L*a*b*. The results showed that, without NaF, plate-like nanocrystals were formed on the enamel surface, but with NaF, needle-like nanocrystals were formed. By adding EMD, a layer of well-compacted hydroxyapatite crystals was successfully precipitated onto the natural enamel surface. No significant differences were observed in the L* value of the mineralization surface pre-etching and after mineralization buffer containing NaF and EMD. A new method has been developed to recover the color quality of enamel, as well as to mineralize the tooth enamel by constructing hydroxyapatite crystals with mineralization buffers containing NaF and EMD on the etched tooth surface.This study aimed to demonstrate and compare the accuracy of tooth shade selection due to the remineralized enamel crystal with enamel matrix derivative (EMD) in vitro. Etched enamel slices were immersed in four types of mineralization buffers for 16 h. Sodium fluoride (NaF) was added to final concentrations of 1–100 ppm with the mineralization buffer that demonstrated the highest mineralization efficiency. EMD was added to the mineralization buffer containing NaF to see if it has any remineralization capacities. The remineralized enamel crystal was analyzed by SEM and XRD. The tooth shade was evaluated by CIE  Dental enamel is the hardest mineralized tissue in the human body and is composed of more than 95% hydroxyapatite crystals by weight . A soundIn addition, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste, bioglass materials, have been attempted to promote enamel remineralization at the early stage of demineralization of the enamel surface, but regeneration of the enamel is not established , 8. The Regeneration of enamel provides enough hardness for the oral environment, prevention of initial caries, and improved esthetic features on the surface of the tooth. Enamel demineralization should be restored with the regenerative enamel more than the mineralization. The regenerated enamel is distinguished from the mineralization by the presence of the crystal structure of hydroxyapatite .Various studies of regenerating enamel-like hydroxyapatite crystals have been performed under extreme non-physiological conditions, such as a high-temperature, high-pressure, low acidic pH, or in the presence of surfactants –12. ThesEscherichia coli-based, yeast-based, and virus expression systems, no methods have been widely adopted due to uncertainty, such as involvement of its phosphorylation .Mineralization buffer A was prepared with 2.12 mM calcium chloride dihydrate ; the pH was adjusted to 7.6 with 50 mM Tris–HCl.Mineralization buffer D was prepared with 2.58 mM CaCl2·2H2O, 1.55 mM KH7, (B) 3.85 × 107, (C) 5.50 × 107, and (D) 8.32 × 107 , where L* is the brightness index and a* and b* are indices of perceived chromaticity. The mean values of L*, a*, and b* were measured. For color tone correction separately from the Casmatch, each tooth was partially filled with A2 composite resin  as a control  and recorded in TIFF file format. To standardize color and the exact location each time a photograph was taken with a Casmatch  placed near the tooth examined in Fig.  control .L* indicate greater brightness and whiter colors, higher values of a* indicate more red in the positive (+) direction and more green in the negative (−) direction, and higher values of b* indicate more yellow in the positive direction and more blue in the negative direction. Changes in color over time were determined by measuring the values pre-etching , after etching , and after immersion in mineralization buffer . Color differences (ΔEab) pre-etching and after immersion in mineralization buffer were compared by calculating the values of ΔL, Δa, and Δb at each of these two times. 10 sites were randomly selected in each tooth for the calculation.The CIELAB system systematically arranges colors in a way that correlates with visual perception, allowing them to be shown as a specific color. Higher values of Difference in color was calculated by the following formula:L*, a*, and b* pre-etching, after etching and after immersion in mineralization buffer were analyzed using IBM SPSS Statistics version 21 . Differences in the values were tested using one-way analysis of variance (ANOVA) followed by Scheffe’s test, with p < 0.05 taken to be significant.From the colorimetric results, the values of SEM was used to assess remineralization after enamel slices were immersed in mineralization buffers A, B, C, and D for 16 h. Figure Sodium fluoride was added to mineralization buffer C to final concentrations of 1, 10, and 100 ppm, and remineralization procedures were carried out. The plate-like remineralized structures were transformed into needle-like structures as a result of the addition of NaF. The needle-like depositions were thicker at higher fluoride concentrations Fig. . On the EMD  or 6% PGA (control) was added to the NaF-containing mineralization buffer, and mineralization was evaluated by SEM. No differences in the deposited crystals were observed between the mineralization buffer containing-NaF and the mineralization buffer containing-NaF with 6% PGA Fig. .Fig. 3SENeedle-like deposits were formed with the mineralization buffer containing-NaF with 0.05% and 0.1% EMD. In contrast, the needle-like deposits aggregated to form columnar shapes with the mineralization buffer containing-NaF with 0.5% and 1% EMD. At higher EMD concentrations, these aggregated columnar structures were larger Fig. .Fig. 4SEFigure θ = 25.9°, 32.2°, and 34.0° in the enamel slices pre-etching, the intensity of the peaks was less, and the width of the peaks was greater in enamel slices after etching. With the enamel slices after immersion in mineralization buffer alone, mineralization buffer containing-NaF, and mineralization buffer containing-NaF and EMD,: HAp peaks were found at 2θ = 25.9°, 31.7°, 32.2°, 32.9°, and 34.0°. With the mineralization buffer, high-intensity HAp peaks not present in enamel after etching were found at 2θ = 31.7° and 32.9°. With mineralization buffer containing-NaF, high-intensity HAp peaks were found at 2θ = 31.7°, 32.2°, and 34.0°. With mineralization buffer containing-NaF and EMD, high-intensity HAp peaks were found at 2θ = 31.7°, 32.2°, and 32.9°.While clear HAp diffraction peaks were visible at 2L*a*b* values are shown in Figs. L* value pre-etching was 84.5, after etching was 93.1, and after immersion was 91.2; the difference with respect to the value pre-etching was significant after etching (p = 0.001) and after immersion (p = 0.04). The mean a* value pre-etching was 6.1, after etching was 1.4, and after immersion was 2.9, with no significant difference after etching (p = 0.4) or after immersion (p = 0.9). The mean b* value pre-etching was 22.4, after etching was 11.4, and after immersion was 15.4; the pre-etching value was significant after etching (p = 0.00) and after immersion (p = 0.025).Photographs of immersed teeth and changes in the L* value pre-etching was 87.7, after etching was 92.9, and after immersion was 92.0, the difference was significant after etching (p = 0.002) and after immersion (p = 0.048). The mean a* value pre-etching was 4.4, after etching was 0.6, and after immersion was 0.5; the difference was significant after etching (p = 0.048) and after immersion (p = 0.037). The mean b* value pre-etching was 31.0, after etching was 21.2, and after immersion was 25.5; the pre-etching value was significant after etching (p = 0.000) and after immersion (p = 0.000).With the mineralization buffer containing-NaF, the mean L* value pre-etching was 89.7, after etching was 95.4, and after immersion was 89.9; the difference was significant after etching (p = 0.000), but not significant after immersion (p = 1). The mean a* value pre-etching was − 0.8, after etching was − 3.7, and after immersion was 0.2; the difference was significant after etching (p = 0.000) and after immersion (p = 0.000). The mean b* value pre-etching was 25.7, after etching was 22.5, and after immersion was 23.7; the pre-etching value was significant after etching (p = 0.000), but not significant after immersion (p = 0.716).With the mineralization buffer containing-NaF and EMD, the mean L*a*b* system after being immersed in mineralization buffer C, mineralization buffer C + 100 ppm NaF, and mineralization buffer C + 100 ppm NaF + 1% EMD , and they may be a precursor of hydroxyapatite (HAp) –33. In aF-might acts on the surface of the plates that make up the plate-like structure, resulting in loss of the surface and the formation of needle-like structures [Addition of NaF to the mineralization buffer caused the remineralization to take on a needle-like structure. The needle-like structures became thicker at higher fluoride concentrations. Enamel HAp exhibits a needle-like structure, and the similar structure in the enamel probably possess similar mechanical properties , 35. At ructures . This suEMD consists of proteins extracted from 6-month-old porcine molar and premolar germs, containing more than 95 wt% amelogenin fragment, and it is used clinically for induction and regeneration of alveolar bone . The EMDθ = 31.7° and 32.9°, indicating that there was formation of deposits, but from the lack of change in the intensity, it appears that the site occupancy of the deposits was low. With mineralization buffer containing NaF, clear peaks were found at 2θ = 31.7°, 32.2°, and 34.0°, with an increase in intensity and a decrease in peak width, suggesting a high site occupancy in the deposit and increased crystallinity. With mineralization buffer containing NaF and EMD, clear peaks were found at 2θ = 31.7°, 32.2°, and 32.9°, with an increase in intensity and a decrease in peak width, thus also suggesting a high site occupancy in the deposit and increased crystallinity. With the enamel slices pre-etching, an HAp peak was observed at 2θ = 25.9°. Peak width is used to evaluate crystallinity, and the decreased peak width of the immersed enamel slices in the present results therefore suggests high crystallinity of the deposits [XRD intensity is considered a quantitative estimation and of the site occupancy of the crystal layer –44. In tdeposits . Amelogedeposits , 47, anddeposits , and theL* value increased significantly after etching. A higher value of L* indicates a brighter, whiter color, and the increase in L* was therefore the result of demineralization of the surface layer of the enamel by 40% phosphoric acid. After immersion in mineralization buffer and in mineralization buffer containing NaF, the value of L* increased significantly with respect to the value pre-etching, but there was no significant difference in L* after immersion in mineralization buffer containing NaF and EMD with respect to the value pre-etching. SEM observation showed that the deposit formed a columnar structure with mineralization buffer containing NaF and EMD, suggesting that this columnar structure affects the color tone.The mean E from pre-etching to after immersion was 52.06 with mineralization buffer, 32.9 with mineralization buffer containing NaF, and 3.525 with mineralization buffer containing NaF and EMD. Most studies set the proposed acceptance limit for color matching to 3.7, beyond which the differences are clinically visible [E was 3.525 with mineralization buffer containing NaF and EMD, suggesting that the appearance of the enamel was restored to the acceptable levels as pre-etching. It is, however, not known that this method is the best for the color tone among the methods previously reported. Further investigations are needed to clarify about it.The value of Δ visible –51. In tθ = 25.9° might restore the color tone of the enamel after demineralization. The present method may provide a new method to regain shiny enamel after initial demineralization of enamel.In conclusion, the present study demonstrated that calcium phosphate containing NaF and EMD successfully precipitated hydroxyapatite crystals onto the natural enamel surface in 16 h. These mineralization structures that exhibited HAp structures at 2


To investigate the predictive power of mono-exponential, bi-exponential, and stretched exponential signal models of intravoxel incoherent motion (IVIM) in prognosis and survival risk of laryngeal and hypopharyngeal squamous cell carcinoma (LHSCC) patients after chemoradiotherapy.f) by bi-exponential model, distributed diffusion coefficient (DDC), and diffusion heterogeneity index (α) by stretched exponential model were measured. Survival data were collected for 5 years.Forty-five patients with laryngeal or hypopharyngeal squamous cell carcinoma were retrospectively enrolled. All patients had undergone pretreatment IVIM examination, subsequently, mean apparent diffusion coefficient (ADCmean), maximum ADC (ADCmax), minimum ADC (ADCmin) and ADCrange (ADCmax − ADCmean) by mono-exponential model, true diffusion coefficient (D), pseudo diffusion coefficient (D*), perfusion fraction . D* had the greatest AUC of 0.802, with sensitivity and specificity of 77.4 and 85.7% when D* was 38.85 × 10–3 mm2/s. Kaplan–Meier survival analysis showed that the curves of N stage, ADCmean, ADCmax, ADCmin, D, D*, f, DDC, and α values were significant. Multivariate Cox regression analysis showed ADCmean and D* were independently correlated with progression-free survival (PFS) .Thirty-one cases were in the treatment failure group and fourteen cases were in the local control group. Significantly lower ADCmean, ADCmax, ADCmin, D, The pretreatment parameters of mono-exponential and bi-exponential models were significantly correlated with prognosis of LHSCC, ADCmean and D* values were independent factors for survival risk prediction. Laryngeal and hypopharyngeal cancers are common head and neck tumors, and squamous cell carcinoma is by far the most common type with a dominant proportion of nearly 95%. According to National Cancer Institute (NIH) statistics, oral cavity and pharynx cancer account for 2.8% of all new cancer cases and 1.8% of all cancer deaths, while laryngeal cancer accounts for 0.7 and 0.6%, respectively. Overall, the 5-year relative survival rate for cancers of the oral cavity, pharynx, and larynx is approximately 60–66% , 4. One With superb soft tissue resolution, no radiation, and multiple morphological and functional sequences, magnetic resonance imaging (MRI) has been widely used for the detection and diagnosis, TNM staging, treatment response evaluation, and prognosis prediction of laryngeal and hypopharyngeal squamous cell carcinoma (LHSCC) , 8. Intrf) values, and stretched exponential model for the distributed diffusion coefficient (DDC) and diffusion heterogeneity index (α) values. The measurement of ADC values can be affected by multiple factors, among which tissue perfusion and extracellular water molecular movement are the most important. The bi-exponential model idealizes the irregular motion in the human body into perfusion-related fast dispersion and slow dispersion of water molecular motion. The stretched exponential model is a new method to fit the apparent diffusion attenuation characteristics that reflect a continuous distribution [f, and DDC values and higher D* value than adenocarcinoma; the poorly differentiated group of SCC had lower D and DDC values, and adenocarcinoma had lower ADC and D values than the well-moderately differentiated group [The IVIM exponential model assumes the collective motion of blood water molecules in the network, flowing from one randomly-oriented capillary segment to the next (at the voxel level) during the diffusion encoding time, mimicking a collective random walk (“pseudo-diffusion”) . The monribution . To dateribution . To our ribution –21 or onribution –24. Someribution , 21, 22.ed group .Hence, the aim of this study was to compare the value of multi-parameters derived from IVIM mono-exponential, bi-exponential, and stretched exponential signal models in predicting long-term outcomes of patients with laryngeal and hypopharyngeal carcinoma after chemoradiotherapy.This retrospective study was approved by the local institutional review board , and informed consent was obtained from all the enrolled patients. The inclusion criteria were as follows: 1) patients who had undergone baseline MRI including IVIM examination, before any anti-tumor treatment within 2 weeks; 2) laryngoscopy revealed a mass in the larynx or hypopharynx and biopsy confirmed the histology of squamous cell carcinoma; 3) clinical workup confirmed the absence of distant metastasis; 4) each patient had received standard chemoradiotherapy after MRI examination. We excluded those patients: 1) in whom IVIM images were degraded by severe swallowing artifacts; 2) whose tumor was too small (size less than 1 cm); 3) who had received previous anti-tumor treatment of any kind, or surgery after MRI examination. All patients received concurrent chemoradiotherapy comprising intensity modulated radiotherapy (IMRT) + concurrent chemotherapy + tumor radiosensitizer sodium glycididazole (CMNa). Radical radiation therapy was performed with 6 MVX linear accelerators, 2 Gy per day, five times per week, for a total of 33 irradiations. Concurrent chemotherapy drugs were paclitaxel + liposome 270 mg (IV drip day 1) and cisplatin 40 mg , starting from the first day of radiotherapy. Each cycle lasted for 21 days. Based on the inclusion and exclusion criteria, 50 patients were enrolled in this study between December 2014 and March 2016.All enrolled patients were followed up after the completion of standard treatment every 3 months in the first 2 years and every 6 months in the third to fifth years by at least one of the following examination methods: MRI, CT, ultrasound examination, laryngoscopy, and biopsy at our hospital or other medical institutions. Among them, 34 patients were followed up at our hospital to obtain definitive imaging examination results and prognosis conditions, while 11 patients that could not visit the hospital in-person were followed up over the telephone to obtain information regarding the development of any new complications or adverse events. The primary endpoints were treatment failure and local control over 5 years. “Treatment failure” was defined as tumor progression, tumor recurrence, distant metastasis identified using follow-up imaging methods, and tumor-related death information obtained from telephone inquiries. In contrast, “local control” was defined as absence of tumor relapse, metastasis, or tumor-related death. The time from treatment initiation at our hospital to the endpoint of the study was regarded as progression-free survival (PFS).All patients underwent 3.0 T MRI  using an 8-channel phased array head and neck combined coil. The routine clinical MR protocol included conventional fast spin echo T1-weighted imaging with fat suppression  before and after administration of gadodiamide contrast agent , and fast spin echo T2-weighted imaging with fat suppression (TR/TE = 5760/88.3 ms), field of view (FOV) = 260 mm × 260 mm, slice thickness of 4 mm, and slice spacing of 0.4 mm.2). The number of collections (NEX) was set to 2 when the b value was between 0 and 200 s/mm2 and 3 when the b value was between 400 and 1000 s/mm2 (TR/TE = 250/79 ms). The remaining parameters were as follows: bandwidth = 250 kHz, FOV = 260 mm × 260 mm, matrix = 160 × 160, slice thickness = 5 mm, and slice spacing of 1 mm.IVIM sequence scanning was performed using echo planar imaging (EPI) sequence with 12 b values  referring to the axial T1-weighted contrast enhanced images, which were then reviewed by a senior radiologist (Dehong Luo with more than 30 years’ experience), with any disagreement resolved through discussion. A fixed circular ROI with an area of 25–50 mm2 was placed on the slice of the largest area of solid tumor tissue. In reference to T1-weighted, T2-weighted and T1-weighted contrast-enhanced imaging, cystic changes, necrotic areas, and hemorrhage were avoided. Measurements for each lesion were repeated for three times to obtain the average value. The model calculation was derived from D Le Bihan’s study [f values. In addition, stretched exponential model was used to generate DDC and α values.To reduce artifacts, patients received oral codeine 20 min prior to examination. To enhance the stability of the signal-to-noise ratio (SNR), the signal from each voxel within the region of interest (ROI) was integrated to obtain the total signal intensity distribution. The mean signal intensity for each b value was derived. On the GE AW 4.6 post-processing workstation, the Functool software was used to draw the ROI on DWI images with b value of 800 s/mm’s study . Mono-ex2. 2, D* is obviously greater than D so that the effect on signal attenuation is negligible.Formula  is a mont-test or Mann–Whitney U test. Receiver operating characteristic (ROC) curve analysis was performed for all continuous variables and the area under the curve (AUC) was calculated. Kaplan–Meier survival curve univariate analysis was performed to determine the predictors of PFS. The parameters with p < 0.1 were selected into the multivariate Cox regression model, and progressive forward selection was used to determine independent predictors. p < 0.05 (double-tailed) was considered statistically significant.All data were statistically analyzed using SPSS 21.0 software . The measured results were represented as the mean ± standard deviation. Chi-square test was used for categorical variables and rank sum test was used for grade data. Continuous data were tested by independent sample Student’s n = 5; glottic region, n = 4) and 36 cases of hypopharyngeal carcinoma . Thirty-one cases were assigned to the “treatment failure group” because of local recurrence and progression (n = 22) and metastasis (n = 9), of which 29 cases had tumor-related death. Fourteen cases were assigned in the “local control group”  are summarized in Table f, DDC, and α values from the primary tumors of the two groups are summarized in Table –3 mm2/s vs 1.31 ± 0.26 × 10–3 mm2/s, p = 0.005), and lower ADCmax , and lower ADCmin , compared to local control group; however, ADCrange did not significantly differ between the two groups. The pretreatment D value was 0.92 ± 0.45 × 10−3 mm2/s, D* value was 88.86 ± 66.48 × 10−3 mm2/s, and f value was 28 ± 17% in the treatment failure group. In the local control group, D value was 1.17 ± 0.32 × 10−3 mm2/s, D* value was 33.27 ± 52.10 × 10−3 mm2/s, and f value was 45 ± 20%. The pretreatment D and f values in the treatment failure group were lower than those in the local control group, and D* value was significantly higher than that in the local control group (p < 0.05). Two representative cases with different survival outcome were shown in Figs. The detailed parameters including ADCmean, ADCmax, ADCmin, ADCrange, and D, D*, –3 mm2/s, sensitivity and specificity of 74.2 and 71.4% were obtained for predicting the local long-term prognosis. Similarly, ADCmax was set at 1.74 × 10–3 mm2/s with the sensitivity and specificity of 74.2% and 71.4%, respectively. Furthermore, ROC analysis showed that D, D*, and f values had significant performance with AUCs of 0.747, 0.802, and 0.741, respectively, whereas DDC and α values had fairly inferior performance with AUC of less than 0.7 (p > 0.05). D* value had the greatest diagnostic efficacy among these parameters, with the sensitivity and specificity of 77.4 and 85.7% when the threshold of D* was set at 38.85 × 10–3 mm2/s . However, there were significant differences in N stage . The patients were divided into two groups based on the cut-off value determined by ROC analysis. Kaplan–Meier survival analysis showed that the two survivor curves based on the cut-off values of ADCmean, ADCmax, ADCmin and D, D*, f, DDC, and α values were found significantly different , except ADCrange (p = 0.126) (Table p = 0.001) and D* values  were independently correlated with PFS.The Kaplan–Meier survival curve showed no significant difference in T stage . The f value reflects the magnitude of the low b component, which is associated with perfusion, and is negatively correlated with the high b component, which is associated with diffusion. A study by Thomas Hauser et al. showed that the initial f value was significantly higher in patients with locoregional failure than locoregional control, and the initial D value did not differ significantly [f value was lower in the failure group than that in the local control group, which meant that the treatment failure group had lower perfusion and higher diffusion, and D value was significant in the survival analysis. This is most likely due to the fact that our failure group comprised not only local recurrence, but also metastasis and death. Furthermore, follow-up intervals were different between the two studies. In addition, since the f value is related to the b value’s setting and calculation formula, the b values we used (0–1000 s/mm2) produce Gaussian and non-Gaussian effects, which may artificially exaggerate the value and efficiency of the parameters.DWI and ADC values have gradually been applied in clinical practice. Their function in clinical diagnosis, differential diagnosis, and short-term efficacy evaluation have been confirmed by many studies , 26–28.  control , which wficantly . Howeverf, and DDC values than low-grade patients; additionally, D and DDC were more efficient than f in differentiation [The studies of Kim and King et al. , 30 showntiation . These rntiation . Meanwhintiation . As confntiation . The stu2. A non-Gaussian effect might appear from 800 or even 600 s/mm2 in many tissues, including DWI data at higher b values in the IVIM analysis, which might lead to the exaggeration of f value, if not analyzed properly. Thus, the next step of our work is to use open-source tools such as “https://github.com/slevyrosetti/ivim-toolbox” to assess the differences in b values and model fitting [There were some limitations in our study. First, this was a retrospective study with a relatively small sample size. Second, the data were imbalanced, with the majority of patients having advanced-stage malignancies. Third, there were problems of recall bias and loss of follow-up, which warrants a study with larger sample size and more balanced data. Finally, the study did not deeply explore intra-tumor heterogeneity, or the correlation with the in vivo IVIM models’ functional parameters. We explored IVIM parameters with b values of 0 to 1000 s/mm fitting .f, and D* values were significantly correlated with PFS, and pretreatment ADCmean and D* values were independent predictors of survival risk.The quantitative parameters of the IVIM mono-exponential and bi-exponential models can predict the long-term prognosis of laryngeal and hypopharyngeal carcinomas after chemoradiotherapy, and are more efficient than the stretched exponential model. Pretreatment ADC, D,


There was an error in the original publication . There wAbstract, lines 4–5. The correct text appears below.A correction has been made to Bacillus species cultures were tested.”“An industrially used microbiological concentrates and their components of mixed 1. Introduction, paragraph 4, lines 1–3. The correct text appears below.A correction has been made to “In this paper, we examinate a microorganisms species devised for industrial and household use, as a potential prototype preparation, possibly subjected certain modifications, for dietary supplementation.”2.1. Bacteria, Reagents and Equipment. The correct text appears below. A correction has been made to 2.1. Bacteria, Reagents and Equipment2 [5 mM/L], MgCl2 [10 mM/L] from WarChem sp z o.o.  and D-fructose [0.5%/L] from BTL Species z o.o. company  were added. For agar TYM plates, 20% agar from BD™ Difco™ company was added. The pH of the microbial mediums was 6.7 (±0.2). All other reagents were purchased from Sigma-Aldrich .The microbiological samples were from Piotr Skowron’s private collection. Before analysis, the long-stored samples were thoroughly mixed prior to analysis. The TYM nutrition was prepared from Pepton K [20 g/L] and yeast extract at [4 g/L], which were purchased from BTL Species z o.o. company . At the end, CaCl3.1. Selection and Quantification of Bacterial Cultures, paragraph 1. The correct text appears below.A correction has been made to Bacillus group  were determined, as shown in Figure 1. For the tested product, the temperature 45 °C was used as the most adequate. Environmental Bacillus strains were counted quantitatively at a range of temperatures (Supplementary Table S1), while a quantitative analysis of their growth depending on temperature is presented in Figure 2A–F. At a temperature of 40 °C, Bacillus strains were less numerous, and fever species were identified. Here, B. subtilis dominated, followed by B. licheniformis and B. cereus. Interestingly, at a temperature of 54 °C, B. subtilis and B. cereus were the most abundant, which suggests their facultatively thermophilic character despite their fuzzy shape and smaller size of their colonies. None of the Bacillus strains grew at 57 °C. Furthermore, the optimal cultivation time for colonies features evaluation, in the temperatures from 37 °C to 57 °C, turned out to be 17 h. A longer incubation time (24 h) resulted in bigger colony size.”“We have previously analyzed a few of commercial ‘industrial probiotics-like’ preparations intended for the use as ‘friendly bacteria’ supplements in various cleaning preparations [19]. The use of bacteria cocktails in industrial and household products inevitably leads to their contact with humans. For sheer safety reasons, they are very likely to penetrate the human gastrointestinal tract, their producers had to test them and thus, unintentionally, they provided them as human probiotics. In this study, we have found that some of those species are also common in a numerous other applications, which may be a basis for the development of probiotic food preparation. Six different species of the 3.1. Selection and Quantification of Bacterial Cultures, paragraph 2, lines 9–12. The correct text appears below.A correction has been made to B. pumilus presented in Figure 2E and B. amyloliquefaciens presented in Figure 2F only grow as single colonies and only at the temperature of 45 °C. We suggest that perhaps these bacteria may be just a trace contaminant present in the tested Bacillus preparations.”“Furthermore, we found out that 3.2. Characteristics of Bacterial Cultures, Colonies and Bacterial Cells, paragraph 2, lines 1–2. The correct text appears below.A correction has been made to B. subtilis colonies  present to other species of the Bacillus group.”“We compared 3.2. Characteristics of Bacterial Cultures, Colonies and Bacterial Cells, paragraph 4, lines 2–3. The correct text appears below.A correction has been made to Bacillus mixtures, B. cereus were flat, growing as large single colonies or forming clusters.”“Our study showed that, in the tested 3.2. Characteristics of Bacterial Cultures, Colonies and Bacterial Cells, paragraph 5, line 5. The correct text appears below.A correction has been made to Bacillus species mixture diluted 10−7).”“ mass spectroscopy analysis in MALDI-TOF assays as described . These data allowed the distinguishing of closely related 3.5. Antibiotic Resistance, paragraph 7. The correct text appears below.A correction has been made to B. amyloliquefaciens have been determined due to the very low content of this bacterium in the potential Bacillus probiotic source.”“No zones of inhibited growth for 4. Discussion, paragraph 1, lines 7–10. The correct text appears below. A correction has been made to Bacillus species isolated from a commercial concentrate, and showed their sensitivity/resistance to antibiotics.”“Thus, in this study, we characterized the antagonistic activities and probiotic potential of 4. Discussion, paragraph 4, lines 8–21. The correct text appears below. A correction has been made to Bacillus species. However, in the analyzed conglomerate only a small number of B. cereus were recorded as growing on a solid medium at temperatures of 40 °C and 45 °C , and thus they may be an unintended contamination during large scale cultivation of mixed Bacillus species population. In addition, B. cereus colonies are typically larger, thus this species might actually be a closely related B. cereus-like species. Furthermore, it the MALDI-TOF factor of 1.99 is low-to-medium as shown, this isolate may represent a variant of a ‘human-friendly’ species B. atrophaeus, containing some more common proteins with B. anthracis. Within such a closely related ‘Bacillus group’, horizontal gene transfer undoubtedly is a common phenomenon. The above analysis points to the possibility of exploring Bacillus preparations used for other than human use purposes for valuable species, which eventually could be used for new formulations as human probiotics upon further extensive testing.”“Thus these bacteria should not be included in the development of bimodal probiotic from A correction has been made to Funding. The updated text appears below.“The work was supported by TECHMATSTRATEG2/410747/11/NCBR/2019 grant to University of Gdansk, Faculty of Chemistry, Molecular Biotechnology Department.”Figure 1, Figure 4, Figure 5, Table 1. The correct legends appear below.In the original publication, there was a mistake in the legend for Figure 1. Identification of probiotic bacteria colonies detected in the course of commercial preparation analysis in the growth temperature of 45 °C. Dilution of 10−7. Cultures marked: (1) B. subtilis, (2) B. atrophaeus, (3) B. cereus, (4) B. licheniformis, (5) B. pumilus, (6) B. amyloliquefaciens.Figure 4. Inhibition of foodborne pathogenic bacterial growth in the presence of CFS from Bacillus strains isolated from potential probiotic source. Escherichia coli O157:H7 ATCC 35150 (A), Salmonella Enteritidis KCCM 12021 (B), or Staphylococcus aureus KCCM 11335 (C) were incubated in the presence of CFS from the Bacillus strains  at 37 °C for 24 h. The bacterial growth was determined at OD600. The growth rate of foodborne pathogenic bacteria without CFS was assigned to 100% (Control).Figure 5. Antibiotic resistance and susceptibility of the Bacillus derived from a potential probiotic source. Data for triplicate.Table 1. Antibiotic resistance of the Bacillus species derived from a potential probiotic source. The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated.


Kiwifruit is very popular for its unique flavor and nutritional value, and for its potential health benefits, which are closely related to its richness in a variety of natural antioxidant substances, in which polyphenolics play a non-negligible role. This study investigated changes in the fruit quality, phenolic compounds, and antioxidant potential of Chinese red-fleshed kiwifruit “Hongshi No. 2” during postharvest ripening at room temperature (20 ± 1 °C). Results showed that the weight loss rate slowly increased, the firmness rapidly decreased, and the soluble solid concentration gradually increased during the postharvest ripening of red-flesh kiwifruit. In addition, the total phenolic (TPC), total flavonoid (TFC), and total proanthocyanidin (TPAC) contents gradually increased during postharvest ripening. The most abundant phenolic compounds in kiwifruit throughout postharvest ripening were catechin (CC), proanthocyanidin B1 (PB1), and proanthocyanidin B2 (PB2). Furthermore, the methanolic extracts of red-flesh kiwifruit exhibited remarkable antioxidant activities throughout postharvest ripening stages. Indeed, some phenolic compounds showed good correlations with antioxidant activities; for instance, chlorogenic acid (CHL) showed a significantly positive correlation with ferric reducing antioxidant power (FRAP), and isoquercitrin (IS) showed a significantly negative correlation with DPPH free radical scavenging ability. The findings from this study are beneficial to better understanding the quality profile of red-flesh kiwifruit “Hongshi No. 2” during postharvest ripening. Actinidia), is subordinated to the genus Actinidia in the family Actinidiaceae, and has been domesticated and cultivated by humans for more than 100 years, since the beginning of the 20th century [Actinidia arguta (AP) had antidiabetic effects, and isoquercitrin derived from AP might be useful in preventing type 2 diabetes mellitus. In addition, Xie et al. [Kiwifruit . They were harvested on 26 September 2020, when the concentration of soluble solids reached 7–9%, and were stored at room temperature (20 ± 1 °C). The firmness was estimated every 2 days. The samples were divided into five groups according to 5 post-ripening stages, according to the hardness and shape of the fruit. The division principle is shown in The 1 represented the average weight of the fruit on the first day and A2 represented the average weight of the fruit on the day of post-ripening stages.The evaluation of WLR referred to the study by Lu . All fre−1; measurement velocity of 2 mm·s−1 and trigger strength of 5.0 g. Firmness results are expressed in Newton (N).The firmness was measured based on Maarten LATM’s method, with appropriate modifications . Six kiwAbout 50 g of fresh kiwifruit at each stage was collected, ground evenly using a blender and filtered with four layers of gauze, and the clarified filtrate was collected. SSC was determined with a handheld digital refract orometer. The products were manifested as percentage content (%).C was as described in previous research [w/v) on ice, then centrifuged at 4000× g at 4 °C for 10 min and the volume adjusted to 50 mL. TCA was used as blank control. A Varioskan Flash Multimode Reader was manipulated to take stock of the absorbance at 534 nm. A standard curve of VC was used for calibration. The results were expressed as mg VC per gram weight of fresh kiwifruit (mg·g−1).The determination of Vresearch . Brieflyv/v). Ultrasound-assisted extraction  was performed twice at room temperature, and the solvent was halved for the second extraction with 60 min each time. Following centrifugation , the supernatant solution was pooled and concentrated at 45 °C with a rotary evaporator, and then the volume was adjusted to 25 mL with methanol for subsequent determination.The extraction of polyphenolics was followed by Li  with som2CO3  over 3 min. Finally, the reaction was carried out in the dark for 30 min at room temperature (25 °C). The absorbance of the sample was identified at 765 nm with a microplate reader, and methanol was used as the blank. TPC values were calculated by regression curves fixed from gallic acid standards . The outcome was presented in mg of gallic acid equivalents per gram weight of dry kiwifruit powder (mg GAE·g−1).TPC was judged with the Folin–Ciocalteu procedure, which was explained by Everett  with sli2 solution (w/v) was added to 100 μL of kiwifruit extract or rutin standard solution. After 6 min of reaction, 30 μL of 10% Al(NO3)3 (w/v) was added. Subsequently, 400 μL of 4% NaOH (w/v) was added and the reaction was carried out for 25 min at room temperature and protected from light. The absorbance of the sample was measured at 510 nm, and methanol was applied as the blank. TFC values were finished by a linear fitting equation fixed from rutin standards . The outcome emerged as mg of rutin equivalents per gram weight of dry kiwifruit powder (mg RE·g−1).The TFC was determined by Lin  with par−1). The outcome was provided in mg of catechin equivalents per gram weight of dry kiwifruit powder (mg CE·g−1).The TPAC was determined with the Vanillin sulfuric acid method . A total−1). HPLC analysis was performed using the column Phenomenex Gemini C18 110A . The mobile phase was 0.5% acetic acid (A)-acetonitrile (B). The flow rate and injection volume were 0.8 mL/min and 20 μL, respectively. The detection wavelength was 280 nm. Eight standard substances were selected, including gallic acid (GA), protocatechuic acid (PA), procyanidin B1 (PB1), (+)-catechin (CE), chlorogenic acid (CA), procyanidin B2 (PB2), (−)-epicatechin (EC), and isoquercitrin (IS).Qualitative and quantitative interpretations of individual phenolic compound were carried out as in Li . The con50 values were summarized in the concentration of the kiwifruit sample solution (mg·mL−1). For FRAP capacity [−1).The antioxidant activity of kiwifruits at each storage time was detected by the free radical scavenging capacity (DPPH and ABTS) and FRAP. For DPPH scavenging capacity ,29, the capacity , FRAP won ≥ 3). ANOVA was used to compare statistically with Duncan’s multi-range test. The disparity was found to be statistically significant at p < 0.05. Pearson’s relevance coefficients were finished with Origin 2023 software.Data analysis was accomplished with IBM SPSS Statistics 27. The results were given by the mean ± standard deviation , which was much higher than other red-flesh kiwi varieties with VC content of 0.644 mg·g−1 [The variations in WLR, firmness, SSC, and V studies . Kiwifru content . The VC 4 mg·g−1 , indicat−1 (stage A) to 10.00 mg GAE·g−1 (stage E). The total phenolic content of kiwifruit during storage is reported to be related to storage conditions; when stored at a low temperature, it may increase with the storage time [−1 among all ripening stages. In most previous studies, the TFC of kiwifruit decreased during postharvest storage, and with some preservation, treatment could inhibit the reduction of total flavonoids [−1 to 14.56 mg CE·g−1 during post-ripening stages. It was evident in The changes in TPC, TFC, and TPAC are shown in age time . Howeverage time . The higavonoids . Howeveravonoids . This phavonoids . Proanthavonoids . In this2 = 0.9991, PA: y = 0.5422x + 0.0195, R2 = 0.9990, PB1: y = 0.1463x + 0.0219, R2 = 0.9999, CE: y = 0.2637x − 0.3900, R2 = 0.9993, CA: y = 0.6606x − 0.1152, R2 = 0.9994, PB2: y = 0.1546x − 0.0062, R2 = 0.9995, EC: y = 0.2315x + 0.0271, R2 = 0.9990, IS: y = 0.3663x + 0.0247, and R2 = 1.0000. The contents of the eight phenolic compounds detected at different stages are shown in −1 (stage A) to 90.34 μg·g−1 (stage E) (p < 0.05). The EC content showed an overall decreasing trend from 282.23 μg·g−1 (stage A) to 230.18 μg·g−1 (stage C), followed by a fluctuation in stages D and E. The PA content obviously increased from 23.80 μg·g−1 (stage A) to 92.72 μg·g−1 (stage E). Similar trends were found in PB1 and PB2; both showed the minimum content at the edible stage (stage D) with 414.51 μg·g−1 and 346.46 μg·g−1, respectively, and then significantly increased at the decay stage (stage E). IS content increased from 24.09 μg·g−1 (stage A) to 41.65 μg·g−1 (stage E). CE was the most abundant phenolic compound detected, and it enhanced remarkably with storage time (p < 0.05). Unlike other phenolic compounds, the content of CHL peaked at the edible stage (stage D) at 92.23 μg·g−1. Other phenolic compounds in kiwifruit varieties were mentioned in the study and found in comparison under similar chromatographic conditions [A. deliciosa cv. Hayward, A. chinensis cv. Hongyang, etc. [50 values of DPPH and ABTS scavenging capacities and FRAP capacity are displayed in −1, and then decreased after over ripening. These results indicated that the cumulative or synergistic effects of secondary metabolites of kiwifruit might increase the antioxidant activity of the fruit [As shown in he fruit .In this study, the quality profile of red-fleshed kiwifruit “Hongshi No. 2” during postharvest ripening was revealed according to the dynamic changes in fruit quality, phenolic compounds, and antioxidant activity. Results showed that the weight loss rate of kiwifruit slowly increased, and the soluble solid concentration gradually increased. In addition, the total phenolic (TPC), total flavonoid (TFC) and total proanthocyanidin (TPAC) contents gradually increased. Furthermore, the methanolic extracts of red-flesh kiwifruit exhibited remarkable antioxidant activities throughout postharvest ripening stages. In fact, both TPC and TFC were the major contributors to the DPPH radical scavenging ability and FRAP capacity. Additionally, some phenolic compounds, such as CHL and IS, showed good correlations with antioxidant activities. The findings from this study can provide theoretical bases for controlling the quality of red-fleshed kiwifruit “Hongshi No. 2” during postharvest storage.


Estimation of subjective quality of life in schizophrenic patients using speech features By Shibata Y, Victorino JN, Natsuyama T, Okamoto N, Yoshimura R and Shibata T. (2023) Estimation of subjective quality of life in schizophrenic patients using speech features. Front. Rehabil. Sci. 4:1121034. doi: 10.3389/fresc.2023.1121034A Corrigendum on In the published article, there was an error in In the published article, there was an error in Text CorrectionIn the published article, there was an error.3. Results, 3.4. Model comparison on test set for longitudinal measurement, Paragraph Number 1. This sentence previously stated:A correction has been made to “The RMSE and MAE for each subscale were 9.94 for the “Psychosocial” subscale, 6.433 for the “Motivation and Energy” subscale, and 11.008 for the “Symptoms and Side-effects” subscale . The minThe corrected sentence appears below:“The RMSE and MAE for each subscale were 9.607 for the “Psychosocial” subscale, 4.767 for the “Motivation and Energy” subscale, and 9.508 for the “Symptoms and Side-effects” subscale . The min3. Results, 3.4. Model comparison on test set for longitudinal measurement, Paragraph Number 2. This sentence previously stated:A correction has been made to “Fold 3 had the lowest RMSE and MAE at 4.557 and fold 2 had the highest at 20.540.”The corrected sentence appears below:“Fold 3 had the lowest RMSE and MAE at 4.557 and fold 2 had the highest at 13.540.”4. Discussion, 4.2. Estimation of scale scores by longitudinal measurement, Paragraph Number 1. This sentence previously stated:A correction has been made to “However, the RMSE and MAE for the model with longitudinal measures were less than 10 for “Psychosocial” at 9.940 and “Motivation and Energy” at 6.4334.”The corrected sentence appears below:“However, the RMSE and MAE for the model with longitudinal measures were less than 10 for “Psychosocial” at 9.607, “Motivation and Energy” at 4.767, and "Symptoms and Side-effects” at 9.508.”The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Nasal‐type extranodal natural killer (NK)/T cell lymphoma (ENKTL) is a rare and aggressive type of lymphoma. The optimal chemotherapy regimen for ENKTL has not yet been established. In this study, we compared the LVDP  and GLIDE  chemotherapy regimens for the treatment of ENKTL.A total of 267 patients with newly diagnosed ENKTL were included in this retrospective study. Propensity score matching (PSM) was used to adjust for confounders between the LVDP and GLIDE groups. Treatment responses, survival outcomes, and toxicities between the two groups were compared before and after PSM.p = 0.212; CR, p = 0.996). With a median 71 months follow‐up, the 5‐year progression‐free survival (PFS) and overall survival (OS) rates were 64.3% and 68.5%, respectively. The 5‐year PFS and OS were 65.6% and 70.1% for the LVDP group compared with 61.6% and 64.6% for the GLIDE group, respectively . After PSM, no significant differences in short‐term efficacy  or long‐term efficacy  between the two groups were identified. However, treatment‐related toxicities were milder in the LVDP group compared to the GLIDE group, even after adjusting for confounders via PSM.At the end of therapy, the objective response rate (ORR) and complete response (CR) were 83.5% and 62.2%, respectively, for all patients. The ORR and CR were 85.5% and 62.2% for the LVDP group compared with 79.3% and 62.2% for the GLIDE group, respectively, and no differences between the two groups were found  and GLIDE  chemotherapy regimens showed similar short‐term and long‐term efficacies, with 85.5% ORR and 62.2% CR rates, and 64.3% PFS and 68.5% OS rates at a median 71‐month follow‐up. However, LVDP was associated with milder treatment‐related toxicities compared to GLIDE, even after adjusting for confounders via PSM. These findings suggest that LVDP could be a preferable option for patients with nasal‐type extranodal NK/T‐cell lymphoma. The following criteria were used for patient inclusion: (1) confirmed as ENKTL from January 2012 to January 2017 in accordance with the 2008 WHO lymphoma classification;Patients' baseline characteristics and pre‐treatment laboratory data were obtained for analysis, including age, gender, Eastern Cooperative Oncology Group performance status (ECOG) score, B symptoms, white blood count (WBC), Hb (Hemoglobin), platelet count, lactate dehydrogenase (LDH), plasma EBV DNA, and bone marrow examination. Imaging examination was used for staging, including computed tomography (CT) and positron emission tomography/computed tomography (PET/CT). Response assessment was also carried out using PET/CT. However, some patients chose CT for response assessment due to individual economic circumstances. The PINK‐E scoring system was utilized in this study. Regional lymph node (RLN) involvement was classified as N1, N2, or N3, according to the definitions of the tumor‐node‐metastasis (TNM) staging system.2.22 intravenously on days 1–5), etoposide (80 mg/m2 intravenously on days 1–3), dexamethasone (40 mg/day intravenously on days 1–4), and cisplatin (25 mg/m2 intravenously on days 1–3). An L‐asparaginase skin test administered to each patient prior to each cycle; if the result was positive, pegaspargase replaced L‐asparaginase on day 4. The schedule and dose of the GLIDE regimen were given as described in a previous study.In this study, 267 patients with ENKTL were included. Among these, 63 patients were treated through chemotherapy only, and 204 patients were treated by chemoradiotherapy. Within this group, LVDP combined with radiotherapy was administered to 166 patients, and GLIDE combined with radiotherapy was given to 38 patients. The LVDP regimen was repeatedly administered every 21 days in this way: L‐asparaginase (5500 IU/mPatients with confirmed early‐stage ENKTL were treated by chemotherapy combined with involved‐field radiation therapy (IFRT), and in our study, 191 of 213 early‐stage patients received radiotherapy. Patients with confirmed advanced‐stage ENKTL were treated by consolidation radiation therapy at the primary tumor site or local residual lesion following the completion of the patient's chemotherapy schedule. However, only 13 of 54 advanced patients received radiation therapy consolidation after chemotherapy. Moreover, patients with complete response (CR) or partial response (PR) after initial treatment were allowed to receive elective hematopoietic stem cell transplantation (HSCT). Owing to a lack of standardized treatment protocols, treatment regimens varied and were decided primarily by physician discretion.2.3Treatment responses of all patients were assessed according to the response criteria of the Lugano Classification, which included the following terms: CR, PR, stable disease (SD), and progressive disease (PD).2.4The 5th version of the Common Terminology Criteria for Adverse Events (CTCAE) was used for toxicity evaluation. All toxicity data were obtained via physical examinations and blood tests including complete blood cell count, kidney, and liver tests.2.5All categorical variables are displayed in the form of frequencies with associated percentages, and associations between categorical variables were elucidated through the chi‐square test. The Kaplan–Meier method was employed to calculate survival results, and the log‐rank test was conducted to compare survival curves. Cox regression analyses were carried out to determine independent prognostic variables for ENKTL. Two‐sided P‐values less than 0.05 were considered statistically significant.We performed nearest‐neighbor 1:2 propensity score matching (PSM)  without replacement to adjust confounding factors between the LVDP and GLIDE groups. Covariates were considered well‐balanced when the absolute value of the standardized difference was lower than 10%. After PSM, baseline covariates and survival outcomes were compared between chemotherapy groups. PSM was conducted by SPSS 22 (IBM Corp). SPSS was used for performing all statistical analyses and for constructing visual figures.33.1A total of 267 patients with ENKTL were included in this study. Within this cohort, 185 patients received LVDP and 82 patients received GLIDE. Fourty‐three years was the median age of patients , with 227 (85%) patients less than 60 years, and 174 (65.2%) patients were male . Most patients (92.1%) had ECOG PS scores of 0 or 1. Approximately 144 (53.9%) patients had B symptoms. Seventy‐eight (29.2%) patients presented with RLN. Most patients (70.8%) were classified as low‐risk (0 or 1) according to the PINK‐E system. Elevated serum LDH levels were detected for 94 (35.2%) patients, and 176 (65.9%) patients were positive for EBV‐DNA. Before PSM, there were significant differences in B symptoms, AASS, PINK‐E, ECOG PS, and LDH between the LVDPand GLIDEgroups. However, after 1:2 PSM at a caliper value of 0.02, no significant differences in clinical parameters between the LVDP and GLIDE groups were identified . Baseline characteristics for included patients are presented in Table 3.2p = 0.996; ORR, p = 0.212). After PSM, there were 68 (57.6%) CRs, 30 (25.4%) PRs, 3 (2.5%) SDs, and 17 (14.4%) PDs for the LVDP regimen, and 48 (65.8%) CRs, 11 (15.1%) PRs, 5 (6.8%) SDs, and 9 (12.3%) PDs for the GLIDE regimen. The ORRs for the LVDP and GLIDE regimens were 83.0% and 80.9%, respectively. Similarly, no difference in short‐term efficacy between the two groups was found  cycles of chemotherapy. The entire cohort was assessed (230 of 267 patients were assessed by PET‐CT), including 166 (62.2%) CRs, 57 (21.3%) PRs, 8 (3.0%) SDs, and 36 (13.5%) PDs, with an ORR of 83.5%. Twenty patients received upfront autoHSCT after achieving CR. Before PSM, the CR and ORR for patients who received the LVDP regimen were 62.2% and 85.5%, respectively, and the CR and ORR for patients who received the GLIDE regimen were 62.2% and 79.3%, respectively. No significant differences between the LVDP and GLIDE groups were identified   , and the median survival time (including PFS and OS) was not reached. After the final follow‐up, 87 of 267 patients died. For 267 patients, the 3‐year PFS and OS rates were 67.3% and 72.1%, respectively, and the 5‐year PFS and OS rates were 64.3% and 68.5%, respectively Figure . The 3‐ ) Figure . Likewis) Figure .χ2 = 46.514, p < 0.001; OS, χ2 = 38.480, p < 0.001; Figure Approximately 76.4% of patients received chemoradiotherapy, and their PFS and OS rates were higher than those of patients who were administered chemotherapy alone  or OS   or OS   Figure . After P) Figure . Similar) Figure . No stat) Figure .3.4χ2 = 3.833, p = 0.051; Figure n = 2) and SMILE (n = 1).In total, 95 PFS events were recorded after the last follow‐up. During the upfront treatment, 36 cases had progressive disease. After the upfront treatment, 57 cases had disease relapse. For patients who experienced the first relapse or progression, the median OS was short with 2 months only, and the 5‐year OS rate was 13.1% Figure . The OS 3.59 L−1) <4, and Hb level <110 g/L. With the exception of EBV‐DNA, all of the above parameters were found to have a correlation on patient OS.To investigate clinical variables and their associations with survival outcomes for the entire cohort (267 patients), Cox regression analyses were conducted to determine independent prognostic factors. After the univariate Cox regression analyses, the clinical variables that may influence the PFS and OS of ENKTL patients are presented in Table To identify potential independent prognostic factors for PFS and OS, a multivariate analysis was conducted integrating all variables identified as significant in univariate analysis Table . The res3.6Before PSM, treatment‐related toxicities, including hematologic and nonhematologic adverse events, are shown in Table 4In this real‐world retrospective study, we analyzed two L‐asparaginase‐based regimens (LVDP and GLIDE) for 267 ENKTL patients, and we determined that the L‐asparaginase‐based chemotherapy regimens were effective. No differences in PFS or OS between the LVDP and GLIDE regimens were discovered, even after PSM. However, compared with the LVDP regimen, treatment‐related toxicities, including hematologic and nonhematologic adverse events, were more common in the GLIDE regimen. Through the Cox regression analysis, we found that ECOG PS ≥2 and Stage III/IV were correlated with poorer OS and PFS.Recently, owing to the anthracycline resistance of ENKTL, the use of L‐asparaginase‐based regimens has been preferred for improved efficacy. The SMILE regimen was given in newly diagnosed ENKTL patients and achieved a 4‐year PFS of 64% and a 5‐year OS of 50%.p = 0.264; ORR, 83.0% vs. 80.9%, p = 0.696; 5‐year PFS, 63.5% vs. 65.4%, p = 0.794; 5‐year OS, 67.7% vs. 67.6%, p = 0.867). In terms of toxicity, the GLIDE group had a higher incidence of myelosuppression and transaminase and fibrinogen abnormalities than the LVDP group. Treatment‐related infections causing death were reported; therefore, treatment‐associated myelosuppression should be handled carefully. In summary, considering its efficacy and toxicity, LVDP is a promising treatment option for ENKTL.With the advent of L‐asparaginase, the survival outcomes of patients with ENKTL have improved. In a retrospective study developed among patients with stage II ENKTL, 48 patients receiving LOP  had statistically better short‐term efficacy (CR and ORR) and long‐term efficacy (OS and PFS) than patients who received the CHOP regimen.p = 0.045).In addition, chemotherapy combined with radiotherapy plays an important role in ENKTL treatment.In many studies, AASS has been found to be an important prognostic factor for predicting PFS and OS in ENKTL. In 2006, Lee et al reported that the advanced stage (stage III/IV) was a vital prognostic factor for long‐term survival.Moreover, pretreatment hemoglobin has been considered to be a vital prognostic factor for predicting the PFS and OS of ENKTL. In a previous study, 32% of patients with NHL had anemia, and the incidence of anemia varied by lymphoma subtypes, with a lower frequency of indolent lymphomas and a higher frequency of aggressive lymphomas such as T‐cell lymphomas.However, a number of limitations affected this study. First, as this was a single‐center retrospective study, a degree of selection bias was likely present. Second, patients with ECOG‐PS score ≥2 and advanced‐stage patients were comparatively fewer, and this might impact the prognostic analysis. Third, the individual chemoradiotherapy schedules for ENKTL patients in our data varied, and this might cause additional bias in survival outcomes. Considering these limitations, subsequent multicenter randomized controlled trials should be conducted to further support and confirm the results presented in this study.5In conclusion, LVDP and GLIDE regimens are effective for ENKTL. However, treatment‐related toxicities were more frequently observed in the GLIDE group than in the LVDP group. Our study provides substantial evidence that LVDP has the potential as an efficacious treatment regimen for ENKTL.Wanchun Wu: Conceptualization ; data curation (lead); formal analysis (lead); writing – original draft (lead). Kexin Ren: Data curation ; formal analysis ; writing – review and editing . Xi Chen: Formal analysis ; writing – review and editing . Na Li: Data curation ; writing – original draft . Qian Luo: Writing – original draft (supporting). Tao Hai: Writing – review and editing (supporting). Huijie Zhou: Data curation (supporting). Liqun Zou: Conceptualization (lead); supervision (lead).No funding was received for this study.The authors have no relevant financial or non‐financial interests to disclose.Ethical approval was obtained from the Ethics Committee of West China Hospital .Table S1Click here for additional data file.


Arius heudelotii, Sardinella aurita, Ethmalosa fimbriata, and Sardinella maderensis) which were sampled in seventeen (17) localities in Senegal. The compounds targeted in this study were benzo(a)pyrene (B(a)P), benzo(a)anthracene (B(a)A), benzo(b)fluoranthene (B(b)F), and chrysene (Chr). The QuEChERS method was used for the extraction of PAHs, and their contents were quantified by gas chromatography (GC) coupled with mass spectroscopy (MS). The validation method was performed in accordance with the French standard NF V03-110 (2010). Satisfactory linearity (R2 > 0.999), LOD (0.05–0.09 μg/kg), LOQ (0.19–0.24 μg/kg), and precision (1.33–3.13%) of the four PAHs were obtained. The results of analysis in the 17 localities showed that all samples are contaminated by the four (4) PAHs with great variability of the contents between the different species and their origin. The B(a)P and ∑4PAHS contents in the samples ranged from 1.7 to 33 µg/kg and from 4.8 to 1082.3 µg/kg, respectively. Twelve (12) samples showed high levels of B(a)P, ranging from 2.2 to 33 µg/kg, thus exceeding the maximum authorized level (2 µg/kg). Fourteen (14) samples showed an overall ∑4PAHS content varying from 14.8 to 1082.3 µg/kg, which is above the maximum authorized limit (12 µg/kg). The principal component analysis showed that sardinella (Sardinella aurita and Sardinella maderensis) have very low levels of B(a)P, B(b)F, B(a)A, and Chr contents. However, high ∑4PAHS contents characterize smoked fish of the Kong species (Arius heudelotii), from Cap Skiring, Diogne, Boudody, and Diaobé, and of the Cobo species  from Djiffer. Thus, based on the authorized limits for PAHs in smoked fish, it appears that smoked fish of the sardinella species are less carcinogenic for human consumption.Polycyclic aromatic hydrocarbons (PAHs) are compounds resulting from any incomplete combustion process. These are pollutants that have proven toxicity due to their carcinogenic nature and can contaminate food during traditional smoking methods. Their highly toxic effect on human health requires monitoring of their levels in food products and the development of appropriate analytical methods for their determination. Thus, this study was conducted to assess the level of PAHs contamination of four (4) species of smoked fish ( Fishing is one of the most dynamic sectors in Senegal as it has experienced strong growth over the past three decades. Catches have increased considerably eight (8) times in thirty-two 32) years . A sourc years . Among these artisanal processing methods, smoking occupies a prominent place in developing countries. This technique is the main and sometimes the only means of supplying populations with fish, especially when they are far from fishing sites. Smoke not only gives fish a special taste and aroma but also improves preservation due to its dehydrating and bactericidal properties . HoweverIt is in this context that this present study occurs, which had as its objective the determination of polycyclic aromatic hydrocarbons (PAHs) in four (4) species of smoked fish from different areas in Senegal. Thus, the contents of benzo(a)pyrene (B(a)P), benzo(a)anthracene (B(a)A), benzo(b)fluoranthene (B(b)F), and chrysene (Chr), enacted by European Commission (EC) Regulation No. 835/2011, were estimated. A principal component analysis (PCA) was done to assess the effect of fish species, source, and technology of processing on the quality of smoked fish.Arius heudelotii), Round sardinella (Sardinella aurita), Cobo , and Tass or Flat sardinella (Sardinella maderensis). The choice of these four species was justified by the fact that they are more consumed in Senegal.The biological material consists of four (4) species of smoked fish that were chosen from the processors. These were Kong (Smoked fish samples were collected between February and June 2021 in seventeen (17) fish-processing sites in Senegal. Chemicals and solvents used are of analytical reagent grade. The acetonitrile (99.9%) and the acetone (99.8%) were, respectively, obtained from Sigma-Aldrich and VWR Chemicals. QuEChERS extraction tubes were purchased from Thermo Fisher Scientific Inc. (USA) and consisted of 50 mL centrifuge tubes containing 4 g anhydrous magnesium sulfate and 1 g sodium acetate. The 4 standards PAHs (B(a)P (CAS No. 50-32-8), B(a)P (CAS No. 56-55-3), B(b)F (CAS No. 205-99-2), and Chr (CAS No. 218-01-9), manufactured by Supelco, were purchased as pure compounds.μL automatic pipette. Everything was transferred using a Pasteur pipette into a vial and then frozen for 24 hours to freeze the fat. Finally, the supernatant was collected using a Pasteur pipette into another vial. The latter was placed in a gas chromatograph (GC) of the Agilent 7890A type coupled to a mass spectrometer (MS) (Agilent 5975C). The apparatus was equipped with an automatic sampler. Separation of PAHs was conducted using a 5% phenyl-methylsilicone (HB-5MS) bonded-phase fused-silica capillary column . Helium (99.999% purity) was used as the carrier gas with a flow rate of 1.2 mL/min. The injection port was adjusted in splitless mode, and the injection volume was 1.5 µL. The GC was programed as follows: initial temperature 70°C for 5 min and ramped at 25°C/min to 310°C for 13 min and allowed to stay for 10 min giving a total of run time of 28 min. The mass spectrometer (MS) was operated in the electron ionization mode, with an electron energy of 70 eV. The MS transfer line and ion source temperatures were adjusted at 310°C and 290°C, respectively.The QuEChERS method was used for the extraction of PAHs . The proOf the eight (8) PAH molecules recognized as carcinogenic when present in food, four (4) have been determined as benzo(a)pyrene (B(a)P), benzo(b)fluoranthene (B(b)F), benzo(a)anthracene (B(a)A), and chrysene (Chr).In order to increase sensitivity, all the GC-MS measurements of the different samples were carried out in triplicate and the results recorded in the various tables represented the average.The quantification method of PAHs in smoked fish samples was developed and validated according to the indications of the European Commission (no. 836/2011) and the French standard NF-V03-110 (2010). This procedure included successive steps and the determination of different parameters such as the linearity of the calibration interval, the detection and quantification limits, coefficients of variation for the repeatability, and intermediate precision tests.µg/kg) are given by the following formulas:mb is the average of each marker PAHs content in the standard solution and σ is the value of standard deviation for the norm for each PAH marker.The identification of PAHs was verified by comparing the retention times of each standard solution between each test. For the linearity tests, the standard stock solutions were dissolved in acetonitrile and five working solutions  were prepared to distinctly construct the calibration curves. These curves made it possible to establish an adequate correlation between the areas of the peaks and the concentrations of PAHs found in the samples tested. The limits of detection (LOD) and quantification (LOQ) were calculated from the standard PAH solution by separate 3 × 10 analysis according to the repository (NF-V03-110 2010). The values of these limits expressed in  for three consecutive days (interday), the precision (%) expressed as a coefficient of variation (CV) was obtained.R .All analyses were injected in triplicate to obtain a good precision. GC-MS data were acquired and analysed by Agilent Chemstation software for GC (G2070BA) and a Microsoft Excel version 2016 spreadsheet. A principal component analysis (PCA), which is the most widespread of the factorial methods , and a nμg/kg and those of quantification were 0.19, 0.24, 0.2, and 0.23 μg/kg for the B(b)F, B(a)A, Chr, and B(a)P. The LOD and LOQ values were within the acceptable limits of ≤0.3 µg/kg and ≤0.9 µg/kg, respectively, for each of the four PAHs (B(a)A, B(a)P, B(b)F, and Chr). The calculated coefficients of variation of the repeatability tests ranged from 1.33–2.25% to 1.95–3.13%, respectively, for intraday and interday. Corresponding results for the PAH4 are presented in The method validation results as the limit of detection (LOD), limit of quantification (LOQ), linearity, and retention times are shown in The results from the analyses are presented in µg/kg). Thus, it appears from these analyses that the samples taken in the areas of Dionewar (2.3 µg/kg), Fanda (2.2 µg/kg), Joel (3.9 µg/kg), Mbour (2.3 µg/kg), Thiaroye (2.3 µg/kg), Boudody (28.4 µg/kg), Cap Skiring (16.9 µg/kg), Diaobé (5.2 µg/kg), Diogue (13.2 µg/kg), Djiffer (33 µg/Kg), Niddior (6.8 µg/kg) and Sedhiou (14.2 µg/kg) are not B(a)P compliant. The highest concentration of this molecule was observed in Djiffer with a value of 33 µg/kg. However, these values are lower than those obtained by [µg/kg for Clarias gariepinus and 288 µg/kg for the species Ethmalosa fimbriata. The mean value (8.0 µg/kg) of B(a)P observed in the samples is much higher than that found in [µg/kg) but lower than that (52.7 µg/kg) obtained in [The results showed a great variability of the contents between the various species and according to their origin. The B(a)P molecule, representing 40% of the total carcinogenic risk attributed to PAHs , is presBoudody 2.4 µg/kg,found in  whose coained in  on smokeµg/kg. These were observed in samples from Cayar and Boudody, respectively. The average chrysene content is 35.1 µg/kg. The latter is higher than the mean values of B(a)A and B(a)P. Compared to the results in [µg/kg of chrysene was obtained in a species of smoked fish called Thunnus albacares.The analysis of samples of smoked fish from various sites also revealed the presence of chrysene at variable concentration levels between 1.3 and 142.6 sults in , a conteµg/kg and from 1.2 to 121.6 µg/kg. The average contents of B(b)F and B(a)A, in the samples, were respectively evaluated at 120.1 and 26.4 µg/kg. Much lower levels were observed in [µg/kg and 5.79 µg/kg.Regarding B(b)F and B(a)A, the levels in the various samples analysed vary, respectively, from 1.7 to 822.3 erved in  on severµg/kg, respectively, observed in the samples from Cayar and Diaobé. Higher concentrations of the sum of PAHs, between 1295 and 2020 µg/kg, were obtained in [µg/kg. The total PAH content recommended by the European Commission is 12 µg/kg. Thus, only the samples taken in the sites of Cayar (4.8 µg/kg), Dionewar (8.4 µg/kg), and Fass Boye (8.3 µg/kg), with values below the standard, are therefore compliant.The total concentration of the four (4) molecules varies between 4.8 and 1082.3 ained in  which waArius heudelotii) and Cobo .It is clear that also samples from sites, such as Boudody, Dioabé, Diogue, Djiffer, Sedhiou, and Cap Skiring, have the highest PAH contents. The latter has been observed in species such as Kong  was carried out to assess the effects of smoking, the nature of the fish species, and the place of processing on PAH levels. The first two dimensions (Dim 1 and Dim 2) express 98.90% of the total inertia .The first dimension (Dim 1) contributes to 81.08% and the second (Dim 2) at 17.82%. They thus present the highest eigenvalues, respectively, equal to 3.24 and 0.71.sardinella which have very low levels of B(a)P, B(b)F, B(a)A, and Chr. However, high levels of B(a)P and Chr characterize class 2 consisting of smoked fish of the Kong species, from Cap Skiring, Diogue, and Boudody, and of the Cobo species from Djiffer A (0.986), B(b)F (0.818), and Chr (0.981) are positively well correlated with the first dimension (Dim 1). However, the second dimension is characterized by the variable B(a)P 0.539), which is positively correlated. Smoked fish have been grouped into three classes. Class 1 is made up of 9, which The analysis of the results of this study shows that the B(a)P content and the sum of the ∑4PAHS contents in different zones and according to the species present a significant difference compared to the standard.Arius heudelotii), the temperature used and multiple smoking could lead to the high numbers obtained. Moreover, studies have shown that a smoking temperature above 450°C would allow the pyrolysis of the wood to generate more (PAH) [This trend could be attributed, on the one hand, to the differences in fat and moisture composition of each species as well as to the nature of the skin cover . Indeed,re (PAH) , 27.PAHs would also be present in fish products before smoking. Indeed, apart from spatiotemporal parameters, it has been revealed that the levels of PAHs would also be affected by several ecological factors. It has been demonstrated that vehicles, especially diesel engines, could also contribute significantly to the generation and release of PAHs into the environment . In addiArius heudelotii). These results, therefore, indicate that it is necessary to improve the smoking process in order to best limit the rate of contamination of the finished products with PAHs and ensure consumer safety. Moreover, it would be very important to carry out an analysis to assess the possible risks to human health associated with the consumption of these smoked fish in Senegal.This study consisted of highlighting the presence of polycyclic aromatic hydrocarbons (PAHs) in traditionally smoked fish. Indeed, smoking, while helping to preserve fish, could also induce certain carcinogenic substances such as PAHs in processed fish. Sampling, carried out in seventeen (17) production areas in Senegal, revealed the presence of benzo(a)pyrene (B(a)P), benzo(a)anthracene (B(a)A), benzo(b)fluoranthene (B(b)F), and chrysene molecules (Chr). The results showed that twelve (12) or 71% and fourteen (14) sites or 82% present contents higher than the European Commission standards, respectively, for B(a)P and the sum of ∑4PAH. The highest PAH concentrations were observed in the Kong species (


Imaging of the salivary ductal system is relevant prior to an endoscopic or a surgical procedure. Various imaging modalities can be used for this purpose. The aim of this study was to compare the diagnostic capability of three-dimensional (3D)-cone-beam computed tomography (CBCT) sialography versus magnetic resonance (MR) sialography in non-tumorous salivary pathologies.This prospective, monocenter, pilot study compared both imaging modalities in 46 patients (mean age 50.1 ± 14.9 years) referred for salivary symptoms. The analyses were performed by two independent radiologists and referred to identification of a salivary disease including sialolithiasis, stenosis, or dilatation (primary endpoint). The location and size of an abnormality, the last branch of division of the salivary duct that can be visualized, potential complications, and exposure parameters were also collected (secondary endpoints).1 = 0.66, p2 = 0.63, and p3 = 0.24, respectively). The inter-observer agreement was perfect (> 0.90) for lesion identification. MR sialography outperformed 3D-CBCT sialography for visualization of salivary stones and dilatations, as evidenced by higher positive percent agreement (sensitivity) of 0.90 [95% CI 0.70–0.98] vs. 0.82 [95% CI 0.61–0.93], and 0.84 [95% CI 0.62–0.94] vs. 0.70 [95% CI 0.49–0.84], respectively. For the identification of stenosis, the same low positive percent agreement was obtained with both procedures (0.20 [95% CI 0.01–0.62]). There was a good concordance for the location of a stone (Kappa coefficient of 0.62). Catheterization failure was observed in two patients by 3D-CBCT sialography.Salivary symptoms involved both the submandibular (60.9%) and parotid (39.1%) glands. Sialolithiasis, dilatations, and stenosis were observed in 24, 25, and 9 patients, respectively, with no statistical differences observed between the two imaging modalities in terms of lesion identification (pBoth imaging procedures warrant being part of the diagnostic arsenal of non-tumorous salivary pathologies. However, MR sialography may be more effective than 3D-CBCT sialography for the identification of sialolithiasis and ductal dilatations.NCT02883140.The online version contains supplementary material available at 10.1186/s12903-023-03159-9. Non-tumorous salivary gland diseases are common in adults and include salivary stones, ductal strictures, anatomical abnormalities, and chronic inflammation. Sialolithiasis is the main cause, representing 60–70% of all cases of obstructive disease, followed by stenosis in 15–25% . VariousThanks to a direct opacification of the salivary ducts, conventional sialography has long been the gold standard for diagnosing non-tumorous salivary diseases , 5, 10. The purpose of this study was to compare the overall diagnostic outcomes of 3D-CBCT sialography versus MR sialography for the detection of sialolithiasis, ductal dilatation, and ductal stenosis, and the outcome measures for each of these three diseases. The null hypothesis predicts that there are no statistically significant differences between the two techniques in overall or disease-specific outcomes.This prospective, monocenter, pilot study included patients over 18 years of age (no upper age restriction) with suspicion of obstructive or inflammatory disease of a parotid or a submandibular gland based on the medical history and clinical examination. The exclusion criteria comprised having a known allergy to iodinated contrast agent, known or suspected pregnancy, lesion of the oral mucosa preventing catheterization, relative contraindication to MRI , and signs of a salivary tumor . Patients with a head and neck scan in the previous 6 months, either by conventional or three-dimensional sialography, were also excluded from this study. For each patient, the following clinical data were collected prior to the imaging procedures: age, gender, medical history, affected gland, side, complaints , and clinical parameters . After being provided information regarding the study and obtaining informed written consent, the included patients underwent MR sialography followed by 3D-CBCT sialography on the same day. The study was approved, and ethical approval was obtained from our institutional review board CPP Ouest IV-Nantes . This study was registered on ClinicalTrials.gov (number NCT02883140) on 30/08/2016. The STARD checklist for diagnostic studies  and the MR imaging was performed on a 3T scanner  using a head and neck coil. The patients were told not to eat or drink in the hour preceding the scan. The patients were positioned in dorsal decubitus and instructed not to swallow saliva during the acquisition times.For sialo-MR sequence acquisitions, three-dimensional (3D) T2 DRIVE (DRIVen Equilibrium) with sagittal fat-suppressed turbo spin-echo (TSE) sequences were obtained with the following parameters: repetition/echo time (TR/TE) 2000/237 ms; matrix 376 × 267; field of view (FOV) 150 × 150; slice thickness 2 mm; interpolation 0.4; voxel 0.4 × 0.5 × 0.4, number of slices 94; spectral presaturation inversion recovery (SPIR); and scan time 4.34 min.Conventional MR sequences were performed for diagnostic purposes in patients as part of routine care, including axial T2 TSE: TR/TE 2336/80 ms; TSE factor 14; NSA 1.5; matrix 416 × 320; FOV 180 × 180; slice thickness 2 mm; space 0.3; voxel 0.3 × 0.56 × 3 mm; number of slices 24; and acquisition time 2.48 min. Coronal T2 TSE: TR/TE 2726/80 ms; TSE factor 14; NSA 2; matrix 360 × 273; FOV 180 × 180; slice thickness 2 mm; space 0.3; voxel 0.5 × 0.66 × 3 mm; SENSE factor 1.4; number of slices 27; and acquisition time 2.38 min. Axial T1 TSE sequences: TR/TE 662/10 ms; factor TSE 5; NSA 1; matrix 364 × 300; FOV 200 × 180; slice thickness 3 mm; voxel 0.55 × 0.66 × 3 mm; number of slices 23; and acquisition time 2.47 min.The 3D-CBCT sialography procedure was performed by two senior maxillofacial surgeons (H.B. and R.B.), as previously described . BrieflyThe imaging data were anonymized and archived using imaging software . Analysis of 3D-CBCT sialography was performed using maximal intensity projection and multiplanar reconstruction (MPR) with 0.25-mm cuts, isotropic voxel size and image pixels 492 × 492. MRI scans were analyzed on a high-resolution visualization workstation with native 2D image analysis (viewer mode) for TSE/T1/T2 sequences, and MPR for the 3D T2 MR-sialography sequences. Images were analyzed twice by two radiologists experienced in head and neck radiology (A.S.D. and E.M.L.), in separate viewing sessions (wash-out period of at least one month) and following a standardized pathway. The readers were blinded to the clinical data. A reading standardization session was performed on ten cases before starting the analyses. The analysis was based on the ability of the examination to identify a salivary disease including sialolithiasis (L), stenosis (S), or ductal dilatation (D) (primary endpoint). On MR sialography, saliva manifested as a high signal intensity and the salivary glands appeared as a low signal intensity, while only the salivary canal was opacified by the contrast medium in 3D-CBCT sialography. Sialolithiasis appeared in MR sialography as round or oval hypointense structures surrounded by hyperintense saliva, while the presence of a dense calcified concretion could be seen on 3D-CBCT sialography. Ductal stenosis displayed a signal or opacification interruption within the salivary ducts, and dilatations appeared as sialectasis in the ductal salivary system. Salivary diseases were classified according to the Marchal et al. classification , each imaging procedure was compared to the other, assuming that a lesion detected by one modality confirmed the presence of the disease. As a result, unbiased estimates of “accuracy”, “sensitivity”, and “specificity” cannot be calculated and the terms should not be used ). Most of the stones concerned the submandibular gland (n = 24), mainly in the Wharton’s duct (19 stones), and 12 concerned the parotid glands; the concordance was considered to be good regarding the stone location (Kappa coefficient: 0.62 [95% CI 0.36–0.88]). The mean size of the stones was 7.54 ± 3.34 mm by 3D-CBCT sialography vs. 7.03 ± 3.48 mm by MR-sialography (p = 0.04).Two patients presented with multiple stenosis. Most of the stenosis were in the parotid gland (n = 10) and only one involved the submandibular gland. Only one of these abnormalities was detected by both imaging procedures.Dilatation was found in the submandibular gland in 14 cases and in the parotid gland in 11 cases. Most of the dilatations were classified as D1, with a mean diameter of 3.34 ± 0.97 mm as measured by 3D-CBCT sialography vs. 3.30 ± 1.25 mm by MR sialography (p = 0.57).2. We did not observe any adverse effects of either catheterization or iodine contrast injection.3D-CBCT sialography allowed exploration of the ductal system up to branch 4.25 ± 1.55 vs. 3.65 ± 0.96 by MR sialography of the submandibular gland (p = 0.14). In the parotid gland, the exploration reached branch 4.36 ± 1.41 with 3D-CBCT sialography vs. 4.08 ± 1.23 with MR sialography (p = 0.30). Ostium catheterization failed in two patients (4.3%) with 3D-CBCT sialography. The mean DAP generated by CBCT was 247.4 ± 76.7 mGy.cmMultiple imaging techniques are used for diagnosing salivary ductal diseases. Ultrasonography is a first-line examination as it exposes excellent diagnostic capabilities, with high sensitivity (0.899) and NPV (0.808) for exploration of the main salivary pathologies . HoweverOur study aimed to compare the diagnostic capability of 3D-CBCT sialography versus that of MR sialography. Our study population was comparable to those in previous publications in terms of age, gender distribution, and symptoms , 34. Mos2, which is three times higher than the mean of 79.9 mGy.cm2 generated by panoramic radiography [2 reported for jaw imaging with different CBCT devices [Regarding the last salivary branch of division visualized, 3D-CBCT sialography outperformed MR sialography irrespective of the gland studied. Many authors have reported that it is possible to explore up to the fifth-order branches with 3D-CBCT sialography –17, 28. iography . Neverth devices , 40. Jad devices . Duct ca devices . We obse devices , 28. The devices .Our study suffers from some drawbacks. The first lies in our per-protocol analysis whereby we excluded patients in which we experienced a catheterization failure or discordant data regarding the side analyzed for the study. These side errors occurred in patients with multifocal symptoms. The MR images were acquired on a single gland in the sagittal plane, i.e., in the plane of the salivary ducts for better resolution, and they did not allow identification of the affected gland. The second is related to the presence of a high number of normal radiological examinations n = 16/46), which could affect the diagnostic performances of the radiological procedures studied. There was no attempt to eliminate the “normal” observations from our analysis as they reflect the current practice. A third drawback, namely measurement bias, is possible in this type of imaging study, although it was limited by the double reading of the images by experienced radiologists and by the calibration session before starting the study analyses. Finally, it is regrettable that we did not compare our two imaging procedures with a reference imaging modality to decide discordant cases. Sialoendoscopy could represent such a positive control in further studies but may fail in cases of very proximal diseases. Another strategy is represented by the development of a consensus between radiologists and surgeons to establish a final diagnosis . In the /46, whicNotwithstanding the limitations of the study and the absence of statistical differences between the two imaging modalities, MR sialography may be more effective than 3D-CBCT sialography for the identification of sialolithiasis and ductal dilatations. Moreover, this non-invasive procedure is not subject to the failures and adverse effects of salivary catheterization. Both procedures allow exploration of the ductal system at least up to the third or fourth branch of division, irrespective of the study of the parotid or submandibular gland, and warrant being part of the diagnostic arsenal for non-tumorous salivary pathologies.Below is the link to the electronic supplementary material.Supplementary Material: Table 1. STARD 2015 checklist for reporting diagnostic accuracy studies. Table 2. STROBE statement for reporting observational studies


Isolation of African swine fever virus (ASFV) is a critical step towards the identification, titration, characterization, and even modification of the virus. Therefore, it is important to identify a suitable cell line that supports the efficient replication of ASFV for these purposes. This should be achieved even when starting with a low virus load, as in the case of isolating the virus from field samples. This article presents a detailed protocol on the preparation of porcine bone marrow primary (PBMP) cell culture, which has a high sensitivity towards ASFV, resulting in high viral yields with a minimal risk of bacterial contamination. Asfarviridae family genus Asfivirus [African swine fever virus (ASFV) is the only member of the sfivirus . It is tsfivirus . The virsfivirus . Laboratsfivirus .Sus scrofa , green monkey , and rhesus monkey (LLCMK2) [Due to the observed phenomenon of “hemadsorption” represented by the accumulation of red blood cells (RBCs) at the surface of infected cells, the first biological models for ASF virus isolation were cell cultures of the mononuclear-macrophage series (blood leukocytes and bone marrow macrophages of pigs) ,6,7,8. P(LLCMK2) . While i(LLCMK2) . Hurtado(LLCMK2) . Masujin(LLCMK2) .The majority of laboratories and research institutes investigating and performing routine diagnostics on ASFV employ three main cell culture lines. These are primary macrophage cultures prepared from fresh pig blood (PBMC), primary alveolar macrophage cultures (PAMC), and continuous cell culture MA-104 ,26,27. PIn this paper, we describe a step-by-step procedure for the preparation of porcine bone marrow primary (PBMP) cell culture that has a high yield and high sensitivity as well as a low risk of contamination. Class II biological safety cabinet .2 incubator .Humidified 37 ± 2 °C, 5 ± 1% CO−70 °C freezer .−20 °C freezer .Refrigerator, 4 °C, with an acceptable range of 2 °C to 8 °C .Water bath, 37 °C .Portable Pipettor .Micropipettors: Single channel, 1–10 µL, 2–20 µL, 50–200 µL, 100–1000 µL .Micropipettors: Multi-channel, 0.5–10 µL, 5–50 µL, 50–300 µL .TM Pipet-Life XLS, L-1200, 100–1200 µL, catalog number: 17014497).Multi-channel for larger volumes .Tabletop Centrifuge .Shaker incubator .Electronic balance .Countess™ automatic cell counter.Pipette tips: 10 μL, 20 μL, 200 μL, 1000 μL .Sterile serological pipettes: 1 mL, 5 mL, 10 mL, 25 mL, 50 mL .®, catalog numbers: 353110 and 352098)Conical tubes: 15 and 50 mL .T-25 flask .T-225 flask .Millex-HV Syringe Filter Unit, 0.45 µm, PVDF, 33 mm, gamma sterilized .Absorbent paper .®, catalog number: 29442-474).Reservoirs , no phenol red .® cell strainers .EZFlowMinimum Essential Medium (MEM) .ASFV field strain: For example an ASFV-Arm07 field strain (Arm07).Trypan blue solution, 0.4% .Gentamicin 40mg/ml Solution .Penicillin G sodium salt .Streptomycin sulfate .Saline solution .Bacto™ TC Lactalbumin Hydrolysate .70% Ethanol.Fetal Bovine Serum .Clinically healthy piglets, 4–6 weeks of age (8–15 kg), obtained from farms free from ASF, CSF, and other infectious diseases, are used. Animals were housed for one week before necropsy and monitored twice a day by veterinary staff. Water and feed were provided ad libitum.Necropsy must be performed in accordance with BSL-3 guidelines and the approved statement from the Animal Ethics Committee.For the preparation of the cell culture, large tubular bones  are taken during necropsy.Sterile containers with samples are placed in a sterile waterproof metal lab container and transferred to the laboratory, where all subsequent stages of the cell culture preparation are performed.Place isolated tubular bones  in a vessel and wash with 70% ethanol by incubating in a shaker-incubator at room-temperature and 250 rpm for 10–15 min in order to disinfect the surfaces of the samples A.Remove the muscle and tissue mass from the bones with a surgical scalpel and rewash the bones, similar to step 1 B.3.2, thereby exposing the bone marrow as much as possible , after which the vials are carefully inverted 1–2 times. Incubate at room temperature (RT) for 2 min, and then discard the buffer.Add washing buffer  to the b6.m/v) into the flasks with bone pieces, and incubate in a shaker-incubator at 37 °C and 250 rpm for 20 min to remove the bone marrow cells from the bone cavities.Add saline solution in a ratio of 1:1.5 . Step 6 can be repeated in order to gather more cells from the bone marrow. The filtrate is evenly poured into sterile centrifuge beakers and centrifuged at +4 °C and 1400 rpm for 20 min.8.After centrifugation, discard the supernatant and re-suspend the pellet in growth medium  Calculate the cell concentration using a Countess3 of cell suspension; H is the number of living cells indicated by the device in 1 cm3 of a cell suspension sample; 2 is the coefficient of cell suspension dilution with 0.4% trypan blue solution; and 10 is the coefficient of dilution of the cell suspension with the saline solutionCell concentration is calculated by the formula:10.3.Dilute the cell suspension in growth medium, and adjust to an inoculation concentration of 20–30 million cells/cm11.Add the diluted suspension of PBMP cells to cell culture flasks as indicated in 12.2 for 48 h.Incubate the flasks in a humidifier at +37 °C with 5% CO13.After a 48-h incubation, the growth medium is replaced with maintenance medium  as shownAfter changing the medium, cell growth is assessed under the microscope. PBMP macrophages should be round or oval and 40–60 microns in size with a smooth surface and a high density of attachment to the substrate, and they should be located singly and not form coagulates . A cell PBMP cell culture should be used, depending on the purpose of the study, within 2–5 days after adding maintenance medium.Cell culture prepared in the laboratory can be in cell culture plates (48-wells or 96-wells) or in cell culture flasks such as T-25 or T-75, depending on the needs of the researchers. Plates are used for virus titration, while flasks can be used for the isolation of new viruses from biological materials or the accumulation of viruses for further uses, such as full genome sequencing. m/v), followed by centrifugation at 1100 g for 10 min. The supernatant is filtered through a Millex-HV Syringe filter (0.45 µm). Gentamicin sulfate (40mg/mL) is added to the filtrate at a final concentration of 0.01% and incubated for 1 h at a temperature ±37 °C, followed by inoculation into porcine PBMP cell culture. The flasks can be incubated up to 7 days at 37 °C in a 5% humidified CO2 incubator. The results are estimated under the microscope on a daily basis for the presence of hemadsorption. Organ samples  taken from infected animals or corps (wild boars or domestic pigs suspected of ASF) are homogenized, and a suspension is prepared by adding saline solution in a ratio of 1:10 (The cell culture is examined for the presence of hamadsorption under a low magnification microscope (10 × 10). The first observation is performed 15–18 h after inoculation, and, subsequently, on a daily basis for 7 days. Before examination, the cell culture should be shaken slightly so that erythrocytes that are not attached to the surface of the cells will be evenly distributed in the culture medium.The hemadsorption phenomenon is based on the attachment of erythrocytes to the surface of virus-infected macrophages. The result for ASF is considered positive in the presence of haemadsorption .If there are no manifestations of hemadsorption in the inoculated cell culture during the observation period, it is necessary to conduct two additional passages in the PBMP cell culture. After the third passage, if no hemadsorption is observed, the VI result is considered negative . Taking into consideration that not all ASFV are hemadsorbing, if no hemadsorption is detected, VI must be accompanied by RT-PCR in order to detect the virus and confirm its replication by Ct level.50/cm3, and titer (in lg HADU50/cm3) was calculated on the fifth day after inoculation as described by de Leon et al. in 2013 [To validate this cell culture for ASFV isolation and titration, different viruses were used to infect PBMP and porcine spleen cell culture (PSC) (n = 3) ,30. The  in 2013 . The resAfrican swine fever virus isolation in cell culture is a critical step in virus characterization and the study of its biological properties. The rate of mutation in the virus genome due to its natural circulation between susceptible animals resulted in the appearance of 24 genotypes and a novel recombinant variant ,32,33. I