Therapeutic plasma exchange (TPE) is used for several chronic conditions with little evidence on the efficacy and safety of different choice of replacement fluid. Measurement of haemostasis, particularly in vitro thrombin generation, could play a role in determining the immediate efficacy of different fluid replacement.To determine the impact of different TPE replacement fluid regimens on haemostatic assays.Prospective observational multi‐centre cohort study in adult patients 18 years and older evaluating haemostatic changes between four different TPE regimens: (1) 5% human albumin solution (Alb) only, (2) 50:50 mix of 5% Alb + modified gelatin, (3) 70:30 mix of 5% Alb and normal saline (NS), and (4) solvent‐detergent, virus‐inactivated fresh frozen plasma (FFP) . Twenty‐one haemostasis variables were analysed  pre and post TPE sessions, including in vitro thrombin generation. Linear mixed modelling and canonical discriminant analyses were used to examine the effect of TPE fluid type on haemostatic variables.P < 0.05), excluding plasminogen activator inhibitor‐1 antigen and thrombin‐anti‐thrombin. Multivariate Analysis of Variance showed significant differences between the fluid types  and this was supported by a canonical discriminant analysis, which identified the 4 most discriminating markers for fluid types as thrombin generation , fibrinogen and Factor V. In our analyses, the effect of FFP on haemostasis was significantly greater compared with other fluid types. Of the non‐FFP fluids, 5% Alb + NS had a lower effect on haemostasis compared to other fluid types (Alb and modified gelatin + 5% Alb).A total of 31 patients with up to 5 TPE sessions each  were enrolled. Out of 21 markers analysed using linear mixed modelling, the main effects of fluid type were found to be significant for 19 markers (Thrombin generation and fibrinogen discriminated better the effect of different TPE fluids on haemostasis and should be considered as potential markers to evaluate the immediate haemostatic effect of TPE procedures. The use of NS as a TPE replacement fluid had a distinctive impact on thrombin generation and fibrinogen responses compared to other non‐FFP fluids. There have been very few randomised controlled trials (RCT) to compare the efficacy and safety of different replacement fluids.In addition to removing the targeted abnormal plasma proteins, TPE also removes normal plasma proteins with resultant disturbance of the haemostatic balance, which potentially could increase the risks of bleeding or thrombosis. Biological markers of haemostasis could be used to determine the immediate effect of different replacement fluids, and thus they can potentially be used as outcomes to evaluate the short‐term efficacy of TPE. Indeed, studies that have examined the impact of TPE on haemostasis have shown a significant immediate decline in fibrinogen and other coagulation factors after plasma exchange, with the recovery of these factors occurring at 24 to 72 hours later.2This was a prospective multi‐centre cohort study in adult patients (≥18 years) undergoing TPE at three hospitals (two in England and one in Scotland). The study received research ethical committee (REC) approvals from Health Research Authorities in England (Integrated Research Application System number 247935) and Scotland (REC reference 18/LO/1954). All patients undergoing elective TPE procedures at each study site who were able to give consent were approached and asked for research blood samples only. The standard care of patients was not altered during this study. Prior to enrolment, written informed consent was obtained from all patients. Patients who underwent TPE treatment for thrombotic thrombocytopenic purpura, atypical haemolytic uraemic syndrome and related thrombotic microangiopathies were not included in this study, as TPE is a well‐established treatment in these patients.2.1Each hospital used a different replacement fluid regimen: Hospital‐1 used exclusively 5% albumin solution (Alb), Hospital‐2 used a 50:50 mix of 5% Alb and Gelofusine , which use a continuous flow centrifugation‐based plasma separator technique. Each patient underwent 1‐1.5 plasma volume (PV) exchanges per session, calculated using the formula shown in below.Eq. 1 Estimated plasma volume (EPV) calculationResult multiplied by 1.5 for 1.5 PV exchange.2.2g for 12 minutes to prepare platelet poor plasma (PPP) which was stored in aliquots at −70°C ± 10°C until day of processing.Research venous blood samples were collected immediately before and after each exchange procedure for up to five exchange sessions in each patient. Blood samples were collected in 5 mL BD vacutainers  each containing 0.5 mL of 0.109 M buffered tri‐sodium citrate in a ratio of 1‐part anticoagulant to 9‐parts blood. Samples were processed within 4 hours of collection by double centrifugation at 20002.3Haemoglobin, platelet count, creatinine and albumin were analysed on each site as per laboratory standard operating procedures (SOP) using automated Sysmex XN analysers and the spectrophotometry, Roche Cobas 8000 analysers, respectively. All haemostatic tests were performed in one laboratory, and these included: prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, factor (F) II, FV, FVII, FVIII, FIX, FX FXI, FXII, alpha‐2‐antiplasmin (A2AP), plasminogen, prothrombin fragment 1 + 2 (F1 + 2), thrombin antithrombin complexes (TAT), plasminogen activator inhibitor‐1 antigen (PAI‐1) and thrombin generation test.One‐stage clotting factor assays for FII, FV, FVII, FVIII, FIX, FX, FXI and FXII were performed on a Sysmex CS5100 analyser, using the laboratory's SOP and Siemens reagents. Enzyme linked immunosorbent assay (ELISA) were used for F1 + 2, TAT (using Siemens Enzygnost ELISA kits), A2AP, plasminogen assays and PAI‐1 (using Hyphen BioMed ELISA kit) following manufacturer's instructions.2.4, and area under the curve ). All patient samples were tested in triplicate: the analysed data is the average of the replicates. All clotting tests were performed in one laboratory and by one person to reduce variations.The calibrated automated thrombogram method as described by Hemker et al.2.5The analysed dataset consisted of 31 patients with up to five TPE sessions each . Pre‐ and post‐exchange tests were made at each session and on each patient. Haemostatic responses were cleaned using clinical and statistical judgement, and levels of missing data were investigated and deemed acceptable in all cases. To examine the effect of TPE fluid type on haemostatic responses, two statistical approaches were used: (1) a linear mixed model with fluid type as the fixed effect, and (2) a canonical discriminant analysis. Four different fluid types were investigated; 5% Alb, 5% Alb + NS, Gelofusine + 5% Alb and Octaplas (either in isolation or mixed with other fluids). Twenty‐one haemostasis variables were analysed: FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, Fibrinogen, PT, APTT, F1 + 2, TAT, PAI‐1, A2AP, plasminogen, lag‐time, peak‐thrombin height, ETP, ttPeak and VI. We were able to analyse all these variables with the models proposed, as a linear mixed model analyses one haemostatic variable at a time and canonical discriminant analysis is a dimension reduction technique especially robust at dealing with a large number of predictor variables. Tabachnick and Fidell2.6fluid type. The random component was set to patient ID (to identify repeated measurements on the same patient over different exchange sessions) and time interval . The response variable was taken as the difference between the post‐ and pre‐exchange measurements for each of the 21 haemostasis markers at each session and on each patient. For simplicity, our analysis assumed independence amongst haemostasis markers and separate linear mixed models were fitted to each marker.The fixed component of the mixed model was set to 2.7difference between the post‐ and pre‐exchange measurements for each of the 21 haemostasis markers at each session as the predictors. Fluid type was used as the pre‐defined grouping variable of interest. The analysis identified the predictors that are most influential in discriminating between the fluid types. Canonical discriminant analysis is a multivariate statistical technique where independence of haemostatic parameters is not assumed and all parameters are considered simultaneously. As part of the discriminant analysis, we performed a multivariate one‐way analysis of variance (ANOVA).A canonical linear discriminant analysis was performed using the 33.1Of the 31 patients in total, 20 (65%) underwent 5 TPE exchange sessions, 2 (6%) underwent 4 sessions, 6 (19%) experienced 3 sessions, 2 (6%) had 2 sessions and 1 patient (3%) underwent one exchange session only. This analysis did not attempt to determine how the number of TPE sessions affected haemostatic assay results. Characteristics of patients, reasons for needing TPE and the details of each fluid type per hospital are provided in Table 3.2fluid type were found to be statistically significant at the 5% level for 19 markers  for all markers except thrombin generation parameters.Table Table 3.3F63,245.61 = 5.50; P < 0.0001). Figure We used canonical linear discriminant analysis as an alternative statistical approach to identify the haemostatic parameters that are most influential in discriminating between the four fluid type groups. A multivariate ANOVA on the hypothesis that the fluid type groups are equal indicated statistically significant differences between the groups . Raw canonical coefficient estimates (not shown) for CV1 in this analysis again identified thrombin generation parameters (lag‐time and ttPeak), Fibrinogen and FII as the top discriminating markers. Figure x axis in Figure A further canonical linear discriminant analysis of only three fluid type groups (excluding Octaplas) indicated statistically significant differences between the groups . We enrolled 31 patients who received a total of 131 TPE sessions.Consistent with previous studies, our data show that coagulation factors pre and post exchanges reduce with all types of fluids.In both our analyses the difference between Octaplas and other fluid types was statistically significant, which is not surprising when we consider the difference in content between Octaplas and other fluids (the former being a plasma product). Of the non‐Octaplas fluids, the use of NS as a replacement fluid for TPE mixed with 5% Alb had a distinctive effect on thrombin generation and fibrinogen responses (the most discriminating CV1 markers) compared to other non‐plasma fluid types, as shown in Figure 5: being an observational study, and the population of patients being intrinsically heterogeneous with clinical indication for TPE and duration between TPE sessions being different at each of the three hospital sites, although we account for these in our modelling.This is the first multi‐centre study providing a comprehensive haemostatic assessment of more than two different replacement fluid types pre and post TPE. However, there are limitations to this study such asThe multivariate nature of canonical discriminant analysis and multivariate ANOVA—where all haemostatic markers are considered simultaneously, is appropriate when variables are correlated Figure  and inde6Thrombin generation and fibrinogen were the best markers for discriminating between different TPE fluid types. Further studies are needed to evaluate the relationship between these markers and clinical efficacy/safety of TPE procedures, with the view to using them as potential outcome measures for study evaluation or clinical practice. The effect of Octaplas on haemostasis was significantly different compared with other fluid types, while the use of NS in combination with other TPE fluid, had a distinctive impact on haemostasis (vs other non‐Octaplas fluid types), in particular on thrombin generation and fibrinogen responses. Future longitudinal studies are needed to evaluate if this effect has an impact on clinical outcomes, and randomised control trials should compare the cost‐effectiveness of different TPE fluids.The authors declare no conflicts of interest.Appendix S1 Supporting informationClick here for additional data file.Appendix S2 Supporting informationClick here for additional data file.


Transthoracic esophagectomy is associated with significant morbidity and mortality. Therefore, it is imperative to optimize perioperative management and minimize complications. In this retrospective analysis, we evaluated the association between fluid balance and esophagectomy complications at a tertiary hospital in Melbourne, Australia, with a particular focus on respiratory morbidity and anastomotic leaks. Cumulative fluid balance was calculated intraoperatively, postoperatively in recovery postoperative day (POD) 0, and on POD 1 and 2. High and low fluid balance was defined as greater than or less than the median fluid balance, respectively, and postoperative surgical complications were graded using the Clavien-Dindo classification.In total, 109 patients, with an average age of 64 years, were included in this study. High fluid balance on POD 0, POD1 and POD 2 was associated with a higher incidence of anastomotic leak . High fluid balance on POD 2 was associated with more severe complications (of any type)  and severe pulmonary complications . For every 1 L extra cumulative fluid balance in POD 1, the odds of a major complication increase by 15%, while controlling for body mass index (BMI) and American Society of Anaesthesiologists (ASA) class. The results show that higher cumulative fluid balance is associated with worsening postoperative outcomes in patients undergoing transthoracic esophagectomy. Restricted fluid balance, especially postoperatively, may mitigate the risk of postoperative complications – however prospective trials are required to establish this definitively.The online version contains supplementary material available at 10.1186/s13104-023-06574-x. Despite the continuous advancement of operative techniques and perioperative care over recent years, international multicenter studies persistently demonstrate a high complication rate of up to 65% after esophagectomy. Pulmonary and gastrointestinal complications are the leading cause of morbidity, with pneumonia and anastomotic leak the most frequent complications –3. PulmoMultiple factors contribute to the development of pulmonary complications and anastomotic leakage, of which with fluid overload is recognized as a substantial contributor, particularly in the context of pulmonary complications –7. We coThe study conducted was at a university-affiliated tertiary referral center. The protocol was registered in the Australian New Zealand Clinical Trials Registry (ACTRN234344). The study was conducted in accordance with the STROBE guidelines for observational studies . This stThe study evaluated patients aged 18 years or older, who underwent elective esophagectomy  between January 2010 and December 2019 for esophageal cancer, benign tumor, or end-stage motility disorders.A standardized ERAS protocol was applied intraoperatively and postoperatively . IntraopPatients’ data were extracted from the prospectively collected hospital’s electronic health records. Complications severity was graded by the Clavien-Dindo (CD) classification system . In alliThe intraoperative fluid rate was defined as mL/kg/hour of the surgical procedure. The intraoperative balance was calculated as the total volume of administrated crystalloids, colloids, and blood products minus the urine output and blood loss. Postoperative fluid balances for days 0, 1 and 2 were calculated as the cumulative volume surgery to the end of days 0, 1, and 2. High and low fluid balances were defined as above and below the median fluid balance, respectively. Postoperative complications were defined by the European Perioperative Clinical Outcome definitions . Key comStandard inferential statistics were used for comparing continuous and categorical variables. Univariate and multivariate analyses were performed to evaluate the contribution of risk factors to the development of complications. The statistical analysis was performed using R  . To asseThe multivariant analysis models included risk factors identified a-priori in the univariate analysis. POD 0, POD 1, and POD 2 were used in separate multivariate models to avoid multicollinearity. Second, we performed a univariate and multivariate regression model using cumulative fluid balance as a continuous variable. Volume was converted to liters for improved interpretability and postoperative days (POD) 0 to 2 were used in separate multivariate models to avoid multicollinearity. We analyzed the risk factors associated with major complications, major pulmonary complications, and anastomotic leaks.Calculated odds ratios (OR) were provided with 95% confidence intervals (CIs). If required, the probability values were modified using the Bonferroni correction method. A comparative analysis is shown with the corresponding p-value and effect size. Statistical significance was defined as a two-tailed p-value < 0.05.A total of 110 elective esophagectomies were performed during the study period. One case was excluded because of missing fluid balance data for POD 2. Thus, a total of 109 elective esophagectomies were included for analysis. Detailed patient demographic, oncological and perioperative variables are further detailed in the Additional File Table The perioperative fluid balances during surgery to POD 2 are presented in Fig. p = 0.023) was associated with high fluid balance, while hybrid procedures were related to low POD 2 fluid balance. Patients with lower preoperative albumin did not significantly differ in their fluid balance on POD 2. Perioperative factors and their association with POD 2 fluid balance are summarized in Table We used the fluid balance on POD 2 to examine the correlation between perioperative factors and high fluid balance. High and low-volume groups were defined as above and below the median fluid balance of 3918 mL (rounded to 3950mL), respectively. Advanced clinical tumour stage T3 or T4 (Complications were recorded in 107 patients (98%), with 46 patients (42%) experiencing major complications (CD IIIa-V). Cardiovascular, pulmonary, and gastrointestinal complications were the most common categories occurring in 84%, 71% and 52% of the patients, respectively. The single most common complication for each of those categories was hypotension  , pneumonia , and anastomotic leak .p = 0.003) (Table p < 0.001) compared to their corresponding low-volume groups . Similarly, a higher fluid balance on POD 0, POD 1 and POD 2 was associated with an increased overall incidence of major complications, major gastrointestinal complications, and anastomotic leak  Table ; howeverPatients with a high fluid balance on POD 1 and POD 2 were more likely to have severe complications (of any type)  and severe pulmonary complications . There was no statistically significant difference in the incidence of kidney injury between the high and low-volume fluid balance in either of the groups.The risk factors associated with major complications, major pulmonary complications, and anastomotic leak are presented in Table Regarding anastomotic leaks, a high postoperative high fluid balance was an independent risk factor throughout the postoperative monitored period, with a high fluid balance on POD 2 having the most effect , followed by POD 0  and POD 1   class.Our data highlight the significant negative impact of daily high postoperative positive balances on complications severity, the incidence of pulmonary complications, and anastomotic leaks. The association between volume overload and overall morbidity is consistent with a growing body of literature. Eng et al. showed that high intraoperative volume resulted in more complications per patient and increased major complications . Glatz ePulmonary complications, particularly pneumonia, cause substantial morbidity following esophagectomy. In the context of esophagectomy, one-lung intubation and thoracotomy-related respiratory pain are significant contributing factors. In addition, excessive fluid may add risk by inducing extravascular pulmonary tissue oedema, altering the lung physiology . We demoOur findings also demonstrated a significant association between fluid excess and anastomotic leaks. Similarly, Glatz et al. reported that intra- and postoperative overload induces a three-fold incidence of anastomotic leakage. In addition, Kubo et al. showed a similar association in minimally invasive esophagectomy , 6. TherHigher volumes of fluids were given intraoperatively compared to the postoperative daily volume. Rapid hemodynamic changes during the operation can result from analgesia, surgical maneuvers, blood losses, or one lung intubation and require fluid administration in response. Despite that, we could not demonstrate a significant adverse effect of intraoperative fluid overload on complications . In contrast, Eng et al. reported that high intraoperative fluid volume increased the complication rate and severity per patient but did not specifically affect pulmonary complications and anastomotic leak , suggestOur analysis showed that positive fluid balance on POD 0 to POD 2 was linked to adverse outcomes. For every 1 L extra cumulative fluid balance in POD 1, the odds of a major complication increase by 15%, even when controlling for BMI and ASA class. In other studies, examining the early postoperative period, only the POD 1 fluid balance was associated with a higher risk for pulmonary complications or anastomotic leak , 19. In Fluid administration was at the discretion of either the anaesthetist in the theatre, or the surgeon, and was confined to the early postoperative period. Furthermore, we defined ‘volume overload’ arbitrarily and without correcting to patient parameters . This study was conducted in a single-center hospital, which may limit its external validity to other centers and countries. Similarly, our findings are not generalizable to other types of major abdominal and thoracic surgery. We did not collect the number of hypotension epochs, nor did we quantity the magnitude of each hypotensive event. We acknowledge that hypotension is associated with microcirculatory hypoperfusion and adverse outcomes , 22. FinBelow is the link to the electronic supplementary material.Supplementary Material 1


P < 0.05) difference was found in the tumour margin, enhancement pattern, growth pattern, and LD values between the 31 patients with GSs and 62 matched patients with GISTs. The GS lesions were mostly (93.5%) well defined while only 61.3% GIST lesions were well defined.The GS lesions were significantly (P = 0.036) smaller than the GIST lesions, with the LD ranging 1.5–7.4 (mean 3.67 cm) cm for the GSs and 1.0–15.30 (mean 5.09) cm for GIST lesions. The GS lesions were more significantly (P = 0.001) homogeneously enhanced (83.9% vs. 41.9%) than the GIST lesions. The GS lesions were mainly of the mixed growth pattern both within and outside the gastric wall  compared with that of GISTs. No metastasis or invasion of adjacent organs was present in any of the GS lesions, however, 1.6% of GISTs experienced metastasis and 3.2% of GISTs presented with invasion of adjacent organs. Heterogeneous enhancement and mixed growth pattern were two significant (P < 0.05) independent factors for distinguishing GS from GIST lesions. In conclusion: GS and GIST lesions may have significantly different features for differentiation in lesion margin, heterogeneous enhancement, mixed growth pattern, and longest lesion diameter, especially heterogeneous enhancement and mixed growth pattern.To investigate clinical data and computed tomographic (CT) imaging features in differentiating gastric schwannomas (GSs) from gastric stromal tumours (GISTs) in matched patients, 31 patients with GSs were matched with 62 patients with GISTs (1:2) in sex, age, and tumour site. The clinical and imaging data were analysed. A significant ( Among them, leiomyomas tend to occur around the cardia and gastric fundus, and glomus tumours often have significant specific enhancement on imaging examination. The incidence of neurofibroma is very low, and they are mostly reported in case reports. More than 75% of inflammatory fibroid polyps of the stomach manifest as nodules growing in the cavity of the gastric antrum3. In typical ectopic pancreas whose onset site is close to the pylorus, the imaging enhancement characteristics are similar to those of the pancreas and are characterized by intraluminal growth and an oval-shaped mass with a long axis along the gastric wall4. GS is a relatively rare submucosal tumour derived from the gastrointestinal tract and occurs mostly in the stomach, accounting for 2%-6% of all gastrointestinal mesenchymal tumours7. Its histological and imaging features are quite different from those of soft tissues or central nervous system tumours10. As a type of mass with a relatively homogeneous texture, smooth edges but no capsules11, the lesions are mostly benign and slow-growing. GS almost never relapses after surgery12, and endoscopic resection of the tumour can achieve satisfactory therapeutic effects13, which is significantly different from the treatment strategy and prognosis of GISTs14. If GSs can be distinguished from GISTs by imaging features, this will greatly help with the later treatment of the patients. Some studies6 have shown that the imaging features of enhanced computed tomography (CT) scans and tumour morphology can be used to distinguish GSs from GISTs. However, the imaging features of GSs overlap much with those of GISTs, which is one of the challenges in distinguishing the two. It was hypothesized that a combination of clinical characteristics and CT imaging features of GSs could be used to differentiate GSs from GISTs. This study was consequently performed to investigate the clinical characteristics and CT imaging features of GSs in distinguishing the two diseases using matched cases for control.Gastric subepithelial tumours are not rare even though there is no definitive epidemiology of these tumours, which may cover a variety of pathological types, including benign or malignant tumours, a tumour-like lesion or a tumour with malignant potential. Gastrointestinal stromal tumours (GISTs) have the highest incidence and the most significant biological diversity in this large group of tumours. Other tumours or tumour-like lesions similar to GISTs include leiomyoma, gastric schwannoma (GS), neurofibroma, glomus tumour, and inflammatory fibroid polyp of the stomach and ectopic pancreasThis retrospective one-center study was approved by the ethics committee of Hebei Medical University Affiliated Forth Hospital, and the informed consent was waived by the same ethics committee of the Fourth Hospital of Hebei Medical University because of the retrospective study design. All methods were conducted in accordance with the relevant guidelines and regulations. From January 2014 to October 2020, a total of 482 consecutive patients with gastric subepithelial tumours confirmed by surgical pathology and immunohistochemical examinations were identified from the picture archiving and communication (PACS) database in our hospital. Figure The medical information of the patients, including clinical data, surgical findings, and results of pathologic examination, was collected. Abdominal contrast-enhanced CT examinations were performed using a multidetector row CT (MDCT) scanner: a Brilliance IC  and SOMATOM Definition Flash  CT: a Lightspeed Pro 32 . Patients were instructed to fast for at least 6 h before CT scanning. Ten minutes before the scan, each patient was administered 10 mg of anisodamine intramuscularly to reduce peristalsis of the gastrointestinal tract before drinking 500–1000 mL water to expand the stomach fully. For MDCT scanners, the scanning parameters were as follows: detector configuration 0.625–1 mm, pitch 0.891–1.35; rotation time 0.5–0.75 s, 120kVp, and 150–250 mAs. MDCT images were reconstructed with a slice thickness and reconstruction interval of 3 mm. For 3D reconstruction, MDCT images were reconstructed with a slice thickness and reconstruction interval of 1–1.5 mm. In addition to axial MDCT images, coronal and sagittal multiplanar reformatted images were captured.15. To measure the maximal enhancement degree of the lesion, the lesion was measured in the area with the most significant enhancement in the venous phase, judged by the naked eye. In the arterial phase, the same site and the same size region of interest were selected as in the venous phase. When measuring, blood vessels visible to the naked eye and necrotic areas were avoided. The degree of enhancement was divided into good (degree of enhancement over 40 HU), moderate (degree of enhancement ranging 20–40 HU), and poor (degree of enhancement less than 20 HU). For tumour growth, lymph nodes surrounding the lesion with a short diameter of more than 5 mm were defined as positive. Tumour invasion of surrounding organs or distant metastasis was also recorded. Tumour location was defined as follows: 1, the gastric fundus and cardia; 2, gastric body; 3, gastric antrum.The image features of gastric tumours Table  were derP < 0.2 in the bivariate analysis were included into the multivariate regression models. Conditional logistic regression analysis was used to estimate the odds ratios (ORs) and 95% confidence intervals (CIs). The receiver operating characteristics (ROC) curve analysis was performed to investigate the diagnostic ability of relevant parameters. In all tests, a value of p˂0.05 was considered a significant difference.The statistical analysis was performed with the SPSS version 25 . We used a 1:2 matching ratio for selection of the control group to reduce selections bias. To evaluate the differences in patient characteristics, including age, sex, and tumour-specific data, between the two groups, the Mann–Whitney U test, Pearson’s chi-squared test, and Fisher’ exact test were used. Variables with This retrospective one-center study was approved by the ethics committee of Hebei Medical University Affiliated Forth Hospital, and the informed consent was waived by the same ethics committee because of the retrospective study design.Totally, 31 patients with GSs (aged 56 years in mean and 36–77 years in range) and 213 cases with GISTs (aged 58 years in mean and 36–77 years in range) were identified to meet the inclusion criteria (Table The GS group consisted of 9 men aged 62.5 (rang 50–77) years and 22 women aged 54.9 (range 36–77) years. A group of GISTs patients were randomly selected as the control group to match the GS group (n = 31) in the following parameters: 1:2 in number, sex, age ± 3 years, and location of the tumour (Table P < 0.05) difference existed in the sex component, tumour location, tumour contour, margin, surface, enhancement pattern, growth pattern, and LD values between 31 patients with GSs and 213 patients with GISTs  difference was still found in the tumour margin, enhancement pattern, growth pattern, and LD values between the 31 patients with GSs and 62 matched patients with GISTs (Table P = 0.036) smaller than the GIST lesions, with the LD ranging 1.5–7.4 (mean 3.67 cm) cm for the GSs and 1.0–15.30 (mean 5.09) cm for GIST lesions. The LD/VD ratio was not significantly (P > 0.05) different between the two groups. In contrast enhancement, the GS lesions were more significantly (P = 0.001) homogeneously enhanced (83.9% vs. 41.9%) than the GIST lesions even though the enhancement degree in the arterial phase or venous phase was not significantly different . The growth pattern of tumour was significantly (P < 0.05) different between the two lesions, with the GS lesions mainly of the mixed growth pattern both within and outside the gastric wall (74.2% vs. 22.6%). No significant (P > 0.05) differences were detected in tumour contour, presence of mucosal ulceration in the gastric cavity, haemorrhage, calcification, perilesional lymph nodes, metastasis, and invasion of adjacent organs  features for differentiating GS from GIST lesions. In mutlvariate analysis, heterogeneous enhancement and mixed growth pattern were two significant (P < 0.05) independent factors for distinguishing GS from GIST lesions.In univariate analysis of risk factors for different lesions, ill-defined lesion margin, heterogeneous enhancement, mixed growth pattern, and longest lesion diameter were significant . This is similar to the combined diagnostic value using all four indicators (with an AUC of 0.875). This study may suggest that even if gastric stromal tumors and gastric schwannoma have similar onset locations, some reference indicators exist that can provide valuable identification information to distinguish the two tumors.16. Before matching in this study, 83.9% of GSs occurred in the gastric body, whereas only 26.6% of GISTs occurred in the gastric body. Because of the overlapping in the location of disease, it is difficult to distinguish the two types of tumour by location alone. Therefore, the onset location was treated as one of the factors for case matching. The incidence of GSs in females was also higher than that in males in our study, consistent with the findings by Choi et al.17. Because of the similar incidence of GISTs in females and males, sex was also used as one of the matching factors. A 1:2 matching ratio was used to match the GSs and GISTs patients based on age, sex, and tumour location to explore the value of tumour imaging characteristics in the differentiating GSs and GISTs.GSs are a relatively rare benign gastric subepithelial tumour that often occurs in the gastric bodyThe two most valuable indicators in differentiating the two tumours were the growth and enhancement pattern. Among the GSs, 74.2% (23/31) showed bidirectional growth across the gastric wall, whereas only 22.6% (14/62) of GISTs had this growth pattern. GSs rarely had an exogenous growth pattern on the gastric wall , whereas GISTs had a significantly greater proportion in the three growth modes. In enhancement pattern, 83.9% (26/31) of GS cases was relatively homogeneous after enhancement, showing progressive enhancement, whereas only 41.9% (26/62) GISTs exhibited this pattern.6 found that GSs were different from schwannomas in other organs which were more homogeneous on CT images before and after enhancement. In the study by Wang et al.18, 18 of 19 GSs patients (94.7%) showed homogeneous enhancement. Therefore, homogeneous enhancement pattern was typical of GSs. Heterogeneous enhancement of GISTs is more common, consistent with the heterogeneity of these tumours. Although the growth and enhancement patterns were two significant independent factors for distinguishing the two tumours, they were not sufficient because of overlapping of these features in some tumours.Lin et al.7. Overall, GSs are relatively small. This is consistent with the slow growth of benign tumours. Irregular shape and large tumour diameter are more common in GISTs. With the characteristics of tumour enlargement, fast and outward growth, an irregular shape of the tumour is bound to appear besides the feature of a large lesion. However, we did not find a value of the optimal diameter in distinguishing the two types of tumours. Choi et al.17 found that the GS lesion size of smaller than 5 cm might be used to distinguish from GISTs, but their cohort was too small besides some confounding factors.Our results showed that the LD of GSs was in the range of 1.5 cm to 7.4 cm, in line with the findings by Mekras et al.19. Both tumours have the possibility of calcification, but intratumoural bleeding is rare. Only five GISTs showed intratumoural bleeding in this study. Metastasis and surrounding tissue invasion were also only seen in patients with GISTs. These features are external manifestations of the malignancy of GISTs and highly suggested that GISTs are malignant20.In both types of tumours, progressive enhancement was the dominant form of CT enhancement, with similar enhancement pattern and degree. When the tumour grows into the gastric cavity, ulcers may occur on the surface of the tumour, which is the reason why patients with either tumour may have gastrointestinal bleedingIn our study, no enlargement of peripheral lymph nodes was observed in cases of gastric schwannoma which is a benign tumor of the stomach. Even if some researchers have discovered the presence of lymph nodes in gastric schwannomas, it may be just an accidental phenomenon rather than a must because of the benign nature of gastric schwannoma.21 like others did to differentiate GSs from GISTs, which may all affect the outcome of this study. Future prospective, randomized, multi-center studies involving multiple races and ethnicities will have to be performed to reach more valuable and better outcomes.Some limitations existed in this study, including the retrospective and once-center study design, a small cohort of patients, Chinese patients enrolled only, and further investigation using radiomicsIn conclusion, GS and GIST lesions may have significantly different features for differentiation in lesion margin, heterogeneous enhancement, mixed growth pattern, and longest lesion diameter, especially heterogeneous enhancement and mixed growth pattern.


Neisseria meningitidis, the causative agent of meningitis and meningococcal septicemia. The overall fold of NMA1982 closely resembles that of protein tyrosine phosphatases (PTPs). However, the hallmark C(X)5R PTP signature motif, containing the catalytic cysteine and invariant arginine, is shorter by one amino acid in NMA1982. This has cast doubt about the catalytic mechanism of NMA1982 and its assignment to the PTP superfamily. Here, we demonstrate that NMA1982 indeed employs a catalytic mechanism that is specific to PTPs. Mutagenesis experiments, transition state inhibition, pH-dependence activity, and oxidative inactivation experiments all support that NMA1982 is a genuine PTP. Importantly, we show that NMA1982 is secreted by N. meningitidis, suggesting that this protein is a potential virulence factor. Future studies will need to address whether NMA1982 is indeed essential for N. meningitidis survival and virulence. Based on its unique active site conformation, NMA1982 may become a suitable target for developing selective antibacterial drugs.Protein phosphorylation is an integral part of many cellular processes, not only in eukaryotes but also in bacteria. The discovery of both prokaryotic protein kinases and phosphatases has created interest in generating antibacterial therapeutics that target these enzymes. NMA1982 is a putative phosphatase from  About one third of all eukaryotic proteins are phosphorylated, predominantly on serine, threonine, and tyrosine3. In bacteria, phosphorylation on aspartate and histidine is well characterized as part of the two-component system4. However, bacterial proteins are also phosphorylated on serine, threonine, and tyrosine6. Interestingly, phosphorylation on tyrosine is much more abundant in bacterial proteins (9%), relative to serine and threonine, than it is in humans (1.8%)7. Both protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) have been isolated from bacteria10 and have been proposed as anti-bacterial drug targets12.Protein phosphorylation is considered a universal mechanism to regulate protein function during most cellular processes14. All PTPs have a cysteine-based catalytic mechanism 5R that forms the phosphate-binding loop, also known as the P-loop. Catalysis is further assisted by an aspartic acid residue that functions both as the catalytic acid, donating a proton to the leaving group in the first step, and the catalytic base, activating the water molecule in the second step of the PTP reaction. This aspartic acid is part of the WPD-loop, named after the conserved tryptophan–proline–aspartate (WPD) motif in all classical PTPs. In DUSPs, only the aspartic acid is conserved; hence, this loop is also referred to as the D-loop. The WPD-loop fluctuates between an “open” conformation, allowing substrate binding, and a “closed” conformation, necessary for catalysis. Decreased mobility of the WPD-loop can result in substantially lower catalytic activity, as shown by mutational studies17 as well as allosteric inhibitors that decrease WPD-loop flexibility18.The PTP superfamily contains various subfamilies with the two largest being the classical PTPs, which only dephosphorylate phosphotyrosine (pTyr), and the dual-specificity phosphatases (DUSPs), which in addition to pTyr can also dephosphorylate serine, threonine, or non-protein substrates such as phospholipids or RNAechanism , which iNeisseria meningitidis is a human pathogen responsible for causing meningococcal diseases such as meningitis and meningococcal septicemia19. Despite established vaccination strategies, N. meningitidis continues to be an important cause of mortality and morbidity in newborns and children in both developed and underdeveloped countries20. Unlike viral meningitis, meningococcal meningitis can potentially be fatal within 24 h after the first symptoms appear21. While antibiotics typically provide an effective treatment, cases of N. meningitidis antibiotic resistance have been reported22. Thus, the identification of novel drug targets that can be exploited for the treatment of meningitis is an unmet medical need. Previously, we reported the crystal structure of the N. meningitidis protein NMA1982, analysis of which suggested that its overall fold shows strong similarity to the DUSP subfamily of PTPs23. However, the P-loop containing the hallmark C(X)5R PTP signature motif is shorter by one amino acid in NMA1982, forming a C(X)4R motif. This disparity has cast doubts about the catalytic mechanism of NMA1982 and the assignment of NMA1982 to the PTP superfamily. Here, we show that NMA1982 indeed uses a PTP-like catalytic mechanism, demonstrating that the shorter C(X)4R motif is suitable to perform phosphatase catalytic function. Moreover, using pH-dependence, small molecule transition state inhibition, and oxidative inactivation experiments, we confirm that NMA1982 behaves like a typical PTP. Finally, we show that NMA1982, like other virulence factors, is secreted by N. meningitidis, suggesting that this phosphatase may be important for bacterial infection.24 structural alignment of the crystal structures of NMA1982 and human DUSPs, we found that cyclin-dependent kinase inhibitor 3 (CDKN3), also known as kinase-associated phosphatase (KAP), is the most similar human phosphatase to NMA1982, with an amino acid identity of 15% and a Dali Z-score of 14.9 (13 could be identified in NMA1982 (1) The phosphate-binding loop (P-loop), which forms the center of the active site, is present in NMA1982, albeit shorter by one amino acid. In NMA1982 the putative catalytic cysteine (C95) and invariant arginine (R100), which both align very well with the corresponding residues in CDKN3, form a C(X)4R motif instead of the typical C(X)5R PTP signature motif. 2) The general catalytic acid/base aspartate-containing WPD-loop is also present in NMA1982. D71 putatively serves the role of the catalytic acid/base. Notably, the WPD-loop in the NMA1982 crystal structure adopts an atypically open conformation (with D71 far removed from the active site), which also has been observed for several human PTPs25. 3) NMA1982 features a typical PTP E-loop, which contains a conserved glutamic acid that coordinates the side chain of the invariant arginine in the P-loop and positions it for phosphate binding13. Indeed, in the NMA1982 crystal structure, the side chain carboxylic acid of E53 forms a salt bridge with the guanidinium group of R100. Collectively, our comparison with human DUSPs confirms that amino acids and loops critical for phosphatase activity are present in NMA1982.Using pairwise Dali of 14.9 . Importa NMA1982 : 1) The 23. To assess the catalytic parameters of NMA1982 more precisely in a kinetic experiment, we adapted a standard, continuous, fluorescence-based phosphatase assay using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as the substrate27. Using this assay, we performed a Michaelis-Menten experiment to determine the Michaelis-Menten constant and turnover number for NMA1982 , VHR (Km = 39 μM), and MKP-1 (Km = 40 μM); the classical PTPs PTP1B (Km = 4 μM), CD45 (Km = 43 μM), and SHP1 (Km = 52 μM); as well as the low-molecular weight PTPs LMPTP-A (Km = 63 μM) and LMPTP-B (Km = 218 μM). Thus, the Michaelis-Menten kinetic data suggest that the standard PTP substrate DiFMUP is similarly well accepted by NMA1982 and human PTPs. The turnover number for the NMA1982-catalyzed DiFMUP reaction was calculated to kcat = 4.4 × 10−05 s−1. This value was at least two orders of magnitude lower than the kcat values for the human PTPs we tested, suggesting that the catalytic conversion of the substrate is slower for NMA1982 compared to many human PTPs. Interestingly, a comparably low activity has been reported for CDKN3, the human PTP that is most similar to NMA198228.We previously showed that NMA1982 has phosphatase activity using p-nitrophenyl phosphate as a pTyr surrogate substrate in a colorimetric assay NMA1982 . The calC95-R-T-G-T-R100 sequence, is one amino acid shorter than the typical PTP P-loop containing the C(X)5R signature motif. Superposition of the NMA1982 crystal structure with crystal structures of human DUSPs suggested that the putative catalytic cysteine C95 and the putative invariant arginine R100 in NMA1982 align very well with the corresponding residues in human DUSPs . These data demonstrate that C95 and R100 of the C(X)4R motif are essential for NMA1982 phosphatase activity.The putative P-loop in NMA1982, consisting of the 30. However, the degree of this loss in activity can vary widely among the PTPs and depends on whether other, nearby amino acids can compensate for the absence of Asp by providing a similar functionality31. To test the role of D71 for NMA1984 activity, we generated recombinant NMA1982 D71A mutant protein. We then assessed phosphatase activity of the mutant using a Michaelis-Menten kinetic assay with DiFMUP as the substrate. We found a substantial drop of 50% in the catalytic efficiency (kcat/Km) for the D71A mutant protein compared to wt NMA1982, demonstrating that D71 is important for catalysis , a general PTP inhibitor that resembles inorganic phosphate and binds into the catalytic pocket as a transition state analog32. Using our established DiFMUP assay, we found that orthovanadate inhibited NMA1982 activity in a dose-dependent manner with an IC50 value of 161 μM (33 and our unpublished data). Next, we tested the pH dependence of the NMA1982 phosphatase reaction. We subjected wt NMA1982 to catalytic rate measurements under various pH conditions and found that NMA1982 activity peaked at pH 6 , which is released upon proteolysis41, or PorB porin, which is released via outer membrane vesicles (OMVs)43. The similarity of NMA1982 to eukaryotic PTPs strongly suggests that it may act as a virulence factor. We therefore assessed the secretion of NMA1982 in the model organism N. meningitidis 8013, which expresses the NMA1982 ortholog NMV0640 . A derivative of the wt NEM8013 strain lacking NMV0640 was engineered as a control (ΔNMV0640). Both wt and ΔNMV0640 strains were grown with agitation in rich medium before the cells and supernatant were separated, collected, and processed by SDS-Page and immunoblot analysis , and the type I secretion systems (T1S)44. The T1S is devoted to the secretion of the iron-regulated proteins FrpA/C only. Secretory proteins of the autotransporter and TPS first cross the inner membrane via the general secretion (Sec) or the twin arginine translocation (Tat) pathways. Periplasmic and outer membrane components also rely on the Sec and Tat systems to translocate through the cytosolic membrane. We therefore looked for a signal peptide in the sequence of NMA1982 that would indicate secretion by these pathways. Using both PrediSi45 and SignalP6.046, no known peptide signals were found, suggesting that NMA1982 is secreted via a Tat and Sec independent pathway.PTPs encoded by bacteria have been shown to be secreted by many pathogens during infection, particularly by intracellular pathogens that can thereby directly target eukaryotic effectorsanalysis . The antN. meningitidis causes systemic meningococcal disease, the mortality rate of which, even with optimal treatment, is still about 10%21. Here, we investigated an N. meningitidis protein that is highly similar to eukaryotic PTPs, enzymes that are considered crucial for many essential cellular processes. We show that NMA1982 uses a catalytic mechanism that is specific to PTPs, involving a highly conserved phosphate-binding loop that forms the catalytic center. This loop is usually seven amino acids in length and contains the hallmark C(X)5R signature motif. In NMA1982 the P-loop is shorter by one amino acid and contains a novel C(X)4R motif. Despite being shorter, the P-loop can accommodate a PTP substrate equally well as demonstrated by the similar Km values of the pTyr mimetic DiFMUP for NMA1982 and human PTPs. In NMA1982, the catalytic cysteine and invariant arginine of the P-loop occupy space in the 3D structure that is similar to human PTPs. This ensures that the catalytic mechanism is not compromised. Given the existence of several bacterial NMA1982 homologs containing a C(X)4R motif , other phosphatases with that motif may exist in bacteria. Notably, a conserved glycine within the P-loop of eukaryotic PTPs is also 100% conserved in all bacterial homologs (G98 in NMA1982). The relatively low catalytic activity of NMA1982 is not unprecedented among the PTPs. The structurally closest human enzyme, CDKN3, exhibits a similarly low turnover number28. Interestingly, CDKN3 activity, which critically regulates cell cycle progression, is itself regulated by protein-protein interactions, resulting in a substantial increase of phosphatase activity under physiological conditions48. Similarly, activity of NMA1982 might be enhanced in vivo. Our data demonstrates that NMA1982 is a PTP, enzymes that are essential for most cellular processes, not only in eukaryotes but also in bacteria. Importantly, we show that NMA1982, like known essential virulence factors, is secreted by N. meningitidis. The absence of a signal peptide suggests that NMA1982 is secreted by an atypical secretion machinery or via incorporation in OMVs as has been described for PorB. Future studies will need to address the question whether NMA1982 is indeed crucial for N. meningitidis survival and virulence, and thus may validate NMA1982 as a novel therapeutic target for the treatment of meningococcal diseases. Human PTPs have become attractive drug targets for many serious conditions, most notably cancer51. Compared to human PTPs, the shorter P-loop in NMA1982 results in a unique active site conformation, which may allow for the development of small molecule inhibitors with selectivity for the N. meningitidis protein.23, a QuickChange site-directed mutagenesis kit (Stratagene) was used for generating the NMA1982 mutants D71A, C95S, R100A, R136A, and R136A-D71A. Mutations were confirmed by DNA sequencing. The plasmids were transformed into Rosetta 2 (DE3) competent cells. Protein was expressed in LB media by induction with 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). Wild-type and mutant NMA1982 proteins were purified as previously described23. The purity of the proteins was confirmed to be > 95% using SDS-PAGE and Coomassie staining. Differential scanning calorimetry (DSC) was used to confirm that all proteins were folded. Recombinant VHX, VHR, PTP1B, LMPTP-A, and LMPTP-B were expressed and purified as described previously53. Recombinant MKP-1 was purchased from Upstate. Recombinant CD45 and SHP1 were purchased from Biomol.Utilizing our previously described pSpeedET plasmid containing the gene encoding NMA198227. The NMA1982-catalyzed hydrolysis of DiFMUP was assayed at room temperature in a 60 μL 96-well format reaction system in 50 mM Bis-Tris, pH 6.0 assay buffer containing 1.7 mM DTT and 0.005% Tween-20. Various fixed concentrations of DiFMUP  were added to recombinant wt NMA1982 (11 μM) or NMA1982 mutants D71A (13 μM), C95S (60 μM), R100A (60 μM), R136A (3.8 μM), or R136A-D71A (4.8 μM). The initial rates were determined from the slopes of the fluorescence emission readings over 30 min using an FLx800 micro plate reader  with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control without addition of enzyme. The Michaelis-Menten constants (Km) and turnover numbers (kcat) were determined by fitting the data to the Michaelis-Menten equation, using non-linear regression and the program Prism . A similar assay setup was used for Michaelis-Menten kinetic assays with PTP1B, VHX, VHR, MKP-1, CD45, SHP1, LMPTP-A, and LMPTP-B.NMA1982 phosphatase activity was measured using a standard fluorescence intensity phosphatase assay utilizing DiFMUP (Invitrogen) as the substrate50 value of orthovanadate for NMA1982 was determined from the initial rates using the program Prism  as described previously27.NMA1982 phosphatase activity was measured using the DiFMUP kinetic assay system described above. NMA1982 (15 μM) was preincubated with various fixed concentrations of sodium orthovanadate  for 10 min at room temperature. The reaction was initiated by the addition of the substrate . The IC2O2  in 300 μL assay buffer  on ice for 1 or 2 h. Samples were then either subjected to DTT treatment (10 mM) for 20 min at room temperature or no treatment before DiFMUP  was added to start the reaction (30 min). Fluorescence intensity was measured and initial rates determined as described above.NMA1982 (22 μM) was incubated with various fixed concentrations of HNMA1982 (22 μM) was assayed at room temperature in a 60 μL 96-well format reaction system in 50 mM Bis-Tris buffer  containing 1.7 mM DTT, and 0.005% Tween-20. DiFMUP  was added to start the reaction (30 min). Fluorescence intensity was measured and initial rates determined as described above.The NMA1982-catalyzed hydrolysis of DiFMUP was assayed at various fixed temperatures  in 50 mM Bis-Tris pH 6.0 assay buffer containing 1.7 mM DTT and 0.005% Tween-20. NMA1982 (22 μM) was incubated with DiFMUP  for 30 min in reactions tubes at the specified temperature, before transferred to a 96-well plate for fluorescence measurements. The fluorescence emission was determined at two time points, 0 and 30 min, using an FLx800 micro plate reader  with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control without addition of enzyme. NMA1982 activity of each sample was determined from the difference in emission intensity between the 0- and 30-min time points.Fig. S1). Anti NMA1982 antibodies also recognized the NMA1982 ortholog NMV0640.Polyclonal rabbit anti NMA1982 antibodies were generated by Abnova (Taiwan). Full-length recombinant NMA1982 protein (6 mg/mL) was used for rabbit immunization. Antibody performance from two batches (Lot #11229 and Lot #11230) was tested against recombinant His-NMA1982 (N. meningitidis 8013 was used to engineer a strain lacking the NMA1982 ortholog NMV0640. NEM8013 is a piliated and capsulated Opa-variant of the serogroup C strain 2C4.3. The NMV0640 gene was disrupted by inserting an aphA-3 cassette (kanamycin resistance) in the coding region using PCR overlap and the following primers: Upstream to NMV0640, 5’-GCCGTCTGAAACTCGTGGAACGTCAAATCC-3’ and 5’-CAGCTCATTACTGTTTGATTTGGGCGAAG-3’; aphA-3 cassette, 5’-CAAACAGTAATGAGCTGATTTAACAAAAATTTAACGCG-3’ and 5’-GACGGGATATCAGAAGAACTCGTCAAGAAGGCG-3’; downstream to NMV0640, 5’-TCTTCTGATATCCCGTCCTTGCCTATTG-3’ and 5’-TTCAGACGGCGCAGACAAACAAGCAGGACA-3’. Insertion of the aphA-3 cassette was verified by PCR and the lack of protein expression was confirmed by SDS-Page and Western blotting.The model organism 600 = 0.05 to OD600 = 0.2). Bacteria were centrifuged for 15 min at 4,000 rpm and the resulting media were filtered . Supernatants were concentrated 200x using an UltraCell-3K column (Amicon). The resulting fraction contained proteins and potentially outer membrane vesicles. 10 μg of a whole cell lysate and 5 μL of concentrated supernatant were studied by SDS-Page and immunoblotting using the following antibodies: polyclonal rabbit anti NMA1982/NMV0640 (#11230), monoclonal mouse anti-PilE , monoclonal mouse anti-RMP4 (Coureuil/Nassif laboratory), monoclonal mouse anti-nicotinamide adenine dinucleotide phosphate (NADP) glutamate dehydrogenase (Coureuil/Nassif laboratory), and secondary antibody anti-IgG  coupled to Horse Radish Peroxidase.Wild type 2C4.3 strain and its derivative lacking NMV0640 were grown in 50 mL of Dulbecco’s Modified Eagle Medium (DMEM) for 2 h (from OD


This study determined the S. aureus (47.3%). Other organism groups included streptococci (13.6%), enterococci (7.5%), and Enterobacterales (18.5%). MIC testing of OMC and comparators was conducted according to CLSI M07 (2018) and M100 (2023) guidelines. MIC results were interpreted using FDA or CLSI breakpoints.876 bacterial isolates from bone/joint infections were received from 39 medical centers in the USA and 16 European countries (2015–2022). Identifications were confirmed by MALDI-TOF MS. The top BJI pathogen was staphylococci , including in vitro activity against S. aureus  isolates from BJI, including MRSA (96.5%S) and coagulase-negative staphylococci  (Table). All (100%) S. pyogenes , 98.4% of Enterococcus spp. , and 84.9% of other streptococci  were S to OMC, whereas comparator agent susceptibilities ranged from 20.3% to 57.9%S. Against Gram negatives, ≥90.0% of Acinetobacter spp. , Citrobacter spp. , E. coli , Klebsiella spp. , and S. maltophilia  isolates were S to OMC (FDA breakpoints applied to similar organism groups for comparison). Comparator agent susceptibilities ranged from 46.2%S to 100%S against these organism groups.OMC demonstrated potent in vitro activity against staphylococci, streptococci, enterococci, and Gram-negative isolates from bone/joint infections, with activity generally more potent than or equal to the other tetracycline class comparator agents.OMC demonstrated potent Michael D. Huband, BS, BARDA: This study has been funded in part by BARDA under Contract No. 75A50120C00001.|Entasis: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support Michael A. Pfaller, MD, Paratek: Grant/Research Support Kelley Fedler, BS, Melinta: Grant/Research Support|Paratek: Grant/Research Support Helio S. Sader, MD, PhD, FIDSA, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support


The effect of normal stress variations on fault frictional strength has been extensively characterized in laboratory experiments and modelling studies based on a rate-and-state-dependent fault friction formalism. However, the role of pore pressure changes during injection-induced fault reactivation and associated frictional phenomena is still not well understood. We apply rate-and-state friction (RSF) theory in finite element models to investigate the effect of fluid pressurization rate on fault (re)activation and on the resulting frictional slip characteristics at the laboratory scale. We consider a stepwise injection scenario where each fluid injection cycle consists of a fluid pressurization phase followed by a constant fluid pressure phase. We first calibrate our model formulation to recently published laboratory results of injection-driven shear slip experiments. In a second stage, we perform a parametric study by varying fluid pressurization rates to cover a higher dimensional parameter space. We demonstrate that, for high permeability laboratory samples, the energy release rate associated with fault reactivation can be effectively controlled by a stepwise fluid injection scheme, i.e. by the applied fluid pressurization rate and the duration of the constant pressure phase between each successive fluid pressurization phase. We observe a gradual transition from fault creep to slow stick–slip as the fluid pressurization rate increases. Furthermore, computed peak velocities for an extended range of fluid pressurization rate scenarios (0.5 MPa/min to 10 MPa/min) indicate a non-linear (power-law) relationship between the imposed fluid pressurization rate and the peak slip velocities, and consequently with the energy release rate, for scenarios with a fluid pressurization rate higher than a critical value of 4 MPa/min. We also observe that higher pressurization rates cause a delay in the stress release by the fault. We therefore argue that by adopting a stepwise fluid injection scheme with lower fluid pressurization rates may provide the operator with a better control over potential induced seismicity. The implications for field-scale applications that we can derive from our study are limited by the high matrix and fault permeability of the selected sample and the direct hydraulic connection between the injection well and the fault, which may not necessarily represent the conditions typical for fracture dominated deep geothermal reservoirs. Nevertheless, our results can serve as a basis for further laboratory experiments and field-scale modelling studies focused on better understanding the impact of stepwise injection protocols on fluid injection-induced seismicity. Fluid injection can be associated with an increased seismic hazard, especially for injection wells located in the vicinity of critically stressed faults, i.e. for faults that are stressed close to their static shear strength, and are favourably oriented with respect to the in-situ stress field2. Thus, hydraulic stimulation treatments must enhance reservoir permeability with minimum induced seismic hazard3. To achieve this, it is crucial to gain knowledge of the dynamics governing the frictional sliding of reactivated faults, induced by fluid injection6. It is a matter of debate whether magnitudes of the largest possible induced earthquakes can be effectively controlled and limited by tuning operational parameters during hydraulic stimulation treatments 7, or if they are controlled primarily by the tectonic stress field and the local geology of the reservoir, as it is discussed for tectonic earthquakes8. Recent laboratory experiments and numerical studies have assessed the effects of pressurization rate on injection-induced seismicity12. These studies suggest that pressure-increase rates play an important, albeit not yet fully quantified, role in fault reactivation and the promotion of subsequent seismic/aseismic slip.Hydraulic stimulation treatments are performed in geothermal projects to enhance the permeability of the targeted reservoir by fluid injection11. In a second stage, we perform a parametric study on the imposed fluid pressurization rate and selected model properties. Our study suggests that a deeper understanding of these processes could help to develop operational methods to better control injection-induced seismicity on pre-existing natural and induced faults.The goal of this study is to further examine the effect of imposed fluid pressurization rates on induced seismicity in laboratory-scale injection-driven shear slip tests using numerical simulations based on an RSF formalism. We consider a stepwise injection scenario in which each fluid injection cycle consists of a fluid pressurization phase and a subsequent constant fluid pressure phase. Our goal is to explore the transition characteristics of the injection-induced fault slip mode from fault creep towards slow stick–slip behaviour as a function of the imposed fluid pressurization rate. In particular, we focus our investigation on how the applied fluid pressurization rate affects the stress drop on the laboratory fault during an induced slow slip event. To achieve this goal, we first calibrate our model formulation to recently published laboratory results of injection-driven shear tests11. In these experiments, two different stepwise fluid pressurization schemes were applied to homogeneous and isotropic Bentheim sandstone samples with a critically stressed sawcut fault to examine the effect of varying fluid pressurization rates on the induced fault slip behaviour. The initial porosity of the samples is ~ 23%, and their permeability is ~ 1 Darcy.We calibrate our model formulation to two injection-driven shear tests conducted by Wang et al. (2020a)11 to ensure a relatively homogeneous pressure distribution across the fault surface during the fluid injection test. Assuming that the effective stress law holds irrespective of the rock type, fault reactivation should depend only on the evolution of the friction coefficient and the effective stress on the fault13. Although low permeability rocks are more representative of enhanced geothermal reservoirs, experiments performed on such rocks  with fluid conduits through boreholes12 are more difficult to interpret, considering the competing propagation of both the pressure and rupture front under locally undrained conditions. Therefore, we consider the data from Wang et al.11 as the simplest but physically relevant input for our numerical study to gain a better understanding of injection-induced seismicity.We note that the high porosity and permeability of the Bentheim sandstone may not be fully representative of typical conditions in enhanced geothermal reservoirs. However, it was chosen for the original experimentsIn the original experiments, two cylindrical samples were prepared with a length of 100 mm and a diameter of 50 mm, each having a saw-cut fault oriented at approximately θ = 30° to the longitudinal axis of the sample, resulting in an elliptical fracture surface 50 mm along the minor axis and 100 mm along the major axis  in the first test, the 4 MPa pressure steps were achieved at a pressurization rate of 2 MPa/min—high fluid pressurization rate experiment (test SC1 hereafter); (ii) in the second test, performed on a separate sample, the pressurization rate was lowered to 0.5 MPa/min—low fluid pressurization rate experiment (test SC2 hereafter). After each pressurization phase, the fluid pressure was held constant for the remaining period of the 10-min cycle, which is referred to as the constant pressure phase, as opposed to the prior pressurization phase  parameter determines whether a fault governed by a rate-and-state formalism is in a regime of rate strengthening or rate weakening, the former characterised by positive values and the latter by negative values of (a–b). The value of (a-b) was chosen in agreement with the available experimental data, which indicate that sandstones behave as slightly rate weakening materials. This is a necessary, though not exclusive, condition for the development of stick–slip instabilities as observed in both laboratory and numerical experiments, at least at high fluid pressurization ratesIt is worth noticing that in our modelling approach we downsize the model dimension to a 2D case with a one-dimensional saw-cut fault Fig. . The fau15. This assumption is supported by the cumulative number of acoustic events recorded in the laboratory, which positively correlates with measured slip rates17.During model calibration we aimed to match the maximum slip velocities of the 2 MPa/min fluid pressurization rate scenario (test SC1), as the injection-induced slip velocity is a good indicator of the induced seismic hazard18.Based on the peak velocities recorded in the two laboratory tests, we describe the observed aseismic slip events as fault creep when characterised by peak slip velocities < 1 µm/s and a longer duration of slip, and as slow stick–slip events for peak slip velocities < 1 mm/s (1 mm/s is considered the lower threshold velocity for seismic slip events) and a quasi-instantaneous slip compared to fault creepComputed peak slip velocities are in good agreement with the experimental data recorded for test SC1 Fig. a. The hiBased on the calibrated model we carried out a detailed parametric study by gradually varying the applied fluid pressurization rate between a maximum of 2 MPa/min and a minimum of 0.5 MPa/min in steps of 0.25 MPa/min Fig. a. This wThe modelling results do not exhibit a sharp transition in velocity marking the onset of slow stick–slip. Instead, we observe a gradual transition in fault behaviour through the evolution of slip velocity Fig. b, stressIn Fig. In agreement with the laboratory experiments, we found that the computed total stress drop, given by the sum of the two contributions, is independent of the applied fluid pressurization rate  are in good agreement with the laboratory data , thereby resulting in a larger stress drop than the one modelled.We simulated two injection-induced shear tests performed on high-permeability Bentheim sandstone samples intersected by saw-cut faults and compared our models with available laboratory resultsIt is worth noting, that laboratory and modelled stress drop values are matched relatively well for the constant pressure phase. One potential reason for this is that in the constant pressure phase, the stress relaxation of the sample occurs much slower than in the pressurization phase, so the MTS machine has sufficient time to respond to the deformation of the system-sample assembly, unlike in the pressurization phase.19 demonstrated that for cases in which the temporal variation of porosity and permeability (i.e. the shear-induced dilation or compaction of the fault) is not coupled to the rate-and-state controlled aseismic slip, the onset of the computed slip is delayed and the final cumulative slip is likely to be overpredicted, as it is the case in our models.In addition, the model is also limited in describing the dynamics of the injection-induced fault slip of the laboratory experiments given our assumption of constant hydraulic properties and, most importantly to the case at hand, constant rate-and-state parameters. A previous modelling study by Yang et al.20 or slip velocity21 has been considered as a potential mechanism to explain the transition from fast to slow earthquakes in subduction settings, where the asperities within a megathrust front gradually move towards a rate strengthening regime over time as pressure or slip evolve. However, there is no direct evidence for such behaviour, as different laboratory studies give different, sometimes even contradictory results23. Linker and Dieterich24 discussed how the rate of change in the normal stress acting on the fault (stressing rate term) can additionally influence the fault frictional behaviour. Following these studies, we additionally tested our model by integrating the stressing rate term following the formalism by Nagata et al.25  parameter with respect to pore pressure.25 Fig. . The incTo gain a better insight into how the rate of pressure-increase can affect the mode of the induced fault slip, we performed a numerical parameter study by varying fluid pressurization rates. Our results indicate a gradual transition from fault creep towards slow stick–slip with increasing fluid pressurization rate (from 0.5 MPa/min to 2 MPa/min), rather than an abrupt change in the sliding mode. We additionally found that with lower fluid pressurization rates, a larger portion of the total stress drop occurs during the active pressurization phase. Furthermore, a lower fluid pressurization rate decreases the maximum and average slip velocities in the pressurization phase, and leads to a slight increase of the minimum slip velocity in the constant pressure phase. Overall, both the maximum slip velocity and the difference between slip velocities during pressurization and constant pressure phases are lower for lower fluid pressurization rates. A non-linear correlation between slip velocity magnitudes and pressurization rate implies a faster drop in the frictional strength for higher pressurization rates and therefore a higher probability that the finite-size instability would trigger a run-away rupture. Run-away instabilities are hindered in our study that is based on a finite sample size, which, given the hydromechanical property of the rock investigated, is smaller than the critical nucleation length. Therefore, further reservoir-scale simulations are required to upscale these results to real case conditions.12 performed a series of injection-driven shear tests on sawcut faults in low-porosity (0.26%) granite samples. They showed that in the low-permeability (~ 1.3 μD) rock, fluid injection causes a heterogeneous pressure distribution in the fault, strongly influencing its frictional stability. Specifically, the rupture front propagates beyond the pressurized area of the fault, in contrast with the experiments performed on Bentheim sandstone, where the rupture front is constrained within the pressurized zone due to the high permeability (~ 1 D) of the sample17. The difference in the fluid pressure distribution on the fault associated with fluid injection is mainly attributed to the permeability contrast between high-permeability sandstone and low-permeability granite. This may lead to differences in the amount of elastic strain energy (indicated by the stress drop) releasing during the pressurization and constant pressure phases on the fault subject to stepwise increasing fluid pressure. To gain insight into the role of rock matrix permeability in delaying the stress drop, we conducted an additional set of simulations assuming a low permeability matrix (10–6 D); see Supplementary Fig. The conclusions derived in this study are valid for the particular rock investigated, a porous and permeable Bentheim sandstone. Low permeability crystalline rocks may exhibit a different behaviour during a similar fluid injection test. Ji & Wu27 . Our results therefore suggest that it is theoretically possible to achieve the same permeability enhancement  with lower seismic hazard by imposing lower fluid pressurization rates. The applicability of our findings to deep geothermal reservoirs and EGS systems is limited by the difficulty of upscaling the conditions studied in the laboratory to field scale conditions. Therefore, further numerical investigations are needed to determine the potential practical utility of our results.The simulated experiment is analogous to a step rate test featured by stepwise increasing fluid pressure. Our results suggest that provided a high permeability reservoir and a direct hydraulic connection between the injection well and a high-permeability fault, by choosing the pressure steps and the fluid pressurization rates carefully, operators might be able to exercise some degree of control over the maximum slip velocity of the fault, as well as the slip and stress drop distribution between pressurization and constant pressure phases, while the total fault or fracture displacement remains the same. The permeability of self-propped, displaced fractures is found to be correlated to the resolved shear displacement along the same faultIn the experiment, fault slip displacement, The shear strength of the fault is determined by plotting the shear stress as a function of slip displacement. After the sample yields, the plateau of the steady-state shear stress value corresponds to the shear strength of the sample.When comparing the results of our 2D model to the experimental data; slip, slip velocity and shear stress are always represented as average values along the fault line.31. The rate-and-state constitutive equations describe the evolution of the friction coefficient along the fault as a function of resolved slip rate and the temporal variation in the internal state of the fault surface29.For the characterization of fault reactivation and dynamic slip we use a rate-and-state friction (RSF) formalism in our simulations29 linked the state variable to the average contact time of the asperities. Given a constant slip velocity, the contact time is proportional to the size distribution of asperities and inversely proportional to the slip rate32.By considering a single internal state variable  determines the slope of the static frictional dependence on the logarithm of the slip velocity. If 16. In a rate-and-state framework, these processes are integrated into an internal dependency of the fault friction coefficient  on the sliding velocity and the internal state variable. Mathematically, these dependencies are expressed by the system of Eqs.  the accumulated elastic energy, while frictional weakening would indicate the processes that control the loss of frictional strength, which can occur either co-seismically or inter-seismically of Eqs. –11)..16. In a11 and 35. To obtain the shear displacement, 36. The code is built on the object-oriented numerical framework, MOOSE37, providing parallel implicit coupling to solve multiphysics problems.To simulate fault reactivation, the stress balance along the fault is described asg of Eq.  and 11)11\documeSupplementary Information 1.Supplementary Information 2.


The aim was to validate previously developed This was a retrospective cohort study in patients collected from a prospective multicentre study in four Dutch institutes. Patients underwent nCRT followed by oesophagectomy between 2013 and 2019. Outcome was tumour regression grade (TRG) 1 (0% tumour) versus TRG 2-3-4 (≥1% tumour). Scans were acquired according to standardised protocols. Discrimination and calibration were assessed for the published models with optimism-corrected AUCs >0.77. For model extension, the development and external validation cohorts were combined.Baseline characteristics of the 189 patients included  were comparable to the development cohort. The model including cT stage plus the feature ‘sum entropy’ had best discriminative performance in external validation , with a calibration slope and intercept of 0.16 and 0.48 respectively. An extended bootstrapped LASSO model yielded an AUC of 0.65 for TRG 2-3-4 detection.The high predictive performance of the published radiomic models could not be replicated. The extended model had moderate discriminative ability. The investigated radiomic models appeared inaccurate to detect local residual oesophageal tumour and cannot be used as an adjunct tool for clinical decision-making in patients. The combination of endoscopy with bite-on-bite biopsies and endoscopic ultrasound with fine-needle aspiration (EUS-FNA) of suspected lymph nodes is 90% sensitive to detect >10% locoregional residual tumour , with a calibration slope of 0.16 (95% CI −0.05 to 0.37) and intercept of 0.48 (95% CI 0.0–0.96) . Following, 49 variables with pair-wise absolute correlations >0.9 were removed. A LASSO model was developed that included five variables with non-zero coefficients, three of which were radiomic features and the clinical variables cT stage and histology . The extended LASSO model yielded an optimism-corrected AUC of 0.65 (uncorrected 0.75) (Table http://links.lww.com/NMC/A248) yielded and optimism-corrected AUC of 0.71 (uncorrected 0.73)  Table . The LAShttp://links.lww.com/NMC/A248. Performance distributions of the models combining the selected radiomic features  with clinical variables are shown in Supplemental Table 9, Supplemental digital content 1, http://links.lww.com/NMC/A248. Over the 100 validation datasets, a Naive Bayes classifier achieved a mean AUC of 0.73 (95% CI 0.63–0.84). Since an independent dataset was not available, optimism of this model could not be further investigated.The LASSO modelling strategy was compared to another workflow for radiomic feature selection and model development. The first step of this workflow indicated that a generic radiomic analysis was suitable, as shown in Supplemental Appendix 2 and Figure 8, Supplemental digital content 1, http://links.lww.com/NMC/A248).Models were also trained to detect substantial residual tumour i.e. TRG 3-4), but this did not result in improved diagnostic accuracy compared to the models trained for the primary endpoint (i.e. TRG 2-3-4)  residual tumour after nCRT. This was different than in the derivation sample alone, in which radiomic features, representing, for example, orderliness of the voxels, appeared to distinguish residual tumour from normal tissue (with treatment effects) [The performance of the investigated models in this study was evaluated at a probability threshold to obtain 90% sensitivity, which is the benchmark for detection of residual TRG 3-4 tumour using gastroscopy with bite-on-bite biopsies and EUS-FNA of suspected lymph nodes . The reseffects) .18F-FDG PET/CT has been shown inaccurate for distinction between residual tumour and inflammation at the primary tumour site at 12 weeks after nCRT. Radiomics was expected to capture more complex information within the image, for example, reflecting underlying biology. Such information did not appear to be retrievable from the investigated imaging dataset.The finding that radiomics did not improve detection of residual tumours may not be surprising in the context of a previous study . Qualitahttp://links.lww.com/NMC/A248) [A strength of this study is the homogeneous multicentre external validation cohort collected from two prospective trials ,5. This MC/A248) , since tMC/A248) . This wohttp://links.lww.com/NMC/A248) and tumour delineation methods [The limited generalisability of the published models is probably due to overfitting to the derivation sample. Another possible cause for decreased performance in the validation cohort is unintentional dependency of radiomic features on scanning protocols, scanner models  and The Netherlands Organisation for Health Research and Development (ZonMw) . P.L. discloses, within and outside the submitted work, grants/sponsored research agreements from Radiomics SA, ptTheragnostic/DNAmito and Health Innovation Ventures. He received an advisor/presenter fee and/or reimbursement of travel costs/consultancy fee and/or in-kind manpower contribution from Radiomics SA, BHV, Merck, Varian, Elekta, ptTheragnostic, BMS and Convert Pharmaceuticals. P.L. has minority shares in the companies Radiomics SA, Convert Pharmaceuticals, Comunicare and LivingMed Biotech, and he is co-inventor of two issued patents with royalties on radiomics , licensed to Radiomics SA, and one issued patent on mtDNA (PCT/EP2014/059089), licensed to ptTheragnostic/DNAmito, three non-patented inventions (softwares) licensed to ptTheragnostic/DNAmito, Radiomics SA and Health Innovation Ventures and three non-issued, non-licensed patents on Deep Learning-Radiomics and LSRT . He confirms that none of the above entities or funding sources were involved in the preparation of this article. H.C.W. discloses minority shares in the company Radiomics SA, unrelated to this work. For the remaining authors, there are no conflicts of interest.


Present the results of the secondary obliteration of chronically discharging radical cavities using S53P4 bioactive glass (BAG).Retrospective cohort study.Single‐center study.A single‐center retrospective cohort study was conducted of all patients that underwent secondary obliteration of persistently draining radical cavities using S53P4 BAG between 2011 and 2022. Patients with middle ear cholesteatoma were excluded. The main outcome was postoperative otorrhea, as indicated by Merchant grading.In total, 97 patients were included. The median postoperative follow‐up time was 3.9 years (range 0.5‐10.4). Average time between the original canal wall down surgery and the secondary obliteration was 25.3 years . At the most recent follow‐up visit, a Merchant grade of 0 to 1 was observed in 95% of the cases. There were no cases of sensorineural hearing loss or facial palsy, one case developed a retro auricular skin defect and 1 patient developed CSF leakage. Minor complications were seen in 10 patients (10%). Ossicular chain reconstruction with a titanium prosthesis was performed in 42 cases, resulting in a median improvement of 11.2 dB in air conduction thresholds. In 9/42 cases (21%), closure of the postoperative air‐bone gap to ≤20 dB was achieved. Twenty‐five percent of cases could be discharged from out‐patient visits.Revision of persistently draining radical cavities with BAG obliteration is feasible and results in a dry and safe ear in 95% of the patients, thereby enabling wearing of a conventional hearing aid. Out‐patient visits could be ceased in 25% of the cases. In canal wall down surgeries, the posterior bony wall of the external auditory canal (EAC) is removed to increase exposure. The creation of a so‐called radical cavity comes with several possible disadvantages, such as higher rates of otorrhea and purulence, pain, adherence to water precautions, and dizziness.Various materials can be utilized as obliteration material, including autologous such as bone dust and bone pâté, allogenic such as bone chips from a donor and synthetic material such hydroxyapatite or ceramic granules.Previous studies, including from our own hospital, have demonstrated the use of S53P4 BAG for indications such as cholesteatoma and chronic otitis media.This retrospective cohort study was conducted at a secondary referral center in the Netherlands. The study was in accordance with the ethical standards of the hospital institutional review board  and the 1964 Declaration of Helsinki. Formal consent was not required for this type of retrospective study. The S53P4 BAG, produced by BonAlive Biomaterials Ltd., has received clearance for clinical use by the CE in 2004 and is currently awaiting Food and Drug Administration (FDA) approval for mastoid surgery. BAG has received FDA approval for use in orthopedic surgery, mainly as filler material for bone defects such as nonunions.Eligible for inclusion were all patients with troublesome radical cavities due to a previous CWD surgery that underwent secondary obliteration using S53P4 BAG in the period of January 2011 to September 2022. All patients were initially treated conservatively, but symptoms would persist despite maximal treatment. Patients were excluded if the cause of the troublesome radical cavity was middle ear cholesteatoma. The patients reported in our previous publication were not included in this study, as they were diagnosed with cholesteatoma.All patients received a CT‐scan preoperatively for surgical planning The canal wall down tympanomastoidectomy with mastoid obliteration (CWD + MO) was utilized as surgical treatment of the troublesome radical cavities. Care was taken to remove all possible causes of the troublesome mastoid, such as infected mucosa or mastoid cells. The posterior wall of the EAC would be reconstructed with cartilage. The BAG granules would be moistened inside their container with 0.9% sterile saline solution. Subsequently, the material is carefully applied to both the epitympanic area and the mastoid cavity. Attention is paid that no cavities exist within the obliteration. Thereafter, the fluid is suctioned from the material and the obliteration is covered with periosteal or fascia flaps.The following outcome parameters were included: indication for secondary obliteration, time between original CWD surgery and secondary obliteration, procedure safety, postoperative Merchant grade at 8 weeks, 6 months, 1 year, and most recent follow‐up moment, secondary cholesteatoma development, and audiometric performance. Possible indications for secondary obliteration were defined as the presenting symptoms of patients and included chronic discharge, dizziness, hearing improvement, and pain. Procedure safety was defined as the absence of perioperative and postoperative complications in the complete follow‐up and need for any revision surgery. Merchant's grading system was used to evaluate postoperative otorrhea .23 PatU test were used for univariate analysis. For audiometric evaluation, Wilcoxon signed‐rank test was used for comparison of preoperative and postoperative AC, BC, speech recognition, and ABG. Mann‐Whitney U test was used to compare postoperative ABG between different subsets of cases. Scattergrams were constructed by combining the word recognition at 50 dB and average AC.sankeymatic.com. P < .05 was considered statically significant.Data were analyzed using SPSS statistics . Continuous data were presented as mean ± standard deviation (SD) or median with interquartile range (IQR), depending on normal or nonnormal distribution. Fisher's exact test, Chi‐squared test and Mann‐Whitney In total, 97 cases were included in this study with a mean age of 51 years . A previous revision of the radical cavity without obliteration had been performed in 17 cases. Indications for secondary obliteration were otorrhea , dizziness , pain , improvement of hearing , stenosis of the external auditory canal  and severe complications of the existing radical cavity such as encephalitis .A CWD + MO surgery was performed in all cases. During surgery, erosion of the bony covering of important structures was observed in 47 cases. Exposed dura was seen in 19 cases, exposed sigmoid sinus in 7 cases, a partially eroded labyrinth in 6 cases, and exposed facial nerve in 30 cases, of which 18 were exposed in the tympanic segment and 12 in the mastoid segment. The exposed structures would be covered with fascia or cartilage before obliteration of the mastoid cavity. Covering of the obliteration and reconstructed external auditory canal with an additional flap was conducted in 49 cases: 5 with temporal muscle, 14 with temporal muscle fascia, and 30 with a mid‐temporal artery/periosteal flap. The status of the ossicular chain at the end of surgery is displayed in P < 0.001). No cases of Merchant grade 3 were seen at the most recent follow‐up moment. This results in a dry‐ear rate of 95%. Univariate analysis was conducted of patients that developed Merchant grade 2 to 3 at the most recent follow‐up visit or before an additional surgery . Following secondary obliteration out‐patient visits were ceased in 24 patients (25%). Postoperative complications were seen in 12 cases (12%), of which the majority  was minor . There wSome type of additional surgery was necessary in 12 cases (12%). Seven cases underwent revision surgery for the following indications: insufficient obliteration due to loss of obliteration material requiring re‐obliteration (n = 2), insufficient obliteration as insufficient material was placed during surgery requiring re‐obliteration (n = 3), CSF leakage (n = 1) or cholesteatoma (n = 1). Therefore, the revision rate of the obliteration itself was 5% (n = 5). The symptoms of patients with insufficient obliteration requiring revision varied: three cases had a small residual cavity without self‐cleaning capabilities causing recurrent infections, one case had an pre‐existent labyrinth dehiscence exposed due to insufficient obliteration which caused dizziness and one case developed a resistant skin infections, causing a skin defect and loss of the obliteration material. Removal of the obliteration material and subsequent reobliteration of the cavity was necessary in 6 of the revision surgeries. Additionally, 2 cases underwent meatoplasty and 3 patients underwent a procedure for hearing improvement. The single cholesteatoma occurred in a patient who refused middle ear surgery, therefore the epitympanum could not be obliterated and a retraction pocket developed.P = 0.026), respectively. Closure of the postoperative ABG ≤ 20 dB was possible in 9/42 cases with PORP/TORP reconstruction. Univariate analysis of 51 patients that underwent some form of ossicular chain reconstruction revealed no factors significantly associated with an ABG ≤ 20 dB postoperatively (Complete tone audiometry was available for 78 cases (80%). The median BC did not change significantly preoperatively to postoperatively . Both thratively .In this retrospective cohort study, we have presented the effectiveness and safety of secondary obliteration of troublesome radical cavities using S53P4 BAG. At the most recent follow‐up visit, 95% of the patients had a Merchant grade of 0 to 1, which is the main objective of secondary obliteration. Additionally, postoperative complications were only seen in 12 cases and they were seldom major. The main strengths of this study are the follow‐up time, the homogeneity in obliteration material and technique, and the fact that it is the first systematic article on S53P4 BAG for secondary obliteration of troublesome radical cavities.Our study has demonstrated excellent dry ear rates as indicated by the Merchant grade at the most recent follow‐up visit. There are several articles to which our results should be compared to. First, Yung et al, in 2011, presented a case series on the use of hydroxyapatite, presenting a dry ear rate of 98% after 3 years.Overall, our dry ear rate seems to be comparable to the available literature. This is especially important as the average time between original CWD and secondary obliteration was 25.3 years, showing the chronic nature of the symptoms of our patients. Although outpatient visits should not be fully ceased postoperatively in order to detect potential delayed problems, obliteration allows for the frequency of visits to be reduced to once per year or even less.When investigating possible factors that would predict failure of the surgery, solely postoperative complications were found to be significantly associated with frequent postoperative otorrhea. Perhaps it shows that difficulties in the healing process result in surgical failure. No other variables such as time between original CWD surgery and secondary obliteration were significantly associated with surgical failure. The origin of postoperative otorrhea is possibly multifactorial and not all factors could be scored in this retrospective study, such as the frequency of previous surgeries performed. The rate of Merchant grade 2 was more than threefold higher eight weeks following surgery compared to the most recent follow‐up visit. This could be because the external auditory canal and tympanic membrane would still be healing 8 weeks postoperatively.Few complications were seen and all except one were minor. This is especially relevant because surgery on troublesome radical cavities can be challenging. Most cavities had been purulent for years and exposed structures such as the facial nerves or labyrinth were seen in 37% of patients. Fortunately, no deaf ears or damage to the facial nerve was observed. The rate of revision surgeries is comparable to the currently available literature.Hearing rehabilitation was challenging. The overall improvement in AC and ABG was small and probably with limited clinical relevance. Similar results are found in other studies.There are several limitations to be mentioned. First, the retrospective nature of the study limits the data validity. Nevertheless, as we choose to include all patients that underwent secondary obliteration in our institute, we think our study gives a realistic picture of the effectiveness of secondary obliteration using S53P4 BAG. As almost all patients since 2011 with troublesome radical cavities were treated similarly, there is minimal risk of selection bias. Second, there was no nonobliterative cohort to compare the obliteration technique to. As our hospital seldom performs revision surgery of radical cavities without obliteration, no adequate cohort could be formed. A prospective, randomized trial of obliterative versus nonobliterative techniques is necessary for a definitive conclusion. Third, no data was available on QoL. QoL should also be included in future studies to further identify the benefit of secondary obliteration beyond Merchant grading.In conclusion, secondary obliteration of troublesome radical cavities using S53P4 BAG is safe and effective. Complications were seldom seen, even in these challenging cases. Even though patients suffered from otorrhea for an average of 25 years since the initial CWD surgery, secondary obliteration was successful in resolving the symptoms of 95% of them.Victor Kroon, gathered the data, performed the analysis and wrote the first draft of the manuscript; Steven W. Mes, designed the study, performed analysis, and gave critical feedback on the manuscript. Pepijn A. Borggreven, designed the study, gave critical feedback on the manuscript; Rick van de Langenberg, designed the study, gave critical feedback on the manuscript; David R. Colnot, designed the study, gave critical feedback on the manuscript; Jasper J. Quak, designed the study, interpreted the data and gave critical feedback on the manuscript.None.Diakonessenhuis Research grant 2021, De Cornelis Visser stichting.