There is a constant need to maintain the quality of consumed food. In retrospect to the recent pandemic and other food-related problems, scientists have focused on the numbers of microorganisms that are present in different food items. As a result of changes in certain environmental factors such as temperature and humidity, there is a constant risk for the growth of harmful microorganisms, such as bacteria and fungi, in consumed food. This questions the edibility of the food items, and constant monitoring to avoid food poisoning-related diseases is required. Among the different nanomaterials used to develop sensors to detect microorganisms, graphene has been one of the primary materials due to its exceptional electromechanical properties. Graphene sensors are able to detect microorganisms in both a composite and non-composite manner, due to their excellent electrochemical characteristics such as their high aspect ratios, excellent charge transfer capacity and high electron mobility. The paper depicts the fabrication of some of these graphene-based sensors, and their utilization to detect bacteria, fungi and other microorganisms that are present in very small amounts in different food items. In addition to the classified manner of the graphene-based sensors, this paper also depicts some of the challenges that exist in current scenarios, and their possible remedies.  The inclusion of sensors in different aspects of the lives of human beings has helped improve the quality of life to a great extent. In earlier times, after the initial use of complementary metal-oxide semiconductor (CMOS) circuits  for sensAmong the different applications that the sensors have had a positive impact on, detecting food quality has been of great importance ,45,46. FA lot of research has been conducted on devising methodologies to be used by graphene-based sensors to detect microorganisms in food items. Some of the common techniques include potentiometric sensing , electroThe succeeding examples describe the fabrication process of some types of graphene sensors, and their subsequent utilization for detection purposes of microorganisms in very small amounts. Each of these sensors are formed using different microfabrication techniques, chosen on the basis of the processed materials and final dimensions of the sensors. The processed materials chosen to integrate with graphene to form the prototypes included various polymers and other enzymes that were selective to the target bacteria and other microorganisms present in the food items.Mohanty et al.  showcaseAnother study that highlighted the use of graphene-based sensors to detect microorganisms in food was shown in the research of Gouvêa et al. . These cIn a study conducted by Kotsilkov et al. , nanocomThe research carried out by Moustafa et al.  can be r−5 S/m in-plane and through-plane, respectively. The prototypes had additional advantages such as enhanced antioxidant activity and a mechanically reinforced chitosan matrix. Around 50% of rGO in the nanocomposites increased the tensile strength from 13 MPa to 27 MPa, and the Young’s Modulus from 0.47 GPa to 2.73 GPa. The resultant sensors displayed high tensile strength and decreased water solubility.Barra et al.  showcase2 dimension at 90 °C by treating the GO solutions with HI fumes. Finally, the PLA-graphene composite films were formed by cutting them into dimensions of 15 × 15 cm2, and then treating them with PVP solutions. The solution was prepared as a binder and the free-standing rGO films were delaminated from the composite films. Finally, the free-standing rGO films were placed between two PLA films and heat pressed at a temperature of 65 °C for half an hour. Two types of films, including PLA-rGO and PLA-GO films, were formed and deployed for sensing purposes. The sensors showed excellent permeability capabilities for water and air, as the water vapor permeability was reduced by up to 87.6% due to the synergy between the two processed materials of rGO and PLA. The air permeability of the prototypes also changed as the oxygen permeability was reduced, by a factor of two in both dry and humid conditions. The extensive and tortuous diffusion pathway of rGO allowed the nanocomposites to be formed with excellent processability.Another interesting study in this area is reference , where s−1. The presence of GO in 0.1 wt. % in the composite increased the initial tensile strength from 22.7 ± 1.2 to 6471.6 ± 1775.5 MPa. As the GO content was increased, there was a subsequent increase in the endothermal peak during thermal studies. It also increased the cross-linking degree between the film and water interaction. The nanocomposites were examined for antimicrobial properties using E. Coli K-12 MG 1655 and B. subtillis 102. The composite of 0.6 wt. % showed prime inactivation, which increased by 22.83% for E. Coli and 54.93% for B. subtillis.Grande et al.  investigManikandan et al.  synthesiNaskar et al.  also repHuang et al.  explaineAnother study explaining the use of graphene was highlighted in , where c2) substrates having a thickness of 300 nm. The electrodes were developed using chromium and gold, with 15 nm and 90 nm thicknesses, respectively. The final part included the formation of a monolayer of graphene using a chemical vapor deposition (CVD) process. The repeatability of these G-FETs was achieved using a photolithography process to form samples of 100 × 100 µm. The patterning of graphene was carried out using a PMMA-assisted transfer process. This was executed via chemical treatment, spin-coating, curing and UV-light treatment. The interaction between graphene and the volatile molecules occurred due to the latter’s absorption on the graphene surface via π-π stacking. The sensors could detect indole for a concentration ranging between 10 ppb and 250 ppb with an LOD of 10 ppb.Another study that highlighted the use of graphene for food-related devices was in the research carried out by Lin et al. . GraphenEscherichia coli (E. Coli) through the stemming of anti-E. Coli antibody-coated sensing surfaces. E. Coli in food poisoning, carried out using synthesized anti-E. Coli, followed by using it as a coating on the selective layer on the sensing area of the prototypes.Another study that involved graphene to develop sensors for detecting microorganisms in food is reference . These sE. Coli antibodies. The sample was tested via injection-and-stop and constant injection modes, and showed a resistance change when E. Coli binded to the electrode surface. It was seen that the resistance values increased with the bacterial concentration, with the sensors obtaining an LOD of 12 cfu/mL. The change in resistance can be inferred from the formation of a kinetic barrier for the electron transfer process when the bacteria-antibody conjugation process took place on the surface of the graphene film. This led to intercellular bioactivity on the sensing surface and, in turn, polarizing of the charges on the cell’s surface. As a result, there was an increase in the formation of electron clouds due to the steric hindrance, resulting in a change in the carrier hole density. The resistance study was carried out from 0 to 50 min with increments of 5 min, and showed a logarithmic increase in resistance with an E. Coli concentration that was enhanced from 2.4 × 5 to 2.4 × 56 cfu mL−1.Antibody-coated graphene sensors were used for detection purposes, with the substrate being coated on the two-electrode assembly with a gold/palladium alloy target. Electrical measurement of the resistance values was conducted with respect to the bacterial concentration. This was followed by creating a polydimethylsiloxane (PDMS) microchannel using soft lithography, maintaining the dimensions at 800 µm × 8 mm × 1 mm, perfectly suitable for low-cost technological applications. After rinsing the PDMS microchannels with deionized (DI) water, the sample was incubated with 15 μL of streptavidin, followed by washing it with buffer and DI water and treating it with biotinylated anti-Maskey et al.  synthesiBased on the reviewed examples and some other studies, This paper presented a substantial review of the fabrication of graphene-based sensors, and their utilization as electrochemical sensors. These prototypes were used to detect different chemicals in food. The graphene sensors were fabricated in composite and non-composite forms. The former involves the inclusion of specific nanomaterials and polymers to improve the sensors’ overall sensitivity and mechanical integrity. Apart from detecting the target analyte, some other advantages of these sensors include their wide operating range, low LOD and biocompatible nature. Microorganisms have been detected in very small amounts due to the excellent morphological characteristics of these sensors, such as their high surface area and superior carrier mobility of graphene. One of the requirements of these sensors would be to detect multiple analytes with equal efficiencies. Different polymers, such as PDMS, poly (lactic) acid and others, have been considered to form composites and substrates due to their excellent capability of forming interfacial bonding with graphene allotropes. The sizes of the sensors also vary based on certain factors such as the size of the target analyte, the working mechanism of the prototypes, and the alignment of graphene in the sensing area.While a lot of research has been carried out on developing graphene-based sensors to detect various bacterial samples, some research areas are still ongoing. The production of some of these sensors is on a limited scale and are being tested in controlled environments; their ubiquitous utilization in real-time scenarios would help to minimize food wastage to a great extent. Sensors being tested for a single target should opt for more microorganisms that can affect food. This is because when the food samples are transferred in real-time scenarios between the storage unit and superstores, there is a high probability for the growth of microorganisms over a period. The types of wireless communicational protocols associated with graphene-based sensing devices should be increased and optimized on the bases of the maximum and minimum transmission rates, bandwidth and network size. The types of food samples that can be kept safe and edible in different airtight containers integrated with these graphene-based sensors should also be increased to decrease overall food wastage. The temperature and humidity specifications for the sensors should be further optimized to determine the correlation between the detected microorganisms and available food storage facilities. Implementing some of these propositions effectively will benefit mankind regarding production pressure and food hunger.


The Ni–Fe–graphene composite coating shows an increment of 393.0% in hardness and a decrement of 55.9% in friction coefficient in comparison with 7075 aluminum alloy substrate. The Ni–Fe–graphene composite coating binds tightly to 7075 aluminum alloy with adhesion strength of higher than 6.895 MPa. These make contributions to provide effective protection for aluminum alloys. Surface morphology and corrosion morphology, as well as morphology of the side bound to the substrate, were characterized. The scattered asperities on the surface were proven to be graphene nanoplatelets being wrapped by Ni–Fe, which comprehensively reveals the formation of asperities.The process parameters of electrodeposited Ni–Fe–graphene composite coating on the 7075 aluminum alloy were optimized by the orthogonal experiment. The optimized process parameters were determined as follows: graphene concentration of 1 g L Aluminum and its alloys are extensively applied to aeronautical and aerospace, automobile, and vessel domains because of their merits, including high specific strength, superior electrical and thermal conductivity, and low cost. However, as the demands for materials possessing low friction, high strength, and lightweight continue to escalate, the weak spots of low surface hardness and poor wear resistance for aluminum alloys are not only increasingly prominent, but also limit their application range, and even lead to huge economic loss due to wear failure. Therefore, it is very crucial for aluminum and its alloys to improve surface hardness and reduce friction and wear, utilizing surface modification and reinforcement methods.2, TiN, WC, Cr2O3, AlN, and MWCNT is beneficial to increase hardness and decrease friction coefficient. The properties of different Ni–Fe composite coatings are listed in Electrodeposited Ni–Fe alloy coating has been extensively applied to electronic sensors and micromechanical systems due to its merits such as homogenous structure, eminent magnetic properties , corrosiGraphene, an atom-thick of carbon, has extraordinary mechanical property, including elasticity modulus of 0.5~1.0 Tpa , tensileUp to now, there have been quite limited reports about electrodeposited Ni–Fe–graphene composite coating on the 7075 aluminum alloy and process optimization. In this work, the process parameters of Ni–Fe–graphene composite coatings are optimized by orthogonal design. The morphology, structure, and adhesive strength are investigated as well. 2SO4 to further remove impurities and then were ultrasonic-cleaned in deionized water again.An aluminum alloy (#7075) plate with size of 25 × 25 × 3 mm was used as a cathode for coatings deposition, and a pure nickel plate was used as an anode. The cathode and anode were positioned parallel at an angle of 45 degrees with the plating solution level. The aluminum alloy substrates were polished with 600# emery paper to remove the surface scratches and obtain a uniform surface, and then were sandblasted using 280 mesh glass beads under pressure of about 0.15 MPa to eliminate the oxidation film and increase the coating’s adhesion. Subsequently, the samples were cleaned ultrasonically and chemically treated for a few minutes in 3%NaOH and 3%H4) is shown in In this study, graphene concentration, current density, agitation speed, and temperature were sieved by the orthogonal designing method, and the orthogonal test table of L16 , were about 4~20 nm in thickness and 5~10 μm in diameter. An SEM image of the graphene nanoplatelets is shown in Prior to electrodeposition, in order to ensure the dispersion of graphene in the electrolyte, the bath solution was stirred for 30 min using an ultrasonic emulsification distributor and subsequently stirred mechanically for about 1 h. To maintain the suspension of graphene nanoplatelets in the electrolyte during the deposition process, mechanical agitation was kept constant. After the deposition, samples were cleaned to remove the residual solution, then dried, and then measured. The adhesion strength between the Ni–Fe–graphene composite coating and the aluminum alloy substrate was evaluated by the Elcometer model 106 adhesion tester . It employs the pull-off method to measure the lift-off force required to pull a small area of coating away from the base material. First, the surface of the dolly and Ni–Fe–graphene composite coatings were roughened with 1200 mesh abrasive paper, then degreased in absolute ethyl alcohol by ultrasonic cleaning. E-44 epoxy was mixed with low-molecular-weight 650 polyamide resin in a mass ratio of 1:1. The mixture, namely, adhesive, was applied as an even film to the surface of dolly. The dolly was placed onto the surface of the Ni–Fe–graphene composite coating and pressure was applied to squeeze out the excess adhesive being removed. Then, we placed them in the drying oven with a temperature of 60 °C for 3 h. After curing, the support ring was placed over the dolly, ensuring it lay flat. The pull-off force was applied to the test dolly until the dolly was removed from the surface. The pull-off force was recorded by means of a dragging indicator on an engraved scale. Three samples were prepared under the same process parameters and were measured.The morphology and element composition were characterized using a scanning electron microscope  equipped with energy-dispersive spectroscopy (EDS). −1, keeping the applied normal load  of 1.0 N for 30 min. The test was repeated three times for each material under the same test conditions. The Vickers hardness of the Ni–Fe–graphene composite coatings was examined using a microhardness tester  with a load of 100 g applied for 10 s. Five data points were averaged for each sample. All coatings and substrate suffered from friction testing on a ball-on-disk tribometer  under dry air environment at room temperature. The counterpart ball for wear test was a GCr15 steel ball with diameter of 3 mm. The test was carried out at the sliding velocity of 0.1 m s−1, current density of 9 A dm−2, agitation speed of 250 r min−1, and temperature of 60 °C, corresponding to experiment No. 4 in −1, current density of 5 A dm−2, agitation speed of 100 r min−1, and temperature of 60 °C, corresponding to experiment No. 6 in The process parameters of the Ni–Fe–graphene composite coatings were optimized according to the hardness and friction coefficient (COF). The orthogonal test results are shown in In addition to the process parameters’ optimization by analyzing orthogonal test results visually, range analysis was conducted on the basis of orthogonal test results, and the range analysis results are shown in 1, A2, A3, and A4 are responding to the average hardness of the same level under different factors . B is representative of COF, and B1, B2, B3, and B4 are responding to the average COF of the same level under different factors . Ri is representative of hardness range and Rj is representative of COF range. The influence degree of different factors on properties is confirmed and the higher the value of the range, the greater the degree of influence. A is representative of hardness, and AThe influence degree on the hardness of the Ni–Fe–graphene composite coating of different factors, from the highest to the lowest, is graphene concentration, current density, agitation speed, and temperature. The optimized parameters according to the average hardness under the different levels are responding to experiment No. 4, which agrees with the optimized parameters by analyzing orthogonal test results visually. −1, current density of 9 A dm−2, agitation speed of 100 r min−1, and temperature of 40 °C, which is different from any experiment in However, the influence degree on the COF of the Ni–Fe–graphene composite coating of different factors, from the highest to the lowest, is current density, agitation speed, graphene concentration, and temperature. The optimized parameters according to the average COF under the different levels are graphene concentration of 3 g L−1, current density of 9 A dm−2, agitation speed of 250 r min−1, and temperature of 60 °C. The hardness and friction coefficients of the Ni–Fe–graphene composite coating prepared under these parameters are 912.6 HV and 0.1990, respectively. In comparison with 7075 aluminum alloy substrate, the Ni–Fe–graphene composite coating shows an increment of 393.0% in hardness and a decrement of 55.9% in friction coefficient.As a result, the optimized process parameters are graphene concentration of 1 g LThe surface morphology and element distribution of Ni–Fe–graphene composite coatings are shown in The element compositions of the Ni–Fe–graphene composite coatings from EDS analysis are shown in 3. The corrosion morphology is presented in In order to further prove the asperities being scattered on the surface, the Ni–Fe–graphene composite coatings were corroded in 30 vol% HNO2+ and Fe2+ ions are reduced preferentially on the GNPs after GNPs are deposited on the cathode, which realize the incorporation of GNPs into the composite coating. Due to excellent electrical conductivity and the large specific surface area of graphene, the deposition rate of metal ions on the GNP surface exceeds that on the reduced Ni–Fe alloy, which modifies the surface morphology and induces the formation of asperities by offering more active sites for metal atoms [2+ and Fe2+ ions continue to be reduced on the surface as electrodeposition proceeds, which generates a layer structure on the wall of holes. The results reveal the formation of asperities and electrodeposited graphene composite coatings. Nial atoms ,34,35. WDollies were separated from between the Ni–Fe–graphene composite coating and the substrate. The separated area is equivalent to 30%, as shown in −1, current density of 9 A dm−2, agitation speed of 250 r min−1, and temperature of 60 °C. The Ni–Fe–graphene composite coating shows an increment of 393.0% in hardness and a decrement of 55.9% in friction coefficient in comparison with the 7075 aluminum alloy substrate. The Ni–Fe–graphene composite coating binds tightly to the 7075 aluminum alloy and the adhesion strength is higher than 6.895 MPa. The incorporation of graphene modifies the surface morphology and induces the formation of asperities. These asperities are graphene being wrapped completely or incompletely, with Ni–Fe alloy being verified by surface morphology, composition, and corrosion surface morphology. Simultaneously, layered structures in the corrosion holes clarify the formation of electrodeposited graphene reinforced metal matrix composite coating.A Ni–Fe–graphene composite coating was prepared on the 7075 aluminum alloy using the electrodeposition technique, and the process parameters were optimized by the orthogonal experiment. The optimum process parameters are graphene concentration of 1 g L


The combined score of PD-L1-positive cells including tumor cells and tumor-associated macrophages (TAMs) was significantly associated with preoperative plasma sPD-L1 levels. In patients with high levels of preoperative plasma sPD-L1, the probability of 5-year RFS was significantly poor for patients with low PD-L1 expression intensity of tumor cells (tcPD-L1) compared with those with high tcPD-L1 . In former group, PD-L1-positive TAMs were markedly infiltrating compared with those from latter group . In NSCLC, plasma sPD-L1 can reflect the accumulation of PD-L1-posotive TAMs, not just PD-L1-positive tumor cells. In patients with high levels of preoperative plasma sPD-L1, the prognoses after surgery depends on which PD-L1-positive cells, tumor cells or TAMs, are the primary source of the sPD-L1. Thus, measuring both plasma sPD-L1 levels and PD-L1 expression status of tumor cells and TAMs is of benefit for assessment of postoperative prognosis in operable NSCLC.Programmed cell death-ligand 1 (PD-L1) on tumor cells can be degraded to soluble form (sPD-L1) and enter circulation, however, the clinical significances of sPD-L1 in peripheral blood remains to be elucidated in non-small-cell lung cancer (NSCLC). We monitored plasma sPD-L1 levels during perioperative periods and evaluated PD-L1-positive cells in tumor tissues in patients with operable NSCLC. Then the correlation between preoperative plasma sPD-L1 levels and relapse-free survival (RFS) was analyzed retrospectively. In patients who underwent radical surgery (The online version contains supplementary material available at 10.1007/s00262-023-03527-y. Since the emergence of immune checkpoint inhibitors as one of the standard therapeutics for non-small cell lung cancer (NSCLC) –4, thereIn patients with NSCLC, measuring the percentage of PD-L1-positive cancer cells in cancer tissues by immunohistochemistry (IHC) is useful for predicting the efficacy and application of anti-PD-1 and -PD-L1 therapeutic antibodies –9. In thPrevious studies reported that soluble PD-L1 (sPD-L1) could be detected in the peripheral blood of NSCLC patients –20, howeTo reveal the biological and clinical significances of sPD-L1 in peripheral blood and answer these questions, in this study, we measured sPD-L1 levels in peripheral blood in settings that were different from previous studies. We compared plasma sPD-L1 levels before and after surgery in patients with NSCLC, meaning a comparison of sPD-L1 levels in the presence and absence of tumors. Additionally, we investigated association of preoperative plasma sPD-L1 with RFS after surgery, and explored the PD-L1-expressing cell type that could influence the prognosis of patients.In total, 69 patients who underwent surgery for pulmonary tumors at Shiga University of Medical Science Hospital between November 2013 and October 2015 were enrolled in this study. Among them, 63 patients were pathologically diagnosed with NSCLC . The 75th percentile value in the investigated cohort was used as the cutoff value, and a high level of plasma sPD-L1 was defined as cases where the level was higher than the cutoff.Whole tumor Sects. (4-μm-thick) of formalin-fixed paraffin-embedded tissue were deparaffinized in xylene and rehydrated in ethanol and distilled water. Antigen retrieval was performed by microwaving sections in Universal HIER antigen retrieval reagent (Abcam) for 20 min. Endogenous peroxidase activity was blocked by treatment with Peroxidase Block  for 10 min, and non-specific binding was blocked by treatment with Protein Block serum-free Ready-to-use (DAKO) for 10 min at room temperature. The sections were then incubated overnight at 4 °C with anti-human PD-L1 monoclonal antibody . The sections were then incubated with Rabbit-specific IHC polymer detection kit and visualized using DAB substrate (both Abcam). Finally, sections were counterstained with hematoxylin. For negative control staining, the anti-PD-L1 primary antibody was replaced with a rabbit IgG monoclonal antibody (Abcam).To evaluate the PD-L1 expression status of NSCLC cells following IHC staining, we used a semi-quantitative scoring method that reflects both the intensity and extent of PD-L1 expression of tumor cells and is expressed as the PD-L1 expression score (H-score), as described previously . BrieflyTo evaluate the cell density of PD-L1-positive TAMs, the total number of PD-L1-positive TAMs was counted in three randomly selected fields under 400 × magnification, and the cell density of PD-L1-positive TAMs was calculated for each field of view by dividing the cell counts by the area of the field of view. Sections were independently examined by two researchers, including a pathologist, and the average of the cell densities in each field of view was calculated.Human CD14-positive peripheral blood mononuclear cells (PBMCs)  were cultured in AIM-V medium  in the presence of granulocyte macrophage colony-stimulating factor for Type 1 (M1) and macrophage colony-stimulating factor for Type 2 (M2) macrophages  for 6 d. Then, the cells were stimulated with 1 μg/mL lipopolysaccharide for 24 h to generate activated M1 and M2 cells. The culture supernatants were collected, and levels of sPD-L1 were measured by ELISA as described above. The expression levels of specific markers for each type of macrophage were evaluated by flow-cytometry using a BD FACS Calibur, and the data were analyzed using BD CellQuest Pro software . Antibodies used for detection were PE mouse anti-CD80  and anti-CD229 (interferon-gamma receptor)  for M1, PE mouse anti-CD163  and anti-CD206  for M2, and PE mouse anti-PD-L1 antibody  for PD-L1.U test, and comparisons between more than three groups were analyzed using ANOVA. Pearson’s product-moment correlation coefficient was used to analyze the correlation between two continuous variables. Relapse-free survival (RFS) after surgery was calculated using Kaplan–Meier analysis and was compared with the log-rank test. All analyses were performed using SPSS Statistics 25.0 software .For continuous variables, comparisons between two groups were analyzed using the Mann–Whitney R = 0.0071, P = 0.293)  , and the median plasma sPD-L1 level was 63.6 pg/mL, with a range of 0.0 to 204.6 pg/mL. Using the H-score as a semi-quantitative evaluation of the expression intensity of PD-L1 on tumor cells (tcPD-L1), the median tcPD-L1 expression score was 97.1, with a range of 0.0 to 244.9. Then, we analyzed the correlation between preoperative plasma sPD-L1 levels and tcPD-L1 expression score. However, a significant correlation was not observed between two  and in smokers compared with never smokers . However, sPD-L1 levels were not associated with age , tumor size , pathological type , postoperative pathological stage , tumor cell grade , lymphatic invasion , or microvessel invasion of tumor cells .Next, we analyzed correlations between preoperative plasma sPD-L1 levels and clinicopathological factors . The data demonstrated that preoperative plasma sPD-L1 levels were significantly higher in male compared with female patients (median: 65.7 n = 61) (Supplementary Table 1). The median preoperative serum sPD-L1 level was 63.4 pg/mL, and 1 month after surgery, the median sPD-L1 level (n = 55) had significantly increased to 72.2 pg/mL (P < 0.001)  had significantly decreased to the preoperative level     were positive for PD-L1, especially those that had gathered in specific regions Fig. a. On then = 55) (Supplementary Table 2) were classified into two groups according to preoperative plasma sPD-L1 levels: the high sPD-L1 group (≥ 80 pg/mL) and the low sPD-L1 group (< 80 pg/mL), as described earlier. Kaplan–Meier analysis revealed that postoperative RFS tended to be shorter in the high sPD-L1 group (n = 18) than in the low sPD-L1 group (n = 37), which had 5-year relapse-free probabilities of 58.8% and 67.9%, respectively   than for the low tcPD-L1 expression group (n = 38), which had 5-year relapse-free probabilities of 76.5% and 59.3%, respectively   and the low tcPD-L1 expression group (< 150), as in a previous report . Kaplan–2) and the low PD-L1-positive TAMs group (< 150/ mm2) (Area under the curve = 1.000). Kaplan–Meier analysis revealed that postoperative RFS tended to be longer for the low PD-L1-positive TAMs group (n = 30) than for the high PD-L1-positive TAMs group (n = 25), which had 5-year relapse-free probabilities of 72.8% and 54.5%, respectively   compared with those in the high sPD-L1–high tcPD-L1 group (n = 8), which had 5-year relapse-free probabilities of 33.3% and 87.5%, respectively  , the cell density of PD-L1-positive TAMs was significantly high compared with that in the high sPD-L1–high tcPD-L1 group (n = 8)    and 2 (M2) macrophages were induced from human PBMCs, and these cells were confirmed to express PD-L1 . ELISA demonstrated that both M1 and M2 cells released sPD-L1 into the culture supernatant, and the levels significantly increased following activation with lipopolysaccharide , which are a dominant cell type in the stroma, can express PD-L1 following stimulation by interferon-gamma (IFN-γ) and that PD-L1-positive CAFs are present in NSCLC tissues . In thatIn addition to the data that plasma sPD-L1 levels were significantly elevated 1 month after surgery, we found no correlation between preoperative plasma sPD-L1 levels and tcPD-L1expression intensity, which is consistent with the results of previous reports . In thisNext, we investigated the clinical significance of sPD-L1 in peripheral blood. Previous papers have reported that high sPD-L1 levels in peripheral blood are associated with poor prognosis in patients with advanced NSCLC (reviewed in ref. 24). In addition, in our study, patients with high preoperative plasma sPD-L1 levels tended to have shorter RFS after surgery. However, when the high sPD-L1 group was further classified into two subgroups by tcPD-L1 expression intensity, it was found that prognosis was significantly different between the subgroups with high and low tcPD-L1 expression. These data demonstrated that RFS after surgery was significantly shorter in the group with high plasma sPD-L1 and low tcPD-L1 expression compared with the group with high plasma sPD-L1 and high tcPD-L1 expression. This may depend on which cells, tumor cells or TAMs, are the primary source of the sPD-L1 in peripheral blood.We have previously reported that high tcPD-L1 expression is a good prognostic biomarker for patients with early-stage NSCLC . This isWe suggested that PD-L1-positive TAMs contributed to the elevated plasma sPD-L1 levels and poor prognosis. However, there are several limitations to this study. First, the number of PD-L1-positive TAMs in tumor tissues was assessed by their density in the tissue. The sPD-L1 level in peripheral blood can be affected by the product of the cell density of PD-L1-positive TAMs and tumor volume; we did not consider tumor volume. Although we examined correlations between plasma sPD-L1 levels and the density of PD-L1-positive TAMs in combination with maximum tumor diameter, no clear data were obtained (data not shown). Second, as mentioned earlier, there are other PD-L1-positive cells in the tumor stroma , 23, andIn conclusion, sPD-L1 levels in peripheral blood were significantly elevated 1 month after surgery compared with preoperative levels despite the absence of tumors. Among patients with high levels of preoperative plasma sPD-L1, those with low tcPD-L1 expression had significantly worse postoperative prognoses than those with high tcPD-L1 expression. In the former group, the cell density of PD-L1-positive TAMs was significantly higher, and suppression of antitumor immune responses by TAMs may be the cause of poor prognosis. The sPD-L1 in peripheral blood can be derived from PD-L1-positive TAMs, not just PD-L1-positive tumor cells. Thus, in patients with operable NSCLC, measuring both plasma sPD-L1 levels and PD-L1 expression status of tumor cells and TMAs is of benefit for assessment of postoperative prognosis.Supplementary file1 (PPTX 96 kb)Supplementary file2 (PPTX 71 kb)Supplementary file3 (XLSX 10 kb)Supplementary file4 (XLSX 10 kb)Below is the link to the electronic supplementary material.


Background: Radiation-induced dermatitis (RID) is one of the most prevalent side-effects of conventional cancer therapies; however, there is no standard treatment for its prevention. Therefore, this study aimed to evaluate the comparative efficacy and safety of Jaungo (mainly composed of Lithospermum erythrorhizon Siebold & Zucc. and Angelica sinensis (Oliv.) Diel) and the water-in-oil-type non-steroidal moisturizer for the prevention of RID in patients with breast cancer (BC).Methods: This is a prospective, single-blind, pilot randomized controlled trial. Between March 2021 and July 2022, 50 patients were randomly selected to use Jaungo or the moisturizer while undergoing postoperative radiation therapy (RT). Assessments were conducted nine times—every week while the patients underwent RT and 2 weeks after the end of therapy. The primary outcome was the incidence rate of RID grade ≥2. The secondary outcomes were the incidence rate of maximum grade RID, time to RID onset, RID-related quality of life (QOL) score, pain intensity, and adverse events.Results: The incidence rate of RID grade ≥2 was 24.0% and 20.0% in the Jaungo and the moisturizer groups, respectively, with no significant difference between the groups (p = 0.733). Regarding the secondary outcomes, the incidence rate of maximum grade RID (p = 0.890), mean time to RID onset (p = 0.092), cumulative incidence rate of RID (p = 0.280), RID-related QOL score, and maximum pain intensity (p = 0.844) also did not differ significantly between the groups. None of the subjects in either group experienced severe adverse effects, although one participant in the moisturizer group had mild fever and insomnia.Conclusion: These findings suggest that Jaungo has preventive efficacy without severe side-effects against RID in patients with BC that is comparable to that of the water-in-oil type non-steroidal moisturizer. Further extensive randomized controlled trials with larger sample sizes are warranted to validate our findings.Clinical Trial Registration: Clinical Research Information Service (CRIS), https://cris.nih.go.kr, identifier KCT0005971. Radiation therapy (RT) is one of the most common conventional therapies for patients with cancer . More thSeveral studies have been conducted on possible strategies for reducing the occurrence of RID after RT in patients with BC; however, no standard preventive treatment for RID has been clearly demonstrated to be effective . TherefoA water-in-oil type non-steroidal moisturizer cream containing hyaluronic acid as the main component showed significant efficacy in preventing and reducing RID compared to an emollient base cream in terms of ameliorating skin toxicity, burning sensations, and erythema in the irradiated area in patients with BC . It was Lithospermum erythrorhizon Siebold & Zucc.  and Angelica sinensis (Oliv.) Diels [Apiaceae/Umbelliferae; Angelica sinensis), is registered for use as a topical drug for dermatitis in Korea. The symptoms approved by the Korean Ministry of Food and Drug Safety for the use of Jaungo are rough skin, frostbite, heat rash, anus laceration, and skin inflammation. It is commonly used to treat skin injuries due to abrasions, frost, or burns. It is also widely used in clinical practice to prevent RID in cancer patients (Jaungo (Shiunko in Chinese and Japanese), a traditional herbal ointment mainly composed of patients . Severalpatients . A priorpatients . Case sepatients . HoweverConsidering the limited options for preventing RID and the results of previous studies, we designed this prospective, randomized, single-blind pilot study (CRIS registration number KCT0005971) to compare two topical agents commonly used for RID—Jaungo and the water-in-oil type non-steroidal moisturizer. This is the first randomized controlled trial (RCT) to evaluate the preventive efficacy of topically applied Jaungo and compare it with that of the water-in-oil type non-steroidal moisturizer in RID patients with BC.The patient eligibility criteria were as follows: pathologically confirmed invasive BC, having undergone breast-conserving surgery, and adjuvant RT planned after surgery. Patients with a history of RT for any reason, unhealed scars in the breasts, soft tissue disease, or uncontrolled diabetes mellitus with HbA1c ≥ 6.5% were excluded. Patients with a metastatic cancer stage were also excluded. This study was approved by the Institutional Review Board (IRB) of Kyung Hee University Hospital at Gangdong, Seoul, Republic of Korea (IRB number KHNMCOH-2020-12-001-002).Between March 2021 and July 2022, 50 patients were prospectively enrolled and randomly allocated to the Jaungo group or the moisturizer group based on random numbers generated using a 2 × 2 randomized permuted block design by an independent statistician. The sample size was estimated based on the grade of Radiation Therapy Oncology Group (RTOG)  and NatiL. erythrorhizon, Angelica sinensis, sesame oil, beeswax, and lard. The detailed compositions and the quality of control of Jaungo are presented in the ®, Pharmbio Korea Corporation, Seoul, Republic of Korea; expiration date 04.23.2023) in the same manner. Owing to the possibility of bolus effect, neither agent was to be applied within 4 h after an RT session, and both were cleaned off from the irradiated region immediately before each session. Jaungo and the moisturizer were contained in individually packaged tubes, and each tube was stored at room temperature between 1 and 30° in an airtight container. If skin irritation, a local rash, or itching occurred, the application of the agents was stopped by the assessors. No other prophylactic topical agents for RID were allowed to be used in either group, but topical and intravenous antibiotics were allowed as rescue medicine.Participants allocated to the intervention group, i.e., the Jaungo group, were instructed to apply Jaungo . Jaungo is an external ointment consisting of 10) ≥ 60 Gy according to the National and International Guidelines. For participants who had risk factors for regional recurrence, such lymphovascular or axillary lymph node invasion, regional lymph node irradiation was also performed.All subjects underwent 3-dimensional conformal RT (3D-CRT) or intensity-modulated RT (IMRT) for 5–7 weeks with five fractions each week, and the tumor bed was treated with a biological equivalent dose  performance status, final educational background, disease history, smoking and diabetes mellitus status, was collected. Information on clinical characteristics, such as breast volume, irradiation side (right or left), histology results and malignant tumor cell molecular subtype, cancer stage based on TNM staging, lymph node irradiation (Yes/No), and concurrent chemotherapy (Yes/No) was also recorded. For the assessment of outcomes, the severity and date of onset of RID, QOL, skin reaction symptoms, and pain intensity, including that of maximum pain related to RID, were evaluated based on the scale of Radiation Therapy Oncology Group (RTOG) criteria . OutcomeThe incidence rate of RID of RTOG grade ≥2 was evaluated as the primary outcome in this study. As secondary outcomes, the time to RID onset, incidence rate of maximum grade RID, CSSP, Skindex-29, and NRS scores, and adverse events (AEs) based on the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 were assessed at the start (visit 1) and end (visit 8) of RT and 2 weeks later (visit 9) to compare between the Jaungo and moisturizer groups .t-test or Mann–Whitney U test. The types, frequencies, and severity of AEs were also assessed; events that were determined to be caused by each intervention are separately analyzed and reported. All analyses were performed using SPSS version 25.0  based on the intention-to-treat (ITT) set. All tests were two-sided, and differences with a p value <0.05 were considered statistically significant.The characteristics of all participants are reported as mean and 95% confidence interval (95% CI) values for continuous variables or frequencies and percentages for categorical variables. The difference in incidence rate of RID of RTOG grade ≥2, the primary outcome, between the two groups was assessed using the Chi-square test. Among the secondary outcomes, the difference in incidence rate of maximum grade RID between groups was assessed using the Chi-square test, between time to RID onset using Kaplan-Meier analysis, and between CSSP, Skindex-29, and maximum NRS scores using the independent samples p = 0.672). Three participants in the Jaungo group and one in the moisturizer group refused to use topical agents during the study and declined to participate. One more participant in the Jaungo group was excluded from the analysis due to loss to follow-up. Therefore, 21 participants in the Jaungo group and 24 in the moisturizer group completed treatment, and all 50 participants were included in the efficacy and safety assessment based on ITT analysis . The body mass index (BMI) was 25.0 ± 5.0 and 24.2 ± 3.3 kg/m2, respectively, with no significant differences in height, weight, or BMI. The mean breast volume calculated using the RT planning computer was 671.1 ± 316.2 versus 642.9 ± 321.8 cc, respectively. The number of participants who underwent concurrent chemotherapy during RT was five (20.0%) and three (12.0%) in each group, and all received taxol-based chemotherapy. There was no statistically significant difference (p = 0.702). The most common type of histology was invasive ductal carcinoma, followed by ductal carcinoma in situ. In both groups, there was one participant (4.0%) with confirmed triple-negative cancer cell molecular subtype, which is usually more aggressive than the estrogen receptor-/progesterone receptor-positive or human epidermal growth factor receptor-positive types (The characteristics of the participants are summarized in ve types . Regardip = 0.733). There were no subjects with RID over grade 2, and 12.0% (three subjects) and 16.0% (four subjects) did not experience RID during the entire trial period. There was no significant difference in the incidence of maximum grade RID between the two groups. The mean time to RID onset was 26.5 ± 11.8  versus 30.4 ± 10.3  days, respectively (p = 0.092). In addition, the cumulative incidence of RID, as assessed using the log-rank test, did not differ significantly between the groups (p = 0.280) (p = 0.536).The outcome results are summarized in = 0.280) . Among pp = 0.045), but pain scores at other visits and maximum pain score did not differ significantly between the groups.CSSP and Skindex-29 were used to assess the effects of RID on participant QOL. The CSSP scores at visit 1, 8, and 9 did not differ significantly between the two groups. The Skindex-29 total score  also showed no difference at any visit. However, when the Skindex-29 subscale scores were separately analyzed, there were significant differences between the groups with regard to the scores for the following items: ‘My skin condition burns or stings,’ and ‘My skin itches’ on the symptoms scale at visit 8, ‘My skin condition affects how well I sleep’ at visit 8 and ‘I tend to stay at home because of my skin condition’ at visit 9 on the functioning scale, and ‘My skin condition makes me feel depressed’ at visit 9 on the emotion scale. The scores for 29 Skindex-29 items are presented in the In the safety analysis, only one participant in the moisturizer group had mild fever and grade 1 insomnia based on CTCAE version 5.0. Both symptoms were evaluated as likely unrelated to the topical agent and resolved without discontinuing treatment or RT. Additionally, there were no severe AEs during the entire trial period.RID is one of the most common side-effects of RT. Patients with RID can experience various symptoms, such as edema, pruritis, erythema, moist desquamation, ulceration, necrosis, and hemorrhage in the irradiated regions, and have delayed or discontinued conventional cancer therapies because of the events . Accordi10 ≥ 60 Gy. The mean age in the Jaungo group was significantly higher than that in the moisturizer group. Based on the prior background, since it has been reported that the incidence of RID increases with age, participants who used Jaungo could be expected to be at a higher risk of RID incidence than those who used the moisturizer. However, our results showed no significant differences in the incidence rate of RID grade ≥2, maximum grade, mean time to RID onset, or cumulative incidence of RID between the two groups. These outcomes support the potential benefit of Jaungo in preventing RID in patients with BC independently of risk factors, and also indicate that it is comparable to the water-in-oil type non-steroidal moisturizer.There are various risk factors for RID in patients with BC. Regarding therapy-related factors, the dose and duration of RT and concurrence with other conventional cancer therapies have been reported to affect the incidence of RID on irradiated skin. In addition, high breast volume, older age, high BMI, poor ECOG performance status, smoking, and alcohol consumption are patient-related factors associated with the early development of RID . Considep < 0.0001), burning sensations (p = 0.039), and moist desquamation within radiation lesions (p = 0.02) compared to the other topical agent without the key components  is a traditional herbal ointment standardized according to the Korean Pharmacopeia. Its main components are Lithspermi radix and Angelica sinensis. The Korea Food and Drug Safety Administration (KFDA) has approved its use as a topical drug for dermatitis. The active ingredients have been identified as shikonin, acetylshikonin, ferulic acid, and decursin, which have been reported to promote soft tissue formation, wound regeneration, and skin healing due to their antibacterial and anti-inflammatory properties (ytokines . Accordiytokines . Jaungo ytokines . Therefoytokines . In addiytokines . TherefoThis study provides evidence that Jaungo has comparable efficacy to the moisturizer for RID in patients with BC. However, it has some limitations. First, this trial was a pilot study with a small sample size. Although the number of patients in each group was estimated statistically based on previous research, further larger sample size RCTs are needed. Second, this study was conducted in a single-blind manner, which means that the subjects knew whether they applied Jaungo or the moisturizer. This may have been a confounding factor; therefore, placebo-controlled trials are required to ensure double-blinding. Despite these limitations, this is the first pilot RCT to evaluate the comparative efficacy and safety of Jaungo and the moisturizer for RID in patients with BC. We believe that the results of this study will encourage further well-designed, double-blind, large-scale RCTs on preventing RID related to conventional cancer therapies.L. erythrorhizon and Angelica sinensis extracts, has comparable efficacy in preventing RID in patients with BC to a water-in-oil-type non-steroidal moisturizer cream currently used in clinical practice. However, large-sample, double-blind RCTs with placebo-controlled are required to fully define the efficacy and safety of Jaungo in preventing RID in patients with BC. (Jaungo, a traditional herbal ointment which mainly consists of with BC. .


Total joint arthroplasty (TJA) is one of the most common surgical operations performed in the US. Periprosthetic joint infection (PJI) is a daunting complication of TJA and is associated with high morbidity. Previous studies have shown that racial disparities exist in postoperative complications of TJAs, including readmissions and mortality. However, it is unclear if such disparities also exist for PJI.A single large hospital system database was used to identify all patients who underwent either primary total knee arthroplasty (TKA) or total hip arthroplasty (THA) between January 2018 and December 2021. Patients were stratified by self-identified racial categories. PJI was defined by ICD-9/10 coding. We present descriptive data as frequencies and percentages for categorical variables and means and standard deviations for continuous variables. We used chi-square tests to estimate crude risk ratios (cRR) and 95% confidence intervals (CIs) for PJI by race.A total of 11,818 patients were included in the final analysis. The majority (96.6%) of patients identified as non-Black (Table 1). The mean age was 69.4 (±10.3) years for non-Black patients and 65.5 (±11.5) years for Black patients. Females represented the majority of non-Black and Black patients who underwent TJA . The majority of TJAs were TKAs for both non-Black and Black patients . The incidence of PJIs was 1.6% among non-Black patients compared to 3.3% among Black patients (cRR (95% CI): 1.99 (1.17 – 3.39); p=0.0112). The majority of PJIs occurred post TKA (Table 1).Preliminary findings suggest that racial disparities exist in PJIs. These findings were not adjusted for possible confounding factors such as age, sex and comorbidities. Future studies are needed to determine if such disparities persist when other factors are considered.Antonia Chen, MD, MBA, Adaptive Phage Therapeutics: Advisor/Consultant|Adaptive Phage Therapeutics: Grant/Research Support|Avanos: Advisor/Consultant|BICMD: Advisor/Consultant|Convatec: Advisor/Consultant|Elute: Grant/Research Support|Ethicon: Advisor/Consultant|GLG: Advisor/Consultant|Guidepoint: Advisor/Consultant|Heraeus: Advisor/Consultant|Hyalex: Stocks/Bonds|IlluminOss: Stocks/Bonds|Irrimax: Advisor/Consultant|Irrimax: Stocks/Bonds|Journal of Bone and Joint Surgery: Deputy Editor|Osteal Therapeutics: Advisor/Consultant|Osteal Therapeutics: Stocks/Bonds|Peptilogics: Advisor/Consultant|Peptilogics: Grant/Research Support|Pfizer: Advisor/Consultant|SLACK Incorporated: royalties|Smith and Nephew: Advisor/Consultant|Sonoran: Stocks/Bonds|Stryker: Advisor/Consultant|Taylor & Francis Group: Royalties|UpToDate: Royalties


The audience response systems are being implemented to support active learning in nursing degree programs. The benefits of audience response systems have been studied in lecture-based classes and seminars, but their advantages or inconveniences when included in the debriefing phase of a high-fidelity clinical simulation have not been explored. The study aim was to discover student´s experience about using of interactive questions during debriefing, and the self-perceived effects on attention, participation and motivation.A Mixed-methods study was used exploratory sequential design in a university. The participants were 4th-year students enrolled in the Nursing Degree in a university in Southern Spain. (1) Qualitative phase: a phenomenological approach was utilized, and focus groups were used for data-collection. (2) Quantitative phase: cross-sectional descriptive study using a questionnaire designed “ad hoc”, on the experiences on the use of interactive questions in the debriefing phase and the Debriefing Experience Scale.(1) Qualitative phase: the students highlighted the facilitating role of the interactive questions during the reflection part of the debriefing, and mentioned that the interactive questions helped with stimulating attention, participation, and motivation during the analytical part of the debriefing; (2) Quantitative phase: it was observed that the best evaluated dimension was “Motivation”, with a mean of 4.7 (SD = 0.480), followed by the dimension “Participation”, with a mean of 4.66 (SD = 0.461), and lastly, the dimension “Attention”, with a mean of 4.64 (SD = 0.418).The use of interactive questions contributed the attention, participation, and motivation of the students during the debriefing, contributing towards a highly satisfactory experience of high-fidelity clinical simulation.The online version contains supplementary material available at 10.1186/s12912-023-01499-z. Clinical simulation as a teaching method for nursing degree students has been used since the start of the 20th Century . HoweverMany debriefing techniques and models exist, although the evidence does not point to the superiority of one over the other . One of In the last few years, the use of new technologies has allowed the implementation of electronic resources, virtual classes, applications with multimedia elements, etc. Multimedia elements have provided advantages through the gamification of the classroom and active learning, as shown by a recent systematic review that described how gamification improved the acquisition of knowledge and the attitude towards learning . To stimThe development of multimedia elements has led to the emergence of other methods of debriefing in high-fidelity clinical simulation, such as synchronous virtual debriefing, or self-debriefing, which utilizes chats on the internet, blogs, or discussion forums , 17. In Until now, multimedia elements have been used during virtual debriefing, but they have not been implemented as a classroom method to contribute to debriefing. The use of interactive questions as a teaching support during debriefing has not been explored in previous studies either. However, their use could provide the advantages offered by the gamification of the content in the high-fidelity clinical simulation debriefing through the ARS in the classroom. To provide new evidence, a study was designed whose objectives were: (1) To discover the experience of students with respect to the use of the Wooclap platform of interactive questions during debriefing, and (2) To quantify the impact of the interactive question on students’ attention, participation, and motivation during debriefing.Mixed-methods study with an exploratory sequential design, in which the qualitative and quantitative phases were conducted in parallel and the conversion of the results from both phases was performed during the interpretation of the results , 21.For the qualitative phase, a phenomenological approach was used to try to capture both individual and collective lived experiences , during The participants were 4th-year students enrolled in the Nursing Degree at the Faculty of Nursing at the University of Murcia, who took the Practicum 4 class during academic year 2021/2022.The following inclusion criteria were defined as: (1) Having attended the 4 high-fidelity clinical simulation sessions that were part of the class, and (2) Accepting to participate in the study. The exclusion criteria were students enrolled in the European Region Action Scheme for the Mobility of University Students (ERASMUS) program, and students enrolled in national mobility programs (SICUE/SENECA) who would take the class at another university.To select the participants, students who met the inclusion and exclusion criteria were invited during the months of February and March to participate in the focus groups. The students who wanted to participate were included in a group until data saturation was reached. Student participation was voluntary, and they did not receive any compensation for it. As for the sample size, it was determined as a function of information power , and theThe quantitative phase used the same participation criteria as the qualitative phase, although only 160 students, from the initial 212, who met the inclusion and exclusion criteria of the entire class, correctly completed the instruments, for a response rate of 75.4%.During their 4th year in the Nursing Degree, the students must take the compulsory class Practicum 4, which deals with services such as delivery room, neonatal intensive care unit, hemodialysis, and mental health centers. It is worth 12 European Credit Transfer and Accumulation System (ECTS) credits, and 300 h of work for the students, of which 10 h correspond to high-fidelity clinical simulation and 240 h of real clinical practice. Those students had received 10 h of high-fidelity simulation in a previous year of the program. The simulation laboratories were structured into 4 sessions composed of 18–20 students. In the first session, the scenarios and the principles of simulation were presented, with the structured simulation of 2 scenarios each taking place in the rest of the sessions. The content of the scenarios were mother-child health, mental health, and hemodialysis (Annex I). For the creation of the scenarios, the standards from the (INACSL) were followed .The Wooclap tool was utilized during the debriefing  , which oIn the qualitative phase, the data collection process took place through focus groups guided by a sole interviewer who was the class’ professor and had previous experience in managing focus groups. For the development of the focus group, a guide with a list of themes was created Table . Each foQuantitative data collection was carried out by means of an online questionnaire. To complete the questionnaire and the DES, a single form was created in the Google Forms platform, including both instruments. This form was distributed through the internet platform Aula Virtual from the University of Murcia, to the students who met the inclusion and exclusion criteria of the study. The data collection took place between May 15th and June 5th, 2022. In the quantitative phase, in addition to the variables gender and age, the following data collection instruments were used:To assess the use of interactive questions in the debriefing phase, an “ad hoc” questionnaire was created. It was based on the results obtained from the focus groups, specifically focusing on topic 2, “Interactive questions in the dynamics of the debriefing.“ with 3 dimensions and a total of 12 items with a Likert scale with 5 response options . Dimension 1 “Attention” measures the attention of the students during the debriefing, and was composed of items A1, A2, A3, and A4. Dimension 2 “Participation” measures the participation of the students, and was composed by items P1, P2, P3, and P4, and dimension 3 “Motivation”, measured the student’s motivation, and was composed by items M1, M2, M3, and M4. Each of the dimensions had a maximum score of 20 and a minimum of 4. All the items were written in the positive sense, so that a higher score indicated a better evaluation of the variable studied. The questionnaire was created and designed by a panel of 3 experts with experience in high-fidelity clinical simulation. To obtain the content validity, 10 educators with more than 2 years of experience in high-fidelity clinical simulation were used, with a resulting content validity index (CVI) being adequate for all the items (CVI between 0.8 and 1), as well as for the total questionnaire (CVI = 0.975). As for the reliability analysis, the internal consistency of the scale was analyzed (Cronbach’s α), which provided a value of α = 0.616 for dimension 1, α = 0.621 for dimension 2, α = 0.796 for dimension 3, and α = 0.875 for the total scale .Debriefing Experience Scale. To evaluate the experience during the high-fidelity clinical simulation, we used the DES developed by Reed , and valThe focus groups were transcribed verbatim, and afterwards, a preliminary analysis was conducted, composed of a summary of the findings, the interpretations, and the observations with respect to the dynamics of the meeting. This preliminary summary served to afterwards proceed with the thematic analysis following the inductive-deductive procedure proposed by Fereday and Muir-Cochrane , for theTo process the quantitative information, a database was created in the statistical package JAMOVI version 2.3.9 for Windows. The descriptive statistical values (mean and standard deviation) of the quantitative variables were calculated, as well as frequencies and percentages for each of the answers provided in each of the items of the questionnaire.The study was approved by the Ethics Committee from the University of Murcia (3640/2021). Informed consent was approved by ethics committee and obtained from all the participants. All the procedures were performed in agreement with the ethical guidelines from the Declaration of Helsinki . The parIn addition, at the start of the focus groups, information was provided about the ethical aspects and the commitment with confidentiality and privacy. A pact was made to provide each participant with a personal code, and a verbal commitment was agreed upon by the participants.The sociodemographic characteristics of the sample show that the average age of the participants was similar in both the quantitative and qualitative phases. The majority of the subjects were women Table .Two main themes emerged from the analysis of the data: (1) Interactive questions as support during the debriefing, and (2) Interactive questions in the dynamics of the debriefing. These themes contributed towards the understanding of the experience associated with the use of interactive questions during the debriefing phase of the high-fidelity clinical simulation.Interactive questions as support during the debriefing.“Also, right when you are here in the debriefing, the questions make you focus more on what you have done, because sometimes you don’t remember if you only start to talk in the debriefing to see what happened.”.“Sometimes you don’t remember what happened in the scenario, so the questions help you remember what happened and what you did in the simulation room.”.The interviewees highlighted the facilitating function of the interactive questions during the reflective part of the debriefing. The interactive questions helped in the mental reconstruction of the important events during the simulation of the scenario, stimulating their analysis through its recreation through a mental image.“In the debriefing, they can tell you a thousand things, that you may not remember, but the questions make you aware about what happened in the scenario, and you see what you did well, and you could improve.”.Likewise, the interactive questions helped as support for the plus/delta resource, as they directed the analysis towards more specific aspects, and helped in the more precise analysis of events in the clinical simulation.“[…] we learn with the questions, because you ask them as the debriefing moves forward, and we truly focus on the most important aspects and concepts, […], it’s like we focus on the specifics, and we do not leave anything important out. And there are always things that we should remember, and I think the questions work very well for this […].”.“Also, when the analysis is being done in the debriefing, asking the questions makes us focus on the most important aspects of the scenario, and we remember these in the end, reflecting about the most important things instead of the entire scenario […].”.The analysis of the scenarios through interactive questions as the guiding thread in the debriefing served for the assimilation of learning objectives defined for each scenario through a process of self-reflection focused on the important events of the scenario.As for the type of interactive question utilized, the participants affirmed that the combined use of various formats favored the reflective process during the analysis of the events. The free response questions, and prioritizing the interventions, allowed for more time for self-reflection.“[…] for example, the ones about prioritizing […] they help a lot, because on many occasions we have so much knowledge that you have to classify and prioritize them according to the scenario and the actions, and these questions make you reflect about it.” .“I think that the multiple-choice ones give you more security, because it’s either one or the other, but the free response ones, as she said, give you more possibilities, and allow you to see what the other colleagues wrote down. And the prioritizing ones, I personally think that they make you learn the most.”.“For me, the ones about prioritizing are harder […] but you learn more, because when you have to take part in the scenario, or you are working […], you know what comes first and what is the most important. I also like the free response ones, because, for example, you can review what you know without looking at the possible options, and so you think more.”“With the question, you can see what could happen according to what the studies say, because comments are made on each of the answers that are shown.”.On the other hand, the interactive questions allowed the students to see the other student’s answers at the same time on a screen, thereby enriching the process of the reflective phase of the debriefing, as other options were explored.2)Interactive questions in the dynamics of the debriefing.The interviewees indicated that the interactive question helped to stimulate their attention, participation, and motivation, during the analytical phase of the debriefing within a safe environment, while also serving as support for the plus/delta resource.“What motivates me is that it is interactive […], all of them are answering some questions, and we can see everyone’s opinions, and also we go step by step, it’s a more dynamic way of doing things [subject 2 agrees], so that for me […] they invigorate the entire debriefing when it’s done this way.”.The use of interactive questions added a series of extrinsic rewards associated to dynamism, competitiveness, or an attractive interface of the tool, to the intrinsic motivation of the students.“It is the first time in four years of the degree that I come prepared to a simulation, in the end, we are all participating, because you know what you have to answer and that there will be a part in which they will ask you questions about the scenario, and you say I’ll take a look and write down the main aspects […].”.“[…] as for the questions, I think of them as being very motivating, the fact of asking questions and knowing what to answer. Hey, look, I don’t know this. But in the end, I’m not that bad.”.The fact of knowing how to answer the questions and feel valid and valued by their peers helped with competitiveness in a safe and controlled environment. This promoted the independent work of the students through their prior preparation of the theoretical cross-sectional content of the scenarios.“[…] the top [places] came out, and this motivated me a lot, you want to go out there a do well, and maybe otherwise you say you will answer, and well… […].”.“[…] if someone comes out, he or she will be in the top places, but the order of the bottom ones does not come up. You see who is moving up, and that can motivate, because with the silly thing of who is winning, you try to answer […].”.The interactive questionnaire tools allow for the establishment of classifications during and after completing the questionnaire, increasing the extrinsic motivation of the students through competition between them.“[…] we really focused on the most important things, and the most relevant concepts that we had to learn when we finished the scenario, so it really helps, it’s like we focus a lot on the specifics and we don’t leave anything important out.”.“I think that what it does is to improve attention in the simulation, because on many occasions, once you have finished the simulation, you stop paying attention, and with the questions, I think you are more involved”.This increase of motivation during the debriefing stimulated attention and participation. Programming the question around the most important events and the learning objectives of the scenario helped to center the focus of attention on the reflection around these objectives.“[…] although we answer with Wooclap, and although we make mistakes, since we can answer later, […] it makes use talk about the subject, and then […] the professors make us feel comfortable, so that we can talk and not be embarrassed to do it, so that we are comfortable throughout the debriefing.”.In this sense, the students affirmed that the interactive questions on some specific aspect of the simulation stimulated their participation in the debriefing, so that related themes emerged, within a safe environment promoted by the facilitator.“Since we don’t know when they question will be asked, we pay more attention, and we are more attentive during the debriefing, in case they ask through Wooclap, and as they ask about the concepts, you pay more attention […].”.Likewise, the uncertainty about the exact moment in which the question appeared maintained the attention of the students for a longer period. Also, the linearity of the sessions was broken up through a change in type of media utilized , and center of attention. Each question could introduce a theme.“[…] on many occasions, if you are only […] observing, or afterwards only listening during the debriefing, that is, if your group did not do the simulation, it doesn’t mean that it doesn’t interest you that much, but you are not waiting for the corrections as if it was you, but since there are questions, and having to stop and think, makes you become more involved, because in the end you are more attentive, because in the end you answer and see what the others have answered.”.“With the [interactive] questions, you feel obligated to answer, so that not only those who took part in the scenario get to participate in the debriefing, because on many occasions, only those who took part in the simulation get to participate. This makes you become more involved.” .The students who were not directly involved in the simulation scenario, and who played the role of spectators, also saw their attention promoted, which fomented their integration into the debriefing dynamic. The participants affirmed that the interactive questions avoided the disconnection of the observing students.“To increase motivation, I supposed that close-ended [questions], because if you have no idea, you are not going to answer anything [in the open-ended questions] […]. With the open-ended question, you see that people answer less, because, for example, if you ask something about a subject that no one knows, you are not going to answer anything […]. If it’s something that you could sense, or more specific things, then you can give more answers.”.The configuration and format of the interactive questions was an important element of analysis in the dynamics and functioning of the debriefing guided by these types of questions. In this sense, the multi-answer question format stimulated participation, as compared to open response questions or “word cloud”. The open response questions assumed minimum knowledge of cross-sectional theoretical knowledge about the scenario, and therefore could have inhibited the response if this knowledge was not present.“I think that perhaps it is embarrassing to speak in public, but when you answer in Wooclap, as it is anonymous, sometimes you answer, and in person, that is, directly, you would not provide an answer in case it is wrong, but as it is anonymous, you do it.”.This factor was able to be corrected using anonymous responses, given that they created a safe learning environment and favored the complete participation of the group, independently if the student had a more extroverted or introverted personality, aside from reducing the fear of answering incorrectly.“And obviously having Wooclap on the phone, in the end you have the phone on the table or with you, and you pay attention to the next question that will come up, in the end you are not going to use it on other things that may result in you losing the thread of the simulation.”.“I think that the more interesting the techniques you use to maintain our attention, it’s true that we have the phone in our hands, but I don’t feel the need to use for anything else or to send messages, I am waiting to answer.”.Lastly, it is necessary to highlight that the use of mobile devices to answer the questions did not greatly interfere with the students’ attention during the debriefing, as the questions stimulated the increase in student participation.The descriptive analysis of the items showed that all the item and dimensions obtained a score higher than 4.5, except for item A4 “The use of the mobile phone to answer the interactive questions does not decrease my attention during the debriefing”, which obtained the lowest score, with a mean of 4.25 (SD = 0.890). On the contrary, the item with the highest score was A2 “I believe that viewing the most important aspects of the scenario, together with the interactive questions, favor my attention during the debriefing”, with a mean of 4.85 SD = 0.375). When analyzing the results according to dimensions, it was observed that the best evaluated dimension was dimension 3, “Motivation”, with a mean of 4.7 (SD = 0.480), followed by Dimension 2 “Participation”, with a mean of 4.66 (SD = 0.461), and lastly, dimension 1 “Attention”, with a mean of 4.64 (SD = 0.418). More than 90% of the students polled evaluated all the items with a 4 or 5 (in agreement/completely agree) , dimension 2 “Analyze thoughts and feelings” obtained a mean of 4.69 (SD = 0.439), dimension 3 “Facilitator skills in conducting the debriefing”, with a mean of 4.69 (SD = 0.443), and dimension 4 “Appropriate facilitator guidance”, with a mean of 4.76 (SD = 0.437). All of the students polled scored all the items with a 4 or 5 (in agreement /completely agree) (Table The results from our study provide evidence on the good acceptance of the use of ARS in the classroom, in agreement with similar studies , 33. LikContributions towards the increase in attention, participation, and motivation of the students during debriefing, using interactive questions, is highly important in university education, as the debriefing phase is the cornerstone of high-fidelity clinical simulation, in which the students consolidate the knowledge and competences they just learned.Attention, the results show that 95.6% of the students considered that the use of interactive questions favored their attention during the debriefing phase. In the focus groups, the students related the increase in attention with the use of interactive questions. The interactive questions maintained the observing students connected to both the development of the scenario, as well as the debriefing, to correctly answer the questions. As for attention in the classroom, studies on ARS indicate that these types of interactive questions, and the use of applications, favor the attention of the students [As for dimension 1 students , 39. Likstudents , 40. TheParticipation, the results obtained through both methodologies applied coincided in that the use of interactive questions fomented the participation of the students, and that anonymity increased it. Our study points out that the interactive questions increased participation through three mechanisms: increasing the attention of students who did not take part in the simulation scenario; favoring emergent themes; making use of the multi-response and anonymous questions. Our results coincide with those obtained in studies on the participation through ARS in classrooms [As for dimension 2 assrooms , 42. Theassrooms .Dimension 3 Motivation obtained the highest score, with a total of 4.7/5. Most of the students believed that the use of interactive questions contributed towards increasing their motivation to learn how to correctly answer, to reflect, and to actively participate in the debriefing, with similar results obtained in studies on the use of interactive questions –43. The In addition, the results obtained in the DES scale show that using the intervention in our study, a highly satisfactory experience was achieved during debriefing. The score of the DES was higher as compared to previous studies, so that the use of interactive questions could be related to an improvement in the debriefing experience .The limitations of the present work, which could restrict possible conclusions, include the lack of representativeness, due to the convenience sampling method and the cross-sectional study design utilized. Due to the cross-sectional design, a pretest was not employed on the sample before the intervention, which restricts the outcomes. In addition, the study utilized a more observational methodology, as an experimental design was lacking, which does allow for the establishment of a causality relationship between the use of interactive questions with the benefits described.Gamification through ARS during the debriefing phase significantly enhances the overall experience of nursing students. The use of interactive questions increases the attention, participation, and motivation of the students during the debriefing, contributing to a highly satisfactory experience during the high-fidelity clinical simulation. It is important to have innovative tools available, which can be adapted to the new needs of students and allow for critical thinking and the optimization of the debriefing phase in clinical simulation.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2