SF addition involves both physical contribution and chemical action in concrete. TDFA contains 25–30% SiO2 and 30–35% CaO, and is considered a suitable secondary pozzolanic material. In this review, special emphasis is given to the various chemical additives and their role in protecting rebar from corrosion. Specialized concrete for novel applications, namely self-curing, self-healing, superhydrophobic, electromagnetic (EM) wave shielding and self-temperature adjusting concretes, are also discussed. Supplementary cementitious materials (SCMs) and chemical additives (CA) are incorporated to modify the properties of concrete. In this paper, SCMs such as fly ash (FA), ground granulated blast furnace slag (GGBS), silica fume (SF), rice husk ash (RHA), sugarcane bagasse ash (SBA), and tire-derived fuel ash (TDFA) admixed concretes are reviewed. FA (25–30%), GGBS (50–55%), RHA (15–20%), and SBA (15%) are safely used to replace Portland cement. FA requires activation, while GGBS has undergone in situ activation, with other alkalis present in it. The reactive silica in RHA and SBA readily reacts with free Ca(OH) TThe main focus of this review is to demonstrate the effects of supplementary cementitious materials and chemical additives on the physical, mechanical and durability properties of hydraulic concrete. It aims to elaborate the effective utilization of SCMs  and CA  wave shielding and self-temperature adjusting) on the distinct properties of concrete. This review is intended to the researchers to select those SCMs and CA most suitable for ordinary and hydraulic concrete thanks to their better physical, mechanical and durability characteristics. SCMs such as FA, GGBS, SF, RHA, SBA, and TDFA are typically added to concrete. Besides cost reduction and enhancement of the workability of fresh concrete, they can be successfully employed to improve the resistance of concrete to thermal cracking, alkali-aggregate reaction, resistance to chloride diffusion, and sulphate resistance ,7,8,9. AFly ash is a finely divided residue resulting from the combustion of ground or powdered coal. The worldwide production and utilization of FA is given in Specifications for fly ash are given in the ASTM C-618  and AASHFly ash used in air-entrained and non-air entrained concrete mixes usually reduces the bleeding due to greater fineness volume and lower water content for a given workability ,30. Both3A-rich cement containing fly ash in the presence of magnesium [2SO4, pH≈3) and 1.5% nitric acid concentration  for a period up to 90 days. In the sulphuric acid environment, the compressive strength loss was minimal for a concrete mix in which cement was replaced with 30% fly ash and 10% ultra-fine fly ash. The mass loss was less in this mix compared to the mix without fly ash. However, mass loss was also less in mixes containing higher amounts of fly ash. In the nitric acid environment, concrete mixes containing 20% fly ash and 10% ultra-fine fly ash and 30% fly ash and 10% ultra-fine fly ash had the minimum compressive strength loss. However, the mass loss in the mix containing 30% fly ash and 10% ultra-fine fly ash was less than the mix containing 20% fly ash and 10% ultra-fine fly ash [Fly ash increases the cementitious compounds, minimizes water demand, and reduces bleeding channels, and thereby yields concrete with low permeable internal voids. Through the pozzolanic activity, fly ash chemically combines with water and calcium hydroxide, forming additional cementitious compounds which result in denser higher strength . This reagnesium . Kim et agnesium  and Mbesagnesium  comparedagnesium  studied  fly ash .2 is present, the solubility of SiO2 in fly ash markedly increases. Thermal activation affects both fly ash reactivity and the kinetics of dissolution; significantly faster glass breakdown occurs at elevated temperatures by thermal activation. The binding mechanism of activated and inactivated fly ash is given in 2, which is advantageous to coagulative structure formation.FA needs activation due to two factors. First, the glassy surface layer of glass beads is dense, chemically stable and protects the inside constituents, which are porous, spongy and amorphous. Second, the silica–alumina glassy chain of high Si, Al, and low Ca is stable; the chain must be disintegrated if activity is to take place. The schematic diagram for the fly ash activation process is given in Ground granulated blast furnace slag is the granular material formed when molten iron blast furnace slag is rapidly chilled by immersion in water. The worldwide production of GGBS is given in GGBS is a cementitious material and can be substituted for cement on a 1:1 basis. The blast furnace cement is made by inter-grinding the granulated slag with Portland cement clinker. The grade of a GGBS is based on its activity index, which is the ratio of the cube compressive strength of a mortar made with 50% GGBS to a mortar made with cement ,48. The HAI = Hydration activity index of GGBS in %;t = Compressive strength of test mortar at “t” age in MPa;f0t = Compressive strength of reference mortar at “t” age in MPa.fGrade 80 has a low activity index and generates less heat than Portland cement. Grade 100 has a moderate activity index, which is similar in cementitious behavior to Portland cement. Grade 120 has a higher activity index, which is more cementitious than Portland cement . The use2 and CaO content in GGBS is 10–27% and 35–45% respectively. Hence, the active ingredients in GGBS readily react with cement and undergo hydration reactions, resulting in the formation of so-called C-S-H gel, which imparts strength to the concrete and leads to a pore blocking effect. Due to the inherently cementitious and pozzolanic nature of GGBS, it does not require any external activation methodology to increase the number of connecting points, as fly ash does. However, GGBS has undergone in situ activation with other alkalis present in it. In pore solution chemistry, GGBS endures both consumption and generation of calcium hydroxide during hydration reactions. However, GGBS favors the formation reaction rate, and hence undergoes self-activation, which leads to the usage of GGBS in high-performance concrete.GGBS exhibits both glassy and crystalline phases. The SiOSilica fume has been widely used as a supplementary cementitious material in high-performance concrete. Silica fume is also known as “micro-silica”. It is a by-product obtained from the reduction of high purity quartz with coal in electric furnaces during silicon and ferrosilicon alloy production. Silica fume is also collected as a by-product in the production of other silicon alloys such as ferrochromium, ferromanganese, ferromagnesium and calcium silicon . Silica 2/kg, which is approximately 100 times smaller than the average cement particle. Due to its extreme fineness and high silica content, it is effectively used as a pozzolanic material [Silica fume consists of very fine vitreous particles with a surface area on the order of 20,000 mmaterial ,72. Mohamaterial . AASHTO material  standardmaterial ,76. Silimaterial ,78. It hmaterial ,80. The material  and Karamaterial , showingmaterial ,83. Rapi2. Thus, it is a highly reactive pozzolanic material and readily reacts with calcium hydroxide to form C-S-H gel and provide hardened concrete. The mechanism of action of silica fume in fresh and hardened concrete is given in The reaction of silica fume in concrete involves both a physical contribution and chemical action. As a physical contribution, adding silica fume fills the spaces in the interfacial region of the cement grains. This phenomenon is called particle packing. As a chemical contribution, silica fume contains more than 90% very highly amorphous SiORice husk ash (RHA) is one of the highly reactive supplementary cementitious materials for the construction of concrete structures ,85. The Mechanical grinding is essential to achieve the necessary fineness and high surface area of the RHA particles. Corderiro et al. studied the suitable grinding time of RHA and reported that a minimum of 120 min of grinding time was sufficient to produce a fine particle with a high surface area which increases the pozzolanic activity index . Song et2SiO42−) from the reactive amorphous silica and calcium hydroxide (Ca(OH)2). The pozzolanic reaction mechanism are given below.The pozzolanic reaction is favorable in RHA when reactive silica is in an amorphous state. In general, the amorphous silica that is ubiquitous in pozzolanic material reacts strongly with lime compared to the material in the crystalline phase. The pozzolanic reaction mechanism is an acid–base reaction between silicium acid  is a major residue of the sugar industry. Bagasse is an agro-waste material that remains after the extraction of sugarcane juice in the sugar industry. After extraction, fibrous sugarcane (bagasse) is partly used as the main fuel in the sugar industry for power generation ,112. The2 [2O3 and Fe2O3. SiO2, Al2O3 and Fe2O3 are pozzolanic substances, and when is mixed with cement act as a cementitious material [Studies have revealed that the bagasse ash contains 63–70% silica ,118,119;2 ,121,122,material . Bahurudmaterial  reportedmaterial . It has material ,124,125.material . Singh ematerial .2SO4 and HCl (1% to 5%). They concluded that acid resistance with up to 10% SBA replacement level was better than control concrete [2SO4. They found improved acid resistance properties in the blended cement when compared to the control mix at a 10% replacement level [2SO4 solution. They concluded that 10% of SBA blended concrete reduces the sulphate attack when compared to control concrete [2 in a cement matrix, which is owing to the pozzolanic reaction. The optimum replacement level of cement is 10% SBA, which improves strength and durability, decreases the acid attack, chloride diffusion and corrosion rate of embedded steel rebar.It has been reported that M20 concrete made using 5–25% of SBA showed greater compressive strength even at a 5% replacement level when compared to control concrete ,127. Theconcrete . Singh ent level . Gupta econcrete . Based o2 in cement matrix, which produces the secondary C-S-H gel and improves the compressive strength of concrete. Moreover, SBA contains Al2O3, which reacts with Ca(OH)2 and leads to the formation of C-A-H, which can resist sulphate attack.SBA contains 77–88% silica, which is favorable for pozzolanic reaction due to the availability of amorphous silica. The presence of amorphous silica in SBA reacts with free Ca(OH)Furthermore, the unreacted silica in SBA acts as a pore filler, which can reduce porosity and voids in the concrete. It is also helpful for resistance to chloride penetration, and reduces the corrosion rate of steel rebar. Increasing the addition of SBA by more than 10% increases the water demand and reduces workability and compressive strength, due to the fineness and surface area of SBA.2, Al2O3, and CaO, and these oxides are suitable for pozzolanic materials. According to the ASTM C-618-08a, TDFA is considered as a class C pozzolana because the sum of the ratio of oxides is more than 50% [2 content compared to other ashes like fly ash, RHA, and SBA. Al-Akhras and Smadi investigated the effects of TDFA as a sand replacement in cement mortar. In their study, 2.5%, 5% 7.5%, and 10% TDFA replaced sand weight [As the automobile industry has grown exponentially worldwide, the usage of tires has continuously increased. Some 1.6 billion new tires are manufactured every year, generating around one billion used tires which require proper disposal . Tires dthan 50% . TDFA had weight . The autd weight . Al-Akhrd weight . In addid weight  by castid weight .3 content in concrete, as higher SO3 contents are present in TDFA.Other research has investigated the use of crumbed and shredded waste tires in fine aggregate and coarse aggregate ,145. Kar2, 24–36% CaO, 4–14.6% Al2O3 and a small amount of carbon material , disodium hydrogen phosphate (Na2HPO4) and trisodium phosphate (Na3PO4) and evaluated them in OPC paste and OPC mortar with steel rebar specimens [2HPO4 showed improved corrosion resistance properties and lower icorr values. Bastidas et al. examined the influence of three soluble phosphates, sodium monoflurophosphate  react with CO2 to form CaCO3, healing cracks in the concrete [Concrete is one of the most generally used construction materials worldwide, but it is easily susceptible to cracking ,217,218,w cracks . In the concrete . On the concrete ,228. Forconcrete . This isconcrete . In theiconcrete . In thei2 and monofluorophosphate as healing materials. They used the X-ray micro-computed tomography (XCT) technique to monitor the corrosion status of rebar in concrete. Based on the experimental results, they reported that the two kinds of self-healing systems showed better performance in healed the cracks caused by corrosion. However, autogenous and autonomous healing is limited to small cracks and was not suitable for wider cracks, and its effectiveness depends on the water content [Du et al. investigated the application of paraffin microcapsules with toluene di-isocyanate for self-healing concrete . They re content . 3 in cracks [3 are able to heal cracks in concrete. This bacteria-based self-healing is very effective, and able to repair wider cracks [Another method involves the utilization of bacteria in self-healing concrete, and can be applied to external cracks by spraying or injection into concrete . Ureolytn cracks ,239,240.r cracks . Howeverr cracks . As a rer cracks . There ir cracks ,242.Reinforced concretes can be vulnerable to cracking depending on the environmental conditions ,217,218.Superhydrophobic materials are admixed in the concrete during casting as a water resistant material. A biomimetic superhydrophobic surface can be developed using silane and silicone hydrophobic materials. For example, Song et al. investigated the influence of fluoroalkylsilane with filler materials admixed in concrete . In theiWireless electronic devices are proliferating rapidly in the contemporary technological world, and the electromagnetic waves emitted from these devices are likewise increasing . EM waveLiu et al. examined the effect of helical CNT-coated carbon fiber composite, which exhibited reflectivity of 32 dB in the frequency range of 8.2 GHz to 18 GHz. It has been reported that an SE of 30 wt% graphene oxide can be obtained by incorporating ferrofluid in concrete, leading to an SE of 46 DB in the frequency range of 8.2–12.4 GHz . Jung et2 with clay admixed into the concrete and reported that TiO2-containing concrete showed good EMI absorption and excellent compressive strength [3O4 fluids were used by He et al. to prepare concrete with 5% Fe3O4, which had a better SE in the frequency range of 8–18 GHz [In the EMI absorption shielding method, concrete is made using highly magnetic materials such as magnetite, hematite and other ferromagnetic materials ,283,284.strength . Zhang astrength ,287. Micstrength . Nano-Fe8–18 GHz . Ogunsol8–18 GHz . EM shie8–18 GHz ,292. The8–18 GHz ,304,305.8–18 GHz . TherefoIndoor concrete buildings consume more energy for cooling during the summer seasons, which amounts to one-third of energy consumption in most countries . Hence, Marrani and Madhkha have studied the thermal properties of microencapsulated PCMs admixed in sandwich concrete panels . They reThe following conclusions can be drawn from the present review: SCMs such as fly ash at levels of up to 25 to 30% can be safely used to replace Portland cement, resulting in better properties. A significant level of replacement of Portland cement by fly ash is not only beneficial to the concrete but also minimizes the production of cement, and thereby reduces the greenhouse gas effect as per the following equation:Mechanical grinding, thermal activation, and chemical activation accelerates the hydration reaction of fly ash with cement. Activation of fly ash is essential in order to obtain better performance in both mechanical and corrosion-resistant properties. Activation methodology increases the number of connecting points in fly ash and binds with cement within a short period. Activated fly ash-admixed cement yield designs showed compressive strength within 28 days on par with ordinary Portland cement.GGBS at levels up to 50 to 55% can be safely used to replace Portland cement, with better properties. GGBS has undergone in situ activation, with other alkalis present in it. The active ingredients in GGBS are highly reactive; hence, it undergoes a hydration reaction with cement which forms bulky calcium silicate hydrate, leads to a pore blocking effect.2 in the cement matrix, which produces secondary C-S-H gel and improves the compressive strength of concrete. SBA contains Al2O3, which reacts with Ca(OH)2, leading to the formation of C-A-H, which can reduce sulphate attack. Unreacted silica in SBA acts as a pore filler, which can reduce porosity and voids in concrete, which is helpful for increasing resistance to chloride penetration and reducing the corrosion rate of steel rebar.Bagasse ash at levels up to 10–15% can be safely used to replace Portland cement, with better properties. The presence of amorphous silica in SBA reacts with free Ca(OH)2. Thus, it is a highly reactive pozzolanic material and readily reacts with calcium hydroxide to form C-S-H gel and provide hardened concrete.The reaction of silica fume in concrete involves both a physical contribution and chemical action. As a physical contribution, adding silica fume fills the spaces in the interfacial region of cement grains. As a chemical contribution, silica fume contains more than 90% very highly amorphous SiORHA is an effective SCM, and at an optimal replacement level up to 15–20% of Portland cement improves strength and reduces the sulphate reaction and chloride diffusion, as well as the corrosion rate of embedded steel rebar. The pozzolanic reaction is favorable in RHA when reactive silica is in its amorphous state, leading to the formation of additional C-S-H gel, which can improve the strength and durability of concrete.SCM-blended concrete resists both chloride and sulphate attack. Hence, concrete with SCMs is highly useful to construct civil infrastructure such as bridges, nuclear reactors, ports, seashore and marine environment areas, etc.st century, tire-derived fuel ash may find applications in the construction industry. TDFA contains 25–30% SiO2 and 30–35% CaO, and thus is considered a suitable pozzolanic material. Furthermore, TDFA can be used as a partial replacement in cement and also used as a coarse aggregate in concrete.With the rapid growth of the automobile industry in the 21The purpose of corrosion-inhibiting admixtures is to protect rebar from corrosion. They are added to the concrete during the casting stage itself. Hence, in the construction of any new concrete structures the use of corrosion inhibitors is inevitable. For an existing concrete structure, a migrating or electro-injection methodology may be adopted to safeguard the embedded steel in concrete.Self-curing concrete is essential in arid and semi-arid regions where scarcity of water is a critical problem. Self-curing concrete minimizes cracking and shrinkage in concrete, and thereby improves its durability. The biggest advantage of self-curing concrete is the elimination of labour requirements, bypassing the laborious curing process of 28 days (for OPC) or 90 days (for PPC) post-construction.The self-healing process in concrete can be achieved by autonomous healing, autogenous healing, and microbial methods. In all three, the concrete is rendered capable of repairing cracks automatically without any external inspection or human involvement. However, this methodology is limited to smaller cracks only.Superhydrophobic chemical admixtures can be applied to concrete either through surface coatings or by admixture in concrete. In both methods, hydrophobic materials resist water and offer protection to both concrete and rebar.Electromagnetic shielding concrete has applications in both military buildings and civil applications. EMI shielding can be achieved by either absorption or reflection. Carbon materials, special types of conducting polymers, and metal fibers play important roles in making EMI-shielded concrete.Self-temperature adjusting concretes are very useful in tropical countries because indoor concrete buildings consume more energy for cooling during the summer seasons. This can be achieved by utilising phase changed materials (PCMs) in the concrete and thereby mitigating thermal effects.The integration of innovative technologies such as nano, geopolymer, 3D printing/digital production, bio, self-assembly, and organic–inorganic copolymerization may soon align with existing technologies to promote the growth of multipurpose structures, and provide a boon to the construction industry.


The concrete workability was reduced with the presence of BSA . The compressive strength increased with the number of curing days. The concrete microstructures were improved; interfacial transition zones (ITZ) decreased with an increase of BSA. The optimal percentage of BSA obtained was at 1.25%. The established model showed significant model terms  with probability of 0.01% for the F-value to occur due to noise. The established model is useful for application in construction industries.The potassium (K) and sodium (Na) elements in banana are needed for hydration reaction that can enhance the strength properties of concrete. This research aims (a) to determine the material engineering properties of banana skin ash (BSA) and concrete containing BSA, (b) to measure the strength enhancement of concrete due to BSA, and (c) to identify optimal application of BSA as supplementary cement materials (SCM) in concrete. The BSA characterization were assessed through X-ray fluorescence (XRF) and Blaine’s air permeability. The workability, compressive strength, and microstructures of concrete containing BSA were analysed using slump test, universal testing machine (UTM) and scanning electron microscope (SEM). A total of 15 oxides and 19 non-oxides elements were identified in BSA with K (43.1%) the highest and Na was not detected. At 20 g of mass, the BSA had a higher bulk density (198.43 ± 0.00 cm It is rich in fibres containing high amount of nutrients such as phosphorus and nitrogen, residues of pesticides and organic carbon6. Banana skin is waste biomass that can be adopted into various uses such as traditional medicine, livestock feeds, medium for mushroom cultivation and slow-release fertilizer for home garden plants7. It can be extracted into raw cellulose fibres as a reinforcing agent in composite polymers8. The banana skin can be processed into starch9. Incineration produces banana skin ash (BSA). Banana peel has been used for water treatment; especially for dye removal10 due to its physical adsorption capacity. BSA is comparable to other agricultural wastes11 for removal of various carcinogenic pollutants in water.Agricultural waste is biomass produced by agriculture industries12 and compressive strengths with improved modulus of elasticity in concrete higher than glass, steel and polymers14. Approximately 95 million tons agricultural waste of banana origin was produced since 201215. It showed that, this waste has a high disposal rate throughout the year providing a sustainable supply for the construction industries. Similar to coal ash, this agriculture waste has low density, appropriate stiffness and satisfactory durability17, possesses pozzolanic properties making it a good candidate for partial replacement of cement, admixture or supplementary cement material (SCM) in concrete15; thus reducing construction costs. It has gained interest in civil construction and materials engineering fields ever since. Table 18, coal bottom ash (CBA)19 and banana by-products from literatures (e.g. banana leaves ash (BLA)15, banana skin powder (BSP)20 and palm oil fuel ash (POFA)20, banana stem fibre (BSF)14) with the current research.In material engineering properties, integration of natural fibres ash in concrete are continuously reported to be able to increase tensile18 and normal (20 to 40 MPa)20 concretes. The reported physicochemical properties from literatures were water absorption index15, scanning electron microscopy (SEM)15, energy dispersive X-ray analysis (EDS)15, adherence resistance15, X-ray fluorescence spectrometry (XRF)20, particle size analyser (PSA)20, fibre linear density14, and single fibre tensile test14. Current research reported physicochemical properties on XRF, Blaine’s air permeability, and SEM.The types of concrete used were high strength (> 40 MPa)18. The integration of CBA in normal concrete produced strength enhancement from 28 (10% CBA) to 33 MPa (15% CBA)19. The addition of BLA in normal concrete yielded strength enhancement from 33 (5% BLA) to 36 MPa (7.5% and 10% BLA)15. The mixture of BSP in normal concrete produced strength enhancement from 22.3 (0.2% BSP) to 31.7 MPa (0.8% BSP)20. Meanwhile, the combination of 0.4% BSP + 15% POFA yielded 29.7 MPa and 1% BSP + 15% POFA yielded 32.7 MPa. The inclusion of BSF in normal concrete yielded strength enhancement to 28.9 MPa, 32.0 MPa and 29.7 MPa for 0.1%, 0.2% and 0.3% BSF accordingly.The application of BDP in high strength concrete produced strength enhancement from 50 (10% BDP) to 61 MPa (1% BDP)21 reported that the use of BSA in conventional concrete improved the tensile modulus for optimum use by 5% as well as flexural strength but with considerably lesser compressive strength. It is noted that, different properties of various natural fibres are able to increase the strength properties of composites accordingly12. The application of banana leaf ash is technically feasible because not only does it improve the concrete performance but also contribute to reduction of cement utilization in construction industries; approximately up to 10% leading to construction cost reduction15. The building material cost reduction has been in good agreement with Prakash et al.14 who used banana stem fibres as an alternative to steel and artificial fibres to increase strength properties in mortar and concrete.Mohamad et al.19 in their study to optimize the amount of CBA and strength enhancement in concrete. Thus, current research presents studies on the strength enhancement of concrete using incinerated agricultural waste namely, BSA as SCM. The optimization on the application of BSA as SCM in concrete was modelled via RSM.In addition, the current research presents observations on the microstructure improvement in the concrete after the integration of BSA via interfacial transition zone (ITZ). Response surface methodology (RSM) was used by Mohd Kamal et al.pisang nipah or Musa sp. were randomly collected from a local supermarket near Universiti Tenaga Nasional, rinsed several times with water, oven-dried (100 °C) for 24 h and incinerated at 300 °C for 30 min to produce banana skin ash (BSA). The resulting BSA was sieved to 75 μm sieve size. The preparation of BSA is shown in Fig. Banana skins locally called en-dried 00 °C forThe material characterization was conducted based on X-ray fluorescence test (XRF), SHIMADZU CORPORATION, Tokyo, Japan) and workability test. The chemical composition of BSA samples was determined using XRF (RaynyEDX-700/800).Blaine’s air permeability test or fineness test based on EN 196 ASTM C204 was conducted to obtain the fineness of BSA. The fineness of cement has a significant effect on the rate of hydration that will increase the concrete strength and the rate of heat evolution. Using Blaine’s apparatus, the specific surface for banana peel ash and cement was obtained.The slump test was conducted to assess the workability  of freshly made mix concrete focusing on the water-cement ratio based on ASTM C143 / C143M – 20 standards. The mix concrete contained 0 (control mix), 1 and 2% of BSA.The compressive strength (MPa) of concrete was determined based on ASTM C39/C39M-21 using a universal testing machine (UTM). The curing days were ranged from 0 to 28 days. The structural composition of concrete was observed and recorded using scanning electron microscope (SEM) . The interfacial transition zone (ITZ) was also observed.Y =  Predicted response variable, β =  Regression coefficient, k = Number of factors or process variables in the experiment, e =  Random errorCentral composite design (CCD) with second order polynomial equation Eq.  was usedThe ranges of process variables and code factors are shown in Table r) was used to measure linear association between two variables  + aluminium oxide (Al2O3) + iron (III) oxide (FeO3) ≥ 80%); while Class C FA comes from lignite, and possesses both pozzolanic and cementitious properties (SiO2 + Al203 + FeO3 ≥ 50%)24. As compared to other materials, the silicon dioxide (SiO2) content of BSA (14.62%) was in the range close to OPC (20.6%). In the presence of heat during curing, the silicate-based material reacts with calcium hydroxide (Ca(OH)2) generated by hydrating cement to form compounds possessing cementitious properties of better high strength performance25. Meanwhile, the calcium oxide (CaO) (4.12%) content of BSA in the current study was comparable to FA (3.32%)23, bottom ash (BA) (7.37%)23 and class F FA (5%)24. The chemical compositions of BSA such as SiO2 (14.62%) + Al2O3 (0.26%) + FeO3 (0.21%) was 15.09%. Accordingly, BSA is a pozzolan but cannot be classified according to ASTM standards classification for coal ash pozzolans because the percentage composition were below the standards. Nevertheless, BSA can be classified as SCM that contributes to the properties of hardened concrete through hydraulic or pozzolanic activity. SCM are often added to concrete to make concrete mixtures more economical, reduce permeability, increase strength, or influence other concrete properties. Banana skin is high in potassium (K) content followed by manganese (Mn), sodium (Na), calcium (Ca), iron (Fe), bromine (Br), rubidium (Rb), strontium (Sr), zirconium (Zr) and niobium (Nb) (mg/g)21. The ‘K’ content in the BSA was 43.15%, the highest compared to other non-oxides elements. The non-oxide element Na was not detected. However; the chemical composition was found to be lower than that reported by Anwahange et al.26. The ‘K’ and ‘Na’ are crucial for hydration reaction in cement mortar for the enhancement of strength properties. These chemicals react with calcium hydroxide to form potassium hydroxide (KOH) and sodium hydroxide (NaOH), which will accumulate in the aqueous phase of the solution and contribute to a change in the composition of the pore fluid. This leads to a change in the pH of the medium and accelerates the hydration of the cement27.The XRF analysis (wt. %) for BSA, (a) percentages of oxide elements (%) and (b) percentages of non-oxides elements (%), is shown in Table 29. The volume obtained for the BSA and OPC at 20 g of mass were 36.32 ± 0.00 cm3 and 198.43 ± 0.00 cm3 respectively. At the same mass, the BSA possessed a higher bulk density as compared to OPC. However, bulk density can be changed depending on the material’s handling (i.e. loose or compact)33. Time taken for the manometer liquid to drop from the second to the third level of the manometer during the test  were 8.33 ± 0.70 s (BSA) and 15.10 ± 0.26 s (OPC). The value measured during testing is the time required to pass a certain volume of air through a packed bed of solids with given size and porosity. The surface area is directly proportional to 34. The higher the surface area, the shorter it takes for the time taken for air to flow through the compacted BSA and OPC beds.Blaine’s air permeability results for BSA and OPC are shown in Table The percentages of BSA (%), slump (mm) and types of slump are shown in Table 35. Nevertheless, ash is a good water absorbent36. Adding ash will reduce water content in the concrete mix and its workability. Although workability of the concrete was reduced, further test such as compressive strength and SEM were conducted to measure the strength and to observe the microstructure changes upon BSA addition as SCM.Theoretically, the increase of more water content in the concrete mix will not only increase its workability but also may increase the potential of segregation between coarse aggregate particles, bleeding, drying shrinkage and cracking as well as decrease in the concrete strength and durabilityThe percentages of BSA (%), ITZ (µm) and comparison of compressive strength (MPa) of mortar is shown in Table 38. It has a strong influence on the mechanical properties of mortar and concrete39. BSA contains potassium that can enhance the properties of mortars in fresh and hardened conditions such as microstructure. The addition of BSA to mixture enhances the properties of the microstructure as the thickness of ITZ is decreased , 1.32 ± 0.33 µm (1% of BSA) and 1.02 ± 0.15 µm (2% of BSA) Table . Figure The 13 experimental runs pertaining to 2 process and 1 response variables were conducted for the RSM process optimization using CCD Table . The proR2) for quadratic model and model terms for actual factors are 17.16711 (y-intercept), − 2.82036 (A), 0.342091 (B), − 0.068095 (AB), 1.77299 (A2) and 0.007079 (B2). Quadratic model and model terms B, A2, and B2 are significant   represents goodness of fit close to 141.The β coefficient or regression coefficient of determination , which followed the usual strength development trend of concrete. The amount of BSA present in concrete also had an impact on its compressive strength. For a given days of curing, the compressive strength was highest at 1 to 1.5% BSA. Hence, 1.25% BSA has been adopted as optimum.43. As discussed previously, the optimal percentage of BSA is equivalent to 1% and can be observed from the minimal error percentage obtained such as 2.4%, 14.3%, and 18.5% for 7, 14 and 28 days of curing accordingly. Although the error percentage for control mix design is the lowest compared to 1% of BSA , the microstructure was improved with concrete containing BSA. This improvement, reducing porosity and pore connectivity, leads to reduce the permeability and finally improve the durability38.Table 2: 0.955) as well as between curing days (day) and predicted value for compressive strength (MPa) (R2: 0.871). The experimental and predicted values were positively correlated with R2 equals to 0.849 indicating the optimised model is validated.The Pearson proximity matrices for BSA (%), curing days (day), experimental and predicted values for compressive strength (MPa) is shown in Fig. 3) has a higher bulk density than OPC (198.43 ± 0.00 cm3) indicating large surface area for water absorption. The compressive strength of concrete increased with the number of curing days. The ITZ improved with the increased in BSA . Overall, the optimal percentage of BSA was 1.25% at which the with the compressive strength was maximum for all curing days studied. The optimal number of curing days obtained was 28 days. The established model for strength enhancement of concrete using BSA as SCM was statistically significant and showed that it adequately represented the design space. The Pearson’s proximity matrices showed correlation between curing days and compressive strength . The established model can assist in the application of BSA in construction industries.Banana skin is an agriculture waste and BSA possesses pozzolanic property. BSA has high ‘K’ content crucial for hydration in concrete mix. The XRF analysed oxides and non-oxides elements of BSA that contribute to strength enhancement. Slump test showed that the workability of concrete reduced with addition of BSA. At the same mass of 20 g, BSA (36.32 ± 0.00 cmSupplementary Information.


The assessment of already installed anchorages for a possible exceeding of the service load level is a question that is gaining more and more importance, especially in building maintenance. Bonded anchors are of particular interest here, as the detection of a capacity reduction or load exceedance can cause damage to the concrete-bonded mortar behavior. This article investigates the extent to which ultrasonic methods can be used to make a prediction about the condition of anchorages in concrete and about their load history. A promising innovative assessment method has been developed. The challenges in carrying out the experimental investigations are the arrangement of the transducers, the design of the test set-up and the applicability of direct, indirect or semidirect ultrasonic transmission. The experimental investigations carried out on a test concrete mix and a bonded anchor system show that damage to the concrete structure can be detected by means of ultrasound. The results indicate the formation of cracks and therefore a weakening of the response determined by means of direct, indirect and semidirect ultrasonic transmission. However, for application under non-laboratory conditions and on anchors with unknown load history, the calibration with a reference anchor and the identification of the maximum load is required. This enables a referencing of the other loaded anchors to the unloaded conditions and allows an estimation of the load history of individual anchors. Fastening technology has developed into a considerable and demanding technical discipline. The requirements for the various anchoring systems are becoming more complex and more extensive as the fields of application expand. In addition to the common destructive test methods for concrete, non-destructive material testing methods are increasingly requested for the assessment of already installed fastening systems. The non-destructive detection of the condition of the component enables the determination of the externally non-visible defects and damages in the concrete structure. Within this contribution the question is discussed, how ultrasound techniques can be applied for the assessment of already installed and loaded post-installed bonded anchors. The main question is if after an extreme loading situation, or within a quality insurance verification after the maximum designed lifetime the anchor is still in a safe status. By means of ultrasound characterization processes it is suggested to be able to assess the deterioration process and the crack development process of single bonded fasteners respectively. It is the main idea to test these non-destructive measurements in a first step on a bonded system due to the working principle over the whole load transfer zone. In contradiction to post-installed mechanical fasteners, the load transmission and hence the ultrasound transmission area are known for bonded fasteners with a higher certainty. As a result of this, it shall be possible to determine deteriorations or influences caused by the crack formations in the contact zone between bonded anchor and concrete specimen.Research conducted by Hofmann and Laumann  show thaIn a more advanced field of research, Wolf, Pirskawetz and Zang  have shoThe research provided within this contribution presents results of a tested bonded anchor system in a concrete slab under various loading situations (loaded in percentage of the ultimate load/unloaded after loading/ultimate load). Ultrasound measurements on different positions  and evaluation processes have been used and are reported. Hence as a result a method will be suggested, how the deterioration state of an already installed bonded anchor system could be determined. B.-C. and Kim, J.-Y. [The ultrasonic measurement method has traditionally been used for the quality control of materials and is very effective in these fields as shown exemplarily in ,8. The cm, J.-Y. .1 and the path length of the signal to be penetrated through the component. The waveform is displayed directly on the measuring device, enabling a first rough analysis after the measurement.For the experiments presented the ultrasound device Pundit Lab+  is used. The portable device from the Swiss company Proceq is especially suitable for tests on concrete. The ultrasonic pulse velocity is determined via the signal propagation time tThe resolution of the signal is shown in the waveform display on the abscissa as time in μs, whereby it can be set in which time period signals are emitted or how long a signal is emitted by the measuring device. On the ordinate of the waveform display, the amplitude, i.e., the deflection of the measured echo, is indicated. This is unitless but is provided as a percentage value. The entire representation of the waveform corresponds to an A—image since the measurement is made at a point.In order to be able to carry out measurements in the different frequency ranges, different transducers were used as listed in T denominates the transmitter and R denominates the receiver. In terms of physical principle, direct transmission is preferable to the others. In practice, however, the opposite side of a mounting surface  is rarely freely accessible. Therefore, a verification of the direct method with semidirect or indirect sound transmission is appropriate.The tests were carried out using direct, indirect and semidirect sound transmission, see The measuring method used (in particular direct sound transmission) requires a structure that presses the probes with uniform pressure against the underside of the component.Theoretically, the probes can also be pressed against the underside of the component by hand. However, this method ensures inaccurate and not replicable measurements. Therefore, a designed-in-house wooden construction is used on which all probes can be clamped.Wood was deliberately chosen as the material for this fixture in order to keep the disturbing influence on the ultrasound wave as small as possible. With a constant contact pressure of 1 kg, the transducers are equally loaded. In addition to making it easier for the transducers to stand alone and stable in the wooden frame, repeatable and stable measurements can be made, see As a loading device, a hydraulic cylinder with a corresponding hydraulic aggregate was used, see Two rubber soundproofing panels are placed directly on the surface of the concrete slab and a steel plate of similar geometric dimensions is placed on top of them. This ensures full-surface pressure distribution and decupling of the contact area between cylinder and concrete. This is threaded into the threaded rod through a central opening before loading. In addition, the load was measured by means of a load cell positioned centrically in the axis of the threaded rod of the bonded anchor.l = 100 cm, width b = 100 cm and height h = 15 cm and a nominal strength class of C20/25 in accordance with EN 206 [c,cube150,testf = 25.4 N/mm2 measured on cubes. The mixture of the concrete consists of round aggregates with a maximum size of maxd = 16 mm with resistances to fragmentation of a Los Angeles abrasion value of 28 and an impact value of 21. Used cement type was CEM I 32.5 R in accordance with EN 197 [w/z = 0.7. According to the scale separation principle, the maximum grain size that occurs specifies the wavelength of the ultrasonic waves. All tests of this contribution have been performed with concrete slabs of the same size, manufacturing date and mixture. This is due to the decision that in a first step influences from different concrete mixtures shall be excluded to focus on the behavior of the deterioration process of the bonded fastener under load itself. The considered concrete specimens had a size of length h EN 206 . The conh EN 197 , the watl = 50 cm have been used (tensile strength uf = 1200 N/mm2 and yield strength yf = 1080 N/mm2), see efh = 60 mm for all considered tests to avoid steel failure of the threaded rod in testing. The minimum thickness of minh = 10 cm for size M12 was fulfilled with the height of the concrete member of h = 15 cm. In the considered tests, the drill holes were hammer drilled with a four-cutting edge driller of cutd = 14.35 mm. The drilled holes were cleaned by blowing in of compressed, oil-free air (2×), brushing by a cleaning brush (2×) and blowing in of compressed, oil-free air (2×). Tests have been performed after a minimum considered curing time of curet ≥ 24 h. A typical failure mode after conduction of the tests is shown in Post-installed bonded anchors are acting by a load transmission over the whole embedment depth of the anchorage. In the considered test schedule a two-component epoxy resin mortar was used. This mortar consists of epoxy resin as binding material, which hardens through polyaddition, hardening material and fillers. The mortar is intended to be used in non-cracked and cracked concrete for applications with an embedment depth from 60 to 600 mm with various steel elements as exemplarily reinforcement bars or threaded rods of the diameter M8 to M30. This mortar consists of an injection cartridge and an appropriate static mixer and is intended to be used for structural applications in reinforced or nonreinforced concrete of strength classes C20/25 to C50/60 in accordance with EN 206 . In caseThe tests reported here are listed in The load levels were determined in such a way that the ultimate loads of the used fastening system are approximately 70–95 kN. Therefore, the last load level of 60 kN is still sufficiently far away from failure, but already significantly above the service load level.In the zero measurements, only the unloaded threaded rod is sounded. Slight deviations from each other result from the gluing process in the drill hole. Small air inclusions in the adhesive can interfere with the full bonding of the threaded rod and cause a deviation of the transmission signal.The 1 kN measurement is taken after all the test equipment has been set up and serves as a reference value for the other load level measurements and as a reference value for the unloaded state.The other load levels are, as mentioned, 20 kN and 40 kN. At the last load level of 60 kN, acoustic signals indicate that first cracks are appearing within the concrete structure. Visually recognizable, superficial damage could not be detected. After the last measurement, the threaded rod is loaded until failure. The schematic loading procedure is depicted in The presentation of the results is based on the one hand on the signal propagation times and on the other hand on the detected waveforms. For comparison with direct transmission, where a larger number of tests are available, the tests with indirect and semidirect transmission are also shown.The test results of the signal propagation time for the different load steps (loaded and unloaded) for direct transmission are shown in The loaded (red) and unloaded (blue) measured values plotted in The first assumption suggests an increase in the signal propagation time with increasing load level. This is probably due to the fact that the ultrasonic wave can only penetrate the concrete structure more slowly as the pull-out load is approached. Both loaded and unloaded measurements show this phenomenon. The data from the shear wave transducer confirm the trend.In order to not only rely on the signal propagation times, an additional method is attempted for the evaluation. Therefore, the wave shapes are compared with each other. In order to enable a quantitatively comparison of different shapes of the waves, the total area of each wave was calculated. As a result of an unknown root function, the method of numerical integration was used.In The areas of the waveforms seem to become smaller with increasing load level. Due to the damping of the wave and the losses from the sound attenuation, information is lost through the structural component. This means that changes in the concrete structure cause these strong deviations. The values of the surface areas themselves, plotted on the ordinates of the diagrams, are not comparable with each other, as the measurements are carried out with the probes in different signal amplifications, see In a further step, indirect and semidirect transmission was applied. Only selected results are provided here, but the results are similar for all used transducers.The test results of the signal propagation time for the different load steps (loaded and unloaded) are shown in In The aim of this work show how an ultrasonic method can be used to make an evaluation about the condition of already installed bonded anchors in concrete. Different measuring methods are to show to what extent changes in the running time and the wave processes are in direct correlation with the fracture pattern that is forming within the concrete. The goal of successfully applying non-destructive testing by means of ultrasound examinations can be seen as achieved in principle. A damage pattern can definitely be determined, whereas no quantification can be made.It is explicitly stated that a change in the running time is directly correlated to the developing fracture pattern as it is shown in The use of the experimental results shown here in reality is possible in principle. At first glance, the realization appears to be difficult. It could be impressively shown that a statement about the load history of an anchor can be made from the measurements. However, the unloaded condition  is known for the tested specimens.In a real application of this method on installed anchors, where it is to be investigated whether they have already been under a load that has caused damage in the microstructure of the concrete, this zero state is, however, difficult to realize. One possibility would be the targeted application of a reference anchor, which is not loaded and can thus be used as a reference specimen. This reference anchor experiences the same environmental conditions and ageing processes, but always remains unloaded and allows the calibration of the other fasteners to the unloaded state later.The validation of the tests carried out with indirect or semidirect sonication is particularly important. In general, the fastening substrate is not equally accessible from all sides. Therefore, direct sound transmission is usually not suitable.The anchor must be accessible and at least indirect through-sounding to the fixing substrate must be feasible;The measurements should be validated on newly installed systems in the same substrate, in particular to exclude the effects of different aggregates, different cementitious matrix or present reinforcement;An unloaded reference anchor of the same age as the anchors to be tested shall be available;The ultimate load should be determined on a “victim anchor” to be able to evaluate the damage;The assessment can be made on the basis of the signal propagation time and on the basis of the integrated area of the sound wave, which can be seen as assessment criteria.Summarizing as a conclusion, the following conditions must be considered for an assessment of the load history of a bonded anchorThis method presented in this article is certainly not to be regarded as already completely ready for final use. However, the fundamental applicability of this method has been properly proven by the research conducted. Further investigations and considerations on the technical implementation in practice should follow.


The neuropeptide urocortin 3 (UCN3) has a beneficial effect on metabolic disorders, such as obesity, diabetes, and cardiovascular disease. It has been reported that UCN3 regulates insulin secretion and is dysregulated with increasing severity of obesity and diabetes. However, its function in the adipose tissue is unclear. We investigated the overexpression of UCN3 in 3T3-L1 preadipocytes and differentiated adipocytes and its effects on heat shock response, ER stress, inflammatory markers, and glucose uptake in the presence of stress-inducing concentrations of palmitic acid (PA). UCN3 overexpression significantly downregulated heat shock proteins  and ER stress response markers  and attenuated inflammation (TNFα) and apoptosis (CHOP). Moreover, enhanced glucose uptake was observed in both preadipocytes and mature adipocytes, which is associated with upregulated phosphorylation of AKT and ERK but reduced p-JNK. Moderate effects of UCN3 overexpression were also observed in the presence of 400 μM of PA, and macrophage conditioned medium dramatically decreased the UCN3 mRNA levels in differentiated 3T3-L1 cells. In conclusion, the beneficial effects of UCN3 in adipocytes are reflected, at least partially, by the improvement in cellular stress response and glucose uptake and attenuation of inflammation and apoptosis. The CRF family consists of ligands, CRF, CRF-bp, and urocortins (UCN) 1,2,3, with their receptors, CRHR1 and CRHR2. These neuropeptides are expressed in discrete areas of the brain and in the adipose tissue, pancreas, skeletal muscle, and heart4. They may play a significant role in various endogenous pathways, including stress adaptation1. For instance, the administration of UCN1 exerts cardioprotective effects on cardiomyocytes5. The activation of CRFR2 by UCN2 and UCN3 also activates the Ras and Raf-1 kinase pathways that are critical for cardioprotection5. UCNs are affected by environmental challenges, such as oxidative stress7 and alcohol consumption8. Interestingly, the disruption of CRF2 signaling causes distortion of the endoplasmic reticulum (ER) morphology, accompanied by ER stress signaling. Uncontrolled protein ubiquitination affects protein synthesis, secretion, and mistargeting under this condition9.The corticotropin-releasing factor (CRF) family of neuropeptides and receptors is implicated in the suppression of food intake and inflammation as well as in the regulation of energy balance11. UCN3 and CRHR2 are highly expressed in pancreatic β cells, where UCN3 modulates insulin secretion through the regulation of somatostatin secretion4. Moreover, they are co-expressed in the adipose tissue and adipocytes12, suggesting local activity in this tissue. UCN3 is involved in energy homeostasis14, and mice with global UCN3 overexpression exhibit resistance to metabolic impairment that is related to a high-fat diet2. The local overexpression of UCN3 in the muscle improves local glucose homeostasis2. Furthermore, impaired UCN3 expression is reported in various metabolic syndromes, such as type 2 diabetes (T2D), obesity, polycystic ovary syndrome (PCOS), and sleep apnea15. Interestingly, UCN3 and CRHR2 are proposed as potential anti-obesity targets owing to their co-location with quantitative trait loci for obesity on chromosome 10p15.116. Recently, we reported that the plasma UCN3 levels decrease with increased body weight in human subjects without T2D; however, such levels increase with concomitant T2D12. Conversely, the UCN3 levels in the same subjects in the subcutaneous adipose tissue (SAT) increase with body weight and decrease with T2D12. These findings indicate the complex and crucial roles of UCN3 as a marker/target for controlling metabolic disease progression.UCN3 is a recently characterized CRF family member and binds specifically to receptor CRHR217. In addition, impaired defense mechanism and ER stress response are observed in obesity and diabetes18. This impairment includes also heat shock response (HSR) considered as a second cellular defense response—critical for protein refolding, repair, and cellular homeostasis—in individuals with obesity19 and diabetes20. Some HSR proteins (HSPs) are ubiquitously expressed, whereas others are expressed under stress conditions. HSP27 inhibits IKKβ and regulates TNFα-induced NF-κB activation21. HSP72 activation is protective through the binding and repression of JNK phosphorylation23. In our previous work, we investigated the impairment of HSPs regulation in obesity and T2D25. HSR may play a significant role in endoplasmic reticulum quality control and could relieve ER stress through various pathways26. GRP78 protein, a HSP and master regulator for ER stress canonical markers, is key to crosstalk between HSR and ER stress responses27. Three ER resident proteins, protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor-6 (ATF6), and inositol-requiring enzyme-1α (IRE1α) are primarily responsible for sensing ER perturbations caused by physiological changes in extracellular conditions29.In obese individuals, the adipose tissue is infiltrated with macrophages and is characterized by chronic metabolic stress and low-grade inflammation, which are reflected in disrupted cellular host defense mechanismsWe hypothesized that the potential protective role of UCN3 in obesity and diabetes is exercised at least partially by facilitating cellular stress response in the adipose tissue. We overexpressed UCN3 in 3T3-L1 adipocytes and treated them with palmitic acid (PA) to induce stress. Then, we investigated the status of HSR and ER stress response, glucose uptake, and inflammation.p < 0.05)  the mRNA expression levels of these proteins. However, the increase of HPS60 and HSP72  was attenuated by UCN3 overexpression. We then evaluated the effects of UCN3 overexpression at the protein levels via western blotting. Despite the more pronounced downregulation of HSP90, GRP78 and HSP72 levels due to this overexpression observed in the presence of PA, HSP60 protein levels were not decreased as shown above at mRNA levels  as shown in Figure p < 0.05), although this attenuation was observed only in the absence of PA for other ER stress regulators, ATF6 and IRE1α . This increase in glucose uptake was further enhanced (p < 0.01) after continuing 3T3-L1 cell differentiation up to 4 days with ectopic UCN3 overexpression. This enhancement was observed despite a part of the increase being caused by differentiation  using an indirect co-culture method to mimic inflammatory conditions. We evaluated the expression levels of UCN3 and HSPs. Under normal conditions, UCN3 mRNA expression was increased with differentiation, as we previously reported12. However, a clear downregulation of the UCN3 mRNA levels (p < 0.05) was observed in the presence of MaCM .Low-grade chronic inflammation is a characteristic of adipose tissue in patients with obesity and insulin resistance-resistant patientsThese observations prompted us to investigate the effect of UCN3 overexpression on representative inflammatory markers. Overexpression significantly decreased the expression levels of TNFα mRNA in the absence and presence of PA Figure . Moreove12. Based on the effects of UCN3 overexpression on HSPs and ER markers in adipocytes, and the opposite trends in the UCN3 and HSPs levels in MaCM, we revisited our previous data and conducted correlation analyses to evaluate the circulating levels of UCN3, HSP60, and GRP78. A description of the population study, the methods used, and the results are detailed in previous reports31. Interestingly, significant negative correlations between the UCN3 levels and the HSP60 and GRP78 levels were observed  using data from 144 non-diabetic humans with various body mass indices  in subjects with diabetes, which was not present in the non-diabetic patients. Consistent with these findings, we report that indirect co-culture with MaCM induces a decrease in UCN3 expression and an increase in ER stress, HSPs, and CHOP levels. Conditioned medium represents low-grade inflammation in the adipose tissue, as observed in obesity. Hence, the local microenvironment may affect the expression levels of UCN3, and UCN3 may participate in adaptive responses to maintain homeostasis. However, with chronic stress, this effect is attenuated.We also observed a significant negative correlation between the UCN3 level and HSP60 and GRP78 levels in human plasma. However, this association is not observed in subjects with diabetes. Moreover, in a previous reportOur findings are in agreement with those of previous studies, which demonstrate that increased UCN3 levels may exert protective effects against moderate or transient metabolic insults. Still, the physiological role and mechanism of action of UCN3 are complex, and its beneficial effects require signal integration among cell types and crosstalk among numerous adaptive mechanisms, both centrally and peripherally. Hence, a better understanding of the molecular mechanisms that regulate UCN3 expression and action is expected to provide critical insight into its role in the pathophysiology of various metabolic disorders.To the best of our knowledge, the present study is the first to report enhanced heat shock and ER stress responses and glucose uptake with UCN3 overexpression in adipocytes under metabolic stress. In vitro work is supported with data from adult human plasma. However, despite our novel findings, this study has some limitations, such as lack of knockdown experiments for the validation and assessment of the status of the CRHR2 receptor in used cells. A detailed characterization of insulin signaling pathways under metabolic stress following UCN3 overexpression is planned, and the results are expected to further elucidate the action of UCN3 on adipocytes in the context of obesity and diabetes.44. Briefly, 3T3-L1 cells were normally differentiated into mature adipocytes until day 8, and in indirect co-culture, 3T3-L1 cell differentiation was induced in conditioned media from RAW264.7 cells. Oil red O staining of lipid droplets was used to monitor cell differentiation as previously described12.Mouse preadipocyte (3T3-L1) and macrophage (RAW264.7) cells were purchased from the American Type Culture Collection  and then cultured as previously described6 cells were electroporated with 2 μg of plasmid using Nucleofector program, A-033. About 24 h after transfection, the cells were treated with 400 µm of PA  in low glucose (1 g/l) and 1% (w/v) serum medium for 24 h with and without insulin (20 nM for 10 min). PA was prepared in 10% (w/v) fatty acid free bovine serum albumin (BSA). The cells were then processed for RNA and protein analyses or plated for glucose uptake assay.Human UCN3 gene cloned in pCMV6 vector tagged with Myc-DDK  was used for transfection. Myc-DDK tagged pCMV6 vector without UCN3  was used as a control. 3T3-L1 adipocytes were transfected via electroporation using the Cell Line Nucleofector Kit  according to the manufacturer’s instructions. Briefly, 2 × 10Adipocytes were placed into a 96-well plate for glucose uptake assay using a cell-based assay kit  according to the manufacturer’s instructions. The culture medium was replaced with 100 µL of glucose-free medium 24 h after plating to starve cells. After 1 h of starvation, the medium was replaced with fluorescently labeled deoxy-d-glucose analog (2-NBDG) (150 µg/mL) and with and without insulin (0.1 µm) at 37 °C for 1 h. The cells were then washed with the buffer provided, and fluorescence was measured using a Synergy H4 plate reader  at 485/535 nm. Glucose uptake experiments were conducted on both preadipocytes and differentiated adipocytes transfected with UCN3 and pCMV6 plasmid. Experiments and measurements were also conducted on transfected preadipocytes differentiated until day 3.TRIzol reagent was used for total RNA extraction according to the manufacturer’s instructions. Isolated RNA was quantified using an Epoch spectrophotometer  prior to reverse transcription using a High-Capacity cDNA Reverse Transcription Kit . cDNA was then amplified with TaqMan gene expression assay or custom primers Table  and normWhole protein extracts from cells were prepared in RIPA buffer  Triton X-100, 1 mM EDTA, 0.5% sodium deoxycholate, and 0.1% (w/v) SDS). Protein concentrations were determined using the Bradford method and normalized to β-globulin. Samples (20 µg) were prepared in loading buffer containing β-mercaptoethanol and resolved on 10% (w/v) SDS-PAGE gels. Then, proteins were transferred onto PVDF membranes (100 V for 75 min) and blocked for 2 h at room temperature (RT) using 5% (w/v) nonfat dried milk in Tris-buffered saline containing 0.05% Tween-20. Membranes were probed with the primary antibodies at 4 °C overnight . Membranes were then washed and incubated with rabbit horseradish peroxidase (HRP)-conjugated secondary antibody  for 2 h at RT. Detection was performed using super sensitivity West Femto ECL reagent , protein bands were visualized by chemiluminescence, gel images were captured using the VersaDoc 5000 system , and band intensities were measured using the Quantity One software . GAPDH was used as an internal control for protein loading.12. Participants with prior history of major illness, use of supplements or medications affecting body composition or bone mass and those who performed any physical exercise within last 6 months before enrollment to study were excluded. The physical, clinical and biochemical characteristics have been previously reported. All subjects had provided informed written consent approved by ethics committee of Dasman Diabetes institute, in line with the principles of the Declaration of Helsinki.A total of 144 non-diabetic normal weight and overweight subjects (n = 37 and 107 resp.) and 98 T2D subjects were recruited for our study as previously reportedPhysical-clinical measurements including body weight, systolic and diastolic blood pressure were made. Peripheral blood samples were obtained, and plasma was collected to analyze various inflammatory and metabolic markers. Glucose and lipid levels were measured using Siemens Dimension Chemistry Analyzer . Haemoglobin A1c (HbA1c) levels were determined with the Variant™ device . Levels of plasma UCN3 , HSP60 , and GRP78  were assayed using ELISA kits, according to manufacturer's instructions. Absorbances were measured on H4 Synergy plate reader .p-values < 0.05 were considered statistically significant.Statistical analyses were conducted using the SPSS software v25.0 . All descriptive statistics in the study are expressed as mean ± standard error and at least 3 repeats were performed for each experimental condition. Nonparametric Mann–Whitney U tests were employed to evaluate the differences in the means between pairs of cell conditions. Correlations among variables were estimated using Spearman’s correlation coefficient. All Supplementary Information.


Publication numbers reporting that ultrasound can stimulate immune reactions in tumors steadily increase. However, the presented data are partially conflicting, and mechanisms are difficult to identify from single publications. These shortcomings can be addressed by a systematic review and meta-analysis of current literature. As a first step, we here present the methodology and protocol for a systematic review to answer the following research question: Does ultrasound alter the immune reaction of peripheral solid tumors in humans and animals compared to control conditions without ultrasound?We designed a protocol to perform a systematic review and meta-analysis. The suitability of the protocol to detect and sort relevant literature was tested using a subset of publications. We extracted study characteristics, ultrasound parameters, and study outcomes to pre-evaluate the differences between publications and present the data as a scoping review.2). Finally, study outcomes included reports on cells of the innate  and adaptive immune system .From 6532 publications detected by our preliminary literature search, 320 were selected for testing our systematic review protocol. Of the latter, 15 publications were eligible for data extraction. There, we found large differences between study characteristics  and ultrasound settings (e.g., wavelength 0.5–9.5 MHz, acoustic pressure 0.0001–15,000 W/cmWe designed a protocol to identify relevant literature and perform a systematic review and meta-analysis. The differences between extracted features between publications show the necessity for a comprehensive search and selection strategy in the systematic review to get a complete overview of the literature. Meta-analyses of the extracted outcomes can then enable evidence-based conclusions.The online version contains supplementary material available at 10.1007/s11307-021-01686-x. The commonly known narrative reviews provide a descriptive overview of the current literature, such as published by Wu et al. , van denOne field of cancer research presenting a large difference between experiments is diagnostic and therapeutic ultrasound. Although ultrasound-induced effects on tumors are still a subject of ongoing research, especially the suitability of focused ultrasound as a tool to induce anti-tumorigenic immune responses and to enhance the efficacy of immunotherapy is currently under intensive investigation –3. DepenMechanical stress can also occur in diagnostic ultrasound settings, where the stable oscillation of microbubbles is detected in contrast-specific imaging. Furthermore, destruction-replenishment methods, including violent microbubble destruction, are often used to assess tissue perfusion  or detecIndependent of the underlying mechanism, the ultrasound-induced immune response in tumors originates from cell stress or cell death. In detail, cells of the innate immune system  are activated via danger-associated molecular patterns (DAMPs) released from damaged or dying cells . After eAlthough the prediction of the immune response is difficult because of the multitude of parameters that can have an influence, this information would be important to refine specific ultrasound settings to trigger the best anti-tumor immune response but also to avoid immune effects in diagnostic applications. In addition, slight immune reactions possibly influence study outcomes when using ultrasound for therapy monitoring in preclinical drug discovery. This can lead to a false interpretation of data which can be a source for low reproducibility between experiments and finally impair clinical translation.As a first step in creating a meaningful summary of the relevant literature, we present a new protocol for a systematic review to answer the following research question: Does ultrasound alter the immune reaction of peripheral solid tumors in humans and animals compared to control conditions without ultrasound? We registered the protocol in the international prospective register of systematic reviews PROSPERO (registration number CRD42021248743). Next, we tested the suitability of the protocol to detect and sort relevant literature on a defined subset of publications from a preliminary literature search. Finally, a scoping review of the selected literature on study characteristics, ultrasound parameters, and study outcomes was performed to assess the differences in study parameters between publications.We registered the systematic review protocol in PROSPERO (registration number CRD42021248743). The items covered by the protocol are described in more detail in the following paragraphs.We formulated the research question following the PICO  format : does ulPublications need to fulfill the following requirements for inclusion: Studies of all animals and humans, of both sexes, and any age with a solid peripheral tumor that can be visualized with ultrasound. Tumors of the central nervous system will be excluded, as the immune system in the brain is different from the rest of the body . FurtherThe search strategy will be designed according to M. Leenaars et al.  and perfWe will use the SYRCLE filter  to identAfter merging all search results and removing duplicate publications, the study selection will be conducted in two phases. First, two out of three reviewers will perform a title/abstract screening using the systematic review software Rayyan . PublicaIn the second selection phase, two out of three reviewers will perform a comprehensive full-text screening of the remaining publications for eligibility for data extraction. In this phase, the exclusion criteria are prioritized as described in Table After completing both screening phases, all remaining publications will be divided between two reviewers for data extraction. A third reviewer will verify the accuracy of extracted data for a subset of included publications. The extraction contains relevant information to identify each study , study design characteristics , ultrasound settings  and all immune-related outcomes  in tumor or blood. Data presented in the text or tables will be preferentially recorded for quantitative data. If this is not possible, a digital ruler  will beThe risk of bias (RoB) of all included publications will be evaluated using SYRCLE's RoB tool  for asseThe number of identified publications in each screening phase and reasons for exclusion will be summarized in a flowchart according to the PRISMA statement . ExtractI2 statistic, ranging from 0 to 100 %, with values below 25 % considered low heterogeneity, indicating that effect sizes are very similar and not influenced by other study characteristics. Values above 75 % indicate high heterogeneity between effect sizes, which means that the effect is influenced by an unknown factor that warrants further investigation  [For the meta-analysis, a random-effects model is used to compare effect sizes even with substantial differences in species, tumor models, and ultrasound settings among the included publications. The effect sizes of all included publications are considered as a random distribution of all possible effects. The random-effects model is used to calculate the mean effect size of all included publications. In addition, the heterogeneity of effect sizes between included publications is assessed using the ression) . The folFurther necessary corrections for meta-analyses  will be performed according to Borenstein et al. .X-axis and the standard error on the Y-axis. Without small-study effects, the effect sizes of all publications will spread symmetrically around the mean. Quantitatively, this will be assessed with a trim-and-fill analysis. An asymmetry in the funnel plot will be compensated by removing (trimming) asymmetric values and re-adding them with a mirror image (filling). Comparing the trim-and-fill analysis’ result with the original mean effect size will show the potential impact of a small-study effect.In a last step, we will assess small-study effects, which describe the problem that small studies with larger effects seem more likely to be published than those with smaller or zero effects  . Potentik = 300) were selected, and the suitability of the systematic review protocol was tested on this subset. The title/abstract screening was performed by three reviewers using Rayyan [We conducted a scoping review for a defined subset of publications to provide an overview of the available literature regarding the potential number of publications and the expected differences in study characteristics between publications. The 100 newest and 100 oldest publications and 100 publications from the middle of the list , using a wrong population (k = 4), wrong intervention (k = 3), wrong comparator (k = 2), not including a proper study design (k = 2), or the full texts were not available (k = 2). An overview of the included publications in the different screening phases can be seen in Fig. Full-text screening of 38 publications (18 from title/abstract screening and 20 from recent review articles) resulted in 15 publications for data extraction. Seven of these 15 publications originate from the subset included in the title/abstract screening  and 8 from recent review articles. Twenty-three publications were excluded in the full-screening phase because of the following reasons: a lack in reporting an immune-related outcome  of included publications used mice, whereas two showed results of female human breast cancer patients aged 46–47 years , 37. Reg2, duty cycle 0.5–75 %), although all included publications used therapeutic ultrasound applications.Furthermore, the ultrasound settings differed substantially between publications , followed by CD4-positive T cells (10/15). Information on tumor macrophages could be found in six and information on dendritic cells in five publications. Furthermore, tumor-associated natural killer cells, B cells, and CD4 Foxp3-positive T cells were reported by two and myeloid-derived suppressor cells, granulocytes, mast cells, and leukocytes by one publication. Finally, two publications report various inflammatory cytokines, chemokines, and DAMPs in tumors and blood. An overview of all outcomes can be found in supplementary Table The number of publications on new cancer treatments is steadily increasing; however, their success rates in clinical applications are still considerably low. According to Begley and Ellis, the poor reproducibility and robustness of published preclinical data contribute to this limitation . This siUsing a published and pre-registered protocol to perform the systematic review and meta-analyses minimizes the risk of introducing a methodological bias, as the protocol cannot easily be adapted retrospectively to suit the authors’ preference . Therefok = 6532), we will have around 130 publications eligible for data extraction in the systematic review, which will be enough to perform subgroup analyses and identify potential publication bias in most cases.The criteria defined in the protocol enable us to identify and sort relevant publications for data extraction. We analyzed a subset of publications as a scoping review, which is defined as a preliminary exploratory assessment of the available literature on a given topic  to estimThe extraction of study characteristics showed large differences in mouse strains, sex, tumor types, and age. Age is a crucial aspect in investigating immune-related parameters, as the immune system naturally changes over time. Therefore, interventions performed in juvenile animals can lead to different results than those performed in elderly animals. In this small subset, the mouse age varied between 4 and 10 weeks, which can be critical, as, e.g., the number of white blood cells in mice increases between 2 and 9 weeks of age . In contThe extraction of study outcomes showed that various cells contributing to the innate and the adaptive immune reaction were investigated after ultrasound treatment. Therefore, changes in both stages of the immune reaction can be assessed by the systematic review.In conclusion, we present a protocol to find and sort relevant literature and perform a systematic review to answer our research question. A scoping review on a subset of the literature revealed a large difference between study characteristics, ultrasound settings, and reported outcomes, demonstrating the need to include a comprehensive sample of publications to generate a clear conclusion from the data. In the future, the results from the complete systematic review and meta-analysis may then assist in unraveling the influence of ultrasound on the tumors’ immune response. In addition, these data can help to refine therapeutic ultrasound applications or highlight the risk for inducing an experimental bias in diagnostic ultrasound settings.Supplementary file1 (DOCX 24 kb)Below is the link to the electronic supplementary material.


Heroin use disorder is a chronic and relapsing disease that induces persistent changes in the brain. The diagnoses of heroin use disorders are mainly based on subjective reports and no valid biomarkers available. Recent researches have revealed that circulating miRNAs are useful non-invasive biomarkers for diagnosing brain diseases such as Alzheimer's disease, multiple sclerosis, schizophrenia, and bipolar disorder. However, studies on circulating miRNAs for the diagnosis of heroin use disorders are rarely reported. In this study, we investigated the differential expression of plasma miRNAs in 57 heroin-dependent patients. Based on literature research and microarray analysis, two candidate miRNAs, miR-320a and let-7b-5p, were selected and analyzed by quantitative real-time RT-PCR. The results showed miR-320a and let-7b were significantly upregulated in plasma of the heroin-dependent patients compared to that in healthy controls. The area under curves (AUCs) of receiver operating characteristic (ROC) curves of miR-320a and let-7b-5p were 0.748 and 0.758, respectively. The sensitivities of miR-320a and let-7b-5p were 71.9 and 70.2%, while the specificities of miR-320a and let-7b-5p were 76.1 and 78.3%, respectively. The combination of these two miRNAs predicted heron dependence with an AUC of 0.782 (95% CI 0.687–0.876), with 73.7% sensitivity and 82.6% specificity. Our findings suggest a potential use for circulating miRNAs as biomarkers for the diagnosis of heroin abuse. Heroin, a morphine derivative, is one of the most powerfully addictive drugs abused in the world. People who abused heroin are among the most marginalized and disadvantaged drug users. They experience poor health outcomes with a greater chance of premature death, increased risk of both non-fatal and fatal drug overdoses, and high rates of potentially life-threatening infectious diseases, such as tuberculosis, hepatitis, and HIV. Heroin addiction is a relapsing and chronic brain disorder that causes persistent alterations in synaptic plasticity –3. HowevMicroRNAs (miRNAs) are a class of stable non-coding small RNA molecules able to regulate gene expression by binding to 3'-UTR regions of their mRNAs targets . MiRNAs Identification of addiction-related human-specific circulating miRNAs will be of important clinical significance. Recent studies have shown that heroin exposure upregulated a panel of plasma miRNAs related to immunity and virus infection , 15. PreOur work is to study the plasma expression pattern of specific miRNAs highly related to drug addiction, which is quite important for the diagnosis and treatment of this disorder. To clarify certain microRNA expression levels in humans and their relationships with heroin use, plasma samples from 4 heroin-dependent patients and 5 normal healthy controls were collected and screened by genome-wide profiling microarray analysis, two distinctly expressed plasma miRNA let-7b-5p and miR-320a were identified. Then, we measured them in the 57 cases of heroin-dependent patients.A total of 61 male heroin-dependent patients (case group) were recruited from Ningbo city through the Ningbo Addiction Research and Treatment Center of Zhejiang Province, the People's Republic of China from June 2014 to June 2018, and performed through the Structured Clinical Interview of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The general demographic data of these subjects were collected. We also collected detailed information about drug use history  as well as the smoking habit by interview. We did not include female subjects in this study because there were fewer female heroin-dependent patients. All subjects were males of Han-Chinese origin and had no other psychiatric disorders such as schizophrenia, bipolar disorder, major depressive disorder, anxiety disorder, or a history of chronic systemic illness such as cardiac, renal, pulmonary, hepatic, endocrine, metabolic, and autoimmune disorders, or a history of chronic pain problem treated with opioid analgesics. The heroin-dependent patients then had urine tests screened for heroin metabolites, methamphetamine, cocaine, or ketamine. All subjects were morphine positive and had no other illicit drug positive except heroin or tobacco. A total of 51 healthy individuals (control group) were recruited from among voluntary blood donors in the Ningbo Blood Center of Zhejiang Province. The general demographic data and smoking habits were also recorded and documented. A multi-stage, case-control study was designed to identify a panel of plasma miRNAs related to heroin dependence. The characteristics of the study subjects were summarized in 8 copies/μl working solution). RNA is eluted in 13 μl of nuclease-free water. The concentration of RNA was measured using a NanoDrop ND-2000 (Thermo Scientific).The venous blood samples of all subjects were obtained after overnight fasting (for at least 8 h) in the morning that were demonstrated to not significantly affect the overall circulating miRNA profiles . For her*60K) V19.0 array] which containing probes for 2006 human microRNAs. The NanoDrop ND-2000 (Thermo Scientific) was used to quantify total RNA. And Agilent Bioanalyzer 2100 (Agilent) was used to assess RNA integrity. Sample labeling, microarray hybridization, and washing were implemented according to the manufacturer's instructions. Briefly, total miRNA was dephosphorylated and denatured, followed by pCp-Cy3 labeling. After purification, labeled RNAs were hybridized onto the microarray according to the array manual. After washing, the slides were scanned into image files with the Agilent Scanner G2505C (Agilent Technologies) and the scanned images were analyzed using Agilent Feature Extraction Software (Agilent Technologies). The resulting data have been deposited in NCBI's Gene Expression Omnibus (GSE160275).Plasma miRNAs profilings of 5 healthy controls and 4 heroin-dependent patients were determined using microRNA microarray  is used. Values range between 0.5 and 0.8 were regarded as an intermediate effect, while ≥0.8 was regarded as a large effect  except Chi-square test, Fisher's exact test, and Spearman's rank correlation analysis, which were analyzed by the SPSS 19.0 . Enumeration data were indicated as percentages, whereas measurement data were expressed as mean ± standard error of the mean (SEM). Normality analysis of each group was performed by using the D'Agostino & Pearson test. For comparing two independent groups, the statistical significance was evaluated by unpaired e effect . ROC curp > 0.05). The average age of the heroin group was 35.7 years and had used heroin for an average of 9.7 years. Forty-one participants of the heroin group use heroin by smoking while 20 by injection. The heroin group had a significantly lower level of education than the healthy control group (p < 0.001).The clinical characteristics of the participants are shown in *60 K) V19.0 Array. The microarray screen result showed that plasma miRNAs differed significantly among the two groups. MiRNAs showing a mean fold-change >2.0 and p-value < 0.05 were illustrated in the heat map generated from the unsupervised clustering analysis  of these distinct miRNAs were shown in To determine the differential miRNA levels in heroin-dependent patients, we comparatively profiled plasma miRNA expression of four heroin-dependent patients and five healthy controls using Agilent Human miRNA . Calculating the effect sizes showed heroin dependence had a large effect on plasma miR-320a (r = 0.94) and let-7b-5p (Hedges' g = 1.072) level.qRT-PCR assay was used to confirm the expression level of the selected miRNAs. The study participants were divided randomly into two sets: a training set and a validation set. The demographic information for the two sets was showed in p < 0.0001) with a cut-off value of −1.829. A sensitivity of 71.9% and a specificity of 76.1% were achieved for identifying heroin addiction (p < 0.0001) with a cut-off value of −1.573. A sensitivity of 70.2% and a specificity of 78.3% were achieved for identifying heroin addiction (P < 0.0001) with 73.7% sensitivity and 82.6% specificity in distinguishing heroin-dependent patients from healthy subjects. We can see that the discrimination power of the two miRNAs combined was superior to that of a single miRNA signature.ROC curve analyses were employed to evaluate the predictive power of circulating miRNAs for differentiating between the patients and healthy controls. The combined cohort of the training and validation sets that consisted of 57 heroin-dependent patients and 46 matched controls were used for analysis. Both miR-320a and let-7b-5p turned out to be significant predictors. The AUC of miR-320a was 0.748  and the heroin smoking group (n = 18). As shown in p-value > 0.05)  .n = 15) and smoker group (n = 31). All heroin-dependent patients enrolled in our study are also tobacco users and were classified into one group (n = 57). As shown in p-value > 0.05), while the levels of both miRNAs were found to be significantly increased in the heroin-dependent patient group.To further investigated whether tobacco using has any impact on the plasma miRNA expression pattern, our total healthy participants enrolled in the training and validation sets were classified into the non-smoker group  can be found in the article/The studies involving human participants were reviewed and approved by Ethics Committee of Ningbo Addiction Research and Treatment Center. The patients/participants provided their written informed consent to participate in this study.HaL and WX designed and performed research, analyzing the data, and writing the paper. WX, JF, HM, JZ, XX, DZ, and WS performed research and data collection. WZ and HuL were responsible for the study concept and design, revising the paper, and organizing the discussion. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.