Porphyromonas gingivalis can persist below the gum line, induce ecological changes, and promote polymicrobial infections remains limited. P. gingivalis has long been described as a highly proteolytic and asaccharolytic pathogen that utilizes protein substrates as the main source for energy production and proliferation. Here, we report that P. gingivalis displays a metabolic plasticity that enables the exploitation of non-proteinaceous substrates, specifically the monocarboxylates pyruvate and lactate, as well as human serum components, for colonization and biofilm formation. We show that anabolism of carbohydrates from pyruvate is powered by catabolism of amino acids. Concomitantly, the expression of fimbrial adhesion is upregulated, leading to the enhancement of biofilm formation, stimulation of multispecies biofilm development, and increase of colonization and invasion of the primary gingival epithelial cells by P. gingivalis. These studies provide the first glimpse into the metabolic plasticity of P. gingivalis and its adaptation to the nutritional condition of the host niche. Our findings support the model that in response to specific nutritional parameters, P. gingivalis has the potential to promote host colonization and development of a pathogenic community.Our understanding of how the oral anaerobe  Various etiological models suggest that the Gram-negative anaerobe Porphyromonas gingivalis may play a central role in the onset of periodontitis via orchestrating a pronounced ecophysiological change below the gingival margin7; however, the mechanistic basis of such a microbial shift has not been elucidated. P. gingivalis can persist in the subgingival ecosystem adjacent to the epithelium, even in the absence of periodontitis in an otherwise healthy individual11; hence, it is speculated that an unknown ecophysiological condition allows P. gingivalis strains to transition toward a pathogenic state, and to produce an array of pathogenicity factors, including the type IX secretion system (T9SS), proteases, and fimbrial adhesions, that promote bacterial proliferation, persistence, and invasion13. Therefore, understanding the environmental factors that affect the physiological state of P. gingivalis is crucial.An orchestrated series of dysbiotic polymicrobial interactions can promote the colonization of a pathogenic microbial community below the gum line, leading to periodontitis, an infection-driven inflammatory disease of the periodontal tissues and alveolar bone, imposing adverse systemic effects on human healthP. gingivalis has been known as a highly proteolytic and asaccharolytic anaerobe that secretes potent proteases known as gingipains via the T9SS constituted by at least ten interacting proteins, including PorK/L/M/N/P/Q/T/U/V/W and SprA , that span the cell envelope14. T9SS and gingipains are essential determinants of P. gingivalis virulence and are necessary for breaking down the host proteinaceous substrates into small peptides, which are then transported into the cytoplasm for fermentation and energy production; intriguingly, the availability of free amino acids and carbohydrates does not meet this requirement18.Historically, P. gingivalis strain 381 (a highly fimbriated and robust biofilm-forming strain) is capable of switching between sessile growth and surface translocation, when sandwiched between two surfaces19. Although the T9SS and secreted proteases were found necessary for biofilm-to-migration transition, such a lifestyle transition was also dictated by nutritional stimuli and signals. As a proof of concept, specific substrates including the key monocarboxylates pyruvate and lactate, along with bovine serum albumin (BSA) were tested and found to impede the transition of P. gingivalis to a migration state. Instead, these nutrients promoted biofilm development under a migration-inducing condition through a yet unknown mechanism19. Pyruvate and lactate are not only key carbon and energy sources for living organisms, but also have other biological functions. For instance, sodium pyruvate is an efficient peroxide scavenger that confers protection to host cells and oral streptococci by detoxifying hydrogen peroxide in the environment27. In the aerobic and saccharolytic lifestyle, pyruvate is produced from glucose metabolism during glycolysis that eventually enters the tricarboxylic acid (TCA) cycle and the electron transfer chain, while it can be reduced to lactate under anaerobic conditions28. However, except for anaerobic sulfate reducing bacteria  that couple lactate oxidation to sulfate reduction, metabolism of pyruvate, and lactate in obligately anaerobic and asaccharolytic organisms has largely remained unknown. The increase of pyruvate and lactate in the intracellular and extracellular milieus of human cells and serum have been reported to be due to a variety of factors, including microbial fermentation29, inflammation30, physiological diseases33, the Warburg effect35, stresses37, and diets40; yet how the availability of pyruvate and lactate and serum components impacts the physiology of P. gingivalis and other oral residents is not clear.Our previous work demonstrated that P. gingivalis can utilize pyruvate and lactate in the presence of human serum. Our data show that when combined with human serum, exogenous pyruvate is an important energy substrate for P. gingivalis that affects not only central carbon metabolism, but also the expression of fimbrial adhesion, a requirement for surface colonization, biofilm development, and invasion. Overall, our findings indicate that such plasticity in nutrient utilization allows the phenotypic adaptation of P. gingivalis to the nutritional parameters of the host environment.In this work, we report that P. gingivalis and the transition between sessile growth and surface translocation19. First, our preliminary assessment using static liquid cultures showed that P. gingivalis strain 381 can efficiently utilize BSA protein for proliferation, but BSA-only medium progressively induced the auto-aggregation and settling of P. gingivalis cells over time when compared with a nutrient-rich laboratory medium, such as TSBHK  restored the pyruvate-induced phenotypic change and significantly increased biofilm formation , rhs (a major component of contact-dependent inhibition module), and mfa1 (the major protein subunit of minor fimbriae); the expression of sprA (the main constitutive component of T9SS) remained unchanged. Further, we determined that higher concentration of BSA in the medium has a positive correlation with the expression of sprA, ragA, rhs, and a cysteine protease (PGN_0508), while the expression of mfa1 remained unchanged , when compared with the BSA-only medium  analysis on RNA samples extracted from biofilms grown in BSA-based medium with and without adding pyruvate or lactate. Figure dia Fig. . On the ged Fig. . Consistium Fig. . Overall+] cofactors and cellular redox status, which in turn affects the dynamics of cellular metabolism, energy production, and numerous biological processes52. Here, we used a resazurin fluorometric (RF) assay to assess the redox status of P. gingivalis, when pyruvate, lactate, and serum components are concomitantly utilized. In this assay, nonfluorescent resazurin molecules (an oxidation–reduction indicator) are irreversibly converted to highly fluorescent resorufin molecules when coupled with the oxidation of NADH, and this conversion is used to infer the metabolism of different substrates. The NADH turnover and conversion of resazurin to resorufin is measured as the relative fluorescent units (RFU)53. Upon the addition of different substrates to the cell suspensions, redox changes were fluorometrically measured over a 6-h incubation period. RFU calculation showed that the conversion of resazurin to resorufin was trivial upon the utilization of various concentrations of BSA, indicating no significant change was induced in the redox status of P. gingivalis upon BSA utilization, but exogenous lactate and pyruvate greatly altered cellular redox status, resulting in a high level of fluorescence yield , trifluoperazine  inhibitor in Mycobacterium tuberculosis), and myxothiazol  strongly inhibited the lactate-induced redox change; others including antimycin A, rotenone, carbonyl cyanide 3-chlorophenyl hydrazone, or thenoyltrifluoroacetone did not impact redox changes , NADH oxidoreductases -dependent dehydrogenase), and cytochrome bd ubiquinol oxidases (PGN_1041-42), which also mediate the engagement with the metabolism of pyruvate and lactate -treated cells to compare with untreated control cells. The quantification of relative gene expression showed that even though HQNO reduces the expression levels of P. gingivalis during biofilm growth. To this end, we performed a global metabolomic analysis on the metabolome of the biofilm cells grown in HSA or human serum with and without pyruvate addition. For multigroup analysis, one-way analysis of variance (ANOVA) followed by post hoc analysis  in addition to variable importance in projection values >1.0 revealed that 74 metabolites of canonical pathways  were differentially produced in biofilm-growing cells in response to the given substrates  analysis were executed and statistically significant metabolites  were presented in the volcanic plots  and gluconeogenesis , this enzyme does not act extracellularly; further, bioinformatics databases predicted the presence of a transmembrane domain in its structure that may guide its localization in the inner membrane. Overall, these findings support our notion that exogenous pyruvate is tied to the metabolism of serum components toward the enhancement of the PPP and biosynthetic processes that are required during the biofilm development (see “Discussion” for the proposed model).Given that our data showed that n-acetylneuraminate is also a pronounced metabolite in the biofilm cells when grown in the human serum medium, and it is a predominant sialic acid in human serum that can serve as a nitrogen/carbon source via entering the PPPP. gingivalis and other potential pathogens5. Given that our findings on mono-species biofilms have demonstrated that among different oral species only P. gingivalis can efficiently exploit such a metabolic plasticity to upregulate fimA expression, our working hypothesis was that such a metabolic plasticity of P. gingivalis may foster biofilm formation of oral species upon co-existence and adherence to P. gingivalis, as previously reported60. To test this hypothesis, we applied a model multispecies system comprised of P. intermedia, T. forsythia, S. gordonii, and F. alocis, but in the presence or absence of P. gingivalis strain 381; respective biofilms were assessed in the human serum-based medium with and without adding pyruvate. In the absence of P. gingivalis, these species were unable to form biofilms in the human serum-based medium, but robust biofilms could develop in the presence of P. gingivalis that were significantly augmented upon addition of pyruvate  cells. First, we confirmed that P. gingivalis could form biofilms in RPMI 1640 medium supplemented with 5% human serum, a defined medium used to propagate the primary HGEP cells; still, addition of pyruvate to this medium could increase the biofilm growth of P. gingivalis strain 381 by eight times  of 100 for 1 h) was performed under both anaerobic and aerobic conditions with and without adding pyruvate. Our data showed that exogenous pyruvate significantly increased the efficacy of the adhesion of P. gingivalis to the primary HGEP cells by 2.7 times under anoxia and 2.1 times under normoxia , 5 μg/ml hemin, and 1 μg/ml menadione was used for the cultivation of Bacteroidetes species, and N-acetylmuramic acid (10 μg/ml) was added to the medium for the cultivation of T. forsythia. For solid culture cultivations, BAPHK [referring to TSBHK supplemented with 5% defibrinated sheep blood ] or BHI  were applied. Desired concentrations of agar were made using Bacto™ Agar or Agarose . For BSA-, HSA-, or human serum-based media, HyClone™ BSA , HSA , and CELLect® Human Serum  were applied, respectively, in the BS buffer containing 14 mM Na2HPO4, 10 mM KCl, and 10 mM MgCl2, pH 7.3, and 5 μg/ml hemin and 1 μg/ml menadione was added to these media in all experiments. Incubation of all bacterial species was performed at 37 °C in an anaerobic chamber  with an atmosphere containing 5% hydrogen, 10% carbon dioxide, and 85% nitrogen. The primary HGEP cells (Celprogen) were cultured either in Human Oral Epithelial Cell Culture Complete Growth Media with Serum  or in RPMI 1640  supplemented with 5% human serum (v/v) at 37°C in 5% CO2. Corning® 24-well microplates (CLS3527), pre-coated with poly-L-lysine solution , were used for HGEP cultivation. For performing assays under microaerophilic condition, BD GasPak™ EZ container system and Thermo Scientific™ AnaeroPouch™-MicroAero Gas Generator were applied. All chemicals, reagents, and chemical uncouplers were purchased from Sigma-Aldrich unless otherwise mentioned.19. For biofilm assay, an overnight culture was washed with pre-reduced BS buffer and pelleted inside anaerobic chamber. Bacterial cells were resuspended in the working medium supplemented with 5 μg/ml hemin and 1 μg/ml menadione to reach the optical density at 600 nm (OD600) = 0.25 (5 × 109 cells/ml). A total of 200 μl of the cells was applied in each well and anaerobically incubated for 48 h. The plates containing bacterial biofilms were washed twice by immersing in distilled water to remove free cells, and air dried. A total of 200 μl of 0.1% safranin in water was used to stain each well for 30 min. The plates were washed by immersing twice in distilled water, and air dried. The extraction of the stain from biofilms was performed using 200 μl of 90% ethanol containing 1% sodium dodecyl sulfate for 30 min followed by reading the absorbance at 492 nm. For multispecies biofilm assays, an overnight culture of each species was harvested, washed, and prepared in the working medium as explained above. A total of 40 μl of each species (at OD600 = 0.25) was added to each well in a 96-well plate to the final volume of 200 μl (~2 × 109 cells/ml); for those conditions without P. gingivalis, the only medium was applied. Incubation was performed anaerobically for 48 h. Biofilms were subjected to safranin staining, as explained above or genomic DNA (gDNA) isolation, as described later.The biofilm and surface translocation assays were performed at 37 °C, inside an anaerobic chamber (COY Lab Products), respectively, using our standard 96-well biofilm protocol and our designed motility chamber, as previously described19. Briefly, a motility chamber slide was made by compressing several layers of sterilized parafilm M (Bemis) on a glass slide followed by fixing of the parafilm with slight heat. The sides of the slide were then sealed with nail polish. The center of the parafilm layer was removed with a sterile scalpel to create a chamber (0.5 cm H × 1.5 cm W × 3.0 cm L). This chamber was then filled with soft agar medium, i.e., BHI medium containing 0.3% agar. The medium was allowed to solidify, and a coverslip inoculated with a tiny dot of cells at the center was inverted and placed on chamber filled with medium and mounted with nail polish. The slide was anaerobically incubated for more than a week. Imaging was performed using an inverted Nikon Eclipse Ti microscope system  inside an anaerobic chamber19.Surface translocation assay was performed using the motility chamber slide, as described previously86 with some modifications. Inoculum was prepared as described above for biofilm assay. Cells were diluted to OD600 = 0.2 and 1 ml of the cells was added to each well of a 12‐well microtiter plate (Greiner Bio‐one) containing a circular glass coverslip. Plates were incubated for 24 h in the anaerobic chamber. Then glass coverslips were gently washed with distilled water to remove free cells and stained with the Invitrogen Live/Dead BacLight Bacterial Viability Kit (Thermo Fisher Scientific). For staining each coverslip, 1 µl of SYTO 9 and 1 µl of propidium iodide (PI) were mixed and added to 1 ml of distilled water. The coverslips were treated with the staining solution for 15 min, followed by washing with 1 ml of distilled water. The coverslips were then removed, placed face down on a glass microscope slide, and biofilms were visualized by a laser scanning confocal microscopy on a Leica SP8 confocal microscope (Leica Microsystems Inc.), using 507/545 nm (SYTO 9) and 614/745 nm (PI) lasers, and ×20 objective. Images were analyzed using IMARIS image analysis software (Bitplane AG).Sample preparation was performed as described previously600 = 0.15 were found ideal for monitoring redox state over 6 h. Cells at the exponential phase in TSBHK medium were cooled down on ice and pelleted at 4700 r.p.m. for 15 min. Cells were washed once with pre-reduced BS buffer and resuspended and diluted (OD600: 0.15) in BS buffer containing 5 µg/ml hemin and 1 µg/ml menadione, as working cell suspension. Substrates were prepared as a 25% stock solution in BS buffer (pH 7.3). Redox state was measured fluorometrically  on the Synergy H1 Hybrid Microplate Reader (BioTek). This experiment was conducted using at least three repetitions for each treatment.This assay was performed in 96-well microtiter flat-bottom plates made of polystyrene . Upon optimization procedure, 2 µl of 50 mg/ml resazurin (filter sterilized) in 200 µl of cell suspension with OD600:0.5) in TSBHK medium were cooled down on ice and pelleted at 4700 r.p.m. for 15 min. Cells were washed once with pre-reduced BS buffer and resuspended in the same volume of BS buffer containing 5 µg/ml hemin and 1 µg/ml menadione to retain OD600 at 0.5. Cell suspensions (4 ml) were supplemented with desired substrates and incubated anaerobically at 37 °C for 6 h (cell doubling time is 3 h). Then, cells were placed on ice, pelleted, and subjected to [NAD+] and [NADH] extraction and quantification, using the EnzyChrom NAD+/NADH assay kit (BioAssay Systems). The protocol of this kit was followed for separate extraction of NAD+ and NADH, and 100 µl of each extraction buffer and 20 µl of assay buffer were applied for 8.7 mg of cells. The standard curve was prepared according to the manufacturer’s protocol using NAD+ as standard. The intensity of generated colors was measured on the Synergy H1 Hybrid Microplate Reader (BioTek) at 565 nm.Cells at the exponential phase (OD19. RNA extraction was performed in the anaerobic chamber to avoid aerobic stress using Direct-zol™ RNA MiniPrep Kit (Zymo Research) with slight modification. Briefly, RNA extraction was performed for biofilm cells after removing free and planktonic cells from 96-well plates. Biofilm cells were lysed using 200 µl of TRI Reagent® (Zymo Research) for each well. Debris were removed from collected samples by centrifugation at 14,000 × g for 10 min and equal volume of 100% ethanol was mixed with each sample. Each mixture was loaded into a Zymo-Spin™ IIC column and centrifuged at 14,000 × g for 30 s followed by washing using 400 µl of RNA wash buffer. DNA residues were completely digested and removed from columns in two rounds, each by adding 80 µl of DNase I and DNA Digestion Buffer (see Direct-zol™ RNA MiniPrep Kit protocol), and incubating at 30 °C for 1 h followed by centrifugation at 14,000 × g for 30 s. Second round was followed by washing columns using 400 µl of Direct-zol™ RNA PreWash and 700 µl of RNA Wash buffers. Pure RNA crude was eluted twice using 15 µl of DNase/RNase-free water. Quality control of RNA samples was performed using Qubit® 2.0 Fluorometer . For RT-qPCR, the reverse transcription reactions were performed by using the RNA to cDNA EcoDry Premix (Random Hexamers) kit . Briefly, for each sample, 2500 ng of isolated RNA was added into 20 µl of reaction solution as final volume. Reaction was performed at 42 °C for 60 min using a Bio-Rad T100TM thermocycler followed by stopping it at 70 °C for 10 min. For relative quantification of desired genes, qPCR assays were conducted in a 20 µl total volume containing 1 µl of 1:10 diluted cDNA, 0.5 µM of each primer . Using serial dilutions of gDNA (10–0.001 ng DNA) and 16S rRNA gene primers  was used in a total reaction volume of 20 μl for qPCR analysis, as described19. From the obtained Cq values, absolute DNA concentration was calculated for each species by plotting on the linear regression, and the relative abundance was calculated from the total gDNA that was obtained in each biofilm biomass.Supplementary Table g for 30 min at 4 °C, collected samples were freeze-dried, weighed, and subjected to further purification using the solvent system consisting of acetonitrile:methanol:acetone (8:1:1)19. For global metabolomic analysis, extracts were pre-normalized to the sample protein content of 500 µg/ml prior to LC–MS/MS analysis19. LC–MS/MS analysis was performed on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler. All samples were analyzed in positive and negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 18-pfp 100 × 2.1 mm, 2 μm column with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. The flow rate was 350 μl/min with a column temperature of 25 °C. A 4 and 2 μl samples were injected for negative and positive ions, respectively. Data from positive and negative ion modes were separately subjected to statistical analyses, and all subsequent data analyses were normalized to the sum of metabolites for each sample. The open-source software MZmine89 was used to identify features and deisotopes, and the MetaboAnalyst software90 was applied for comparative and statistical analyses.Preparation of biofilm cells for metabolomic analysis was similar to the procedure explained in the biofilm preparation section. HSA (0.4%) and human serum (5%) in the BS buffer, with or without pyruvate addition, were used as the working media. After 48-h incubation of 96-well plates, the medium was removed from wells. Biofilm cells were washed with cold phosphate-buffered saline , collected by pipetting, and transferred into tubes. After a centrifugation at 4700 × 91. To this end, we used P. gingivalis strain 381 and primary HGEP cells growing in RPMI 1640 medium (without antibiotic) supplemented with 5% human serum. First, we confirmed that P. gingivalis can form biofilms in RPMI 1640 medium supplemented with 5% serum as explained in the biofilm preparation section. For invasion assay, an overnight culture of P. gingivalis strain 381 cells grown in 2 ml of TSBHK was harvested and washed with RPMI 1640 medium. The bacterial pellet was resuspended in 2 ml of RPMI 1640 medium that was used as the stock bacterial culture (colony-forming unit (CFU) = 1.5 × 107). On the other hand, primary HGEP cells were grown to 90% confluence in RPMI 1640 medium supplemented with 5% serum in 24-well microplates (pre-coated with 0.01% poly-L-Lysine solution). The primary HGEP cells in each well were washed with 1 ml of RPMI 1640 medium. Two sets of plates containing the primary HGEP cells were used to infect with P. gingivalis cells from the stock bacterial culture at a MOI of 100. In each set, a group of wells (n = 4) were supplemented with pyruvate to the final concentration of 0.5%. After adding the bacterial cells to each well, a set of these plates was aerobically incubated, while another set was incubated in an anaerobic chamber; both at 37 °C for 1 h. After incubation, all plates were washed three times with pre-reduced PBS buffer (1 ml per well) under anaerobic condition. For total adhesion and invasion assessment, cells were lysed with 1 ml of sterile distilled water for 15 min, and serial dilutions of the lysates were plated on BAPHK and were anaerobically incubated for CFU measurement. For invasion assessment, first, extracellular bacteria were killed using metronidazole (200 μg/ml) and gentamicin (300 μg/ml) for 1 h, then infected cells were lysed for CFU enumeration. The results were calculated from recovered CFU values as a percentage of total bacteria that were applied as the initial inocula.This assay was performed using a method described by Inaba et al.p > 0.05); therefore, the results were statistically analyzed by ANOVA followed by post hoc Tukey’s honestly significant difference (HSD) test for pairwise comparisons, using XLSTAT statistical add-in software for Microsoft Excel 14.0. GraphPad Prism version 8.0.1 was applied for performing Student’s t test in pairwise comparisons. All experiments were conducted using at least three repetitions. Differences in the data were considered significant when the probability value was <5.0% .Statistical analysis for metabolomics data was based on univariate analysis by ANOVA that was performed separately on the positive and negative ion data from all data sets. For other data, the Shapiro–Wilk test was used to evaluate the normality of the distribution of the data. It was indicated that the data were normally distributed (Shapiro–Wilk test: Further information on experimental and research design is available in the Supplementary InformationReporting Summary


The clinical characteristics and prognosis of primary intestinal diffuse large B‐cell lymphoma (PI‐DLBCL) are rarely reported. We aimed to explore the role of surgery in patients with PI‐DLBCL.Adult PI‐DLBCL patients were included from the Surveillance, Epidemiology, and End Results database. The effect of surgery was evaluated by Kaplan–Meier and Cox proportional regression analyses. Propensity score matching (PSM) was used to reinforce our results. Lasso regression was utilized to determine independent risk factors of overall survival (OS) for a nomogram and a novel web‐based calculator. The performance of the model was measured via concordance index, receiver operating characteristic curve, and calibration plots in both cohorts.p = 0.007, p < 0.001, respectively). Before PSM, local tumor destruction (LTD) displayed a survival advantage over resection in patients without chemotherapy (p = 0.034). After PSM, surgery was still identified as a beneficial factor for OS (p = 0.0015). However, there was no statistical difference between LTD and resection (p = 0.32). The nomogram for 3‐, 5‐, and 10‐year OS predictions exhibited dependable consistency between internal and external validation.Overall, 1602 patients with PI‐DLBCL were analyzed. Surgery significantly improved survival in both univariate and multivariate analyses (This study approves the beneficial effect of surgery on clinical endpoints in PI‐DLBCL patients. For those who are not suitable for resection, LTD may also be a practical option. The predictive nomogram and the web‐based calculator could help clinicians individually evaluate the prognosis and optimize personalized treatment decisions for these patients. This study approves the beneficial effect of surgery on clinical outcome in patients with primary intestinal diffuse large B‐cell lymphoma. The predictive nomogram and the web‐based calculator could help clinicians to predict the prognosis and optimize individualized treatment decisions for these patients. Besides, we established a practical web‐based calculator for individual survival evaluation of patients with PI‐DLBCL.22.1We extracted patients diagnosed with PI‐DLBCL from 2004 to 2016 in the SEER 18 registries. PI‐DLBCL was identified according to the histology code (9680) and primary anatomic site (C17‐21.8) of the International Classification of Diseases for Oncology Third Edition (ICD‐O‐3). Patients who were microscopically confirmed with PI‐DLBCL were included. We excluded these patients: (1) under 18 years old; (2) DLBCL was not the first primary malignancy; (3) with no information on Ann Arbor stage, race, or marital status; (4) unknown surgery treatment, A symptom or B symptom; (5) survival time was recorded as zero.2.2The demographics and disease characteristics of patients included age at diagnosis, sex, race, marital status, primary site, Ann Arbor stage, symptom, surgery, chemotherapy, survival time, and vital status. Age was divided into four categories , race into white, black, and other. Marital status was classified into married, single, and other. Stage was categorized as early (Ann Arbor Stage I/Ⅱ) and advanced stage. Primary site was divided into three sites: small intestine (C17.0–17.9), colon (C18.0–18.9), and anorectal (C19.9–21.8). Surgery group was partitioned into local tumor destruction  and resection .2.3In retrospective studies, selection bias is inevitable, resulting in uneven distribution of confounding factors between two groups. To reduce selection bias and adjust for the confounding factors, we carried out a propensity score matching (PSM) for the surgery variable accounting for all the covariates mentioned above.2.4Summary statistics were applied to depict the basic characteristics of the included population. The survival curves were plotted with Kaplan–Meier method and evaluated via log‐rank tests. To analyze independent prognostic factors related to the OS, we used both univariate and multivariate Cox regression to compute hazard ratio (HR) and the 95% confidence interval (CI).p < 0.05.Furthermore, a nomogram model and a web‐based application were established to predict the 3‐, 5‐, and 10‐year survival probabilities for PI‐DLBCL patients. L1‐penalized (Lasso) regression was implemented to filter factors for the OS nomogram.33.1Upon applying the inclusion and exclusion criteria, we included 1602 adults confirmed with PI‐DLBCL in the SEER database from 2004 to 2016 . Although not statistically significant (p = 0.093), B symptom manifested worse survival. According to the previous research, B symptom is an important factor affecting clinical outcome,In total, 3‐, 5‐, and 10‐year survival rates in the whole cohort were 68.7% , 62.4% , and 50.2% . The 3‐, 5‐, and 10‐year OS probabilities were 71.1%, 65.0%, and 52.0%, respectively, in patients undergoing surgery, and 64.5%, 57.9%, and 46.8%, respectively, for patients without surgery. Kaplan–Meier survival curves for surgery and no‐surgery groups indicated that surgery was related to better survival  , and there was no difference between partial resection and radical resection . Considering the possible confounding effects of chemotherapy, we divided the population into the chemotherapy group and the non‐chemotherapy group. We performed a stratified analysis of the surgery variable in both groups. We concluded that the way of surgery had no significant effect on patients receiving chemotherapy . However, for patients without chemotherapy, LTD showed better OS than resection .Furthermore, we aimed to investigate the effect of surgery mode on the clinical endpoint of PI‐DLBCL. Firstly, we analyzed the entire population who underwent surgery. We found that LTD was associated with a survival benefit over resection . The Kaplan–Meier survival curve of the new matching data was depicted in Figure PSM was introduced to optimize the imbalance among the aforementioned baseline variables in all cohorts. As shown in Figure 3.6Furthermore, a nomogram was produced to predict 3‐, 5‐, and 10‐year OS for PI‐DLBCL patients. 1122 patients were randomly divided into the training set and 480 patients to the validation set. We utilized Lasso regression model in the training cohort to identify independent risk factors affecting OS Figure . A total3.7https://tumor.shinyapps.io/PI‐DLBCL/ to predict the OS of patients with PI‐DLBCL based on our nomogram (Figure Finally, we established a dynamic web‐based calculator accessible via m Figure . It is c4Helicobacter pylori eradication alone.PI‐DLBCL is a rare and heterogeneous disease entity. The lack of prospective randomized clinical trials results in the undetermined optimal therapeutic strategy. Treatments of PI‐DLBCL vary from chemotherapy alone to multimodality combined with surgery and radiotherapy. As surgical excision is not better than chemotherapy combined with radiotherapy, the role of surgery has been weakened in primary gastric DLBCL, and treatment attention has shifted to organ preservation.In our study, 63.4% of patients underwent surgery and 43.4% received surgery combined with chemotherapy, the multimodal treatment related to a better outcome than chemotherapy alone. As the correlation between variables tends to cause confounding bias in measuring baseline variables, PSM is often utilized to eliminate bias in observational studies.Considering the heterogeneity of PI‐DLBCL, we verified the survival benefit of surgery in critical clinical subgroups. Stage and age were prognostic factors as indicated in many reports.In multiple regression analysis, the multicollinearity between variables will affect the research conclusions. Lasso Cox regression analysis was introduced to screen variables during the nomogram construction in our study to cope with potential collinearity. In the Lasso regression model, variables are assigned to different penalties. The more important variables are punished less, making them more likely to be retained in the model, while the less critical variables are punished more and tend to be discarded. Therefore, this method can select the most important prognostic factors to build a model to predict survival.This study presents several limitations. Firstly, although PSM could attenuate the bias derived from the uneven distribution of measured covariates, the bias originating from unmeasured ones is unavoidable. Secondly, it is well known that the lactic dehydrogenase level and performance status are important components of lymphoma prognostic factors, but they are not recorded in the SEER database, so we could not include these variables for analysis. Besides, important factors such as whether intestinal lymphoma is germinal center subtype or non‐germinal center subtype,In conclusion, this is the first population‐based real‐world analysis to evaluate the role of surgical treatment in PI‐DLBCL. Our study approves the beneficial effect of surgery on survival outcome in patients with stage I/Ⅱ PI‐DLBCL. For patients who are not suitable for resection, LTD may also be a potential option. The predictable nomogram and its convenient online version could help clinicians evaluate the prognosis and optimize personal guidance for patients with PI‐DLBCL.The authors declare that there is no conflict of interest.MW and SM designed the study, collected the data, and wrote the paper. WS and YZ contributed to data analysis. SL edited the paper and YH revised the paper. All authors reviewed the paper and approved the final manuscript.The identifiable patient information is not contained in the SEER database, so the approval of an ethics committee was not required.Figure S1‐S2Click here for additional data file.Table S1‐S2Click here for additional data file.


M = 11.43 ± 3.06 years), correlations between drawings scores and Raven’s Matrices scores, age, and academic achievement were examined. Although older children (> 11 years) obtained higher drawing scores than younger ones (p < 0.001), age significantly correlated with DAP:QSS scores only in children younger than 11 years , indicating conflictive evidence for construct validity and a possible ceiling effect. No correlations emerged between DAP:QSS scores and grades . DAP:QSS scores were significantly associated with Raven’s Matrices score, but low correlation coefficients (0.156–0.498), low sensitivity (0.12), and high false negative (87.9%) and positive (82%) rates suggest poor DAP:QSS validity as an intelligence measure. The researchers concluded that DAP:QSS failed to produce a psychometrically sound assessment of children’s intellectual functioning.To assess the psychometric properties of the Draw a Person: A Quantitative Scoring System (DAP:QSS), in 2543 children ( The use and usefulness of human figure drawing tests as measures of intellectual ability have generated considerable debate –3. AmongEvidence suggests that the DAP:QSS generally yields high coefficients for intra- and inter-rater reliability –7, interModerate-to-high correlations with measures of verbal intelligence  , 8, 9 anHowever, other researchers have found little support for the validity of the DAP:QSS as a tool for evaluating children’s intellectual ability and assessing children with mental disabilities , 16, 17.In examination of the utility of any measure of intelligence, relevant importance has been given to its ability to predict scholastic performance , 19; conIn recent investigations of the strengths and weaknesses of the DAP:QSS, researchers have analyzed whether DAP:QSS scores can be improved with practice , can be N = 200, 6–15-year-olds; ; woman, t(2541) = − 6.727, p = 0.000, d = − 0.27 ; self, t(2541) = − 4.876, p = 0.000, d = − 0.2 ), as well as total score (t(2541) = − 5.511, p = 0.000, d = − 0.22 ). By contrast, RCPM and RSPM mean scores did not differ between boys  and girls  in the sample (RCPM t(1037) = 0.700, p = 0.484; RSPM t(1502) = − 1.368, p = 0.172) (data not shown).Girls obtained higher DAP:QSS scores than boys for all drawings  = − 21.469, p < 0.001, d = − 0.85 ; woman, t(2541) = − 24.844, p < 0.001, d = − 1.01 ; self, t(2541) = − 27.235, p < 0.001, d = − 1.1 ; total, t(2541) = − 27.002, p < 0.001, d = − 1.09 )  and girls .As shown in Table However, in an analysis of the sample grouped by age, correlations between DAP:QSS scores and age emerged only for participants aged 11 years or younger but not older ones, even when the effect of SES was considered, as shown in Table z = 8.643, p < 0.001) than those between DAP:QSS total score and RSPM score . In the second step of analysis, the addition of SES in the regression equation increased the explained variance by 1.7% . In a subsequent stepwise regression analysis testing DAP:QSS scores and SES as predictors, SES surfaced as a significant predictor of RSPM scores by explaining approximately 3.9% of the variance . In the second step of that analysis, the addition of DAP:QSS total scores in the regression equation increased the explained variance in RSPM scores by 3.2% .In stepwise regression analysis testing DAP:QSS scores and SES as predictors, DAP:QSS total score emerged as a significant predictor of RCPM scores by explaining approximately 24.7% of the variance . In the second step of the analysis, the addition of DAP:QSS scores in the regression equation increased the explained variance by 1.7% . Testing DAP:QSS scores, RSPM score, and SES as predictors of grades revealed that RSPM score was a significant predictor , as was SES  and DAP:QSS total score .In another stepwise regression analysis testing DAP:QSS scores, RCPM score, and SES as predictors of grades, SES emerged as a significant predictor by explaining approximately 8.8% of the variance .A comparison of the percentile ranks obtained by participants on the RCPM or RSPM and the DAP:QSS with the Wilcoxon signed-rank test revealed that scores obtained on the RCPM or RSPM differed significantly from those on the DAP:QSS  did not have borderline deficient or deficient intellectual functioning as measured by the RCPM or RSPM, for a high false positive rate. According RCPM and RSPM scores, of those 131 participants, 40 (30.5%) had low average intelligence, 36 (27.4%) had average intelligence, 37 (28.3%) had high average intelligence, 14 (10.7%) had superior intelligence, and four (3.1%) had very superior intelligence (Table As shown in Table n = 369 of 420) were not identified as having borderline intellectual functioning according to the DAP:QSS, for a high false negative rate. The DAP:QSS erroneously evaluated those participants by classifying 75 of 369 (20.3%) as having low average intelligence, 229 (62.1%) as having average intelligence, 49 as having high average intelligence (13.3%), and 16 (4.3%) as having superior intelligence  were not of borderline or deficient intellectual functioning as measured by the RCPM or RSPM, for a high false positive rate  were not identified as having borderline intellectual functioning by the DAP:QSS, for another high false negative rate  across a broad age range, with the aim of examining the psychometric properties of the DAP:QSS. To date, the American norms collected in a sample of 2622 individuals aged from 5 to 17 years have constituted the only available guide for scoring and interpreting Naglieri’s drawings. Therefore, the results obtained provide Italian norms for the DAP:QSS as well.The study presented here was conducted with a large sample of Italian children . Accordingly, a small amount of variance in a child’s Raven’s Matrices scores is predicted by their drawings.In terms of concurrent validity, the findings indicate positive and significant—albeit modest—correlations between children’s scores on the DAP:QSS and their scores on standard measures of nonverbal intelligence  regardless of the effect of SES. In particular, correlation coefficients ranging from 0.156 to 0.498 were comparable and sometimes higher than the correlation levels between the DAP:QSS and analogous measures of nonverbal intelligence , as reported by previous studies —may not be a valid measure of cognitive ability , 51.Several limitations in the study warrant attention. First, the sample, despite its size, was recruited out of convenience and did not include children with mental disabilities. Moreover, the construct validity was examined by analyzing the increase in mean scores as a function of age; therefore, aspects of the construct validity of the DAP:QSS remain to be evaluated, especially discriminant validity. Data of the drawings was interpreted with reference to the original score norms of Naglieri’s drawings. It should be taken into account that DAP:QSS was normed in 1988, and adjustment for the Flynn effects—although observed in human figure drawings —is actuaIn this study we aimed at assessing the psychometric properties of the DAP:QSS. Although our results produced encouraging evidence of the reliability of Naglieri’s drawings, support for the validity of the drawings as a measure of nonverbal intelligence was rather weak. The DAP:QSS also appeared to be an inaccurate measure of academic performance and ineffective in screening for intellectual ability.In line with the criticism of psychometric qualities shown by previous and more recent versions of DAP, DAP:QSS consistently failed to produce a psychometrically sound assessment for children’s intellectual functioning. Taken together, this evidence indicates that the utility of human figure drawings as a measure of intelligence is particularly poor, leading to the conclusion that practitioners should not rely on human figure drawing tests as a projective measure of intelligence.


Tributyltin, a well-known endocrine disruptor, is widely used in agriculture and industry. Previous studies have shown that tributyltin could cause deleterious effects on bone health by impairing the adipo-osteogenic balance in bone marrow.− 1) or corn oil by gavage once every 3 days for 60 days in this study. Then, we analyzed the effects of tributyltin on geometry, the polar moment of inertia, mineral content, relative abundances of mRNA from representative genes related to adipogenesis and osteogenesis, serum calcium ion and inorganic phosphate levels.To investigate further the effects of tributyltin on bone, weaned male SD rats were treated with tributyltin (0.5, 5 or 50 μg·kg− 1 tributyltin caused an obvious decrease in femoral cortical cross sectional area, marrow area, periosteal circumference and derived polar moment of inertia in rats. However, other test results showed that exposure to tributyltin resulted in no significant changes in the expression of genes detected, femoral cancellous architecture, ash content, as well as serum calcium ion and inorganic phosphate levels.Micro-computed tomography analysis revealed that treatment with 50 μg·kgExposure to a low dose of tributyltin from the prepubertal to adult stage produced adverse effects on skeletal architecture and strength.The online version contains supplementary material available at 10.1186/s12891-021-04298-2. After 3 days of acclimatization, rats were randomly assigned into four groups (10 rats per group) based on body weight to achieve similar average weights in different groups. Rats were treated with corn oil or TBT  by introperitoneal injection. After reaching the surgical level of anesthesia, blood was obtained from the aorta ventralis. The serum was separated and stored at − 80 °C for further analysis. Both femurs were isolated modestly, and then the muscular tissue was removed. Five left femora were used to assess gene expression, and the other left femora were stored at − 20 °C for ash content analysis. All right femurs were weighed and measured as described in a previous study [Urethane was dissolved in saline solution at a final concentration of 20% (w/v). The rats were anesthetized with urethane , and strained through a 70-μm cell strainer. After centrifugation, total RNA was extracted using the QIAamp RNA Blood Mini Kit  according to the manufacturer’s recommendation. The concentration and quality of RNA were determined with a NanoDrop 2000c spectrophotometer . After quality inspection, RNA was stored at − 80 °C for QPCR.Bone marrow (BM) was flushed from the left femur (–ΔΔCT method [QPCR was performed using QuantiTect SYBR® Green RT-PCR Kits (QIAGEN) with reactions scaled to 25 μl, and 25 ng mRNA was used in each reaction. PCRs (in duplicate) were performed using a 7500 Fast Real-Time PCR System  with a program consisting of 95 °C for 2 min, 40 cycles of 95 °C for 10 s and then 60 °C for 20 s and 72 °C for 30 s, followed by melting curve analysis. The primer sequences for each gene are listed in Table S1. Genes of interest were normalized to the GAPDH gene in the same sample following the 2The fixed femurs were scanned with a Scanco μCT100 scanner , at a 20 μm isotropic resolution using an integration time of 300 ms, energy of 70 kVp, and intensity of 200 μA. Gaussian filtering  was used to reduce background noise. For analyses of trabecular bone within the distal femur, the specimen was scanned in 700 slices and a region of interest (ROI) of trabecular bone was selected between 230 slices and 300 slices to the growth plate. Trabecular bone parameters of the femoral metaphysis, including bone volume fraction (BV/TV), connectivity density (Conn.D), structural model index (SMI), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp), were determined using Scanco’s 3D analysis tools (direct model). For cortical bone analysis, the femur specimens were scanned similarly. One millimeter thick sections immediately distal to the mid-diaphysis were used as ROI. In addition, total cross sectional area (TCS.Ar), cortical cross sectional area (Ct.Ar), cortical thickness (Ct.th), periosteal circumference (Ps.Cf), endocortical circumference (Ec.Cf) and marrow area (Ma.Ar) were obtained from the analysis.Jo was calculated as the medio-lateral (IML) + antero-posterior (IAP) axes, following the method of Jepsen et al. .Five left femora per group were used to detect the ash content. The dried, and ash weights were determined as described previously . Ash con2+ and Pi levels were detected by colorimetry, following the manufacturer’s instructions.Serum Ca(P < 0.05), Dunnett’s or Dunnett’s T3 test was performed to make multiple comparisons among the groups based on the homogeneity of variance.The statistical analyses were performed by SPSS software, version 21.0 for Windows . Two-tailed Student’s t-test was used to analyze the bone structural phenotype and Jo, which were compared between the two groups. Other indicators were analyzed by ANOVA. The significance level was set at 0.05. Once significance was established The mean body-weight of rats is shown in Fig.PPARγ is a key transcriptional regulator of fat formation , while FP = 0.153). Although the expression of Angptl4 showed a significant decrease in the 0.5 μg·kg− 1 and 5 μg·kg− 1 TBT groups compared with the control (P < 0.05), the degree of the decline was minor .Analysis with QPCR analysis showed no notable TBT-related changes in the expression of PPARγ, Fabp4, ALP and OC at 85 d Fig.. The relP = 0.153) along with a decreasing trend in Conn. D , Tb. N , and a subtle increase in Tb. Sp  and SMI , in TBT-treated rats compared to controls, although the differences were not significant , but had no effects on other indexes.Quantitative measures of trabecular bone quality showed a decreasing trend in the BV/TV  significantly increased the CD45−CD31−Sca1+CD24+ (a tri-potent population with stem cell-like characteristics) and adipogenic progenitor cells (APCs) frequencies for 1 day but not for 10 days [Mesenchymal stem cells (MSCs) are multipotent cells contributing to osteoblast and adipocyte progenies in adult bone marrow . As comm 10 days . Our rec 10 days . In addi 10 days . TherefoThe different effects of TBT between this study and in vitro studies , 41 were− 1 TBT caused a significant decrease in Ct. Ar and Jo, but not on ash content, which might be one possible explanation for the reduced but no significantly different biomechanical strength change induced by TBT in our previous study [Jo is a quantity used to predict an object’s ability to resist torsion. Because structures possessing the same Ct. Ar but different moments of inertia  will exhibit different mechanical characteristics in bending and torsion , so measus study .2+ and Pi, mainly in the form of crystalline hydroxyapatite (HA) [2+ and Pi levels in this study, which was in accordance with the data of ash content.The skeleton is made of collagen fibers that form a scaffold where mineralization is initiated by the accumulation of Caite (HA) . Calciumite (HA) , 54. TBT− 1 TBT caused a significant decrease in femoral Ct. Ar, Th. Ar and Jo of rat. All the data suggest that exposure to a low dose of TBT from the prepubertal to adult stage produces adverse effects on skeletal architecture and strength.In conclusion, treatment with 50 μg·kgAdditional file 1: Table S1. Primers used for QPCR analysis of gene expression.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.


Cognitive-behavior therapy (CBT) is known to be an effective treatment for the anxiety and related disorders, with exposure therapy being a key component of this treatment package. However, research on the use of exposure therapy in clinical practice has presented mixed results, potentially due to differences in samples and training programs across countries. The present study aimed to extend upon existing research by examining the use and predictors of use of exposure therapy in a sample of psychologists working in clinical practice in Australia who treat clients with an anxiety disorder, obsessive compulsive disorder (OCD) or post-traumatic stress disorder (PTSD).Mage = 40.60; SD = 10.78; range 23 to 71 years; 84% female) participated in an online study investigating their clinical practices.One hundred registered psychologists (Results suggested that while the general use of exposure therapy is high, the use of disorder specific techniques was considerably lower, particularly for anxiety disorders and PTSD but not OCD. Psychology registration status and level of training were positively associated with use of exposure therapy as was the experience in treating anxiety disorders.These findings suggest that further or ongoing professional training may be required to optimize the use of disorder specific techniques.The online version contains supplementary material available at 10.1186/s40359-021-00613-7. Anxiety, obsessive compulsive disorder (OCD) and post-traumatic stress disorder (PTSD) are among the most frequently experienced mental health problems , 2, tendWhilst the present research focuses primarily on the use of exposure therapy for the anxiety and related disorders, research to date largely considers and evaluates the use of exposure therapy as a key component of treatment in combination with cognitive interventions, particularly given that the two can be difficult to disentangle  and are Despite the substantial research supporting the efficacy and effectiveness of exposure therapy, research on its use in clinical practice has produced mixed results. Much of this research has come from the US , 36–42. This pattern of use has been observed throughout Europe and the United Kingdom –45 and hWith homogenous samples of psychologists with specialist training in CBT however, the use of exposure therapy is considerably higher. In the US, 65% reported use of interceptive exposure for panic disorder , 88.4% uTaken together, there is mixed evidence for the use of exposure therapy in clinical practice, particularly evidence to suggest that use of disorder specific exposure interventions may warrant particular attention. This is consistent with literature which argues that dissemination and implementation efforts must be considered a key priority for our profession . HoweverThis study sought to examine the use of exposure therapy in a homogenous sample of psychologists working in clinical practice in Australia who treat clients with an anxiety disorder, OCD or PTSD. The use of a homogenous psychologist sample is particularly important to the Australian context where psychologists are the primary group providing psychological intervention to individuals with mental health problems. Consistent with prior research, it was hypothesized that: (1) use of general exposure therapy techniques will be high amongst practicing psychologists; and (2) the following variables will be positively related to frequency of use: registration status, experience, training, and cognitive-behavioural orientation.Mage = 40.60; SD = 10.78; range 23 to 71 years; 84% female). As recruitment source and completion rates were not monitored in this study, response rate is unavailable. This sample was also utilized to examine barriers to the use of exposure therapy in a study reported elsewhere. To be included in the study participants were required to: (1) be a registered or provisionally registered psychologists, (2) have more than 1 year of experience in the practice of psychology, (3) have worked clinically, to any frequency, in the last 12 months, and (4) regularly see clients with a diagnosis of or symptoms consistent with an anxiety disorder, OCD and / or PTSD. Table A total of 164 participants commenced this study between December 2018 and December 2019. Participants who met exclusion criteria and those with incomplete data were not included in final analyses. Figure The demographic questionnaire asked participants to indicate their 1) gender; (2) age; (3) years of clinical experience; (4) current registration status; (5) whether they have worked clinically in the last 12 months; (6) the country they currently practise in; (7) highest level of completed training; (8) how frequently they see clients with anxiety and related disorders; and (9) primary theoretical orientation. The demographic questionnaire can be found in the Additional file  gender; never’, ‘sometimes’, ‘half the time’, ‘often’, and ‘very often’. It then asked questions about treatments considered most essential to treatment effectiveness, in order of priority when working with clients with anxiety, OCD and PTSD, highlighting the top three techniques. The exposure therapy use questionnaire can be found in the Additional file The exposure therapy use questionnaire was adapted from Hipol and Deacon  and FreiParticipants were identified and invited to participate in the online study via university psychology training programs, advertisements on relevant social media websites/public list serves, and direct approach to psychologists via email. Recruitment source was not monitored in this study and participants were invited to enter a prize draw to win one of four $100 gift vouchers for participation. Potential participants were directed to a link to the online questionnaire which opened to the participation information sheet and consent form, followed by the exposure therapy use and optimal use questionnaire. Importantly, only those who indicated that they work with each presentation were asked questions relating to frequency of intervention use. The questions appeared in fixed order and took approximately 15 min to complete.t test and Pearson product-moment correlations were performed to determine if frequency of working with the population was predictive of exposure use.The use of therapy techniques was investigated through descriptive statistics, one-way repeated MANOVA univariate analysis and planned comparisons to determine differences in exposure therapy use by registration, training and years of experience. To understand the use of techniques considered essential to treatment, Chi square analyses were undertaken. Group differences in use were analyzed via multiple linear regression, independent sample f) were interpreted as small = .10, medium = .25 and large = .40; effect sizes for chi-square (Cohens w) were interpreted as small = .10, medium,. = 30 and large = .50; effect sizes for regression (Cohen’s f2) were interpreted as small = .02, medium = .15, and large = .35; effect sizes of independent means (Cohen’s d) were interpreted as small = .20, medium = .50, and large = .80, and effect sizes for correlations were (Cohen’s r) were interpreted as small = .10, medium = .30 and large = .50 [Effect sizes were calculated and interpreted as suggested by Cohen . Effect ge = .50 .An a priori power analysis indicated that with a medium to large effect size, alpha of .05 and power of .95, 108 participants were required to assess observed and expected data; 87 participants were required to assess group differences in use by registration, training and experience; and 111 participants were required to determine if frequency of work with population is predictive of use. All analyses were conducted using IBM SPSS Statistical Software Version 25.Participants were asked to provide data on techniques used for presentations reported as frequently treated over the preceding 12 months. When assessing general use, 95.88% of participants reported general use of exposure therapy when working with clients with an anxiety disorder, 91.67% reported use when working with clients with OCD, and 82.35% reported general use when working with clients with PTSD. Table F = 6.00, p < .004; Wilk’s Ʌ = .85; imaginal exposure F = 15.34, p < .00; Wilk’s Ʌ = .69; motivational interviewing; F = 4.39, p < .02; Wilk’s Ʌ = .89; EMDR F = 6.79, p < .002; Wilk’s Ʌ = .83; and ERP F = 62.41, p < .00; Wilk’s Ʌ = .35. Univariate analysis and Tukey post hoc test revealed that use of elimination of avoidance and safety behaviours was statistically higher with the anxiety disorders  than OCD  and PTSD ; use of imaginal exposure was statistically higher for PTSD  than with anxiety  and OCD ; use EMDR was statistically higher for PTSD  than with anxiety  and OCD ; and use of ERP was statistically different across all disorders, greatest use with OCD , followed by anxiety , and PTSD . No statistically significant differences were found in use of motivational interviewing across disorders. See Table Results of one-way repeated MANOVA revealed a statistically significant difference in use across disorders for the elimination of avoidances and safety behaviours F,68 = 6.0Participants were asked to indicate, in order of priority, the three techniques they see as most essential to treatment effectiveness. Results are presented in Table Χ2 (2) = 76.69, p = .00, representing a large effect, where ERP was considered most essential to the treatment of OCD . Imaginal exposure Χ2(2) = 41.64, p = .00 (large effect) was considered most essential to the treatment of PTSD . Elimination of avoidance and safety behaviours Χ2(2) = 9.21, p = .01,  was considered most essential to the treatment of anxiety  and EMDR Χ2(2) = 25.42, p = .00, (medium effect) was considered most essential to the treatment of PTSD . Results are presented in Table When considering the essential use of specific techniques for each disorder, Chi-square analysis revealed a significant relationship between disorder and essentiality of ERP D 58, 77.%. ImaginF = 8.14, p .00, R2 = .21, representing a medium effect. Registration and training added statistically significantly to the prediction.A multiple linear regression was undertaken to predict use of general exposure techniques for anxiety by registration, training, and years of experience. These variables statistically significantly predicted use t test was conducted to examine whether group differences in the use of exposure therapy were observed between clinicians who reported a CBT versus all other orientations. No statistically significant differences between CBT and other reported orientations was found t(95) = 1.11, p = .27.An independent r = .24, n = 97, p = .02).A Pearson product-moment correlation was run to determine the relationship between time spent working with clients with anxiety, OCD and PTSD and the general use of exposure therapy with clients with an anxiety disorder. There was a small, positive correlation between time spent working with the population and use, which was statistically significant  = 3.41, p .02, R2 = .13, representing a medium effect.A multiple linear regression was undertaken to predict use of general exposure techniques for OCD by registration, training, and years of experience. These variables statistically significantly predicted use t test was conducted to examine whether group differences in the use of exposure therapy was observed between clinicians who reported a CBT versus all other orientations. No statistically significant difference for the use of ERP between participants who identified their theoretical orientation as being CBT versus other theoretical orientations, t(70) = .32, p = .21 was found.An independent sample r = .08, n = 72, p = .53).A Pearson product-moment correlation was run to determine the relationship between time spent working with clients with anxiety, OCD and PTSD and use of general exposure techniques with clients with OCD. No statistically significant correlation was found  = 3.87, p .01, R2 = .15, representing a medium effect. Level of training added statistically significantly to the prediction.A multiple linear regression was undertaken to predict use of general exposure techniques for PTSD by registration, training, and years of experience. These variables statistically significantly predicted use t test was conducted to examine whether group differences in the use of exposure therapy were observed between clinicians who reported a CBT versus all other orientation for the treatment of PTSD. Results indicated no statistically significant differences between use t(66) = .09, p = .96.An independent sample r = .27, n = 68, p = .03).A Pearson product-moment correlation was run to determine the relationship between time spent working with clients with anxiety, OCD and PTSD and use of exposure therapy with clients with PTSD. A small positive correlation was found  = 4.28, p .01, R2 = .12, representing a small effect. Registration added statistically significantly to the prediction.A multiple linear regression was undertaken to predict use of imaginal exposure for anxiety by registration, training, and years of experience. These variables statistically significantly predicted use F = 7.47, p .00, R2 = .19, representing a medium effect. Registration added statistically significantly to the prediction.A multiple linear regression was undertaken to predict use of interoceptive exposure for anxiety by registration, training, and years of experience. These variables statistically significantly predicted use t test was conducted to examine whether group differences in the use of interoceptive exposure was observed between clinicians who reported a CBT versus all other orientations. No statistically significant difference for use between participants who identified their theoretical orientation as being CBT versus all other theoretical orientations, t(95) = .16, p = .88 was found.An independent sample r = .42, n = 97, p = .00). Similarly, a medium positive correlation was found for the use of imaginal exposure with clients with anxiety .A Pearson product-moment correlation was run to determine the relationship between time spent working with clients with anxiety, OCD and PTSD and use of interoceptive exposure with clients with anxiety. A medium positive correlation was found  = 5.84, p .00, R2 = .22, representing a medium effect. Registration added statistically significantly to the prediction, p = .01.A multiple linear regression was undertaken to predict use of imaginal exposure for PTSD by registration, training, and years of experience. These variables statistically significantly predicted use t test was conducted to examine whether group differences in the use of imaginal exposure was observed between clinicians who reported a CBT versus other orientation. No statistically significant difference for use between participants who identified their theoretical orientation as being CBT versus other theoretical orientations, t(51) = .36, p = .72 was found.An independent sample r = .01, n = 68, p = .95).A Pearson product-moment correlation was run to determine the relationship between time spent working with clients with anxiety, OCD and PTSD and use of imaginal exposure with clients with PTSD. No statistically significant correlation was found  = 7.11, p.00, R2 = .24, representing medium effect. Level of training added statistically significantly to the prediction, p = .00.A multiple linear regression was undertaken to predict use of ERP for OCD by registration, training, and years of experience. These variables statistically significantly predicted use t test was conducted to examine whether group differences in the use of ERP was observed between clinicians who reported a CBT versus other orientation. No statistically significant difference for the use of ERP between participants who identified their theoretical orientation as being CBT versus other theoretical orientations, t(70) = .59, p = .55 was found.An independent sample r = 0.14, n = 72, p = 0.24).A Pearson product-moment correlation was run to determine the relationship between time spent working with clients with anxiety, OCD and PTSD and use of ERP with clients with OCD. No statistically significant correlation was found  general use of exposure therapy will be high amongst clinicians; and that, (2) registration status, experience, training and cognitive-behavioural orientation would be positively related to frequency of use.In this study, participants’ general use of exposure therapy was high with 95% reporting use with anxiety, 91% reporting use with OCD and 82% reporting use with PTSD. While the general use of exposure therapy was high, the frequency of use of disorder specific exposure techniques varied. Of note, the use of interceptive exposure for anxiety symptoms was reported by only 26% of participants, while the frequent use of imaginal exposure for PTSD was reported by 47% of participants. Importantly, this was not the case in relation to the treatment of OCD, where ERP was reported to be frequently used by 83% of participants. These findings are similar to research from the Netherlands  and US , 54, whiWhen specifically considering use compared across disorders, imaginal exposure was found to be used more frequently for PTSD, and ERP was used most frequently for OCD. This is a pleasing finding, suggesting that there is increasing awareness of the importance of using disorder specific interventions with particular disorders. Further to this, consistent with existing research on treatment effectiveness , 26, 29,Across disorders, registration and training were most frequently found to be positively associated with the use of exposure therapy, although importantly, this association was not consistenly observed across disorders. This finding is consistent with prior research which suggests that training is positively correlated with use . This fiYears of experience and theoretical orientation were not found to be associated with the use of general or specific exposure therapy use across disorders. The former may be explained by therapist drift , whilst Whilst this study did not seek to assess the quality of exposure techniques utilised, results do suggest that other techniques, particularly cognitive restructuring, are frequently used and typically considered essential components to treatment effectiveness. Results may suggest that these techniques are used in combination with exposure techniques, suggesting modified use of exposure therapy. This is again a similar finding to that reported in previous research , 56 wherTo the authors’ knowledge, this study is the first of its kind to assess the use of exposure therapy in an Australian sample of psychologists. Results are promising, suggesting that many clinicians in Australia utilise general exposure techniques, both within and between sessions with the exception of PTSD. However, work remains necessary to improve the use of disorder specific interventions. In particular, the low percentage use of interoceptive exposure with anxiety, and the low percentage use of imaginal exposure with PTSD needs to be better understood. To address this, we recommend that future research considers the barriers to the use of these techniques, and how to address these within training and professional development programs. To be most effective, training programs should be evaluated to ensure effectiveness of dissemination and implementation, which is currently not routinely undertaken.Notwithstanding the important findings reported in this study, there are some notable limitations. Firstly, this study has utilized a relatively small and female dominated sample, although importantly is representative of psychologists in Australia on both gender and registration status . SimilarIn conclusion, the findings in this study suggest that the general use of exposure therapy by psychologists is high. However, the use of disorder specific exposure therapy techniques varies considerably amongst practicing psychologists, with the exception of ERP for OCD. Understanding when and how those techniques are used may inform knowledge and practice gaps, and formal and ongoing professional training needs. In particular, further training may be required in disorder specific exposure therapy interventions, to build on the fundamentals of the general exposure techniques and optimize disorder specific therapeutic outcomes. It is hoped that this research will contribute to the very important dissemination and implementation efforts currently being discussed in the broader psychological community.Additional file 1. Exposure therapy use questionnaire.


Myxovirus resistance protein 1 (MxA) is a biomarker that is elevated in patients with viral infections. The goal of this study was to evaluate the diagnostic value of MxA in diagnosing COVID‐19 infections in the emergency department (ED) patients.This was a single‐center prospective observational cohort study including patients with a suspected COVID‐19 infection. The primary outcome of this study was a confirmed COVID‐19 infection by RT‐PCR test. MxA was assessed using an enzyme immunoassay on whole blood and receiver operating chart and area under the curve (AUC) analysis was conducted. Sensitivity, specificity, negative predictive value, and positive predictive value of MxA on diagnosing COVID‐19 at the optimal cut‐off of MxA was determined.p < .001) in COVID‐19 patients compared to non‐COVID‐19 patients, respectively 1933 and 0.1 ng/ml. The AUC of MxA on a confirmed COVID‐19 infection was 0.941 (95% CI: 0.867–1.000). The optimal cut‐off point of MxA was 252 ng/ml. At this cut‐off point, the sensitivity of MxA on a confirmed COVID‐19 infection was 94% (95% CI: 85%–98%) and the specificity was 91% (95% CI: 72%–99%).In 2021, 100 patients were included. Of these patients, 77 patients had COVID‐19 infection and 23 were non‐COVID‐19. Median MxA level was significantly higher  levels accurately distinguish COVID‐19 infections from bacterial infections and noninfectious diagnoses in patients with suspected COVID‐19 infection in the emergency department. MxA measurements may be of added value in the diagnostic workup and patient flow in the emergency department. The study was conducted at the Laboratory Medical Immunology and ED of Erasmus University Medical Center, an academic hospital with annually 40.000 ED visits. The study was approved by the local institutional review board and registered under number: NL73846.078.20.Inclusion criteria were: referral because of clinical suspicion of COVID‐19 infection, age ≥18 years, and written informed consent. Exclusion criteria were: insufficient knowledge of the Dutch language, non‐COVID‐19‐related incapacitated subjects, or absence of informed consent.Suspected COVID‐19 infection was defined as patients presented at the ED with any of the following symptoms: dry cough, fever, headache, diarrhea, dyspnea, rhinitis, or lack of taste or scent.The sample size consisted of a convenience sample. Due to the availability of the research personnel, screening for eligibility and enrollment of patients took place from Monday to Friday during working hours. Any patient visiting the ED was screened for eligibility during this period.2.1Patient data including demographics, comorbidities, duration of symptoms, vital signs, and laboratory tests were collected during the ED visit. The use of immunosuppressive medication, defined as the use of systemic corticosteroids >7.5 mg prednisone equivalent per day, use of disease‐modifying antirheumatic drugs (DMARDs), biologicals, or anti‐rejection medication after organ transplantation, was recorded. Standard laboratory testing during the ED visit included white blood cell count, CRP, procalcitonin, lactate, ferritin, and lactate dehydrogenase (LDH). Patients were followed up for 30 days after hospital discharge.After obtaining informed consent, extra blood was drawn in a sodium heparin tube. MxA enzyme immunoassay was conducted as previously described.2.2The primary outcome of this study was a confirmed COVID‐19 infection by RT‐PCR test. An RT‐PCR test was part of the standard clinical workup at the ED and ordered by the treating physician. Patients were classified as COVID‐19 patients when the RT‐PCR test was positive and non‐COVID‐19 when the RT‐PCR test was negative.2.3Normally distributed variables were reported as mean with standard deviation (SD), non‐normally distributed variables as median with interquartile range (IQR). Cases with missing data that were not used in the primary or secondary analyses were deletedχ2 test. Differences in continuous variables were analyzed using an independent sample t test for normally distributed data and a Mann–Whitney U test for nonnormally distributed data.Differences in dichotomous variables between the confirmed COVID‐19 and non‐COVID‐19 patients were analyzed with a For the primary outcome, we calculated a receiver operating chart (ROC) and calculated the area under the curve (AUC) using MxA as continuous variable. With the AUC, we calculated the optimal cut‐off point of MxA using the Youden's Index.For the secondary analysis, we used the cut‐off point of 20 and 423 ng/ml, as previously described in literature, and calculated the sensitivity and specificity of MxA on diagnosing COVID‐19.Statistical analyses were performed using “R” version 4.1.0.3Between March and June 2021, a total of 240 patients were eligible for inclusion, of which 100 patients were included in this study . Other vital parameters were not significantly different between groups. In laboratory parameters, ferritin (p = .001), LDH (p < .001), and WBC (p < .045) were higher in COVID‐19 patients. COVID‐19 patients were admitted to the general ward more often (p = .001) and required ICU admission more often (p < .001). The mortality rate in COVID‐19 patients was higher than in non‐COVID‐19 patients (p < .001).Baseline characteristics are shown in Table p < .001) in COVID‐19 patients compared to non‐COVID‐19 patients, respectively 1933 and 0.1 ng/ml  in median MxA level in patients using immunosuppressive medication (n = 46) with a median of 1702 versus 1760 ng/ml in patients not using immunosuppressive medication. The median MxA level in patients with immunodeficiency (n = 11) was 932 versus 1756 ng/ml in patients without an immunodeficiency, which was not significantly different (p = .292).The characteristics of patients using immunosuppressive medication and immunodeficiency are shown in File 4In this study, we investigated the diagnostic value of MxA and found an AUC of 0.941 (95% CI: 0.867–1.000) on identifying COVID‐19 infections in patients presenting with suspected COVID‐19 infection in the ED. Based on these results, patients with COVID‐19 can be accurately identified when they have an elevated MxA level in the ED.MxA has previously been investigated as biomarker of other viral infections in the ED, such as influenza.n = 11). These findings may be at risk of a Type 2 error and further studies powered to find a difference in MxA levels in these groups should be performed to validate these findings.MxA is upregulated by type‐I interferons in response to viral infection. Patients using immunosuppressive medication or patients with immunodeficiency may have a reduced type I interferon activity and consequently diminished MxA production.5Our study has several limitations. Our study was conducted during the COVID‐19 pandemic when the SARS‐CoV‐2 prevalence was high. MxA is a mediator in antiviral immune responses in general, and does not specifically reflect the host response to COVID‐19 infection, but also the host response to other respiratory tract infections.The study population was relatively small with 100 patients, included in an academic hospital. These results require international multicenter validation. Furthermore, informed consent needed to be obtained before enrolling patients in this study. Often, critically ill patients or patients with severe respiratory symptoms were not able to give informed consent. Therefore, there may have been a selection bias towards noninclusion of this group of patients. We recommend that MxA is thoroughly validated in critically ill patients. Lastly, our study enrolled patients with a clinical suspected COVID‐19 infection. Therefore, these results are not validated in asymptomatic patients or patients that visit the ED for other reasons than a suspected COVID‐19 infection.6Whole blood MxA levels accurately distinguish COVID‐19 infections from bacterial infections and noninfectious diagnoses in the ED in patients with a suspected COVID‐19 infection. The results of this study indicate that MxA measurements may be of added value in the diagnostic workup and patient flow in the ED. Validation of these findings is recommended before implementation in routine clinical practice is issued.The authors declare no conflicts of interest.Kirby Tong‐Minh, Samantha van Hooijdonk, Virgil A. S. H. Dalm, and Willem A. Dik were involved in the conception or design of the manuscript. Kirby Tong‐Minh, Samantha van Hooijdonk, and Yuri van der Does performed the analysis and interpretation of the data. Kirby Tong‐Minh and Samantha van Hooijdonk drafted the manuscript. Kirby Tong‐Minh, Samantha van Hooijdonk, Marjan A. Versnel, Cornelia G. van Helden‐Meeuwsen, Petrus Martin van Hagen, Eric C. M. van Gorp, Henrik Endeman, Yuri van der Does, Virgil A. S. H. Dalm, and Willem A. Dik were involved in the critical revision of the manuscript and final approval of the manuscript.The study was approved by the local institutional review board and registered under number: NL73846.078.20. This study was conducted in accordance with the Declaration of Helsinki .Supplementary information.Click here for additional data file.Supplementary information.Click here for additional data file.


We examined the relationship between epicardial fat thickness (EFT) measured by echocardiography and left ventricular diastolic function parameters in a Beijing community population.We included 1004 participants in this study. Echocardiographic parameters including E and A peak velocity, the early diastolic velocities (e′) of the septal and lateral mitral annulus using tissue doppler imaging, E/e′, and EFT were measured. EFT1 was measured perpendicularly on the right ventricular free wall at end diastole in the extension line of the aortic root. EFT2 was the maximum thickness measured perpendicularly on the right ventricular free wall at end diastole. Multivariable linear regression was used to analyze the relationship between EFT and the mean e′ and E/e′.p < 0.05), and the correlation coefficient for EFT1 and EFT2 and mean e′ was − 0.138 and − 0.180, respectively. EFT1 and EFT2 were positively correlated with lateral E/e′, septal E/e′, and mean E/e′ (p < 0.05), and the correlation coefficient for EFT1 and EFT2 and mean e′ was 0.100 and 0.090, respectively. Multivariable egression analysis showed that EFT2 was independently and negatively associated with e′ mean . There were no interactions between EFT2 and any covariates, including age or heart groups, sex, BMI, or presence of hypertension, diabetes, or coronary heart disease, in relation to left ventricular diastolic dysfunction.The mean age of the participants was 63.91 ± 9.02 years, and 51.4% were men. EFT1 and EFT2 were negatively correlated with lateral e′, septal e′, and mean e′ (EFT2 was negatively and independently associated with e′ mean, which suggests that more attention to this type of adipose fat is required for cardiovascular disease therapy. The incidence of obesity has progressively risen with the increasing prevalence of unhealthy lifestyles in recent years. An increase in visceral fat is the main manifestation of the abnormal fat distribution in obesity, which is closely related to cardiovascular disease and metabolic syndrome . TherefoTwo types of fat are present around the heart: epicardial and pericardial fat . EpicardLeft ventricular (LV) diastolic dysfunction is one of the main factors associated with all-cause mortality . ObesityAll residents who were 40 years of age or older and lived in Shi Jing Shan district of Beijing were invited. Residents were contacted by recruitment advertisements or by telephone, and those volunteering to participate were included. Of the 5593 individuals who were ≥ 40 years of age, 1058 (18.9%) volunteered to participate in this study. The investigation started in July 2004 and ended in June 2005. Participants who had echocardiography were recruited in this study, while participants who had valve disease, evident arrhythmia , regional wall movement abnormal, or LVEF < 50% were excluded. Finally, 1004 participants were included in this study. The study was approved by the institutional review board of Peking University First Hospital, and informed consent was obtained from all participants.Waist circumference (WC) was measured in the standing position using the middle circumference between the lower rib margin and the iliac crest. The body mass index (BMI) was also calculated. After a rest period of 15 min, blood pressure (BP) was measured three times, with 5-min intervals between each measurement, at the right upper arm in a sitting position with a mercury sphygmomanometer. Mean systolic BP and diastolic BP were calculated on the basis of the three measurements. The total cholesterol and total triglyceride concentrations were analyzed by a fasting blood sample using standard techniques at the Beijing Hypertension League Institute. Participants with cigarette smoking history and current smokers were identified as smokers. Hypertension was defined as office systolic BP ≥ 140 mmHg and/or diastolic BP ≥ 90 mmHg, or history or the usage of antihypertensive drugs. Diabetes was diagnosed according to each participant’s interview and use of hypoglycemic drugs. Participants with a fasting glucose ≥ 7.0 mmol/L and a 2-h glucose ≥ 11.1 mmol/L on the oral glucose tolerance test were also defined as diabetic. Stroke, including intracerebral hemorrhage, cerebral infarction, and transient ischemic attack, was determined on the basis of a history of data collected from hospitalizations and outpatient records, which were confirmed by computed tomography (CT) or magnetic resonance imaging (MRI) scan 3) + 0.6 g, where PWTd and SWTd are the posterior and septal wall thickness at end diastole, respectively, and LVIDd is the LV dimension at end diastole  v3.4.3; SPSS v13.0).Continuous data are presented as mean ± standard deviation, while frequency data are presented as percentages. Spearman correlation was used to analyze the correlation of EFT with echocardiographic parameters. Multivariable linear regression was used to analyze the relationship between EFT and e′ mean and E/e′ mean, adjusting for age, heart rate, WC, hypertension, diabetes, and CHD. Subgroup analyses and interaction tests were used to examine the EFT2 and e′ mean according to age (< 65 years or ≥ 65 years), sex , BMI , but not with LVMI and LAVI (p > 0.05). EFT1 and EFT2 were negatively correlated with E and E/A (p < 0.05), and positively correlated with A and DT (p > 0.05). EFT1 and EFT2 were negatively correlated with e′ lat, e′ sep, and e′ mean (p < 0.05), and positively correlated with E/e′ lat, E/e′ sep, and E/e′ mean (p < 0.05)  Table .Table 3CUnivariatable analysis showed that EFT1 and EFT2 were negatively associated with e′ mean, while multivariable regression analysis showed that EFT2 was negatively associated with e′ mean after adjusting for age, sex, heart rate, WC, hypertension, diabetes, and CHD. EFT1 and EFT2 were not independently related with E/e′ mean on univariatable analysis or multivariable regression analysis Table . ResultsMany studies have focused on the association of adipose tissue and LV diastolic dysfunction, including global adiposity , centralEpicardial fat can be assessed by multiple imaging methods, including CT, MRI, and echocardiography. The thickness, area, and volume of epicardial fat can be measured by CT with manual or semi-automated methods. However, the high cost and radiation exposure associated with CT are disadvantageous, especially in large population studies. MRI is considered the gold standard for evaluating heart fat, although its use is also limited because of high cost and high requirement. Conversely, echocardiography is the most convenient method to evaluate epicardial fat, and is particularly suitable for epidemiological studies. The thickness and area of the epicardial fat can be measured with echocardiography , althoug2) than in our cohort. A further study reported that EFT was correlated with atria enlargement in morbidly obese subjects [In the present study, EFT was associated with most echocardiographic parameters. For example, EFT was positively correlated with ventricular septum and LV posterior wall thickness, but not with LVMI. One study showed that an increasing epicardial fat thickness was significantly related to an increase in LVM because of high free fatty acids levels, insulin resistance and adrenergic activity, and that increased visceral fat directly affected LV output to perfuse the increased body mass . In thatsubjects . ConversSeveral echocardiographic parameters used to evaluate LV diastolic function. Mitral inflow patterns, including E and A wave velocity, E/A, and E wave DT, are affected by many factors. A significant association of e′ with LV relaxation was reported in human subjects . The E/eIn our subgroup analysis, there was no effect of CHD on the relationship between EFT2 and e′ mean. Cavalcante and Konishi reported an independent association of epicardial fat and E/e′, although some patients may have advanced diastolic dysfunction since ischemia because that study included patients suspected of CAD . HyperteThere are several limitations to our study. First, epicardial fat was measured by echocardiography rather than MRI or CT. However, EFT measured by echocardiography was shown to correlate with volumetric measurements. Second, because of the cross-sectional nature of our study, a causal relationship between EFT and e′ cannot be determined. Prospective studies examining whether increased EFT is predictive of LV diastolic dysfunction are required. Finally, most patients were > 40 years of age, and thus our findings may not reflect the characteristics of epicardial fat in a younger population.In summary, EFT measured by echocardiography was independently correlated with the e′ mean, but not with the E/e′ mean, in a high-risk community population with an LVEF ≥ 50%. Subgroup analysis showed there were no interactions between EFT and any covariates, including age and heart groups, sex, BMI, or presence of hypertension, diabetes, or CHD.


The prognostic factors for survival in patients with ependymoma (EPN) remain controversial. The aim of this study was to establish a prognostic model for 5‐ and 10‐year survival probability nomograms for patients with EPN.Clinical data from the Surveillance, Epidemiology, and End Results (SEER) database were used for patients diagnosed with ependymoma between 2000 and 2018 and were randomized 7:3 into a development set and a validation set. Factors significantly associated with prognosis were screened out using the least absolute shrinkage and selection operator (LASSO) regression. The calibration chart and consistency index (C‐index) are used to evaluate the discrimination and consistency of the prediction model. Decision curve analysis (DCA) was used to further evaluate the established model. Finally, prognostic factors selected by LASSO regression were evaluated using Kaplan–Meier (KM) survival curves.p < 0.001).A total of 3820 patients were included in the prognostic model. Seven survival predictors were obtained by LASSO regression screening, including age, gender, morphology, location, size, laterality, and resection. The prognostic model of the nomogram showed moderate discriminative ability in the development group and the validation group, with a C‐index of 0.642 and 0.615, respectively. In the development set and validation set survival curves, the prognosis index of high risk was less effective than low risk  remain controversial. Based on the SEER database, we constructed prognosis models for EPN 5‐year and 10‐year survival rates. It may provide a reference for clinicians to personalized treatment. It is common in the posterior fossa in children and supratentorial and spinal cord in adults.The prognostic factors of ependymoma are still controversial. Previous studies are often based on the cohort statistical analysis of a small number of people, and the prognosis results are quite different. Even experienced clinicians still have great challenges in predicting the survival time of patients. Moreover, there are differences in medical technology among different medical institutions, which brings greater challenges to the prognosis prediction of ependymoma patients. For neurosurgeons, it is very important to use the clinical data of patients to build an accurate tool to predict the survival probability. Physicians and patients will benefit from a readily available and intuitive predictive model tool that can assess survival outcomes through demographic, histopathology, and surgical approaches in clinical practice.The nomogram is a common clinical statistical method, which scores the risk factors and then plays a role in predicting the prognosis of the tumor. Unlike previous studies, we included more prognostic factors and a large sample of patients with EPN. The Surveillance, Epidemiology, and End Results (SEER) database is a cancer population registry in the United States that collects basic patient information, clinicopathological characteristics, and treatment‐related data covering nearly one‐third of the U.S. population.22.1We obtained the complete 2000–2018 dataset online from the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute (released in Nov 2020). These data sets contain basic patient information from the following 18 registries in the United States (the year of data collection): San Francisco‐Oakland SMSA (2000), Connecticut(2000), Detroit (Metropolitan) (2000), Hawaii(2000), Iowa (2000), New Mexico(2000), Seattle (Puget Sound) (2000), Utah(2000), Atlanta (Metropolitan) (2000), San Jose‐Monterey(2000), Los Angeles(2000), Alaska Natives(2000), Rural Georgia(2000), California excluding SF/SJM/LA(2000), Kentucky(2000), Louisiana(2000), New Jersey(2000), and Greater Georgia(2000). The data of patients with ependymoma, coded 9391–9394 in the International Classification of Diseases for Oncology, third edition (ICD‐O‐3), and confirmed by microscopical diagnosis were screened. And the primary site topography codes were C70.0–C72.9. The flowchart of case inclusion and exclusion criteria is shown in Figure 2.2SEER demographic data were extracted including patient age, gender, race, morphological diagnosis, location, size, tumor laterality, surgical status, overall survival time, and the survival status at the time and of diagnosis. The ICD‐O‐3 code was used to define the tumor morphological diagnosis: 9391 for Ependymoma, NOS; 9392 for Ependymoma, anaplastic; 9393 and 9394 for Myxopapillary ependymoma, malignant. The primary tumor site was defined by topography codes: supratentorial , posterior fossa , spine , and other/unknown . Tumor size: 0–9 mm, 10–29 mm, 30–59 mm, 60 mm+, unknown. Tumor laterality: only one side and bilateral side. RX Summ‐‐Surg Prim Site (1998+): No surgery and biopsy only for codes 00 and 20; partial resection for codes 21, 22, 40, and 90; gross total resection for codes 30 and 55. We divided the population into three groups according to age: the children group (0–19 years), the adults’ group (20–49 years), and the elderly group (50+ years). The race includes white, black, and other groups.2.3p < 0.05 was considered to be statistically significant.EPN patients meeting the criteria were randomly divided into the development group and the validation group in a 7:3 ratio (createDatapartition package). Factors selected from least absolute shrinkage and selection operator (LASSO) regression analysis were associated with the prognosis of ependymoma.https://www.r‐project.org) was used to construct the nomogram and statistical analysis of all data. All datasets come from the SEER∗Stat software .R software 4.0.5  were placed in the development set and 1144 (30%) in the validation set. There were 1947 males (51.0%) and 1,873 females (49.0%). In addition, 1262 cases (33.0%) were older than 50 years old, 1538 cases (40.3%) aged 20–49 years old, and 1020 cases (26.7%) aged 0–19 years old. In the development group, the median follow‐up period was 78 months (range 1–227 months). The 5‐ and 10‐year survival rates were 59.2% and 32.3%, respectively. For the validation group, the median follow‐up period was 80 months (range 1–227 months). The 5‐year and 10‐year survival rates were 58.4% and 31.9%, respectively. The detailed clinical data of the patients are shown in Table 3.2We reduced the initial eight characteristics of 2676 patients in the development cohort to seven potential predictors of survival: age ,gender , morphology ,location ,size ,laterality ,and resection  . (Figure In this study, seven variables were used to calculate the PI, and the survival time was used as the cut point to calculate the optimal cut point of PI. The optimal PI cutoff points of the development set and validation set are 5.4 and 6.4. The development validation cohort was divided into high‐risk group and low‐risk group according to different PI cut‐off values. The 5‐year and 10‐year survival curves were drawn and the individual survival number and time data were included. Log‐rank test results showed that there were significant differences between the high‐risk group and the low‐risk group (4In this study, we used potential prognostic factors in patients with pathologically diagnosed ependymoma to construct clinical prognostic models of 5‐year and 10‐year probability of survival by obtaining patient data from the SEER database. Our results suggest that age, gender, morphology, location, size, laterality, and resection may be important predictors of survival in patients with ependymoma. Although these clinical features are also prognostic factors for other cancers, their role in the prognosis of patients with ependymomas remains controversial.Our study constructed an integrated predictive model for patients with ependymoma. Consistent with previous reports, we finally found that seven clinical variables were predictors of survival.In addition, the correlation between gender, tumor size, and surgical condition has been confirmed by relevant studies.We developed a new nomogram using retrospective clinical cohort data from the SEER clinical database with moderate C‐index and calibration curve results. The results showed that the model constructed by seven prognostic factors obtained from LASSO was relatively stable and reliable.This study has some limitations. First, we only carried out internal verification of the data, hoping to get external verification in the real world in the future. Second, as it is a retrospective cohort study, potential selection bias is inevitable. Data screening may exclude patients with missing information on the variables collected, which may lead to selection bias and lower C‐index in this study compared with other models. Third, some treatments, such as radiotherapy and chemotherapy, have not been included in the prognosis model, so their differentiation ability is limited. Therefore, further prospective studies are planned to verify the accuracy of the prognosis model.5We constructed and internally validated a more broadly applicable nomogram for predicting 5 ‐ and 10‐year survival in patients with EPN. The new nomogram can be used as a simple clinical prediction tool to provide personalized service for patients.6None declared.Additional file S1Click here for additional data file.Additional file S2Click here for additional data file.Additional file S3Click here for additional data file.