Ovariohysterectomy (OHE) induces inflammation and stress in female dogs. The anti‐inflammatory effects of melatonin have been reported in several studies.The goal of this study was to assess the effects of melatonin on the concentrations of melatonin, cortisol, serotonin, α‐1‐acid glycoprotein (AGP), serum amyloid A (SAA), c‐reactive protein (CRP), interleukin‐10 (IL‐10), interleukin‐8 (IL‐8), interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) before and after OHE.n = 5): melatonin, melatonin+anaesthesia and melatonin+OHE and received melatonin  on days –1, 0, 1, 2 and 3. Ten dogs were assigned to the control and OHE groups (n = 5) without melatonin treatment. OHE and anaesthesia were performed on day 0. Blood samples were obtained via jugular vein on days –1, 1, 3 and 5.The total number of animals was 25 and aligned in 5 groups. Fifteen dogs were divided into three groups  and inflammatory cytokines significantly increased after OHE. The CRP, SAA and IL‐10 concentrations decreased significantly in the melatonin+OHE group compared with the OHE group. The concentrations of cortisol, APPs and proinflammatory cytokines increased significantly in the melatonin+anaesthesia group compared with the melatonin group.The oral administration of melatonin before and after OHE help controlling the high levels of inflammatory APPs, cytokines and cortisol induced by OHE in female dogs. OHE surgery induced inflammation in female dogs. Oral melatonin administration before and after surgery can be useful to control the high levels of inflammatory acute‐phase proteins, cytokines and cortisol induced by OHE in female dogs. Melatonin, as an endogenous hormone, is secreted by the pineal gland rhythmically during the dark phase of the light‐dark cycle Haldar, . In addiSerotonin is a neurotransmitter with various regulatory functions in physiologic, emotional and behavioural processes. There is an association between aggressive behaviour and low levels of serum serotonin and 5‐hydroxyindoleacetic acid, the primary metabolite of serotonin in dogs, in the cerebrospinal fluids of dogs  was used to feed all dogs (300 g/dog/day). All dogs had free access to water and were kept in the same cage with a 12 h:12 h light/dark cycle. During the two weeks of preparation and adaptation, deworming and anti‐parasitic treatments were performed using tablets containing fenbendazole (150 mg), pyrantel embonate (144 mg) and praziquantel (50 mg). The overall health of dogs was evaluated daily during the study by checking their body temperature, heart rate, respiratory rate, appetite and behaviour while feeding and cleaning their shelter. All dogs were ovariohysterectomised at the end of the study and kept in a nongovernmental organisation shelter for adoption.Twenty‐five adult mixed‐breed and clinically healthy female dogs were selected in the anaestrous phase of the estrous cycle. The selected female dogs were aged 1–3 years and weighed 15–20 kg. Commercial dog food (Nutripet2.2n = 5). The melatonin+OHE group was ovariohysterectomised at day 0 and received melatonin  on days −1, 0, 1, 2 and 3  and xylazine  were used as premedication; moreover, ketamine  and diazepam  were used as induction agents. Isoflurane (1.2%), vaporised in oxygen, was administered after intubation to maintain the general anaesthesia. Ampicillin  and ketoprofen  were injected at the end of surgery  was used to measure CRP , SAA , tumour necrosis factor‐α (TNF‐α) , interleukin‐1β (IL‐1β) , interleukin‐8 (IL‐8) , interleukin‐10 (IL‐10)  and serum melatonin . Alpha‐1‐acid glycoprotein (AGP) was measured by a commercial canine solid‐phase sandwich ELISA kit . Serum serotonin was measured using a commercial solid‐phase sandwich ELISA kit ; also, serum cortisol was measured using another commercial solid‐phase sandwich ELISA kit . All laboratory methods and assays (kits) have been validated in the clinical pathology laboratory.2.5p < 0.05.The Kolmogorov–Smirnov test evaluated the data distribution. For statistical significance and comparison, two‐way repeated‐measures ANOVA , followed by post hoc Tukey's multiple comparisons), was performed using GraphPad Prism version 6 . All data are presented as mean ± SEM. The level of significance was set at 3There was no significant difference between the study groups regarding the concentrations of acute‐phase proteins, inflammatory cytokines and hormones on day −1.3.1p = 0.01), but their interaction did not significantly affect melatonin concentration. On days 1 and 3, melatonin concentration increased significantly in melatonin, melatonin+OHE and melatonin+anaesthesia compared to the control and OHE groups (p < 0.02). On day 5, melatonin concentration was significantly higher in the melatonin+anaesthesia group compared to the control and OHE groups  in the melatonin group, on days 1 and 3 versus day −1 (p = 0.01) in the melatonin+OHE group and on days 1, 3 and 5 versus day −1 (p < 0.01) in the melatonin+anaesthesia group  and group (p = 0.003) had significant effects on cortisol concentration, but the interaction between them did not significantly affect cortisol concentration. Cortisol concentration in the OHE group was significantly higher than in the other groups on day 1 but significantly lower in the melatonin group compared with the control group on day 3 (p = 0.02). Moreover, cortisol concentration in the OHE group was higher than in the control group on day 1 (p = 0.02). No significant difference was observed between the groups on day 5  and decreased on day 3 versus day 1 (p < 0.005) in the OHE group . Serotonin concentration was significantly higher in the melatonin and melatonin+anaesthesia groups compared with the other groups on day 3 (p < 0.001). On day 5, serotonin concentration in the melatonin+anaesthesia group was significantly higher than that of the control, melatonin and melatonin+OHE groups  and decreased on day 1 versus day −1 in the melatonin+OHE group (p = 0.01). Furthermore, serotonin concentration on day 3 was significantly higher than that on days 1 and −1 in the melatonin+anaesthesia group . Maximum CRP concentration was observed in OHE, melatonin+OHE and melatonin+anaesthesia groups, respectively, and the CRP concentration was significantly different between these groups on days 1 and 3 (p < 0.0001). CRP concentrations increased significantly in the OHE, melatonin+OHE and melatonin+anaesthesia groups compared with the melatonin and control groups  and decreased on days 3 and 5 versus day 1 in the OHE, melatonin+OHE and melatonin+anaesthesia groups . On day 1, a significant increase in SAA concentration was observed in the OHE, melatonin+OHE and melatonin+anaesthesia groups compared with control and melatonin groups (p < 0.001). On day 3, SAA concentration in the OHE group was significantly higher than in the melatonin+OHE group (p < 0.004). SAA concentration was significantly higher in the OHE and melatonin+OHE groups compared to the control, melatonin and melatonin+anaesthesia groups , and on day 1 versus other sampling days in the melatonin+anaesthesia group . AGP concentration was higher in the OHE group compared with other groups on day 1. AGP concentrations increased significantly in the melatonin+OHE and melatonin+anaesthesia groups compared with the control and melatonin groups on day 1 (p < 0.001). On day 3, AGP concentration was significantly higher in the OHE group compared with the control, melatonin and melatonin+OHE groups (p < 0.002) and in the melatonin+OHE group compared with the control group . Moreover, AGP concentration in the melatonin+anaesthesia group increased significantly on day 1 versus days −1, 3 and 5 . TNF‐α concentration was significantly higher in the OHE and melatonin+OHE groups compared with the other groups on day 1 (p < 0.0001). In addition, on day 1, TNF‐α concentrations significantly increased in the melatonin+anaesthesia group compared with the control and melatonin groups. They decreased in the melatonin+anaesthesia group compared with the OHE and melatonin+OHE groups (p < 0.0001). On day 3, TNF‐α concentration was significantly higher in the OHE group compared with the other groups and in the melatonin+OHE group compared with the control and melatonin groups . In addition, TNF‐α concentration increased significantly on day 1 versus other sampling days in the melatonin+anaesthesia group . IL‐1β concentration was significantly higher in the OHE, melatonin+OHE and melatonin+anaesthesia groups compared to the control and melatonin groups on day 1. The maximum concentration of IL‐1β was observed in the OHE group compared to the melatonin+OHE and melatonin+anaesthesia groups on day 1 (p < 0.0001). IL‐1β concentration was significantly higher in the OHE, melatonin+OHE and melatonin+anaesthesia groups compared to the control and melatonin groups on day 3  and the interaction between time and group (p < 0.0001) had a significant effect on IL‐8 concentration. IL‐8 concentration was significantly higher in the OHE, melatonin+OHE and melatonin+anaesthesia groups compared with the control and melatonin groups on day 1. The highest concentration of IL‐8 was observed in the OHE group compared with the melatonin+anaesthesia group (p < 0.0001). IL‐8 concentration was significantly higher in the OHE, melatonin+OHE and melatonin+anaesthesia groups compared with the control and melatonin groups on day 3 . IL‐8 concentration significantly increased on day 1 versus other sampling days in the melatonin+anaesthesia group  in intact and ovariohysterectomised female dogs. There was a correlation and dose dependency between the oral administration of melatonin and its bioavailability in dogs. Furthermore, it was reported that first‐pass hepatic metabolism did not affect the bioavailability of oral melatonin and that an oral dose of 10 mg/kg of melatonin showed a high absolute bioavailability in dogs  melatonin increases serotonin concentration, and increased serotonin, in turn, decreases cortisol concentration in animals with aggressive behaviour and during stress conditions  challenge was performed for each of the four groups. A temporary increase in serum cortisol concentrations was observed in all groups, but cortisol concentrations in the group undergoing OHE were four times higher than presurgery levels in other groups. This increase in cortisol concentrations lasted about 6 h and then returned to pretreatment levels after 24 h  versus day 3 in female dogs that received melatonin. Similarly, in another study, the intraperitoneal administration of melatonin to rats increased serotonin concentration in the midbrain within 20 min. The mechanisms involved were not clear, but it was suggested that melatonin might alter the function of neurons containing serotonin or increase serotonin concentration in the brain by inhibiting the release of the amine, increasing serotonin reuptake or preventing serotonin intra‐ or extraneural metabolism  and SAA concentrations. These two positive acute‐phase proteins are reliable markers for assessing the complications in the postoperative period of OHE . CRP concentration on days 1 and 3 and SAA and AGP concentrations on day 1 were higher in the melatonin+anaesthesia group compared with the melatonin group. Intravenous general anaesthesia with propofol and remifentanil in human patients increased IL‐6, IL‐7, IL‐8 (a major factor in acute inflammation), IL‐15 and other biomarkers such as monocyte chemoattractant protein 1, placental growth factor, macrophage inflammatory protein‐1β and vascular endothelial growth factor‐A. The neuroinflammatory response was dominated by IL‐6, IL‐8 and modulatory calcineurin interacting with protein‐1  and inflammatory cytokines . The oral administration of melatonin before and after OHE significantly decreased cortisol, CRP, SAA and IL‐10 concentrations but increased serotonin concentration. However, oral melatonin did not affect the concentrations of inflammatory proteins and cytokines in intact female dogs.Sina Salavati: conceptualisation; data curation; investigation; methodology; resources; writing – original draft. Asghar Mogheiseh: conceptualisation; data curation; funding acquisition; investigation; methodology; project administration; resources; supervision; writing – original draft; writing – review & editing. Saeed Nazifi: conceptualisation; funding acquisition; investigation; methodology; resources; supervision; validation; writing – original draft; writing – review & editing. Atefeh Amiri: data curation; methodology; visualisation.The authors have no conflict of interest to declare.Experimental protocols were performed in accordance with the Iranian animal ethics framework under the supervision of the Iranian Society for the Prevention of Cruelty to Animals and Shiraz University Research Council (IACUC no: 4687/63). The recommendations of European Council Directive (2010/63/EU) of September 22, 2010, regarding the standards in the protection of animals used for experimental purposes, were also followed.https://publons.com/publon/10.1002/vms3.1112.The peer review history for this article is available at


Objectives The aim of thisin vitrostudy was to test the influence of the amount of toothpaste on enamel cleaning efficacy.Materials and Methods The hydrated silica-based test toothpaste (radioactive dentin abrasion: 60.19 ± 1.35) contained all ingredients of a regular fluoride toothpaste. The cleaning efficacy of four different toothpaste amounts  diluted in 1.00 mL water were each tested for different brushing times  using a standardized staining model on human molars with a brushing machine. Photographic documentation and colorimetric measurements were conducted, respectively, initially, after staining and after each brushing step. Colorimetric measurements were used to calculate the stain removal (in %).Statistical Analysis Results were analyzed by one-way analysis of variance with post hoc Tukey test and Levene's test for analysis of homogeneity of variance. The level of significanceαwas set at ≤ 0.05.Results The cleaning efficacy decreased significantly when using smaller toothpaste amounts. Stain removal after 120 seconds brushing time was: 77.4 ± 5.0% (1.00 g toothpaste), 75.7 ± 3.4% (0.50 g toothpaste), 54.1 ± 6.7% (0.25 g toothpaste), and 48.2 ± 7.1% (0.125 g toothpaste), respectively.Conclusion In thisin vitrostudy the cleaning efficacy of a medium-abrasive, hydrated silica-based toothpaste was analyzed. Note that 1.00 g toothpaste showed for all brushing times a significantly higher cleaning efficacy than 0.25 g toothpaste and 0.125 g toothpaste. Caries and gum disease (gingivitis/periodontitis/peri-implantitis) are plaque-associated diseases.In addition to delivering therapeutic agents to combat caries and gingivitis, toothpastes are formulated to remove dental plaque, as well as stains, as effectively as possible while being gentle to teeth and gingiva.Toothpastes are highly complex semisolid pastes that contain several active ingredients. One well-known active ingredient for remineralization and caries prevention is fluoride.Additionally, some toothbrush heads, particularly electric brushes, have become smaller, so also adults may be using lower amounts of toothpaste.in vitrostudy was to analyze the influence of different toothpaste amounts  and ground to 1,200 grit (Struers). To facilitate the adhesion of the polyphenols during storage in the staining media, an etching procedure was included. The samples were slightly etched by immersing in 1% hydrochloric acid solution , followed by saturated sodium carbonate solution  and 1% phytic acid solution .The test toothpaste  had a RDA value of 60.19 ± 1.35 . Note that ISO/ADA reference material  was used for testing.The toothpaste composition was as follows:-), Sodium Cocoyl Glycinate.Aqua, Hydrated Silica, Glycerin, Hydrogenated Starch Hydrolysate, Xylitol, Silica, Cellulose Gum, Sodium Sulfate, Sodium Methyl Cocoyl Taurate, 1,2-Hexanediol, Aroma, Caprylyl Glycol, Sodium Fluoride (1450 ppm Fn = 8)  were investigated on eight tooth samples in each group (n = 8) .The cleaning efficacy was tested by means of stain removal. Tooth samples were stained according to a modified protocol from Lath et alArtificial saliva was prepared as previously described.The specimens were fixed in the sample holder and placed in the mechanical brushing device . Before brushing, the toothbrushes  were wetted with deionized water under standardized conditions for 5 seconds. The toothbrushes were moved in reciprocating motions on the sample surfaces with following parameters: Brushing load 1.5 N and brushing frequency 2.5 Hz. Samples were brushed with horizontal movements for 10, 30, 60, 120, 180, and 300 seconds with toothpaste slurry with the respective dilution degree . A continuous movement of the slurry and its presence between toothbrush and specimen was always ensured. The samples were rinsed thoroughly in deionized water after brushing treatment.L*a*b* colorimetric measurements  conducted on initial, after staining, and after brushing samples using a measurement area of Ø 4 mm.Photographic images of the enamel samples were taken before the experiment began, after staining and after brushing under standardized conditions using a reflex camera . Stain removal was calculated from1ΔE: colorimetric difference between brushed and stained specimen2ΔE: colorimetric difference between initial situation and stained specimenαwas set at ≤ 0.05.The results were analyzed by one-way analysis of variance with post hoc Tukey test and Levene's test for analyses of homogeneity of variance . The level of significanceInAt brushing with the dilution degree of 1:1 and 1:2, the progression of the stain removal in dependence of the brushing time shows an asymptotic behavior. While the stain removal increases strongly at the beginning (until 120 seconds), the values increase only slightly after a brushing time of 120 seconds .At all brushing times, lower (but not significant) stain removal was determined for dilution degree of 1:2 compared to 1:1. At dilution degree 1:1 for all brushing times significantly higher cleaning efficacy was measured compared to dilution degree 1:4 and 1:8. No statistical significance has been determined for the dilution degree of 1:4 and 1:8 after 10, 30, 60, and 120 seconds .in vitrostudy shows that the cleaning efficacy of a typical fluoride toothpaste is significantly depended on its amount and on the brushing time. There is a positive correlation between cleaning efficacy and toothpaste amount as well as between cleaning efficacy and brushing time.The presentin vitroenamel and dentin wear of two different fluoride toothpastes (with 1450 ppm F-) with RDA = 90 (“Toothpaste A”) and RDA = 200 (“Toothpaste B”), respectively.in vitro. They found that the dentin wear caused by a toothpaste with a dilution fraction of 1:1 (toothpaste:diluent) is higher than the dentin wear caused by a toothpaste with a dilution fraction of 1:4 (toothpaste:diluent). The diluent they used was deionized water as well as a carboxymethylcellulose-based artificial saliva.in vitrostudy and showed that the tested whitening toothpastes had a higher stain removal efficacy than the tested nonwhitening toothpastes .Some studies analyzed the effect of different toothpaste amounts  on abrasion. For example, Franzò et al tested both theTo date, however, no study has focused on the stain removal efficacy of varying toothpaste amounts on stained enamel. The strength of the present study is that only the toothpaste amount was varied; all other parameters, for example, the toothbrush type (including filaments) and toothpaste formulation as well as the dilution medium were kept constant. Furthermore, this cleaning efficacy study was carried out using a modified staining model from Lath et al.The present study clearly demonstrates the influence of different amounts of a test toothpaste with a medium abrasion (RDA: 60.19 ± 1.35) on cleaning efficacy. This study shows that using higher toothpaste amounts and increased brushing time will lead to better cleaning efficacy. However, families have been advised to use smaller amounts of toothpaste for children. For example, the amount of fluoride toothpaste for children is limited to “pea” and “grain of rice” sizes.Another argument for using higher amounts (≥ 0.5 g) of toothpaste is that the toothpaste is quickly diluted by saliva during tooth brushing in the oral cavity,One could argue that toothpaste is not needed to remove dental plaque. However, this seems to be only true for young and soft biofilms , as shown in a systematic review by Valkenburg et al who analyzed the additional instant effect of toothpastes on mechanical plaque removal.in vitroresults of this study show that there is a clear influence of toothpaste amount on its cleaning efficacy of clinically relevant plaque and stain. Tellefsen et al, for example, showed in anin vitrostudy that the influence of the toothbrush plus toothpaste on abrasion is more pronounced than the influence of toothbrush alone.in vitrostudy, Wang et al showed that brushing with tap water alone (used as a control) is by far less efficient in stain removal than brushing with a toothbrush and toothpaste.Thein situdemineralization study that the toothpaste amount multiplied by concentration (“intensity of treatment”) is more important than just focusing on the fluoride concentration.It is also important to note that, when using a reduced amount of toothpaste, the absolute amounts of all ingredients, which are present in the oral cavity during tooth brushing, especially active, or medicinal ingredients, are also reduced. Paiva et al, for example, found in anThis study shows that, in addition to the toothpaste amount, brushing time is also crucial. Cleaning efficacy can be significantly increased by the amount of time taken during tooth brushing. Consequently, patients should be trained to brush their teeth as recommended by many dentists and dental hygienists, and also other dental professionals for at least 2 minutes. Slot et al, for example, show in their systematic review that a longer brushing time using manual toothbrushes increases the mean plaque reduction .in vitro,in situ, andin vivostudies.A limitation of the study is that not all relevant parameters for cleaning efficacy of toothpastes were tested in this study. For example, there are many different toothpaste formulations on the market  and there are different individual brushing habits . The influence of these parameters could be tested in futurein vitrostudy, the cleaning efficacy of a medium-abrasive, hydrated silica-based toothpaste was analyzed. Note that 1.00 g toothpaste showed for all brushing times a significantly higher cleaning efficacy than 0.25 g toothpaste and 0.125 g toothpaste. It is also important to analyze the cleaning efficacy of different toothpaste amounts in future clinical studies.In this


The present study intended to establish a predictive nomogram for early relapse (ER) (<12 months) after autologous stem cell transplantation (ASCT) in the novel drug era for multiple myeloma (MM).The nomogram was designed and constructed to a retrospective clinical data of newly diagnosed MM patients received novel agent induction therapy and subsequent ASCT at three centers in China from July 2007 to December 2018. The retrospective study was conducted among 294 patients in the training cohort and 126 in the validation cohort. The nomogram's predictive accuracy was evaluated by the concordance index, calibration curve and decision clinical curve.The study cohort included 420 newly diagnosed MM patients, and 100 (23.8%) were identified as having ER, including 74 in the training cohort and 26 in the validation cohort. According to the result of multivariate regression in the training cohort, the prognostic variables included in the nomogram were high‐risk cytogenetics, LDH > UNL, and response less than very good partial response (VGPR) after ASCT. The calibration curve showed good fitness between the nomogram predictions and the actual observations and the nomogram was further validated by a clinical decision curve. The nomogram's C‐index achieved 0.75 , which was higher than that of the Revised International Staging System (R‐ISS) (0.62), ISS (0.59), and Durie–Salmon (DS) staging system (0.52). The discrimination ability of the nomogram in the validation cohort was superior to that of the other staging systems (C‐index: 0.73 vs. R‐ISS (0.54), ISS (0.55), and DS staging system (0.53)). DCA showed the prediction nomogram adds much more clinical utility. Different scores of the nomogram draw a distinction of OS.The present nomogram could serve as a feasible and accurate prediction of ER in novel drug induction transplantation‐eligible MM patients, which could help modify the post‐ASCT strategy for patients at high risk of ER. Early relapse post‐ASCT in MM makes patients suffer adverse outcomes, which is so far unpredictable. This study retrospectively enrolled data of transplant‐eligible MM patients from multicenters in China and established a predictive nomogram for ER. The C‐index demonstrates good predictive accuracy for survival and the calibration curve for the probability of survival showed good agreement between the prediction by the nomogram and actual observations, which could be used to modify the post‐ASCT strategy for those patients. As next‐generation novel drugs with multiple mechanisms of action have already sprung up, a delicate and personalized strategy of novel drugs could help improve the poor outcome of ER patients. To figure out the profile of Chinese ER patients in MM, we analyzed the clinical records of patients who received novel agents involved induction followed by ASCT from three Chinese centers. The objective of this study was to construct an effective prognostic nomogram for ER (<12 months) after ASCT in MM in the novel drug era that reflects important clinical features and can be used to guide real‐world clinical practice.22.1This retrospective study was designed and performed on a cohort of newly diagnosed MM patients who underwent 4–7 cycles of novel agent‐based induction followed by ASCT from July 2007 to December 2018 at Beijing Chao‐Yang Hospital, Guangzhou Zhongshan Hospital, and Shanghai Changzheng Hospital. The exclusion criteria were as follows: salvage ASCT, secondary transplants, allogeneic stem cell transplant, unknown induction treatment, unknown relapse status, and traditional chemotherapy introduction. All patients provided informed consent for the use of their medical records. The Ethics Committee of Beijing Chao‐Yang Hospital approved this study following the Declaration of Helsinki.2.2t , t , and t . Two hundred interphase nuclei were analyzed for each sample. The cutoff values were as follows: 10% for t , t , and t  and 20% for del(17p) and 1q21 gains.Both MM diagnosis and response were evaluated under the International Myeloma Working Group criteria.2.3The response after ASCT was assessed at 80–120 days after ASCT. The ER group was defined as patients who progressed or relapsed within 12 months after ASCT. Time to progression (TTP) was defined as the time from ASCT to progressive disease or death. Overall survival (OS) was defined as the time from the first date of stem cell infusion to death or last follow‐up. In addition, patients who had undergone salvage ASCT and died within 12 months without evidence of myeloma‐related death were excluded. The non‐ER group was defined as patients who did not have disease progression or myeloma‐related death within 12 months after ASCT.2.4t‐test were used to calculate differences between groups for categorical and continuous variables, respectively. Kaplan–Meier Curve and the two‐tailed log‐rank test were used to analyze survival data using SPSS 22.0 and GraphPad Prism 7. Univariate analysis and multivariate analysis were conducted to compare variates among groups over time. Cox regression analysis was to investigate the validity of multivariate analysis for survival data. The multivariable logistic analysis was conducted to identify independent factors predicting ER.Methods of Statistics to determine risk variables were executed by SPSS 22.0. The characteristics of patients were described using the median value for continuous variates and the frequency for categorical ones. A two‐sided Fisher exact test and Student's http://www.r‐project.org/). The process of randomization for patients into the training and validation cohorts was conducted using the formula in R. A final model selection was calculated by a backward step‐down selection process with criteria previously reported.https://www.mskcc.org/departments/epidemiology‐biostatistics/biostatistics/decision‐curve‐analysis] in R which was modified based on “rmda” package in R.p < 0.05 indicated significance.A nomogram was calculated based on the filtered variables of multivariate analysis by the rms33.1n = 297; 70%) and the validation cohort . Median age of the total cohort was 53 years . The median time from diagnosis to ASCT was 6.70 months. The median follow‐up time was 54 months. The patients were separated into ER/non‐ER groups based on the above definition. The study cohort included 420 newly diagnosed MM patients, and 100 (23.8%) were identified as having ER. The training cohort contained 74 (24.9%) and the validation cohort contained 26 (21.1%). The baseline demographics are detailed in Table Total of 420 newly diagnosed MM patients who received novel agents‐involved induction and subsequent ASCT were entered into this study. The study cohort was randomly separated into the training cohort , and the ER group patients had a higher median lactate dehydrogenase (LDH) level  vs. 157 , p = 0.003) and a higher rate of high‐risk cytogenetics .In the training cohort, the rate of stage R‐ISS III group was more in ER than in non‐ER . At 3 month after ASCT, the rate of a VGPR or better response was higher in the non‐ER group . The complete response (CR) rate after ASCT in the non‐ER patients was higher than those in the ER group .The therapeutic response of the training cohort is detailed in Table t , t ) in the ER group . Del17p and t  were more prevalent in the ER group .Cytogenetic profiles based on FISH are listed in Table 3.3n = 38), 6–12 m (n = 62), 12–24 m (n = 119), 24–36 m (n = 83) and > 36 m (n = 118)], the median OS was significantly prolonged   , and the median OS was 41.7 months (95% CI: 14.36–93.8) in the training cohort. The Kaplan–Meier Curve showed that with the rise in the median TTP  at 41.9% compared with 25.6% in the non‐ER patients of the training cohort, which is consistent with previous reports.In this study, ER patients accounted for a significantly higher percentage of high‐risk cytogenetics patients [del17p, Our study showed that serum LDH > ULN was a strong ER indicator, consistent with previous studies.This study has limitations as well, because it was a retrospective and nonrandomized research. The following problems led to a narrow interpretation of our study. First, researchIn conclusion, ER after ASCT still appears to be a vital independent adverse prognostic factor even in the era of novel agents. As presented in this study, the nomogram objectively and accurately predicted ER and OS in transplant‐eligible MM patients undergoing novel agent treatment. High‐risk ER patients may need an updated post‐transplant strategy to achieve better outcomes. Additional researches are obligatory to further validate this model.Huixing Zhou: Conceptualization (lead); data curation (lead); formal analysis (lead); resources ; writing – original draft (lead); writing – review and editing (lead). Yuan Jian: Data curation ; funding acquisition ; resources . Juan Du: Data curation (lead); formal analysis . Junru Liu: Data curation ; methodology ; supervision ; validation . Zhiyao Zhang: Data curation . Guangzhong Yang: Data curation ; software . Guorong Wang: Data curation . Ying Tian: Data curation ; formal analysis . Yanchen Li: Conceptualization ; data curation . Yin Wu: Conceptualization ; data curation ; supervision . Wenming Chen: Conceptualization ; resources ; supervision (lead); writing – review and editing . Weijun Fu: Conceptualization ; data curation ; supervision ; writing – review and editing . Juan Li: Conceptualization ; data curation ; investigation ; validation ; writing – review and editing . Wen Gao: Conceptualization ; data curation ; validation ; writing – original draft ; writing – review and editing .The authors have no conflicts of interest.The Ethics Committee of Beijing Chao‐Yang Hospital approved this study following the Declaration of Helsinki.Data S1Click here for additional data file.


One of the first management tasks we had to grapple with was the issue of consent, which is a particularly important issue for OA journals.After 6 years as the Editor in Chief of Clinical and Experimental Dermatology,https://onlinelibrary.wiley.com/toc/2690442x/2021/1/1). In my last edition as Editor, we published a special collection on Psychodermatology (https://onlinelibrary.wiley.com/toc/2690442x/2022/2/4)Our first manuscripts were published online in July 2020 and we published our first compete edition of the journal in March 2021 ; Writing – review & editing (Lead).This editorial received no specific grant from any funding agency in the public, commercial, or not‐for‐profit sectors.Not applicable.


Staphylococcus aureus aggregates formed in synovial fluid (SynF). We explore whether antibiotic activity could be increased against Staphylococcus aureus aggregates using ultrasound-triggered microbubble destruction (UTMD), in vitro and in a porcine model of septic arthritis. In vitro, when bacterially laden SynF is diluted, akin to the dilution achieved clinically with lavage and local injection of antibiotics, amikacin and ultrasound application result in increased bacterial metabolism, aggregate permeabilization, and a 4-5 log decrease in colony forming units, independent of microbubble destruction. Without SynF dilution, amikacin + UTMD does not increase antibiotic activity. Importantly, in the porcine model of septic arthritis, no bacteria are recovered from the SynF after treatment with amikacin and UTMD—ultrasound without UTMD is insufficient. Our data suggest that UTMD + antibiotics may serve as an important adjunct for the treatment of septic arthritis.Treatment failure in joint infections is associated with fibrinous, antibiotic-resistant, floating and tissue-associated  Coupling ultrasound-triggered microbubble destruction with antibiotics in diluted synovial fluid leads to the effective clearance of S. aureus in models of septic arthritis This problem is exemplified in joint infections, where despite aggressive local and systemic antibiotic treatments, infections can recur in up to 45% of septic arthritis cases2. Staphylococcus aureus (S. aureus) is the causative organism in as many as 42% of these cases of septic arthritis3, a debilitating disease that has an 11% mortality rate4, affects ~20,000 people annually in the United States5 with medical expenses >$1 billion yearly6. S. aureus aggregates in synovial fluid (SynF)12, the lubricious fluid within the joint, and can be sequestered within different joint compartments as well as the fibrin-rich pannus, so that joint drainage is often insufficient for their removal13. Current treatment options include joint lavage/drainage, aggressive local and systemic antibiotic intervention, and surgical debridement. The continued presence of these aggregates is thought to be a cause of treatment failure14. Thus, we explored strategies for restoring antibiotic susceptibility of bacterial aggregates to improve the outcome of acute septic arthritis treatments.Biofilms, whether floating or adhesive, are assumed to be the major cause of infections, and treatment is made difficult by the increased antibiotic tolerance associated with the bacteria that constitute these biofilms15. Reduced antibiotic penetration, slowed metabolism resulting from adaptive changes to a nutrient-depleted environment, and the emergence of persister cells make biofilms tolerant to antibiotics. Unlike many biofilms which can be dispersed with DNase or dispersin B, SynF bacterial aggregates do not disperse with these treatments and require aggressive enzymatic digestion, e.g., proteinase K or tissue plasminogen activator (TPA)18. The use of proteinase K in a physiological site could cause tissue damage19 and the effects of activation of plasminogen by TPA would need to be delineated. We, therefore, turned to mechanical means, specifically, ultrasound-triggered microbubble destruction (UTMD) to perturb aggregate structure. Microbubbles, typically 1–8 µm in diameter, are ultrasound contrast agents that are comprised of a polymer, protein, or lipid shell surrounding a gas core20. UTMD uses ultrasound waves to cause inertial cavitation of microbubbles, producing shockwaves emanating from the microbubbles. UTMD causes simple in vitro biofilms and catheter-associated biofilms to become more susceptible to vancomycin22. Other phase contrast agents of smaller diameters have also been used to enhance antibiotic efficacy against biofilms23. However, ultrasound effects on septic joints and SynF bacterial aggregates are limited to a single equine case study without the use of the augmentation associated with UTMD24. We hypothesize that UTMD will: (1) permeabilize S. aureus aggregates to allow for antibiotic penetration, and (2) restore bacterial metabolic activity. Both would increase antibiotic susceptibility.Biofilms are three-dimensional bacterial communities rich in proteins, polysaccharides, and extracellular DNA (eDNA)28 with human usage predominantly in neonates30. We identified bacterial response to the synovial fluid environment and conditions which resulted in markedly increased S. aureus aggregate susceptibility to antibiotics, in vitro. We used similar conditions in a study of septic arthritis in pigs. Based on the results of this study, we propose UTMD combined with antibiotics as an effective, inexpensive, and readily accessible method for the reduction of bacterial load, especially with regard to the antibiotic-tolerant aggregates that are thought to be a major barrier to successful treatment of septic arthritis. We suggest that UTMD + antibiotics allows increased antibiotic activity in infections associated with floating biofilms and could be used as an adjunct to the systemic antibiotic treatments that are standard for the treatment of septic joints.We investigated the conditions under which UTMD reproducibly increased the amikacin (AMK) susceptibility of bacterial aggregates in SynF, in vitro. AMK was chosen as it is a standard antibiotic used in the treatment of veterinary septic joints8, methicillin-susceptible S. aureus (MSSA) aggregate into free-floating, dense, fibrous, structures that have characteristics of antibiotic-tolerant biofilms  in TSB at 6 h. Overall, antibiotic tolerance was greater in pSynF . Thus, in pSynF, even a 104 CFU/mL inoculum showed some survival after antibiotic challenge.Because antibiotic action is predicated on the inhibition of functions of dividing cells, we first investigated whether antibiotic tolerance of bacteria in SynF was due to the induction of metabolic quiescence. In human SynF and in a recently described pseudo-synovial fluid (pSynF)lms Fig. . This relms Fig.  by 6–7 lynF Fig. , in keept = 0) caused a small increase (~0.5 log) in CFU/mL at 30 min (p = 0.0450) , bacteria are dispersed allowing comparisons between media Fig. . Water b50) Fig. . In pSynp = 0.0002). In pSynF without sonication, AMK showed a dose-dependent effect  and 50 μg/mL AMK caused an ~1.5 log decrease (p < 0.0001) in CFU/mL(trypsin). The log decrease for the 30 μg/mL AMK treatment became slightly larger with sonication i.e., ~1.5 log for 10 (p = 0.0044) and 20 min (p = 0.0047), and ~2 log (p = 0.0004) for 30 min. After sonication of various lengths , the log decrease in pSynF incubated with 50 μg/mL AMK remained at 1.5-2 logs . The large error bars suggest that the overall response of MSSA to sonication was heterogeneous and that antibiotic susceptibility of MSSA was only minimally increased with sonication.We investigated whether sonication increased antibiotic sensitivity Fig. . Indepenect Fig. , where 331. To model treatment immediately following arthroscopy, where a combined saline/SynF mix would be present in the joint, we diluted TSB or pSynF after inoculation/aggregate formation and determined the effects of sonication. Without dilution  . When pSynF and its dilutions (containing pre-formed MSSA aggregates) were sonicated, 100% pSynF showed an ~1 log increase in CFU/mL (p = 0.0031). 50% SynF did not further change with sonication, but 20% pSynF showed an additional increase in recovered counts (p < 0.0017). To test the individual effects of changes in total volume or protein concentration/viscosity, we first used TSB or pSynF as the dilutant to keep the protein/viscosity constant, but changed the volume ; further dilution/volume increases using the same medium did not alter dispersal. Finally, we kept the volume constant, but allowed the protein/viscosity to change by dilution with PBS to a fixed volume . No significant changes were observed in dispersal with the diluted TSB samples and only a small increase at 20% dilution in the pSynF sample , suggesting that increased dispersal from aggregates depends on multiple factors including the protein/viscosity characteristics.Joint lavage through arthroscopy is the minimum standard for treating septic arthritis and consists of a washout of SynF , potential placement of local antibiotics, and ultimately a serosanguinous fluid replacing the saline lavage fluid46) Fig. . Dilutioume Fig. . Recoverume Fig. , S4. CFUp = 0.0449). In 20% pSynF, the effect of 2 h incubation on metabolism was less marked . After sonication, however, the metabolism of the diluted (20%) pSynF increased up to 26-fold (p < 0.0001), which was a significant increase over the unsonicated values (p < 0.0001), and metabolic levels approximated values measured in TSB.Because sonication of the diluted samples increased recovered counts in the pSynF samples, we investigated the effects of sonication and dilution on bacterial metabolism. Using 20% TSB, metabolism increased over a 2 h incubation Fig.  which wap < 0.0001) for both sonicated and unsonicated samples. Sonication significantly increased AMK activity (p = 0.0006) in TSB, albeit the actual decrease in CFU/mL (trypsin) was small (~0.5 log). In pSynF, AMK treatment decreased total CFU by at most 1.5 log, whether the sample was sonicated or sonicated+diluted. In human SynF, AMK treatment caused an ~1 log decrease (p = 0.0053) in CFU/ml (trypsin). With sonication, AMK in SynF displayed a 5.5 log decrease in CFU/mL(trypsin) (p < 0.0001). When human SynF was diluted to 20%, 30 μg/mL AMK caused an ~4 log decrease in CFU/ml(trypsin) (p < 0.0001). Sonication of 20% SynF containing 30 μg/mL AMK decreased recovered MSSA counts by ~7.5 logs (p ≤ 0.0001). The same trends were observed with S. epidermidis in the presence of AMK using TSB and SynF . However, no increase in CFU/mL was measured in pSynF, independent of UTMD or dilution , independent of the presence of microbubbles or application of insonation  in TSB by 5–6 logs , all animals were visibly lame and infected joints were hot and painful to palpation. SynF samples obtained from the septic joints of anesthetized animals showed elevated nucleated cell counts and protein levels . Furthermore, AMK + UTMD resulted in a significant decrease in CFU compared to either AMK alone (p = 0.0204) or AMK + insonation (p = 0.0187); no significant difference was observed between AMK and AMK + US treatments. Necropsy of all joints at time of sacrifice presented with marked hyperemic synovial thickening, associated with fibrinous strands. When synovial tissues were scored (blinded) using the Krenn synovitis score (grade 0–9)32 based on tissue hyperplasia (0–3), inflammatory infiltrate (0–3), and synovial stromal activity (0–3), total scores associated with infected synovia treated with AMK + UTMD were not significantly different than the scores for AMK alone 36. This placement of antibiotics after lavage creates the conditions that we have described for the in vitro studies, i.e., bacterial aggregates that have been put into a dilute environment rich in antimicrobials. Because ultrasound application has been used to amplify the effects of antibiotics against bacterial biofilms in rabbits and horses37, we reasoned that the microbial aggregates in the joint might respond to this mechanical perturbation. A major goal of our investigation was to determine if UTMD could restore antibiotic activity in SynF, both in vitro and in vivo.Treatment of the septic joint includes debridement of the pannus which is laden with fibrinous bacterial aggregates38. In this manuscript, we showed that SynF MSSA aggregates displayed a marked reduction in antibiotic susceptibility and reduced metabolic activity, consistent with acquisition of a “persister” phenotype. Additionally, antibiotic tolerance was largest at higher bacteria numbers. This inoculum effect has been described for antibiotics in the penicillin, cephalosporin, and fluoroquinolone families and is attributed to decreased antibiotic-bacterial target affinity, antibiotic quenching, the inverse correlation between bacterial density and metabolism, and the production of antibiotic degradative enzymes41. For AMK, the inoculum effect is attributable to irreversible binding between AMK and the 30 S ribosomal subunit42. While in vitro results often don’t predict in vivo results, data showing decreased antibiotic sensitivity at higher bacterial numbers is consistent with clinical experience of improved outcome when treatment is initiated early in the time course of septic arthritis44.Most acute septic joint infections are characterized by large numbers of aggregated bacteria. These mucinous aggregates are bacterial biofilms that are rich in serum proteins and decorated with host cells50. We initially turned to a bath sonicator as a means to screen through different treatment possibilities. Once we had determined that dilution was required to increase AMK efficacy against bacteria within SynF, an important finding which would be replicated by the joint lavage that is common in treatment of septic joints, we then investigated conditions that would allow similar effects to be realized using clinically-relevant ultrasound probes and energies. Even then, we were merely assured that an insonation effect was possible. Another transition was then required to the complex, constrained space within the in vivo diarthrodial joint model. As we show, each one of these steps required changes with the final testing in the porcine model requiring the additional energy associated with the rupture of ultrasound contrast agents. In vitro, using MSSA aggregates, water bath sonication only caused minimal bacterial dispersal, but restored antibiotic susceptibility to some extent in pSynF and to a greater extent in human SynF. This increase in antibiotic susceptibility could be due to ultrasonic pressure waves subjecting the aggregates to persistent cycles of expansion and contraction. At a microscopic level, this bioacoustic effect would enhance transportation of antibiotics across the bacterial cell membrane51,52. In vitro, these effects have proven to be frequency and energy-dependent and to some extent, we saw this when we looked at the difference in MSSA responsiveness to AMK vs. vancomycin after sonication. We did not pursue this further as both antibiotics showed US-enhanced antibiotic activity and while VAN sensitivity was not increased with dilution, it remained enhanced by the application of US. Importantly, in this work, we show that the media environment modulates effects of ultrasound on bacteria and that this knowledge can be roughly translated to an in vivo situation.To enhance antibiotic sensitivity in clinically retrieved SynF and a synthetic pSynF and ultimately treat septic arthritis, we turned to ultrasound-mediated methods. Effects of ultrasound on bacterial biofilm phenotype have been shown to depend on the ultrasound parameters, strains studied, and experimental conditions50. In our experiments using the inflamed pig joint, intraarticular injection of antibiotics and microbubbles diluted the SynF, even in the absence of lavage. In the pig joint, however, ultrasound alone was insufficient and required UTMD to restore antimicrobial efficacy.Because lavage removes much of the infected SynF during treatment of septic arthritis, remaining bacteria within the infected joint are exposed to a SynF diluted by antibiotics and saline. Therefore, we mimicked this environment by dilution of aggregate-containing SynF and pSynF. Dilution of the SynF after aggregation in concert with water bath sonication increased MSSA antibiotic susceptibility and metabolism, possibly by allowing nutrient-richer media to infiltrate into the nutrient-depleted aggregate interior53. UTMD is minimally invasive and used for other clinical therapeutic applications56, including permeabilizing tumors for drug delivery57, targeting gene delivery to specific tissues58, and facilitating lysis of fibrin clots59. Together, these mechanisms are all appropriate for the observed in vitro facilitation of antibiotic activity and bacterial metabolism. These effects were most marked after dilution of SynF, even after aggregation had occurred. This suggests a modality that could be coupled with post-arthroscopic lavage to improve outcomes of treatments for septic joints.Microbubble rupture caused by UTMD subjects the aggregates to shockwaves to transiently permeabilize the cell membrane, increase antibiotic transport, increase biofilm extracellular matrix porosity, and decrease biofilm thickness10. The pig anatomy, physiology60, and innate immune system62 are very similar to those of humans, and the microvasculature62 and microarchitecture64 of porcine bone closely resembles that of human bone. 24 h after injection of MSSA, the injected joint showed clinical signs of septic arthritis and the SynF contained aggregates that showed dense bacterial clustering in a proteinaceous, mucinous matrix, surrounded by red and white blood cells.Finally, we tested the ultrasound/antibiotic combination in an animal model of septic arthritis. We chose the pig as we had recently presented the horse and pig as a suitable model for SynF investigations, both for in vitro characterization of effects on bacteria, as well as models of septic arthritisOur in vivo trials with this porcine model of septic arthritis showed no effect of insonation on antibiotic activity in the absence of microbubbles, unlike our in vitro trials where ultrasound was sufficient to increase AMK activity against MSSA aggregates. UTMD in the presence of AMK was required to result in marked reduction in retrieved CFU from the porcine joint where no bacterial counts could be recovered from SynF harvested 6-8 h after the treatment compared to the >2,000 CFU/ml MSSA present in the SynF prior to treatment. We speculate that this requirement for microbubbles is due to the anatomy of the joint. Unidirectional ultrasound waves would be attenuated/blocked by the physical barriers associated with the anatomical complexity of the joint. The inertial cavitation of microbubbles provided multidirectional perturbation, allowing for uniform coverage of the joint spaces, and linked the in vitro findings with these promising in vivo effects.Staphylococcus epidermidis after dilution; US augmentation of vancomycin activity against MSSA aggregates in SynF did not require this dilution. We do predict that other Staphylococci, including strains of MSSA, MRSA, and Staphylococcus epidermidis, will produce similar augmentation of antibiotic activity, although exact antibiotic concentrations and UTMD settings may slightly vary. Finally, our results rely on data from 15 pigs, 7–8 pigs , AMK with insonation , or AMK with UTMD (n = 7); 7 for bacterial analysis and 8 for histological analysis) of which received UTMD + AMK treatment after development of septic arthritis. In the pigs designated for UTMD + AMK, no bacterial recovery was measured in 7 of 7 joints, 6–8 h after treatment with AMK + UTMD. Furthermore, while the synovium showed clear signs of changes associated with septic arthritis, the destruction of the microbubbles by UTMD did not significantly alter the synovial histology, suggesting that the UTMD itself would not be destructive to the joint. Because our analyses were limited to the 8 h after treatment, the histology reflected the infected environment, where longer times would be necessary to determine if the UTMD-treated animals which displayed greatly reduced bacterial load, would recover synovial integrity. Within those analyses would include an assessment of nucleated cell count and immune cell function. However, these assessments would not be informative, based on our previous work which showed that changes in these parameters required several days after the infection had started to resolve65. Thus, additional experiments will be necessary over a longer post treatment survival time to determine if the treatment strategy results in sustained infection clearance and recovery from septic arthritis.There are limitations associated with this study. Our in vitro work initially relied on experiments performed in an artificial SynF (pSynF). However, our findings were reproduced in human SynF, where effects appeared more marked than originally seen in pSynF. As in most in vitro systems, the immune component was absent. Furthermore, the US augmentation of AMK activity could be reproduced in To our knowledge, this is the first report of the application of UTMD to MSSA aggregates for treatment of septic arthritis. Bacterial eradication remains a sought-after treatment; reduction of the bacterial load through local antibiotics allows systemic antibiotics and immune surveillance to control the infection. Our in vitro experiments establish the importance of SynF concentration and ultrasound permeabilization in reduction of bacterial load, especially with regards to the antibiotic-tolerant aggregates that are thought to be a major barrier to successful treatment of septic arthritis. Our in vivo experiments demonstrate the efficacy of this approach and suggest that the combined effects of UTMD and antibiotics may serve as an important adjunct for the treatment of septic arthritides.S. aureus in different fluids, including human synovial fluid and a model synovial fluid. We tested the ability of insonation and UTMD to increase antibiotic sensitivity both in 100% media and diluted media, as would be encountered in the post-lavage environment of the septic joint . We then tested the successful treatments in a porcine model of septic arthritis using AMK and UTMD. After 6–8 h, synovial fluid was harvested and assessed for bacterial counts. The IACUC of the University of Pennsylvania approved this study designed in accordance with the ARRIVE guidelines. Human synovial fluid that would normally be discarded was obtained from therapeutically necessary joint aspirations or operations. This material, designated as “waste” (no identifiers), was retrieved and designated as “not human research” by the Thomas Jefferson University Office of Human Research Protections, in keeping with the revised Federal Policy for the Protection of Human Subjects .We set out to determine how antibiotic sensitivity could be enhanced in the synovial environment, representative of septic arthritis. The experiments in this study were designed to test the conditions under which ultrasound-triggered microbubble destruction (UTMD) would restore the antibiotic sensitivity of synovial fluid with bacterial aggregates. We tested the ability of antibiotics to eradicate Staphylococcus aureus (MSSA) ATCC® 25923TM or methicillin-susceptible Staphylococcus epidermidis ATCC® 14990TM was grown overnight, 37 °C, 180 rpm, in trypticase soy broth  from a single colony, and then diluted 1:5 in TSB and cultured for 2 h. Bacteria numbers were determined by comparison to a 0.5 McFarland standard (~108 CFU/mL) or direct plating. Experimental subculture media consisted of TSB, PBS, human SynF, or pSynF8. All plating was performed on 3 MTM PetrifilmTM aerobic films . When 0.25% trypsin, 2.21 mM EDTA dispersion was used, samples were pelleted, washed, resuspended in trypsin, and incubated for 20 min, 180 rpm, 37 °C, re-pelleted, washed, and serial dilution and plating undertaken8. In all experiments, the countable range of bacteria on PetriFilms is between 30-1000 and dilutions are used that fall within those ranges.Methicillin-susceptible 7 CFU/mL) in TSB, aggregate-containing SynF or pSynF were transferred to 3.81 cm × 6.35 cm sealable LDPE pouches , sealed, and sonicated for 0, 10, 20, 30 min using a bath sonicator (Branson 2800 with 40 kHz transducers). Samples without dispersion were serially diluted, plated, and counted. To dilute the medium, volume and/or viscosity was varied by adding PBS and pSynF in different proportions , operating in cadence pulse sequence harmonic imagining mode with a transmit frequency of 2.0 MHz, peak negative pressure 2.0 MPa, and a mechanical index of 1.06 for 6 min with bursting flash mode (50 frames at maximum power over 4 s). TSB controls were treated in parallel. Additional controls included no insonation, no microbubbles. Samples without dispersion were serially diluted, plated, and counted.7 CFU/mL) or S. epidermidis in TSB or pSynF/SynF (containing aggregates) were incubated with 0, 30, or 50 μg/mL AMK, VAN, or PBS for controls, 6 h, 37 °C, 125 rpm. At the end of the incubation, bacteria were dispersed with trypsin, and after treatment, diluted and plated, followed by direct counting. Bath sonication effects. MSSA in TSB or pSynF (containing aggregates) were treated with 0, 30, or 50 μg/mL AMK, transferred to pouches, bath sonicated for 0, 10, 20, or 30 min, and incubated for 6 h, 37 °C, 125 rpm. Samples were dispersed by trypsin and pipetting/vortexing. Dispersed samples were diluted, plated, and counted.MSSA (104–107 CFU/mL) were aggregated in pSynF, treated with 30 μg/mL AMK or PBS, and incubated, 6 h, 37 °C, 125 rpm. TSB was inoculated with MSSA, 30 μg/mL AMK or PBS, and incubated 6 h, 37 °C, 125 rpm. Samples were dispersed by trypsin and pipetting/vortexing. Dispersed samples were diluted, plated, and counted. Dilution and sonication: MSSA in TSB or SynF/pSynF containing aggregates were transferred to pouches. Undiluted or diluted samples , operating in cadence pulse sequence with a transmit frequency of 2.0 MHz, peak negative pressure 2.0 MPa, and a mechanical index of 1.06 for 6 min with bursting flash mode (50 frames at maximum power over 4 s). Pouches were opened and incubated, 6 h, 37 °C, 125 rpm. Samples were dispersed by trypsin and pipetting/vortexing. Dispersed samples were diluted, plated, and counted.5 CFU/mL) in TSB or pSynF (containing aggregates) (24-well tissue culture plate) were incubated for 1.5 h, 37 °C, 180 rpm, and 90 μL of each were transferred into a 96-well black clear bottom tissue culture plate . Blank wells contained TSB or pSynF alone. To each well, 10 μL of resazurin  reagent was added, samples incubated, 37 °C, 180 rpm, and fluorescence (λex/em = 540/590 nm) measured hourly for 6 h using an Infinite® M1000 plate reader . Metabolic activity is expressed as net fluorescence (bacteria well fluorescence – blank well fluorescence).MSSA  was measured in 100 μL. Following sealing, samples were bath sonicated or incubated at room temperature for 30 min, pouches were opened, incubated for 2 h, 37 °C, 180 rpm, and fluorescence was read in 100 μL. UTMD. 100 μL Definity microbubbles were added, and baseline fluorescence was measured. Following sealing, samples were treated using a Siemens S3000 HELX Evolution ultrasound scanner with a 6C1 curvi-linear array (bandwidth 1.5–6.0 MHz), operating in cadence pulse sequence with a transmit frequency of 2.0 MHz, peak negative pressure 2.0 MPa, and a mechanical index of 1.06 for 6 min at room temperature with bursting flash mode (50 frames at maximum power over 4 s). All pouches were opened, incubated for 2 h, 37 °C, 180 rpm, and fluorescence was read in 100 μL.To measure metabolism after insonation, 200 μL of pSynF samples (including the aggregates) were transferred to pouches. For TSB, 20 μL of 106 CFU/mL MSSA (ATCC 25923) in 1 mL saline with a sterile gloved hand using a 3.81 cm 20-gauge hypodermic needle. After the procedure animals were recovered from general anesthesia under close veterinary supervision until standing and ambulating. Then they were returned to their pen and housed in pairs or single as described above under a natural light-dark cycle. Animals were examined by a veterinarian at 12 and 24 h following joint injections for signs of joint sepsis, systemic health, and gait asymmetry. After 24 h following joint injection, animals were again premedicated and placed under orotracheal inhalant anesthesia as described above. Following an aseptic prep of the skin of the infected joint, ~200–500 µL SynF were collected from the infected joints for microbial plate and nucleated cell counts and total protein content. Then, five pigs received intraarticular AMK (1 mL of AMK (250 mg)) only into the infected femorotibial joint and three pigs received intraarticular AMK (1 mL of AMK (250 mg)) combined with ultrasound treatment using the identical protocol as described below. The remaining nine pigs received intraarticular injection of 100 µL of Definity microbubbles and 1 mL of AMK (250 mg). The microbubble-containing joints were insonated using a S9 Pro scanner  fitted with a curvilinear C353 probe (bandwidth: 2.0–6.8 MHz). Briefly, the access window to the femorotibial joint in this study was via a dorso-medial and dorso-lateral approach using a curvilinear probe. The caudal (posterior) joint space in pigs is very narrow and does not allow for targeted scanning. The joint was taken through multiple range of motion flexion-extension during the UTMD procedure to allow for redistribution of joint fluid. Pulse inversion harmonic imaging with flash replenishment (MI < 0.15) and 4 s destructive pulses (MI > 0.6) was used to burst the microbubbles. Blood was collected pre-inoculation, pre-treatment, and post treatment and submitted for CBC/chemistry analysis. Then animals were recovered from general anesthesia. After 6-8 h post treatment, animals were euthanized with an overdose of Pentobarbital 1 ml/5 kg) according to the guidelines set forth by the current AVMA Panel on Euthanasia and SynF samples were immediately collected from all infected joints for cytological analysis and bacterial quantification. To determine bacterial load, all samples were treated with proteinase K (200 µg/mL) for 10 min and serial threefold dilutions were cultured from all samples and processed for Petrifilm assays to assess bacterial titers (CFU/mL). Then target tissues (synovium and samples of articular cartilage) were harvested and processed for histology.This study was approved by the University of Pennsylvania IACUC committee and was designed and executed consistent with ARRIVE guidelines. Seventeen Yorkshire pigs  castrated male and female; ~2–3 months old; ~35–40 kg body weight) were acclimated and habituated to veterinary care staff for 7–10 days and housed on natural bedding in pairs. Pigs incompatible to pair housing were single housed with visual, olfactory, auditory and touch contact to herd mates. Pigs either received buprenorphine  and fentanyl  or buprenorphine (0.01 mg/kg IM) and morphine (0.2 mg/kg IM) pre- and post-arthrocentesis. Prior to induction, animals were sedated in their pens via intramuscular injection with a combination of tranquilizers and analgesics: Dexmedetomidine (0.02-0.04 mg/kg), Midazolam (0.2–0.4 mg/kg), and Butorphanol (0.1–0.2 mg/kg). Animals were then transported from the pens to the surgical suite. After endotracheal intubation, Isoflurane gas anesthetic (1-4%) was administered with an oxygen carrier, and venous access via an auricular vein was established. Following an aseptic preparation of the skin of one femorotibial joint, the joint was inoculated with 1 × 1032 by a microscopist blinded to treatment.Tissue staining of 10% neutral buffered formalin fixed synovium samples was performed using Brown and Hopps to visualize Gram (+) and (−) bacteria and H&E for tissue ultrastructure. All sections were deparaffinized with xylene and rehydrated in a graded series of ethanol from 100 – 70% (2 × 2 min each) before moving to distilled water. For Brown and Hopps, slides were transferred to 1% Crystal Violet Stain (2 min.) rinsed in DI water to Gram’s Iodine (5 min.), and then rinsed in DI water. Excess water was blotted before decolorizing one slide at a time in acetone until the liquid ran clear, followed by rinsing in running tap water. Basic Fuchsin Stain, 0.25% was applied then rinsed in running tap water. Gallego Solution (5 min.) was used to differentiate then rinsed in running tap water. The slides were then dipped in acetone for 1–2 quick dips, Picric Acid-Acetone 0.05% for 3–10 dips, and Acetone-Xylene 1:1 for 5 dips. Slides were cleared in three changes of xylene, 10 dips each, and then mounted using permount. Sections were visualized by microscopy and scored based on Krenn’s synovial scalet test were used. For those that did not show a normal distribution, either Kruskal–Wallis or Mann–Whitney analyses were used. All measurements were taken from distinct samples.Statistical analyses were performed using Prism statistics. For samples with normal distribution using the Kolmogorov–Smirnov test, ANOVA or Student’s Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


This retrospective study was to evaluate the feasibility and accuracy of coronary artery calcium score (CACS) from virtual non-contrast (VNC) images in comparison with that from true non-contrast (TNC) images.A total of 540 patients with suspected of coronary artery disease (CAD) who underwent a dual-layer spectral detector CT (SDCT) in three hospitals were eligible for this study and 233 patients were retrospectively enrolled for further analysis. The CACS was calculated from both TNC and VNC images and compared. Linear regression analysis of the CACS was performed between TNC and VNC images.r = 0.923, p < 0.001). The CACS from VNC images were lower than that from TNC images . When the regression equation of the overall coronary artery was applied, the mean calibrated CACS-VNC was 221 which had a significant difference from the CACS-TNC (p = 0.017). When the regression equation of each coronary branch artery was applied, the mean calibrated CACS-VNC was 221, which had a significant difference from the CACS-TNC (p = 0.003). But the mean difference between the CACS-TNC and the calibrated CACS-VNC in either way was less than 1. The agreement on risk stratification with CACS-TNC and CCACS-VNC was almost perfect.The correlation of overall CACS from VNC and TNC images was very strong (This multicenter study with dual-layer spectral detector CT showed that it was feasible to calculate CACS from the VNC images derived from the spectral coronary artery CT angiography scan, and the results were in good accordance with the TNC images after correction. Therefore, the TNC scan could be omitted, reducing the radiation dose to patients and saving examination time while using dual-layer spectral detector CT. Coronary artery disease (CAD) is the most common type of cardiovascular diseases globally and the leading cause of death . CoronarA routine non-contrast scan was often taken before the coronary artery CT angiography (CCTA) scan to obtain a CACS . The CCTFurther, several studies demonstrated that the VNC images generated from spectral CCTA data could be used for CACS calculation , 14–16. vol) and dose length product (DLP) of TNC and CCTA scans were analyzed retrospectively. The effective dose (ED) was calculated using the formula: ED (mSv) = DLP × k, where k was the chest (heart) effective dose conversion factor, k = 0.014 mSv•mGy−1•cm−1  were retrospectively included for the analyzes. Among them, 49 patients with coronary stents and 258 patients with a CACS of 0 calculated from the TNC images were excluded after preliminary image analysis. Then, a total of 233 patients were included for further analyzes . Among ty−1•cm−1 .All patients underwent TNC and CCTA ECG-gated scans in the three centers using the SDCT scanner . The main scan parameters of the three centers for TNC were: tube voltage of 120 kVp, tube current ranging from 62 to 191 mA with dose modulation enabled, gantry rotation time of 0.33 s, collimation of 64×0.625 mm, FOV ranging from 206 to 273, reconstruction filter of CB, slice thickness of 2.5 mm, matrix of 512×512. A dose of 50–60 mL of iodine contrast agent was injected at a flow rate of 4–5 mL/s followed by 40–50 mL of saline with same rate. The bolus tracking technique was applied with a trigger threshold of 100 HU in the ascending aorta for the CCTA scan. The scan parameters for CCTA were: tube voltage of 120 kVp, tube current ranging from 147 to 752 mA with dose modulation enabled, gantry rotation time of 0.27 s, collimation of 64×0.625 mm, pitch of 0.16, FOV ranging from 206 to 273, reconstruction filter of CB, slice thickness of 0.9 mm with increment of 0.45 mm, matrix of 512×512. Conventional images were reconstructed using the iterative reconstruction algorithm (iDose-3), and spectral-based-images (SBI) were reconstructed using a spectral algorithm (level 3). For CCTA imaging, Iodinated contrast: Peking Union Medical College Hospital ; The Second Peoples Hospital of Hefei ; Nanjing Drum Tower Hospital .The conventional images and SBI data were transferred onto a dedicated workstation  for post-processing and further analysis. The VNC images were derived from CCTA SBI data with slice thickness setting of 2.5 mm which was same as TNC. Representative samples of TNC and VNC images from the same patient are shown in r < 0.10), weak correlation (0.10 < r < 0.39), moderate correlation (0.40 < r < 0.69), strong correlation (0.70 < r < 0.89), and very strong correlation (0.90 < r < 1.00)  statistical software was used for statistical analysis. Continuous variables were expressed as mean ± standard deviation (SD) and categorical variables were expressed as frequencies or percentages. The Kolmogorov–Smirnov test was used for testing normality of continuous variables. The correlation of the CACS between the TNC and VNC images were determined by Spearman correlation coefficient. Additionally, the Kruskal-Wallis H test was performed to compare the difference between CACS from TNC images from three centers. The agreement was defined as negligible correlation (0.00 <  < 1.00) , 21. Aft < 1.00) . The G*Pvol was 3.9 ± 1.2 mGy•cm, and the ED was 0.7 ± 0.3 mSv for the TNC scan; The CTDIvol was 38.7 ± 16.8 mGy•cm, the ED was 8.0 ± 3.5 mSv for the CCTA scan. Details of the study population and radiation dose are shown in A total of 233 patients  with CACS of >0 were included for further analysis. While for risk stratification analysis, additional 258 patients with CACS of 0 included. The CTDIp < 0.001), but with a very strong positive correlation  respectively. The detailed results are presented in p = 0.274, respectively).For the overall coronary artery, CACS-TNC was significantly higher than CACS-VNC . But the difference was less than 1 (p < 0.001). The overall regression equation was also applied to calibrate each coronary artery branch calcium score and compared with the CACS-TNC. The detailed results were listed in The regression equation for the overall coronary artery was Y = 68.48 + 2.2*X. After the regression equation was applied, the mean CCACS-VNC was 221 which had a significant difference from CACS-TNC , Y = 56.76 + 1.7*X(LAD), Y = 31.62 + 2.31*X (LCX), and Y = 48.4 + 2.02*X (RCA), respectively. After correction with the corresponding coronary artery branch regression equation, the CCACS-VNC for LM, LAD, LCX, and RCA were 96 ± 105,132 ± 122, 83 ± 80, and 107 ± 131, respectively. The CCACS-VNC of LM and LAD arteries had no significant difference with the CACS-TNC (both p > 0.05). The CCACS-VNC of LCX and RCA arteries had significant differences from the CACS-TNC (both p < 0.05). The sum of CCACS-VNC from all the branches was 221 ± 254, which was significantly different with CACS-TNC (p = 0.003), but the difference was less than 1 (p < 0.001). The detailed results are listed in The regression equations for all branch arteries, LM, LAD, LCX, and RCA were p = 0.553); but the CCACS-VNC of all branch arteries showed significant differences . The detailed results are listed in Additionally, no significant statistical difference was found for CCACS-VNC of overall coronary artery calculated by two ways   were regrouped to the adjacent category compared with the results using the CACS-TNC . The agrp = 0.023). And the difference in attenuation value of ascending aorta between the TNC and VNC images was less than 1 HU (p < 0.001). Image noise from the VNC images was lower than that from the TNC images . The statistical power of this study was 0.99.The mean attenuation value of the ascending aorta in CCTA images was 405.93 ± 70.21 HU, and the mean attenuation value of the ascending aorta on the VNC images was slightly higher than that on the TNC images  with the CACS derived from TNC scan. After the regression equation was applied, the corrected CACS from VNC was similar to the CACS-TNC (mean difference of <1). The agreement of risk stratification using the CACS-TNC and CCACS-VNC was almost perfect . Therefore, the TNC scan before CCTA can be omitted to reduce radiation dose to patients and save examination time.This retrospective multi-center study with large cohort patients demonstrated that the CACS derived from the VNC images of CCTA scan had a very strong correlation  patients were assigned to the same category; 432 (88.0%) patients were with branch regression way. Our results were comparable with the outcomes of previous studies conducted by Gassert et al. (83.3%) and Dan Mu et al. (93%). Although Mu et al. proposed a deep learning method to calculate CACS from the CCTA images , the metThere were several limitations in our study. First, the reference standard in this study was the TNC image, which was more likely affected by beam hardening artifacts or calcium blooming artifacts. Second, only the Agatston method was used to calculate the CACS, and other methods such as mass integral or volume integral methods were not used in this study. Second, the contrast medium injection protocols in this retrospective study were not the same, which might impact the VNC results. Although previous studies using different contrast medium injection protocols , 13, 27,In conclusion, this multicenter study with dual-layer spectral detector CT showed that it was feasible to calculate CACS from the VNC images derived from the spectral coronary artery CT angiography scan, and the results were in good accordance with the TNC images after correction. Therefore, the TNC scan could be omitted, reducing the radiation dose to patients and saving examination time while using dual-layer spectral detector CT.zhaorenlxh@126.com.The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by The First Affiliated Hospital of Anhui Medical University. The ethics committee waived the requirement of written informed consent for participation.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported in part by the National Natural Science Foundation of China (82071897 and 81970446) and National Science Foundation for Distinguished Young and Scholars of the Higher Education Institutions of Anhui Province, China (2022AH020071).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.