Recycle concrete aggregates (RCA) consist of natural aggregates and remnant mortar adhered to their surface. The amount, size, and morphology of the adherent remainder paste influences quality aspects of RCA, such as their bonding potential with new cement matrix in an RCA-based concrete, as well as the concrete’s overall rheological and performance characteristics. The objective of this research was to study the effect of reducing the adhered mortar in RCA, by means of a mechanical treatment method, on the performance of concrete containing RCA at different percentages. The treatment process was conducted within a concrete mixer truck drum at specific time intervals, the effect of which was determined by means of image analysis, mass loss recordings, and circularity determinations. The effect of size of treated and field RCA, as well as replacement percentages on mechanical performance and durability of high and normal strength concrete mixes, were also investigated. It was concluded that the optimal treatment duration where no further significant removal of adhered paste occurred thereon was 3 h, and concrete mixes containing 3 h treated RCA exhibited comparable performance characteristics to those of the reference concrete mix. The needs of the worldwide infrastructure would have never been met without concrete. Due to rapid global urbanization, driven by upturns in construction sectors in both developed and developing countries, there is indeed an exponential increase in concrete production demand, which currently reaches over 55 billion cubic meters globally . With suTo provide for the infrastructural needs of future generations without compromising on global natural resources, the abovementioned issue may be tackled through the utilization of recycled concrete aggregates (RCA) in concrete. This approach, in addition, would alleviate the emerging issue of construction waste disposal, especially in already congested urbanized regions. Research on RCA indeed allows for the development of effective recycling approaches, further reducing waste, as well as reducing the costs associated with disposal and storage of waste. Backed by a combination of acquired knowledge on their inherent properties, practical implementation of RCA in concreting practice may allow for natural resources to be conserved and exploited for a prolonged period of time. On the other hand, there are certain challenges that need to be addressed and tackled before adopting a large-scale utilization of RCA in the concrete industry. In particular, RCA have been known to possess certain disadvantages compared to natural aggregates, such as lower density, higher water absorption, weak adherent paste on the aggregate surface, poor mechanical properties, and durability ,3. In coKazemian et al.  have rep2 carbon footprint), the cost embedded with the usage of relevant equipment , and the overall yielded performance.Based on the previous findings, it is therefore important to provide insights on the effect of the adhered mortar paste by understanding how its inherent quality characteristics affect the overall performance of conventionally utilized concrete. Several enhancement methods for eliminating the bound mortar have been previously suggested, such as mechanical grinding ,13,14,15The objective of this research is to determine, by utilizing a previously suggested mechanical treatment found in our previous work , whetherNomenclature used thereon within the text: RCA = Recycled concrete aggregates; NA = Natural Aggregates; RTC = Recycled Treated Coarse Aggregate; RFC = Recycled Field Coarse Aggregate (untreated); RFF = Recycled Field Fine Aggregate (untreated); PSD = Particle size distribution.Natural and recycled aggregates were obtained from a local quarry in Cyprus, at sizes 0/4 mm, 4/10 mm, and 8/20 mm, of which the properties were determined in accordance with the relevant EN Standards, as shown in 1). Each sample was placed in the mixer drum together with water (1 part of aggregates to 1 part of water) at a speed of 0.5 rotations per second for 1, 2, 3, 4, and 5 h, respectively. The water was added to improve this method by weakening the adhered mortar. When the treatment was completed, the RCA were sieved and washed using the 2.36 mm aperture sieve. Following that, they were dried to constant mass at a temperature of 110 °C and the final mass was recorded (m2). The mass reduction of the RCA was calculated by using the following equation:1mis the dried sample mass before treatment (g);2mis the dried sample mass after treatment (g).The treatment method was based on the mechanical grinding of the adhered mortar from the surface of the RCA. The aggregates were placed inside a 100 L capacity concrete drum mixer along with water, where the rotating motion allowed the aggregates to collide with each other and with the metal walls of the drum. The collision subsequently caused the material’s surface to wear, altering the geometrical and morphological characteristics by removing the loose adhered mortar. Five random samples of RCA (samples of 4/10 mm and 8/20 mm) were dried to constant mass at a temperature of 110 °C. Approximately 3 kg of each sample were selected and sieved using the 2.36 mm aperture sieve. The samples were then weighted ;is the area of the aggregate (mmPis the perimeter of the aggregate (mm).To characterize the circularity of the aggregates, digital image processing was applied initially to obtain high resolution images of the samples on a digital form and then for the relevant software to perform a mathematical procedure and obtain the necessary information . Both GICircularity values are ranging between 0.0 to 1.0 with 1.0 corresponding to a perfect circle. As the value approaches 0.0 is an indication of an increasingly elongated shape.Samples of RCA having a mass of 3 kg were initially collected and placed on a black felt  with a measuring tape and photographed. The obtained images were then edited using GIMP removing the background and keeping the aggregates and the measuring tape as the only visible objects. Following editing, the image was analyzed using ImageJ software. Initially, the scale was set, and the image was converted into a 32-bit image. The threshold was then adjusted, and the image was analyzed for the determination of circularity. The overall circularity of each sample was taken as the average of the circularities of all the analyzed aggregate particles.Qis the amount of 2-propanol absorbed (g);ρ3);is the density of propanol (g/mmA2);is the cross-section area exposed to propanol (mmtis the time (min);k0.5).is the sorptivity coefficient , placed in a vacuum desiccator, and subjected to a pressure of 2.0 ± 0.7 kPa for a duration of 2 ± 0.2 h. The desiccator was then completely filled with demineralized water and atmospheric pressure was restored. The samples were kept in the desiccator for 24 h and both immersed (mh) and saturated surface dry condition (ms) masses were recorded. The open porosity (%) was then expressed as the ratio of the volume of open pores and the apparent volume of the specimens according to the following equation:Open porosity was determined based on BS EN 1936:2006 , where sChloride resistance of 28-day cylindrical concrete disc specimens of 100 mm diameter and 50 mm height was determined in accordance with ASTM C-1202 using GERMANN PROOVEit apparatus system . The specimens were placed in cells containing 3% sodium chloride solution and 0.3 N sodium hydroxide solution and were subjected to a potential difference flow for 6 h. The value of the electric charge (Coulombs) was recorded during the test. Resistivity tests were conducted on 9 points on the surface of the same cylindrical samples prior to chloride resistance testing to assess these measurements. The resistivity test was carried out using a 38 mm-probe PROCEQ Resipod surface resistivity meter, which applies a current to the sample’s surface and measures the potential difference between its outer probes.“R” for RCA.“F” for fine aggregates replacement (second letter in formulation nomenclatures).“C” for coarse aggregates replacement.“F” for field non-treated (third letter in formulation nomenclatures).“T” for treated.“50” or “25” represent the replacement percentage of NA.A total of 12 mixes were prepared in accordance with the mix proportions presented in Within this paper and throughout the discussions, the w/c ratio is suffixed at the end of each code name.3 and 864 kg/m3 in order to evaluate the effect of RCA in both normal and high strength concrete categories.The mixtures were designed with cement contents of 400 kg/mMixes were prepared and cast using a Tecnotest AT 205 pan mixer of 150 L capacity and in accordance with BS 1881-125:2013. All types of aggregates were maintained in the laboratory, in air-dried conditions and the necessary water content corrections were made based on aggregates’ moisture content and absorption values. The samples were demoulded 24 h after casting and water-cured at a constant temperature of 22 ± 2 °C until the day of testing.Changes in mass loss and circularity for both 4/10 mm and 8/20 mm RCA and NA, along with the effect of the presence of adhered mortar on the changes in circularity across the treatment hours, are shown in cu value was observed in REF1 and the highest early strengths were exhibited by RFF25 (0.25), RTC25 (0.25), and RTC50 (0.25), reaching up to 96% of their 28 day strength values at 7 days. For low w/c ratios, higher replacement contents of coarse recycled aggregates, either treated or untreated, reduced both compressive and splitting tensile strengths at 28 days, although the compressive to splitting tensile strength ratio for each of those combinations appeared to be maintained relatively constant. This is apparent in When considering the w/c ratio of 0.5, it can be seen that there is a good and almost linear rate of reduction in both compressive and splitting tensile strengths when 25% of either treated or untreated aggregates were incorporated. In this case, RTC25 exhibited slightly improved mechanical properties compared to that of RFC25. This behavior may be underpinned by the fact that treatment of aggregates contributed to a slightly improved microstructure compared to utilization of field aggregates at the same contents, in agreement with the results of the open porosity values of the same concretes, shown in It was also observed that RFC50 concrete exhibited the lowest 28 day splitting tensile strengths compared to the reference (up to 22% reduction), regardless of the w/c ratio. These reductions indicated that the adherence between the untreated RFC and the new mortar matrix was most probably poorly sustained. It can be therefore suggested that the presence of remnant mortar on the surfaces of untreated RFC, when this is present in high amounts within the mixes, negatively affects the bonding characteristics of the concrete in the interfacial transition zone.Open porosity, sorptivity, chloride resistance, and resistivity results of the investigated concrete mixtures at 1, 3, 7, and 28 days are summarized in The relationships between the relevant durability properties of mixtures at 28 days are shown in It was observed that the microstructural development of almost all the investigated mixtures followed a reasonable trend, i.e., pore volumes and permeabilities were mainly higher at day 1, 3, and 7 days, while being reduced towards the age of 28 days. This behavior was evident apart from the cases of RFF50 (0.25). Based on the results shown in The relationship between the chloride resistance and capillary water absorption coefficient of mixes at 28 days, is shown in An opposing behavior was observed when the incorporated amounts of either fine or coarse RCA (treated or untreated) in low w/c concrete mixtures was 50%, instead of 25% by mass. The 3 hs mechanical treatment of RTC led to an increase in the sorptivity coefficients of RTC50 concrete mixture by almost 20% compared to REF, although it maintained the chloride resistance at a comparable level to that of RFC50.−0.5 difference for each increment). These changes were more drastic than those that occurred in concretes of low w/c ratios. Such reduction indicates a beneficial effect that the mechanical treatment offered, which empowered the pore network of the investigated concretes. This was also reflected in the compressive strength development of the aforementioned concretes presented in It was observed that in concretes of w/c ratio 0.50, the incremental addition of 3 h mechanically treated aggregates at 25% and 50% by mass, reduced the sorptivity coefficients at a linear rate  was found to be 3 h. This was further evidenced through a large-scale treatment process that yielded comparable circularity differences.At low w/c ratios, higher replacement contents of coarse RCA, either treated or untreated, reduced both compressive and tensile strengths of concrete mixtures at 28 days. When incorporating recycled fine aggregates, and particularly at a replacement content of 25% by mass, the compressive and tensile strengths of concrete mixtures were the closest to those of the reference.Durability properties seem to be slightly affected by the addition of recycled aggregates. A 3 h treatment on aggregates, which were incorporated at high replacement percentages within low w/c ratio mixes, led to an increase in the sorptivity coefficients by almost 20%, although it maintained the chloride resistance at comparable levels to those of concretes containing untreated aggregates at high amounts.−0.5 difference for each increment). These changes were more drastic than those that occurred in concretes of low w/c ratios. Such reduction indicates a beneficial effect due to the mechanical treatment which enhanced the pore network of the investigated concretes.It was observed that in concretes of w/c ratio 0.50, the incremental addition of 3 h mechanically treated aggregates at 25% and 50% by mass reduced the sorptivity coefficients at a linear rate (approximately a 0.0011 mm/sAn optimal treatment duration for recycled concrete aggregates was determined based on the observations in mass loss and geometrical alterations of recycled aggregates. Following the treatment process, the aggregates were utilized in concrete combinations at w/c ratios 0.50 and 0.25, aiming to study their effect on the mechanical properties and durability of concrete with natural crushed and recycled aggregates, at strategic increments in the mix design. The following conclusions were drawn:


High titanium heavy slag is one kind of solid waste that exists in large amounts in the southwest of China. In this paper, this high titanium heavy slag is used as natural pre-wetted material in concrete because of its porous structure. Three kinds of aggregates are used in this concrete. The first one is natural limestone and river sand. The second one is dry slag fine aggregate and coarse aggregate. The third one is pre-wetted coarse slag aggregate and dry slag fine aggregate. The strength, dry shrinkage, autogenous shrinkage, relative humidity, pore size distribution, stress–strain relationship, micro-hardness and chloride penetration of concrete composed of the above three aggregates are tested in this study. The results show that pre-wetted slag aggregate is a suitable internal curing material. The concrete with pre-wetted slag aggregate shows higher strength, lower shrinkage and smaller porosity. The water absorbed in the slag aggregate can be released effectively to increase the relative humidity, accelerate hydration, improve porosity and increase the interface strength. High titanium heavy slag is the solid waste product after smelting vanadium titanium magnetite, which exists in large quantities in Guizhou Province in China. This high titanium heavy slag has a porous structure with no harmful ingredients . This slThe volume stability is a matter of concern for the durability of concrete structures. In order to enhance the volume stability of concrete, several methods are used. Firstly, the volume deformation of concrete is controlled by adding shrinkage-reducing admixture ,4,5,6,7.Aggregate in concrete occupies 60 to 80 percent of the volume, so it plays a major role in concrete. High titanium heavy slag is a natural porous aggregate, and its mechanical properties are similar to natural gravel. If it can be used as internal curing material, it can not only improve the volume deformation of concrete but also not increase the cost of concrete. Therefore, in this paper, the feasibility of this problem is discussed.High titanium heavy slag is solid waste with no harmful components. Originally, this waste was used as foundation filling material. In this paper, the feasibility of the use of this slag as an aggregate in concrete is studied. Furthermore, slag has a porous structure and higher crushed indexes than light porous aggregate. Therefore, the suitability of the use of this slag as an internal curing agent in concrete is discussed firstly.Two kinds of aggregates are used in this study. One is the high titanium heavy slag, which is crushed into fine aggregate (≤4.75 mm) and coarse aggregate (5–20 mm), from Guizhou Province. Another one is limestone and river sand. The appearance of slag aggregate with high content of titanium is shown in The concrete is composed of aggregate, fly ash and P·O 42.5 ordinary Portland cement, which meets the Chinese standard GB 175-2007. The chemical compositions of cement and fly ash are shown in In our experimental program, three kinds of concrete are molded. First one is natural aggregates concrete (NAC), including limestone and river sand. Second one is dry slag aggregates concrete (DSAC) containing dry slag fine aggregate and coarse aggregate. The third one is pre-wetted slag aggregate concrete (WSAC) with coarse slag aggregate and dry slag fine aggregate. The mix proportions of concrete with these three different types of aggregate are shown in The three species of samples are demolded a day after casting and cured in a fog room  for different curing ages. The samples with the size of 100 × 100 × 100 mm are used to cubic compressive strength and splitting tensile strength testing. The 150 ×150 × 550 mm prism specimens are used to flexural strength, as shown in The prism concrete specimen with size of 150 × 150 × 550 mm is formed, and it is cured for 28 days before being put into the shrinkage testing room, where the temperature is kept at 23 ± 2 °C and humidity is about 50 ± 3% according to GB/T 29417-2012. The shrinkage deformations of specimens are measured by dial gauge, as presented in The cylinder concrete specimens of 100 mm in diameter and 200 mm in length are casted in PVC tube, which are used to obtain the autogenous shrinkage deformation and relative humidity in concrete up to 125 days according to SL/T 352-2020. The whole cylinder sample is coated by the insulation film to isolate the temperature and humidity exchange between samples and environment, and the vibration-wire strain gauge and the moisture meter shown in  are bothIn order to analyze the porosity of samples, the specimens curing for 120 d are subjected to measurements of pore volume and pore size distribution using intrusion porosimetery .The samples are taken out after curing for 28 days. The stress and strain relationships under uniaxial compressive loading of concrete made of different groups of aggregate are tested by 300 KN material test system . The axial deformation is measured by LDVT , as shown in In order to analyze the compactness of the samples, the concrete cores of φ100 × 50 mm are drilled out from 150 × 150 × 150 mm cubic samples, which consist of three various kinds of aggregate, at 7d and 28d. Then, each core sample is cut into three parts with the same size of φ100 × 50 mm. The cutting samples are subjected to chloride penetration test according to ASTM C1202 and the relative device, as shown in In order to study the interface strength between aggregate and paste in concrete consisting of different types of aggregates, the concrete sample is cut and the cross section is polished. The average micro-hardness values in interface transition zone and paste matrix are at least measured at 10 points around each aggregate and paste zone, respectively.The compressive strength, flexural strength and splitting tensile strength of six samples with each kind of mix proportion were tested. The average values of strength of NAC, DSAC and WSAC samples at 7 days and 28 days are shown in Three samples with each mix were used for the dry shrinkage measurement. The temperature change and autogenous shrinkage in the concrete samples tested by vibration-wire strain gauge embedded in concrete are presented in 3/g and 0.0594 cm3/g, respectively. The volume percent of the pore below 50 nm was about 76.1% and 69.3% of the WSAC and NAC samples separately according to the pore size distribution results, and the number of the pores larger than 50 nm, which is considered harmful for the durability of concrete, was higher in NAC than WSAC. It is illustrated that the structure of the concrete improved because of the pre-absorbed technology.The pore volume and pore size distribution of the NAC and WSAC samples curing for 120 days are shown in 3 dry density) and DSAC (with 2480 kg/m3 dry density) was stronger than that of NAC (with 2300 kg/m3 dry density).The cubic concrete sample was cut to analyze the interface between paste and aggregate, and the results are shown in Three samples with each mix were used for the stress and strain relationship analysis. The average stress and strain relationships of concrete with various aggregates are presented in Three samples with the size of φ100 × 50 mm of each mix were used for chloride diffusion testing. The average electric flux of concrete is shown in The microhardness of the ITZ and matrix results are displayed in High titanium heavy slag aggregate, one kind of solid waste, occupies a great deal of land and causes environmental pollution in Guizhou Province in China. Originally, this slag was used as foundation filling material. Preliminary research by our team indicates that there is no harmful and alkali active component in this slag. In this paper, the feasibility of using this slag as an internal curing aggregate in concrete is studied for the porous and rough structure of this kind of slag.The compressive strength, flexural tensile strength and splitting tensile strength of the samples with dry slag aggregate and pre-wetted slag aggregate were all higher than those of normal aggregate. Additionally, the interfacial micro-hardness testing results show that the interface strength between the paste and slag aggregate was obviously higher than that between paste and limestone because of the surface roughness of the slag. Similarly, the failure morphology of concrete under compressive loading results indicates that the ITZ improved by adding this porous slag aggregate. Furthermore, because of this interface enhancement, the high titanium heavy slag aggregate concrete has better resistance to chloride ion penetration. Therefore, this porous slag can be used as fine and coarse aggregate in concrete.To make use of this porous structure in slag aggregate, the pre-absorbed technology was adopted in this study. The internal relative humidity and pore size analyses results present that the pre-wetted slag aggregate can play a role as an internal curing agent to increase the relative humidity and decrease the porosity in concrete. Meanwhile, the concrete with pre-wetted slag aggregate can decrease the deformation in drying shrinkage and autogenous shrinkage effectively. Furthermore, using pre-wetted slag aggregate instead of limestone in concrete increases the interface strength in the samples and improves the mechanical properties. Thus, this porous high titanium heavy slag can be used as an internal curing agent in concrete.In this paper, the properties of strength, volume stability, interfacial strength and pore structure of NAC, DSAC and WSAC were studied. From the above investigation, the following conclusions can be drawn.(1) The high titanium heavy slag, one kind of solid waste, can be widely used as an aggregate instead of natural aggregate in concrete. It not only decreases environmental pollution but also improves the strength of concrete.(2) The high titanium heavy slag is a good internal curing agent in concrete. Concrete composed of pre-wetted coarse slag aggregate and dry fine slag aggregate not only had higher strength and lower dry shrinkage but also had smaller expansion in autogenous shrinkage measurement.(3) The relative humidity in concrete can be improved by using the slag aggregate as the pre-absorbed material. Furthermore, the porosity and amount of harmful pores decreased because the pre-absorbed water could accelerate the hydration of the cement.


Metformin, the first-line drug for type 2 diabetes mellitus (T2DM), has additional effects on improvements of nonalcoholic fatty liver disease (NAFLD); however, there are no treatments for both T2DM and NAFLD. Previous studies have shown hepatoprotective effects of a mixture of lemon balm and dandelion (LD) through its antioxidant and anti-steatosis properties. Thus, combination effects of metformin and LD were examined in a high-fat diet (HFD)-induced metabolic disease mouse model. The model received an oral administration of distilled water, monotherapies of metformin and LD, or a metformin combination with LD for 12 weeks. The HFD-induced weight gain and body fat deposition were reduced more by the combination than either monotherapy. Blood parameters for NAFLD , T2DM , and renal functions  were reduced in the combination. The combination further enhanced hepatic antioxidant activities, and improved insulin resistance via the AMP-activated protein kinase and lipid metabolism pathways. Histopathological analyses revealed that the metformin combination ameliorated the hepatic hypertrophy/steatosis, pancreatic endocrine/exocrine alteration, fat tissue hypertrophy, and renal steatosis, more than either monotherapy. These results suggest that metformin combined with LD can be promising for preventing and treating metabolic diseases involving insulin resistance. Obesity is the most prevalent metabolic disorder caused by a high-calorie diet intake and physical inactivity, and the rising global prevalence is an emerging public health concern . A signiMetformin, an AMP-activated protein kinase (AMPK) activator, is a biguanide drug, which is recommended as the first-line treatment for T2DM . It not Taraxacum officinale [L.] Weber ex F. H. Wigg) is a traditional herb used for treating various diseases, including diabetes and liver disease, because of the diverse pharmacological effects including antioxidant, anti-inflammatory, diuretic, antidiabetic, and anti-hyperglycemic properties [Melissa officinalis L.) is also a traditional herb, involved in treating hyperglycemia, hyperlipidemia, and insulin resistance-related metabolic alterations [w/w) has hepatoprotective effects on ethanol- and CCL4-induced liver damage by enhancing the antioxidant and anti-inflammatory properties via activation of the nuclear factor (erythroid-derived 2)-like (NRF)2 pathway, which are greater than those of monotherapies of dandelion and lemon balm alone [Common dandelion . The mice that adapted to the high-fat diet for a week were regrouped based on their body weights. All animals were fasted overnight prior to the initial and last treatments to avoid the diet effects, and the body weights were measured daily.All animal experiments were conducted according to the national regulations of the usage and welfare of laboratory animals, and approved by the Institutional Animal Care and Use Committee in Daegu Haany University . Six-week-old female SPF/VAF CrljOri:CD1 (ICR) mice were obtained from Orient Bio Inc. . They were housed five per polycarbonate cage in a temperature (20–25 °C) and humidity (40–45%)-controlled room, with a light/dark cycle of 12/12 h. Feed and water were supplied free to access. After a week of acclimatization, the mice with similar body weights were divided into one normal-fat diet group with Purinafeed  and six high-fat diet model groups with Rodent Diet with 45 Kcal%  (w/w) (LD), as described previously [2O and 28.5% O2, and the mice were euthanized using a CO2 gas. Then, the liver, pancreas, left periovarian/abdominal fat pads, and left kidney were sampled, and the organ weights were measured. The blood and tissue samples were subjected to biochemical and histopathological analyses.Leaf extracts of lemon balm (LB) and dandelion (DL) were supplied by Evear Extraction . The extracts were dissolved in distilled water (DW) and prepared as a mixture of LB and DL at 2:1  and the Total Cholesterol Assay Kit (colorimetric) , according to the manufacturers’ protocols. The levels were examined in duplicate using a plate reader . Fat density was assessed in total body and the abdominal regions using dual-energy X-ray absorptiometry .A small volume of whole blood was analyzed for the blood glycated hemoglobin (HbA1c) using EasyA1c . The other samples were collected in sodium fluoride glucose vacuum tubes  to assess the blood glucose level, and clotting-activated serum tubes for serum levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), gamma-glutamyltransferase (GGT), blood urea nitrogen (BUN), creatinine, triglyceride, total cholesterol, and low-/high-density lipoprotein cholesterol (LDL/HDL). The levels were assessed using Dri-Chem NX500i . Insulin level was measured using a mouse Insulin ELISA Kit .2O2 (pH 7.8) per min at 25 °C was measured at 240 nm. Superoxide dismutase (SOD) activity was estimated as the generation of superoxide radicals produced by xanthine and xanthine oxidase which react with nitrotetrazolium blue to form formazan dye, and measured at 560 nm. One unit of SOD enzyme activity is the amount diminishing the initial absorbance of nitrotetrazolium blue by 50% for 1 min. All levels were measured using a plate reader (Tecan), and normalized to the tissue protein contents.Some liver tissue (about 0.3 g) was prepared as 10% homogenates in ice-cold 0.01 M Tris-HCl (pH 7.4) for assessing the antioxidant activities, as described previously . Brieflyw/v) in 0.1 M triethanolamine, 0.2 M EDTA, and 2 mM dithiothreitol. The supernatant was collected after centrifuging at 1000× g and then at 10,000× g for 15 min at 4 °C, and used for assessing the hepatic enzyme activities. For glucokinase (GK) activity, the supernatant (10 μL) was incubated with the reaction buffer (0.98 mL) containing 50 mM HEPES-NaGT (pH 7.4), 100 mM KCl, 7.5 mM MgCl2, 2.5 mM dithioerythritol, 10 mg/mL albumin, 10 mM glucose, and 4 units of glucose-6-phosphate dehydrogenase, at 37 °C for 10 min. The reaction was initiated by incubating with 5 mM ATP (10 μL) at 37 °C for 10 min, and measured at 340 nm. For glucose-6-phosphatase (G6pase), the supernatant (5 μL) was incubated with the buffer containing 765 μL of 131.58 mM HEPES-NaGT (pH 6.5), 100 μL of 18 mM EDTA (pH 6.5), 100 μL of 265 mM glucose-6-phosphate, 10 μL of 0.2 M NADP+, 0.6 IU/mL of mutarotase, and 0.6 IU/mL of glucose dehydrogenase at 37 °C for 4 min, and measured at 340 nm. For phosphoenolpyruvate carboxykinase (PEPCK), the supernatant (10 μL) was mixed with the buffer (0.99 mL) containing 72.92 mM sodium HEPES (pH 7.0), 10 mM dithiothreitol, 500 mM NaHCO3, 10 mM MnCl2, 25 mM NADH, 100 mM inositol-1,4-diphosphate, 200 mM phosphoenolpyruvate, and 7.2 units of malic dehydrogenase. The activity was measured according to the reduced absorbance at 340 nm using a UV/Vis spectrophotometer . All the reagents were purchased from Sigma-Aldrich .Liver tissue (about 0.3 g) was homogenized as 10% . The contaminating DNA was removed using recombinant DNase I . The RNA was reverse-transcribed using the reagent High-Capacity cDNA Reverse Transcription Kit . The PCR conditions were as follows: 10 min at 94 °C, 39 cycles of 15 s at 94 °C, 20 s at 57 °C, and 30 s at 72 °C. The expression was analyzed by the ABI Step One PlusTM Sequence Detection System (Applied Biosystems), and normalized to that of GAPDH using the comparative threshold cycle method [Expressions of mRNAs, acetyl-CoA carboxylase 1 (ACC1), AMP-activated protein kinase (AMPK)α1, and AMPKα2 were measured in the liver, and gene expressions of leptin, adiponectin, uncoupling protein (UCP)2, CCAAT-enhancer-binding protein (C/EBP)α, C/EBPβ, sterol-regulatory-element-binding protein 1c (SREBP1c), PPARα, PPARγ, and fatty acid synthase (FAS) were measured in the periovarian fat tissue as a predominant region of white adipose tissues, as described previously ,25. Totae method . The oliiSolution FL ver 9.1, IMT i-solution Inc., Vancouver, BC, Canada) by a histopathologist blinded to the groups.Samples of the liver, pancreas, fat mass, and kidney were fixed in 10% neutral buffered formalin, paraffin-embedded, and serially sectioned at 3–4 μm. The sections were stained with hematoxylin and eosin (HE), and examined for diameter of the hepatocyte and adipocytes (in at least ten cells), thickness of fat tissues, area occupying pancreatic zymogen granules, number and diameter of pancreatic islets, and numbers of vacuolated renal tubules. A portion of the liver was dehydrated in 30% sucrose solutions, and frozen and sectioned at 20 μm. The sections were stained in oil red O, and the stained regions were examined. The histomorphometric analyses were performed using a computer-assisted automated image analyzer  for 1 h. The sections were incubated with guinea pig anti-insulin  and rabbit anti-glucagon antibodies  overnight at 4 °C. The next day, they were incubated with biotinylated universal secondary antibody  for 1 h and ABC reagents  for 30 min. The immunoreactivity was visualized with the peroxidase substrate kit  for 3 min, and counterstained with hematoxylin. All sections were incubated in a humidity chamber, and rinsed in 0.01 M phosphate-buffered saline three times between each step. The cells occupying more than 20% of immunoreactivities were regarded positive, and they were counted using an iSolution image analyzer by a histopathologist blinded to the groups.Other serial sections of the pancreas were treated with 0.3% Hp-value less than 0.05 is considered statistically significant.All data were expressed as the means ± standard deviations (SDs) of ten sample sizes. Since the Kolmogorov–Smirnov tests showed a normal distribution of the variables, data were examined by one-way analysis of variance (ANOVA). Kinetic changes of body weights were examined by two-way ANOVA with main factors for the groups and the time-points measured, and the time-point was treated as a repeated measure. Based on homogeneity of variance by Levene’s test, the multiple comparison was examined by Tukey and Dunnett’s T3 post-hoc tests in cases of equal and non-equal variances, respectively. A p < 0.01, p < 0.01); however, they were reduced in three metformin combination groups vs. the HFD control, and further reduced in the Met + LD(H) vs. the Met group (p < 0.05). There were also significant interactions between the groups and the time-points (p < 0.01), meaning time-dependent group differences. The body weights were increased in all of the HFD models regardless of the treatments vs. the Intact group. However, compared with the HFD control, they were reduced in week 9 to 12 post-treatments in the Met and LD(H), in week 4 to 12 in the Met + LD(H), in week 5 to 12 in the Met + LD(M), and in week 8 to 12 in the Met + LD(L) (p < 0.05). The body weights were further reduced in week 7 to 12 and week 8 to 12 in the Met + LD(H) vs. the Met and the LD(H), respectively, and they were also reduced in week 10 to 12 in the Met + LD(M) vs. both monotherapy groups (p < 0.05). The total weight gains were increased in the HFD control vs. the Intact group; however, they were reduced in all treatment groups vs. the HFD control, and further reduced in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups . Further, the fecal triglyceride was excreted more in the Met + LD(H) and Met + LD(M) than both monotherapy groups, and the fecal cholesterol was higher in the Met + LD(H) than the LD(H) group and in the Met + LD(M) than both monotherapy groups (p < 0.05).There were no differences in daily food consumption among the groups c; howevep < 0.01); however, the densities were reduced in all treatment groups vs. the HFD control, and further reduced in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups ; however, they were reduced in all treatment groups vs. the HFD control, and further reduced in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups ; however, the relative weight of the abdominal fat mass was reduced in all treatment groups vs. the HFD control, and further reduced in the Met + LD(H) vs. both monotherapy groups and in the Met + LD(M) vs. the LD(H) group (p < 0.05). The relevant weight of the periovarian fat mas was reduced in the Met and the combination groups vs. the HFD control, and further reduced in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups (p < 0.05). Although the absolute weight of the pancreas was not different among the groups, the relative weights were significantly reduced in the HFD control vs. the Intact group (p < 0.05). However, they were increased in the Met and the combination groups vs. the HFD control, and further increased in the Met + LD(H) vs. both monotherapy groups and in the Met + LD(M) vs. the LD(H) group (p < 0.05).Absolute weights of the liver, kidney, and abdominal/periovarian fat mass were increased in the HFD control vs. the Intact group . The levels were further reduced in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups (p < 0.05). The level of HDL was increased in the Met and the combination groups vs. the HFD control, and further increased in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups.Among the 14 parameters in blood biochemical analyses, serum HDL was reduced in the HFD control vs. the Intact group, while the others were increased ; however, they were increased in all treatment groups vs. the HFD control, and further increased in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups (p < 0.05). The level of GK, as a glucose sensor, was reduced in the HFD control vs. the Intact group; however, it was increased in all treatment groups vs. the HFD control, and further increased in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups (p < 0.05). Levels of G6pase and PEPCK, as regulators of hepatic gluconeogenesis, were increased in the HFD control vs. the Intact group (p < 0.01); however, they were reduced in all treatment groups vs. the HFD control, and further reduced in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups (p < 0.05).The level of MDA for lipid peroxidation was increased in the HFD control vs. the Intact group; however, it was reduced in all treatment groups vs. the HFD control, and further reduced in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups . The regulation was greater in the Met + LD(H) and Met + LD(M) groups than in both monotherapy groups. In the fat tissue, lipogenesis-related gene expressions of leptin, C/EBPα, C/EBPβ, SREBP1c, PPARγ, and FAS were upregulated in the HFD control vs. the Intact group, while lipolysis-related gene expressions of adiponectin, UCP2, and PPARα were downregulated (p < 0.01). However, compared to the HFD control, the expressions of leptin, C/EBPα, C/EBPβ, PPARγ, and FAS were downregulated in all treatment groups, and the adiponectin, UCP2, and PPARα were upregulated (p < 0.05). The expression of SREBP1c was downregulated in the Met, Met + LD(H), and Met + LD(M) vs. the HFD control (p < 0.05). The lipid metabolism-related gene regulation in the fat tissue was also greater in the Met + LD(H) and Met + LD(M) groups than in both monotherapy groups (p < 0.05).In the liver, mRNA expression of ACC1, an enzyme that catalyzes carboxylation of acetyl-CoA to malonyl-CoA for fatty acid synthesis, was upregulated in the HFD control vs. the Intact group, while expressions of AMPKα1 and AMPKα2, as master regulators of cellular energy homeostasis, were downregulated ; however, the sizes and areas were reduced in all treatment groups, and further reduced in Met + LD(H) and Met(M) vs. both monotherapy groups ; however, the number was reduced in all treatment groups, and the size was reduced in the metformin combination groups ; however, they were also reduced in all treatment groups vs. the HFD control. The inhibitory effects on the pancreatic islets and the immunoreactive cells were greater in the Met + LD(H) and Met(M) than both monotherapy groups (p < 0.05). Further, the ratio of the insulin- to glucagon-immunoreactive cells was increased in the HFD control vs. the Intact group (p < 0.01); however, it was reduced in all treatment groups vs. the HFD control, and further reduced in the Met + LD(H) vs. both monotherapy groups and in the Met + LD(M) vs. the LD(H) group (p < 0.05). The acinar area containing pancreatic zymogen granule was reduced in the HFD control vs. the Intact group (p < 0.01); however, it was increased in all treatment groups vs. the HFD control, and further increased in the Met + LD(H) and Met(M) vs. both monotherapy groups (p < 0.05).The pancreatic islets were significantly increased and enlarged in the HFD control vs. the Intact group (p < 0.01); however, it was reduced in all treatment groups vs. the HFD control, and further reduced in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups ; however, they were reduced in all treatment groups vs. the HFD control, and further reduced in the Met + LD(H) and Met + LD(M) vs. both monotherapy groups (p < 0.05).Tubular vacuolation of the kidney was increased in the HFD control vs. the Intact group (Obesity is accompanied by excessive fat accumulation, and visceral fat mass is a particularly important factor contributing to the development of metabolic diseases involved in lipid metabolic alterations and insulin resistance . Indeed,Excessive intake of fatty acids leads to hepatic insulin resistance, which reduces the insulin-mediated gluconeogenesis and activates lipogenesis, leading to hepatic triglyceride accumulation as a hallmark of NAFLD . MetformThe HFD control showed increased blood glucose, insulin, and HbA1c levels, indicating insulin resistance and hyperglycemia characterized in T2DM . TogetheChronic exposure to fatty acids leads to fat tissue dysfunction with increased lipolysis and FFA releases, resulting in lipotoxicity-induced insulin resistance in the fat tissue . The exaThere have been limitations in mono-targeting to the specific pathways in insulin resistance-related metabolic diseases. For example, targeting PPARs has been investigated as an antihyperglycemic medication for treating NAFLD and T2DM; however, the PPARα agonist has only weak effectiveness, or the PPARγ agonist shows undesirable adverse effects, including weight gain . ACC1/2 These results demonstrated that metformin combined with LD further enhanced the effectiveness of either monotherapies in HFD-induced NAFLD, T2DM, dyslipidemia, and renal steatosis. It suggests that combination of metformin and LD can be a promising therapeutic option for the prevention and treatment of metabolic diseases involving insulin resistance.


The Central Sensitization Inventory (CSI) measurement properties in patients having nonspecific, noncancer pain are well-established. However, studies examining the reliability and validity of either the CSI or the Central Sensitization Inventory short-form version (CSI-9) in breast cancer survivors (BCS) are scarce. The purpose was to evaluate convergent validity and internal consistency of the CSI and CSI-9. Additionally, the relevance of a new cluster calculator using the CSI was explored. The cross-sectional multi-center study included 65 BCS and 37 healthy volunteers. Patients filled out multiple questionnaires assessing pain, number of painful areas, anxiety, depression and quality of life. The relevance of a cluster calculator was explored by known-group comparisons and boxplot description. All hypotheses were formulated before data analysis. The majority of hypotheses on the correlations between the CSI or CSI-9 and other health outcomes were confirmed (22 out of 27). The CSI and CSI-9 have excellent (α = 0.92) and good (α = 0.86) internal consistency, respectively. The CSI cluster calculator might be an interesting tool to use to have a patient’s overall condition snapshot. Generally, the study findings support the construct validity and internal consistency of the CSI, which underline the use of this self-reported instrument in BCS. The CSI-9 shows promising results, but should be further evaluated. The Global Cancer Observatory estimated that around 2.3 million new female breast cancer cases were diagnosed worldwide in 2020 . Among wAmong these consequences, pain is a frequently occurring symptom in cancer survivors, with a prevalence higher than 20% after curative treatment . Pain isThe Central Sensitization Inventory (CSI) was initially designed as a self-report measure to assess CS-related symptoms severity and screen for the presence of Central Sensitivity Syndromes (CSS) . The ratThe aims of the present study in BCS were to evaluate the internal consistency of both questionnaires and evaluate their convergent validity with related health outcomes in BCS. A secondary aim was to investigate how the three subgroups formed with the CSI-CC differ on various health outcomes.Prior to the analysis, we hypothesized that an excellent and a good internal consistency would be found, respectively, for the CSI and CSI-9 in BCS. Given its factor structure and items, it was hypothesized that the CSI scores would show a very strong correlation with the CSI-9; a moderate correlation with the pain Visual Analogue Scale (VAS), Widespread Pain Index (WPI), Hospital Anxiety and Depression Scale (HADS) anxiety, HADS depression and the European Organization for Research and Treatment of Cancer Quality of Life-Questionnaire C30 (EORTC) physical, role, emotional, cognitive, fatigue, pain, insomnia and global health status; and a weak correlation with the EORTC social. The CSI-9 may yield correlation coefficients lower than the original CSI, but the reduction should not drop by more than 0.10 from the CSI . FinallyThe COSMIN consensus on health-related patient reported outcome was used as guidelines and used to report this study ,25.BCS were consecutively recruited by independent physiotherapists and physiotherapists from our research team (J.NTS and P.S) at several private practices in the Netherlands. Female healthy volunteers were conveniently recruited by approaching people from the general population in the environment of the researchers . All participants and participating physical therapists were unpaid volunteers.BCS were included if they were: native Dutch speakers; had been affected by a breast cancer diagnosis with or without pain related to their cancer or cancer treatment. Patients were excluded if: they were still receiving any type of cancer treatment except for hormone therapy or immunotherapy in the last 30 days before assessment, or they had metastases in their medical history, or pain was unrelated to cancer or cancer treatment. All data that were collected were used to assess if the pain was related to cancer or cancer treatment.Healthy volunteers were included if they were: native Dutch speakers, were not experiencing any pain and were not diagnosed with any (chronic) disease at the time of their assessment. The female healthy volunteers were only used for the CS-related symptoms subgroups analysis. All participants were provided with study information and informed consent was obtained prior to study enrollment.Each physical therapist that participated in the study received a protocol for administering the questionnaires and informational packages intended for participants consisting of: written information regarding the research protocol, an informed consent sheet, a social demographic questionnaire and the questionnaires of interest in a random order.BCS were asked to fill out the following questionnaires in their Dutch version: the CSI, VAS, WPI, HADS, EORTC and a social demographic questionnaire. The CSI-9 was extracted from the full CSI. The same questionnaires were administered to female healthy volunteers except for the VAS and WPI. Measures were taken to limit missing data. That is, during data collection, each participant’s questionnaires were checked for completeness, and, in case of incomplete responses, the participant was encouraged to complete the missing questions. To ascertain valid data processing, all data were entered in two different numeric files by two blinded researchers and were later checked for discrepancies.The CSI is a two-part questionnaire, of which part A measures a full array of 25 somatic and emotional symptoms associated with CS, scored with a 5-point Likert scale from 0 (never) to 4 , resulting in a total possible score of 100. Higher scores denote a higher degree of self-reported CS-related symptomatology. Part B asks if the patient has been previously diagnosed with seven CSS and three CSS-associated diagnoses, but it was not considered in our study. It was shown that a bifactor model containing one general factor representing “CS-related symptoms” and four orthogonal factors of “physical symptoms,” “emotional distress,” “headache/jaw symptoms” and “urological symptoms” fits the CSI structure the best ,22. The The CSI-9 was developed using Rasch Analysis . It measThe VAS is a non-specific psychometric response scale used to measure the mean pain intensity experienced by the patient. The patient answers by marking a 100 mm line, where 0 mm is equivalent to “no pain” and 100 mm equals “worst imaginable pain”. The distance measured from the “no pain” end to the patient’s mark is the VAS score. The VAS is a valid and reliable tool to assess pain in cancer patients .The WPI or formerly the “Regional Pain Scale” consists of a body diagram divided into 19 regions . PatientThe HADS is a self-assessment scale developed to detect levels of anxiety and depression . The HADThe EORTC is a questionnaire with 30 items scored with a 4-point Likert scale . It was The social demographic questionnaire collected the following information: age in years, cancer type with diagnosis date, date of end of medical treatment, cancer treatment received among a list, comorbidities and current medication.The CSI-CC is a tool to cluster patients into CS-related symptom severity subgroups. This tool has been developed in a large pooled multicountry sample of chronic pain patients and healthy subjects by hierarchical cluster analysis and latent profile analysis . The CSIAll statistical analyses were performed using the SPSS version 25 for Mac . Data distribution was visually inspected by Q–Q plots and the Kolmogorov–Smirnov test. Associations were calculated with Pearson correlation coefficient for parametric variables and Spearman’s correlation coefficients for nonparametric variables. If the correlation coefficient was >0.9, this was considered as a very strong association, between 0.7 and 0.9 was a strong association, 0.5 and 0.7 was a moderate association, between 0.3 and 0.5 was a weak association and <0.3 was a negligible association. The Cronbach α was used to measure internal consistency. An α between 0.50 and 0.60 was considered poor, between 0.60 and 0.70 was considered acceptable, between 0.70 and 0.90 was considered good and higher than 0.90 was considered excellent.p < 0.05 for all other tests.To examine the differences between the three cluster subgroups on the VAS, WPI and EORTC global health, ANOVA or Kruskal–Wallis tests were applied, with Bonferroni correction. The alpha for Bonferroni correction for multiple comparisons was set at 0.016 to control for the family wise error rate. Boxplots were made to compare the CS-related symptom severity clusters for the remaining measured variables. Patient responses were inspected for missing data. Statistical significance was set at Data collection took place from November 2015 until March 2016. The present exploratory cross-sectional multi-center study included 69 BCS and 37 female healthy volunteers. Data of 65 BCS were used for analysis, because four were excluded before analysis: three because of metastasis and one because of pain unrelated to cancer. Data were missing for the subscale EORTC insomnia for only one patient, who was excluded for the corresponding analysis. For the other subscales, no missing data were present. The participants characteristics of the study sample are presented in p < 0.01). A strong association between the CSI and EORTC emotional  was found as well. A moderate association between the CSI and VAS , WPI , HADS anxiety , HADS depression , EORTC physical , role , cognitive , social , fatigue , pain , insomnia  and global health status  was found. The CSI-9 correlations did not drop by more than 0.10 from the CSI correlations, except for HADS anxiety , EORTC physical  and EORTC fatigue .Cronbach’s α coefficient for internal consistency was 0.921 for the full CSI and 0.860 for the CSI-9. All associations for the CSI and CSI-9 are presented in n = 33, 63.5%), whereas female healthy volunteers were a minority in the medium level of the CS-related symptom severity cluster . No female healthy volunteers were found in the high level of the CS related symptomatology cluster.Descriptive statistics for the three clusters of CS-related symptom severity are presented in p < 0.001, dl = 2). The low cluster statistically differed from the medium  and high  cluster, but the medium and high groups did not differ from each other .The differences between the three cluster subgroups on the VAS, WPI and EORTC global health are presented in p < 0.001, dl = 2). The low cluster statistically differed from the medium  and high  cluster, but the medium and high groups did not differ from each other .For the WPI scores, the Kruskal–Wallis H test showed that there was a statistically significant difference between the different CS-related symptom severity clusters . The low cluster statistically differed from the medium  and high  cluster. The medium and high groups also statistically differed from each other .Furthermore, the Kruskal–Wallis H test showed that there was a statistically significant difference in EORTC Global health status scores between the different CS-related symptom severity clusters , depression (rs = 0.67) and EORTC emotional (rs = 0.72), which is consistent with the factor “Emotional distress” and previous studies [s = −0.66), which is consistent with the items 13 and 23 and the general association between pain and cognitive difficulties.Our results regarding the measurements properties of the CSI are in line with previous studies in non-cancer chronic pain populations . The CSI studies ,36,37,38 studies . A moders = 0.62) [s = −0.56) and role (rs = −0.60). Several authors have reported moderate correlations between the CSI and physical functioning measures [s = 0.68) and insomnia (rs = 0.59), which are symptoms commonly associated with CS [The WPI has consistently shown moderate correlations with the CSI in all studies to date, and the present study is the first to show this in BCS (r = 0.62) ,36,39. T = 0.62) ,40. On t = 0.62) ,36,38,41measures ,38,41. T with CS ,35. Agais = −0.51) between the CSI and EORTC social was found in this sample of BCS, whereas a priori, a weak association was hypothesized. This is remarkable as items measuring this social component are not included in the CSI. This suggest that symptoms assessed by the CSI might impact social functioning in BCS.A moderate association , which is in line with previous reports that found values ranging from 0.87 to 0.91 ,22. A res = −0.55), absence or negligible associations with quantitative sensory testing and the moderate to strong association with psychological distress [Although often presented as such, the CSI does not reflect a direct measure of CS, but is a measure of general physical and emotional distress originating from symptoms attributed to CS. This is further supported by the moderate association observed between the CSI and global health status (rdistress ,37,41,42distress . The CSIdistress . NonetheFinally, the limitations of the CSI arise from the lack of agreement about the definition of CS. While some authors consider it as a clinical picture, a diagnosis or a set of changes in the central nervous system, others advocate to reserve the term CS for the neurophysiological phenomenon related to its discovery ,14,40,44Regarding the CSI-CC, the goal was to explore the extent to which the CSI cluster classification correlates with patient-reported outcomes . In thisThe small sample size and the broad definition of CS were the main limitations of the study. Another limitation was that the CSI-9 answers were extracted from the CSI, which might have impacted CSI-9 scores. Study strengths include using COSMIN guidelines to validate this tool. Additionally, the CSI was evaluated in clinical setting during regular rehabilitation in several private practices. Furthermore, the use of validated and widely used measures along with adequate statistical tools are important study strengths.In summary, the study findings support the internal consistency and convergent validity of the CSI in BCS, and the internal consistency and convergent validity of the CSI-9 in BCS. This study, therefore, complements the already existing body of evidence for the validity and reliability of the CSI in cancer and non-cancer chronic pain populations, which underlines the use of this self-reported instrument to have a BCS’ CS-related condition overview. However, the CSI responsiveness and interpretability should also be investigated in order to use it as a follow-up tool. The CSI-9 is still in its early validation phase and should be further evaluated, as well as compared to the CSI, especially with regards to structural validity and content validity.


Escherichia coli and Vibrio cholerae, can become viable but nonculturable (VBNC) following exposure to specific stress conditions. Corynebacterium diphtheriae, a known human pathogen causing diphtheria, has not previously been shown to enter the VBNC state. Here, we report that C. diphtheriae can become VBNC when exposed to low temperatures. Morphological differences in culturable and VBNC C. diphtheriae were examined using scanning electron microscopy. Culturable cells presented with a typical rod-shape, whereas VBNC cells showed a distorted shape with an expanded center. Cells could be transitioned from VBNC to culturable following treatment with catalase. This was further evaluated via RNA sequence-based transcriptomic analysis and reverse-transcription quantitative PCR of culturable, VBNC, and resuscitated VBNC cells following catalase treatment. As expected, many genes showed different behavior by resuscitation. The expression of both the diphtheria toxin and the repressor of diphtheria toxin genes remained largely unchanged under all four conditions . This is the first study to demonstrate that C. diphtheriae can enter a VBNC state and that it can be rescued from this state via the addition of catalase. This study helps to expand our general understanding of VBNC, the pathogenicity of VBNC C. diphtheriae, and its environmental survival strategy. Many pathogenic bacteria, including  More than 60 species of pathogenic bacteria have been reported to enter the viable but nonculturable state (VBNC) during different kinds of stress , includiCorynebacterium diphtheriae is a rod-shaped gram-positive bacterium [C. diphtheriae is recognized as a reemerging human pathogen.acterium  that cauacterium . Diphtheacterium . ECDC daacterium . This diacterium . CurrentVibrio cholerae O1 into rabbit ileal loops resulted in fluid accumulation and that culturable V. cholerae O1 samples could be isolated from this fluid. Furthermore, human volunteer studies have shown that VBNC V. cholerae O1 converts to its culturable state following ingestion [C. diphtheriae, and to examine its effects on morphology and the diphtheria toxin gene. The results of this study can be used to understand the pathogenicity of VBNC C. diphtheriae and its bacterial survival strategy during stress.Colwell et al.  showed tngestion . These rC. diphtheriae strains. The toxigenic strain is equivalent to NCTC13129 and was the first C. diphtheriae strain to have its whole genome sequenced [This study used both toxigenic (ATCC 700971) and non-toxigenic (ATCC 27010) equenced , making equenced . ATCC270equenced ,24. BothC. diphtheriae strains were grown on brain heart infusion agar plates (BHIA) at 37 °C for 3 days and then each plate was tightly wrapped to keep moisture and transferred to 4 °C for 28 (ATCC700971) or 39 days (ATCC27010) in a dark condition. C. diphtheriae cells were then collected and resuspended in PBS at an optical density of 0.5 at 600 nm (OD600), representing approximately 108 CFU/mL on BHIA. This suspension was confirmed to contain VBNC cells (see Results) and used as the “VBNC C. diphtheriae suspension”. Viability of the prepared VBNC cells was examined using CTC staining  which evaluated the respiratory activity of the VBNC cells [C. diphtheriae cells were enumerated using DAPI stain (Dojindo Laboratories). All of the staining procedures were completed using the manufacturer’s instructions.NC cells  and the 7 CFU/mL) were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde. The fixed cells were dehydrated through a gradient of 50, 70, 80, 90, 95, and 100% acetone for 30 min each and then dried using a critical point dryer , coated with osmium  and observed under a scanning electron microscope . Culturable and VBNC C. diphtheriae ATCC700971 were harvested by centrifugation and washed three times with PBS. The cells  was added and incubated at room temperature for 6 h without light and agitation. After incubation, the sample was inoculated onto a BHIA plate and incubated at 37 °C for 3 days and resuscitated colonies were counted. The negative control, in which PBS was added instead of catalase, did not show resuscitated colonies. Both strains were obtained from the American Type Culture Collection .VBNC resuscitation was assayed using the Senoh et al. ,27 methoC. diphtheriae were then evaluated as follows. C. diphtheriae from each state  were suspended in 10 mL of PBS at an OD600 of 0.5, and collected by centrifugation at 3000× g for 10 min and then used for total RNA extraction via a High Pure RNA Isolation kit . rRNA was depleted using the Bacteria Ribo-Zero kit  and sequencing libraries were prepared using the TruSeq Stranded mRNA library Prep kit (Illumina) and sequenced on an Illumina NextSeq sequencer (Illumina). cDNA quality and concentration were evaluated using the Agilent High Sensitivity DNA kit and the Agilent 2100 Bioanalyzer . Paired-end reads with a length of 2 × 75 bases were used to generate the whole transcriptome libraries. Read processing and mapping was carried out by the CLC Genomic Workbench version 20 software  and the expression levels were evaluated following their normalization . Differentially expressed genes were identified using a multi-factorial statistical model based on a negative binomial GLM [Transcriptional profiles for culturable, VBNC, VBNC + catalase , and resuscitated (cells that were not subcultured following resuscitation) mial GLM  model. TC. diphtheriae from each state  was prepared as described in Transcriptome analysis section. For RT-qPCR, a PrimeScript RT reagent Kit with gDNA Eraser  was used to synthesize cDNA from 0.005 mL of total RNA. Then qPCR was performed using QuantStudio 5  and TB Green Premix Ex Taq II (TaKaRa Bio Inc.). Primers used for qPCR were listed in (t) values was obtained by qPCR. Standard curves were generated by plotting DNA concentration and their corresponding C(t) values for individual genes. The C(t) values of qPCR products of each gene were compared to those of standard target DNA for quantification. The molar ratio of cDNA copy numbers of each gene was determined using the following equation: the molar ratio of cDNA copies = the amount of cDNA (ng) / the molecular weight of expected PCR product of dsDNA (ng). The amount of cDNA was determined based on the cDNA concentration after multiplying by the fold-dilution of each gene. The molecular weight of the expected PCR product was calculated using the Sequence Manipulation Suite (https://www.bioinfomatics.org/sms2/ last access 18 February 2021). The cDNA copy numbers for each gene of C. diphtheriae were compared to the number of cDNA copies of the culturable C. diphtheriae.Total RNA of t-test. A p value of < 0.05 was considered statistically significant.The data are expressed as the mean ± standard deviation (SD). Differences between two groups were assessed using the unpaired two-tailed Student’s C. diphtheriae ATCC700971 cells gradually decreased and finally reached zero at day 28. Culturable cells in 0.1 mL of undiluted VBNC C. diphtheriae suspension were not detected in 10 replicates. In contrast, at this point in time, the number of viable cells as measured by the respiratory activity test  was still in excess of 106 CFU/mL  and elongation factors (tuf) were increased in culturable and resuscitated cells, whereas helicase (DIP0827 and DIP1423) and IS3 family transposases (DIP1266 and DIP1366) were increased in VBNC and VBNC + catalase samples , diphtheria toxin repressor gene , DIP0751, DIP1120, and 16S rRNA) of culturable, VBNC, VBNC + catalase, and resuscitated C. diphtheriae were measured by RT-qPCR. tox and dtxR were expressed almost equally under all four conditions   A,B. The d cells) C–E.C. diphtheriae, despite the fact that C. diphtheriae is recognized as a reemerging human pathogen. Therefore, we confirmed whether C. diphtheriae enters VBNC and analyzed its morphology and genetic characteristics before, during, and after this response.The term VBNC is used to describe when bacterial cells remain viable but do not grow or divide on, or in, routinely used bacteriological media. VBNC was first reported by Xu et al. , and thiC. diphtheriae ATCC700971 in PBS according to the previously described methods. After C. diphtheriae was kept at 4 °C for several weeks in PBS it became microscopically undetectable (data not shown), making this method unreliable for characterization. We then tried to produce VBNC C. diphtheriae using the BHIA plate method. This was shown to work well and we were able to implement this method with a high degree of reproducibility, confirming our belief that C. diphtheriae can enter the VBNC state.First, we prepared VBNC C. diphtheriae did not form coccoids but did experience some degree of cellular injury demonstrated by its distorted shape, expressed as an expanded center. VBNC morphology is linked closely to the species of the bacteria being evaluated and the general environmental conditions used to induce this stasis.After the majority of the bacteria enter the VBNC state, the dominant cell morphology is known to switch to coccoid ,36. HoweC. diphtheriae was produced in VBNC cells and culturable cells (data not shown). However, it is not likely that the endogenous catalase contributes to VBNC formation and/or resuscitation, because gene expression levels of the catalase gene (cat) were essentially the same under different conditions  and elongation factors, was decreased in VBNC when compared to culturable cells, and increased again in the resuscitated cells (Vibrio cholerae [Escherichia coli [tox) and the diphtheria toxin gene repressor (dtxR), a transition metal ion-dependent regulatory element that controls the expression of diphtheria toxin and several genes involved in the synthesis of siderophores in C. diphtheriae [C. diphtheriae pathogenicity and the implementation of this state as a survival strategy under challenging environmental conditions.In an effort to identify the critical genes necessary to induce VBNC and their resuscitation, we decided to compare the transcriptome of these bacteria under different conditions . Hundreds of genes showed increased or decreased expression levels. Among these, dozens showed >5-fold changes . Expressed cells . These rcholerae  and Eschhia coli . Whetherhtheriae , between


Immunoglobulin G4-related disease (IgG4-RD) is a systemic fibro-inflammatory disorder. Laryngotracheal manifestation is very rare; therefore, it is usually associated with complex diagnostic and therapeutic problems. Herein, we report the case of a 35-year-old woman with idiopathic subglottic stenosis (ISGS) treated with one-step laryngotracheal reconstruction surgery. Postoperatively, the lesion was found to be a part of the IgG4-RD spectrum. Objective and subjective phoniatric tests, spirometry, and Quality of Life Questionnaire were used for the evaluation of postoperative functional results. Slide laryngotracheoplasty as a one-step surgery without stenting and tracheostomy ensured a sufficiently wide subglottic space with no adverse effect on voice quality. During a follow-up period of 22 months, endoscopy and computed tomography scan revealed no significant restenosis. The patient was able to return to premorbid activities of daily living without any further medical treatment. The laryngeal involvement of IgG4-RD is uncommon; however, it is a manifestation that should be included in the differential diagnosis of subglottic stenoses (SGS). Furthermore, subglottic IgG4-RD might be a potential etiological factor of ISGS and acquired airway stenosis after short-term intubation. Slide laryngotracheoplasty might be a favorable solution without stenting and tracheostomy even in special cases of SGS. Immunoglobulin G4-related disease (IgG4-RD) is a rare immune-mediated fibro-inflammatory disorder of unknown origin with heterogeneous pathologic features and variable disease manifestations. IgG4-RD is a systematic condition characterized by abundant IgG4 positive plasma cell infiltration and tumor-like lesions of the involved organs and consequently is often misdiagnosed as either malignancy or granulomatous conditions -4. SinglA 35-year-old Caucasian woman presented with an 8-year history of progressive dyspnea on exertion. She had no history of prolonged intubation, airway trauma, or gastroesophageal reflux disease (GERD) and was a lifetime non-smoker. Apart from insulin-dependent diabetes mellitus, she had no other chronic disease. The breathing problem started after a septorhinoplasty under general anesthesia, performed due to a spontaneous deformation of the cartilaginous nasal septum. To identify a supposed underlying autoimmune disease, blood tests and histopathologic examinations were performed. Autoimmune panel  showed negative result, and the histopathology findings of the nasal cartilage and mucosa were normal. In the next 8 years, no symptoms occurred other than dyspnea with slow progression. Finally, a computed tomography scan revealed an approximately 10 mm long, subglottic, circumferential soft-tissue mass with a lumen diameter of 4 mm . No clin0; anti-hFinally, an anastomosis was created between the trachea, anterior cricoid, and midline incised thyroid cartilage with double-armed continuous locked sutures .Histologic findings revealed the replacement of the respiratory epithelium by metaplastic non-dysplastic squamous epithelium. The submucosa was thickened by dense lymphoplasmacytic infiltrates and focally storiform fibrosis. The plasma cell infiltrates proved to be polyclonal, with the abundance of IgG4-positive plasma cells. The ratio of IgG4-positive plasma cells per IgG-positive plasma cell was 75%. Obliterative phlebitis, tissue eosinophilia, or granulomas were not detected .th postoperative day with good swallowing and breathing. The postoperative ultrasound did not show any fibro-inflammatory disorder in the thyroid and salivary glands. The serum IgG4 level of the patient was 366 mg/l one week after the surgery. In addition, 6 weeks after the intervention, the patient was able to return to her premorbid way of life. During the follow-up period of 22 months, endoscopy revealed no significant restenosis (th postoperative month). No major perioperative or postoperative complications occurred. The patient was discharged on the 9stenosis . The sernd postoperative month: Fundamental frequency: 250.1 Hz; Jitter%: 0.33; Shimmer%: 2.82; Harmonics-to-Noise Ratio: 26.2 dB; Maximum Phonation Time: 19.6 s  . All the: 19.6 s . The Voi: 19.6 s . Approximately, 5% of all acquired SGS considered idiopathic after all possible causes  have been ruled out . The truThe IgG4 antibody has a low antigen-binding affinity and accounts for less than 5% of total IgG concentration, with IgG4 concentrations within the range of 0.01-1.4 mg/dL and variability of 5-50x among healthy individuals . ImportaVölker et al. described the first laryngeal manifestation of IgG4-RD in 2009. Since then, isolated subglottic involvement and tracheal and hypopharyngo-laryngeal involvements have been reported , 25-30 . It is assumed that due to the local IgG4-RD presentation, even short-term intubation was enough for the initiation of the mucosal inflammation process and fibrotic remodeling of the airway mucosa. This might be the way how the former asymptomatic low-grade stenosis became manifest. The typical tumefactive appearance of the lesion remained hidden by the metaplastic non-dysplastic squamous epithelium, caused by the mechanical injury by the endotracheal tube . The earThe use of other more intense drugs, such as bortezomib, fludarabine, and cyclophosphamide, which are efficient in patients with other organ involvement, has not yet been investigated in airway patients -34. In pIn addition, the surgical solution should be primarily chosen in cases of lesions involving critical airway stenosis . In caseRelapse may develop in the originally affected organs; however, it can also occur in completely different sites. Furthermore, there is no consensus on the systematic follow-up of IgG4-RD patients, and it is still not clear whether IgG4 level can be used for the assessment of treatment response . DespiteThe laryngeal involvement of IgG4-RD is uncommon; nonetheless, it is a manifestation that should be included in the differential diagnosis of SGS. Furthermore, subglottic IgG4-RD might be a potential etiological factor of ISGS and acquired airway stenosis after short-term intubation. Herein, we presented an easily performed reconstruction option using readily available vascularized local tissues. Slide laryngotracheoplasty might be a favorable solution without stenting and tracheostomy even in special cases of SGS.


Laser-induced breakdown spectroscopy (LIBS) is a technique which enables the analysis of material components with precision and spatial resolution. Furthermore, the investigation method is comparatively fast which enables illustrating the distribution of elements within the examined material. This opens new possibilities for the investigation of very heterogeneous materials, such as concrete. Concrete consists of cement, water, and aggregates. As most of the transport processes take place exclusively in the hardened cement paste, relevant limit values linked to harmful element contents are specified in relation to the cement mass. When a concrete sample from an existing structure is examined, information on the concrete composition is usually not available. Therefore, assumptions have to be made to convert the element content analyzed in the sample based on the cement content in the sample. This inevitably leads to inaccuracies. Therefore, a method for distinction between cement paste and aggregates is required. Cement and aggregate components are chemically very close to each other and therefore, complex for classification. This is why the consideration of a single distinguishing feature is not sufficient. In this paper, a machine learning method is described and has been used to automate the distinction of the cement paste and aggregates of the LIBS data to receive reliable information of this technique. The presented approach could potentially be employed for many heterogeneous materials with the same complexity to quantify the arbitrary substances. Laser-induced breakdown spectroscopy (LIBS), also sometimes called laser-induced plasma spectroscopy (LIPS), is an analytical atomic spectrometry technique which identifies elemental compositions of a material from its plasma emission. The plasma is formed with a high-power laser beam focused on the surface of the sample. The high thermal energy in the focal point brings the atoms to an excited state through evaporating and atomizing the sample and generate the plasma. When the atom relaxes to ground state, photons reveal the atoms’ unique identity ,2. At thThe determination of chloride content in reinforced concrete structures is a very important component in the field of maintenance and vulnerability analysis of existing reinforced concrete buildings. Chlorides can reach a building component, if exposed to deicing salt or sea water. Then they are able to penetrate into the concrete and over time may reach the reinforcement. If a critical chloride content is exceeded at the reinforcement bar, it may start to corrode. This has led to extensive damages and repair in the past. A reference value for this critical chloride content is defined as 0.5 wt.%/cement . TherefoThe problem in identifying the aggregate and further distinguishing it from the hardened cement paste is due to the fact that the aggregate itself is very heterogeneous. In general the contained minerals and thus the chemical elements of the aggregates depend on the geology of the surrounding landscape where the concrete factory is located. Therefore, a fast measuring method is required for simultaneous multi-element detection. With LIBS it is possible to scan a surface and provide an element distribution of the scanned surface. This is one of the fundamental advantages of LIBS over conventional methods. Despite the capabilities of the LIBS method, further processing is needed to identify aggregate in concrete. The data acquired by LIBS is usually large and the higher spatial resolution of the measurement leads to a higher amount of data and longer processing time to translate it to information. To speed up the processing and increase the accuracy in aggregate classification, an application of machine learning (ML) has been used to identify the aggregates.2 to form the plasma. The laser beam is focused to a spot size of about LIBS’ main experimental instruments include lasers, spectrometers, detectors, and computers, as shown in tion see . For detType 1Aggregate specimen, comprised of aggregates and epoxy resin to exclusively characterize the aggregate and its variation by selected element distributions. Aggregates from various types with grain size in a range of 8–16 mm were filled in a paper cup and epoxy-resin was added to cover the gravels. Epoxy is a homogeneous polymer which similar to concrete contributes to the stabilization of the aggregates in the mixture. The homogeneity and the chemical composition of the epoxy resin allows an easy differentiation of the analyzed data in terms of epoxy or aggregate. This provides the basis for an adjusted data set. Due to this approach it was possible to investigate the different types of aggregate simultaneously, which facilitated the analysis. The used aggregates are classified as pyrogenic rock, migmatite, and gneiss with the main minerals feldspar (plagioclase), quartz, and mica.Type 2Cement specimen with Portland cement (CEM I 42.5 R) prepared with deionized water and a water-cement ratio of 0.50 to characterize solely the hardened cement paste by its element distributions.Type 3Concrete specimen with cement and water-cement ratio as described for the sample type (2) together with the aggregate measured within specimen type to verify the trained algorithm.For investigation three sample types have been prepared with different mixtures leading to a set of 15 samples in total see . The samThe sample types (1) and (2) have been used to characterize a virtual concrete whereas sample type (3) was used to verify the trained model in real application.In order to show what results can be obtained with the method presented here, a drill core taken from an existing structure was also examined (in situ sample). The concrete core had been taken from a part of a structure, which had been exposed to deicing salt that contains chlorine. Before the measurement of this sample the core was split parallel to its axis to examine how far and to what extent chlorides have penetrated into the concrete.2 to obtain a higher number of data points to detect the potentially large scatter due to the various aggregate types. The slices of the specimen belonging to samples types (2) and (3) have been measured in an area of 35 × 35 mm2.After curing, all test specimens were cut into slices and the cut surfaces were analyzed via LIBS. The slices with aggregate and epoxy resin (sample type (1)) were scanned over an extended area of 60 × 40 mmThe measurement was performed in while motion mode. That means that the measurement head continuously moves with a specific speed over the surface of specimen. The speed of the measurement head is adjusted internally in the measurement system so that with a repetition rate of 100 Hz the mentioned grid size is met.Major minerals that form the aggregates of concrete are: plagioclase, quartz, hematite, magnetite, amphibole, pyroxene, olivine, mica, and calcite. The element distribution in these minerals among each other, as well as compared to cement, have a lot similarities. With data taken from  the massThe spectral lines for the selected elements H, Ca, C, and O  and ultra violet (UV) but due to limitation of the control box of the experimental setup, it was not possible to cover this entire spectral range at the same time.kNN is still the better choice for applications where accuracy is in higher priority and predictions are not requested frequently [In classical programming a rule is being defined and data is provided as input. With this information processing takes place under defined rules leading to the answer, which is the output. In machine learning (ML) there is a shift in this paradigm. An answer replaces the rule in order to have a rule as an output . The difequently .kNN is a non-parametric classifier, which means that the model has a fixed number of parameters [K nearest points, to the input x, in the training set with the Euclidian distance. Then the members of each class within this set are counted and a vote is raised based on empirical fraction to estimate and predict. More formally,K nearest points to x in D and rameters . This alThe spectrometers disperse the emitted radiation of the laser-induced plasma to capture a spectrum in terms of intensity as a function of the wavelength . In thisSince cement consists essentially of burnt stone, such as limestone, clay, and dolomite, aggregates and cement show a large overlap in the element distributions. The comparison of density distributions derived from the measurement results show this. A comparison of these functions in relation to cement and aggregate illustrates the difficulties of differentiation. It is easy to discern between cement and aggregates in the areas, where the intensity of aggregates is lower or higher than minimum or maximum intensity of cement. The material allocation of the measuring points, where the intensity lies in this overlapping area, requires special attention in order to be able to determine whether the material measured is cement or aggregates. Due to overlapping of the common elements, it may not be sufficient to use a single distinguishing element and therefore several elements should be considered simultaneously. These findings are in good agreement with the literature study of element distribution in minerals discussed in the previous section.To train the algorithm a virtual concrete consisting of the two data-sets of sample type (1) and (2) have been measured and labeled. The first data set represents aggregates and the second one hydrated cement paste. From the sample type (1), the epoxy resin has been removed with thresholding the intensity for each element  as is shown in In order to avoid losing data points after data removal linked to epoxy, the empty points within the aggregates have been filled up with the value of zero, by masking the oxygen layer to them see . By thisIn this study, the prepared data for learning is labeled as cement/aggregate and therefore, supervised learning with classification algorithms has been used. An advantage of supervised learning is the possibility of measuring the accuracy of the trained model with the test data set. The virtual concrete is not only used for training purpose but it has also been used for tracking the predictions and determine the accuracy of the model.The virtual concrete has been divided randomly into two sets for training and for testing with the size of 118,400 and 29,600, respectively.A cross-validation for the evaluation the performance of the model has been performed and compared with different algorithms. The kNN algorithm showed an outstanding performance among Logistic Regression, Decision Tree, Ridge, and Random Forest. Mean absolute error (MAE), mean squared error (MSE), and the coefficient of determination ). The quality of the material differentiation was then checked visually, which was possible without much effort due to the grain size of the aggregate being between 8 and 16 mm. It is expected that the entire area between the aggregates will be detected as cement, as no sand was added to the concrete mixture. To train the model, different variations of elements have been tried in an iterative process until the segregation has been completed visually. Here, as before, it became apparent that no satisfactory result could be achieved by considering only the information from one element. Since in sample type (3), there were only aggregates size from 8 to 16 mm, it is expected that the algorithm shows a complete hollow space among aggregates. As This is illustrated in In In the determined chlorine heatmap of the specimen see  Left, thThe application of the developed algorithm is demonstrated on a concrete core drilled from an existing reinforced concrete structure. Before applying this method, the trained aggregate extractor (Aggex) model was serialized and deployed in the software. This gives an unprecedented advantage over clustering algorithms such as k-mean as studied by . To distWith the outlined approach the mean chloride content within a certain depth can be calculated in wt.-% based on the cement enabling the output of so-called chloride profiles see . This prOne can see that the curve becomes smoother with gravel exclusion and that there is a shift to higher concentrations. With this data, more reliable information linked to the prediction of service life can be achieved compared to conventional methods. This enables more optimized maintenance and repair actions over service life as well as improving the sustainable use of structures. Furthermore, by also providing very localized information, a better understanding of chloride distribution in cracks can be achieved. This is important for future investigations as the cracks directly exposed to chlorides are very severe, causing extensive repair costs. As an example, the decks of parking garage should be mentioned.Concrete is a heterogeneous material that is broadly used in construction. Chlorides from deicing salt or seawater may heavily influence the durability of reinforced concrete structures as it may cause corrosion and subsequently affect the service life. As relevant limit values linked to harmful substances such as chloride are specified in relation to the cement mass uncertainties may occur due to the lack of knowledge of the concrete compositions within the sample examined. By using the laser-induced breakdown spectroscopy technique element distributions of the scanned surface can be given. The acquired data from this technique needs further processing to quantify relevant element contents in materials that are examined. Furthermore, the data can be used for segregating the aggregates from the cement. A machine learning algorithm with LIBS data has been accomplished for this purpose. The model designed in this paper is based on a kNN algorithm with an accuracy close to 100%. To train the aggregate extractor four elements have been selected for stabilizing the accuracy, which are calcium, carbon, hydrogen, and oxygen. Oxygen has been used for internal normalization and the other elements mentioned for automated distinction. Cement and aggregate components can be very close to each other within its element distribution, which leads to huge overlapping in element intensities measured with LIBS. Despite high overlap in some elements such as calcium in cement and aggregate, with multivariate analysis it is possible to differentiate between aggregate and cement. As LIBS also provides a dynamic continuous observation over the scanned area of the specimen this enables achieving detailed information within a fast measuring process. The possibility to obtain locally very detailed information as well as rather global information (e.g. mean penetration depth) is important to design suitable maintenance and repair strategies for reinforced concrete structures. This enables more optimized actions linked to maintenance and repair over service life and therewith improving the sustainable use of structures.


Acute febrile illness in children is frequently treated with antibiotics. However, the inappropriate use of antibiotics has led to the emergence of multidrug-resistant pathogens.We measured use of antibiotics for fever in 4 pediatric cohorts that were part of the Surveillance for Enteric Fever in India (SEFI) network. In this network, 24 062 children were followed up weekly, capturing information on fever and other morbidity between October 2017 and December 2019.An antibiotic was given in 27 183 of the 76 027 (35.8%) episodes of fever. The incidence of fever-related antibiotic use was 58.0  per 100 child-years. The median time to initiation of antibiotics was 4 days, and in 65% of those who received an antibiotic it was initiated by the second day. Antibiotics were continued for <3 days in 24% of the episodes. Higher temperature, younger age, male sex, joint family, higher education, internet access, and availability of personal conveyance were associated with antibiotic treatment for fever.In developing countries where antibiotic use is not regulated, broad-spectrum antibiotics are initiated early, and often inappropriately, in febrile illness. Frequent and inappropriate use of antibiotics may increase risk of antimicrobial resistance. Acute febrile illness with or without other symptoms is common in children . While aThe Surveillance for Enteric Fever in India (SEFI) network established pediatric cohorts in 4 locations in India to study the burden of culture-confirmed enteric fever . Each coThis analysis estimates the frequency and patterns of antibiotic use in children for febrile illness, including incidence of fever-related antibiotic use and the choice, timing, and duration of antibiotics. In addition, we examined the demographic and socioeconomic factors associated with antibiotic usage for fever.The study protocol was approved by the Institutional Review Board of the Christian Medical College and each participating institute. Written informed consent was obtained from parents of participating children and assent from age-eligible children.In the SEFI network, 4 cohorts of children, between 6 months and 15 years of age, were recruited and followed up, in 3 urban  settings and a rural area on the outskirts of Pune. In Delhi, the study was conducted in 2 contiguous blocks of Sangam Vihar, a low-income urban resettlement neighborhood in the south of the city. In Kolkata, the study area consists of wards 58 and 59, a predominantly impoverished slum area located in the eastern part of the city. In Vellore, the study area comprised the contiguous semi-urban settlements of Chinallapuram, Kaspa, Ramnaickanpalayam, and Vasanthapuram. In Pune, the study area, located 30 km from the city, was a rural, agrarian settlement. The urban sites had a slightly higher literacy rate than the rural site  : 20 663 vs 11 268, P < .001). They also had greater access to internet and motorized transportation . Both the duration of fever  and highest temperature documented  were lower at Pune than at the other sites.The antibiotic use at Pune was significantly higher than the other sites. This rural population was more affluent compared with the other sites that were predominantly in urban settlements  fever-related antibiotic courses per 100 CYO. More than half of the children (55.9%) had at least 1 fever-related antibiotic course during the study. While other studies have extrapolated antibiotic data from Demographic and Health Surveys , this stOf those who received antibiotics, 64% had antibiotics initiated before the third day of fever. Very early initiation may reflect the lack of diagnostic tests and the demand from families paying out of pocket for healthcare for prompt resolution of symptoms. Based on the provisional diagnosis assigned by the treating physician, we note that the antibiotic usage was unlikely to have been warranted in 65.4% of the episodes where antibiotics were initiated. Fink et al, from a study on 8 LMICs, predominantly in Africa, reported unnecessary antibiotic usage rates ranging between 49% and 81% [Our study reports the widespread use of broad-spectrum antibiotics like amoxicillin and azithromycin for treating febrile illnesses. This aligns with observations in the World Health Organization report on surveillance of antibiotic consumption that noted that amoxicillin and azithromycin were the most commonly used antibiotics in most regions . BecauseWe observed a higher risk of antibiotics among those with temperatures >38.4°C, which parents and local physicians consider a marker of severity of illness. Younger children, households with higher education, internet literacy, and the availability of personal conveyance were independently associated with a higher risk of antibiotic usage. Similarly, male children and those belonging to joint families were more likely to be initiated on antibiotics during a fever episode. Thus, both deep-rooted sociocultural and economic factors contribute to the prevailing antibiotic use practices. Though the systematic review by McKay et al on the factors associated with antibiotic usage among respiratory tract infections identified that clinical diagnosis and evidence from clinical examination were the principal drivers of antibiotic prescription , in the The shorter duration of fever and lower temperature in febrile episodes noted in Pune might partly be explained by early and more aggressive management of febrile illness at this location.This study has several limitations. We estimated incidence in children >6 months of age and do not provide information the first 6 months, when antibiotic use is likely to be relatively high , 18. TheThe high incidence of antibiotic use and the inappropriate use of antibiotics pose a significant risk and can contribute to worsening antibiotic resistance in India. Both education at all levels of healthcare and communication to the public regarding inappropriate antibiotic use are needed to complement cost-effective diagnostic tests for an early identification of fever etiology in order to reduce inappropriate antibiotic usage.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiab115_suppl_Supplementary_Figure_1Click here for additional data file.jiab115_suppl_Supplementary_Tables_1-3Click here for additional data file.