Construction and demolition (C&D) waste was considered as zero value materials and, as a result, most C&D waste materials ended up in landfills, eventually constituting environmental issues. Therefore, it is important to explore the potential of such C&D waste materials for concrete production. Thus, this research effort aims to find a new method to improve sustainable concrete properties exposed to elevated temperatures at the lowest costs and identify the relationship between temperature change and a change in colour in a heat-exposed concrete structure. Therefore, this study explored the behaviour of three types of concrete: plain concrete (PC), waste ceramic optimal concrete (WOC), and waste ceramic optimal concrete reinforced by hybrid fibre (WOC-Hybrid) in ambient and elevated temperature. The study shows that colour change in a concrete structure exposed to high temperature has a consistent relationship, where it has been found that the colour alteration is of great interest because its appearance usually coincides with the onset of a significant loss of concrete strength as a result of elected temperatures. Overall, it can be considered that waste ceramic materials reinforced by hybrid fibres can be used for concrete production, and by doing so, the negative impact of these wastes on the environment can be controlled as well as fortify the mechanical properties. Resistance of concrete structures to rising temperatures is a primary global concern today. Therefore, it requires a solid and unified effort to arrive at a heat-resistant economic concrete using readily available resources. A promising source of low-cost materials is the growing waste generated due to excessive urbanisation, global economic development, and rapid industrialisation. Casares, M. L. et al.  noted th3) degrades to form quicklime (CaO), which is extremely hydrophilic. When lime comes into contact with water or humidity in the air, it will absorb a large amount of water. Subsequently, its volume will increase. This volume increase results in cracks in the rocks, thus causing the concrete to collapse [When conventional concrete is exposed to heat, exceptionally elevated temperatures lose significant strength or even wholly degrade. The presence of limestone rocks and ordinary cement in concrete is the main reason for the cracks and the release of certain gases when exposed to elevated temperatures. However, limestone is the primary reason that contributes to this process. Ordinary rocks are primarily composed of lime. Regular lime CaCO degradescollapse ,5,6.Najm and Ahmad  proved tRecent research has focused on using ceramic wastes of various sizes as a whole or partial replacement of concrete aggregates to reduce the adverse environmental effects of waste ceramics . In addiMany concrete structures are prone to crack and collapse after exposure to high temperatures, especially over time because of limestone in the mix. Therefore, such damage to concrete may be avoided if ceramic waste materials replace natural materials. In addition, hybrid fibres can significantly reduce the risk of concrete cracking, increase thermal conductivity, and reduce the concrete’s internal stresses. Najm and Ahmad  used cerThus, among the previous studies, aside from the combination of hybrid fibre and ceramic wastes for making WOC and WOC-Hybrid concrete, there are no available data on the effect of thermal conditions on the physical and mechanical properties of these novel mixtures which can be considered an indication of the novelty of the present study. Therefore, the current work presents a preliminary study on the effect of ceramic material  and hybrid fibre (PVA and CR) on the colour change of WOC and Hybrid-WOC subjected to elevated temperatures. Several laboratory experiments have been conducted to investigate WOC and Hybrid-WOC’s behaviour. In turn, it provides a basis for further research on WOC and Hybrid-WOC concrete and its potential applications.In the present study, the authors propose a new concrete mix named WOC and Hybrid-WOC. Many considerations have been coined based on the new generation of concrete: (1) PVA + crimped steel fibre is used to improve the performances of concrete both before and after exposure to elevated temperatures. (2) The inclusion of ceramic waste material is mainly for environmental protection and ceramic tiles have lower thermal conductivity and better fire resistance after exposure to high temperatures. (3) There is beneficial interaction between hybrid fibre (PVA+CR) and ceramic, so resistance to elevated temperatures makes Hybrid-WOC concrete useful in fire-resistant structures.Finally, the study’s main contribution is to evaluate the potential use of colour change analysis in fire-damaged concrete investigations. Concrete samples were subjected to steady-state heating to be analysed for colour changes. This colour alteration is of great interest because its appearance usually coincides with the onset of a significant loss of concrete strength as a result of heating. Furthermore, we investigate the fundamental properties of a new generation of concrete (WOC and WOC-Hybrid) as sustainable concrete in ambient and under elevated temperatures.This article is divided into the following sections: application of a colour change in fire sciences is briefly introduced in Section 5Colourimetry and the physical phenomenon of solid colour change due to heating are used in many fields of fire science research. It has been documented in the wildfire literature that physical and chemical changes at the soil surface during a fire can result in colour shifts . Colour Temperature and the type of aggregate used in the mix can influence how much colour changes in heated concrete. Concrete containing silicate aggregates  will turn red when heated to a temperature between 300 °C and 600 °C, according to the research of Short, N. R. et al. . The cemAn inherently subjective visual colour analysis is currently used to make a rough estimate of the temperature exposure of the concrete. Felicetti R.  has usedTo be sure, colour shifts are unrelated to concrete’s mechanical and physical properties, but they do serve as a gauge for the fire’s temperature rise. Several techniques should be used together in practice for a complete and accurate picture of the damage done to the concrete member . A thoroFor assessing concrete damage caused by fire, the above-mentioned all agree that the material colour change is a valuable indicator of maximum temperature exposure.WC), 4.75 mm (waste ceramic sand-SWC), and 75 μm (waste ceramic cement-CWC) as shown in WC, SWC, and CWC), OPC, river sand, and natural aggregate are shown in Locally available materials such as ordinary Portland cement (OPC) 43-grade, natural coarse aggregate (NCA)/stones, coarse river sand, and ceramic floor tiles were used in the experimental work , as shown in Overall, 21 cylindrical specimens with a diameter of 150 mm and a height of 300 mm were cast in three groups for testing under high temperatures, as shown in For the present study’s purposes, 21 cylindrical specimens, each 150 mm in diameter and 300 mm in height, were cast in three groups.First group: based on test methods per IS 10262 guidelines , plain cSecond group: based on experimental results of Najm and Ahmad , waste cThird group: based on experimental results of Najm and Ahmad , Hybrid-3) and W/C ratio (0.5) were the same for all the concrete mixes. However, they differed in their cement and aggregate (coarse and fine) content because either waste ceramic was used as partial replacements for cement, coarse, and fine aggregates. The quantities of ceramics material, natural material, and fibres used are reported in WC, SWC, and CWC represent coarse ceramic aggregate, fine ceramic aggregate, and ceramic powder weight per cubic meter of concrete. The mix proportions for different types of concrete are shown in The concrete mixture for reference specimens containing natural aggregate and ordinary Portland cement (43 grade) was designed for compressive strength of 25 MPa. The water quantity   was fabrThe colour changes of PC, WOC, and Hybrid-WOC concrete were determined using the 21 cylinders measuring 150 mm × 300 mm (diameter x height). After 24 h, these specimens were demoulded and cured for 28 days. The specimens were prepared for testing at a single heating-cooling cycle from ambient temperature and exposed to an elevated temperature ranging from 100 °C to 300 °C at an interval of 100 °C after the submerged curing process. The researcher used 300 °C as a maximum range of elevated temperature due to the high water–cement ratio used in the mixed design (0.5 W/C), and this would cause damage to the furnace if we increased the temperature by more than 300 °C.To avoid the cracking of concrete due to high temperature gradient between the core and outside surface, the concrete specimens were heated at a slow rate according to IS:1642 . OtherwiConcrete samples can now be photographed under the same lighting conditions using flatbed scanners , as proposed by previous studies . These oIn addition, Hager, I.  proposesAs an alternative, the visible aggregate on the concrete surface could be observed. The cut surface that results from coring or sawing was examined. Colours can change dramatically due to these processes, revealing aggregate grains that often turn red or pink . This meUnderstanding colour and its measurement can be considered complex (see reference ) and a lThe mentioned primary colours space is commonly used in monitors and cameras since it is good and simple for displaying and generating images. Separate signals for these colours are gained and then the resultant colour is defined by the percentage of blue, green, and red that are present as its elements. People do not think of the colours red, blue, and green, which is considered a disadvantage of this system.In the hue, saturation, and intensity colour space: hue refers to the kind of colour whether it looks green, yellow, red, etc.; it is the characteristic by which the eye discriminates several parts of the spectrum. It is measured in terms of wavelength. It can be represented by its position on a horizontal circle see . The zerIn the hue, saturation, and intensity colour space, the circle circumference denotes 100% saturation with 0% at the centre . IntensiMoreover, in the hue, saturation, and intensity colour space, it is denoted by an axis vertical to the circle plane . The fulThe damage assessment of concrete after heating usually begins with observing the mechanical properties of the concrete, colour changes, and concrete surface cracking. At 100° C temperatures, the specimens’ appearance and colour were the same as those at ambient temperature, with a light grey colour. At 200 °C temperatures, the specimens were grey. Finally, at 300 °C temperatures, the specimen turned to a yellowish-grey colour. The same results were observed by Guo, Y. C. et al. .The physical and chemical transformations in concrete cause the color change in concrete after heating. The cement and fine aggregate components are primarily responsible for the colour change. The whitish colour in concrete after heating is due to the process of dehydration and formation of lime in the cement component. On the other hand, the red colour in concrete after heating is due to the high amount of iron-bearing minerals in the river sand (fine aggregate). Due to the combination colour of cement (whitish colour) and fine aggregate (red colour), the light peach colour of concrete is formed during the heating.Further, Short, N. R. et al.  observedIn this regard, Colombo, M. et al.  observedAs previously stated, the WOC contained a significantly higher percentage of excellent particles, which filled the microvoids in the concrete made with ceramic materials . Consequently, the hydration and micro-filtering effects contributed to the growth in strength. Furthermore, due to the tiny size of WOC particles, they may have enough specific surface area to allow the pozzolanic reaction.Finally, in the case of concrete made with hybrid fibre and waste ceramic (WOC-Hybrid), the strength development is due to the PVA fibres effectively controlling the crack propagation at the bending crack tip. The steel fibres resist the widening of the crack width, thereby greatly enhancing the concrete’s strength.In the presence of water in the mortar matrix, WOC and WOC-Hybrid may react with water-hydrated products of the clinker to form secondary calcium silicate hydrate (CSH) gels. CSH gels would fill the micropores, thereby strengthening the interfacial transition zone (ITZ) and increasing the mortar mix’s strength.According to Netinger, I. et al. , concretKeshavarz, Z. and Mostofinejad, D.  concludeGenerally, the addition of non-metallic fibres (PP or PVA) has no significant effect on improving concrete’s CS after exposure to elevated temperature. However, such improvement is visible to a certain extent when FS and TS are considered, particularly at a temperature below 400 °C. Non-metallic fibres (PP or PVA) can increase concrete resistance to cracking, improving its behaviour under the TS test. However, Peng, G. F. et al.  prove thHan, C. G. et al.  noted thOn the other hand, adding metallic fibres (HK or CR) can improve the concrete mechanical properties at elevated temperatures when CS, FS, and TS are considered. These improvements may result from the testing temperatures not being high enough to melt steel fibre. Furthermore, Gao, D. et al.  concludeThis study provided insight into the use of ceramic wastes as partial replacement of cement and natural aggregates in concrete. The effects of elevated temperatures on the physical and mechanical properties of concrete made with ceramics (WOC) as well as those of concrete made with hybrid fibres (WOC-Hybrid) were evaluated. According to the findings, WOC and WOC-Hybrid concrete outperform standard concrete at high temperatures. Therefore, it is recommended to use these types of local and waste materials in buildings in fire-prone areas. Moreover, ceramic waste can be used to make concrete rather than dumping it in landfills, which pose a risk to the environment’s health and sustainability.(a)The colour change is mainly because of chemical and physical changes in fine aggregates and cement. At 100 °C, the colour and appearance were similar to those at ambient temperature (light grey), and at 200 °C, the examined specimens’ colours were changed to grey, while at 300 °C, specimens became a yellowish-grey colour.(b)Among all the tested specimens, hybrid fibre concrete performed better than waste ceramic optimal (WOC) and plain concrete (PC) due to the increase in the transport properties of heated material. In addition, heat transmits more uniformly in the concrete reinforced with hybrid fibres than the other examined types.Lastly, the relationship between the temperature changes and the accompanying colour changes in the concrete exposed to different high heating temperatures was studied. According to the test results, analysis, and discussions presented in this paper, the following is concluded:In this research, efforts were made to create a new generation of concrete with waste ceramic tiles where it was found that the mechanical and physical properties of concrete would not be harmed if these conventional materials were substituted for waste ceramic ones. Since there are numerous environmental issues that arise from the accumulation of solid waste, the only positive solution is reusing these materials in order to maintain the rapidly depleting environment. Overall, the use of wastes and local materials such as ceramic waste floor or wall tiles remains a positive solution to the sustainability issue of depleting natural aggregate sources.


Soy lecithin (SL) can be used in to prevent spermatozoa cryodamage during cryopreservation by mitigating the efflux of cholesterol or phospholipids, thus reducing the formation of intracellular ice crystals. SL nanoparticles (nano-SL) have a smaller particle size and higher solubilizing capacity as compared with those that have not undergone nanotreatment. Thus, they allow for a better interaction or coating of sperm, decreasing cold shock injury during freezing–thawing processes. The objective of this study was to determine the optimal concentration of nano-SL. In order to achieve this, we assessed the quality of frozen–thawed semen in vitro and in vivo. We found that a nano-SL dosage of 1.0% in the semen extender had an affirmative influence on post-thawing quality in roosters, improving various parameters related to sperm motion, protecting the membrane and acrosome integrities, increasing mitochondrial activity and antioxidant capacity, and reducing the oxidative stress caused by the cryopreservation process. Moreover, enrichment of 1.0% nano-SL in the semen extender improved the fertilizing capacity of rooster sperm after artificial insemination. p < 0.05). The MDA levels in the SL0.5 and SL1.0 groups were lower than the other groups (p < 0.05). TAC activities in SL0.5, SL1.0, and SL1.5 groups were significantly higher than those in the other groups (p < 0.05). It was observed that the concentration of SOD was higher in the SL1.0 group than in the other groups (p < 0.05). The activity of GPx was not influenced in any of the cases (p > 0.05). Moreover, the percentages of fertility and hatchability in the SL1.0 group were higher (56.36% and 58.06%) than those in the control group (42.72% and 40.43%). In summary, the addition of nano-SL to the extenders enhanced the post-thawed semen quality and fertility of roosters by reducing the level of oxidative stress. The optimum nano-SL concentration was 1.0%. These results may be beneficial for improving the efficacy of semen cryopreservation procedures in poultry breeding.The present study aimed to investigate the impact of different concentrations  of nano-soybean lecithin (SL) in the extender on sperm quality, sperm motion characteristics, and fertility outcomes of post-thawed rooster semen. Adult Ross broiler breeder roosters (n = 20) were subjected to semen collections twice a week for three weeks. At each collection, semen samples were pooled and allocated into five treatments corresponding to different nano-SL concentrations . Sperm parameters, including motility , plasma membrane and acrosome integrities, and mitochondrial activity were assessed. Sperm malondialdehyde (MDA) and antioxidant activities ; superoxide dismutase (SOD); glutathione peroxidase (GPx)) were evaluated. The fertility and hatchability obtained with frozen–thawed rooster semen supplemented with the optimum nano-SL concentration were assessed after artificial insemination. The results showed that the addition of 1% nano-SL into the extender led to a higher semen motility in roosters, improved plasma membrane and acrosome integrities, and higher mitochondrial activity of post-thawed rooster semen in comparison to controls ( Sperm freezing is crucial for the preservation of biodiversity in poultry. In poultry in particular, the use of frozen semen may decrease the risk of breed and population extinctions and reduce the cost of transportation . The cryArtificial insemination (AI) with frozen–thawed sperm is associated with low fertility rates, which is also a central problem in commercial applications and the conservation of genetic resources in poultry . Bird spSoy lecithin (SL), which is mainly composed of phosphatidylcholine, has the capacity to protect the integrity of the phospholipid membrane during cryopreservation . It alsoThe particle size of SL in the extenders is generally considered to be an important factor in frozen–thawed semen quality . SL nanoAs far as we are aware, the physiological mechanism of the nano-SL-based extender in the cryopreservation of rooster semen has yet to be systematically explored. As such, this study aimed to explore the influence of nano-SL at various concentrations on sperm motion parameters, plasma membrane integrity, acrosome integrity, and mitochondria function of post-thawed rooster sperm. Considering the antioxidative effect of SL in the extender, we evaluated changes in the enzymatic antioxidant activity of post-thawed sperm, including the malondialdehyde (MDA) level, the total antioxidant capacity (TAC), and the superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels. Importantly, the fertility and hatchability rates after AI with frozen–thawed semen were also assessed.In all animal experiments, the animal procedures were approved by the Ethics Committee on the Use of Animals of Shanghai Academy of Agricultural Sciences, China . All materials were acquired from Sigma Chemical Co.  unless mentioned otherwise.Semen was collected from Ross broiler breeder roosters , which were bred at the Shanghai Academy of Agricultural Sciences facilities. All roosters were kept in separate cages (size = 70 cm × 70 cm × 85 cm), maintained in a 15 h light:19 h dark photoperiod, fed basal diets, and had free access to water.9 spermatozoa/mL concentration, ≥ 80% sperm motility, ≥90% sperm viability, and ≤ 10% abnormal morphology) were accepted for further processing. The semen samples were pooled to eliminate individual differences and then grouped in five treatments according to different SL concentrations . The nano-SL concentration chosen for this study was chosen based on a former study [Semen was collected twice a week for a 3-week period (six replicates), using the dorsoabdominal massage method . From ther study ,17.The SL nanoparticles was prepared using the reverse-phase evaporation method, according to the literature, with minor modifications . In totaThe coarse emulsion was homogenized by sonication  for 15 min to reduce the size of the droplets, and then transferred into a high pressure homogenizer  at 30,000 lb/in2 for 30 cycles at 4 °C. The particle sizes of the prepared nanoparticles were measured by transmission electron microscopy. SL nanoparticles with a certified particle size distribution of <100 nm were used and stored at 4 °C for future use in the semen extender.9 spermatozoa/mL using modified Lake pre-freezing extender [9 sperm/mL with Lake pre-freezing extender containing 18% dimethylacetamide (DMA), leaving to 6% final DMA concentration. After equilibration , the extended semen was aspirated into 0.25 mL plastic straws, and frozen 4 cm over liquid nitrogen for 20 min. Finally, the straws were plunged and kept in liquid nitrogen for at least 1 month. For the post-thaw sperm quality assessments, frozen samples were thawed separately in a 37 °C sterile water bath for 20 s.Semen aliquots were diluted to a definitive concentration of 1.5 × 10extender , supplem6 spermatozoa/mL. Sperm morphology was assessed using Hancock solution  [Sperm concentration was assessed using a haemocytometer (Bürker-Türk) and expressed as 10 K2HPO4) . For thiThe sperm motion was evaluated with a computer-assisted sperm assessment  as described previously . SamplesPlasma membrane integrity was evaluated using the hypo-osmotic swelling test (HOS-t) . In brie6 spermatozoa/mL) was centrifuged for 3 min at 1200× g. A total of 5 µL of sperm pellet were added to 100 µL ethanol (purity: 96%). Next, a 10 µL sample droplet was placed on a glass slide and heated in order to evaporate the solvent. Afterwards, 30 µL of work solution were added and incubated in the dark at room temperature for 5 min. After incubation, the slides were washed and examined with a fluorescence microscope . At least 200 spermatozoa per slide were observed. Sperms with PI were considered to be dead and sperms without PI were considered to be alive. Alive cells were categorized as acrosome-reacted (FITC-PNA-) or as acrosome-intact (FITC-PNA+). Acrosomal status was evaluated by staining with propidium iodide (PI) and fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA), according to the method described by Thananurak et al. . The sta6 spermatozoa/mL) and incubated for 20 min at 37 °C in the dark. Flow cytometry was performed using a FACSCalibur flow cytometer . At least 10,000 events from each sample were recorded at a flow rate of 300–400 spermatozoa/sec. Green fluorescence from R123 was collected through FL1, and red fluorescence from PI was collected through FL3. To determine the correct side scatter and forward scatter of spermatozoa, the samples that were stained with R-123 or PI only and were backgated from the area in which fluorescently stained populations appeared in the green and red channels. The data obtained were analyzed using the CellQuest software . The percentage of live spermatozoa with active functional mitochondria was determined by R123 high fluorescence and no PI fluorescence (R123 + /PI-).Mitochondria membrane potential was assayed using the cationic agent Rhodamine 123 (R123) (0.01 mg/mL) and PI (1 mg/mL) [6 spermatozoa/mL) was added to 500 µL of cold 20% (w/v) tricholoroacetic, and centrifuged for 5 min at 1200 rpm. The supernatant was mixed with 1 mL of 0.67% (w/v) thiobarbituric acid and incubated in a boiling water bath for 10 min. To stabilize the chromogenic MDA-cTBA complex, samples were incubated for 10 min at room temperature. The supernatant was collected and the absorbance was determined at 532 nm by Shima-dzu UV 2100 spectrophotometer [MDA concentrations in semen samples were measured as an indicator of the lipid peroxidation using the thiobarbituric acid (TBA) test . Each seotometer . The conThe levels of TAC and SOD were evaluated by commercial colorimetric assay kits  according to the manufacturer’s instructions. TAC was measured by monitoring hydrogen peroxide decomposition. The TAC value was measured using the absorbance at 600 nm, and the concentration was calculated using the calibration curves. The concentration of TAC was expressed as mmol/L. The nitro blue tetrazolium reduction method was carried out to measure SOD activity. Sample concentrations (U/mg) were determined by measuring the mean absorbance values with a pectrophotometer at 630 nm, and converting the SOD activity using a standard calibration curve.IU) per mg of protein.GPx activity was evaluated by NADPH oxidation using a coupled reaction system consisting of GSH . In brie6 spermatozoa/per hen), and then every tree days. The total number of insemination days was five, and all inseminations were performed between 15:00 h and 16:00 h. Collecting eggs for incubation began a day the after the first insemination and was performed daily for nine days. Eggs (n = 220/per group) were incubated at 37.8 °C in the setter for 18 d and then placed in a hatcher for up to 3 days. Fertility rate (fertilized eggs/incubated eggs × 100%) was evaluated by candling the eggs on day 7 of incubation. After 21 days of incubation, the hatching rate of fertile eggs (hatched eggs/fertilized eggs × 100%) was calculated.The reproductive performance of the post-thaw semen was evaluated by AI as described previously by Thananurak et al. . Accordit-test. Data in percentages were arcsine-square root transformed before the statistical analysis. The significance level was set to 0.05. Data are presented as mean value ± standard error of the mean (SEM).A total of three replicates were performed for each in vitro evaluation of sperm parameters. The Statistical Package for Social Sciences (SPSS) V.23 was used for statistical analyses. All data were checked using the Shapiro–Wilk test and were found to fit the normal distribution. The groups were compared using one-way ANOVA, followed by post hoc analysis and the least significant difference test. Differences between two groups were analyzed using Student’s 6 spermatozoa/mL, and 6.19 ± 0.18, respectively. The total sperm motility was higher than 80%, the viability was recorded to be more than 90%, and the sperm deformity rate was 7%.The mean volume, sperm concentration, and pH of fresh sperm from the ejaculate were 1.20 ± 0.26 mL, (742.68 ± 32.12) × 10p < 0.05). The percentage of PMOT was significantly higher in the SL0.5 and SL1.0 groups as compared with the other groups (p < 0.05). The sperm motility VSL and VCL parameters were significantly higher in the SL0.5 and SL1.0 groups than in the other SL groups (p < 0.05). However, differences between the control and the SL1.0 group were nonsignificant (p > 0.05). A significantly higher BCF was observed in the SL1.0 group than the other groups (p < 0.05). The percentages of LIN and STR were significantly higher in the SL0.5 and SL1.0 groups, as compared to the groups at other SL concentrations and the control groups (p < 0.05). For ALH and WOB, no significant differences were found among the groups (p > 0.05).The results of the sperm motility and motion parameters are presented in p < 0.05). A significant decrease in the percentages of plasma membrane and acrosome integrities were observed in the SL1.5 and SL2.0 groups as compared to the SL0.5 groups (p < 0.05). Percentages of sperm with high mitochondrial activity significantly increased in the SL0.5 and SL1.0 groups, as compared to the controls (p < 0.05). As regards mitochondrial activity, no significant differences were found between the control and the SL1.5 or SL2.0 groups (p > 0.05).The values of plasma membrane integrity, acrosome integrity, and mitochondrial activity of post-thawed rooster semen are shown in p < 0.05). A significant increase in TAC activity was observed in the SL1.5 group as compared to the SL2.0 and control groups (p < 0.05). However, the value was not different for the SL1.5 group and the SL0.5 and SL1.0 groups (p > 0.05). The SOD concentration in the SL1.0 group was significantly higher than that in the other groups (p < 0.05). As regards GPx activity, no significant differences were found between the groups (p > 0.05).p < 0.05). It was observed that the hatching rate percentage was significantly higher in the SL1.0 group than in the controls (58.06% vs. 40.43%) (p < 0.05).The results of the fertility and hatching rates after insemination with frozen–thawed semen are listed in Semen cryopreservation is a useful tool to preserve genetic resources. These resources are used to prevent extinction by infectious disease, to maintain genetic diversity, and to increase AI success . It is wMany studies confirm that SL can prevent spermatozoa cryodamage during cryopreservation by mitigating the efflux of cholesterol and phospholipids. Further, it was reported that using 2% nano-SL in the extender improved the sperm cryosurvival of goats, and the results of cryopreservation of sperm are better than any SL suspension . In thisOur findings revealed that among the test concentration range of SL (0.5–2%), the best concentration of SL for cryopreservation of rooster semen was 1.0%. However, previous sperm studies for semen cryopreservation in other species demonstrated that the optimal SL concentration ranged from 0.8 % in canines , 1.5% inThe CASA is an effective, optimized, comprehensive, and detailed technique that is used to assess the quality of sperm in several mammalian species, including humans . Sperm MOur study suggested that high doses of nano-SL had a negative effect on motion characteristics. A similar finding was previously reported in which 1.5% SL added to the extender increased the extender’s viscosity. It was also reported that particular SL debris may reduce sperm motility . MoreoveAs the first sperm cell components, membranes are associated with the resistance of sperm to cooling, which is influenced by the chemical composition or temperature changes of the extender . In thisSemen quality is not only linked to the cell membrane stability but also to acrosome integrity. Acrosomal integrity has a decisive effect on the sperm penetration and fusion ability, which thereby affect the fertility of frozen semen . From ouSperm mitochondria represent the regulatory center of energy metabolism and oxidative stress, and are the hub of signaling pathways that regulate cell survival and apoptosis . In partCryopreservation damage of sperm is partly related to ROS and oxidative stress generation. The antioxidant effect of SL has been attributed to phospholipids, phosphatidic acid, lyso-phospholipids, and vitamin E (the main components of lecithins) through multiple mechanisms ,44. We nThe results of our in vitro experiments suggest that a supplementation of 1.0% nano-SL to the extenders is optimum for improving the quality of frozen–thawed rooster semen. Moreover, another main goal of this study was to investigate the impact of nano-SL on sperm function, i.e., on fertility and hatchability rates. Our in vivo results showed that the fertility and hatchability percentages of the eggs were improved by adding 1.0% nano-SL to the extender, as compared with controls. This result further demonstrates that SL had a good effect in terms of improving the post-thaw functionality of rooster semen. According to previous studies, the use of SL nanoparticles may eliminate the toxicity of cryoprotectants, thus helping preserve the spermatozoa functional properties for producing viable offspring . The resIn conclusion, a nano-SL dosage of 1.0% in the semen extender had a positive influence on the post-thawing quality in roosters, improving various sperm motion parameters, protecting membrane and acrosome integrities, increasing mitochondrial activity, augmenting antioxidant capacity, and reducing the oxidative stress caused by the cryopreservation process. Moreover, the enrichment of the semen extender with 1.0% nano-SL increased the fertilizing efficiency of rooster sperm after AI. Furthermore, the results from the in vitro and in vivo experiments can serve as a resource for further studies concerning the use of nano-SL as an antioxidant in the extenders for the cryopreservation of rooster semen. We believe that future experiments should assess how nano-SL interacts with spermatozoa and how to preserve or improve this system to achieve the optimum protective efficacy.


Background: The human gut microbiota are important to health and wellness, and disrupted microbiota homeostasis, or “dysbiosis,” can cause or contribute to many gastrointestinal disease states. Dysbiosis can be caused by many factors, most notably antibiotic treatment. To correct dysbiosis and restore healthier microbiota, several investigational microbiota-based live biotherapeutic products (LBPs) are in formal clinical development. To better guide and refine LBP development and to better understand and manage the risks of antibiotic administration, biomarkers that distinguish post-antibiotic dysbiosis from healthy microbiota are needed. Here we report the development of a prototype Microbiome Health Index for post-Antibiotic dysbiosis (MHI-A).Methods: MHI-A was developed and validated using longitudinal gut microbiome data from participants in clinical trials of RBX2660 and RBX7455 – investigational LBPs in development for reducing recurrent Clostridioides difficile infections (rCDI). The MHI-A algorithm relates the relative abundances of microbiome taxonomic classes that changed the most after RBX2660 or RBX7455 treatment, that strongly correlated with clinical response, and that reflect biological mechanisms believed important to rCDI. The diagnostic utility of MHI-A was reinforced using publicly available microbiome data from healthy or antibiotic-treated populations.Results: MHI-A has high accuracy to distinguish post-antibiotic dysbiosis from healthy microbiota. MHI-A values were consistent across multiple healthy populations and were significantly shifted by antibiotic treatments known to alter microbiota compositions, shifted less by microbiota-sparing antibiotics. Clinical response to RBX2660 and RBX7455 correlated with a shift of MHI-A from dysbiotic to healthy values.Conclusion: MHI-A is a promising biomarker of post-antibiotic dysbiosis and subsequent restoration. MHI-A may be useful for rank-ordering the microbiota-disrupting effects of antibiotics and as a pharmacodynamic measure of microbiota restoration. Disruptions to the human gut microbiota, known as dysbiosis, can contribute to gastrointestinal, neurologic, epithelial, genitourinary, and oncological disorders . To counClostridioides difficile infections (CDI) or infections by multidrug-resistant Enterobacteriaceae or Enterococci  with confidence limits were generated by fitting a Dirichlet-multinomial (DM) distribution to OTU data using maximum likelihood estimation . Dirichlp values, and odds ratios showed that each class by itself separated the baseline and RBX2660 groups. The odds ratios for Gammaproteobacteria and Bacilli indicated high value of these classes to predict post-antibiotic dysbiosis and low odds ratios for Bacteroidia and Clostridia indicated high value of these classes to predict healthy or non-dysbiotic samples  analyses were conducted within GraphPad Prism using baseline and RBX2660 MHI-A data from the PUNCH CD2 trial. All pairwise hypothesis testing between MHI-A data groups, which used Mann–Whitney tests unless otherwise specified, was conducted within GraphPad Prism.The MHI-A was designed to differentiate post-antibiotic dysbiosis from healthy microbiota. Literature teaches that many antibiotics reduce the relative abundance of Bacteroidetes and Firmicutes phyla compared to healthy populations, with a concomitant increase in Proteobacteria , the median MHI-A was 0.0013, whereas the median MHI-A for RBX2660 was 183 , and the majority were > 7.2 (p < 0.05). Analysis of non-responders (CDI recurrence) was limited because most recurred prior to the earliest 7-day time point, at which point participants received additional antibiotic and/or RBX2660 treatment and were therefore excluded from subsequent microbiome analysis. Among the five 7-day and two 30-day samples that were included from non-responders, most had MHI-A < 7.2  and similar to baseline participants in RBX2660 and RBX7455 trials. This MHI-A decrease was driven by lowered Bacteroidia and Clostridia abundance concurrent with a bloom of Gammaproteobacteria. By 6 weeks and 6 months after the start of treatment, MHI-A had largely recovered to pre-treatment levels. Thus, MHI-A was clearly diagnostic of a post-antibiotic microbiota shift with subsequent restoration in this study.To evaluate how well MHI-A can be generalized as an indicator of post-antibiotic dysbiosis, published studies of antibiotic treatments were evaluated. A literature search revealed three such studies for which longitudinal samples collected before and after antibiotic treatment were sequenced and data publicly available. For each study, sequencing data was processed to OTU from which MHI-A values were calculated. The first study characterized a 4-day course of a broad-spectrum cocktail of meropenem, gentamycin, and vancomycin in 12 healthy adult males using whole-genome sequencing . Prior tp > 0.05, p < 0.05) but converged thereafter. The only antibiotic that significantly decreased MHI-A was clindamycin, and only at 1 month after treatment; notably, clindamycin is known to increase CDI risk . Thus, in this study MHI-A clearly differentiated between the narrower- and broader-spectrum CDI antibiotics.The third study characterized the microbiome before, during, and after a 10-day course of vancomycin or ridinilazole for CDI in a Phase 2 clinical trial , and decreased Gammaproteobacteria correlated with reduced CDI recurrence, trends that are highly consistent with MHI-A. They also cited the MHI-A described here, showing that a small change (1%) in the MHI-A reported herein was more effective at predicting CDI recurrence than changes in individual taxa alone or than the Shannon index. It may be that the ability of MHI-A to account for interconnected positively and negatively correlated taxa increases its diagnostic potential. Overall, the taxonomic changes measured by MHI-A are quite consistent with other metrics reported in the literature.Two additional metrics were proposed to predict response to or recurrence after CDI antibiotics, and both are consistent with MHI-A. In one, elevated abundance of six taxa prior to CDI antibiotic was positively associated with successful antibiotic response . All sixcurrence . They foIt was conceivable that taxonomic levels other than class could lead to a similar index. At the phylum level, the F/B ratio was less effective, as already described. At the order level, Clostridiales shifted concurrently with Clostridia, Lactobacillales with Bacilli, Enterobacterales with Gammaproteobacteria, and Bacteroidales with Bacteroidia. However, the accuracy of MHI-A was not increased when assessed at the order level. At the family or genus taxonomic levels, patient-to-patient variability was higher, which would decrease diagnostic accuracy. Thus, MHI-A appears simplest to calculate while retaining high accuracy.Enterococcus, Streptococcus, or Gammaproteobacteria dominance of the intestinal microbiome was correlated with increased risk of bacteremia, and these microbiome signals preceded infection by up to 7 days  measure of microbiota restoration for LBPs. Typically, PD is defined as the action of a drug in the body, manifested as biochemical or physiologic effects, but debate remains about how to define PD for microbiota-based LBPs . In the A few limitations of this work bear noting. First, it is an apparent limitation that MHI-A was not decreased by all antibiotics evaluated herein, specifically in healthy volunteers that received clindamycin, ciprofloxacin, moxifloxacin, or amoxicillin. This prompts consideration of what is the minimum level of disruption detectable with MHI-A. It bears noting that other studies using culturomics or pyrosequencing methods suggested these antibiotics may be more impactful than was observed in the data set used to calculate their MHI-A shift here . NeverthDespite the potential limitations, there are several key strengths of our work: the large number of samples and studies assessed, the consistency of patient definitions and sample collection processes across three controlled clinical trials, the evaluation of antibiotics as well as investigational microbiota-restoring treatments, consistent performance of MHI-A among three sequencing methodologies, and the inclusion of publicly available data sets to assess the generalizability of observations. Overall, the MHI-A provides a novel perspective and promising prototype microbiome marker that merits further evaluation and development as a tool for monitoring post-antibiotic dysbiosis.The datasets presented in this article are not readily available because they are restricted by an ongoing commercialization process. Requests to access the datasets should be directed to KB.IRB approval was obtained at each study center for NCT02299570, NCT02589847, and NCT02981316. The patients/participants provided their written informed consent to participate in this study.KB and CJ contributed to conception and design of the study. KB, DW, CG, and WS performed the statistical analysis. KB wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.The work was supported by Rebiotix, a Ferring Company, as part of a commercial clinical development program for microbiota-based investigational live biotherapeutics RBX2660 and RBX7455.KB, CJ, and DW are employees of Rebiotix, A Ferring Company. CG and WS are employed by BioRankings, LLC, which received fees for this analysis from Rebiotix.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Respondents’ knowledge on effective use, unnecessary use or associated side effects of antibiotics was high (>90%), compared with their knowledge on the spread of antibiotic resistance (62.2%). For ease of access to the required guidelines on managing infections, and to materials that advise on prudent antibiotic use and antibiotic resistance, prescribers agreed in 62% and 46.1% of cases, respectively. 28.4% of respondents had prescribed antibiotics when they would have preferred not to do so more than once a day or more than once a week. Among respondents who prescribed antibiotics, 63.4% would never or rarely give out resources on prudent use of antibiotics for infections. The findings are of importance to inform antibiotic stewardships about relevant interventions aimed at changing prescribers’ behaviors and improving antibiotic prescribing practices.More research is needed on the drivers of irrational antibiotic prescribing among healthcare professionals and to ensure effective prescribing and an adequate understanding of the issue of antibiotic resistance. This study aimed at evaluating prescribers’ knowledge, attitudes and behaviors about antibiotic use and antibiotic resistance. A cross-sectional study was conducted utilizing an online questionnaire and included physicians and dentists from all sectors in Jordan. A total of 613 prescribers were included (physicians  Antimicrobial resistance (AMR) poses a significant problem to public health that threatens the provision of effective treatment for a range of infections and undermines the efforts to control the development and spread of multidrug-resistant (MDR) bacteria . The devAntibiotic prescribing is a complex process influenced by several factors including the healthcare system, physicians, other healthcare providers, patients and the general public . A lack More than 90% of the respondents correctly answered the questions on effective use, unnecessary use or associated side effects of antibiotics; however, lower percentages of correct answers were observed for questions on the spread of antibiotic resistance .p = 0.0031), middle-age groups (36–55 years) (p = 0.0140) and specialists (p < 0.0001) (p = 0.0350), and more years of experience (p = 0.0183), as shown in Knowledge average scores were found to be significantly higher among physicians ( 0.0001) . Higher p < 0.0001; estimate: 0.27, CI: 0.09–0.37, p = 0.0152, respectively).Further, a multivariable stepwise regression model was conducted, and only profession and age were found to be statistically significant predictors of higher knowledge scores. The results of the univariate analysis are presented in The results of perceived knowledge  show thaAround two thirds of the prescribers agreed or strongly agreed that they have easy access to the required guidelines on managing infections (62.0%), and around half agreed or strongly agreed that they have easy access to materials that advise on prudent antibiotic use and antibiotic resistance (46.1%). Moreover, 67.3% agreed or strongly agreed they had a good opportunity to provide such advice . Neverthn = 210, 34.3%), patient education  and new patient consultation . However, 73 (11.9%) prescribers would not follow any strategy.The majority of prescribers (85.6%) agreed or strongly agreed that they would consider antibiotic resistance when treating a patient. Around 75% of prescribers were confident in making prescribing decisions and confident about the available antibiotics guidelines on managing infections . In addin = 262, 42.7%) or weekly , with 10.8% (n = 66) monthly, 6.0% (n = 37) quarterly and 6.4% (n = 39) yearly. Although 27.6% of prescribers did prescribe antibiotics more than once a day, more than 50% would never or rarely give out resources on prudent use of antibiotics for infections , “Patient uninterested in information” , “Insufficient time” , “Difficulty getting patient to understand diagnosis” , “Language barriers” , “No resources available” , “I was not sure what advice to provide”  and “I was able to give out advice or resources as needed” .Most of the respondents would prescribe antibiotics daily  of respondents could not decide whether environmental factors contribute to antibiotic resistance (n = 339) answered that action at all levels is required. Others responded at the individual level (prescribers) , regional/national level , global level  and environmental or animal health level , and 6.0% (n = 37) did not know the answer.Concerning the One Health question, most of the respondents were unsure if it is legal to use antibiotics to stimulate growth in farm animals, with only 14.2% answering correctly . Furthersistance . When asTo our knowledge, this is the first study in the Middle East area to assess the knowledge, attitudes and behaviors about antibiotic use and antibiotic resistance among physicians and dentists using the ECDC’s validated instrument ,23. AntiA study conducted by Wushouer and colleagues in China reported that the higher the knowledge of the practitioners, the lower the rate of antibiotic prescribing . This waAdditionally, we found that most of the participating physicians and dentists achieved a high score in questions related to effectiveness, inappropriate use and adverse effects but scored less well in questions addressing antimicrobial resistance. This is similar to a study conducted in the United States in which the authors compared the prescribing behavior of those prescribing high rates versus those prescribing low rates of antibiotics . Higher Multiple factors were found to contribute to antibiotic prescribing decisions including psychological and socioeconomical factors, in addition to clinical aspects ,43,44,45Despite the findings that both physicians and dentists knew about strategies to promote prudent antibiotic use, these were not implemented effectively. It is known that delaying antibiotic prescribing can control inappropriate antibiotic use without affecting the health outcomes for patients ,47; howeThe majority of the prescribers indicated that they tend to prescribe antibiotics on a daily basis, similar to high rates reported in other studies ,47,49. WThis study has some limitations. First, this study used an online survey that may introduce selection bias. However, this is less likely to affect our results, particularly as the survey was sent to physicians and dentists (literate population) who are increasingly using the internet and smartphones in their practice ,56,57. IIn conclusion, the results of this study indicate that antibiotic resistance should direct our plans to a more robust system to educate our practitioners and update them about local and global antibiotic use status and antimicrobial resistance. The findings are of importance to inform antimicrobial stewardship with relevant interventions aiming at changing prescribers’ behaviors and improving antibiotic prescribing practices.A cross-sectional study survey of Jordanian physicians and dentists was conducted using an online questionnaire. This survey was conducted using the European Centre for Disease Prevention and Control’s (ECDC) validated instrument ,23.Physicians and dentists from all sectors were eligible to participate in the study. A web-based survey software (Google Forms) was used to collect data, applying the “required” and “Limit to one response” validation options. Physicians and dentists were recruited through a variety of means, including direct mail, online recruiting, online professional conferences and social media. Participants received an invitation leading them to a Google Forms-based online survey with more comprehensive details about the study. Participants were provided with a brief description of the study and informed that their participation was voluntary and that their responses would be anonymized and treated as confidential.A 10 min online questionnaire was adapted from the questionnaire developed and tested by the European Centre for Disease Prevention and Control (ECDC) ,23 Ques. The surSection three consisted of a 5-point Likert scale (strongly agree to strongly disagree), in addition to not applicable/I do not understand the question. A level of agreement was obtained on 14 statements, including two statements on antibiotic resistance in the context of food and environmental factors, three statements that assessed perceived knowledge, three statements that assessed opportunities that may affect prudent prescribing and management of infections, three statements that assessed available opportunities to enact positive behaviors for preventing and controlling AMR and six statements that were on prescribers’ motivations to initiate antibiotic prescriptions and antibiotic prescribing decisions, support and accessibility to guidelines.The fourth section included 12 questions on the frequency with which prescribers provided antibiotics or resources related to the prudent use of antibiotics (3 questions), behavior on drivers for initiating prescriptions (6 questions) and antibiotic prescribing behaviors (3 questions). Questions on the frequency were asked concerning the last week, with options that included more than once a day, more than once a week, once a day, never and rarely, in addition to “not applicable” and “I do not remember” options. The frequency of antibiotic prescribing, in general, was also obtained from respondents. Prescribers were also asked about why they were unable to provide any resources to their patients, strategies employed to prescribe antibiotics prudently and the level of implementation they think would be most effective to tackle resistance to antibiotics.The questionnaire was administered in both Arabic and English languages for better interpretation. Prescribers had the option to choose any of the two languages. An initial online pretest of the questionnaire was performed with 10 physicians and dentists. Each participant was interviewed to determine whether the questions were correctly interpreted and if the answer options given were adequate. Based on the pretest interviews, minor adjustments were made to the questionnaire. The pilot interview data were not included in the final sample.http://www.raosoft.com/samplesize.html), based on a margin of error of 5%, confidence level of 95%, population size of 36,000  and a response distribution of 50%. The calculated sample size was 381. Google Forms was connected to a Google spreadsheet for further analysis and data organization. The IBM SPSS  version 24.0 software was used to analyze the collected data. The percentages, frequencies, means and standard deviations were used to display the results. An analysis of variance (ANOVA) test or t-test was used to examine the effect of demographic characteristics on respondents’ knowledge scores. The chi-square test or Fisher’s exact test was used to examine differences among respondents with a full knowledge score. Finally, a multivariable linear regression model was conducted to assess predictors of knowledge, adjusting for confounding variables. The significance of all results was determined using a p-value of less than 0.05.The sample size was determined, using Raosoft sample size calculator (


Cancer immunotherapy is limited by the immune escape of tumor cells and adverse effects. Photo-immunotherapy, the combination of immunotherapy and phototherapy (such as photodynamic therapy (PDT) and photothermal therapy (PTT)), can improve the effectiveness of immunotherapy in cancer treatment. Here, we first explored mesoporous hexagonal core–shell zinc porphyrin-silica nanoparticles (MPSNs), which are composed of a zinc porphyrin core and a mesoporous silica shell, and exhibit high laser-triggered photodynamic and photothermal activity, as well as outstanding drug loading capacity. In other words, MPSNs can be used not only as excellent photosensitizers for photo-immunotherapy, but also as an ideal drug carrier to achieve more efficient synergy. After loading with R837 , MPSNs@R837 will elicit high-efficiency immunogenic cell death via PDT and PTT, and promote dendritic cell maturation after the PH-responsive release of R837, thereby, inducing tumor-specific immune responses. When combined with a programmed death ligand-1 checkpoint blockade, the photo-immunotherapy system markedly restrains primary tumors and metastatic tumors with negligible systemic toxicity. Therefore, the therapeutic strategy of integrating PTT, PDT and checkpoint blockade, shows great potential for suppressing cancer metastasis.The online version contains supplementary material available at 10.1186/s12951-022-01388-8. Cancer immunotherapy serves as a clinical modality against tumor growth and metastasis by stimulating host immunological responses, which has achieved great progress in the field over the past few years –4. HowevPhototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is one of the least invasive treatments, especially compared to chemotherapy –17. PDT Photo-immunotherapy-the combination of phototherapy and immunotherapy-can effectively enhance treatment effectiveness compared with a single treatment modality –30. In rIn this study, we first developed a multifunctional nanoplatform based on mesoporous hexagonal core–shell zinc porphyrin-silica nanoparticles (MPSNs) loaded with R837 (a toll-like receptor-7 agonist), which could be used to integrate PDT, PTT, and tumor-specific immunotherapy for breast cancer. MPSNs with ZnP as the core and a mesoporous silica framework as the shell can effectively generate singlet oxygen and convert photons to heat energy under only one light source, making the operation easier and safer. Meanwhile, the excellent mesoporous structure of the silica shell can facilitate efficient R837 loading. Taken together, MPSNs are not only excellent photosensitizers, but also efficient drug carriers. The immune adjuvant R837, functionalized together with tumor-associated antigens derived from primary tumors, is used to promote DC maturation, eliciting a strong immune response. Furthermore, combined with a programmed-death ligand-1 (PD-L1) checkpoint blockade, the novel nanoplatform showed more conspicuous anti-metastatic performance in 4T1 tumor-bearing mice. Therefore, the therapeutic strategy based on MPSNs@R837 not only eradicated primary tumors via phototherapy modalities (PDT and PTT), but also effectively inhibited distant metastasis due to the strong immune response triggered by the two-way mechanistic interaction  and reacted for 24 h to form pre-MPSNs at room temperature during the first step. Subsequently, tetraethyl orthosilicate (TEOS) and a small amount of 3-aminopropyltriethoxysilane (APS) were slowly injected into the pre-MPSNs aqueous solution and stirred for 1 h at 40 ℃. TEM images of pre-MPSNs reveal fragmented structures in a 15-nm in diameter following the self-assembly of ZnP monomers  was estimated by the ROS sensitive green fluorescent probe, named singlet oxygen sensor green (SOSG), whose fluorescence enhancement can characterize the content of ROS. Compared with the water sample, the MPSNs and MPSNs@R837 exhibit a noticeable fluorescence enhancement of SOSG, which proves their efficient ROS generation ability, indicating the potential for PDT in cancer treatment  by performing MTT assays. As expected, no significant decrease in cell viability occurs after 24 h or 48 h co-incubation, and all results indicate that the MPSNs are almost non-toxic , whereas nearly no fluorescence was observed both in the PBS groups and in the non-irradiated groups. The mean fluorescence intensities of the MPSNs (+) and MPSNs@R837 (+) groups are significantly higher than those of the other four reference groups  was used to measure ROS levels, based on the rapid oxidation of the nonfluorescent DCFH molecule into the fluorescent molecular dichlorofluorescein in the presence of ROS. As illustrated in Fig. 50 value of MPSNs@R837 (+) is the lowest among all groups  at different times post-injection. Photothermal images in Additional file 2DCFDA, followed by laser irradiation for 5 min. It is noticed that the tumor sections from the MPSNs and MPSNs@R837 treated groups exhibit brighter green fluorescence than those from the control group  was denoted as the primary tumor, and the left tumor (without laser irradiation) was designated as a distant abscopal tumor Fig. A. SimilaIn summary, we prepared a novel MPSNs with three functions, PDT, PTT and drug loading, compared with other known nanosystems with relatively limited functions. Usually, integrating the synergistic effect between PDT and PTT requires two photosensitizers with different absorption wavelengths, while MPSNs can efficiently achieve both effects at the same time under only one light source. More notably, MPSNs are not only excellent photosensitizers, but also efficient drug carriers because of the rich mesoporous structures in the silica shell. After loading the immune adjuvant R837, the multifunctional nanoplatform broke through the inhibition of the tumor immunosuppressive microenvironment, effectively promoting DC maturation. The R837-loaded MPSNs efficiently destroyed primary tumors, leading to tumor cell death and promoting the release of tumor-associated antigens, which triggered an immune response by integrating with mature DCs. Furthermore, in combination with PD-L1 checkpoint blockade, photo-immunotherapy could augment PDT and PTT-induced ICD and elicit systemic antitumor immunity, causing regression of primary and metastatic tumors with minimal adverse effects. MPSNs@R837 displayed great potential for combination therapy with PDT, PTT, and immunotherapy, offering a promising and safe photo-immunotherapy strategy for metastatic cancer.Additional file 1. Experimental Section. Fig S1. Characterization of MPSNs. Fig S2. Fluorescence stability of MPSNs. Fig S3. The generation of singlet oxygen of MPSNs determined by the increased SOSG fluorescence. Fig S4. Cytotoxicity of MPSNs. Fig S5. 4T1 cell viability. Fig S6. CLSM images of 4T1 cells stained with Calcein-AM and PI. Fig S7. 4T1 cell viabilities after different treatments. Fig S8. A Photothermal imaging after intravenous injection of saline, MPSNs and MPSNs@R837 in tumor-bearing mice. B Photothermal heating curves. C The ex vivo fluorescence images of tumors and major organs. Fig S9. Representative H&E staining images of heart, liver, spleen, lung, and kidney. Fig S10. Change of body weights of mice after different treatments; Serum biochemistry indicators. Fig S11. A Tumor volume. B-D cytokine levels of TNF-α, INF-γ and IL-12 in sera from mice. Fig S12. Change of body weights of mice after different treatments; Serum biochemistry indicators. Fig S13. Representative H&E staining images of heart, liver, spleen, and kidney. Fig S14. Tumor volume of A primary and (B) distant tumors of each group. C the images of tumors. Fig S15. Representative immunofluorescence staining for CD8a (green) and IFN-γ (red) of spleen sections. Fig S16. A-C cytokine levels of TNF-α, INF-γ and IL-12 in sera from mice. Fig S17. Representative H&E staining images. Fig S18. Change of body weights of mice after different treatments; Serum biochemistry indicators.


Proteus spp., Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli. The usage of prophylactic antibiotics for surgical procedures was high and varied between hospitals. Hospital B had significantly lower concordance to antibiotic therapeutic guidelines, resulting to a higher infection rate, compared with hospital A. ASHP adherence components were then further investigated, after which antibiotic dosing interval and injection time was found to be a significant predictor for positive bacterial growth based on logit–logistic regression. Further interventions and strategies to implement antibiotic stewardship is needed to improve antibiotic prescriptions and their use.Clean surgery without contamination does not require prophylactic antibiotics, but there are high-risk surgical procedures that may cause infection and/or involve vital organs such as the heart, brain, and lungs, and these indeed require the use of antibiotics. This study aimed to determine the quantity of antibiotic use based on the defined daily dose (DDD) per 100 bed days and the quality of antibiotic use based on the percentage of concordance with The American Society of Health-System Pharmacists (ASHP) therapeutic guidelines . This includes the profiles of surgical site infection (SSI) in surgical patients from January through June 2019. The study subjects were 487 surgical patients from two hospitals who met the inclusion criteria. There were 322 patients out of 487 patients who had a clean surgical procedure. Ceftriaxone (J01DD04) was the most used antibiotic, with a total DDD/100 bed days value in hospitals A and B, respectively: pre-surgery: 14.71, 77.65, during surgery: 22.57, 87.31, and postsurgery: 38.34, 93.65. In addition, 35% of antibiotics were given more than 120 min before incision. The lowest concordance to ASHP therapeutic guideline in hospital A (17.6%) and B (1.9%) was antibiotic selection. Two patients experienced SSI with bacterial growths of  Antimicrobial resistance threatens global health; antibiotic use in surgical specialties is an infection control indicator and may reflect the incidence of surgical site infection. In most developing countries, particularly Indonesia, antibiotics are given before the incision but also during the time from admission to discharge from the hospital which may result in higher chances of antimicrobial resistance. A new strategy is therefore needed to overcome this problem.Surgery is a medical procedure in the form of an incision made by a surgeon with the aim of preventing or treating an illness. In general, all surgical procedures can pose a risk of infection. Thus, antibiotic prophylaxis is needed to prevent postoperative infection. However, not all types of surgery require antibiotic prophylaxis. Clean surgery without contamination does not require prophylactic antibiotics unless there are high-risk procedures that may cause infection and/or involve vital organs such as the heart, brain, and lungs. Prophylactic antibiotics are generally recommended in some clean and all contaminated operations . In geneThe mismatch of antibiotic use with therapeutic guidelines is reported to be high in surgical ward , ICU 3,,3,4, andPrescribing antibiotics preoperatively can reduce the occurrence of wound infection. Whereas not following the guidelines, for example, by using different spectrum antibiotics or different tissue antibiotic concentrations at the start and during surgery, can reduce antibiotics’ effectiveness. Moreover, antibiotic selection pressure leads to antibiotic resistance. The incidence of SSI is significantly higher when prophylactic antibiotics are given postsurgery than when given presurgery with an odds ratio (OR) of 1.89 (95% CI 1.05–3.4). Prophylactic antibiotics more than 120 min preincision showed a higher prevalence of SSI than those within 120 min ) ,8. The uWorld Health Organization 2019) states that implementing antimicrobial stewardship by evaluating the use of antibiotics can optimize the use of antibiotics, encourage behavior change in prescribing antibiotics, improve quality of care and patient outcomes, save unnecessary health care costs, and reduce the emergence, selection, and further spread of antimicrobial resistance (AMR)  states t,12,13,14A total of 487 patients in both hospitals met the inclusion and exclusion criteria. Pre-, During, and Postsurgeries = 66.124, p < 0.001).Based on the recapitulation of antibiotic prophylaxis (DDD/100 bed-days) in hospitals A and B, respectively, between January and June 2019, the antibiotic use prophylaxis presurgery was 44.26 and 133.58; during surgery was 33.32 and 132.4; postsurgery was 66.65 and 148.68. Ceftriaxone had the highest DDD/100 patient days in the pre-, during, and postsurgery, with sequences of 14.71 and 77.65, 22.57 and 87.31, 38.34, and 93.65 . Overall2 = 283.62; p < 0.001) and dosing interval  = 4.165; p < 0.041) were significant predictors for infection cases in hospital B.Other than the route of administration, compatibility with ASHP guidelines was less than 50%. Order from highest to lowest conformity, the concordance to the ASHP guidelines was route of administration, injection given time, dosage, dosing interval, and selection . In geneProteus spp. and S. aureus, while the second patient had E. coli and S. epidermidis. Profiles of the types of bacteria that cause SSI are shown in Surgical site infection occurs related to surgical incision procedures within 90 days of follow-up. Only two patients experienced SSI in this study. Both patients experiencing postoperative SSI were identified after undergoing a third review in the first patient, and after the first review in the second patient. The results of the first patient’s pus culture showed the presence of Escherichia coli bacteria are still sensitive to several types of antibiotics, such as cefoxitin, chloramphenicol, fosfomycin, oxacillin, tetracycline, and vancomycin. Bacteria Proteus spp. were sensitive to antibiotics such as amoxicillin, amoxicillin–clavulanate, ampicillin, ciprofloxacin, cotrimoxazole, fosfomycin, norfloxacin, ofloxacin. Proteus spp. and had intermediate sensitivity to cefoxitin and chloramphenicol. Staphylococcus aureus bacteria were sensitive to chloramphenicol, oxacillin, and vancomycin and had intermediate sensitivity to amoxicillin, amoxicillin–clavulanate, and cefoxitin.The results of antibiotic sensitivity tests showed that The assessment of the quantity of antibiotic use was conducted by calculating DDD/100 bed-days based on the classification  that has been approved worldwide by the World Health Organization (WHO). DDD is defined as the use of antibiotics based on the average daily dose given to 70 kgs adult patients according to main therapeutic indications. However, DDD cannot provide information pertaining to the suitability of the actual daily dose prescription. DDD calculation is influenced by the amount of antibiotic use (grams) and the total length of stay (bed-days). The higher percentage of antibiotics use leads to the greater the DDD value .The calculation of DDD/100 bed-days between January and June 2019 was divided into pre-, during, and postsurgeries. Overall, the average of prophylactic antibiotic use in pre, during, and postsurgeries in hospital B was higher than in hospital A. Ceftriaxone is the most widely used antibiotic including surgery. Tolbert’s 2019 evaluative study on the use of ceftriaxone in tertiary hospitals in Tanzania showed 322 (51.1%) of 630 research samples used ceftriaxone antibiotics. A total of 44 samples (40.7%) received ceftriaxone for surgical prophylaxis purposes that were divided into 32 (72.7%) samples receiving antibiotics before surgery, 3 samples (6.8%) samples receiving ceftriaxone antibiotics during surgery, and 9 samples (20.5%) receiving antibiotics after surgery . Anotherp = 0.047). In clean-contaminated operations, the incidence of SSI in ceftriaxone was 4.6% and comparator 6.4%  [Prophylactic antibiotics can significantly reduce the risk of postoperative infections. A meta-analysis of 40 studies testing the effectiveness of ceftriaxone compared with the comparators as the surgical prophylaxis  showed that in clean operations, the incidence of SSI in ceftriaxone was 5.14%, and comparator 6.2%  .The average value of DDD/100 prophylactic antibiotic bed-days in this study is higher than other studies in one hospital in Indonesia but is still lower than that in the study of the use of prophylactic antibiotics in one hospital in Turkey. The ceftriaxone antibiotics dosage used in hospital B was 87 DDD/100 bed-days, which means there were 87 out of 100 patients who received ceftriaxone 1 DDD antibiotics per day. A primary study of surgical patients in the hospitals in Surabaya assessed the total use of ceftriaxone prophylactic antibiotics using the DDD/100 bed-days unit, and the total use of these antibiotics was 30–54 DDD/100 bed-days ,20. ThesAccording to Permenkes RI RI No. 2406/MENKES/PER/XII/2011 on General Guidelines for the Use of Antibiotics in 2011, the basis for selecting antibiotics should be drawn from the patterns of bacterial sensitivity to antibiotics, the clinical condition of patients, the selection priorities of first-line antibiotics or antibiotics with a narrow spectrum, cost, and adjusted according to the hospital diagnosis and therapy (PDT) Guidelines [Based on ASHP therapeutic guidelines, to ensure adequate plasma and tissue concentration during surgery, it is recommended that the drug to be given intravenously within one hour of an incision. In this study, more than 95% of patients received prophylactic antibiotics intravenously, but only around 50% in concordance with the preoperative dosage timing recommendation.Some antibiotics, such as vancomycin and fluoroquinolones, must be given 120 min before surgery due to the influence of the half-life and protein binding of each antibiotic ,23. Allep = 0.012). In addition, antibiotic resistance was observed in most types of bacteria in the surgical ward. One of these bacteria, Escherichia coli, was resistant to the antibiotics such as ceftriaxone, chloramphenicol, ciprofloxacin, and gentamicin [The incidence of SSI in surgery patients in hospital B from January through June 2019 was 2 out of 323 patients (0.62%). The SSI patients in this study both had ceftriaxone at intervals and doses of 2 × 1 g. The administration time was more than 2 h preincision. Mismatched timing of prophylactic antibiotics can cause SSI and the growth of resistant bacteria. Research conducted by Billoro in 2019 to evaluate the use of prophylactic antibiotics in 255 surgical patients in southern Ethiopia, using a prospective cohort study, showed that administration of surgical prophylactic antibiotics earlier than 1 h before the incision was associated with 20% of SSI cases, whereas administration within 1 h had a lower incidence, at 11.4%  [p = 0.012) [p = 0.002)). Our SSI patients were known to have a record of concomitant diabetes mellitus with preoperative blood sugar levels of 149 mg/dL and 138 mg/dL. Patients with blood sugar level that exceeded 100 mg/dL preincision had shown a 1.7 times greater risk of SSI [Extended preoperative waiting times for hospitalized patients increased the risks of SSI. Preoperative waiting time >7 days could increase the risk of SSI 2.48 times greater than preoperative waiting time <7 days (ARR = 2.48 (95% CI 1.28–4.79); = 0.007) . Both ou= 0.012) . Gachaba= 0.012)  found thk of SSI .The strength of this study is its large number of patients (n > 400) and follow-up. The reported cases of surgical site infection were not when the patient was in the hospital (inpatient) but during 30–60 days after the day of surgery when they returned to the outpatient clinic. The limitation of this study is that severe cases in ICU and those who died were not accounted for, and there were laboratory limitations, and therefore, the bacteria that cause SSI were not identified.A retrospective observational study in both hospitals was conducted by observing patients who underwent surgical procedures during hospitalization at a private hospital in Surabaya  and West Nusa Tenggara  in the period January–June 2019. Hospital A is a private hospital with 235 beds, whereas hospital B is a government hospital with 411 beds. Both hospitals are referral hospitals. Surgical procedures classifications were (1) clean surgery—surgery performed on an area without preoperative infection and without opening the tract , planned surgery, or primary skin closure with or without the use of closed drains, (2) clean-contaminated operations—operations performed on the ducts  or operations without significant contamination, and (3) contaminated surgery—surgery that opened the digestive tract, bile duct, urinary tract, airway to the oropharynx, or reproductive tract except for ovaries with gross spillage .Inclusion criteria included patients aged >18 years old who received prophylactic antibiotics, underwent surgery, and hospitalization in the surgical ward, including surgical wound control within <90 days postoperatively. The exclusion patient criteria were patients in the intensive care unit (ICU), patients who had an infection before surgery, those discharged “against medical advice” (AMA), and those who died.Slovin’s formula was used to calculate the appropriate sample size (n) from a population using the known population size (N) and the acceptable error value (e). In this study, simple random sampling was used from the population of admitted patients in the study period; N = 267 participants, e = 0.05; therefore, n = 160 participants hospital A; N = 1597 participants, e = 0.05; therefore, n = 320 participants hospital B.Slovin calculations used Equation (1) as follows:Antibiotic usage data were obtained from patient medical records and antibiotic use records compiled by pharmaceutical software, which stores all patient data. Data were collected from inpatient medical records, including diagnosis, surgery, length of hospitalization, drug name, dose, and the number of antibiotics received. The dataset was anonymized prior to analysis.In Indonesia, antibiotics can be prescribed to the patient at admission, during patient care, until the patient’s discharge, and usually two weeks after discharge (next outpatient consultation). In this study, antibiotic use classifications were pre-, during, and postsurgery. Presurgery represents antibiotics given before the day of surgery; antibiotics during surgery are given on the day of surgery (120 min preincision to <24 h after surgery), and antibiotics postsurgery are given >24 h after surgery.The obtained data were evaluated for antibiotic uses quantitatively and qualitatively. The quantitative evaluation used DDD per 100 bed-days, while the qualitative analysis was the percentage of antibiotic concordance to the American Society of Health-System Pharmacists (ASHP) Therapy Guidelines in (1) route of administration, (2) time, (3) type, (4) dosage, and (5) duration.Data on bacterial culture test results were obtained from clinical pathology laboratories, and prevention and control committees of infection to follow up regarding the bacteria that cause SSI in postsurgical patients. Patients with SSI were observed in the time they were in the hospital, and when they went to the doctor for review, until the patient was declared cured.All patient data were anonymized prior to analysis. Data analysis was performed descriptively in the form of an analysis of the quantity of prophylactic antibiotic use with the DDD/100 bed-days, which was calculated using the following formula:The percentage of compliance with guidelines of antibiotic use with the ASHP therapeutic guidelines was analyzed using the 2014 SIGN criteria as a quality analysis, including the sensitivity profile of the bacteria causing SSI.A generalized ANOVA model was also carried out on the aggregated DDD dataset to identify the overall difference between the two hospitals in this study. Z-test, in conjunction with Monte Carlo simulation, was also used to investigate which hospital was most adhering to ASHP guidelines. In addition, logit–logistic regression was utilized in this study to further investigate which ASHP adherence components that would be the significant predictor for reported infection cases. Participants’ demographics , comorbidities, and antibiotic usage were included as covariates in the model.The usage of prophylactic antibiotics for surgical procedures was high and varied between hospitals. The low concordance to antibiotic therapeutic guidelines suggested injudicious antibiotic use. Further surveillance is needed to identify the incidence of antibiotic-resistant bacteria in the future. It is highly recommended that surgeons abide by guideline recommendations, using only a single antibiotic dose for clean surgery. Further intervention and strategies in implementing antibiotic stewardship are needed to improve antibiotic prescribing and its use.


Thyroid hormone autoantibody (THAb) is a common antibody in autoimmune disease and can interfere with the detection of thyroid hormone (TH). There was no research reporting the prevalence of THAb in Chinese and the rate of THAb interfering with TH detection.We collected 114 patients with autoimmune thyroid disease (AITD) , 106 patients with nonthyroid autoimmune diseases (NTAID), and 120 healthy subjects. According to the presence or absence of thyroid antibodies, patients with NTAID were divided into two groups: NTAID‐AITD and NTAID groups. Radioimmunoprecipitation technique was used to detect THAb in all subjects. TH was detected on Abbot and Roche platforms in patients with positive THAb.p = 0.014; 34.2% vs. 71.3%, p < 0.01). Among total 98 patients with positive THAb, TH levels of 9 patients were falsely elevated (9.18%).The prevalence of THAb was 22.8% in Hashimoto's thyroiditis and 45.6% in Graves’ disease. The prevalence of THAb in AITD group was lower than that in NTAID or NTAID‐AITD groups (34.2% vs. 61.5%, The prevalence of THAb in AITD patients was lower than that in NTAID patients. Although THAb had a high frequency in various autoimmune diseases, the prevalence of THAb interfering with TH detection was only 9.18%. We firstly investigated THAb in Chinese patients, and we found the prevalence of THAb in AITD patients was lower than that in NTAID patients. Although THAb has a high frequency in various autoimmune diseases, the rate of THAb interfering with thyroid hormone detection was only 9.18%. Previous results of thyroid function showed that the thyroid‐stimulating hormone (TSH) and free thyroxine (FT4) levels were simultaneously higher than the upper limit of normal value in multiple periods. We suspected that there was interference in FT4 detection, because when we detected FT4 on another platform, the result was significantly different . THAb is the most common substance interfering with thyroid hormone detection. So, we suspected that THAb interfered with the detection of FT4. However, there was no report on THAb in China, and the normal value of THAb in Chinese was unknown.Collectively, the current study aimed to establish the reference value of THAb in the Chinese population, determine the prevalence of THAb in Chinese patients with AITD, and evaluate the rate of THAb interfering with thyroid hormone detection in patients with positive THAb.22.1We collected 114 patients with AITD who visited in the endocrine clinic of Shandong Provincial Hospital from 2019 to 2020 (57 cases for HT and 57 cases for GD), in which 76 patients were newly diagnosed and 38 patients were under treatment. The exclusion criteria included the following: NTAID, thyroid surgery, and malignancy. We recruited 106 NTAID patients with thyroid dysfunction or thyroid autoantibody positivity who were admitted to the Department of Rheumatology and Immunology, Shandong Provincial Hospital, in which 38 cases were systemic lupus erythematosus (SLE), 27 cases were rheumatoid arthritis (RA), 16 cases were primary Sjogren syndrome (pSS), and 25 cases were other collagenases. Patients who had a history of thyroid surgery or malignancy were excluded. At the same time, we collected 120 healthy subjects who made health examinations in Shandong Provincial Hospital : 45.28 (10.36) years). The exclusion criteria included the following: NTAID, thyroid disease, thyroid surgery, and malignancy. The study was approved by the medical ethics committee of Shandong Provincial Hospital, Jinan, Shandong, China.2.2AITD included HT and GD. Diagnosis of HT was based on TgAb and/or thyroperoxidase antibody (TPOAb) positivity. GD was defined by thyrotropin receptor antibody (TRAb) positivity and TSH below normal value. The diagnostic standards of SLE and RA were the American Rheumatology Association (ACR) 1997 revised SLE classification criteria2.3125I‐T3 or 125I‐T4  for 60 min at 23 C. Twenty microliters of this mixture was then incubated with 150 μl anti‐human IgM‐Agarose or Protein G, both prediluted 1:10 with saline containing bovine serum albumin (BSA) (Sigma) at a final concentration of 0.5%. After 24‐h incubation at 4℃, tubes were centrifuged at 2,000× g for 1 min and the supernatant was aspirated. Finally, the radioactivity of immunoprecipitation was detected. The percentage of THAb was equal to total radioactivity divided by immunoprecipitation radioactivity.THAb was detected by radioimmunoprecipitation technique described in previous researches,2.4Thyroid function and autoantibodies were detected by electrochemiluminescence . The reference ranges for FT4, free triiodothyronine (FT3), TSH, TgAb, and TPOAb are 12.0−22.0 pmol/L, 3.1−6.8 pmol/L, 0.27−4.20 µIU/ml, 0−115 IU/ml, and 0−34 IU/ml, respectively. We used the Architect immunoassay (Abbot Diagnostics) to re‐evaluate FT3 and FT4 for patients with positive THAb. The reference ranges for FT4 and FT3 in Abbot are 9.01−19.05 pmol/L, 2.43−6.01 pmol/L.2.5p < 0.05 indicated that the difference was statistically significant.Continuous variables were expressed as the mean (M) ± standard deviation (SD). The Kolmogorov‐Smirnov test was used to examine whether the data of THAb in the healthy obeyed Gaussian distribution. Categorical variables were expressed by percentages. Categorical variables were compared by the chi‐squared test and Fisher tests. Continuous variables were compared by t tests and Kruskal‐Wallis test. SPSS version 25 was applied in statistical analysis. 33.1T3‐IgG, T3‐IgM, T4‐IgG, and T4‐IgM in the healthy obeyed Gaussian distribution. We used M + 2.56SD to calculate the normal value of four types of THAb: T3‐IgG, 0–4.45%; T3‐IgM, 0–4.62%; T4‐IgG, 0–3.98%; and T4‐IgM, 0–4.20%.3.2p = 0.002). There was no significant difference in mean age between the NTAID‐AITD group and the NTAID group or between the AITD group and the NTAID group. The prevalence of THAb in AITD group was lower than that in NTAID or NTAID‐AITD groups . As to T3‐IgG, there was no significant difference among the three groups. AITD group had a lower frequency of T3‐IgM, T4‐IgG, and T4‐IgM than NTAID‐AITD group . Both NTAID and NTAID‐AITD groups had higher prevalence of T4‐IgM than AITD group . With the classification base on specificity thyroid hormone bound, both NTAID‐AITD and NTAID groups had a higher prevalence of T3‐T4Ab than AITD group . As to classification based on Ig type, both NTAID‐AITD and NTAID groups had a higher prevalence of IgG and IgM‐Ab than the AITD group . NTAID‐AITD group also had a higher prevalence of isolated IgM‐Ab than the AITD group . There was no significant difference in the distribution of THAb between NTAID‐AITD and NTAID groups . Both RA and pSS groups had a higher prevalence of isolated T3Ab than HT group . RA group had a higher prevalence of isolated T4Ab than HT group (p = 0.048). The prevalence of T3‐T4Ab was higher in SLE than that in HT (p = 0.012). RA and pSS groups had a higher prevalence of isolated IgM‐Ab than HT group . SLE group had a higher prevalence of IgG and IgM‐Ab than HT group (p < 0.001). GD group had a lower prevalence of T3‐T4Ab and IgG and IgM‐Ab than SLE group  and had a lower prevalence of overall THAb and isolated IgM‐Ab than pSS group   . One patient had interference in FT3 detection, and 8 patients had interference in FT4 detection. The patient with interference in FT3 detection had T4‐IgG. In patients with falsely elevated FT4, 3 patients had T4Ab, 4 patients had T3Ab, and 1 patient had T3‐T4Ab. There was no difference in the prevalence of thyroid hormone detection interference among different diseases (7) Table .4In our research, we calculated the normal value of THAb: 0–4.45% (T3‐IgG), 0–4.62% (T3‐IgM), 0–3.98% (T4‐IgG), and 0–4.20% (T4‐IgM) and determined that the prevalence of THAb was 22.8% in HT and 45.6% in GD. In 1997, Benvenga et al. firstly used radioimmunoprecipitation technique to detect THAb and obtained the normal value of THAb: 0–3.6% (T3‐IgG), 0–3.9% (T3‐IgM), 0–3.9% (T4‐IgG), and 0–3.4% (T4‐IgM).Although THAb can bind to thyroid hormone, thyroid hormone is a hapten and cannot directly elicit the production of antibody. In 1972, Yukio et al. reported that rabbits immunized with mild denatured thyroglobulin (Tg) produced antibodies that could bind to thyroid hormone in the gamma‐globulin region.THAb can interfere with thyroid hormone detection, which may cause laboratory results inconsistent with clinical manifestations, and lead to misdiagnosis of clinicians. Since THAb was first discovered in 1956,There were some strengths of this study. It was the first research that investigated THAb in the Chinese population. We selected 120 healthy subjects with a sex ratio of one to one and used a 99% reference interval to establish normal values, which made our reference values more credible. The normal value of THAb established by us could provide a reference for future research on THAb in China. The prevalence of THAb in SLE was described for the first time. Furthermore, we determined the rate of THAb interfering with thyroid hormone detection. However, this study also had some limitations. The sample size of patients in this study was small, and there might be selection bias. On the other hand, the use of glucocorticoids and immunosuppressants  was not collected.5The prevalence of THAb in the general population was lower than that in AITD patients, and the prevalence of THAb in AITD patients was lower than that in NTAID patients. Although THAb has a high frequency in various autoimmune diseases, the rate of THAb interfering with thyroid hormone detection was only 9.18%.