and Met in combination prevent ethanol-induced cytotoxicity by inhibiting oxidative stress and ROS generation and protect human HepG2 cells and the in level . We hypoResults showed in the current study lead to numerous conclusions showing the contribution in the improvement of direct therapies by the combined presence of probiotic V and Met. Results from the present study suggested that co-administration of probiotic V and Met with ethanol exposure was efficient in improving the cellular injury and liver histology in the ethanol-exposed HepG2 cells, RAW 264.7 cells, and ethanol-fed rats. Also, co-administration of probiotic V and Met with ethanol considerably reduced the inflammatory response and lipid accumulation, upregulated antioxidant levels, and regulated the lipid metabolism in an in vitro and in vivo model of ethanol-induced hepatic injury. The present study inferred the influence of combined treatment of probiotic V and Met in the presence of ethanol at the cytokine expression as well as protein levels. Therefore, reduced levels of ROS and oxidative stress as well as inflammatory markers might be the potential targets for the preventive effects presented by probiotic V and Met in combination. The activation of the MAPK/Nrf-2/HO-1 signaling pathway might be a possible mechanism contributing to the combined protecting effect of probiotic V and Met against ethanol-induced HepG2 cytotoxicity and hepatic injury . Among a


The reliability and durability of reinforced concrete structures depend on the amount of concrete cracking. The risk associated with cracks generates a need for diagnostic methods for the evaluation of reinforced concrete structures. This paper presents the results of a study of 10 single-span reinforced concrete beams to follow the process of crack formation and changes in their width. The beams were loaded to failure with two forces in a monotonic manner with unloading and in a cyclic manner. Continuous observation of the crack formation process was provided by the digital image correlation system. The simplified method for estimating the maximum crack width is proposed. The presented results confirmed the stochastic character of the process of crack formation and development. The maximum crack widths calculated on the basis of the proposed formula were on the safe side in relation to those calculated according to Eurocode 2. It was also confirmed that the distances between cracks do not depend on the loading manner. Hence the density function describing the distribution of distances between cracks can be used to assess the condition of reinforced concrete elements. The research has also shown the suitability of the DIC system (ARAMIS) for testing concrete elements. Concrete is the most extensively used material in the construction process, with the annual global consumption level at 4.2 billion tonnes by volume . Its proThe main benefits of this composite material include strength, durability, shock resistance, relatively low deformability, high fire resistance, and resistance to temperature and moisture fluctuations. It provides a high degree of stiffness and is easy to make into any shape. These properties make concrete a safe and durable option for various construction purposes—from civil engineering to residential to hydraulic engineering projects.Reinforced concrete (RC) structures, most common among the structures built from concrete, are exposed to static and dynamic loads, extreme weather and cyclic temperature changes. As a result of physical, chemical, mechanical, physicochemical and biological impacts, the structural materials used in making RC structures deteriorate, thereby shortening their safe use.Advances in technology have resulted in a trend towards the use of increasingly higher strength concrete and steel. Economical use of high quality and high ductility steel leads to correspondingly larger deformations, contributing to an increase in displacements and crack widths. These effects are not relevant for the load-bearing capacity of reinforced concrete elements, but they have a decisive influence on the structure’s performance, durability included. Not surprisingly, with the changing performance characteristics of concrete and steel, the state of cracking is still a subject of research.The multitude of factors contribute to concrete cracking ,6,7,8,9.One relies on the fracture mechanics and concrete resistance modeling of concrete ,15,16,17The second approach, applied in this paper, involves tracking the changes in crack formation and crack width to develop diagnostic methods for the evaluation of reinforced concrete structures whose condition depends on the intensity of cracking. Cracking intensity is the number of cracks per unit length a member and the crack width which determines the reinforcement corrosion risk. Random variables influence the crack width size; hence the crack width is also a random variable of which the probabilistic or the evaluation properties follow a normal or log-normal distribution. In terms of the limit states, In the limit states view, the maximum crack width is a quantile at β, where the probability of this event β is fixed a priori and can be a function of the purpose and durability of the structure. From a practical point of view, of particular importance is the maximum crack width estimation accuracy, which decides the risk of corrosion . CorrosiAlthough various crack width estimation methods have been developed over the years, research needs to be continued ,7,13,21.This paper presents the results of a broad-range program of testing reinforced concrete beams subjected to monotonic loading with unloading and cyclic loading. The research was conducted with a view to developing a method of diagnosing reinforced concrete bridges.In parallel, work was carried out on diagnostics using acoustic emission technique ,25.Since the durability of reinforced concrete structures depends on the intensity of cracking and crack widths, a parallel topic of tracking the process of crack formation and development was undertaken. A known density function for inter-crack distances and crack widths under loading will allow the development of a method for RC structure condition diagnosis based on artificial intelligence. A diagnosis that relies on methods, i.e., acoustic emission and artificial intelligence will be much more reliable, particularly in difficult cases.The formation of cracks and their development was recorded using the ARAMIS digital image correlation (DIC) system. The system provides a 3D real time display of strain field overlaid on live image, thereby allowing a continuous measurement of crack widths. The use of a power supply system with a controller, which was synchronized with the measuring equipment, enabled the implementation of cyclic loads.The crack width test results were compared with those calculated according to EC2  and withTen reinforced concrete (RC) beams manufactured at the prefabrication plant producing bridge elements were subjected to bending tests. The beams were simply supported and had two concentrated loads applied at a distance of 1 m from the supports. The beams, each 120 mm × 300 mm in cross section and 3300 mm in length (3000 mm effective span), were ordered from concrete C40/50, and the bars were made of B500S (class B) steel. The longitudinal reinforcement ratio at the beam tension zone was 2%. Four of the beams were designed without stirrups, and two had no compression reinforcement in the central section. Different loading regimes were applied: monotonically increasing load until failure, loading and unloading and low-cycle loading. The tested members are shown in Cement CEM I 52,5N HSR NA Chełm—390 kgWater-cement ratio 0.4Water—155 kgAggregates—basalt 8/16–694 kg (35.2%), basalt 2/8–617 kg (31.3%), river sand 0/2–660 kg (33.5%)Superplasticizer—1.84 kgAir entraining agent—0.47 kg.Material composition and properties of the concrete mixture obtained from prefabrication plant for concrete grade C40/50:The beam reinforcement design with or without longitudinal bars at the compression zone and with or without stirrups in the midspan section of the beam is illustrated in Three different loading regimes were used, marked as M—monotonic loading progressively increased to failure, O—loading and unloading, and C—low-cycle loading, as compiled with loading rates in The strength of the concrete was tested on the 150 mm × 150 mm × 150 mm cuboids fabricated while casting particular concrete beam pairs in the multi-cavity mould. The failure force test results for the concrete cubes, the calculated compressive strength of the concrete and the relevant statistics are presented in Average concrete compressive strength estimated according to (1)Characteristic concrete compressive strength according to (2)Class of concreteModulus of elasticity according to (3)For example, p0.2, upper yield point ReH and tensile strength Rm.A total of 124 bars used for the main reinforcement were examined, including 58 bars of ϕ 12 mm and 66 of ϕ 14 mm. An example of the stress–strain relationships obtained during the tensile test of BS500 steel bars is shown in The test setup for the reinforced concrete beams was as shown in During the loading, displacements were continuously measured using the HBM measuring system and the field deformation of the middle section of the beam (between forces) by the Digital Image Correlation (DIC) system. The HBM—Hottinger Baldwin Messtechnik system was used to record deflections in the midspan of the beam, under forces and near the supports, while the DIC system was used to measure strains in three directions of the previously prepared central surface, including cracking on the side surface of the beams. The actuator’s analogue outputs allowed synchronizing the entire test apparatus, and the recorded force was used to control the HBM and DIC systems measurements. Apparatus synchronization enabled assigning deflection results and the image of strains within the tested field for each force value in the middle section of the beam under load.DIC is widely used in many fields of science, e.g., biomechanics, astronautics, microelectronics and now also in civil engineering ,36,37,38Analysis of the cracking process in tests conducted with the DIC system is performed after the test has been performed, using the results of calculations for subsequent images presented in the form of strain maps. The system is used for non-contact three-dimensional strain measurements. It performs calculations and documents deformations. The system uses a series of images from two digital cameras on one tripod. It recognizes the surface texture of the sample area in the images. The first photo in the series is treated as a photo of the object before loading and is used as a reference “object”. After taking the measurements, the system generates a grid on the first photo. The grid consists of small square or rectangular planes called facets. It assigns each facet a single and unique texture of grey dots. The unique texture makes it possible to find facets in subsequent images. Based on the similarity of the facets in the successive images, the strain of the facets is calculated.Consecutive images in the form of strain distribution maps are used in the analysis of the cracking process. Analysis of the strain map indicates local strain accumulations at the lower zone of the beam in tension, identified as cracks in concrete .i ≥ 0.05 mm.The measurement of the crack width  included computing the change in the length of section ΔL at a random force level ,39 throuTo illustrate the correctness of the calculated crack widths, The results obtained with the magnifier and the DIC system fall within the magnifier’s error area. Measurement uncertainty, described in , is ±0.0This satisfactory agreement made it possible to use the DIC system to record and measure the quantities describing the cracking state of the beams based on consecutive images. The strain measurements were recorded over the entire prepared area of the beam, thereby enabling the measurement of distances between the cracks and crack widths at the height corresponding to the centroid of the tension reinforcement during loading to failure. The frequency of image triggering events was adjusted to each loading program. The images were captured either at a specified time or at appropriate load values. A single tripod with two cameras was used to capture images every 30 s for A2M beams and every 20 s for C2M and D2M beams.When testing A2O beams, the so-called “triggerlist” was used, and a program for taking images triggered by a defined voltage corresponding to a given load was written. The photos were taken with one DIC system tripod with two cameras at defined force levels, as shown in For beams A2C, the images were captured intermittently due to the limited system memory. In this way, with each image, one actuator achieved the maximum load for a given phase, and the other actuator—the minimum load, because the actuators worked with a phase shift, i.e., alternately, as shown in cr, the distance between the cracks sr and the crack width w. The last two of these quantities are functions of primarily the bending moment M, which can be written as:Three quantities typically characterize the cracking state. These are the cracking moment Mcr, the distance between the cracks would be greater than the beam length, and the crack width would be zero. The crack width and the distance between the cracks are thus strongly interrelated. Accordingly, the results of the RC beam test are presented for these three quantities.All these characteristics are interrelated. It can easily be shown that the cracking moment is a special property of these functions (4) and (5), because for M < MThe use of the DIC system in this study enabled the analysis of crack formation and development processes  for the loading program specified in As an example, The history of crack formation versus the bending moment in the middle segment X = 1000 mm in length on the A2M-2 beam side surface is shown in Deterministic crack formation criteria lead to a description in which all cracks are formed simultaneously over the entire section of the beam, where the moment reaches the critical value for cracking. The cracking on the middle section of the beam, between the forces, proceeds gradually, despite the constant value of the bending moment in this section, and new cracks appear even at the load that significantly exceeds the cracking load. Therefore, it can be concluded that cracks in the beam subjected to pure bending action under increasing load initiate successively as a result of a random spread of strength and forming limit in tension, which proves that both the distance between cracks and crack width are, in fact, stochastic processes. The deterministic description of the distance between the cracks can only refer to the final state of the cracking process.The relationship in A crack formation diagram similar to that in The results confirm that the crack formation and development are stochastic, regardless of the loading program. The non-parametric tests  used to analyze the results obtained, lead to the conclusion that there is no correlation between crack distances and loading method.The crack formation graphs were used to determine the third quantity describing the cracking state, i.e., the cracking moment. Analysis of the results confirms that the crack width and the distance between the cracks depend on the moment value, while the cracking moment is not moment-dependent. A small cracking moment in beams A2M-1, A2M-2 and A2O-1 proves that during concrete hardening, some action  resulted in the formation of an internal network of micro-cracks in its structure. Therefore, immediately after the load was applied, the internal microcracks developed into cracks visible on the side surface of the beams. This shows that many factors, some difficult to establish, affect the formation of cracks in concrete elements and that it is essential to test early age concrete.Crack width is a crucial characteristic for the cracking limit state because it is the crack width, or rather its maximum value that decides the durability of concrete structures. Checking the cracking limit state of a structure consists of calculating whether cracks will appear in the structure under the influence of loads or, more generally, actions on the structure, or whether the crack width exceeds the permissible size. Thereby the calculations involve the estimation of the maximum value, which, as it depends on many factors, is characterized by a substantial random scatter and thus, the estimate will be more or less accurate. From a practical point of view, it is therefore important to estimate the maximum crack width in a safe and relatively simple manner.w—crack width,a′, a″—passive section measured from the cross-section with the crack,2a = a′ + a″s—steel strain in the section with the crack,εa.ω ≤ 1—geometric factor for steel and concrete strain differences in section As proposed in , the proThe size of the area surrounding the crack, where stress decreases, is generally a random function. However, in search of a simplified formula, the continuous changes of the crack width were measured using the DIC system. The measurement of the crack width development under loading indicates the linear increase of up to 0.9 of the failure force , as showM—bending moment values1—cross sectional area of tension reinforcementAs—steel modulus of elasticityEz—lever arm of internal forces of the flexure section.Known crack width enables the estimation of the 2aω value which is the product of the length of the bond loss section 2a = a′ + a″ and the geometric factor for strain differences between steel and concrete d—effective depth of a cross-section2—distance of compression edge of the beam section to the centroid of the compression reinforcement.dThe lever arm of internal forces in cracked flexure sections does not change significantly in the loading process, so, without much error, it is possible to take this value as a constant and calculate it based on the work of the section in bending in phase IIa . Since BS500 steel reinforcement was used in the compression zone of the tested RC beams, the maximum internal force arm was assumed to be:2aω was calculated from Formula (7). The results of the calculations are given in The maximum crack width was obtained from the DIC system at selected load levels for the tested five beams with a constant moment in the central segment, and the value of The obtained results are presented graphically in When analyzing the Analysis shows that estimating the crack width from the simplified Formula (6), assuming the value of 2aω from Dependence (9) based on the concept of the probabilistic model of crack formation presented in The reliability and durability of a reinforced concrete element is closely related to the extent and severity of concrete cracking, which has a profound effect on reinforcement protection against corrosion. Concrete cracking research follows two approaches.One approach is based on the fracture mechanics and modeling of concrete resistance including cracking. This approach looks at the influence of concrete mix components, aggregate size, internal mage, additives used and stress. Fracture mechanics and fracture toughness modeling have been covered in many papers ,15,16,17The obtained results of continuous measurement of crack width changes during the loading process allowed the formulation of a simplified formula for estimating the maximum crack width;The maximum crack widths estimated on the basis of the proposed simple relation were on the safe side in relation to those calculated according to the Eurocode 2;The results of the analysis of the distances between cracks developed in the form of drawings and tables illustrate that the formation and development of cracks is a stochastic process, and also allows the conclusion that there is no relationship between the distances between the cracks and the method of loading;The optical system consisted of a tripod with an arm (distance between cameras) that ensured the observation of surfaces of dimensions 1.5 m × 1.0 m for use in the study of the process of crack formation and development in reinforced concrete beams.The second approach, which is presented in this paper, is the tracking of crack pattern and crack width changes, utilized toward the development of methods for the assessment of reinforced concrete structures. The process of the loading of reinforced concrete beams is accompanied by the process of crack formation. As the load increases, the initially rapid changes in the crack pattern decrease and the pattern gradually stabilizes. The crack formation process depends on many factors, but for a given element it is a function of the load. It is thus a function of one variable, and the quantities such as shape, section dimensions, type of reinforcing steel, concrete mix composition, etc., act as parameters. The combined effect of these factors determines the intensity of the process which is a function of load. The crack intensity is the number of cracks per unit length of the element and the crack width, which determines the corrosion protection of the reinforcing bars. The paper presents the results of an extensive research program on reinforced concrete beams subjected to monotonic loads, and cyclic loads. The research presented in this paper was carried out with a view to developing a method for diagnosing reinforced concrete bridges; in particular, using the acoustic emission method ,25. SincThe obtained results are the basis for further work, that will include consideration of diagonal cracks that arise at the section of the combined action of the bending moment and transverse forces, and the preparation of the experimental function of crack density and its verification.


The subject of the research was the production of silsesquioxane modified rigid polyurethane (PUR) foams (POSS-Cl) with chlorine functional groups  characterized by reduced flammability. The foams were prepared in a one-step additive polymerization reaction of isocyanates with polyols, and the POSS modifier was added to the reaction system in an amount of 2 wt.% polyol. The influence of POSS was analyzed by performing a series of tests, such as determination of the kinetics of foam growth, determination of apparent density, and structure analysis. Compressive strength, three-point bending strength, hardness, and shape stability at reduced and elevated temperatures were tested, and the hydrophobicity of the surface was determined. The most important measurement was the determination of the thermal stability (TGA) and the flammability of the modified systems using a cone calorimeter. The obtained results, after comparing with the results for unmodified foam, showed a large influence of POSS modifiers on the functional properties, especially thermal and fire-retardant, of the obtained PUR-POSS-Cl systems. Foams are the main group of polyurethane materials that dominate the market with over 65% of the world’s polyurethane production ,2. They The main drawback of PUFs is their easy ignition and high flame spreadability, which makes their use limited in many engineering applications . It is kRecently, there has been an increasing amount of research into a multifunctional group of flame retardants, polyhedral oligomeric silsesquioxanes (POSSs), that combine characteristics of organic and inorganic materials ,23. TheiWhen considering the effect of POSS on flame retardancy, two main types of these nanofillers are distinguished: containing eight identical R groups or seven R groups and one functional group R’ which can be an alcohol, ester, epoxy, isocyanate, amine, or silane. The wide range of these substituents enables their selective selection to achieve compatibility of dispersed nanofillers with the polymer matrix. As with the use of other fillers, the incorporation of POSSs into a polymer matrix can affect both the melt viscosity, mechanical strength, and thermomechanical and electrical properties of the polymers . MoreovePolyol premixes are typical non-Newtonian shear-thinning fluids for which the viscosity decreases with increasing shear rate. The viscosity of the polyol premixes is a parameter that determines the rate of foam growth. Therefore, it is important that the viscosity is not too high due to the addition of solid excipients, as this would drastically limit the foam growth process, resulting in a composite with a very small pore size and high density.As expected, the addition of POSS particles increased the viscosity of the polyol , with thThe polymerization reaction of PUR foams is a strongly exothermic reaction with the release of a large amount of heat. For the reference foam, the maximum temperature was 118.2 °C. On the other hand, the introduction of POSS-Cl significantly increased the reactivity of the system, which was manifested by an increase in the maximum temperature during foam synthesis. The largest increase in Tmax was recorded for the PUR-POSS-Ar-Et-Cl system (141.2 °C). In addition, the individual times of foam synthesis have also changed. Due to the higher viscosity value, the initial foaming was slower, which was manifested in the longer creaming time of the composition with the addition of POSS. Compared to PUR-0, creaming time increases from 40 s to more than 45 s. However, well-dispersed solids can act as an additional nucleation center, leading to the formation of more vesicle cells during the nucleation process, which could be manifested by accelerating foam expansion, where the rising time shortened from 270 s for PUR-0 to even 215 s for PUR-POSS-Pr-Cl. The introduction of the modifier also leads to a shortening of the tack-free time. This means that the POSS-Cl particles can act as a hardening accelerator during the foaming process.The cellular structure is an important parameter of porous materials, determining their mechanical and thermal properties. One of the most important parameters determining the use of PUR foams as a thermal insulation material is the apparent density, which indirectly affects the mechanical properties, such as compressive strength and hardness. Changes in the structure as a result of the introduction of POSS are manifested by an increase in the apparent density of foams . The reaTo determine the mechanical properties of the PUR foam, the compressive strength at 10% deformation was tested. Since in rigid PUR foams there is anisotropy of cells manifested by cell elongation in the direction of foam growth, the samples were tested in two directions: parallel and perpendicular to the direction of foam growth . By analThe obtained results of the highest bending stress transmitted by the sample (δfm), called the flexural strength, are shown in The presence of Cl in the POSS particle resulted in its low surface energy, which could translate into a reduction in the hydrophobicity of the entire system . TherefoThe measurement of short-term water absorption showed a positive effect on the reduction of water absorption by foam. In all modified foams, it was possible to reduce the water absorption, where the use of POSS-Ar-Et-Cl allowed for a 30% reduction in this parameter. This effect was caused not only by the nature of the halide POSS themselves but also by the more irregular structure which also has a strong influence on the hydrophobicity of the surface.The reduction in affinity for water is also well illustrated by the results of the measurements of the contact angle of the foam surfaces, which show that the foams with the addition of POSS-Cl are characterized by much lower wettability by water compared to PUR-0. This test also confirmed the most hydrophilic nature of the sample with the addition of POSS-Ar-Et-Cl, for which a contact angle of 140° was obtained .To evaluate the influence of the applied flame retardants on thermal stability, TGA measurements were performed. 5 is the temperature corresponding to a 5% weight loss, similarly T10, T50 and T70 corresponding to 10, 50 and 70% weight loss to the initial mass of samples. Comparing the foams containing the POSS additives with the reference PUR-0 foam, it has been noticed that modified PUR foams required a higher temperature to pass between the degradation steps. The results summarized in the table show that the PUR-POSS-Pr-Cl foam reached temperatures slightly higher than the reference sample during the subsequent stages of degradation. On the other hand, PUR-POSS-Ar-Cl and PUR-POSS-Ar-Et-Cl samples show increased thermal stability, which is especially noticeable in the analysis of weight loss over 50%.The determination of char residue by TGA is a relative method of assessing the fire resistance of a polymer . The foaThe fire resistance properties of PUR based on combustion in a cone calorimeter are represented by the ignition time (TTI), the peak heat release rate (pHRR), and total smoke release (TSR), total heat release (THR), the maximum average rate of heat emission (MAHRE) as well as the total smoke release (TSR) and char residue.2, respectively, compared to 268 kW/m2 for the reference sample. A similar trend was observed for MARHE. Such an improvement in the fire resistance of PUR composite foams may be related to the formation of a protective carbon layer in the initial decomposition phase, which is an insulating barrier for the material beneath it and prevents the transfer of necessary heat and gases for further combustion. The carbon layer can also reduce the amount of smoke and harmful gases released during combustion. It is also believed that POSS may migrate to the surface during initial ignition and be degraded by homolytic breakage of Si-C bonds. The resulting ceramic layer consisting of stable Si-O bonds creates a reinforced carbon layer, which protects the material against further combustion [Analyzing the data in mbustion . In addiAmong all series of PUR composite foams, the greatest improvement was observed for PUR composite foams containing POSS with aromatic structures a,b—the pN,N-Dimethylcyclohexylamine), flame retardant (Tris(2-chloro-1-methylethyl) phosphate), a chain extender  and water as a blowing agent. PUR foams were modified with silsesquioxanes functionalized with halogen: chloropropylisobutyl-POSS (POSS-Pr-Cl), chlorobenzylisobutyl-POSS (POSS-Ar-Cl), chlorobenzylethylisobutyl-POSS (POSS-Ar-Et-Cl) from Hybrid Plastics, Inc . Their structural formulas are shown in The water-blown RPUFs used in this study were obtained from a two-component system supplied by Purinova Sp. z o. o., after mixing the polyol (Izopianol 30/10/C) and the diphenylmethane diisocyanate (Purocyn B). The polyol is a mixture of components containing polyester polyol , catalyst  was placed in a plastic cup and intensively mixed at 1000 rpm with a mechanical mixer for 60 s. The appropriate amount of POSS-Cl modifier was then added to the cup and mixed for another 60 s to obtain a homogeneous dispersion. The calculated amount of isocyanate (Purocyn) was added to the reaction mixture and thoroughly mixed for 30 s. The reaction mixture was allowed to grow freely by measuring the individual cream, growth and tack-free times, and the maximum reaction temperature (Tmax). The grown PUR foams were left at room temperature for 24 h to ensure complete curing of the composites.The viscosity of the polyol systems was evaluated using a Viscometer DVII+  in the function of a shear rate according to ISO 2555. The measurement was performed at ambient temperature.The apparent density of the analyzed foams was measured according to the standard ASTM D1622 . The density was tested on five samples of each foam and expressed as an average.Cell size distribution and foam morphology were examined based on the cellular structure pictures of foams taken using JEOL JSM-5500 LV scanning electron microscopy . The microscopic research was carried out in a high-vacuum mode and at the accelerating voltage of 10 kV.A three-point bending test was carried out accordingly to the standard ASTM D7264  using a Zwick Z100 Testing Machine . The analyzed samples were bent with a speed of 2 mm min−1. For each series of foams, at least five measurements were made. Obtained flexural stress at the break results for each sample was expressed as a mean value and averaged.−1. The compression strength was examined as a ratio of the load causing 10% deformation of samples cross-section in both parallel and perpendicular direction to the square surface. The compressive strength was measured in five samples of foam (8 cm × 8 cm × 5 cm) and expressed as an average.The compressive strength (σ10%) of the foams was determined according to the standard ASTM D1621 . The measurement was conducted using a Zwick Z100 Testing Machine  with a load cell of 2 kN and a speed of 2 mm minSurface hydrophobicity of PUR foams was measured using contact angle goniometer OEC-15EC  with software module SCA 20. Water absorption of PU foams was performed according to ISO 2896:2001.−1) and in the temperature range between 25 and 600 °C (heating rate 10 °C min−1). The measurement included an analysis of the mass change as a function of temperature during thermal decomposition of the polyurethane foams. The initial temperatures of the following decomposition stages were noticed and designated as T5%, T10%, T50%. These temperature values corresponded to the percentage of weight loss.The thermal stability of the foams was analyzed using a Mettler Toledo thermogravimetric analyzer TGA/DSC1. A thermal decomposition examination was conducted in air . The measurement for each foam was repeated on three samples and averaged. Each specimen with dimensions of 10 cm × 10 cm × 5 cm was wrapped with aluminum foil and burned at an external heat flux of 35 kW mThe paper presents the effect of POSS-Cl as modifiers of the functional properties of rigid PUR foams, with particular emphasis on the flame-retardant properties of silsesquioxanes. The conducted research shows that the introduction of the silicate cage modifier significantly influences the properties of the tested systems, starting from the rheological properties of the polyol premixes, which resulted in the subsequent characteristics of the obtained PUR-POSS-Cl foams. Starting from a change in structure to a more heterogeneous one with smaller and more irregular cells, they simultaneously resulted in a higher density of the molded foams. This, in turn, translated into an increase in the mechanical properties and hardness of the modified PUR foams. It was found that the addition of POSS-Cl had a significant effect on increasing the hydrophobicity of the system as compared to the PUR-0 reference sample. However, the most important aspect of the presented research is the improvement of thermal stability and reduction of flammability of modified PUR-POSS-Cl systems. Among all the silsesquioxanes used, POSS-Ar-Cl can be indicated as the best, as it had the greatest impact on increasing thermal and mechanical properties.


In the current study, the utilization of glass and nylon fibers in various percentages are added to enhance the mechanical performance of peach shell lightweight concrete. Glass and nylon fibers were added at 2%, 4%, 6%, and 8% by cement weight. The results showed that, as we added the glass and nylon fibers, the density of peach shell concrete was reduced by 6.6%, and the compressive, split tensile and flexural strength were enhanced by 10.20%, 60.1%, and 63.49%. The highest strength that was obtained in compressive, split tensile, and flexural strength at 56 days was 29.4 MPa, 5.2 MPa, and 6.3 MPa, respectively, with 6% of glass fiber in peach shell concrete. Mechanical test results showed that post-failure toughness and modulus of elasticity of peach shell concrete is enhanced with the utilization of fibers. To verify our lab results, a statistical analysis, such as response surface methodology, was performed to make a statistical model, it was confirmed by both lab results and statistical analysis that the mechanical performance of peach shell concrete could be significantly improved by adding glass fibers as compared to nylon fibers. With the use of fibers, the water absorption and porosity were slightly increased. Hence, the glass and nylon fibers can be used to improve the peach shell concrete mechanical properties to make concrete eco-friendly, sustainable, and lightweight. The utilization of modern agricultural or industrial waste materials to substitute the regular raw material in concrete has accomplished eco-friendly and sustainable improvement by decreasing raw material production expense ,2. The l3 (ACI 213R) [Lightweight aggregate affects lightweight concrete properties . TypicalCI 213R) . Adding CI 213R) . The funCI 213R) ,26. PastCI 213R) ,28. Yab CI 213R)  declaredCI 213R) .3 and lesser as compared to a maximum recommended unit weight set for lightweight aggregate concrete. There are not many writings on the examination of nylon fiber-and glass fiber-reinforced lightweight peach shell concrete [Even though there are different studies on the inclusion of fibers in lightweight aggregate concrete, there is very limited research on the utilization of fibers in peach shell concrete. To make peach shell concrete like other lightweight aggregate concrete applied to reasonable structure components, for example, partition walls, parking garages, sidewalks, and roadblocks, the durability and mechanical properties of peach shell concrete must be additionally improved. The inclusion of fibers into peach shell concrete can make it a sustainable structure material and also make it ductile. Usually added fibers are nylon fiber, PP fiber, and steel fiber, and there are not many writings available on the utilization of glass fibers in lightweight aggregate concrete. The primary disadvantages of adding steel fibers to (LWAC) are decreased workability and a huge increase in unit weight. Polypropylene fiber is primarily utilized in concrete to improve impact resistance, ductility, and toughness, but do not expect to increase the strength . Mastaliconcrete ,33The reason for the current examination is to study the influence of two kinds of fibers (glass fiber and nylon fiber) and every fiber in different percentage of 2%, 4%, 6%, and 8% by weight on the mechanical performance of lightweight peach shell concrete. An examination of glass fiber-and nylon fiber-reinforced lightweight peach shell concrete is explored, the experimental tests on concrete include compressive, split tensile and flexural strength, density, workability, water absorption, residual compressive strength, porosity, and modulus of elasticity. Statistical analysis, i.e., response surface methodology (RSM) was adopted to make a statistical model to verify the research performed in an experimental trial to assists with growing information on the impact of glass fibers and nylon fibers with the different percentage to improve the mechanical properties of (PSFRC) peach shell fiber-reinforced lightweight concrete.For cement, 53-grade type I OPC  PortlandCrushed limestone is used as a normal-weight aggregate in the control sample and the peach shell is used as a replacement for crushed limestone. Unit weight of peach shells was 59% less as compared to typical coarse aggregate. Lightweight aggregates are not only good for reducing the dead load in buildings, but also have positive thermal properties (insulation). For fine aggregate, normal river sand was utilized and obtained from a nearby material supplier. Water for blending in cement had the pH value someplace in the scope of 6 and 8. In this study, clean drinking water was utilized, which was acquired from tube well property.A high-range superplasticizer (SP) was utilized in the examination to increment the fresh concrete workability. At 1% of the cement weight, a superplasticizer is used in all blends.Two different types of fibers are used in the present study, glass fiber and nylon fiber. The image and properties of glass fiber and nylon fiber are provided in A sum of eight concrete blends was set up with various fiber proportions, and a control mix without the inclusion of fibers was additionally cast as a reference sample. The total sum of materials for all blends was equivalent, except for two different fibers (glass and nylon) and four fiber percentages. Fibers were added in different percentages like 2%, 4%, 6%, and 8% by weight, accordingly. Mix proportion of all concrete samples are provided in To make peach shell fiber reinforced concrete the following method was followed: initially, peach shell and sand from the river were filled into a mixture and dry blended for 3 min. Then, fibers and silica fume and cement were added to the blend and dry blended for 2 min. Fibers were manually distributed before they were blended. After that, 70% water blended in with a superplasticizer was incorporated into the blend and blended for 5 min. Following this, 30% water was added to the blend and blended for 7 min. After blending was over, then the test for the slump was performed right away, and afterward, the samples were cast in lubricated steel molds. Entire samples were compacted by a vibrator. Following compaction, all samples alongside the molds were enclosed by a sheet of plastic to forestall dampness. Samples were removed from the molds after 24 h. Lastly, samples were put away in a controlled environment with an overall moistness of 42% ± 3% and a temperature of 22 °C ± 3 °C till the experiment. The slump test was used to determine the workability of all blends as indicated by ASTM C143 . The uniM2 represents air surface-dried sample mass after drenching and M1 represents an air oven-dried sample mass. All samples’ porosity was determined as per ASTM C642 [In the above equation, STM C642 . To deteSTM C642 ; subsequSTM C642 . The vol3) and 3).Here, M1 is the oven-dried sample mass; M2 is the surface-dried sample mass of the in-air after inundation; M3 is the surface-dried sample mass in air subsequently submersion and heating; and M4 is the sample apparent mass in water subsequently drenching and boiling.Here, The relationship between slump value and the fiber amount is presented in 3 and more than 15 MPa of compressive strength [3 to 1914 kg/m3, which satisfied the (LWAC) necessity because the peach shell is about 67% lighter than the normal weight aggregate. The inclusion of nylon and glass fibers in concrete decreased the oven-dried density of PSC because the specific gravity of fibers is low. When the percentage of nylon fibers is included at rates of 2%, 4%, 6%, and 8% by weight, concrete oven-dried density is reduced by 1.6%, 3.5% 5.3%, and 6.6%, separately, as in comparison to reference sample.Lightweight aggregate concrete (LWAC) is described as concrete that has an oven-dried density in the scope of 1700–200 kg/mstrength . The con3, and it was just 1.2% more than N8. It is noticed that though nylon fibers and glass fiber’s specific gravity is fundamentally diverse, its effect on the PSC oven-dried density is not self-evident. Nonetheless, compared with a unit weight of around 2410 kg/m3 for conventional concrete, PSC oven-dried density is reduced around 31%, which is a huge decrease in the weight of concrete.In From For the most part, the beginning and cracks spreading in concrete are because of the ceaseless expansion in compressive loading. Since the pressure produced by the fiber is opposite to the break spread way, the debonding starts at the interface among the cement paste and fiber. The fiber resembles an extension in this cycle that captures the propelling breaks and thus improves the concrete strength . TherefoResponse surface methodology (RSM) is a statistical tool, and its main purpose is to predict response or output from the experimental trial tests, (RSM) can be influenced by several factors or input variables. When there is more than one response, then it is important to find the combined optimum dosage of both materials that is not possible individually . In thisEntire concrete samples split tensile and flexural strength have appeared in Oven dry densities of N8 and G8 were lesser in the present study, and the flexural strength and split tensile strength of N8 and G8 were more as compared to the control sample. The ductile behavior of glass fibers is more prominent as compared to nylon fibers, glass fibers reinforced peach shell concrete led to higher flexural strength and split tensile strength. At percentages of 6% and 8%, flexural strength and split tensile strength of peach shell concrete that contain glass fibers were discovered to be about 8.1–16.8%, and 12.0–12.5% individually, and higher than those of nylon fibers. Flexural strength and split tensile strength of G6 were 6.3 MPa and 5.3 MPa, which improved by 63.49% and 60.37%, as compared with the control sample. At the same fiber percentage, the diameter of glass fiber is smaller, prompting more fibers to join the breaks . Due to To evaluate the optimum dosage of glass fibers and nylon fiber for concrete split tensile strength, 6.0% glass fibers and 5.5% nylon fibers were selected from the contour plot giving split tensile strength 7.0 MPa as shown in To assess optimum dosage glass fibers and nylon fiber for flexure strength of concrete, 6.0% glass fibers and 5.5% nylon fibers were selected from the contour plot giving flexure strength 10 MPa, as shown in The modulus of elasticity (MOE) of all concrete samples is displayed in Water absorption and porosity for all blends of 24 h have appeared in In Nylon and glass fibers reduce the PSC slump values. Fresh Peach shell concrete with nylon and glass fibers slump values are decreased to 60–70 mm. Concrete that has glass fibers had higher slump values as compared to nylon fibers.3 to 1914 kg/m3 and satisfies the lightweight aggregate concrete requirement.The oven-dried density of peach shell concrete is reduced by adding nylon and glass fibers. Concrete oven-dried density ranges from 1801 kg/mThe early compressive strength of whole concrete samples was high. PSC compressive strength was increased with nylon fibers and glass fibers, and peach shell concrete compressive strength increments as we increase the fiber amount. Peach shell concrete compressive strength at 28 days was around 7–20%.Peach shell concrete split tensile strength is increased with adding of nylon fibers and glass fibers. Split tensile strength gets higher as the number of fibers is increased. Split tensile strength is increased by 9.8–57.2% and 8.0–60.3% by adding nylon and glass fibers from 2.0% to 8.0%.Peach shell concrete flexural strength improved as fiber content is increased. Flexural strength is increased by 6.2–39.4% and 7.1–63.4% by adding nylon and glass fibers from 2.0% to 8.0%.The flexural and split tensile strength of glass fiber-reinforced peach shell concrete is high. At the percentage of 6.0% and 8.0%, peach shell concrete containing glass fibers the split tensile and flexural strength are between 63.49% and 60.37% more as compared to nylon fibers.Statistical models (response surface methodology) confirmed the lab results, that glass and nylon could be utilized to improve the mechanical performance of peach shell concrete.Peach shell concrete modulus of elasticity is also improved with the addition of nylon fibers and glass fibers. All concrete samples’ modulus of elasticity ranges somewhere in the range of 10.2 GPa to 12.5 GPa. At percentages of 2.0%, 4.0%, 6.0%, and 8.0%, nylon and glass fiber-reinforced PSC modulus of elasticity enhanced by 11.2%, 13.1%, 14.6%, and 15.4 for nylon fibers and 22.6%, 23.9%, 26.5%, and 29.8% for glass fibers.No residual compressive strength was produced in a control sample. PSC residual compressive strength is increased by adding nylon and glass fibers. Furthermore, the higher the fiber amount, the higher the residual compressive strength. At a percentage of 8.0%, the residual compressive strength of nylon and glass fiber-reinforced PSC increases by 47% and 79%.Adding fibers marginally builds peach shell concrete porosity and water absorption. Despite that, the amount and type of fibers insignificantly affect water absorption and porosity.Effects of joining two different kinds and percentages of fiber  on the peach shell lightweight concrete mechanical properties have been studied in the present research. Following conclusions are obtained from the current study:The final conclusion about results obtained from the utilization of glass and nylon fibers is that both the fibers are suitable material to be used in concrete; however, in the present study, glass fibers were shown to be more effective in improving concrete mechanical properties.


It is time to stop referring to the antibiotic resistance pandemic as ‘silent’. Continuing to use such a term denies the reality that antibiotic-resistant bacterial infections, driven by misuse and abuse of antibiotics by humans against microbial ecosystems that we should be living in symbiosis with, is wrong. Both our terminology and who the real ‘enemy’ is in relation to antibiotic resistance demands serious reconsideration. Words matter, as do the meaning they convey and the aspiration that lies behind them.1  and was top of the health agenda for G7 and G20 meetings,3 the subject of calls-to-action and a focus area for Ministries of Health from Albania to Zimbabwe, has been sidelined.In the shadow of COVID-19, antibiotic resistance (ABR), one of the world’s worsening pandemics, has been ‘silenced’. A pandemic that, pre-COVID-19, commanded a high-level meeting of the United Nations General Assembly4 the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC) clinical database attests to the inordinate amount of misuse and overuse of antibiotics, with 61% of persons admitted to hospital receiving one or more antibiotics.5De-prioritizing efforts to mitigate antibiotic resistance has enabled yet another respiratory virus—SARS-CoV-2—to drive inappropriate antibiotic use worldwide. Despite a low overall pooled proportion of patients who had laboratory-confirmed bacterial coinfections of 7% ,6 in political speeches7 and on social media. But what does that convey to those who we are trying to encourage to join the efforts to save antibiotic efficacy for future generations? We believe that most people have adopted this term to convey comparison with the very ‘loud’ COVID-19 pandemic and its prioritization.In response to reduced global attention to ABR, the term ‘the silent pandemic’ has gained traction in publications,qui tacet consentire videtur [he who is silent seems to consent]’, is apt. By propagating the use of the term ‘silent pandemic’ we are at risk of encouraging the very people we want to galvanize to action to consent silently to the status quo.However, something that is labelled as ‘silent’ is often kept so. Although said outside of the ABR context, Sir Thomas More’s quote, ‘COVID-19 has reminded us of the power that threat has as a driver for action, be it behaviour change  or designating unprecedented funds to research and development of new antivirals, vaccines and diagnostics. By conveying ABR as a silent pandemic, are we tacitly propagating that silence when we should be espousing completely the opposite?8If we are to ramp up the ABR decibels, then we need to (i) speak to truth and (ii) understand what people relate to. For far too long, we have exhorted people to join the ‘fight’ against antibiotic-resistant bacteria; our fight is not with bacteria, it’s with humans. It is us who overuse and misuse antibiotics, us who feed antibiotics to animals for food production, and us who pollute the environment through antibiotic manufacturing and other means as far-fetched as incorporating antibiotics into antifouling paint to prevent barnacle attachment on ships.9 we have an opportunity to focus people’s attention on the harm of destroying our own personal environment and the critical role that bacteria and other microbes play in our immune response, in outcompeting pathogens by creating barriers for entry to our body’s surface and internal environment, and in digestion. The importance of this environment to health and disease has been recently reviewed.10Climate change and emerging infectious diseases have focused attention on the importance of protecting the environment. This is equally relevant to ABR, and it affords us an opportunity to focus on a relatable concept—the destruction of our own body’s environment when we abuse antibiotics. As symbionts, living an interdependent relationship with bacteria and other microbes, which collectively outnumber our own human cells,11 call time on the use of ‘silent’, and point out even more clearly that, as for climate change, humans need to take responsibility for the destruction of a  environment, which should be protected from antibiotics except when clearly necessary, in order to limit further emergence of bacterial resistance and maintain health through critical human–microbial and animal–microbial symbiotic relationships.If we are to shout from the rooftops to regain the attention that antibiotic resistance deserves, then it’s time, once again, to think more carefully about the terminology we use,


In order to improve the utilization efficiency of road runoff and the remove effects of heavy metals, porous asphalt pavements have been used as an effective measure to deal with heavy metals in road runoff. However, the removal effect on dissolved heavy metal is weak. In this paper, basic oxygen furnace (BOF) slag was used as aggregate in porous asphalt concrete to improve the removal capacity of heavy metal. Road runoff solution with a copper concentration of 0.533 mg/L and a zinc concentration of 0.865 mg/L was artificially synthesized. The removal effect of BOF slag porous asphalt concrete on cooper and zinc in runoff was evaluated by removal tests. The influence of rainfall intensity and time on the removal effect was discussed. The results obtained indicated that BOF slag porous asphalt concrete has a better removal effect on copper. The removal rate of copper is 57–79% at the rainfall intensity of 5–40 mm/h. The removal rate of zinc is more susceptible to the changes of rainfall intensity than copper. The removal rate of zinc in heavy rain conditions (40 mm/h) is only 25%. But in light rain conditions (5 mm/h), BOF slag porous asphalt concrete maintains favorable removal rates of both copper and zinc, which are more than 60%. The heavy metal content of runoff infiltrating through the BOF slag porous asphalt concrete meets the requirements for irrigation water and wastewater discharge. The results of this study provide evidence for the environmentally friendly reuse of BOF slag as a road material and the improvement of the removal of heavy metal by porous asphalt concrete. With the rapid development of urbanization and the continuous increase of highway mileage and traffic volume, soil pollution and water pollution caused by highways have gradually attracted attention. Various pollutants from vehicles or the atmosphere accumulate on the road during non-rainfall periods, and when it rains, the accumulated road pollutants are easily washed by rainfall runoff and carried into the groundwater or soil, causing potential harm to the surrounding environment ,2. In ad2+ and Pb2+ to form carbonate precipitates [In order to remove road runoff pollutants more efficiently, advanced measures have been applied to road engineering such as vegetation control, wet retention ponds, and infiltration systems . The vegipitates . The negipitates .Basic oxygen furnace (BOF) slag is a kind of steel slag. As a by-product in the steelmaking process, steel slag is often recycled for road construction due to its good mechanical properties ,19. Stee2+ and Zn2+ was artificially synthesized. The removal rate of copper and zinc by porous asphalt concrete under different conditions was measured by removal tests. The effects of steel slag porous asphalt concrete on the removal of copper and zinc in road runoff and the influence of rainfall intensity and duration were evaluated. The results of this study provide evidence for the reuse of steel slag as a road material and the improvement of the removal of heavy metal by porous asphalt concrete.Although there are a variety of methods to deal with pollutants in road runoff, only a few have obvious effects on heavy metals, and include the disadvantage of low efficiency. Using steel slag as the aggregate of asphalt concrete to precipitate heavy metals by increasing the pH of the solution is a more efficient way to take effect. As steel slag is a kind of solid waste, the reuse of resources is realized at the same time. In this study, steel slag and basalt were used as aggregates to prepare porous asphalt concrete, and their volume properties and road performance were characterized. According to the observation data of road rainwater runoff in the literature, a rainwater runoff solution containing CuIn this study, BOF slag and basalt from Hubei Province were used as the aggregates. BOF slag generally contains a higher content of calcium oxide than other steel slags, which results in a higher alkalinity. The chemical composition of BOF slag and basalt was obtained by X-ray fluorescence analysis (XRF), as shown in 3)2 solution, 0.05 mol/L Cu(NO3)2 solution and deionized water were used to artificially synthesize road runoff solution because nitrate ions would not interfere with the removal tests.In order to prepare porous asphalt concrete with large porosity, modified asphalt with higher viscosity is required to provide sufficient cohesive strength. SBS modified asphalt has better viscosity and high temperature stability than base asphalt. In this study, SBS modified asphalt with a penetration of 46.8 , ductility of 45.0 cm , and softening point of 78.3 °C was used as the binder. 0.05 mol/L Zn2 and Zn(NO3)2, which are hydrolyzed to make the solution weakly acidic. When the solution infiltrates through the BOF slag porous asphalt concrete, the f-CaO in the BOF slag hydrates and the solution becomes alkaline. The BOF slag with the size of 5–10 mm can make the pH of the solution up to 11–12.5 [The value of 0 h represents the pH of the solution before tests. The synthetic runoff solution is composed of Cu on the removal of zinc only after 168 h storage with the initial concentration of 0.51 mg/L, while the BOF slag porous asphalt concrete provides a stabilizing effect (45–70%) for the removal of zinc from the beginning with the similar initial concentration (0.865 mg/L). It is indicated that BOF slag, as the aggregate of porous asphalt concrete, provides an efficient removal effect on zinc and copper. The final concentrations of copper and zinc are 0.096–0.227mg/L and 0.337–0.649 mg/L, respectively, under different rainfall conditions, which meet the Chinese standards for wastewater discharge and irrigation water .In addition to zinc and copper, multiple heavy metals exist in BOF slag aggregate, which may leach into the runoff. The leaching concentration of heavy metals in steel slag asphalt mixture shows a downward trend with time . Therefo(1)The BOF slag porous asphalt concrete can turn the pH of the runoff solution up to 10–11, which significantly increases the removal rate of copper. The pore structure of porous asphalt concrete plays an important role in the removal of zinc.(2)With the increase of rainfall intensity and time, the removal rate of copper and zinc gradually decreases. The removal rate of zinc is more susceptible to the changes of rainfall intensity than copper. However, in light rain conditions, BOF slag porous asphalt concrete maintains favorable removal effect on both copper and zinc.(3)While removing the original heavy metal in the runoff, the BOF slag porous asphalt concrete leaches little additional heavy metal. The heavy metal content of runoff solution infiltrating through the BOF slag porous asphalt concrete meets the requirements for irrigation water and wastewater discharge.In this paper, the removal effect of BOF slag porous asphalt concrete on copper and zinc in runoff was evaluated by removal tests, and its influencing factors were explored. On the basis of the data obtained in this study, the following conclusions are drawn:The results obtained indicate that BOF slag porous asphalt concrete has favorable pavement performance and significant removal effect on heavy metal in road runoff. The environmentally friendly reuse of BOF slag as a pavement material is feasible.


Sphenophorus levis  is one of the main pests of sugarcane in Brazil. Although its major digestive proteases are known, its complex digestive process still needs to be further understood. We constructed a transcriptome from the midgut of 30-day-old larvae and identified sequences similar to its major digestive protease (cysteine cathepsin Sl-CathL), however, they presented a different amino acid than cysteine in the active cleft. We identified, recombinantly produced, and characterized Sl-CathL-CS, a pseudo cysteine protease, and verified that higher gene expression levels of Sl-CathL-CS occur in the midgut of 30-day old larvae. We reverted the serine residue to cysteine and compared the activity of the mutant (Sl-CathL-mutSC) with Sl-CathL-CS. Sl-CathL-CS presented no protease activity, but Sl-CathL-mutSC hydrolyzed Z-Phe-Arg-AMC  and was inhibited by a cysteine protease inhibitor E-64 (Ki = 38.52 ± 1.20 μM), but not by the serine protease inhibitor PMSF. Additionally, Sl-CathL-CS interacted with a sugarcane cystatin, while Sl-CathL-mutSC presented weaker interaction. Finally, protein ligand docking reinforced the differences in the catalytic sites of native and mutant proteins. These results indicate that Sl-CathL-CS is a pseudo-cysteine protease that assists protein digestion possibly by interacting with canecystatins, allowing the true proteases to work.The  Sphenophorus levis  is one of the main pests of sugarcane crops (Saccharum spp.) in Brazil. S. levis is a particularly challenging pest to control because the larvae develop inside the stem, opening galleries inside the plant, making the use of commercial pesticides inefficient [The sugarcane weevil fficient ,2. ThereS. levis has revealed that its major protease is Sl-CathL, a cysteine cathepsin L-like, followed by other proteases, e.g., trypsin-like enzymes [Coleoptera insects mainly use cysteine proteases as digestive enzymes ,4,5,6,7, enzymes ,5, inver enzymes , and pec enzymes ,12. Mean enzymes . This re enzymes .S. levis larvae, one sequence was found to be related to Sl-CathL, although it presented a serine residue instead of cysteine in the putative protease active site. A deeper analysis revealed some other sequences that presented amino acids other than serine in the putative catalytic site. Some proteins, called pseudoenzymes, share homology with other proteases but present at least one amino acid in the catalytic site different from the conserved protease, resulting in a protein without the original enzymatic activity [When analyzing the transcriptome from the midgut of activity . Early ractivity ,16,17, bactivity . S. levis and the insect tissues, then cloned, expressed, and characterized the pseudo-cysteine protease (Sl-CathL-CS) and the mutant (Sl-CathL-mutSC), in which we reverted the Ser138 to Cys138 and analyzed their interaction with CaneCPI-1 [In this regard, we decided to investigate the role of the pseudo-cysteine protease Sl-CathL-CS in the insect digestive process. We identified the expression pattern in developmental stages of aneCPI-1 , a sugarS. levis larvae is summarized in The transcriptome information of pooled RNA from 30-day-old 272 from the putative catalytic site. The final sequences were divided in three groups, according to the shared identity with Sl-CathL, as follows: group 1, 99 to 75% shared identity; group 2, between 74 and 50% and group 3, between 49 and 30%  and in different parts of the 30-day-old larvae . Among the developmental stages, Sl-CathL-CS showed high expression in 30-day-old larvae, and this stage was used for further analysis regarding the gene expression levels in the tissues. This stage shows higher protease and hydrolase activity. The pseudoenzyme was expressed mainly in 20- and 30-day-old larvae and the midgut and hindgut, but with very low expression in the latter . Sl-Cathv/v) methanol in P. pastoris yielding 7 and 2 mg/L. As both proteins were cloned in frame with a C-terminus 6x His tag sequence (N-AAASFLEQKLISEEDLNSAVDHHHHHH-C), they could be purified in a single step through nickel immobilized metal affinity chromatography (IMAC). An analysis in 15% SDS-PAGE revealed bands of around 45 kDa for Sl-CathL-CS  with Sl-CathL [The initial analysis of the transcriptome of the larval midgut revealed around 1220 sequences related to Sl-CathL, which were further filtered by considering the identity percentage, and the final sequences were split into three groups according to their similarity with Sl-CathL. Group 1 shared between 75 and 99% similarity, group 2 shared between 50 and 74%, and group 3 shared between 30 and 49% . Groups lalanine . The putA gene expression analysis through RT-qPCR of the different larval body tissues displayed that Sl-CathL-CS was mostly expressed in the midgut, with the highest expression levels in 20- and 30-day-old larvae . These rRelationships between plants and their insect predators are good examples of a fine-tuned co-evolutionary process. Insects produce a rich arsenal of enzymes, including digestive proteases, while host plants produce micromolecules and protein inhibitors ,22,23. PLeptinotarsa decemlineata diet altered the profile expression of digestive proteases, which increased the expression of cysteine and other proteases (aspartic and serine proteases) that are insensitive to the inhibitors [L. decemlineata digestive process revealed that insects that fed on inhibitors produced different enzymes that were structurally similar to those found in the control group, however, they differed by up to 50% in sequence identity [Plant protease inhibitors (PIs) can positively select and alter the expression profiles of digestive proteases in the insect midgut. In several Lepidoptera, a diet containing serine protease inhibitors led to the expression of more types of digestive proteases, which were both sensitive and insensitive to the inhibitors ,30. The hibitors ,32,33. Aidentity . Insect identity . The comidentity . Sl-Cath138 was reverted to a cysteine residue. Surprisingly, even though the only difference between cysteine and serine amino acids is reliance on their nucleophile side chain , the two proteins presented different activities. The first difference was noted during protein expression. Sl-CathL-CS reached the highest expression level in 24 h  and entire individuals (n = 4) in several development stages  were used to extract total RNA with TRIzol Reagent , according to the manufacturer’s instructions.The different parts  of 30-day-old The midgut RNA from 30-day-old larvae (n = 4) was treated with RNAse-free DNAse I  in reactions with 3 μg of RNA to a final volume of 20 μL. Poly (A) mRNA was isolated with oligo(dT) beads and transcriptome construction was conducted with TruSeq Small RNA Library Preparation Kits , according to the manufacturer’s instructions. The mRNA was fragmented in a Fragmentation Buffer 1 ×  and used as a template in a reaction with SuperScript II  with random hexamer primers to obtain the first-strand cDNA, followed by a reaction with DNA polymerase I and RNAse H  to synthesize the double-strand cDNA. After pair-ending (2 × 100 bp), adenylation, and ligation, suitable fragments (200 ± 30 bp) were selected and sequenced using HiSeq1000 . The raw data from the sequencing runs were submitted to the Sequence Read Archive (SRA) repository of the National Center for Biotechnology Information under BioProject number PRJNA694547, BioSample: SAMN17526444 and SRR13518685.The raw read quality control was obtained using the FASTQC v.0.11.9 program . TrimmomSphenophorus levis midgut transcriptome we used the OmicsBox  platform to run the local BlastP tool  against a local database with the Sl-CathL sequence as reference. Approximately 1220 sequences were retrieved, and we used a minimum coverage of 40% to filter the fragments. Then, sequences were individually analyzed and the putative protein that presented the Cys138 and His272, which are two residues from the catalytic triad, were chosen for further exploration. The final sequences were divided into three groups, according to their shared identity with Sl-CathL: group 1, between 99 and 75% identical to Sl-CathL; group 2 between 74 and 50% shared identity; and group 3, between 49 and 30%. In group 3, we identified one sequence that presented Ser138 instead of Cys138, and one was named Sl-CathL-CS. The identity matrix was calculated using Clustal Omega [To identify putative enzymes in −ΔΔCT method [Approximately 600 ng of pooled RNA was treated with DNAse I  and used to synthesize cDNA in a reaction using the ImProm-II Reverse Transcription System  with 0.5 μg of Oligo(dT)  in a 20 µL reaction. The primers for Sl-CathL-CS and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), that were used as the internal calibrator, were designed with the aid of the Primer3 program, version 4.0 , and areT method . For theEcoRI and NotI (indicated in bold), respectively. The signal peptide was identified using the SignalP 4.1 server [2, 10 pM of each primer, 1 × PCR buffer, and 1 U of Platinum Taq DNA Polymerase High Fidelity  in a 25 μL reaction. The reaction began with the denaturation for 1 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C with final extension for 10 min at 72 °C. The amplicon was analyzed in 1% agarose gel and the product was digested with EcoRI and NotI and cloned into a pPICZαA vector  cleaved with the same enzymes, resulting in the plasmid pPICZαA-Sl-CathL-CS. The open reading frame (ORF) for the putative protease Sl-CathL-CS (GenBank: JZ136744.1) was used for designing primers and cloning Sl-CathL-CS into the expression vector pPICZαA. Forward and reverse primers  were des1 server  and then138 to Cys138 was performed using the Quick-Change II Site-directed Mutagenesis kit  to generate plasmid pPICZαA-Sl-CathL-mutSC. Primers Sl-CathL-mutSCforw and Sl-CathL-mutSCrev  skim milk powder and the sodium acetate buffer , to a final volume of 1 mL, at 37 °C for 24 h. Aliquots were obtained every hour and protein degradation was analyzed in 15% SDS-PAGE [First, Sl-CathL-CS and Sl-CathL-mutSC protease activity was explored using a complex substrate. Around 1.5 µg of each protein was incubated with 0.5% (SDS-PAGE . ex = 380 nm and λem = 460 nm. Approximately 4.3 nM of the proteins was incubated with 4 mM of DTT in a 500 μL reaction with sodium acetate 10 mM buffer pH: 5.0 for 5 min at 37 °C. The proteolysis was measured using increasing concentrations (40 µM to 1.2 mM) of cysteine cathepsins fluorogenic substrates Z-Phe-Arg-AMC , Z-Leu-Arg-AMC , and Z-Arg-Arg-AMC  for both proteins, and the Sl-CathL-CS was tested on serine protease substrates Abz-Lys-Leu-Phe-Ser-Ser-Lys-Gln-EDDnp , Abz-Phe-Ser-Lys-Gln-EDDnp , Abz-KNRSSKQ-EDDnp , and Abz-Gly-Ile-Val-Arg-Ala-Lys-Gln-EDDnp .The next stage included assessing the activity of the two proteins on specific serine and cysteine protease substrates. This assay was performed as described before  with a fi) was calculated according to Morrison [Both E-64, a classic cysteine protease inhibitor, and PMSF, a serine protease inhibitor, were also tested against Sl-CathL-mutSC. Increasing concentrations of E-64 (10–100 μM) were tested and the inhibition constant  on substrate Z-Phe-Arg-AMC (20 mM) was measured as described in the previous section through a kinetic reaction measured in a Hitachi F-2500 spectrofluorometer. Then, CaneCPI-1 (5 nM) was added to the reaction and the remaining proteolytic activity of Sl-CathL was calculated. Increasing concentrations of Sl-CathL-CS (0.01–0.3 μM) were mixed to the reaction and the re-establishment of the protease was measured. The assay was performed in triplicate and the protease activity is presented as relative activity (%). Statistical analyses were performed with the aid of GraphPad Prism software, version 6.00 .g, followed by the 2 washing steps with 200 µL of the lysis buffer . Finally, the elution was performed twice with 200 μL of lysis buffer with 250 mM of imidazole. Samples were analyzed in SDS-PAGE 15% and Western blotting. The CaneCPI-1 was produced and purified as previously described . One uniw/v) skim milk powder in TBS buffer  and washed with a TBS buffer. Then, the membranes were incubated with an anti-His antibody  or anti-CPI-1 antibody (1:5000 dilution) and washed with a TBS buffer. The membranes were incubated with an anti-Mouse IgG Antibody , washed, and detected with a Clarity Max ECL Substrate .The Western blotting was conducted using 2 primary antibodies: monoclonal antibody anti His-tag  and polyclonal antibody anti-CaneCPI-1 . The proThe structures of Sl-CathL, Sl-CathL-CS, and Sl-CathL-mutSC were simulated by MODELLER  using th