The study investigated the effects of high-pressure processing (HPP) (600 MPa/5 min), pasteurization (PT) (85 °C/30 s), and high-temperature short time (HTST) (110 °C/8.6 s) on physicochemical parameters , sensory-related attributes , antioxidants , and sensory attributes of yellow passion fruit purée (PFP). Compared to the PT and HTST, HPP obtained the PFP with better color, sugar, and organic acid profiles. Although PT was equally effective preservation of antioxidants and antioxidant capacity of PFP compared to HPP, high temperature inevitable resulted in the greater degradation of the aroma profile. The amounts of esters, alcohols, and hydrocarbon in PFP were significantly increased by 11.3%, 21.3%, and 30.0% after HPP, respectively. All samples were evaluated by a panel comprising 30 panelists according to standard QDA  procedure, and the result showed that HPP-treated PFP was rated the highest overall intensity score with 7.06 for its sensory attributes, followed by control (6.96), HTST (6.17), and PT (6.16). Thus, HPP is a suitable alternative technology for achieving the good sensory quality of PFP without compromising their nutritional properties. Passion fruit is one of the most popular species in the Passiflora family and is widely planted in the subtropical and tropical regions of Asia, America, Africa, and Australia . RecentlThe passion fruits are usually commercialized in the form of fresh fruit, purée, juice, or concentrated pulp, which are appreciated for their unique exotic aroma and color. Among them, passion fruit purée (PFP) exhibits an increasing market value because it is a convenient food product or ingredient with a natural fresh appearance and aroma. The aroma of passion fruit contributes to the great popularity of this fruit and directly affects the sensory quality of fresh passion fruit and its products, which arise from a complex combination of several secondary metabolites, such as formaldehyde, alcohols, ketones, esters, and terpenes . Porto-FThermal pasteurization is one of the most important procedures in juice and purée processing and has been widely used in recent years to improve food safety and extend the shelf life of juice products . Heat, hHigh-pressure processing (HPP) is a non-thermal technology that uses pressure to inactivate microorganisms and enzymes, while also reducing the damage to the nutrients and aroma . NumerouIn the present study, we comprehensively evaluated and compared the physicochemical parameters , sensory-related attributes , antioxidants , and sensory attributes of yellow PFP treated with HPP, PT, and HTST. The knowledge obtained will help develop PFP as a new food ingredient, to improve the sensory attributes, consumer acceptability, and functional characteristics of these products.The yellow passion fruits were purchased from a local passion fruit planting orchard  and stored in a refrigerator at 4 °C for further experiments.The passion fruits were opened, the pulp was separated from the seeds to make purée using a home juicer, and the resulting homogenization was filtered with two layers of nylon gauze to remove residue . The obtIn the food industry, HPP at 500–600 MPa yielded food products with good quality and safety . The obtThermal pasteurization was conducted in a multipurpose ultrahigh temperature UHT sterilization unit . Two levels of processing intensity were selected: pasteurization (PT) (85 °C/30 s), and high-temperature short time (HTST) (110 °C/8.6 s). To effectively destroy pathogens and inactivate endogenous enzymes, purée was preheated to 65 °C and pasteurized at 85 °C for 30 s , and prew/w) in sterile saline solution. The plate count agar was used for counting the total aerobic bacteria (TAB) after incubation at 36 ± 1 °C for 48 ± 2 h. The number of yeast and mold (Y&M) samples were detected after incubation in rose bengal agar at 28 ± 1 °C for 5 d. Then, the microorganism numbers of the samples were enumerated as a log of CFU/mL.Microbial analyses were performed for HPP, PT, and HTST treated and untreated samples. One milliliter of purée was diluted (1:10 w/v) insoluble PVPP, 1 M NaCl, and 1% (w/v) Triton X-100 in 0.2 M sodium phosphate with a final pH of 6.5, and centrifuged at 14,000× g and 4 °C for 30 min. More details of the methodology can be found in The extraction of polyphenol oxidase (PPO) and peroxidase (POD) and analysis of activity were performed using the method described by Yi et al.  with minTSS was determined with a Brix refractometer  at 25 ± 1 °C and the results were expressed as °Brix. TS was determined by Fehling reagent titration method . The res4 [Fe(CN)6]) and 250 µL Carrez II (1.86 mol/L ZnSO4) were added to 5 mL purée. The mixture was homogenized with a vortex mixer for 3 min. After standing at room temperature for 30 min, the mixture was centrifuged  for 15 min. The supernatant was diluted (1:9) in HPLC-grade water and filtered through a 0.45 µm nylon membrane for determination of individual sugar by using HPLC  with evaporative light scattering detection . More details of the methodology can be found in The sugar profile was analyzed by high-performance liquid chromatography (HPLC) using the procedure of Pham et al.  with somTA content was calculated using Equation (1) [C is the NaOH concentration (0.1 mol/L), m (g) is the weight of purée, V0 (mL) is the total volume of purée, V1 (mL) is the purée used, V2 (mL) is the volume of NaOH used, and K is the conversion factor of citric acid (0.07).The determination of pH value was carried out by an Orion 868 pH meter  at 20 ± 1 °C. TA was determined by titrating with standardized 0.1 mol/L NaOH, reaching pH 8.1 by an automatic potentiometric titrator  . TA was tion (1) .(1)TAg/The extraction procedure for organic acids was the same as individual sugars described in 2.4. Extraction solution was diluted (1:4) using HPLC grade water and filtered through a 0.45 µm syringe filter. Following the procedure of Wibowo et al. , the orgE) was calculated by Equation (2) [The color of PFP was determined by using the CIE L*a*b* system and a colorimeter  with D65 illuminant and 10° observer angle. Total color difference (Δtion (2) .(2)ΔE=LThe L*, a* and b* signify the measured brightness value, redness value, and yellowness value of the PPF by different treatment, respectively, and the subscript ‘0’ stood for untreated samples.The extraction of aroma compounds was following the method described by Pan et al.  using div/v), sonicated for 20 min, and centrifuged at 9000× g and 4 °C for 5 min. The obtained supernatant was used for the TPC, TFC, and antioxidant capacity determination on a microplate reader , according to Wang et al. [According to the method of Wang et al.  with slig et al. .2CO3 (0.71 mol/L), then reacted at room temperature in the dark for 1 h. A 200 µL solution was pipetted into the microplate and measured at 765 nm. The results were expressed in gallic acid equivalent per liter of purée (mg GAE/L). At 6 min, 100 µL of 1.0 mol/L NaOH was added and mixed. The absorbance was determined at 510 nm.The determination of TPC was carried out by using the classical Folin–Ciocalteau assay with slight modifications. A total of 50 µL extract was mixed with the 10-fold-diluted Folin–Ciocalteu reagent (500 µL) and 450 µL Na3 colorimetry with some modifications. Firstly, 100 µL of extraction solution and 5 µL of 0.72 mol/L NaNO2 were added to the microplate, and 0.37 mol/L AlCl3 (15 µL) was added 5 min later. After incubation for 30 min at room temperature, the absorbance was measured at 510 nm. The result was expressed in rutin equivalent per liter of purée (mg RE/L).TFC was determined by AlClSix independent repetitions were executed for the extraction and analyzed by a Thermo Fisher Ultimate 3000 UHPLC system equipped with a Q-Exactive Orbitrap mass spectrometer . More details of the methodology can be found in DPPH reaction solution was prepared by adding water/methanol (1:4) to adjust its absorbance to 0.90 ± 0.05 at 517 nm. Forty microliters of diluted samples (1:10) and 160 µL DPPH reaction solution was added to the microplate, then reacted for 30 min in the dark. Absorbance value was measured at 517 nm. The results were expressed as mmol Trolox equivalent (TE)/L of purée.•+ solution was diluted with methanol to an absorbance of 0.70 ± 0.02 at 734 nm. The ABTS•+ solution was produced by reacting 7 mmol/L ABTS stock solution with 2.45 mM K2S2O8 and kept in dark at room temperature for 12–16 h before use. Diluted methanolic extract (1:9) was mixed with ABTS•+ solution (A734 = 0.70 ± 0.02) and incubated for 6 min at room temperature, then the absorbance was measured at 734 nm. Results were expressed in mg Trolox equivalent (TE)/L of purée.The ABTS3 and 0.08 mol/L CH3COOH, pH 2.0), and centrifuged at 10,000× g for 30 min at 4 °C. The obtained extract was divided into two parts: one was used for AA analysis and the other was used for vitamin C analysis, which was further analyzed by HPLC . The details of the methodology can be found in Total vitamin C includes ascorbic acid (AA) and dehydroascorbic acid (DHAA). According to Cao et al. , 5 mL of3OH/EtAc/CH2Cl2, 25:25:50, v/v/v, containing 0.005 mol/L of BHT), stirred for 5 min, and placed in an ultrasound bath for 5 min to enhance extraction. The mixture was centrifuged at 17,000× g at 4 °C for 5 min. These operations were repeated until color exhaustion was found with extracting the solvent. The organic phase containing carotenoids was separated and pooled. Finally, the organic phase was concentrated to dryness by rotary evaporation at 30 °C and analyzed by HPLC . More details of the methodology can be found in The extraction of carotenoids was according to the method of Giuffrida et al. , with soSamples after different treatments were evaluated by a panel comprising 30 panelists by following standard QDA  procedures . The samp < 0.05 is significant. Variable importance in projection (VIP) coefficients were calculated to select discriminant compounds, and those values with the largest |VIP| > 1 were selected for principal component analysis (PCA) by bioinformaticsa free online data processing software. Metaboanalyst, a free online data processing software, was used for heatmap analysis.Statistical analysis was performed using Origin 8.0  and SPSS 20.0, and results are expressed as mean ± SD. A one-way analysis of variance (ANOVA) was used to perform Tukey’s significant difference test, and As shown in p > 0.05) on those values of the PFP. Similar results were also found by Yi et al. [As can be seen in i et al.  and Wu ei et al. , where ti et al. ,29. The i et al. . A simili et al. , where tA total of six organic acids were detected in PFP, including oxalic acid, malic acid, lactic acid, acetic acid, citric acid, and quinic acid. Citric acid, with a content of 25.90 ± 1.82 mg/mL, was the main organic acid in control PFP, accounting for 81.8% in the total organic acids and consistent with a proportion of 85.3% reported for yellow passion fruit juice in the Xie et al. ,29. A siAs shown in A total of 51 aroma compounds were identified in PFP, including 23 esters, 12 alcohols, 7 hydrocarbons (6 terpenes and 1 hydrocarbon), 5 ketones, 3 acids, and 1 aldehyde . Most ofAs compared with control PFP, the amounts of esters, alcohols, and hydrocarbon in the HPP-treated sample were significantly boosted by 11.3%, 21.3%, and 30.0%, respectively, while those values in the PT- and HTST-treated samples were significantly decreased by 40.7–48.0%, 80.3–81.8%, and 66.7–71.3%, respectively. Previous results also reported that HPP could better preserve the aroma compounds of fruit products, such as apple juice and mango juice ,19.Octadecanoic acid, 1-heptanol, nerylacetone, linalool, and linalool oxide were significantly increased by 647.8%, 201.2%, 97.9%, 76.4%, and 58.5% after HTST, respectively. The formation of these potentially temperature-induced compounds could be linked to the Maillard reaction and oxidative reactions  . Some in•+ in PFP after HPP and PT was significantly higher than those values of HTST treated sample.As compared to the control PFP, HPP did not change the contents of antioxidants, except that TPC was decreased by 8.3%. This was possibly explained by an increase in condensation reactions of the phenolic compounds in the PFP promoted by HPP . The iniTo better understand the individual phenolic compound changes in PFP after different technologies, the phenolic compounds were identified and quantified by using LC-MS. A total of 15 phenolic compounds were identified in control PFP . The maiO-glucoside, and luteolin-4-O-glucoside in PFP were significantly increased by PT, while the contents of protocatechuic acid, galloyl-glucoside, and p-coumaric acid were slightly decreased. The different stability of phenolics towards PT was caused by the different structures of these phenolics. Moreover, some phenolics might bind tightly with food substrates through covalent bonds, and PT would have limited ability to destroy these glycoside bonds [O-glucoside was significantly increased by 40.1% after HTST. The increase of glycoside content was possibly due to the breakdown of a cell wall structure and hydrolysis of polysaccharides induced by a high temperature (90 °C/60 s) [As shown in de bonds . HTST ca°C/60 s) . A simil°C/60 s) .There were four carotenoids detected in PFP, namely, β-cryptoxanthin, zeaxanthin, β-carotene, and lycopene. The highest content of 45.36 ± 2.88 µg/mL was found for β-cryptoxanthin in fresh purée, which accounted for 46.8% of the total carotene. As compared to the control sample, HPP had no significant effect on β-carotene, lycopene, and total carotenoid contents in PFP, whereas zeaxanthin in HPP-treated PFP was increased by 56.8%, and correspondingly β-cryptoxanthin was decreased by 39.3%. It was reported that β-cryptoxanthin was (R)-isomer of β-carotene, which could be converted into zeaxanthin by the enzymatic action of the β-ring hydroxylase . It was O-glucoside, octadecanoic acid, 1-heptanol, and ethyl hexanoate.As shown in Sensory evaluations of PFP processed by HPP, PT, and HTST were shown in In summary, HPP-treated PFP retained better color quality and more antioxidants of PFP compared to PT and HTST and had high similarity in the sensory descriptive profiles with the control sample. Although PT was equally effective at preservation within antioxidants, including TPC, TFC, vitamin C, β-carotene, zeaxanthin, and lycopene, as well as antioxidant capacity of PFP as compared to HPP, the high temperature inevitably resulted in the greater degradation of aroma profile and sensory descriptive profiles of PFP. The amounts of esters, alcohols, and hydrocarbon in the HPP-treated sample were especially significantly increased by 11.3%, 21.3%, and 30.0%, respectively, while most of the aroma compounds were significantly decreased by thermal pasteurization. Furthermore, the changes in sensory evaluation agreed with the changes in physicochemical characteristics such as color, pH, TSS, sugar, acid, and the aroma profile of PFP. Interestingly, zeaxanthin in HPP-treated PFP was increased by 56.8% and, correspondingly, β-cryptoxanthin was decreased by 39.3%, which was explained by the fact that HPP promoted the enzymatic action of the β-ring hydroxylase in PFP. Hence, HPP proved to be a promising preservation method of PFP, in contrast with thermal processing. The sensory analysis performed with consumers could provide more information related to HPP-treated purée acceptability in the future. Furthermore, the effect mechanism of HPP on antioxidants, such as carotenoids and phenolics, is worth studying based on the enzymatic reaction in future work.


After the deposition in a document, it starts to evaporate over time. This ageing process potentially allow to estimate the date of an ink in a document , however−1 to 100 ºC, and increased to 240 ºC at a rate of 30 ºC min−1 during 4.67 min. The MS analysis was carried out in SIM mode, which m/z of PE was 77, 94, 138 and PE-TMS was 151, 195, 210.In order to compare peak resolution between PE and derivatized PE, a solution of 50 µg/ml of PE was prepared in hexane. The derivatized sample was prepared using chemical derivatization with MSTFA:TMCS (95:5) at a 80ºC during 30 min. The samples were analysed using GC/MS with an Agilent Technologies 5973 − 6890 N. GC MEGA-5 MS; 0.25µm, 0.32mm, 30 m capillary column was used. Chromatographic analysis was carried out under the following conditions: injection volume 2 µl, split ratio of 2:1 and injection temperature at 280 ºC. The oven temperature program starting at 90 ºC during 8 min, then ramped at 10 ºC minDiscussion and conclusions: This preliminary study showed an increase of the sensitivity of the PE-TMS. Our results revealed that PE derivatization with MSTFA:TMCS, might be useful in ink dating determination and can contribute to the decreasing of LOQ. However, more work has to be done. In conclusion, derivatization proves to be promising in the field of ink ageing, allowing getting sensitivity to estimate the age of an ink, in a long period of time.The chromatogram results  showed t


Background: Kratom (Mitragyna speciosa Korth), a popular opioid-like plant holds its therapeutic potential in pain management and opioid dependence. However, there are growing concerns about the safety or potential toxicity risk of kratom after prolonged use.Aim of the study: The study aimed to assess the possible toxic effects of kratom decoction and its major alkaloids, mitragynine, and speciociliatine in comparison to morphine in an embryonic zebrafish model.Methods: The zebrafish embryos were exposed to kratom decoction , mitragynine, speciociliatine, and morphine (100–3.125 μg/ml) for 96 h post-fertilization (hpf). The toxicity parameters, namely mortality, hatching rate, heart rate, and morphological malformations were examined at 24, 48, 72, and 96 hpf, respectively.Results: Kratom decoction at a concentration range of ≥500 μg/ml caused 100% mortality of zebrafish embryos and decreased the hatching rate in a concentration-dependent manner. Meanwhile, mitragynine and speciociliatine exposure resulted in 100% mortality of zebrafish embryos at 100 μg/ml. Both alkaloids caused significant alterations in the morphological development of zebrafish embryos including hatching inhibition and spinal curvature (scoliosis) at the highest concentration. While exposure to morphine induced significant morphological malformations such as pericardial oedema, spinal curvature (lordosis), and yolk edema in zebrafish embryos.Conclusion: Our findings provide evidence for embryonic developmental toxicity of kratom decoction and its alkaloids both mitragynine and speciociliatine at the highest concentration, hence suggesting that kratom consumption may have potential teratogenicity risk during pregnancy and thereby warrants further investigations. Mitragyna speciosa Korth. (Rubiaceae), is known as Ketum or Biak-biak in Malaysia and Kratom in Thailand. This plant holds various therapeutic potentials especially in pain management and opioid dependence (pendence . People pendence . Given ipendence . In factpendence .Ki = 54.5 nM) towards the human mu-opioid receptor compared to its diastereoisomer—mitragynine (Ki = 161 nM). This alkaloid constitutes 9% of the total alkaloid present in kratom leaves. Further to this, speciociliatine demonstrated a better antinociceptive effect in rats when compared to mitragynine , a small aquatic vertebrate, is a valid translational model in the field of neuroscience research, toxicology, or translational medicine , wild-type AB strain were used in this study. The zebrafish were maintained in the automated housing system  that automatically regulates the pH (7.5 ± 0.5), temperature (28°C ±0.5), salinity, and water flow with 14 h light:10 h dark cycle . They were fed daily with tetraMin® tropical flakes and live brine shrimp twice per day. Embryos were obtained from spawning sexually matured male and female adult zebrafish at a ratio of 2:2 through natural mating. According to European legislation , no animal ethics permission was requested for zebrafish larvae below 120 h post-fertilization (hpf) (Zebrafish (on (hpf) .Mitragyna speciosa Korth.) leaves were collected from a local farm located at Permatang Rawa, Penang, Malaysia. The plant was authenticated by a botanist, Dr. Rosazlina binti Rusly from the School of Biological Sciences, Universiti Sains Malaysia. A voucher specimen [NEL-(K2)-2019(02)] was deposited at the Herbarium of School of Biological Sciences, Universiti Sains Malaysia.Approximately 4 kg of fresh kratom (The collected fresh kratom leaves (1 kg) were washed with tap water and ripped into small pieces before placing them into a boiling pot of water (4 L). The leaves were brewed for approximately 2 h at constant heat until the volume was reduced to approximately one-third of the initial volume. After that, the solution (1 L) was left to cool, filtered, and freeze-dried to yield a lyophilized kratom decoction extract. The lyophilized extract was kept at −80°C before high performance liquid chromatography (HPLC) analysis and toxicity evaluation.M. speciosa leaves according to the method described in our previous study (Mitragynine (1) and speciociliatine (2) were extracted and purified from the fresh us study . The detThe reference standards: mitragynine (1) and speciociliatine (2) (purity ≥97%)  were extThe content of mitragynine (1) and speciociliatine (2) in the prepared kratom decoction sample were determined using a validated HPLC method as described in our previous study . The detn = 20) that reached the blastula stage with normal cleavage pattern were randomly transferred to 24-well plates, one embryo in each well with 1.5 ml of the test sample. Next, well plates were incubated at 26 ± 1°C under a 14 h light: 10 h dark cycle. The test samples were renewed on the daily basis (semi-static exposure). The tests were performed in triplicate. Lethality parameters, such as coagulation of embryos, lack of somite formation, non-detachment of the tail, and lack of heartbeat and sub-lethal parameters include pericardial oedema, yolk sac oedema, spinal curvature , heartbeat and hatching rate at 24, 48, 72 and 96 hpf were examined under zoom stereo microscope. The heart rate of larvae (n = 5) was counted for 15 s using a stopwatch under the stereomicroscope when the larvae were immobile, and then multiply by 4 to obtain the beats per minute. The sub-lethal morphological effect was expressed as the percentage of embryos with malformation over total alive embryos at 24, 48, 72, and 96 hpf (The stock solutions of kratom decoction (2 mg/ml), mitragynine (200 μg/ml), speciociliatine (200 μg/ml), and morphine (200 μg/ml), were prepared in 0.1% dimethyl sulfoxide (DMSO). A series of working concentrations ranging from 1,000–62.5 μg/ml (kratom decoction) and 100 to 3.125 μg/ml  were prepared by serial dilution of the stock solution. System water was used as negative control and 0.1% DMSO as solvent control, whereas 20 μg/ml doxorubicin was used as a positive control. The FET test was performed according to the Organization for Economic Co-operation and Development (OECD) TG 236 guideline . Brieflyd 96 hpf .50 value. The mortality rate at 24, 48, 72, and 96 hpf were analyzed using a two-way repeated-measure ANOVA, followed by Bonferroni post hoc test. The hatching rate, heartbeat, and sub-lethal morphological effect data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. Probability values of less than 5% (p < 0.05) are considered significant.Statistical analysis was performed using the software Graph Pad Prism. 5. Data were expressed as mean ± standard deviation (SD). Concentration-response curves were used to determine the lethal concentration, LCBased on HPLC analysis, the amount of mitragynine (1) and speciociliatine (2) detected in the prepared kratom decoction  were 37.63 ± 3.01 and 5.49 ± 0.37 μg/ml, respectively. The HPLC chromatograms of lyophilized kratom extracts and its detected mitragynine 1) and speciociliatine 2) are provided in The test was validated according to OECD 236 guideline criteria, as shown by less than 5% mortality rate in system water (negative control) and 0.1% DMSO (solvent control), whereas 100% mortality rate in 20 μg/ml doxorubicin (positive control) at 96 hpf .p < 0.001, versus negative control group). With regards to alkaloid compounds, both mitragynine and speciociliatine at the highest concentration (100 μg/ml) killed 100% of the embryos at 72 hpf. Besides that, the mortality rate of embryos in both mitragynine and speciociliatine groups at concentrations 50 and 25 μg/ml was significantly increased at 96 hpf . Mitragynine at concentrations of 50 and 25 μg/ml killed 93.33% and 21.67% of the embryos, respectively, whereas the mortality rate of speciociliatine at 50 and 25 μg/ml were 16.67% and 13.33%, respectively. These results indicate that speciociliatine is safer than mitragynine. In morphine-exposed embryos, the highest concentration (100 μg/ml) significantly increased the mortality rate at 96 hpf in comparison to the negative control group (p < 0.001).As shown in 50 of kratom decoction and mitragynine at 96 hpf were 260.68 μg/ml and 32.01 μg/ml, respectively. We were unable to calculate the actual LC50 value of speciociliatine since it showed an exponential increase of mortality rate at 100 μg/ml concentration, hence the estimated LC50 of speciociliatine at 96 hpf from the concentration-response curve was 79.86 μg/ml. For morphine, the highest mortality rate recorded was less than 20%, and it is not possible to calculate the LC50 value.The concentration-response curve of kratom decoction, mitragynine, speciociliatine, and morphine for mortality rate at 96 hpf are shown in p < 0.001, versus negative control group). In addition, the percentage of embryos hatched was only 9.06% and 28.36% in the group treated with 125 and 62.5 μg/ml of kratom decoction at 96 hpf, respectively (p < 0.001). These data indicated concentration-dependent delayed hatching in the kratom decoction-treated groups in comparison to the control group. Hatching inhibition was also found in higher concentrations of mitragynine and speciociliatine (50 and 25 μg/ml) treated groups at 72 and 96 hpf, respectively . Morphine did not affect the hatching rate of zebrafish embryos at 72 and 96 hpf.In general, zebrafish embryos started to hatch from 48–72 hpf . As showp > 0.05). The heart rate of kratom decoction , mitragynine, and speciociliatine (100 μg/ml) was not determined, as there were no surviving embryos at 96 hpf.The heart rate per minute of zebrafish embryos at 96 hpf is shown in p > 0.05, p < 0.01, versus negative control group). Meanwhile, the embryos exposed to speciociliatine at the concentration ≥25 μg/ml exhibited signs of scoliosis  are far higher compared to kratom decoction (as a mixture). The embryotoxicity observed in kratom decoction might be due to the presence of other phytochemicals such as other indole and oxindole alkaloids, terpenes, flavonoids, phenolics, plant peptides, polysaccharides, etc. The single or synergic effect of these compounds in the overall embryotoxicity of kratom decoction cannot be ruled out as well, therefore this warrants further investigation. For morphine, we could not determine the LC50 value because the highest mortality rate recorded was less than 20%, indicating that morphine was relatively safe even at a higher concentration range up to 100 μg/ml. Altogether, kratom decoction, mitragynine, and speciociliatine at the highest dose display more toxic effects in terms of embryo survival, when compared to morphine.With regards to alkaloids, the LCHatching is the most important process in the development stage of zebrafish and its retardation following exposure to chemical/drugs like environmental pollutants, nanomaterials, or natural product is a sign of sub-lethal toxicological effects on zebrafish embryos . In the Morphological malformations such as spinal curvature, yolk oedema, and pericardial oedema are important sub-lethal parameters observed in zebrafish embryos when exposed to toxic chemicals . An examOverall, we have demonstrated that kratom (≥500 μg/ml) and its alkaloids mitragynine and speciociliatine (≥50 μg/ml) have certain undesirable effects on embryonic development by affecting survival, hatching, and body morphology of zebrafish embryos. This finding suggests that the potential risk of kratom intake during pregnancy on the development of the fetus is based on the fact that the early embryo developmental process of zebrafish is similar to humans. However, this notion should be interpreted with caution, and warrants further investigation in other animal models such as rodent.


Ralstonia mannitolilytica, a newly emerging opportunistic pathogen worldwide, has been reported to be responsible for human pneumonia, septicemia and meningitis. This is the first report of a case of Ralstonia mannitolilytica sepsis after elective cesarean delivery.+ 1 weeks of gestation. Sudden high fever and decreased blood pressure occurred a short time after the operation. Ralstonia mannitolilytica was identified in her blood culture 5 days after the operation. Based on the presence of sepsis and septic shock, massive fluid replacement, blood transfusion, vasoactive agents, imipenem/cilastatin and cefoperazone sulbactam sodium were applied. She was discharged after intensive care without complications.A 25-year-old woman, gravida 1 para 0, was scheduled for an elective cesarean delivery at 39Ralstonia mannitolilytica is relatively low, once infection occurs in a puerpera, severe symptoms develop abruptly. Thus, prompt diagnosis and appropriate treatment are key to the cure.Although the incidence of sepsis due to  Ralstonia mannitolilytica (R. mannitolilytica) belongs to the genus Ralstonia of the Burkholderiaceae family and was first isolated by Yabuuchi in 1995 , increased neutrophilia of 96.1% [50–70%], decreased platelets of 83 × 109/L [100–450 × 109], a hemoglobin concentration of 105 g/L [110–155], a hematocrit of 30.7% [35–45%], coagulopathy with a prolonged activated partial thromboplastin time (APTT) of 91.3 s [22.3–32.5] with low fibrinogen (FIB) < 0.25 G/L[2-4], and hypokalemia of 3.26 mmol/L [3.5–5.5]. The sequential organ failure assessment (SOFA) score was above 2 . A central venous catheter and a femoral artery catheter were inserted, red blood cell concentrates and fresh frozen plasma were transfused, low-dose noradrenalin  was administered, and the antibiotic was changed to 2.0 g of ceftriaxone sulbactam sodium  at 12-h intervals. Five hours after surgery, all vital signs except her body temperature stabilized. Eighteen hours after surgery, her temperature decreased to 37.8 °C, with a raised white blood cell count of 23.13 × 109/L, neutrophilia of 91.7%, procalcitonin above 100 ng/mL, a decreased platelet count of 64 × 109/L, hemoglobin of 71 g/L and hematocrit of 19.8%. The antibiotic dose was altered to 500 mg of intravenous imipenem/cilastatin sodium  at 6-h intervals. Five days after surgery, the results of the blood culture and microbe identification confirmed R. mannitolilytica. At that time, the patient’s body temperature had fluctuated to approximately 37.4 °C, and the hemogram decreased significantly . Treatment with imipenem/cilastatin sodium was continued because the drug sensitivity test of the bacteria could not be carried out in our laboratory. Eight days after surgery, the patient’s body temperature returned to normal. Blood analysis showed a white blood cell count of 12.09 × 109/L, neutrophilia of 76.6% and procalcitonin of 2.26 ng/mL. The antibiotic was changed to 2.0 g of intravenous cefoperazone sulbactam sodium at 6-h intervals for 6 days. All the blood results had returned to normal, the repeated blood culture was negative on day 14 postoperatively and she was discharged well on day 15. No abnormality was found during follow-up at half a year after discharge.In the anesthetic resuscitation room, she continued to shiver and complained of general numbness and breathlessness. She was febrile with a temperature of 40.7 °C. Her blood pressure dropped to its lowest point of 78/44 mmHg, she had tachycardia with a maximum heart rate of 177 bpm, and she had intermittent vaginal bleeding. However, her oxygen saturation remained at 98%. Hence, septic shock was suspected, but we were unable to rule out anaphylactic shock. Resuscitation was initiated with massive fluid replacement, intravenous dexamethasone , and a uterotonic agent, namely, 250 μg of intramuscular carboprost tromethamine . Concurrently, a Bakri Postpartum Balloon  was placed. Despite these treatments, her blood pressure remained unstable (< 90/60 mmHg). The blood investigations revealed metabolic acidosis , a raised procalcitonin of 94.5 ng/mL [0.0–0.5], an increased white blood cell count of 15 × 10R. mannitolilytica was first reported in 1995, and since then, there have been case reports on infections involving neonatal, oncological, dialysis and renal transplantation patients [R. mannitolilytica was first isolated in China from a patient with chronic obstructive pulmonary disease [R. mannitolilytica infects humans mainly through water sources and affects mainly immunocompromised groups [R. mannitolilytica infection. However, the source of the infection is still unknown. She had rapid progression to critical septic shock, and there were no subsequent cases in our hospital.patients –7. In 20 disease . R. mannd groups –8. The cd groups , 5, 7. HEscherichia coli, Streptococcus and Staphylococci are the causal bacteria of severe obstetrical infectious events [R. mannitolilytica, and this case confirms the notion that this opportunistic pathogen causes maternal infections. Due to the diversity of infected tissues and organs, the main clinical manifestations of R. mannitolilytica infection are pneumonia, bacteremia, and meningitis, and a few cases develop septic shock. The main symptoms of the current case were fever and hypotension, which progressed to septic shock a short time after the operation. These symptoms were basically consistent with descriptions in the literature [Obstetric sepsis is among the top 3 leading causes of maternal death worldwide, and can lead to multiple organ or system dysfunction and even failure . In mosts events . This isterature , 11, 12.R. mannitolilytica can produce various enzymes that can hydrolyze antibiotics. These compounds can confer resistance to a broad range of antibiotics, including benzylpenicillin, narrow-spectrum cephalosporins, ceftazidime, aztreonam, and carbapenems [When the pathogen was not clear, initial treatment with supportive therapy and an empirical broad-spectrum antibiotic strategy could be selected according to the American Critical Medical Association’s 2016 International guidelines for Treatment of Sepsis and Septic Shock . Concurrbapenems . Additiobapenems . In SaidR. mannitolilytica infection have a good prognosis [After treatment with antibiotics, most patients, though not those with cystic fibrosis, who are infected with rognosis . Howeverrognosis . AccordiR. mannitolilytica, a new opportunistic pathogen, can contribute to critical infectious events. As a special patient group, pregnant and postpartum women should be assessed for the possibility of infection with this pathogen if rapid sepsis and septic shock occur. Early diagnosis and appropriate antibiotics can decrease complications.In summary,


Francisella tularensis causes the deadly zoonotic disease tularemia in humans and is able to infect a broad range of organisms including arthropods, which are thought to play a major role in Francisella transmission. However, while mammalian in vitro and in vivo infection models are widely used to investigate Francisella pathogenicity, a detailed characterization of the major Francisella virulence factor, a noncanonical type VI secretion system (T6SS), in an arthropod in vivo infection model is missing. Here, we use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. tularensis subsp. novicida virulence. We report that G. mellonella larvae killing depends on the functional T6SS and infectious dose. In contrast to other mammalian in vivo infection models, even one of the T6SS effectors PdpC, PdpD, or OpiA is sufficient to kill G. mellonella larvae, while sheath recycling by ClpB is dispensable. We further demonstrate that treatment by polyethylene glycol (PEG) activates Francisella T6SS in liquid culture and that this is independent of the response regulator PmrA. PEG-activated IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK, OpiB1, OpiB2, and OpiB3. The results of larvae infection and secretion assay suggest that AnmK, a putative T6SS component with unknown function, interferes with OpiA-mediated toxicity but not with general T6SS activity. We establish that the easy-to-use G. mellonella larvae infection model provides new insights into the function of T6SS and pathogenesis of Francisella. Francisella tularensis is the causative agent of the deadly zoonotic disease called tularemia  and a high mortality rate if left untreated (up to 60%)  . In Euron humans .Francisella virulence depends on the Francisella pathogenicity island (FPI)  . Interescal FPIs . The FPIcal FPIs –7. T6SS al FPIs –. The T6Sal FPIs –. Contracal FPIs ––11. Dynal FPIs ––, 12, 13.F. tularensis subsp. novicida, six secreted T6SS effectors were identified as follows: PdpC, PdpD, OpiA, and OpiB1, OpiB2, and OpiB3 (OpiB1–3) (1–3 are encoded outside the FPI at different genomic sites (For OpiB1–3) . While POpiB1–3) , 16. Intic sites . OpiA waic sites . Conversic sites . The FPIic sites . Howeveric sites , 18, 19.Francisella is able to infect and survive in a wide range of hosts ranging from amoeba and insects to mammals . In addition, the main T6SS effectors PdpC and PdpD were dispensable for killing G. mellonella larvae. In contrast to mammalian in vivo infection models, individual effectors PdpC, PdpD, or OpiA were sufficient to kill G. mellonella larvae in a manner comparable to the parental strain. We demonstrate that Francisella T6SS can be activated in vitro by polyethylene glycol (PEG) in a PmrA-independent manner and use this to show that AnmK affects OpiA-mediated killing of G. mellonella larvae without altering T6SS activity or IglC secretion. In summary, our results suggest that G. mellonella larvae serve as a suitable model for testing roles of uncharacterized Francisella genes in infection.Here, we show that virulence of G. mellonella larvae as an in vivo infection model for Francisella, we first tested if F. tularensis subsp. novicida establishes infection in a T6SS-dependent manner. We used F. tularensis subsp. novicida U112 iglA-sfGFP  and compared to a phosphate-buffered saline (PBS) control  . Interestingly, the strains with PdpC, PdpD, and OpiA alone were as efficient in killing of larvae as the parental strain  killed larvae at the same rate as the T6SS-negative strain (ΔpdpB mutant)  induces assembly of IglA-sfGFP into dynamic structures in less than 20 min (pdpB mutant) cells  cells .Francisella, the orphan response regulator PmrA is required for regulation of FPI expression and intracellular replication upon environmental cues   . We alsol strain . This sullonella .Francisella is able to infect a broad range of arthropods, it is important to understand Francisella pathogenicity in suitable infection models. Here, we characterized the contribution of the noncanonical T6SS and its known effectors to F. tularensis subsp. novicida pathogenicity in an in vivo arthropod infection model, namely, G. mellonella larvae. Our data show that F. tularensis subsp. novicida robustly kills G. mellonella larvae in a T6SS- and dose-dependent manner  reverts the intermediate phenotype to parental strain-like killing of G. mellonella larvae ., 10.F. tnovicida  to F. ThG. mellonella larvae are an easy to handle and robust in vivo infection model for studying Francisella virulence and its T6SS. Moreover, this model makes it possible to uncover new functions and interactions between T6SS components as shown for AnmK and OpiA. Further investigations about why some effectors are more toxic in one infection model than another will lead to a more detailed understanding of the mode of action of different effectors. Intriguingly, a well-characterized arthropod in vivo model might help to study Francisella traits important for persistence in the environment and in potential reservoir hosts.In summary, we demonstrate that F. tularensis subsp. novicida U112 and derivative strains were grown in brain heart infusion (BHI) broth with aeration or on BHI agar plates at 37°C. The medium was supplemented with 0.1% l-cysteine (Acros Organics) and 100 μg/ml ampicillin (AppliChem) for overnight cultures and plates. Escherichia coli DH5α λpir and derivative strains were aerobically grown in Luria broth (LB) or on agar plates supplemented with 50 μg/ml kanamycin at 37°C. All strains are listed in Table S1 in the supplemental material.F. tularensis subsp. novicida in-frame deletion mutants were created with suicide vector pDMK3  of 1 was reached. Day cultures were washed once in LB and 1 ml of both donor and recipient strain culture was concentrated and mixed together. Conjugation took place on an LB agar plate supplemented with 300 μM 2,6-diaminopimelic acid at 25°C overnight. Then, the mixture was transferred on Mueller-Hinton agar plates supplemented with 0.1% l-cysteine, 0.1% d-glucose (Millipore), 0.1% fetal calf serum (BioConcept), 100 μg/ml ampicillin, and 15 μg/ml kanamycin to select for recipients containing the suicide vector. After incubation at 37°C for 2 days, colonies were restreaked on BHI agar plates supplemented with 0.1% l-cysteine, 100 μg/ml ampicillin, and 15 μg/ml kanamycin. Negative selection was carried out on LB agar plates supplemented with 0.1% l-cysteine, 5% sucrose, and 100 μg/ml ampicillin, which were incubated at room temperature for several days. All plasmids and remaining peptides of in-frame deletions are listed in Table S2. All cloning products were sequenced, and sites of homologous recombination were verified by PCR.or pDMK3  as reporor pDMK3 , 67. In nd Dehio  and conjGalleria mellonella larvae from TruLarv (BioSystems Technology) were used for all infection experiments. For each experiment and condition, 10 randomly chosen larvae were infected as previously described , and OD600 was adjusted to 1 in PBS. Ten-fold dilutions in PBS were carried out. Ten microliters of the 108, 106, or 104 CFU/ml dilution  was used for injection into the second left proleg with a Hamilton syringe . All infected larvae per condition were placed in one petri dish (Greiner Bio-One) and incubated at 37°C for 5 days. Survival was scored manually every 24 h. Death was defined as no movement of legs, head, or body. Pupated larvae were considered alive as long as they exhibited any movement but were censored and not considered for calculating the percentage of surviving larvae. As control for proper handling, each experiment included larvae injected with PBS.Weight and aged defined escribed . F. tulaG. mellonella larvae and after 5 days all remaining G. mellonella larvae were incubated at −20°C overnight before disposal.Petri dishes with 10 dead F. tularensis subsp. novicida strains was also used to determine the actual inoculum concentration. A total of 100 μl of the calculated 103 CFU/ml dilution was plated on Mueller-Hinton agar plates supplemented with 0.1% l-cysteine, 0.1% d-glucose (Millipore), 0.1% fetal calf serum (BioConcept), and 100 μg/ml ampicillin. The plates were incubated for 2 days at 37°C, and colonies were counted afterward.The prepared 10-fold dilution series of 600 of 0.2. After 3.5 h, the OD600 was adjusted to 2, and the bacterial cultures were washed twice with PBS and resuspended in 1 ml of BHI without l-cysteine. Then, 1 ml of 20% polyethylene glycol 4000 (Sigma-Aldrich) in BHI without l-cysteine was added so that a final PEG 4000 concentration of 10% was achieved. The cultures were incubated at 37°C shaking for 1 h. Afterward, 1 ml of the PEG 4000-treated samples was centrifuged at 16,000 × g for 1.5 min. A total of 0.9 ml of supernatant was used for trichloroacetic acid (TCA)/acetone protein precipitation. In brief, 100 μl of 100% TCA (wt/vol) (Sigma-Aldrich) was added to the harvested supernatants, followed by incubation at 4°C for 10 min with mixing in between. After centrifugation at 18,000 × g and 4°C for 5 min, the precipitated proteins were washed twice with cold acetone (Merk Millipore) and left to dry at room temperature. Then, the precipitated proteins were resuspended in 40 μl 1× lithium dodecyl sulfate (LDS) buffer (Thermo Fisher). The remaining bacterial cells were resuspended in 100 μl PBS, boiled at 95°C for 10 min, and sonicated afterwards. Thirty microliters of these samples were mixed with 10 μl 4× LDS buffer.Overnight cultures were washed twice with PBS and then resuspended in BHI and used for inoculation of day cultures without antibiotics at an ODSamples prepared for the T6SS-dependent secretion assay (see above) were supplemented with 4 μl of 1 M dithiothreitol  and incubated at 72°C for 10 min. Then, 20 μl of the samples was loaded on 10% polyacrylamide gels, and proteins were separated by gel electrophoresis. For immunodetection, proteins were transferred to a nitrocellulose membrane (25 V for 45 min). After blocking of the nitrocellulose membrane in 5% milk in Tris‐buffered saline containing Tween 0.1% (TBST) at room temperature for 2 h and three washing steps with TBST for 5 min each, the nitrocellulose membrane was incubated with the primary antibody at room temperature for 2 h. Primary α-IglC antibody  was used at a final concentration of 1 μg/ml in 5% milk in TBST. Incubation for 1 h with secondary antibody α-rabbit conjugated to horseradish peroxidase (Jackson ImmunoResearch) at a final concentration of 30 ng/ml in 5% milk in TBST followed. LumiGLO chemiluminescent substrate (KPL) was used for detection of peroxidase on a gel imager (GE ImageQuant LAS 4000). Exposure time is given in the figure legend.F. tularensis subsp. novicida parental and mutant strains were inoculated from plate at an OD600 of 0.2 without antibiotics. At an OD600 of 1, the cultures were concentrated in phosphate saline buffer to an OD600 of 10. A total of 1.5 μl of the concentrated cultures was then spotted on a pad consisting of 1% agarose in phosphate saline buffer. The agarose pad was covered with a cover slip and incubated at 37°C for 1 h before imaging at 30°C and 95% humidity . To monitor T6SS activation through PEG 4000 treatment (see T6SS-dependent secretion assay), 1.5 μl of liquid culture was spotted on a pad consisting of 1% agarose in BHI, covered by a coverslip, and imaged immediately. Images were collected every 30 s for 5 min.Microscope set up was described previously , 11, 70.Image analysis was carried out with Fiji software  as previG. mellonella larvae batches were performed. Mutants of a given set were tested in the same infection experiments. Pooled and single survival plots were calculated with Prism 8 (GraphPad Software). For more clarity, the graphs contain only data of the indicated mutant and the controls  of the whole experiment. Thus, for a given set of mutants, the controls are the same for individual graphs. Standard errors were calculated for pooled survival plots. The log rank (Mantel-Cox) test in combination with Bonferroni corrected threshold  was used to determine if compared curves are significantly different. P values are given in the figure legends.Three infection experiments with independent Number of T6SS assemblies per bacterium was quantified in biological replicates. The smallest number of analyzed bacteria for a data set is given in the figure legend. Means with standard deviation were calculated. To test for significant differences in means, the Tukey’s multiple-comparison test with a confidence level of 95% in Prism 8 (GraphPad software) was used.


Botrytis cinerea (the cause of grey mould). Production of the associated phytotoxic metabolites botrydial and dihydrobotrydial was measured by our newly developed and validated HRAM UPLC-MS/MS method, and was also shown to be reduced by this treatment. With a light dose of 120 J/cm2, the reduction in spore viability was directly proportional to curcumin concentrations, and the overall concentration of both botrydial and dihydrobotrydial also decreased with increasing curcumin concentration above 200 µM. With curcumin concentrations above 600 µM, the percentage reduction in fungal spores was close to 100%. When the dye concentration was increased to 800 µM, the spores were completely inactive and neither botrydial nor dihydrobotrydial could be detected. These results suggest that curcumin-mediated photosensitization is a potentially effective method to control B. cinerea spoilage, and also to reduce the formation of these phytotoxic botryane secondary metabolites.Photosensitization is a novel environmentally friendly technology with promising applications in the food industry to extend food shelf life. In this study, the natural food dye curcumin, when combined with visible light (430 nm), was shown to be an effective photosensitizer against the common phytopathogenic fungi  Botrytis grey mould. Botrytis cinerea is the causal agent of this disease, which effects not only strawberries, and is considered one of the most important phytopathogens of horticultural crops. This fungus is found in more than 200 ornamental and agriculturally important plants in the world, and ranks second in the list of ‘top 10’ fungal plant pathogens + , further loss of CO  and an additional loss of either H2O to C14H19  or a second CO to C13H21 . Such losses are consistent with a protonated form of the botrydial structure (1). For dihydrobotrydial in ESI+ mode the major MS/MS fragmentation ions of [M+Na]+m/z 335.1825  , further loss of NaOH and H2O . Such losses are consistent with the dihydrobotrydial structure (2), and demonstrate attachment of the Na+ ion within the hydroxy lactol arrangement of this compound.For botrydial in ESI+ mode, the major MS/MS fragmentation ions of [M+H]311.1853  correspo35.1829) d corresp1) of m/z 311.1853 → 205.1586 and for dihydrobotrydial (2) of m/z 335.1829 → 275.1614 were selected for quantification, with secondary transitions utilized for confirmation of botrydial (1) (m/z 311.1853 → 187.1481 and m/z 311.1853 → 233.1534) and dihydrobotrydial (2) (m/z 335.1829 → 217.1586). The calibration curves based on these quantification PRM responses were linear over the range 1.02–9.92 µg/L for botrydial and 9.22–89.28 µg/L for dihydrobotrydial. Rapid separation (<7 min), good reproducibilities (average RSDs < 10%), and good recoveries (77.2% and 83.5%) were obtained in the HRAM UPLC-MS/MS analysis of both botrydial (1) and dihydrobotrydial (2) from spiked agar plates  transitions for botrydial  and D−L+ treatment  produced characteristic prolific greyish-brown spores, and visible differences in fungal growth was readily apparent with increasing curcumin concentrations after photosensitization treatment and incubation for 8 days  reduction in the spores was also observed when the spores were treated with curcumin-mediated photosensitization. Treatment with light or curcumin alone (D−L+ or D+L−) was not effective. The curcumin-mediated photosensitization (D+L+) reduction in spore germination was directly proportionate to curcumin concentrations, with increasing concentrations of curcumin photosensitizer acting to drastically inhibit B. cinerea conidia germination. With curcumin concentrations above 600 µM and light dose of 120 J/cm2, spore germination was completely (100%) inhibited (p < 0.001) (The effect of increasing curcumin dye (D) concentrations with a photosensitization light dose (L) of 120 J/cmr 8 days . With in survive . As show< 0.001) .p < 0.05), compared to control or light alone (D−L− or D−L+). The control treatment (D−L−) contained botrydial and dihydrobotrydial at measured concentrations of 5.57 ± 1.13 µg/g and 140.50 ± 45.87 µg/g, respectively, providing a dihydrobotrdial/botrydial ratio of 25:1  and under control conditions (D−L−). The percentage reduction in total spores reached 100% with the curcumin-mediated light doses of 120, 240, and 360 J/cm2 (+L+) are used at the same time with light doses of 120, 240 and 360 J/cm2 (+L−), comparing to the control (D−L−) (p < 0.05). However, the dihydrobotrydial concentration was not significantly different between the curcumin-only treatment (D+L−) and the control (D−L−) (As shown in 60 J/cm2 . Similar60 J/cm2 . The botl (D−L−) .B. cinerea with increasing incubation time (−L−) mycelial growth was fast with white hyphae apparent within two days incubation. With prolonging incubation time, the three treatments  display the expected regular fungi morphology. In contrast, with the curcumin treatment (D+L+), the spores did not germinate during the 16 days incubation. As shown in −L+) and (D+L−) demonstrated a general increase in production of both secondary metabolites reaching 8.31 ± 0.54 and 11.18 ± 1.53 µg/g of botrydial, and 179.69 ± 30.04 and 205.29 ± 53.71 µg/g of dihydrobotrydial, respectively, at 15 days. The control treatment (D−L−) produced a more variable botrydial concentration (+L+) were below limits of detection for both botrydial and dihydrobotrydial  is 19.42 ± 3.62, 21.83 ± 3.52 and 15.34 ± 3.20, respectively.In studies of effect of different treatments on ion time , it was ntration  with a motrydial , which iB. cinerea cultures as described here.High-resolution accurate mass (HRAM) spectrometers, such as the time-of-flight (TOF) and the Orbitrap spectrometer, has become more accessible and affordable to many laboratories in recent years. One of the biggest strengths of a HRAM instrument is its ability to acquire accurate mass data, which allows the user to relate the observed accurate mass to an equivalent elemental composition/s and molecular formula/s. Molecular formulas alone, however, are not sufficient to identify specific compounds. Without applicable standards, erroneous identifications can result due to the many possible compounds with the same molecular formula, and for this reason we have chosen to isolate authentic botrydial/dihydrobotrydial to use as standards in this study. These metabolites are not commercially available but can be obtained from + and [M+NH4]+ molecular ions of botrydial. El Fellah et al. [2 fragmentations of the protonated molecular ion [M+H]+ C17H27O5  which they ascribed to botrydial. These authors used the co-detection of a second component identified on the basis of accurate mass  as abscisic acid, a compound also associated with B. cinerea fungi, to support the identification of botrydial in their water samples. Authentic botrydial and abscisic acid standards were stated to be not available and identification was based on ‘accurate mass method with UHPLC-HRMS’. Notably however according to SciFinder searches, there are 1648 different compounds known with molecular formulae C17H26O5. Each of these 1648 compounds would exhibit the same [M+H]+ 311.1853 Da  but expected fragmentation would be different. On the basis of HRAM, the sequential losses reported by El Fellah et al. + molecular ions reported by Niedzwiecki et al. + 311.1853) and dihydrobotrydial . The source settings were set as follows: spray voltage at 3500 (+), capillary temperature at 250 °C, sheath gas at 45 (arbitrary units), Aux gas flow rate at 10, Aux gas heater temperature at 400 °C, S-lens RF level at 50 and Sweep gas flow rate at 2. A 5 ppm mass accuracy window was set for the mass extraction and detection setting. The HRAM UPLC-MS/MS system was controlled by Xcalibur™ 4.1 software and the MS data were processed using TraceFinder™ 4.1 software.The HRAM MS/MS was acquired on a Q-Exactive mass spectrometer  with a heated electrospray ionization (HESI) probe. The MS data were collected in the parallel reaction monitoring (PRM) positive mode at 35,000 FWHM mass resolution, AGC target at 2.0e5, maximum IT at 100 ms, mass isolation window of 4.0 m/z, Normalized Collision Energy (NCE) at 35 and an inclusion list containing botrydial (C1) of m/z 311.1853 → 205.1586 for quantification (and m/z 311.1853 → 187.1481 and m/z 311.1853 → 233.1534 for confirmation) and for dihydrobotrydial (2) of m/z 335.1829 → 275.1614 for quantification (and m/z 335.1829 → 217.1586 for confirmation).External standards in the range 1.02–9.92 µg/L botrydial and 9.22–89.28 µg/L dihdrobotrydial were used for quantification with PRM transitions for botrydial (p < 0.05 (significant), or p < 0.001 (highly significant) were applied to determine any significant differences between specific means.All experiments were performed in triplicate. The value were expressed as mean ± SD. The analysis of statistical difference was performed using the TTEST function in Microsoft Excel 2010. Two-tails and ‘type’ for the test were choosen by the correspondence and variance of the data with FTEST.


This study aimed to compare the diagnostic value of the single or combined applications of transient elastography (TE) and multivariate indicators with biopsy for the detection of liver fibrosis in children caused by chronic hepatitis B (CHB).This study included 148 CHB children treated at Hunan Children’s Hospital from January 1st 2015 to December 31st 2018, aged from 0.83 to 14.58 years old. All patients underwent liver biopsy (LB), of which 43 patients underwent TE. Multiple clinical data, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), Platelet (PLT), and HBV-deoxyribonucleic acid (HBV DNA) of all patients were collected. The diagnostic values for CHB of TE and its combinations with these indicators were measured. The patients were classified in two ways: no hepatic fibrosis group (F0) versus fibrosis group (F ≥ 1), and no significant hepatic fibrosis group (F < 2) versus significant hepatic fibrosis group (F ≥ 2). The statistical assessment was performed between groups within each classification to compare the diagnostic value of different parameters.AUC) of liver fibrosis diagnosed by liver stiffness measurement (LSM) which obtained by TE, AST-to-PLT ratio index (APRI), and fibrosis-4 index (FIB-4) were 0.740, 0.701, and 0.651, while the corresponding cut-off values were 5.9 kPa, 0.50, and 0.10, respectively. The AUC of significant liver fibrosis diagnosed by LSM, APRI and FIB-4 were 0.849, 0.701, and 0.509, while the corresponding cut-off values were 8.4 kPa, 0.76, and 0.08, respectively. While with the combinations of LSM and APRI, LSM and FIB-4, LSM and APRI and FIB-4, APRI and FIB-4, the AUC of significant liver fibrosis were 0.866, 0.855, 0.869, and 0.684, respectively. The AUC of significant liver fibrosis diagnosed by the LSM was significantly higher than APRI and FIB-4.The operating characteristic area under curve (The diagnostic value of transient elastography was better than that of APRI and FIB-4 for CHB children with significant liver fibrosis. In addition, TE also has relatively high application values on the diagnosis of patients with different degrees of liver fibrosis caused by CHB. Hepatitis B virus (HBV) infection is a widespread public health threat across the world. The hepatitis B serum epidemiological study held by the Chinese Center for Disease Control and Prevention (CDC) in 2014 discovered that the HBsAg detection rate was above 1.26% in children in China –5. In clCurrently, liver biopsy (LB) serves as the gold standard for the diagnosis and staging of liver fibrosis. However, LB is an invasive examination with complications such as right upper quadrant pain, bleeding, infection, pneumothorax, and so on . Further1-α/2 × √se[1] × (1 − se)/0.1)2 = (1.96 × √0.915 × (1–0.915)/0.1)2 ≈ 30 [This study recruited 161 hospitalized patients diagnosed with CHB (aged 0–18 years old) at Hunan Children’s Hospital from January 1st 2015 to December 31st 2018. Among them, 2 patients were excluded due to complicated infections with hepatitis D and 3 patients due to nonalcoholic fatty liver. At the same time, 3 patients who refused liver biopsy and 5 patients with incomplete data were also excluded. Finally, 148 eligible patients were enrolled  and fluorescence probe were used to detect the serum load of HBV DNA with the aid of ankle brachial index (ABI) 7500 instrument  and new HBsAg and anti-HBs kit.Liver function was monitored by using the Bayer-2400 automatic biochemistry analyzer. The reference ranges of ALT and AST were from 0 to 40 U/L. Peripheral platelets were assayed by the ADVIA2120 automated hematology analyzer and its corollary reagent manufactured by Bayer, Germany.The patients were maintained at the supine position or at the slightly lateral recumbent position to determine the puncture with B-ultrasound localization. Percutaneous liver biopsy was performed with Bard needle to obtain the liver tissue with 10% formaldehyde fixation fixed, HE stained and slices routinely prepared. After that, the same senior pathologist should read the slices in a unified manner and observe the pathological changes by the electron microscope to divide the degrees of liver fibrosis into stage F0 to F4 according to the Scheuer scoring system [The ultrasonic liver cirrhosis detector produced by Shenzhen Integrated Medical Technology Co, Ltd. was used, which is also a detection technique based on transient elastography. The intercostal space was chosen to measure the hardenability of liver parenchyma. The value of LSM was obtained after the measurement of effective values for ten times. The accuracy of the LSM value included shall be more than 60% with the deviation value being less than 1/3 of the median. All procedures were performed by professionally trained physicians. We performed the examination of transient elastography when children fell asleep. For some children who cannot cooperate with the operation, we have sedated the children with the consent of their parents.F0: No fibrosis; F1: Fibrous expansion of portal areas; F2: Periportal fibrosis or portal-portal fibrous septa but intact architecture; F3: Fibrosis with architectural distortion but no obvious cirrhosis; F4: Probable or definite cirrhosis.IQR) were calculated for continued non-normal distribution measurement data as APRI, FIB-4 index, AAR, and LSM values. Categorical variables were expressed as the number and percentage of patients. Liver fibrosis stages F0 to F4 were identified by the gold standard for liver fibrosis according to LB.We classified patients into two groups in two ways: no hepatic fibrosis group (F0) and fibrosis group (F ≥ 1), no significant hepatic fibrosis group (F < 2) and significant hepatic fibrosis group (F ≥ 2). Based on the above described classification, the receiver operating characteristic (ROC) curves of the APRI, FIB-4 index, AAR, and LSM values were created and the area under the receiver operating characteristic curve (AUC) was calculated within each group. Discrimination and calibration of the model were assessed by AUC, the predictive value was classified as low (AUC = 0.500–0.700), moderate (AUC = 0.700–0.900), or high (AUC = 0.900–1.000). Calibration across deciles of risk was evaluated using the Hosmer–Lemeshow goodness-of-fit test. For this test, a P-value > 0.05 indicates good calibration [PPV), negative predictive value (NPV), positive likelihood ratio (+ LR) and negative likelihood ratio (− LR). The ROC contrast test was used to compare ROC curves. Excel software was used to calculate the Z value based on the comparison of AUC between different groups through the calculation formula (Z = (S1 − S2)/(SE1 ^ 2 + SE22) ^ 0.5), where S1 and S2 indicate the area under ROC curve while SE1 and SE2 signify the corresponding standard error. Then the value of P was calculated by the formula P value = 1 – NORMSDIST .SPSS 20.0 software and MedCalc software were used for statistical analysis. Median and inter-quartile range  were under 1 year old, 42 (28.40%) were 1–3 years old, 47 (31.80%) were 3–5 years old, 40 (27.00%) were 5–10 years old, and 13 (8.80%) were above 10 years old.There were 28 (18.92%) cases with F0, 94 (63.51%) cases with F1, 19 (12.84%) cases with F2, 5 (3.38%) cases with F3 and 2 (1.35%) cases with F4  were 1–3 years old, 14 (32.50%) were 3–5 years old, 16 (37.20%) were 5–10 years old, and 4 (9.30%) were above 10 years old. There were 9 (20.93%) cases with F0, 23 (53.49%) cases with F1, 8 (18.60%) cases with F2, 2 (4.65%) cases with F3 and 1 (2.33%) case with F4  patients had a history of hepatitis B virus infection.The detailed information was shown in Table AUC for the diagnosis of liver fibrosis in CHB patients was 0.740 (95% CI: 0.543–0.938), and the cut-off value, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (+ LR) and negative likelihood ratio (− LR) for fibrosis were 5.9 kPa, 94.12%, 55.56%, 88.90%, 71.40%, 2.12 and 0.11 respectively. AUC for the diagnosis of liver fibrosis in CHB patients based on APRI value was 0.701 (95% CI: 0.603–0.800), the cut-off value, Se, and Sp for fibrosis were 0.50, 60.00%, and 78.57%, respectively. Based on the FIB-4 index, AUC for the diagnosis of liver fibrosis was 0.651 (95% CI: 0.546–0.755), the cut-off value, Se, and Sp for fibrosis were 0.10, 65.00%, and 64.29%, respectively. As for AAR value, AUC for the diagnosis of liver fibrosis was 0.440 (95% CI: 0.328–0.552), the cut-off value, Se, and Sp for fibrosis were 0.90, 35.83%, and 85.71%, respectively. AUC for the diagnosis of liver fibrosis in CHB patients based on a combination of LSM, APRI and FIB-4 was 0.771 (95% CI: 0.580–0.942), which meant that the combination had a good discrimination of liver fibrosis, and a result of the Hosmer-Lemeshow goodness-of-fit test for logistic regression confirmed that the combination was well calibrated . The AUC value (0.740) of liver fibrosis diagnosed by LSM value was higher than that of APRI value (0.701), the differences of which showed no statistical significance . The AUC value (0.740) of liver fibrosis diagnosed by LSM value was higher than that of FIB-4 value (0.651), the differences of which showed no statistical significance . The AUC value (0.771) of liver fibrosis diagnosed by combining LSM and APRI as well as FIB-4 index was higher than that of LSM value (0.740), which showed no significant difference . The values of Se, Sp, PPV, NPV, + LR and − LR in the diagnosis of liver fibrosis with biochemistry indicators were listed, all of which were shown in the following TableBased on LSM value, AUC for the diagnosis of significant liver fibrosis in CHB patients based on LSM value was 0.849 (95% CI: 0.713–0.986), and the cut-off value, Se, Sp, PPV, NPV, + LR and − LR for significant liver fibrosis were 8.4 kPa, 81.82%, 78.12%, 56.20%, 92.60%, 3.74 and 0.23 respectively. AUC for the diagnosis of significant liver fibrosis in CHB patients based on APRI value was 0.701 (95% CI: 0.591–0.810), the cut-off value, Se, and Sp for fibrosis were 0.76, 61.54%, and 72.95%, respectively. Based on the FIB-4 index, AUC for the diagnosis of significant liver fibrosis was 0.509 (95% CI: 0.388–0.630), and the cut-off value, Se, and Sp for fibrosis were 0.08, 84.62%, and 27.87%, respectively. As for AAR value, AUC for the diagnosis of significant liver fibrosis was 0.458 (95% CI: 0.329–0.586), the cut-off value, Se, and Sp for fibrosis were 1.13, 69.23%, and 48.36%, respectively. AUC for the diagnosis of significant liver fibrosis in CHB patients based on a combination of LSM, APRI and FIB-4 was 0.869 (95% CI: 0.741–0.998), which means that the combination had a good discrimination of significant liver fibrosis, and a result of the Hosmer-Lemeshow goodness-of-fit test for logistic regression confirmed that the combination was well calibrated . The AUC value (0.849) of significant liver fibrosis based on LSM value was higher than that of APRI value (0.701), the differences of which reached statistical significance . The AUC value (0.849) of significant liver fibrosis based on LSM value was higher than that of FIB-4 (0.509),the differences of which reached statistical significance . The AUC value (0.869) of significant liver fibrosis by the combination of LSM value and APRI value as well as FIB-4 index was higher than that of LSM value (0.849). There were no significant statistical differences in this case when Z was equal to 0.208 and P was equal to 0.418. The values of Se, Sp, PPV, NPV, + LR, and − LR in the diagnosis of significant liver fibrosis with biochemistry indicators were listed. They were clearly shown in the following Table AUC value above 0.700. TE and APRI could perform well in differentiating the stages of liver fibrosis in children with CHB. The results of LSM and APRI in the diagnosis of CHB patients with significant liver fibrosis were good, with the AUC values all being more than 0.700. It was shown that TE can better differentiate patients with significant liver fibrosis than APRI. The diagnostic value of TE was better than that of APRI and FIB-4 for CHB children with significant liver fibrosis. The combination of TE and multi-parameter indicators failed to significantly improve their abilities in the diagnosis of varying degrees of liver fibrosis in our study.TE is a relatively mature non-invasive diagnostic technique for liver fibrosis. The ultrasonic liver cirrhosis detector used in our study is an operation instrument based on the principle of TE. It has the same advantages of noninvasiveness and good repetitiveness as Fibroscan and Fibrotouch. This study showed that TE and APRI were effective in diagnosing young CHB patients with liver fibrosis, with an AUC values all being less than 0.700, which was in accord with the findings of Eminler et al. [Our results showed that TE was superior to APRI and FIB-4 index in the diagnosis of hepatic fibrosis in terms of value, which was consistent with the results obtained by Orasan et al.  and Guo r et al. . HoweverAUC values were calculated to analyze the diagnostic value of TE and its combination of different parameters on liver fibrosis. The results showed that the combination of TE and multi-parameter indicators failed to significantly improve their abilities in the diagnosis of varying degrees of liver fibrosis, which was complied with the results by Zeng et al. [The g et al. . Howeverg et al. . The samOur results showed that the best cut-off values of LSM for the diagnosis of liver fibrosis and significant liver fibrosis were 5.9 kPa and 8.4 kPa, respectively. In this case, the sensitivities were 94.12% and 81.82%, and specificities were 55.56% and 78.12%, respectively. Goyal R. found that adults with liver fibrosis in stage F2, F3 can be excluded by FibroScan measured LSM value < 6 kPa, and LSM value > 9 kPa indicated that the degree of liver fibrosis reaches stage F2 or F3, and its sensitivity and specificity were above 90% . Chang PThis study had several limitations. First, the number of patients, especially the sample size of patients with the F3-F4 fibrosis stage was small, and the number of patients we selected who were performed TE was small.Second, in the practical operation of TE testing, the difficulty to cooperate with young children and the impact of related factors such as the age, BMI and laboratory indicators of the children could interfere with the accuracy.Third, there are five scoring systems to evaluate the degree of liver fibrosis. In this study, we used Scheuer scoring systems, which might not be the most accurate one. Further studies to compare the accuracy of the different scoring systems are necessary. Future studies need to utilize larger sample sizes and stratify the patients according to their age, BMI and laboratory indicators.Transient elastography performed better than APRI and FIB-4 index in the diagnosis of young CHB patients with significant liver fibrosis, with a relatively high application value for the clinical diagnosis of CHB patients with varying degrees of liver fibrosis. However, we need to consider the impact of relevant clinical factors on the test results in the process of application.


Some ferns have medicinal properties and are used in therapeutic interventions. However, the classification and phylogenetic relationships of ferns remain incompletely reported. Considering that chloroplast genomes provide ideal information for species identification and evolution, in this study, three unpublished and one published ferns were sequenced and compared with other ferns to obtain comprehensive information on their classification and evolution.Dryopteris goeringiana (Kunze) Koidz, D. crassirhizoma Nakai, Athyrium brevifrons Nakai ex Kitagawa, and Polystichum tripteron (Kunze) Presl were sequenced using the Illumina HiSeq 4,000 platform. Simple sequence repeats (SSRs), nucleotide diversity analysis, and RNA editing were investigated in all four species. Genome comparison and inverted repeats (IR) boundary expansion and contraction analyses were also performed. The relationships among the ferns were studied by phylogenetic analysis based on the whole chloroplast genomes.The complete chloroplast genomes of Pi) values of > 0.02 were identified. A total of 75–108 SSRs were identified, and only six SSRs were present in all four ferns. The SSRs contained a higher number of A + T than G + C bases. C‐to‐U conversion was the most common type of RNA editing event. Genome comparison analysis revealed that single‐copy regions were more highly conserved than IR regions. IR boundary expansion and contraction varied among the four ferns. Phylogenetic analysis showed that species in the same genus tended to cluster together with and had relatively close relationships.The whole chloroplast genomes ranged from 148,539 to 151,341 bp in size and exhibited typical quadripartite structures. Ten highly variable loci with parsimony informative (The results provide valuable information on fern chloroplast genomes that will be useful to identify and classify ferns, and study their phylogenetic relationships and evolution. Study reported three newly sequenced chloroplast genomes of ferns.Study provided more information on understanding evolution and phylogenetic of ferns. A phylogenetic tree was constructed based on the chloroplast genomes of almost all ferns reported thus far. The present study was conducted to achieve the following objectives: a) to sequence and report the chloroplast genomes of D. crassirhizoma, D. goeringiana, A. brevifrons, and P. tripteron; b) to compare the chloroplast genomes of eight fern species to identify useful DNA barcodes for plant identification and evolution analysis; and c) to identify a more comprehensive phylogenetic relationship among ferns.22.1D. crassirhizoma, A. brevifrons, D. goeringiana, and P. tripteron were collected from Maoer Mountain, Maoershan Town, Shangzhi City, Heilongjiang Province , China , 2018–22 (A. brevifrons), 2018–32 (D. goeringiana), and 2018–33 (P. tripteron).Wild specimens of 2.2260/280 of 1.8–2.0 were used for library preparation.Fresh leaves were collected, immersed in liquid nitrogen immediately, and stored at − 80℃ prior to DNA extraction. We isolated chloroplast DNA using an improved extraction method  protocol. The libraries were sequenced using the Illumina HiSeq 4,000 platform  . Then, the chloroplast genomes were assembled in three steps as follows , AUGUSTUS , and EVidenceModeler version 1.1.1, respectively. Protein‐coding genes, tRNA genes, and rRNA genes were predicted using the DOGMA tool  was used to detect the SSRs of D. crassirhizoma, A. brevifrons, D. goeringiana, and P. tripteron. The minimum number of repeats was set to eight, five, and four, for mononucleotide, dinucleotides, and trinucleotides, respectively, and to three for tetranucleotides, pentanucleotide, and hexanucleotides. The distance between two SSRs should be shorter than 100 bp. The SSR primers were designed using Primer3 (http://www.simgene.com/Primer3).MIcroSAtellite identification tool (2.5Pi) characters per‐site values to identify the most variable chloroplast genes using MAFFT 7.123b  and Variscan 2.0 (http://www.ub.es/softevol/variscan). Pi values were calculated with a step size of 200 bp and a slide window of 300 bp. Loci with a Pi value of > 0.20 were considered as highly variable regions.We measured the parsimony informative  in Shuffle‐LAGAN mode. We compared IR boundary expansion and contraction in all the above‐mentioned ferns, except P. glycyrrhiza.Genome comparison analysis was conducted for 2.8Adiantum capillus‐veneris and Myriopteris (Cheilanthes) lindheimeri were included as outgroup species.MAFFT v7.149 was used to align the cpDNAs sequences under default parameters  raw reads were obtained from the four ferns (Table D. crassirhizoma) to 151,341 bp (A. brevifrons). The chloroplast genomes had a circular assembly and exhibited a typical quadripartite structure  contained one intron and three genes  contained two introns 2, (ATCT)1, and (TTTA)1 also detected   Figure . Ten loc3.4D. crassirhizoma, 55 in A. brevifrons, 50 in D. goeringiana, and 78 in P. tripteron. C‐to‐U conversion  was the most prevalent RNA editing event, followed by U‐to‐C , A‐to‐G , and G‐to‐A .RNA editing is defined as the post‐transcriptional modification of precursor RNAs to alter their nucleotide sequences through the insertion and deletion, or specific substitution of nucleotides to introduce or remove start or stop codons or yield functional RNA species , whereas that of P. glycyrrhiza was the smallest . The chloroplast genomes ranged from 148,974 bp to 150,987 bp within Dryopteris genus, and the difference between the largest and smallest genomes was only 2,013 bp. However, this difference was 10‐fold in Polypodium . Additionally, rpoC2, rpoB, psbC, pasA, rbcL, ycf2, ycf1, and ndhB were identified to be divergent among these chloroplast genomes , and that tRNA genes were more diverse than protein‐coding and rRNA genes, which possibly play an essential role in fern evolution.The chloroplast genome provides information that is valuable for species identification, population genetics, plant phylogenetics, and genetic engineering. In the present study, three previously unpublished and one published fern were sequenced and compared with other species. The chloroplast genomes, which were 148,539–151,341 bp in length in the present study, were within the limit of fern chloroplast genomes  reported by Gao et al.,  and RuizclpP, rps12, and ycf3 have been found to be two‐intron genes  had a Pi value of > 0.20, except rpl16. Intron‐containing genes are more highly conserved than exon‐containing genes only in the chloroplast genome. In other words, higher variability was found in exon‐containing genes, which provided more valuable information for species evolution. This result was consistent with that in fern plastomes . Ferns have higher G + C content (41.49 ± 3.27) than gymnosperm (37.87 ± 1.56), angiosperm (37.71 ± 1.10), bryophyte (33.12 ± 4.16), and green algae (32.47 ± 6.07) ; Funding acquisition (lead); Writing‐original draft (lead); Writing‐review & editing . Wei Ma: Methodology (lead); Visualization ; Writing‐review & editing (supporting). Shilei Liu: Software (lead); Writing‐review & editing (supporting). Qingyang Huang: Conceptualization ; Funding acquisition (supporting); Resources (lead); Writing‐review & editing .Table S1Click here for additional data file.Table S2Click here for additional data file.


We aimed to investigate changes in volume and MRI T2‐weighted intensity in desmoid‐type fibromatosis (DF) receiving methotrexate plus vinca‐alkaloids (MTX‐VA) at Istituto Nazionale dei Tumori, Milan.All cases of sporadic DF treated with MTX‐VA from 1999 to 2019 were reviewed. MRIs at baseline, 6 and 12 months of chemotherapy and at treatment withdrawal were retrospectively reviewed, contouring the tumor lesion and measuring diameters, volume, and mean T2‐signal intensity  changes. These parameters were also evaluated according to clinical variables.Thirty‐two DF patients were identified. Best RECIST response was: 25% partial response, 69% stable disease, 6% progression. A ≥65% tumor volume reduction was observed in 38%, <65% reduction in 53%, an increase in 9%. 22% had RECIST stable disease with a ≥65% tumor volume reduction. T2‐signal intensity decreased by ≥50% in 47%, <50% in 41% and increased in 12%. In patients with symptomatic improvement while on therapy and in patients maintaining symptomatic improvement during follow‐up, median T2‐signal intensity showed a reduction along the time points ; in patients without symptomatic improvement and in those clinically progressing during follow‐up, a reduction was not observed. High T2‐signal intensity at baseline was observed in patients showing RECIST progression during follow‐up.In this series, RECIST detected a lower proportion of responses as compared to volumetric and T2‐signal changes. T2‐signal reduction seemed to better reflect symptomatic improvement. High T2‐signal intensity at baseline was related to a higher proportion of further progression. In this series of patients with sporadic desmoid‐type fibromatosis treated with low‐dose chemotherapy and evaluated with MRI, T2 signal reduction was detected in more patients as compared to RECIST responses and volume reductions, and seemed to better reflect symptomatic improvement and worsening. RECIST may have underestimated treatment responses as compared to volumetric and T2 signal intensity changes. A high T2 signal intensity at baseline seemed to be related to a higher risk of RECIST progression after the end of chemotherapy. FAP‐related DF was excluded. Cases were retrieved from a prospectively maintained database including all the DF patients treated at INT. Institutional ethics committee's approval was obtained. Patient medical records were examined to collect the following data: age, gender, tumor site, prior therapies, symptoms at baseline, chemotherapeutic regimen, reason for therapy discontinuation, symptomatic changes during chemotherapy and after therapy withdrawal, and additional treatments during follow‐up. MTX‐VA was administered weekly until reaching a number of cycles between 40 and 50, or until reaching one year of treatment, or the evidence of progression or toxicity.IntelliSpace Tumor Tracking software (Philips); three orthogonal diameters, volume, and mean T2‐signal intensity of each lesion  were obtained from all the available MRI examinations.MRI at baseline, at 6 and 12 months of chemotherapy, at the end of treatment, and yearly during follow‐up were retrospectively extracted from the institutional Picture Archiving and Communication System and analyzed. Follow‐up data were censored at the date of starting a further treatment or at the last patient contact, in case of event‐free patients. Patients evaluated by CT or whose MRI examinations were not available or did not include T2‐weighted sequences were excluded. A radiologist performed manual segmentation of each entire tumor lesion in a slice‐by‐slice fashion using For each of the three pivotal variables , descriptive statistics were used to describe the radiological data at each time point  by considering their original continuous scale. Spaghetti plots were used to graphically depict the patient's trends over time, as well as their median trends. Moreover, the same analysis and graphical representation were performed by considering as pivotal variables the relative change from baseline (i.e. percentage of baseline). Then, for each radiological parameter, the best response during treatment was defined as the maximum reduction observed among the considered time points relative to the baseline values. A bar plot was adopted to graphically depict these patterns among patients. In addition, to aid the interpretation of the results, each pivotal variable was evaluated according to pre‐specified cut‐off values. Specifically, one‐dimensional response was categorized according to RECIST 1.133.1Thirty‐two cases from a pool of 70 sporadic DF patients treated with MTX‐VA at INT from May 1999 to June 2019 were identified. Patient's characteristics and treatment received are shown in Table Thirty patients (94%) completed the scheduled treatment, while 1 (3%) interrupted it for RECIST PD and 1 (3%) for therapy intolerance with stable disease (SD).The median follow‐up duration was 47.6 months (range 11.4–234.0 months). After the therapy withdrawal, among the 20 initially symptomatic patients, 3 (15%) experienced worsening of symptoms, 14 (70%) remained clinically stable and for the other 3 (15%) no data were available. According to RECIST, 4 patients out of 32 (12.5%) had PD during follow‐up, 19 (59.4%) had SD and 7 (21.9%) had a partial response (PR), while data were missing for 2 (6.2%). Eight patients underwent further treatments: 2 (6.3%) had surgery and 6 (18.8%) had systemic therapy.3.2Figure According to RECIST, the best response was: PR in 8 patients (25%), SD in 22  and PD in 2 (6%). A tumor volume reduction of ≥65% was achieved in 12 subjects (38%), a <65% volume decrease in 17 (53%), and a tumor volume increase in 3 (9%). Seven patients (22%) had a small decrease or even an increase in longest diameter , while showing a tumor volume reduction ≥65% (lesions shrank without diminishing in the main diameter). A ≥50% decrease of normalized T2‐signal intensity was observed in 15 patients (47%), a <50% decrease in 13 (41%), and an increase in 4 (12%).As reported in Figure Among the 20 symptomatic patients (62.5%), symptomatic improvement during therapy was experienced in 15 (75.0%), while 4 (20.0%) had no symptomatic relief and 1 (5.0%) had symptomatic worsening. In particular, improvement of pain was observed in 13 patients, no improvement in 4 and worsening in 1. Improvement of functional impairment was found in five patients and no improvement in one.3.33 among the time points; a reduction also in the interquartile range (i.e. IQR =75th – 25th centiles) values  was observed. Similarly, we observed a gradual reduction over time of the normalized T2‐signal intensity of the lesions, both for median values  and interquartile range  at baseline, 6 and 12 months, respectively. Figure The overall trend of the three radiological parameters over time is represented by spaghetti plots and descriptive statistics in Figure 3.43, respectively; a clear trend was not deducible in patients without symptomatic improvement: 110.9, 85.8, 112.9, 81.1 mm and 155.4, 101.4, 183.4, 92.4 cm3, respectively.In the subgroup of patients experiencing a symptomatic improvement during therapy Figure , a reduc3, respectively, in clinically non‐progressing patients; 143.4, 114.1, 121.9, 121.9 mm and 223.7, 160.9, 169.8, 151.2 cm3, respectively, in clinically progressing patients.In the subgroup of patients maintaining the symptomatic improvement during follow‐up Figure , a decre3, respectively, in non‐retreated patients; 130.4, 95.2, 96.0, 107.8 mm and 165.8, 107.4, 96.0, 114.6 cm3, respectively, in retreated patients.As shown in Figure 3, respectively, in progressing patients; 2.7, 2.0, 1.5, 1.6 and 89.5, 69.0, 33.8, 57.5 cm3, respectively, in non‐progressing patients. In the subgroup with RECIST progression, median volumes and T2‐signal intensity at baseline were higher.Looking at progressions according to RECIST during follow‐up Figure , the medFigures 4In this series of sporadic DF treated with low‐dose MTX‐VA, 25% of patients achieved a RECIST PR, while more responses were recorded based on volume and normalized T2‐signal intensity changes . Few cases showed some discrepancies between the three radiological variables. When the radiological variables were evaluated according to the clinical variables, T2‐signal reduction seemed to be more informative about symptomatic improvement during therapy and future clinical progression.This study has some limitations. First, a relatively small number of patients was included. Obviously, this reflects the rarity of this disease. Second, this was a retrospective analysis. Therefore, formal quality of life tests or tumor‐specific outcome tools was not used and there was a lack of standardization. Indeed, MRI examinations had been performed at different institutions  and this may have caused some variations in measured T2‐signal intensity, even with a reference standard.To our knowledge, however, this is the largest series of chemo‐treated patients with DF evaluated by MRI by using all three radiological criteria. In a population of 11 DF patients treated with sorafenib, Gounder et alThe rate of RECIST responses in the present series is superimposable to previously reported data.By looking at the changes of the three radiological parameters altogether Figure , we obseIn the present analysis, an effort to investigate possible relationships between radiological variables and clinical ones has been made, taking into account that tumor response assessment through symptoms evaluation is crucial in this disease, where the quality of life improvement should be the goal of any treatment. A better knowledge of the radiological pattern of response, however, may be useful also to assess tumor response in asymptomatic patients, in which symptoms changes during treatment are not so informative. Interestingly, when we looked at the relation between radiological and clinical parameters, T2‐signal intensity proved to better reflect the symptomatic improvement during therapy and clinical progression during follow‐up. Similarly, Libertini et al,High volumes and high T2‐signal intensity values at baseline were shown to be a possible predictor of further RECIST‐progression during follow‐up. Kamali et alIn conclusion, we think that these results may well lead to validating a standardized MRI‐based algorithm as a tumor response indicator in a larger prospectively evaluated series of DF patients treated with systemic therapy, with the support of disease‐specific quality of life tests.All Authors have no potential conflicts of interest to declare.Edoardo Zanchetta, Elena Palassini, Paolo Giovanni Casali, Chiara Maura Ciniselli, Paolo Verderio, Carlo Morosi, Alessandra Casale and Francesca Gabriella Greco: conceptualization and methodology. Edoardo Zanchetta, Elena Palassini, Silvia Stacchiotti, Giacomo Giulio Baldi, Salvatore Provenzano, Rossella Bertulli, Federica Bini, Alessandra Casale, Francesca Gabriella Greco, Andrea Ferrari, Marco Fiore, Alessandro Gronchi, Chiara Colombo, Paolo Giovanni Casali: investigation. Edoardo Zanchetta, Chiara Maura Ciniselli and Annalisa Bardelli: formal analysis and visualization. Paolo Giovanni Casali, Paolo Verderio and Carlo Morosi: supervision. Edoardo Zanchetta and Elena Palassini: writing‐original draft. All authors: writing review and editing.Institutional ethics committee's approval was obtained.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Fig S4Click here for additional data file.Fig S5Click here for additional data file.