We used verification CTs and prostate motion data calculated from 1024 intra-fractional prostate motion records to develop a voxel-wise based 4-dimensional method, which had a time resolution of 1 s, to assess the dose impact of prostate motion. An example of 100 fractional simulations revealed that motion had minimal impact on planning dose, the accumulated dose in 95 % of the scenarios fulfilled the clinical goals for target coverage (D95 > 37.5 Gy). This method can serve as a complementary measure in clinical setting to guarantee plan quality. This was achieved by utilizing four-dimensional (4D) synthetic computed tomography (sCT) simulations. The method was applied to an example patient anatomy and EHIMPT plan.22.1This study used the CT imaging data  for this patient was generated in RayStation Research 7.99  according to our protocol constraints . Every f2.3Prostate motion data of twenty-six PCa patients treated with conventionally fractionated radiotherapy  in the Department of Radiation Oncology of the University of Pennsylvania were used Among 1055 available treatment fractions’ records, all records longer than four-minute treatment time were selected (1024), since the time interval for every fractional treatment would be at least four minutes in our medical center. The 95th percentile of motion range was calculated in each dimension (left right (x), inferior superior (y), and posterior anterior (z)). Every range in each direction yields two opposite 1-dimensional vectors: , , . Equispaced sparse sampling resulted in eight 3-dimensional (3D) motion vectors: , , , , , , , .Six common motion trajectories were reported by Kupelian et al. 2.4Five weekly vCTs were used to simulate five fractions of EHIMPT. Each vCT was registered to the pCT based on fiducial markers according to the institutional procedure for online verification.Each weekly vCT was used to create eight sCTs based on the eight 3D motion vectors. The CTVs were translated within the patient according to the motion vector . Next, the CTVs and pelvic bones were set as controlling regions of interest (ROIs) for the hybrid deformable image registration  in RayStation Research 7.99) to obtain the deformation vector fields (DVFs), and created a sCT F that il2.5For each beam, a combination of motion vector and motion initiating time was randomly selected from the 128 combinations (8 motion vectors and 16 initiation time) with a time resolution of 1 s. We conducted 100 fractional simulations, with each motion scenario randomly selected using R , to obtain 20 complete treatment simulations. 2.6For each EHIMPT, the sub-plan of every second was calculated on the corresponding sCT. Using the DVFs obtained by ANACONDA, all sub-plan doses on sCTs were warped back to the vCTs of every fraction, and then the five-fraction doses on vCTs were warped and summed to the pCT . The accumulated dose on the pCT G represe3The 95th percentile of prostate motion range were 2.0 mm in lateral direction, 3.2 mm in inferior-superior direction, and 3.9 mm in posterior-anterior directions.In the 20 simulated treatments, the mean simulated accumulated doses met the dose constraints for CTV1 and CTV2 with D95 % of 37.9 Gy [95 % CI 37.1 – 38.1] and 31.6 Gy [95 % CI 31.5 – 31.8] . The worWhen comparing the mean simulated accumulated dose to nominal dose of CTV1, no difference existed, with 37.9 Gy in the mean simulated accumulated dose and in the nominal dose. However, a difference is reported for D95 % dose of CTV2, with 31.6 Gy in the mean simulated accumulated dose versus 30.3 Gy in the nominal dose .3 of the posterior rectal wall and D5cm3 of the rectum. The largest difference was found in the D0.01 cm3 of the posterior rectal wall with 26.8 Gy in the mean simulated accumulated dose and 19.0 Gy in nominal dose . Simulations in this study used perfect setup during beam delivery, but included repeated imaging to include inter-fraction motion and synthetic imaging with a 4D resolution of 1 s to include intra-fraction motion. Therefore, our method can be used additional and complementary to the standard robustness evaluation method of proton plans using setup and range errors.The probability-based motion simulation in our study is based on a randomization procedure applying a range of motion (95 % range derived from 1024 fractions of 26 patients). This was done to simulate a wider range of motion possibilities while excluding the extreme outliers. Other studies In our study, the mean and the minimal dose (worst-case dose) of CTV1-D95 % (prostate) in simulated treatments were 37.9 Gy and 37.0 Gy which are 101.1 % and 98.7 % of the prescribed dose. This is comparable to the results of Su et al. (103.1 % (mean) and 96.7 %  using uniform scanning proton delivery technique) A limitation is that generating sCTs based merely on rigid displacement of the prostate. This problem is inevitable since the Calypso data do not include organ deformation or rotational information. Rotations could result in target dose reduction 3 needed 13 energy layers per beam).Our study was limited to simulating only one patient’s anatomy and treatment plan. However, the focus was on the implementation of the simulation method. The selected patient had the largest inter-fraction motion among all patients with available vCTs. For patients with possible larger prostate volumes, the amount of energy layers and delivery time increase accordingly (e.g. CTV1 volume increased to 54.9 cmWe will be using the presented probability-based simulation method as an additional tool to the setup and range robustness evaluation of EHIMPT plans to gain more insight and confidence in the robustness of EHIMPT against inter- and intra-fraction prostate motion in a larger group of patients and treatment plans.In conclusion, this study introduced a new, 4-D simulation method to assess the dose impact of inter- and intra-fraction motion. This method can be used in addition to the robust evaluation method.Sen-Quan Feng: Conceptualization, Methodology, Software, Data curation, Writing – original draft, Writing – review & editing, Visualization. Charlotte L. Brouwer: Conceptualization, Methodology, Supervision, Writing – review & editing, Visualization, Project administration. Erik W. Korevaar: Methodology, Writing – review & editing. Neha Vapiwala: Resources, Data curation, Writing – review & editing. Ken Kang-Hsin Wang: Resources, Data curation, Writing – review & editing. Curtiland Deville: Resources, Data curation, Writing – review & editing. Johannes A. Langendijk: Conceptualization, Resources, Supervision, Writing – review & editing, Project administration, Funding acquisition. Stefan Both: Resources, Data curation, Writing – review & editing. Shafak Aluwini: Conceptualization, Resources, Methodology, Supervision, Writing – review & editing, Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Osimertinib, as third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is the first-line treatment approved to treat advanced T790M mutation-positive tumors. Triazole antifungals are therapeutic drugs for cancer patients to reduce the risk of opportunistic fungal infections. Our objective was to investigate whether three triazole antifungals  could change the pharmacokinetics of osimertinib in rats.n = 6): control (0.3% CMC-Na), and voriconazole (20 mg/kg), itraconazole (20 mg/kg), or fluconazole (20 mg/kg) combined with osimertinib (10 mg/kg) group. Tail vein blood samples were collected into heparin tubes at various time points within 0–48 h after osimertinib administration. Osimrtinib’s plasma concentration was detected using HPLC-MS/MS system equipped with a Waters XBridge C18 column, with the mobile phase consisting of acetonitrile and 0.2% formic acid water at a flow rate of 0.5 mL/min.The adult male Sprague-Dawley rats were randomly divided into four groups (max of osimertinib by 58.04% and 53.45%, respectively; the AUC0–t increased by 62.56% and 100.98%, respectively. However, when co-administered with itraconazole, the Cmax and AUC0–t of osimertinib only increased by 13.91% and 34.80%, respectively.Co-administration with voriconazole or fluconazole increased the COur results revealed that the pharmacokinetics of osimertinib were significantly changed by voriconazole and fluconazole in rats, whereas it was slightly affected by itraconazole. This work will contribute to a more comprehensive understanding of the pharmacokinetic properties of osimertinib when co-administered with triazole antifungals. Drug–drug interactions (DDIs) play a substantial role in the increased risk of adverse drug reactions (ADRs), accounting for approximately 30% of all reported ADRs, which may consequently influence health outcomes . The occin vivo. Voriconazole (VCZ), itraconazole (ICZ), and fluconazole (FCZ) are common triazole antifungals with moderate to strong CYP3A inhibiton (Patients with cancer are at considerable risk of DDIs. Osimertinib (OSIM) is a third-generation tyrosine kinase inhibitor (TKI) that targets the epidermal growth factor receptor (EGFR) . It is hnhibiton .In clinical practice, as a therapeutic strategy for patients with cancer, OSIM is administered in conjunction with CYP3A inhibitors to decrease the incidence of invasive fungal infections . DDI, hoOSIM (≥98.0%) was purchased from the National Institute for Food and Drug Control . VCZ (≥98.0%), ICZ (≥98.0%), and FCZ (≥98.0%) were provided by Zhejiang Provincial People’s Hospital . Nilotinib (≥99.0%), carboxymethyl cellulose sodium, isopropanol, and formic acid of LC-MS grade were purchased from Aladdin Industrial Corporation . Acetonitrile and methanol were purchased from Merck . Ultra-pure water was produced with a Millipore Synergy system . The rat tail vein fixator  was provided by the Laboratory Animal Center of Zhejiang Provincial People’s Hospital.Two separate stock solutions of OSIM, 0.5 mg/mL, were produced in methanol and stored at −40 °C. The first stock solution was used to prepare working solutions for the calibration curve by serial dilution in methanol at concentrations ranging from 20 ng/mL to 5000 ng/mL. Calibration curve samples consisting of eight non-zero concentrations were obtained by spiking aliquots of 90 µL blank plasma with 10 µL working solutions of calibration solutions . Quality control  samples were prepared in the same manner at concentrations of 20,50, 2000, and 4000 ng/mL. Internal standard (IS) nilotinib was prepared in acetonitrile at a final concentration of 10 ng/mL.18 C18 column  maintained at 45 °C. Mobile phase A was 0.2% formic acid in water, and mobile phase B was acetonitrile. The gradient elution procedure was optimized as follows: 20% B (0–0.5 min), 20–40% (0.5–1.0 min B), 40–90% B (1.0–2.0 min), 90% B (2.0–3.0 min), and 90–20% B (3.1–3.2 min) at a flow rate of 0.50 mL/min for 4.0 min. Carry-over was minimized with methanol-acetonitrile-isopropanol-water solution  before and after the injection procedure.A liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) system coupled to a Jasper™ HPLC and Triple Quad™ 4500MD and equipped with an electrospray ionization (ESI) interface source  was used for the analysis. The separation was achieved on a Waters XBridge Cm/z 500.0 → m/z 72.1 for OSIM , and m/z 530.0 →m/z 289.1 for IS . The MS/MS spectra of the ion fragments are illustrated in The source parameters were set as follows: ion spray voltage: 5.5 kV, source temperature: 550 °C, GS1: 50psi, GS2: 55 psi, curtain gas: 30 psi, collision gas: 9 psi, entrance potential: 10 V. Multiple reaction monitoring (MRM) was performed in the positive ion mod, and monitoring was carried out for two ion pairs: g for 10 min. Finally, each sample was reconstituted with an aliquot of mobile phase A. The sample was transferred to a 0.8 mL 96-well plate, of which 5 µL was injected into HPLC–MS/MS system for analysis.The 100 µL of plasma samples  were precipitated with 100 µL IS (10 ng/mL) and 200 µL acetonitrile. The blank plasma samples were prepared by mixing 100 µL blank plasma and 300 µL acetonitrile. Subsequently, all samples were vortex-mixed for 30 s and were centrifuged at 14,000 × Twenty-four adult male Sprague-Dawley rats (220 ± 20 g) with specific pathogen free were supplied by the Laboratory Animal Center of Zhejiang Provincial People’s Hospital. All rats were healthy and did not involve genetic modification status, genotype, and any previous procedures. The rats were housed at an animal laboratory  for one week as an adaptation period. The study was performed in accordance with the Animal Care and Use Committee of Zhejiang Provincial People’s Hospital (number: A20220030).n = 6): control , VCZ (20 mg/kg), ICZ (20 mg/kg), and FCZ (20 mg/kg) groups. Only those responsible for treatment management researchers know this group well. Each group was housed in a single cage. The rats in the four groups were orally dosed once daily with 0.3% CMC-Na, VCZ, ICZ, or FCZ, respectively; they reached steady-state exposure after five continuous days of dosing. Thirty minutes after the last administration, each rat received a single oral dose of 10 mg/kg of OSIM. All rats received an oral gavage dose of approximately equivalent volume treatment regimens (one mL). Rats were restrained on the rat fixator on the laboratory bench. Blood samples (300 µL) from the tail veins were collected in heparin tubes at 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 12, 24, and 48 h after OSIM administration. Plasma samples were then separated by centrifugation  for 10 min, and the resultant plasma was frozen at −30 °C prior to analysis.OSIM, VCZ, ICZ, and FCZ were prepared in 0.3% carboxymethyl cellulose sodium (CMC-Na). After 12 h of fasting and free water consumption, the rats were randomly separated into four groups, and the sample size was based on a similar study using as few rats as possible . Non-compartmental pharmacokinetic parameters of concentration–time data were processed by PKSolver 2.0 , and the NCA Extravascular module was employed to derive pharmacokinetic parameters for each treatment separately, including Cmax, AUC0–t, AUC0–inf, Tmax, t1/2, MRT0–inf, Vz/F, and Cl/F. SPSS 26.0 was used for statistical analysis . Significant differences among the groups were identified using one-way ANOVA and Tukey test. P < 0.05 was considered significant.Experimental data were given as mean ± standard deviation (SD). The mean concentration levels of OSIM r2) of over 0.99.The chromatographic conditions were carefully optimized to provide the best peak shape and highest response. The retention times were 1.82 and 2.08 min for OSIM and IS, respectively. Representative chromatograms are presented in Intra- and inter-day precision and accuracy were measured in sextuplicate at the LLOQ, LQC, MQC, and HQC levels, on three consecutive days. The precision  and accuracy  for all QC samples should be within ± 15% (± 20% for LLOQ). For all QC samples, the average %RE ranged from 88.45 to 110.41%, and the intra- and inter-day %CV was less than 7.73% .Extraction recovery and matrix effect were determined using three concentrations of QC samples in five replicates. The peak area ratio of QC obtained in blank plasma pre-extraction was compared to that prepared in samples spiked post-extraction for extraction recovery. The matrix factor was estimated by comparing the peak area ratio of QC generated in samples spiked post-extraction to that of pure solutions at the same nominal concentration. The mean extraction recovery of OSIM ranged from 96.25 to 100.67%, with the %CV ≤ 2.50% . The IS-The stability of the method was evaluated by determining the concentrations of LQC, MQC, and HQC; the evaluation was performed in quadruplicate. Bench-top stability was tested by placing the pretreatment samples at room temperature (−25 °C) for 6 h, autosampler stability was assessed by placing the prepared sample in the injector for 24 h, freeze-thaw stability was determined to be adequate over at least three cycles (from −30 °C to 25 °C), and long-term stability was assessed to be at least one month before sample preparation (at −30 °C for 30 d). For all stability results, the average %RE of OSIM ranged from −11.04% to 7.48% across the three QC concentrations, with the %CV less than 6.42% .Carry-over was evaluated by injecting four blank plasma samples over the upper limit of quantitation . The peak area of the blank plasma sample should not exceed 20% of that recorded at LLOQ for OSIM and should be less than 5% of IS. The calculated carry-overs for OSIM and IS were 4.44% and 0.11%, respectively .The above results demonstrated that the assay met the required standards and can be applied to the pharmacokinetic study of OSIM.The mean concentration–time profiles of OSIM alone and OSIM combined with VCZ, ICZ, and FCZ are presented in max, AUC0–t, AUC0–inf, Vz/F, and Cl/F (P < 0.05). Concomitant administration of OSIM with VCZ demonstrated an increase in Cmax and AUC0–t of OSIM by 58.04% and 62.56%, respectively compared with those of administration of OSIM alone. Conversely, the Cl/F of OSIM decreased by 37.74% in VCZ group. The Tmax value of OSIM in the VCZ group increased by 17.67%, but the t1/2, MRT0–inf, and Vz/F values were decreased by 42.56%,10.25%, and 64.03%, respectively compared with the control. These results implied that VCZ contributed to enhanced systemic exposure and decreased clearance of OSIM. When OSIM was co-administered with FCZ, the Cl/F value of OSIM in the FCZ group decreased by 0.49-fold, but the values of Cmax and AUC0–t were enhanced by approximately 0.53- and 1.01-fold, respectively. In contrast, co-administration with OSIM and FCZ did not significantly alter the t1/2 and Tmax of orally administered OSIM. Following the administration of OSIM with ICZ, the t1/2 and Vz/F of OSIM decreased by 25.41% and 38.08%, respectively (P < 0.05), but other parameters  were not significantly different compared with the control group.These results revealed significant differences in pharmacokinetic parameters between the control and the VCZ or FCZ groups, especially for the values of CImmunocompromised patients with lung cancer are more vulnerable to invasive fungal infections than healthy individuals . Triazolmax and a 1.35- to 2.01-fold increase in AUC0–t. VCZ and FCZ have been recommended as CYP3A inhibitors in clinical studies (0–360 of midazolam was significantly increased (133%)  . Anotherd (133%) . Therefomax of capmatinib alone were similar to those of the combination of capmatinib and ICZ, and that ICZ had no significant effect on the formation of the product CMN288 (max and AUC0–t, were not significantly different between the control and ICZ groups. Furthermore, Tmax was similar, and the Cl/F was also similar for the two groups, suggesting that the observed increase in exposure was related to an increase in absorption rather than a change in hepatic clearance. Therefore, we concluded that ICZ had no significant effect on the pharmacokinetic profile of OSIM. Our findings were consistent with those of an earlier study assessing the effect of ICZ on the pharmacokinetics of OSIM, in which the AUC0–t was increased by approximately 8% compared with the case wherein OSIM was administered alone (Likewise, ICZ has also been recommended as a potent CYP3A inhibitor in clinical DDI investigations . Theorett CMN288 . Thus, Ded alone .In conclusion, a detailed understanding of the potential for DDIs between OSIM and VCZ, ICZ, and FCZ was gained in the present study. Both VCZ and FCZ had a significant effect on OSIM exposure in rats; however, the inhibition of OSIM metabolism by the strong inhibitor ICZ had no significant effect on plasma exposure. Further human clinical trials of the effects of VCZ, ICZ, and FCZ on OSIM need to be investigated to confirm the significance and accuracy of the interactions.10.7717/peerj.15844/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15844/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.15844/supp-3Supplemental Information 3Click here for additional data file.


Cucurbita maxima ‘Bambino’, Cucurbita pepo ‘Kamo Kamo’, Cucurbita moschata ‘Butternut’, Cucurbita ficifolia ‘Chilacayote Squash’, and Cucurbita argyrosperma ‘Chinese Alphabet’. The content of polyphenolic compounds was determined by ultra-high performance liquid chromatography coupled with HPLC, while the total content of phenols and flavonoids and antioxidant properties were determined by spectrophotometric methods. Ten phenolic compounds  were identified. Phenolic acids were the most abundant compounds; the amount of syringic acid was found to be the highest, ranging from 0.44 (C. ficifolia) to 6.61 mg∙100 g−1 FW (C. moschata). Moreover, two flavonoids were detected: catechin and kaempferol. They were found at their highest level of content in C. moschata pulp , with the lowest amount detected in C. ficifolia . Analysis of antioxidant potential showed significant differences depending on the species and the test used. The DPPH radical scavenging activity of C. maxima was 1.03 times higher than C. ficiofilia pulp and 11.60 times higher than C. pepo. In the case of the FRAP assay, the multiplicity of FRAP radical activity in C. maxima pulp was 4.65 times higher than C. Pepo pulp and only 1.08 times higher compared to C. ficifolia pulp. The study findings show the high health-promoting value of pumpkin pulp; however, the content of phenolic acids and antioxidant properties are species dependent.Antioxidant properties and phenolic acid content in the pulp of five pumpkin species were evaluated. The following species cultivated in Poland were included:  Antioxidant properties found in plant products have beneficial effects on human health. Their role is to prevent the harmful effects of free radicals, which at high concentrations pose a serious threat to cell structures . Many stCucurbita L.) is an annual plant belonging to the Cucurbitaceae family, which includes about 130 genera and over 800 species [−1, though in some varieties it may exceed 22 mg∙100 g−1 [−1), E (from 1.03 to 1.06 mg∙100 g−1), and B6 (from 0.06 to 0.11 mg∙100 g−1), as well as thiamine (0.05 mg∙100 g−1), riboflavin (0.11 mg∙100 g−1), niacin (0.6 mg∙100 g−1), K (1.1 μg∙100 g−1), and folate (from 0.16 to 0.20 μg∙100 g−1) [E. coli, S aureus, and P. multocida) [Pumpkin  . Moreove100 g−1) . It may 100 g−1) . The bio100 g−1) ,20. A st100 g−1)  confirms100 g−1) . Pumpkin100 g−1) . Extractltocida) . Accordiltocida) , pumpkinltocida) . Numeroultocida) ,20,26. CC. pepo) to 1.73 (100%) (C. maxima) mg∙100 g−1 FW. In the study by Zhou et al. [C. maxima, C. pepo, and C. moschata) grown in China was slightly lower than that in our study, ranging from 0.13 to 1.67 mg∙100 g−1 FW. In the pumpkin species tested in our study but cultivated in Austria, the content of β-carotene ranged from 0.06 to 7.4 mg∙100 g−1 FW [C. maxima pulp was 6.18 mg∙100 g−1.The content of β-carotene in the samples is shown in u et al. , the con0 g−1 FW . Accordi0 g−1 FW , the conC. moschata) to 14.7 (C. maxima) mg∙100 g−1 FW. Likewise, the highest content of vitamin E was found in C. maxima (1.59 mg∙100 g−1 FW), while the lowest content was determined in C. ficifolia (0.38 mg∙100 g−1 FW). Similar results were reported by Zhou et al. [Cucurbita moschata, the content of ascorbic acid was 6.8 mg∙100 g−1 FW. According to Roura et al. [C. moschata Duch. fruits cultivated in Argentina was 22.87 mg∙100 g−1 FW. The L-ascorbic acid content reported by them is significantly higher compared to our findings, which can be attributed to differences in the climate–soil conditions of pumpkin cultivation.The content of L-ascorbic acid and vitamin E in the pulp of the pumpkin species tested is presented in u et al. . In the u et al.  assessina et al. , the conC. pepo) to 8.73 (C. maxima) mg QE∙100 g−1 FW (C. maxima (8.23 mg QE∙100 g−1 FW), C. pepo (0.51 mg QE∙100 g−1 FW), and C. moschata (5.36 mg QE 100 g−1 FW) were reported by Zhou et al. [C. maxima was 2.21 mg CE∙g−1 extract. Furthermore, in the study by Hussain et al. [C. maxima was 139.37 CE∙100 g−1 powder. The content of flavonoids in the pulp of the pumpkin species assessed ranged from 0.75 , while the lowest was found in Cucurbita pepo (62.91 mg GAE∙100 g−1 FW) (−1 FW), Nawirska-Olszańska et al. [−1 FW), and Oloyede et al. [−1). According to Yadav et al. [Cucurbita pepo was characterized by several times higher total polyphenol content compared to Cucurbita maxima. Moreover, in the study by Telesiński et al. [Cucurbita moschata, the highest polyphenol content was demonstrated in ‘Kurinishiki’ (99.86 mg GAE∙100 g−1 FW), while lower content was found in ‘Muskatna’ (80.41 mg GAE∙100 g−1 FW), ‘Butternut Rugosa’ (79.23 mg GAE∙100 g−1 FW), and ‘Muscade de Provence’ (73.70 mg GAE∙100 g−1 FW). The highest total polyphenol content was found in  g−1 FW) . Our fina et al.   to 6.61 mg∙100 g−1 FW (C. moschata). The lowest values were noted for chlorogenic, caffeic, p-coumaric, and ferulic acid, whose levels in C. ficifolia and C. argyrosperma were below detection limits. The next most abundant phenolic acid was protocatechuic acid, whose content varied in individual pumpkin species, ranging from 0.34 mg∙100 g−1 FW (C. ficifolia) to 1.37 mg∙100 g−1 FW (C. moschata). Comparable results were reported by Kostecka-Gugała et al. [Ten phenolic compounds were identified in the study samples of the pumpkin pulp, i.e., protocatechuic acid, p-hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, p-coumaric acid, syringic acid, ferulic acid, salicylic acid, and kaempferol . Phenolia et al. . Pumpkina et al. .C. moschata , while the lowest amount was determined in C. ficifolia . Flavonoids are potent antioxidants characterized by defined health properties: they can protect against oxidative stress-induced diseases, modulate the activity of enzymes, and interact with some receptors [Moreover, two flavonoids, catechin and kaempferol, were detected in the samples of various pumpkin species. The highest content of these compounds was found in eceptors . Moreoveeceptors . Currenteceptors ,41.p = 0.05  was observed in C. maxima. Jiao et al. [DPPH free radical scavenging activity in the pulp of the pumpkin species tested proved to be statistically significant at p = 0.05 . Statisto et al.  also demC. pepo (105.96 mg Troloxu∙100 g−1), while the lowest potential was found in ‘Festival’ (25.54 mg Trolox∙100 g−1) and ‘Snow Ball’ (24.68 mg Troloxu∙100 g−1), also belonging to C. pepo. Telesiński et al. [Cucurbita moschata: ‘Kurinishiki’, ‘Butternut Rugosa’, ‘Muscade de Provence’, and ‘Muscatna’. The highest DPPH radical scavenging ability was demonstrated in ‘Kurinishiki’ (31.39% inhibition). The lowest antioxidative potential was found in ‘Muscatna’ (17.41%). According to Oyeleke et al. [Cucurbita mixta is characterized by higher DPPH radical scavenging ability compared to the pulp of Cucurbita maxima.In a study by Kulczyński et al. , the higi et al.  analyzede et al. , the pulC. pepo and C. argyrosperma. The second cluster was formed by variables characterizing the level of the antioxidative activity of the pulp of C. ficifandlia, C. moschata, and C. maxima, and the pulp of the above species was thus characterized by higher antioxidative activity. A closer look at the clusters in the dendrogram shows that the first cluster contained species whose pulp showed higher antioxidative activity when compared to the pulp in the second cluster. Dendrogram 2a and 2b show clusters of the content of phenolic compounds in the pulp of the pumpkin species tested. The reduction of data revealed their new structure, which amounts to the separation of three clusters showing interdependencies (correlations). Statistical analysis carried out to determine correlations among the quality characteristics of pumpkin fruit pulp a,b allowC. pepo and C. moschata, followed by C maxima. The lowest amount of phenolic compounds was determined in C. ficifolia and C. argyrosperma.However, in this case, the varieties with the highest amount of phenolic compounds in the pulp were found to be Cucurbita maxima “Bambino”, Cucurbita pepo “Kamo Kamo”, Cucurbita moschata “Butternut”, Cucurbita ficifolia “Chilacayote Squash”, and Cucurbita argyrosperma “Chinese Alphabet”.The study material was the pulp of five pumpkin species: Plants were grown in Żyznów (49°49′ N 21°50′ E) (Poland) in 2018 under identical agro-climatic conditions.The pumpkin fruits were harvested on October 20 by cutting off the fruits from the shoot. Immediately after harvesting, the pumpkin fruits were washed and peeled, and seeds were then removed. The resulting pulp was crushed into small pieces, which were frozen at −25 °C and then freeze-dried (0.37 mBa) for 48 h in a FreeZone 12 L freeze dryer . The obtained samples of lyophilizates were stored at −25 °C in dark jars protected from light .The extracts were prepared by crushing 0.75 g of freeze-dried material in a mortar with 80% methanol  poured in portions to a total volume of 25 mL. The extraction time was 4 h. Each sample was filtered through a funnel with a sintered disc into a 25 mL volumetric flask. The extraction process was carried out at a temperature of 22 °C with limited lighting. Three extracts of pulp were prepared for each pumpkin species. The extracts were stored in the dark at −25 °C for one week . v/v/v), with isocratic flow at a rate of 1.0 mL∙min−1. The concentration of β-carotene in the samples was calculated based on the external standard of β-carotene and is presented as a milligram of β-carotene per 100 g−1 FW.The content of β-carotene was determined using an HPLC technique based on the method of Liu et al.  with min−1 oxalic acid solution was added to 50 g of the sample and homogenization was carried out . Subsequently, the sample was centrifuged at 4000 rpm at 4 °C for 15 min. The obtained supernatant (10 mL) was titrated with a solution of 2,6-dichlorophenolindophenol (concentration—0.1%). The concentration of L-ascorbic acid was calculated from the volume of 2,6-dichlorophenolindophenol used for titration and expressed in milligrams per 100 g fresh weight.The content of L-ascorbic acid was determined by titration with 2,6-dichlorophenolindophenol according to the method described by Zhou et al.  with min2CO3, 125 mL of the Folin–Ciocalteu reagent , and 2.25 mL of water and then incubated for 20 min. Absorbance was measured spectrophotometrically at a wavelength of 765 nm . The total phenolic content of pumpkin pulp extracts was expressed as mg of gallic acid equivalents (GAE) per 100 g fresh weight.The content of phenolic compounds in the extracts was determined based on the reaction with the Folin–Ciocalteu reagent . The ext−1), total flavonoid content was determined in all samples in triplicate. The results were expressed in milligrams of quercetin equivalents (QE) per 100 g fresh weight.Total flavonoid content was determined using the method described by Di Marco et al.  with min−1. The identification of phenols was based on the retention times of chlorogenic, caffeic, p-hydroxybenzoic, p-coumaric, ferulic, protocatechuic, syringic (Sigma-Aldrich), and salicylic (LGC Standards) acid, as well as retention of (+)-catechin (LGC Standards) and kaempferol (Sigma-Aldrich) [To identify phenolic compounds in pumpkin pulp extracts, the high-performance liquid chromatography (HPLC) method (Shimadzu LC-10AS chromatograph equipped with a C18 RP column and an SPD-10AV UV-VIS detector) was used. Signal detection was set at wavelengths of 325 and 265 nm. Chromatographic separation was carried out at 33°C using the following solvents: (A) water (Sigma-Aldrich) with acetic acid (0.1%), (B) methanol  with acetic acid (0.1%). The following gradients were used: 90% A, 10% B for 20 min; 75% A, 25% B for 30 min; 65% A, 35% B for 40 min; 55% A, 54% B for 50 min; 50% A, 50% B for 60 min; 30% A, 70% B for 62 min; 100% B up to 80 min; and 80% A, 10% B up to 85 min. The flow rate was 1 mL minAldrich) .●) free radical. The colorimetric method allows for the measurement of changes in the absorbance of a DPPH solution at a wavelength of 517 nm as the result of the antioxidant activity of the sample [L−1 Trolox FW).The antioxidant capacity of extracts was assessed based on the reduction of the synthetic and stable 2,2-diphenyl-1-picrylhydrazyl (DPPHe sample . A volum−1 acetate buffer (pH 3.6), 2.5 mL of 10 mmol∙L−1 of TPTZ (soluble in 40 mmol∙L−1 HCl), and 2.5 mL of 20 mmol∙ L−1 ferric chloride was prepared [−1 at 4 °C for 20 min. The collected supernatants were mixed. For further determination, 0.4 mL of ten-fold-diluted supernatant mixed with 8.0 mL of FRAP solution (0.14 mmol∙L−1) was used, which was mixed and left at 37 °C for 15 min. Absorbance was measured at 593 nm using a spectrophotometer ; the FRAP solution was a blank. The results were expressed as μmol Trolox  per 100 g fresh weight (TEAC μmol∙L−1 Trolox FW).A fresh FRAP solution consisting of 25 mL of 0.3 mol∙Lprepared . The sam3 and 200 mg of Na 2SO4, followed by 10 mL of methanol/tetrahydrofuran  and an internal standard, α-tocopherol acetate , were added to the samples. The extraction was carried out in an ultrasonic bath  in water for 15 min and was repeated two times, with 10 mL of the solvent used each time. Subsequently, the samples extracts were centrifuged at 4000 rpm min−1 for 10 min. The combined extracts were concentrated in a rotary evaporator at 30 ± 1 °C for about 30 min to a volume of about 1 mL and dissolved in 10 mL of n-hexane/methyl-tert-butyl ether . Vitamin E (tocopherols and tocotrienols) was determined according to the method described by Franke et al. [v/w). The volume of injections was 20 μL at a flow rate of 1.5 mL∙min−1 and a total time of 45 min. The measurement was performed at an excitation wavelength of 292 nm and an emission wavelength of 330 nm using a fluorescence detector. To identify the substances, their retention times were compared with those of external standards. The content of individual vitamin E derivatives is presented in mg∙100 g −1 fresh weight, and the content of vitamin E is presented as the sum of all derivatives.To achieve better extraction of vitamin E, 2 mL of distilled water was added to 2 g of the crumbled sample. After 5 min of soaking, 200 mg of MgCOe et al.  using anp < 0.05 was considered significant. In order to establish deeper links between the analyzed variables, a data reduction technique based on cluster analysis and PCA  was used.The results are presented as the mean of three independent measurements. All analyses were performed in Statistica version 13.3 . The significance of means was examined post hoc by the Duncan’s test, which is a part of one-way analysis of variance (ANOVA). C. ficifolia and C. argyrosperma. Moreover, two flavonoids, catechin and kaempferol, were detected in the pumpkin pulp; however, their levels were different in the individual pumpkin species. Analysis of the antioxidant potential of pumpkin pulp showed significant variation depending on the species and the test used. The highest antioxidative potential in all tests was found in C. Maxima, while the lowest values were demonstrated in the pulp of C. pepo. In our study, the content of the compounds analyzed varied in individual pumpkin species. The results of our study are a valuable source of information on the varying levels of antioxidant properties and phenolic acids of the flesh of the studied pumpkin species grown under the soil and climatic conditions of Poland. In addition, the results of our research are of an applied nature, as they provide the pharmaceutical and cosmetic industries with knowledge of the health-promoting properties of pumpkin flesh as a valuable raw material for the production of extracts that can be used in the development of new medicinal products, as well as a valuable source of information for food processing for the development of new food products, including functional foods in both adult and child nutrition. The research results we have presented will increase consumer awareness of the health-promoting properties of various pumpkin species and can also be a source of knowledge regarding the selection for cultivation of pumpkin species with the highest antioxidant properties and the highest phenolic acid content. The published results of our study may also contribute to further research on these pumpkin species for health benefits.Our study findings demonstrate that pumpkin pulp is a source of phenolic acids and has antioxidant properties. However, the total content of the individual compounds varied and was species dependent. Ten phenolic compounds were identified in the pulp of each pumpkin species: protocatechuic acid, p-hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, p-coumaric acid, syringic acid, ferulic acid, salicylic acid, and kaempferol. Phenolic acids were most abundant, including syringic acid. The lowest values were obtained for chlorogenic, caffeic, p-coumaric, and ferulic acid, whose levels were below the limit of detection in


This study aimed to elucidate the magnetic resonance (MR) characteristics of anal fistulas extending to the scrotum, and the applicable rules, and to correlate MR features with surgical findings.We conducted a retrospective study in 150 consecutive patients with anal fistulas extending into the scrotum, who were diagnosed and underwent surgery at University Medical Center Ho Chi Minh City between January 2017 and April 2022. MR findings were evaluated and compared with surgical findings using Cohens kappa coefficient (k) with a 95% confidence interval.k = 0.83 (0.780.87) and k = 0.89 (0.85 0.93) (p<0.001), respectively. There is a significant correlation between the location of internal openings and the type of fistula (p<0.05). Low transsphincteric fistulas were predominant in the anterior group (103/122 patients vs. 10/19 patients), while in the posterior group, it was more common in the high transsphincteric fistulas (7/19 patients vs. 14/122 patients), and the intersphincteric fistulas (1/19 patients vs. 5/122 patients); and the suprasphincteric fistulas were only seen in the posterior group (1 patient).150 patients (mean age 37.6 ± 10.9 years) with 166 fistulas, including 150 anal fistulas with scrotal extension. Most fistulas were low transsphincteric . There was a strong agreement for primary tract classification and detecting the location of internal openings between MRI and surgical findings with Anal fistulas with scrotal extension are exceptions to Goodsalls rule. Albeit long-tract fistulas, most are low transsphincteric and have anterior internal openings. Anal fistulas affect approximately 0.01% of the population. They are more common in men in their fourth decade, which is two- to four-fold compared to women .In some cases of males with anal fistulas, the fistulas can extend into the scrotum. The presentations are variable in those cases. The scrotum usually presents with redness, swelling, pain, and pus discharge from the external opening, while anal symptoms may be absent or minimal –6. Such Goodsall's rule for predicting the trajectory of anal fistulas is popular; however, the long-tract anterior fistula, which may include fistulas with scrotal extension, is an exception to this rule –12. On tAccording to our best knowledge, this is the first study about the role of MR in the preoperative evaluation of anal fistulas with scrotal extension. We conducted this study to elucidate the MR characteristics of anal fistulas extending to the scrotum, the rules applicable to them, and to correlate MR features with surgical findings.2.2.1.A total of 150 consecutive patients with anal fistulas extending into the scrotum who were diagnosed and underwent surgery at the University Medical Center, Ho Chi Minh City between January 2017 and April 2022 were included in this study. All included patients underwent preoperative MRI examination with intravenous contrast. The exclusion criteria were (a): patients having recurrent fistula, or (b) their operative records were not detailed .2.2.MR imaging was performed on either a 1.5 Tesla  or 3 Tesla  system using six-channel body phased-array coil for pelvic imaging and no special patient preparation was required.The MR protocol consisted of the following pulse sequences: sagittal T2-weighted (T2W) turbo spin-echo (TSE), oblique coronal T2W TSE with fat saturation (FS), and oblique axial T2W TSE with the imaging planes oriented orthogonal and parallel to the axis of the anal canal, respectively.The parameters for these T2W sequences were repetition time (TR) 3,000–5,000 ms, echo time (TE) 75–85 ms, field of view (FOV) 20–25 cm, turbo factor (TF) 17, slice thickness 3–4 mm, intersection gap 0.6–0.7 mm, matrix 320 × 256  or 320 × 320 , number of signals acquired (NSA) 2. The T1-weighted (T1W) TSE FS with gadolinium enhancement (post-contrast) (0.2 ml/kg) was obtained in three planes . The parameters for post-contrast T1W TSE FS are TR 600–700, TE 10–12 ms, FOV 20–25 cm, TF 3, slice thickness 3–4 mm, intersection gap 0.6–0.7 mm, matrix 320 × 256  or 320 × 320 , NSA 2.2.3.We retrospectively reviewed the patient's medical records, including their operative records and MR findings. The MR images were analyzed by two radiologists who had more than five years of experience in MR fistulography. We classified anal fistulas in the study according to Park's classification, which includes intersphincteric, transsphincteric, suprasphincteric, and extrasphincteric fistulas. We further classified transsphincteric fistulas as high or low as follows: high transsphincteric fistulas pass through the upper half of the external anal sphincter, whereas low transsphincteric fistulas traverse the lower half , 17.We defined the scrotal extension of the anal fistula  using the physical examination results reported in the operation records. On MR, internal openings were detected when the tract terminated in the intersphincteric space , 18. The2.4.p-value < 0.05 was considered statistically significant.Statistical analysis was conducted using STATA version 16 . Obtained data were presented as mean and standard deviation  or median and interquartile range (with skewed distribution), ranges, numbers, and ratios. Numbers and ratios were analyzed using Fisher's exact test. A k) with a 95% confidence interval. The degree of agreement was interpreted as follows: ≤0 as no agreement, 0.01–0.20 as none to slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1.00 as almost perfect agreement.MR findings were compared with surgical findings using Cohen's kappa coefficient . The age group of 30–50 was the most common (62.0%), followed by the age group of under 30 (26.0%) and over 50 (12.0%). Most fistulas had histological findings of non-specific inflammation , and only 2 patients were due to tuberculosis. The mean duration from MR acquisition to surgery was 1.5 ± 1.8 days (range 0–15 days).3.2.There was a total of 150 patients with 166 fistulas, including 136 patients with one fistula , 12 patients with two fistulas , and 2 patients with three fistulas .3.2.1.The external opening was detected in 142/150 (94.7%) patients with 159 external openings. 129 patients (86.0%) had one, 10 patients (6.7%) had two, 2 patients (1.3%) had three, and 1 patient (0.7%) had four external openings. In the remaining 8 patients without external openings detected, the presence of scrotal involvement was recorded based on surgical reports. Including these 8 cases, the total number of locations where scrotal involvement took place was 167. Among the aforementioned 167 locations, we noticed a left-side predominance , followed by midline  and right-side .3.2.2.p < 0.001), which is demonstrated in Each of the 150 patients had only one fistula-in-ano extending to the scrotum. According to Parks classification, most of the fistulas were classified as low transsphincteric , followed by high transsphincteric , intersphincteric , and suprasphincteric fistula . There was a strong agreement for primary tract classification between MR and surgical findings with a kappa value of 0.83 (95% CI: 0.78–0.87) (3.2.3.p < 0.001), which is shown in 141 internal openings were detected in 150 patients. The most common location of the internal openings was from 11 o'clock to 1 o'clock (74.7%) at the level of the dentate line, followed by 6 o'clock (10.7%). The remaining positions were less common . There wWe observed a significant correlation between the location of the internal opening  and the fistula type . Low trap > 0.05).The mean distance from the external opening to the anal verge was 6.2 ± 2.5 cm (range 3–12 cm). The mean distances from the external opening to the anal verge of the anterior and posterior internal opening group were 6.2 ± 2.5 cm (range 3–12 cm) and 6.4 ± 2.3 cm (range 3–10 cm), respectively. No statistical significance was found between the two groups  . In our In the present study, there was a strong agreement for primary tract classification between MR imaging and surgical findings, qualifying MR as a valuable modality in the preoperative classification of anal fistulas. On MR, high transsphincteric fistulas passed through the upper half of the external sphincter, whereas low transsphincteric fistulas traversed the lower half , 5. The The location of scrotal involvement in our study was most common in the left scrotum 38.9%), followed by the midline (33.5%) and the right scrotum (27.6%). This is consistent with the study of Araki et al. %, follow demonstrProper identification of the internal opening is an integral part of fistula surgery to avoid recurrence . On MR, p < 0.05). This is consistent with the previous reports  and the fistula type ( reports , 23 whic reports , 24 (Fig reports . This st reports . Anal fi reports , 25. AccThough the external openings are more than 3 cm from the anal verge, most anal fistulas with scrotal extension are low transsphincteric and have anterior internal openings, making them exceptions to Goodsall's rule. On the other hand, they are consistent with the Midline rule, so we suggest surgeons utilize the Midline rule when dealing with fistulas extending to the scrotum. We recommend performing magnetic resonance in fistulas with scrotal extension thanks to its excellent performance in classifying the primary tract and detecting the internal opening, which are major contributors to surgical prognosis.


Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), and Cymbidium ringspot virus (CymRSV) represent three important and prevalent orchid viruses. The detection system proposed in this study uses a triplex TaqMan quantitative real-time PCR assay to identify CymMV, ORSV, and CymRSV in a simultaneous manner. We designed specific primers and probes for CymMV, ORSV, and CymRSV, with amplified sequences of 156 bp, 148 bp, and 145 bp, respectively. The minimum detection limit of the triplex qRT-PCR assay for CymMV and CymRSV was 1 copy/assay, and the minimum detection limit was 10 copies/assay for ORSV. The minimum stable detection limits for CymMV, ORSV, and CymRSV were 10, 102, and 102 copies/assay, respectively. Therefore, this system exhibited higher sensitivity (approximately 10 to 104-fold) than RT–PCR. The intra-and interassay CVs of Cq values are less than 0.55 and 0.95%, respectively, indicating that the triplex assay is highly reliable and accurate. In addition, 66 samples from five different orchid genera were analyzed using the established assay and gene chip. The detection results demonstrated that the triplex probe qRT–PCR demonstrated higher sensitivity than the gene chip, indicating that the triplex real-time PCR assay could be used for the detection of field samples. Our findings suggest that the triplex real-time RT–PCR detection system represents a rapid, simple, and accurate tool for detecting CymMV, ORSV, and CymRSV on orchids.Orchids are significant ornamental plants whose viral infection results in substantial economic damage.  The orchids cultivars included “Large chili pepper”, “Asahi”, “Greenbear”, and others. From the Kaiyuan orchids factory, we also obtained Dendrobium nobile, Zygopetalum, Haraella retrocalla, and Oncidium hybridum samples.Samples infected with CymMV and ORSV single virus or both viruses and healthy 2.2.Multiple coat protein sequences of CymMV were downloaded from the NCBI. Based on DNAMAN comparison and according to RT–qPCR primer design requirements, three pairs of specific primers and probes were designed using Primer 5.0 software in highly conserved regions of the virus sequences. Primer-blast comparisons were performed in NCBI to ensure primer specificity. The specific primers were screened through RT-PCR, followed by agarose gel electrophoresis, primers that can produce bright and single amplicons were selected for further screening utilizing the TB Green dye method. For triplex taqman qRT-PCR fluorescence detection, primers given rise to a single dissolution curve and high amplification efficiency in the TB Green dye method were utilized. The method for selecting primers for ORSV and CymRSV was identical to that for CymMV. The three probes were labeled with FAM/BHQ1 (CymMV), HEX/BHQ1 (ORSV), and Cy5/BHQ3 (CymRSV) at their 5′ and 3′ terminals. The primers and probes were synthesized by Sangon Biotech , and the details of these oligos are shown in 2.3.2O 1.0 μL; 37°C 15 min, 85°C 5 s, 4°C insulation.The total RNA was extracted from three samples only infected with a single virus of CymMV, ORSV, or CymRSV using a Mini BEST Plant RNA Extraction Kit , quality and quantity were assessed using a Nanodrop2000 spectrophotometer . Subsequently, cDNA was synthesized employing the PrimeScript RT Reagent Kit  with gDNA Eraser to perform the reverse transcription reaction. The reverse transcription reaction system and the reaction procedure are described as follows: 5 × g DNA Eraser Buffer 2.0 μL, gDNA Eraser 1.0 μL, total RNA 7.0 μL; 42°C 2 min, 4°C insulation; PrimeScript RT Enzyme Mix I 1.0 μL, RT Primer Mix 4.0 μL, 5 × PrimeScript Buffer 2 4.0 μL, RNase Free ddH9 copies/assay and then mixed (109 copies/assay). The mixed plasmids were subsequently diluted to 109 copies/assay to 1 copy/assay at a 10-fold ratio.Using the obtained three cDNA as templates, the annealing temperature of CymMV, ORSV, and CymRSV were optimized based on the base sequence of primers, whereas CymMV and ORSV were optimized over different Tm ranged from 55°C to 60°C at 1°C interval, CymRSV were optimized over different Tm ranged from 52°C to 57°C at 1°C interval. The PCR products were sent to Sangon Biotech  for sequencing, and the results showed 100% identity to the target sequence. The PCR products were then purified utilizing a DNA Gel Extraction Kit . Retrieved purified DNA fragments were cloned into the pMD19-T vector  to construct three plasmids as positive controls and templates named pMD19T-CymMV, pMD19T-ORSV, and pMD19T-CymRSV. Positive plasmid standards for the three viruses were sequenced to ensure sequence accuracy. The positive plasmids were used to establish standard curves. According to the absorbance measurement, the concentration of plasmids was calculated, and the calculation used to determine the copy number of the plasmid in a previous study was used . The sta2.4.To obtain a more sensitive, stable, and efficient qRT–PCR method, the annealing temperature, primer concentration, and probe concentration were carefully optimized for each target gene. The experimental conditions with highest fluorescence signal and the lowest cycle threshold (Cq) value were determined to be the optimal reaction conditions. Using optimized three cDNA (10 < Cq < 35) as templates, CymMV-F, CymMV-R, and CymMV-Probe; ORSV-F, ORSV-R, and ORSV-Probe. CymRSV-F, CymRSV-R, and CymRSV-Probe were used for the real-time quantitative PCR amplification system and program screening. The annealing temperature , primer concentration  and probe concentration  were optimized using the matrix method. The optimal annealing temperature and the concentration of primers and probes were selected for each virus separately. Then, the optimization of triple detection system was further optimized to identify the most optimal conditions for triple detection by further optimizing the proportion of primers and probes added for the three viruses and the annealing temperature based on the optimal detection system for a single virus.9 to 102 copies/assay was used as the template, and ddH2O served as the negative control. These samples were amplified using triplex qRT-PCR based on the optimized reaction systems. When established the dynamics and standard curves, the amplification efficiency (E) and correlation coefficient (R2) were used as parameters to evaluate the triplex assay.The mixed plasmid containing 102.5.Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Carnation mottle virus (CarMV), and Lily mottle virus (LMoV). CMV, CarMV, and CymRSV are spheroids viruses of an equiaxed icosahedron, and CarMV and CymRSV belong to Tombusviridae. Both TMV and ORSV are rhabditiform viruses, and both belong to Tobamovirus. LMoV belongs to Potyvirus, CymMV belongs to Potexvirus, whereas both LMoV and CymMV are linear viruses. The above five viruses extracted RNA from the leaves of infected plants, the reverse transcription for cDNA was verified by PCR, then cDNA was used for fluorescence quantitative specificity detection. In order to validate the sensitivity of the method, a mixed 109 copies/assay to 1 copy/assay plasmid was used as the template and subjected to the sensitivity of the triplex assay. The repeatability of the method was tested using plasmids as templates at 104, 105, 106, 107, and 108 copies/assay, respectively. For the intra-assay repeatability test, each independent experiment was conducted in triplicate, and for the inter-assay repeatability test, triplicate runs were conducted.The specificity of the established qRT–PCR assay was confirmed using 2.6.2O. The cycle program was as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, Tm for 30 s (Tm (CymMV) = 58°C, Tm (ORSV) = 58°C, Tm (CymRSV) = 55°C), and 72°C for 1 min, and a final step of 10 min at 72°C. The PCR products were detected by 1.0% agarose gel electrophoresis. Three standard plasmids were used as positive controls, and ddH2O served as the negative control.The reaction system and reaction conditions of the RT-PCR procedure for sequence amplification and sensitivity verification are as follows (according to the PCR amplification kit instructions). RT–PCR was performed with a standard protocol in a 25.0 μL reaction volume on a PCR cycle . Reaction mixture components included 2.5 μL 10 × Ex Taq Buffer, 2.5 μL dNTP Mixture, 0.25 μL Ex Taq, 0.5 μL 20 μM Primer F, 0.5 μL 20 μM Primer R, 2.5 μL cDNA, and 16.25 μL RNase-free ddH2.7.The CFX96 Real-Time PCR system  was utilized for data generation and data collection. Using Microsoft Excel 2017, data management, analysis, and graphic generation were performed. The intra-and interassay variations were calculated from the Cq values, and the results were expressed as the mean value, standard deviation (SD) and coefficient of variation (CV).2.8.Phalaenopsis, 17 Zygopetalum, 17 Oncidium hybridum, and 16 Dendrobium were collected from the Orchids Plant in Kaiyuan, Yunnan Province, China. The triplex qRT-PCR was used to screen the samples for CymMV, ORSV, and CymRSV. Additionally, ddH2O was used as the negative control, and the three virus mixed plasmids (107 copies/assay) as the positive control.Sixty-six samples, including 16 3.3.1.2) value were determined to be the optimal reaction conditions. We obtained the optimal reaction conditions for CymMV with a primer concentration of 20 μΜ, a probe concentration of 10 μΜ and an annealing temperature of 58°C. The optimal reaction conditions for ORSV included a primer concentration of 10 μΜ, a probe concentration of 10 μΜ and an annealing temperature of 60°C. The optimum system for CymRSV included a primer concentration of 20 μΜ, a probe concentration of 10 μΜ for the probe, and an annealing temperature of 60°C. To ensure adequate efficiency of the TaqMan primer-probe sets in the presence of other oligonucleotides and fluorogenic dyes, we optimized the amount of primers and probes as well as the annealing temperature of three viruses  and correlation coefficient , ORSV , and CymRSV .The mixed plasmids were diluted 10-fold to 10c curves  were obt3.3.After adding the various virus isolates to the reactions individually, we obtained a signal only from the specific reporting dye when testing cDNA. This finding demonstrated that each primer-probe set amplifies cDNA from its target species but not from the other two species. Four nontarget viruses  and cDNA extracted from healthy individuals were not amplified . This fi3.4.9 copies/assay to 1 copy/assay) revealed that the lowest detection limit for CymMV, ORSV, and CymRSV was 1 copy of each. The minimum stable detection limits for CymMV, ORSV, and CymRSV were 10, 102, and 102 copies/assay, respectively , 31.82% (21/66), and 0% (0/66), respectively. The 66 samples were also tested by gene chip, and the test results showed that the positive rate of CymMV, ORSV, and CymRSV was 19.70% (13/66), 12.12% (8/66) and 0% (0/66), respectively. According to the results of the two methods, 13/8 samples were positive for CymMV/ORSV by gene chip, among the 53/58 samples detected negative by the gene chip, 7/13 samples detected positive by the triplex assay we established, and 46/45 samples remained negative. No samples were tested for CymRSV infection using either method. The results demonstrated that the triple real-time quantitative PCR system had a higher detection rate than gene chip detection.The triplex qRT–PCR assay successfully detected CymMV, ORSV, and CymRSV infections. A total of 66 samples of 16 4.Recent years have witnessed an increase in the use of quantitative real-time PCR technology based on TaqMan probes for the uniplex and multiplex detection of viruses and pathogens in plants and animals.4.1.grapevine fanleaf virus (GFLV) is 100-fold that of RT-PCR at a detection limit of 10−5 ng/μL for cDNA (sweet potato latent virus (SPLV) demonstrated that it can detect the positive plasmid at a concentration of 7 × 101 copies/μL, and the sensitivity is 100-fold that of RT–PCR (cucumis melo endornavirus (CmEV) showed that it can detect the positive plasmid at a concentration of 1.8 × 103 copies/μL, and the sensitivity is 100-fold that of RT–PCR , and they optimized the annealing temperature and primer concentration of RT–PCR to describe the most appropriate reaction conditions. 4.3.R2 of 0.994–0.999 and an E value of 87.8–95.6%. 8 copies/μL to 2 × 102 copies/μL). 7 copies/assay to 5 × 101 copies/assay) with an R2 of 0.996–0.999 and an E value of 87.8–95.6%. 9 copies/μL to 103 copies/μL) with an R2 of 0.998 and an E value of 100%. According to our research, a standard curve was derived from eight dilution gradients (109 copies/assay to 102 copies/assay) with an R2 of 0.999 and an E value of 99.4–100.1%.4.4.The quadruplex-specific detection system described by 4.5.4 copies or 5 fg. Moreover, bovine parvovirus (BPV) and bovine coronavirus (BCoV) was 200 copies/μL, and the sensitivity detection threshold of bovine parainfluenza virus (BPIV) was 20 copies/μL. 2 copies or 6.2 × 10−3 fg to 6.2 × 10−1 fg, 10–104-fold that was noted for RT–PCR.4.6.4.7.potato virus Y in 4 lilies with specific gene chips. D. longicolla, D. caulivora, D. eres, and D. novem and healthy soybean stems and soybean seeds . Cymbidium, Brassia, Dendrobium, Phalaenopsis, Masdevallia, and Oncidium) as verification of the system. Phalaenopsis samples in triplicate and calculated the average Cq value and the viral content copy number of CymMV. Our research detected 66 orchids from five different orchid genera  by triplex taqman qRT–PCR and gene chip for plant verification. The detection results demonstrated that the triplex taqman qRT–PCR detection rate is higher than gene chip detection, which means that the sensitivity of the established triplex taqman qRT-PCR was higher than the gene chip.4.8.P. amabilis and other orchids , indicating that the experimental conditions are optimal  and exhibited a linear trend  and by Yunnan Seed Industry Joint Laboratory Project (202205AR070001-05).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


No significant differences were found between the periods for the TL and PL. Significant differences were found between specific positions: Full Back (FB: n = 4), Central Defender (CD: n = 3), Midfielder (MD: n = 4), Winger (WG: n = 4), and Forward (FW: n = 3), both the TL and PL. We conclude that the PL profile based on his playing position is independent of the development of the PL shown during friendly matches. The monitoring, quantifying, and controlling of the TL added to the PL provides a more holistic vision of soccer players in friendly matches. The relative ease IMU application technology offers an alternative with less time-cost and more significant benefits than other types of technologies applied up to now.This study aimed to record, analyze and quantify professional soccer players’ technical (TL) and physical load (PL) in friendly matches to compare their records during the first and second halves and between players with different positions. Eighteen professional soccer players, 24.6 ± 2.7 years, 1.78 ± 0.3 height (m), 74.6 ± 4.5 body mass (kg), 9.8 ± 2.2 body fat (%), and 65.6 ± 2.7 maximal oxygen consumption (VO Soccer performance is multifactorial and requires training programs that combine technical, tactical, and psychological aspects . That´s 2), 9.8% ± 2.2% body fat, and 65.6 ± 2.7 maximal oxygen consumption  belonging to the same team, took part in this study. The inclusion criteria were only data from outfield players who participated a minimum of 45 min in one of the two periods. In addition, participants were required to be in good health and free from any injuries that could affect their performance during the matches. At the same time, the exclusion criteria were one friendly match due to their extension of more than 90 min. Also, goalkeepers were excluded from the study to focus specifically on outfield players and their activity profiles. In the study, 103 records were made, 59 for the first period and 44 for the second one. The players were classified according to their specific position: Full Back (FB: n = 4), Central Defender (CD: n = 3), Midfielder (MD: n = 4), Winger (WG: n = 4), and Forward (FW: n = 3). All of them were previously informed about the object of study and provided their signed informed consent, following the indications of the Declaration of Helsinki (2013). Before starting the study, it was approved by the ethics committee of Pablo de Olavide University with code 0398-N17.A total of 18 professional soccer players (n = 18) aged 24.6 ± 2.7 years, 1.78 ± 0.32 height (m), 74.6 ± 4.5 body mass (kg), 23.54 ± 2.7 body mass index  to 0.05, the desired power (1 - β error probability) to 0.80, and based on the effect size on previous studies  involving a professional soccer team competing in the third tier of Spanish soccer. The preseason period is characterized by a high PL for the players . Further2max) measurements were performed by specialist at the Andalusian Sport Medicine Center  during 24th to 26th August 2020. Laboratory testing was conducted between 9 a.m. and 12 p.m. with ambient temperature between 22°C and 24°C. To measure height and body mass, the participants stood without shoes and with only shorts. For both measurements, a portable stadiometer (accuracy of ±5 mm) and balance weighting scales (accuracy of ±0.1 kg)  were used.The anthropometric, body composition and maximal oxygen consumption (VO2) was calculated using body mass/height2. Measuring the skinfold thickness at seven sites  using a calliper . One experienced anthropometrist carried out all the anthropometric tests following the anthropometric measurement protocols established by the International Society for the Advancement of Kinanthropometry (ISAK). The percentage of body fat was calculated following Body mass index . The initial speed was 9 km/h-1 for 3 min, then increased by 1 km/h every minute until exhaustion occurred within 10–15 min for all subjects. Maximal oxygen uptake was measured during both tests via a breath-by-breath gas analyzing system . The VO2max with the highest VO2 was calculated when a plateau in O2 consumption was reported despite an increased workload. All these measures are considered as dependent variables in this study.A maximal exercise test on a treadmill  with a continuous and incremental protocol was made to calculate VOThe players’ demands during friendly matches were monitored using an IMU technology-based data collection instrument . Smart mThe TL variables analyzed have been: Total Touches (TT: number (#) of times the ball hits the player’s feet); Releases (REL: number (#) of times the player throws the ball with his foot); Total Possessions (TP: number (#) of times the player maintains possession of the ball); One touch (1T: number (#) of times the player contacts the ball without reception); Short Possessions (SP: number (#) of times the player maintains possession of the ball for no more than 2.5 s); Long Possessions (LP: number (#) of times the player retains control of the ball for more than 2.5 s); Receptions (RC: number (#) of times the player receives the ball); Release Velocity (RV: speed quantified in meters per second (m/s), with which the player performs the mechanical gesture at the moment of hitting the ball); Release Index  .2).The PL variables analyzed have been: Top Speed (TS: highest speed peak (m/s) reached by a player); Distance Covered  traveled in meters); Work Rate (WR: indicator (m/min) of load measured in amount of distance traveled in meters (m) between the time measured in minutes (min); High Intensity Distance Covered  covered in a speed range greater than 4.1 m/s; Sprint Distance Covered  traveled in a range greater than speed of 5.83 m/s); Number of Sprints (SP: number (#) of times the player reaches a speed greater than 5.83 m/s); Distance Traveled Zone 1  traveled in meters in a speed range of 0.0–2.5 m/s); Distance Traveled Zone 2  traveled in meters in a speed range of 2.5–4.17 m/s); Distance Traveled Zone 3  traveled in meters in a speed range of 4.17–5.0 m/s); Zone 4 Distance Traveled Zone 4  traveled in meters in a speed range of 5.0–5.83 m/s), Distance Traveled Zone 5  traveled in meters in a speed range of 5.83–6.66 m/s); Distance Traveled Zone 6  traveled in meters in a speed range greater than 6.66 m/s); Acceleration/Deceleration Actions (ADA: number (#) of times the player performs an intense change of direction and speed variation, in an accelerated or decelerated manner, in a speed range greater than 2.6 m/sThe data corresponding to TL variables: TT, LAN, REL, 1T, SP, LP, RC and RV and, to PL variables: HIDC, SDC, SP and ADA, are presented both in absolute values and relative to the time of game. For this, the data obtained in the first half’s friendly matches (average and standard deviation) are differentiated from those of the second period.post hoc test was conducted to further investigate significant differences. A significance level of 95% was employed to determine statistical significance.All variables were presented as mean values and standard deviations. The normal distribution of the data sets was assessed using the Shapiro-Wilk normality test. To analyze the differences between the first and second halves of technical load (TL) and physical load (PL), paired sample t-tests were performed. Additionally, a one-way analysis of variance (ANOVA) was utilized to compare the mentioned variables among playing positions during the preseason and a Bonferroni The effect size (ES) for the difference between variables was evaluated using Cohen’s d . A valuep = 0.02, ES: 0.57), HIDC (m) , HIDC (m/min) , SDC (m) , SDC (m/min) , SP (#) , SP (#/min) , DTZ3 , DTZ4  and DTZ5 , generate significantly higher values in the first part than in the second.The results obtained for the variables TL and PL are shown in p = 0.84) but obtaining significant differences with FB (p = 0.01) and with FW (p ≤ 0.001).p ≤ 0.001) and FW (p = 0.01). In contrast, they do not differ significantly with CD (p = 0.87) or FB (p = 0.80), respectively. In the same way, CD generates the best record for REL without reaching significant differences with FB (p = 0.09) or with MD (p = 0.34) but obtaining significant differences with FW (p ≤ 0.001). CD also generates the best record for TP, with substantial differences with FB (p = 0.04), WG (p = 0.02) and FW (p ≤ 0.001), while no significant differences were found with MD (p = 0.12). In the same way, CD generates the best records for SP, LP, RC, RV and RI.In the same way, MD also generates the best record for 1T, reaching significant differences with WG (p ≤ 0.001), MD (p ≤ 0.001) and FW (p = 0.03). In contrast, they do not reach significant differences with WG (p = 0.92). In the same way, the MD are those that reach the highest records in DC, with significant differences with CD (p ≤ 0.001), FB (p ≤ 0.001) and WG (p = 0.03); WR being the significant differences with CD (p ≤ 0.001), FB (p ≤ 0.001), WG (p = 0.03) and FW (p ≤ 0.001); DTZ2 being the significant differences with CD (p ≤ 0.001), FB (p ≤ 0.001), WG (p ≤ 0.001) and FW (p ≤ 0.001); DTZ3 being the significant differences with CD (p ≤ 0.001), FB (p ≤ 0.001) and FW (p ≤ 0.001); Similarly, the WG reach the highest records in the following variables: HIDC (finding significant differences with CD (p = 0.01), FB (p ≤ 0.001) and FW (p = 0.02); DS (finding significant differences with CD (p = 0.01), FB (p = 0.01), MD (p = 0.01) and FW (p = 0.02)); SP (finding significant differences with CD (p = 0.01), FB (p = 0.01), MD (p = 0.01) and FW (p = 0.02)); DTZ4 (finding significant differences with CD (p = 0.02), FB (p = 0.01) and FW (p = 0.03)); DTZ5 (finding significant differences with CD (p = 0.01), FB (p = 0.01) and FW (p = 0.03)); and finally in DTZ6 (finding significant differences with CD (p ≤ 0.001), MD (p ≤ 0.001) and FW (p = 0.01)); and finally, for FW in DTZ1 (finding significant differences with MD (p = 0.03) and WG (p = 0.04). The number of accelerations (ADA) and accelerations per minute (ADA/min) are the only variables that did not manifest differences by positions.On the other hand, considering PL variables, the highest values by position are found for FB in TS, reaching significant differences with CD  for WR (m/min) in the first period highlights a substantial difference in the work rate during this match phase. These effect sizes demonstrate that the observed changes in performance metrics are not only statistically significant but also of practical importance. On the same way, large effect size (ES: 0.57) for DC (m). Additionally, it is worth noting that the effect sizes can provide valuable context when comparing our results to those of previous studies. For instance, the differences in Total Touches and Releases between our study and the work by This is one of the first studies that shed light on providing information on the activity profile based on TL variables of the soccer player in (friendly) matches applying IMU technology. In our study, we have recorded that the soccer player during each part of a friendly match performs an average of 47.0 ± 18.5 and 48.7 ± 26.4 Total Touches and 16.4 ± 9.6 and 15.6 ± 10.7 Releases, respectively. These values are higher for Total Touches and lower for Releases, compared to those obtained by Previous research has shown that players show lower PL records on Match Day-1, contrary to what occurs with TL in the study conducted by Taking this TL indicator (RI) to the analysis by specific positions, the Central Defenders in our study reach the highest values, followed by the Midfielders. These particular positions in the study carried out by Regarding the analysis of the PL, we found in our study results of Work Rate (m/min) similar to those obtained with GPS technology , both atThis study has the following limitations: Firstly, the study’s sample size was limited to a single professional team, which may affect the generalizability of the findings to a broader population of soccer players. Including multiple teams from different levels of play would enhance the representativeness and reliability of the results. Secondly, the study focused on friendly matches during the preseason, which may only partially capture the intensity and competitive nature of official matches. The findings might differ in different match contexts, such as league matches or cup competitions. Lastly, it is important to note that this study did not consider other potential factors that could influence TL and PL, such as mental conditions , environmental conditions , tactical data  or individual player characteristics . Future studies could incorporate these variables to provide a more comprehensive understanding of the factors influencing TL and PL in soccer matches.The TL profile of professional soccer players, based on their playing position, is found to be independent of the development of PL observed during friendly matches. This profile appears strongly influenced by the game system and the specific role assigned to each position. Therefore, monitoring, quantifying, and controlling TL and PL of soccer players offers a more comprehensive and holistic understanding of the demands in friendly matches compared to solely analyzing PL. Assessing TL during friendly matches enables the differentiation of actions based on players’ positions, which can optimize performance during training sessions.- Designing training tasks with the TL component depending on the player’s specific position.- Adapting the volume and intensity of these variables to the needs of the training session within the microcycle, ensuring adequate tapering for the competition day, as is done with PL.- IMU technology in this context offers a convenient and time-efficient alternative with significant benefits over other existing technologies.The practical applications that this entails are.


Alteration in glycosphingolipids (GSLs) in Parkinson's disease (PD) still needs to be determined.We evaluated if PD subjects show abnormal GSLs levels compared to healthy controls (HC) and if GSLs correlate with clinical features.We analyzed GSLs and glucosylceramide (GlcCer) in plasma using two normal‐phase high‐performance liquid chromatography assays; clinico‐demographic data were extracted.GBA1 mutation carriers. Movement Disorder Society Unified Parkinson's Disease Rating Scale, Part III, correlated with Gb4 and Montreal Cognitive Assessment with GD1b levels.Eighty PD subjects and 25 HCs were analyzed. Levels of GlcCer, GD1b, Gb4, GalNAcGA1, and b‐series were higher in PD patients than in HCs; total GSLs, GT1b, GM1a, GM3, GM2, and a‐series levels were lower in PD patients than in HCs. Changes in GSLs were present in PD subjects, with GlcCer levels similar to those in HCs. The results were similar after excluding certain Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.Multiple GSL abnormalities in plasma were detected in patients with and without GlcCer changes, indicating a broader shift in lipid homeostasis. © 2022 The Authors.  GBA1, encoding the glucosylceramide (GlcCer)‐degrading enzyme glucocerebrosidase (GCase), was identified as the most common genetic risk factor of PD.GBA1 mutation carriers increase the risk of PD is not fully understood. Reduced GCase activity has been described in brain tissues of PD patients with and without GBA1 mutationsGBA1‐related disorder, needs broader examination and further clarification.Parkinson's disease (PD) is the second most common neurodegenerative disease globally.We, therefore, analyzed the plasma levels of GSLs in PD subjects compared to healthy controls (HC). We identified increased GlcCer in a majority of the PD plasma samples, and therefore, we further stratified the PD population to evaluate if changes in other GSLs were affected by high GlcCer levels, because GlcCer is the precursor of all the GSLs. In addition, as an exploratory outcome, we analyzed whether specific GSLs correlate with clinical features.GBA1 gene was sequenced by pyrosequencing or screened by TaqMan PCR and confirmed by Sanger sequencing for the following mutations: N370S, T369M, E326K, and L444P.https://www.protocols.io/view/analysis‐of‐glycosphingolipids‐from‐human‐plasma‐busvnwe6; see Supporting Data for the full nomenclature of GlcCer and GSLs.Patients' blood samples were retrospectively extracted from a cohort of subjects with parkinsonism (BioPark cohort).With the present study, we aimed to evaluate (1) whether PD subjects demonstrate different GSLs levels compared to HCs and (2) whether GSLs differences, relative to HCs, are present in PD subjects with normal GlcCer levels. We also analyzed, as an exploratory outcome, whether specific GSLs levels correlate with clinical features.U test/Fisher's exact test. A comparison between three groups was made using Kruskal–Wallis test and then Dunn's multiple comparison test. Correlations with clinical features were performed with nonparametric partial correlation analysis, adjusted for age, gender, disease duration, and levodopa equivalent daily dose (LEDD); correlations with clinical features stratified by gender were performed with nonparametric partial correlation analysis, adjusted for age, disease duration, and LEDD. For the correlation analysis with clinical features, only subjects with full data were included (LISTWISE exclusion with SPSS). Two‐sided P‐value significance was set at 0.05. Mann‐Whitney U test/Fisher's exact/Kruskal–Wallis tests for normal distribution tests were carried out using GraphPad Prism ; nonparametric partial correlation analyses were carried out with SPSS . Data in tables are expressed in median (interquartile range), frequency, and rho (degree of freedom).Tests for normal distribution were carried out using Shapiro–Wilk and Kolmogorov–Smirnov tests. Given that most of the data in the PD cohort were nonnormally distributed, a comparison between two groups was performed using Mann‐Whitney GBA1 mutations, whereas the GM1a outlier was a GBA1 mutation carrier. The GM1a outlier subject was also removed from total GSLs and a‐series values that could have been affected by the GM1a outlier. GA1 levels were not available in the HC group because the data were below the detection limit of the assay. Hoehn and Yahr was significantly higher in female PD cohorts compared to male PD cohorts ; no other differences in clinico‐demographic data have been found.A total of 80 PD subjects and 25 HCs were analyzed; clinical and demographic data extracted are presented in Table P < 0.0001), GD1b (P < 0.0001), Gb4 (P < 0.0001), GalNAcGA1 (P = 0.0497), and b‐series (P = 0.0070) were higher in PD subjects compared to HCs. On the contrary, total GSLs (P = 0.0285), GT1b (P = 0.0036), GM1a (P = 0.0176), GM3 (P = 0.0050), GM2 (P = 0.0003), and a‐series (P = 0.0042) were lower in PD subjects compared to HCs . Other differences in the GSLs stratified by gender versus control are presented in Table GlcCer levels were similar in male and female PD subjects, but both male and female PD cohorts had higher levels compared to controls , whereas 4 of 27 (15%) in the normal GlcCer group were carriers of GBA1 mutations  (P = 0.55).GlcCer data from HCs were normally distributed ; therefore, we considered the mean plus 2 standard deviations as a good discriminator for subjects with normal versus high GlcCer levels in the general population. Eleven of 50 (22%) subjects in the high GlcCer group were carriers of GBA1 mutation carriers were removed, there were no changes in GSL levels overall , whereas better Montreal Cognitive Assessment (MoCA) score was correlated with higher GD1b levels (ρ (55): 0.333; P = 0.011) and higher levels of GD1 α (ρ (55): 0.281; P = 0.034) . No correlations with clinical features were found when only the female PD cohort was analyzed.Worse score in the Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS‐UPDRS), Part III, was correlated with higher Gb4 levels (ρ (55): 0.319; 4) Table . After tts Table ; in addiWe have previously reported changes in GSLs levels in postmortem substantia nigra (SN), cerebral spinal fluid (CSF), and serum samples from UK and US biobanks.In our previous study, GlcCer levels were increased in the SN of PD patients,GBA1 mutation carriers were excluded. These associations were not present in the female PD cohort; however, this cohort had a lower number of subjects, and this could have masked some clinical correlations. GD1b IgM has been found to be associated with dementia,Our analysis showed a positive linear correlation between GD1b and better MoCA score between Gb4 and worse MDS‐UPDRS Part III score when GBA1 mutations, and therefore, possibly more GBA1 mutation carriers are unrecognized in the cohort; however, this eventuality should not affect the results because of the very small numbers of possible mutation carriers of other variants. Moreover, most of the subjects were carriers of low‐risk mutations. Third, we lack data on the levels of GSLs enzymes in this cohort. In addition, the correlation with clinical features does not consider multiple comparisons, so the results should be interpreted with caution. Finally, the cross‐sectional design does not allow the evaluation of the causality of our associations, and replicating these results in another cohort would be important to validate these findings. Nonetheless, this study suggests that the enrollment of smaller, but biochemically well‐characterized, subjects may lead to a greater chance of success when trialing disease‐modifying therapies by reducing heterogeneity.To the best of our knowledge, this is the first study that extensively analyzed specific GSLs in plasma with clinico‐demographic data. Our sample size is larger than that in previous studies, adding strength to our analysis. However, some limitations are present. First, many of the statistically significant differences are minor, with overlap between patients and controls; moreover, male gender was represented more. The finding of GD1b is, in this aspect, encouraging where a subgroup of patients exhibit clearly elevated levels. Second, our subjects have been tested only for selected Research project: A. Conception, B. Organization, C. Execution.Statistical analysis: A. Design, B. Execution, C. Review and critique.Manuscript preparation: A. Writing of the first draft, B. Critical reading and editing.DtV: 1A, 1B, 1C, 2B, 3BAS: 1A, 1C, 2A, 2B, 3ADAP: 1A, 1B, 1C, 2C, 3BPT: 1C, 2C, 3BEH: 2C, 3BMA: 2C, 3BIM: 1C, 2C, 3BK‐LW: 1A, 1B, 1C, 2C, 3BFP: 1A, 1B, 1C, 2C, 3BPS: 1A, 1B, 1C, 2C, 3AP.S. had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Table S1. Glycosphingolipid levels.Table S2. Glycosphingolipid levels excluding GBA1 mutation carriers.Table S3. Comparison between GBA1 mutation carriers and noncarriers.Table S4. Glycosphingolipid levels in PD subjects stratified by gender and healthy control.Table S5. Glycosphingolipid levels in PD subjects with normal and high GlcCer.Table S6. Glycosphingolipid levels in PD subjects with normal and high GlcCer excluding GBA1 mutation carriers.Table S7. Correlations between GSLs and clinical features in PD subjects.Figure S1. Glycosphingolipid comparison between PD subjects and healthy controls. PD, Parkinson's disease; HC, healthy control. *P ≤ 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Values inside the box are outliers.Figure S2. Glycosphingolipid biosynthetic pathway and representative high‐performance liquid chromatography profile and summary of main findings. HPLC trace of main plasma GSLs not included (GlcCer HPLC profile not shown). Green circles highlight abnormal glycosphingolipids in our analysis; red arrows indicate an increase in PD, whereas blue arrows indicate a decrease in PD.Click here for additional data file.


Homobutein had kinh = (2.8 ± 0.9) × 103 M−1s−1, pairing the relative BDEOH measured by radical equilibration EPR as 78.4 ± 0.2 kcal/mol for BU and estimated as 82.6 kcal/mol for HB. The inhibition of mushroom tyrosinase (mTYR) by HB and BU was also investigated. BU gives a reversible uncompetitive inhibition of monophenolase reaction with KI′ = 9.95 ± 2.69 µM and mixed-type diphenolase inhibition with KI = 3.30 ± 0.75 µM and KI′ = 18.75 ± 5.15 µM, while HB was nearly competitive toward both mono- and diphenolase with respective KI of 2.76 ± 0.70 µM and 2.50 ± 1.56 µM. IC50 values (monophenolase/diphenolase at 1 mM substrate) were 10.88 ± 2.19 µM/15.20 ± 1.25 µM, 14.78 ± 1.05 µM/12.36 ± 2.00 µM, and 33.14 ± 5.03 µM/18.27 ± 3.42 µM, respectively, for BU, HB, and reference kojic acid. Molecular docking studies confirmed the mechanism. Results indicate very potent antioxidant activity for BU and potent anti-tyrosinase activity for both chalcones, which is discussed in relation to bioactivity toward protection from skin disorders and food oxidative spoilage.Butein (BU) and homobutein (HB) are bioactive polyhydroxylated chalcones widespread in dietary plants, whose antioxidant properties require mechanistic definition. They were investigated by inhibited autoxidation kinetic studies of methyl linoleate in Triton™ X-100 micelles at pH 7.4, 37 °C. Butein had  Asteraceae , Asparagaceae , Anacardiaceae , Fabaceae , Pinaceae , Solanaceae , and others  is the initial substrate concentration, while Vmax and Km are, respectively, the maximum reaction rate (at saturating substrate concentration) and the M-M constant, with the substrate concentration yielding half-maximum rate . Results in 50 of butein decreases on increasing the concentration of l-tyrosine for monophenolase inhibition, owing to the uncompetitive mechanism that requires pre-formation of the E-S complex, values variably increase with the concentration of substrate for butein diphenolase inhibition and for homobutein and kojic acid, all showing a mixed-type inhibition with the (variable) prevalence of competitive behavior. This further highlights the limits of IC50 in quantifying inhibitors’ performance.Determination of M-M inhibition constants trations ,35. None50 for butein and homobutein, against both mono- and diphenolase reactions, were sensibly lower, confirming their higher inhibiting potency. Concerning butein, our values are lower but compatible with those of Khatib et al.  = 1 mM as “standard” settings . Resultsb et al.  for monob et al. . While wTo help rationalize the inhibiting mechanism of butein and homobutein, molecular docking simulations were performed against mTYR (PDBID: 2Y9X) to determine binding structures and affinities ,50,51 usl-tyrosine and l-dopa; setting flexibility in the ligand and rigid structure in the protein; and were repeated using AutoDock 4.2, AutoDock GPU, and AutoDock Vina to identify consistent binding poses and the protein residues which are mainly involved in interaction with the ligands. Results guided the setting of the flexible residues in AutoDock Vina, which was used for re-docking all the ligands so as to obtain optimized interaction at a low computational cost [Computations were first performed by individually docking butein, homobutein, and substrates nal cost . This reKI value for homobutein compared to butein measured for diphenolase inhibition , in the absence of other substrates, i.e., without l-tyrosine and l-dopa. Their full UV–Vis spectrum was monitored for 60 min. Results showed a rapid spectral variation attributable to the complexation with the enzyme for butein, followed by a very slow decay of its concentration, which was identical in the presence or absence of the enzyme . The lower BDEOH of catechol butein  compared to homobutein  justifies its higher reactivity. However, it explains the outstanding antioxidant performance in the protection of methyl linoleate micelles only in part. Indeed, butein was able to outperform nature’s premiere antioxidant, α-tocopherol, despite its BDEOH being higher by 1.3 kcal/mol, and, most interestingly, butein was able to trap almost twice as many peroxyl radicals per molecule of antioxidant compared to α-tocopherol. This behavior is reminiscent of the behavior previously found for catechol/quinone antioxidant systems [•) radicals. It is based on the very fast reduction by HOO• of the semiquinone and quinone from the antioxidant to re-generate fresh catechol ready to carry on the inhibition. However, it was observed here without the deliberate addition of a source of hydroperoxyl radicals. We suggest that butein is able to exploit the HOO• radical formed as a side event during the autoxidation of methyl linoleate [2-•/HOO• is produced under both physiologic and pathologic conditions, which lends support to the redox bioactivity reported for butein in previous studies [Both butein and homobutein are effective antioxidants working with a chain-breaking mechanism consisting of the fast trapping of chain-carrying peroxyl radicals , with KI of 3.30 and 2.50 µM, respectively, both outperforming the reference kojic acid. This mechanism, which is supported by molecular docking studies, is investigated for the first time for homobutein and is at variance from previous knowledge for butein. Both chalcones were also active against the l-tyrosine substrate (monophenolase), albeit with a different mechanism, which was uncompetitive for butein (KI′ = 9.95 µM), and it was nearly competitive for homobutein (KI = 2.76 µM). While the mechanism of homobutein is nicely supported by molecular docking, that of butein appears to require further efforts for full rationalization.Along with their antioxidant activity, both butein and homobutein have major efficacy in the inhibition of tyrosinase, showing a prevailing competitive mechanism when Of interest, the combined antioxidant and anti-tyrosinase activities open previously unexplored applications for the two chalcones, particularly in dermatology, to contrast photoaging, melasma, and age-related pigmentation disorders  and in f


Trichonephila clavata  from South Korea is determined and characterized in detail, which is the second mitochondrial genome reported from this species: the first was published from the Chinese sample by Pan et al. (T. clavata (Subfamily Nephilinae) from South Korea and China are clustered together, distinctly separated from the other subfamily Araneinae in the monophyletic family Araneidae.The mitochondrial genome of a golden orb-web spider n et al. . It was  Nephila are tropical and subtropical in distribution and construct exceptionally large and impressive orb webs  is taxonomically acceptable. From the species, Pan et al. , which was stored in 95% ethanol and deposited under the voucher number KNU-ARAR-001 at KNU . Total cellular DNA was extracted from a spider leg using a DNeasy tissue kit (QIAGEN Co.) following the manufacturer’s instructions. First, we amplified the partial region (465 bp long) of 16S rRNA with universal PCR primers: 16SA , consists of 37 genes including 13 PCGs, two rRNA genes, and 22 tRNA genes, with a control region (CR). The overall base composition is 35.99% for ‘A,’ 9.09% for ‘C,’ 14.88% for ‘G,’ and 40.04% for ‘T,’ showing strong A + T biased composition (76.03%). Among 13 PCGs, ‘ATA’ as a start codon was used for ND1, ND2, ND4, ND5, CYTB, and ATP6, ‘ATT’ for ND3, ND4L, ND6, and ATP8, ‘TTG’ for COX2 and COX3, and ‘TTA’ for only COX1. As stop codons, ‘TAA’ was used for COX1, COX2, ND3, ND4, ND4L, ND5, ATP6, and ATP8, ‘TAG’ for ND1, ND2, ND6, and CYTB, and ‘T’ for COX3. When we compared the present result with the mitochondrial genome published from the same species of China  and the concatenated amino acid sequence alignment (AA) from 19 araneid species , and distinctly separated from the monophyletic clade of Araneinae.To determine the phylogenetic position of