Sorghum, long regarded as one of the most underutilized crops, has received attention in recent years. As a result, conducting multidisciplinary studies on the potential and health benefits of sorghum resources is vital if they are to be fully exploited. In this study, the nutritional contents, functional metabolites, and antioxidant capacities of 23 sorghum breeding lines and three popular cultivars were assessed.All of the sorghum genotypes were grown under the same conditions, and mature seeds were hand-harvested. The metabolite contents and antioxidant capacities of sorghum seeds were assessed using standard protocols. Fatty acids were quantified using a gas chromatography-flame ionization detector, whereas flavonoids and 3-deoxyanthocyanidins were analyzed using a liquid chromatography-tandem mass spectrometry method. The data were analyzed using both univariate and multivariate statistical approaches.p < 0.05). Antioxidant capacity, measured using three assays, also differed significantly. Five fatty acids, including palmitic, stearic, oleic, linoleic, and linolenic acids, were found in all the sorghum genotypes with statistically different contents (p < 0.05). Furthermore, the majority of the sorghum genotypes contained four 3-deoxyanthocyanidins, including luteolinidin, apigeninidin, 5-methoxyluteolinidin, and 7-methoxyapigeninidin, as well as two dominant flavonoids, luteolin and apigenin. Compared to the cultivars, some breeding lines had significantly high levels of metabolites and antioxidant activities. On the other hand, statistical analysis showed that total tannin, total phenolic, and antioxidant capacities varied significantly across white, yellow, and orange genotypes. Principal component analysis was used to differentiate the sorghum genotypes based on seed color and antioxidant index levels. Pearson’s correlation analysis revealed strong links between biosynthetically related metabolites and those with synergistic antioxidant properties.Total protein (9.05–14.61%), total fat (2.99–6.91%), crude fiber (0.71–2.62%), dietary fiber (6.72–16.27%), total phenolic (0.92–10.38 mg GAE/g), and total tannin (0.68–434.22 mg CE/g) contents varied significantly across the sorghum genotypes (This research demonstrated the diversity of the sorghum resources investigated. Those genotypes with high levels of nutritional components, functional metabolites, and antioxidant activities could be used for consumption and breeding programs. Sorghum, known for its drought tolerance, stress resistance, low production cost, and high yield, can be grown in water-stressed, arid, and semi-arid environments throughout Africa, Asia, America, and Europe , vanillin, Folin–Ciocalteu phenol reagent, potassium ferricyanide, trichloroacetic acid, ferric chloride, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid were ordered from Sigma-Aldrich .The chemicals and reagents used in this study were of analytical grade (purity >99.8%) and were used as obtained. HPLC-grade water, acetonitrile, methanol, and sulfuric acid were purchased from Fisher Scientific . The remaining chemicals and reagents, including luteolin, apigenin, luteolinidin, apigeninidin, eriodictyol, catechin, gallic acid, L-ascorbic acid, formic acid, anhydrous sodium carbonate . The seeds were sown on June 17, 2021 in an experimental field found at the center (latitude/longitude: 30°49′38.37″ N/127°09′7.78″ E), and the cultivation period lasted until October of the same year. The mature seeds were hand-harvested, and seed samples from each genotype were dried in a post-harvest crop dryer  at 40°C for 7 days. The samples were then ground using a grinder , placed in sealed plastic bags and kept at −20°C pending extraction and subsequent analysis. The color of mature sorghum seeds was also determined by visually grading roughly 10 seeds per genotype with a QP card 203 color code .2.3.Polyphenols were extracted using a previously reported protocol with some modifications . Briefly2.3.1.R2 = 0.9983) at five concentration levels (0.25–8.00 mg/mL). TTC was calculated as milligrams of catechin equivalents per gram on a dry seed weight basis (mg CE/g) using catechin as a standard.The TTC was determined using a method proposed by Price et al.  with sli2.3.2.2CO3 solution after 3 min of incubation at 25°C. Finally, the absorbance at 750 nm was measured after 30 min of reaction in the dark using methanol as a blank. Calibration curve  was plotted using gallic acid at five concentration levels (0.025–3.00 mg/mL). TPC was determined as milligrams of gallic acid equivalent per gram on a dry seed weight basis (mg GAE/g).The Folin–Ciocalteu method, as modified in our recently reported protocol , was use2.3.3.• scavenging activity, 100 μL of the sorghum seed extract was mixed with an equivalent volume of freshly made DPPH solution (150 μM), and the mixture was incubated for 30 min in the dark at 25°C. Then, the absorbance at 517 nm was measured, and ascorbic acid was used as a standard. Ascorbic acid was used as a standard to plot calibration curve  at five concentration levels (0.0025–0.0250 mg/mL). The DPPH• scavenging activity was reported as milligrams of ascorbic acid equivalent antioxidant capacity per gram on a dry seed weight basis (mg AAE/g) (For DPPHg AAE/g) .2.3.4.•+ was initially prepared by dissolving ABTS (7 mM) in potassium persulfate . The mixture was incubated for 16 h at 25°C before being diluted with water to achieve an absorbance of 0.700 ± 0.02 at 734 nm. Then, 10 μL of the sorghum seed extract was mixed with 150 μL of ABTS•+ solution and the mixture was incubated for 3 min at 25°C in the dark. Finally, the absorbance was measured at 734 nm. Using Trolox as a standard, calibration curve  was plotted at five concentration levels (0.005–0.250 mg/mL). The ABTS•+ scavenging activity was determined as milligrams of Trolox equivalent antioxidant capacity per gram on a dry seed weight basis (mg TE/g) (A stock solution of ABTSmg TE/g) .2.3.5.2R = 0.9999) at five concentration levels (0.0025–0.0250 mg/mL), and the FRAP activity result was reported in mg AAE/g  and 150 μL of 1% potassium ferricyanide solution. The mixture was incubated for 20 min at 50°C, followed by the addition of 150 μL of 10% trichloroacetic acid, and centrifugation at 13,000 rpm for 10 min. Then, 100 μL of the supernatant was mixed with an equal volume of distilled water, followed by the addition of 20 μL of 0.1% ferric chloride solution. The absorbance at 700 nm was measured after 10 min of incubation. Ascorbic acid was used as a standard to plot calibration curve  × 100]. Using the AI values, the sorghum genotypes were classified as very low (0–19%), low (20–39%), medium (40–59%), high (60–79%), or very high (80–100%) AI genetic materials.The antioxidant index (AI) was determined according to the method described by Ng et al.  and was 2.5.The total protein, total fat, crude fiber, and dietary fiber contents were determined using standard methodologies . In summ2.6.For fatty acid analysis, fatty acid methyl esters (FAMEs) were synthesized using a direct methylation technique as previously described with a minor modification . In brie2.7.2.7.1.The extraction of 3-deoxyanthocyanidins and flavonoids was performed in methanol containing 1% HCl . In a 152.7.2.m/z), and the UV–Vis spectrum data along with commercially available standards, were used to confirm the identity of the target molecules (Identification of the target metabolites was achieved using a Waters Acquity ultra-performance liquid chromatography (UPLC) system outfitted with a PDA detector and a Synapt XS Q-TOF mass spectrometer . During analysis, 10 μL of sample extract was injected and separation was carried out on a Waters ACQUITY UPLC HSS T3 column  set to 40°C. As a mobile phase, a binary solvent system of 0.1% formic acid (A) and acetonitrile (B) was used. The solvent flow began with 10% B at a rate of 0.3 mL/min, and the gradient conditions were set as follows: 10–25% B (0–15 min), 25–40% B (15–25 min), and 40–90% B (25–30 min). The final condition was equilibrated to 10% B for the last 5 min (30.1–35 min). The mass scanning range was 100–1,200 Da in both positive and negative ionization modes with capillary voltages of 3 kV and − 2.5 kV, respectively. The collision energy was 20–50 V and the cone voltage was 30 V. The ion source temperature was 120°C, while the desolvation temperature was 450°C. The chromatograms and mass spectra were analyzed using MassLynx 4.1 software, and anthocyanins were read at 500 nm and flavonoids at 360 nm. Molecular mass data, fragment ions  equipped with a Shimpack GIST C18 column  under a similar UPLC condition described above was used to quantify the target anthocyanins and flavonoids. Luteolinidin, apigeninidin, luteolin, and apigenin, were quantified using the corresponding external standards. 5-methoxyluteolinidin and 7-methoxyapigeninidin, with no available standards, were quantified as luteolinidin and apigeninidin equivalents, respectively multiplied by the appropriate correction factors . In each2.8.p < 0.05 level. The statistical and principal component (PCA) analyzes were carried out using xlstat software . Scatter grams, scatter plots, and Pearson’s correlation analyzes were computed using R-software .For each experiment, measurements were performed in triplicates and results are expressed as mean ± standard deviation (SD). Significant differences between measurements were statistically determined using analysis of variance (ANOVA) at 3.Sorghum has received a lot of attention in recent years, and a range of food products derived from its seeds are being developed for dietary purposes . As a re3.1.p < 0.05), with mean values of 186.01 mg CE/g and 4.51 mg GAE/g. Previously, several studies have investigated the total tannin and total phenolic content of several sorghum genetic resources, and the reported values were wide-ranging. The TPC range obtained in our study was consistent with a previous study in which TPC ranged from 1.56 to 11.99 mg GAE/g across 11 sorghum varieties . In contrast, IS12611 displayed the lowest TPC, ABTS•+ scavenging activity, and FRAP, while ET 185–2 and ET 36–1 displayed the lowest TTC and DPPH• scavenging activity, respectively. As shown in The TTC, TPC, and antioxidant activities of individual sorghum genotypes are provided in arieties . In othearieties  observedarieties , 37–39. ectively . Among t• scavenging activity, ABTS•+ scavenging activity, and FRAP, respectively. Among them, three breeding lines including IS8009, IS8107, and IS8121 exhibited significantly higher values of each trait (p < 0.05). On the other hand, ET36-1, ET185-2, and IS12611 had significantly lower TTC, TPC, FRAP, and ABTS•+ scavenging activity than all of the cultivars . Total protein and total fat levels were in the ranges of 9.05–14.61 and 2.99–6.91%, with mean values of 11.58 and 4.24%, respectively. Similarly, total dietary fiber and total crude fiber contents were in the ranges of 6.72–16.27 and 0.71–2.62%, with means of 10.51 and 1.51%, respectively. Compared to our study, Htet et al. . In terms of fat content, 39.13% had a higher value than all the cultivars, with eight breeding lines  having a significantly higher total fat content (p < 0.05). Apart from their numerous health benefits, fibers play important roles in improving the quality of gluten-free dietary products and are thus important components of sorghum seeds . The majority of the breeding lines (65.21%) exhibited a higher stearic acid content when compared to cultivars. In contrast, only three breeding lines including IS8017, IS8023, and JN 36 displayed a higher oleic acid level than all the cultivars. Similarly, only Bonita had a significantly higher linoleic acid content than all the cultivars, while IS8112 and IS8107 had a higher linolenic acid content. In general, total polyunsaturated fatty acid (PUFA) content (39.20–52.52%) was higher than total monounsaturated content (MUFA) (25.62–42.31%) and total saturated fatty acid (SFA) content (16.82–21.86%) in all genotypes except those two breeding lines (IS8023 and JN 36). A higher ratio of unsaturated to saturated fatty acids has been linked to reducing the risk of cardiovascular diseases  were observed among the sorghum genotypes . The variations of apigenin and luteolin were likewise not significantly varied between white, yellow, and orange genotypes , ABTS•+ scavenging activity (r = 0.86), and FRAP (r = 0.89), all of which were significant at p < 0.001. Similarly, the associations of TTC to DPPH• scavenging activity (r = 0.95), ABTS•+ scavenging activity (r = 0.97), and FRAP (r = 0.98) were strong and significant (p < 0.001). The positive and significant correlations of TTC and TPC to each of the antioxidant activities demonstrate the roles of tannins and phenols in regulating ROS and RNS, which corroborate multiple other studies (p < 0. 001), luteolinidin and 5-methoxyluteolinidin , and apigeninidin and 7-methoxyapigeninidin  could also be explained by their interrelated biosynthetic pathways (p < 0.001) and linolenic acid  were noteworthy further supporting the contradictory levels of these unsaturated fatty acids  of the National Institute of Agricultural Sciences, Rural Development Administration .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.


Salvia lavandulifolia Vahl essential oil is becoming more popular as a cognitive enhancer and treatment for memory loss. It is high in natural antioxidants and has spasmolytic, antiseptic, analgesic, sedative, and anti-inflammatory properties. Its aqueous extract has hypoglycemic activity and is used to treat diabetic hyperglycemia, but few studies have focused on it. The objective of this work is to evaluate the various biological and pharmacological powers of Salvia lavandulifolia Vahl leaf aqueous extract. Quality control of the plant material was first carried out. Followed by a phytochemical study on the aqueous extract of S. lavandulifolia leaves, namely phytochemical screening and determination of total polyphenols, flavonoids, and condensed tannins contents. Then, the biological activities were undertaken, in particular the antioxidant activity  and the antimicrobial activity. The chemical composition of this extract was also determined by HPLC-MS-ESI. Finally, the inhibitory effect of the α-amylase enzyme as well as the antihyperglycaemic effect was evaluated in vivo in normal rats overloaded with starch or D-glucose. The aqueous extract obtained by use of the decoction of leaves of S. lavandulifolia contains 246.51 ± 1.69 mg EQ of gallic acid/g DE, 23.80 ± 0.12 mg EQ quercetin/g DE, and 2.46 ± 0.08 mg EQ catechin /g DE. Its total antioxidant capacity is around 527.03 ± 5.95 mg EQ of ascorbic acid/g DE. At a concentration of 5.81 ± 0.23 µg/mL, our extract was able to inhibit 50% of DPPH° radicals. Moreover, it showed bactericidal effect against Proteus mirabilis, fungicidal against Aspergillus niger, Candida albicans, Candida tropicalis, and Saccharomyces cerevisiae, and fungistatic against Candida krusei. A marked antihyperglycemic activity (AUC = 54.84 ± 4.88 g/L/h), as well as a significant inhibitory effect of α-amylase in vitro (IC50 = 0.99 ± 0.00 mg/mL) and in vivo (AUC = 51.94 ± 1.29 g/L/h), is recorded in our extract. Furthermore, its chemical composition reveals the presence of 37.03% rosmarinic acid, 7.84% quercetin rhamnose, 5.57% diosmetin-rutinoside, 5.51% catechin dimer, and 4.57% gallocatechin as major compounds. The antihyperglycemic and α-amylase inhibitory activities, associated with the antioxidant properties of S. lavandulifolia, justify its use in the treatment of diabetes in traditional medicine and highlight its potential introduction into antidiabetic drugs. Aromatic and medicinal plants have played a crucial role in the treatment and prevention of a wide range of diseases for thousands of years . The theS. lavandulifolia is a widely distributed species in the Mediterranean basin, in eastern Spain, extending to the western Mediterranean: southeastern Spain and northwestern Africa (Morocco and Algeria)  (0–19 min), “A+B” [70:30] (20–24 min), “A+B” [5:95] (25 min), and “A+B” [98:2] (26–30 min). The detection was carried out using a diode array detector by scanning in the wavelength range of 280–360 nm, as well as by the spectrometer of mass (Exactive) after negative ionization. Data were acquired using MASS LYNX software(Version 4.2). The molecules were identified based on retention time, mass spectrum, molecular weight, and by comparison with standards (injected under the same conditions as the extract): caffeic acid, coumaric acid, ferulic acid, gallic acid, rosmarinic acid, sinapic acid, syringic acid, tannic acid, trans-cinnamic acid, vanillic acid, apigenin, catechin, coumarin, kaempferol, luteolin, myricetin, and rutin.The HPLC-MS analysis was conducted by the Dionex UltiMate 3000 ULC/HPLC system coupled to an Exactive mass spectrometer with an ESI ionization source and an orbitrap analyzer. A volume of 10 μL of extract dissolved in distilled water (C = 100 μg/mL) was injected into a C18 column with 100 mm long, 2.1 mm in diameter, and with 1.7 µm pores. The temperature was programmed at 30 °C, while the flow rate was 0.45 mL/min. The mobile phase contained two solvents: solvent A (Water + formic acid (0.1%), The test sample was mixed with 1 mL of ammonium molybdate (4 mM), 1 mL of sodium phosphate (28 mM), and 1 mL of sulfuric acid (0.6 M) in test tubes. The tubes’ contents were combined and incubated at 95 °C for ninety minutes before being normalized for 20 to 30 min at room temperature. At 695 nm, the measurement was made. The results were expressed in milligram equivalents of ascorbic acid per gram of dry extract (mg EAA/g DE), with ascorbic acid serving as the control .An increasing volume of extract was put into test tubes, and ethanol was added to reach a total volume of 200 µL. A quantity of 2.8 mL of ethanolic solution of DPPH°  was added to the mixture and left to incubate for 30 min in the dark. Notably, UV–Vis absorbance was measured at 515 nm .% inhibC: Absorbance of the negative control, AS: Absorbance of the sample, IC50: Inhibiting concentration 50% of DPPH° radicals (mg/mL), CDPPH: Concentration of DPPH° (mg/mL).A50 effective concentration was used to calculate the antiradical potency. The higher the ARP, the more effective the antioxidant [ECioxidant .ARP=10050: Effective concentration of the sample.ECS. lavandulifolia aqueous extract was tested against nine bacteria and fungi , with the 11th and 12th wells serving as growth and sterility controls, respectively. After incubating for 24 h at 37 °C, 10 μL of resazurin was added to each well as a microbial growth indicator before reincubating for two hours at 37 °C. The change in color from purplish blue to bright pink revealed growth. The MIC is defined as the lowest concentration that prevents resazurin from changing color. To determine the MBC or MFC, 10 µL was taken from each well where no growth could be seen and put on MH agar for bacterial growth or Sabouraud for fungal growth for 24 h at 37 °C. After incubation, MBC/MFC is determined as the lowest concentration inhibiting colony formation on solid agar medium [The minimum inhibitory concentration (MIC) was determined by use of 96-well microplates using the reference method of microdilution . From anr medium . To evalr medium .Wistar rats (200–250 g) and albino mice (25–35 g) were used in this study and were reared under ideal conditions photoperiods of 12 h light/12 h dark and 22 ± 2 °C) with free access to water and food. This test was carried out in accordance with the Organization for Economic Cooperation and Development’s guidelines (OECD) [s (OECD) .n = 6; ♂/♀ = 1). The control group received distilled water (10 mL/kg) and the treated groups received the doses: 0.5 g/kg, 1 g/kg, and 2 g/kg. When the test first began, the mice were weighed. Immediately afterwards, they received a single dose of the test extract, orally. Then, they were continuously monitored for 10 h to report any apparent signs of toxicity. For the remaining 14 days, the mice were kept under daily surveillance for further clinical or behavioral signs of toxicity. This test was performed in accordance with the guidelines of the Organization for Economic Co-operation and Development (OECD) [A quantity of 24 albino mice (20–35 g) on an empty stomach (14 h) were randomly distributed into four groups (t (OECD) .n = 6; ♂/♀ = 1): control group: distilled water (10 mL/kg) and test groups: normal rats force-fed with the extract (400 mg/kg) or glibenclamide (2 mg/kg). First, blood glucose was measured at t0 just before administration of the test product . A total of 30 min later, another measurement of glycemia was carried out then the rats were overloaded with D-glucose (2 g/kg). Subsequently, the variation in blood glucose was measured every half hour up to 90 min and then after one hour.The oral glucose tolerance test was performed to evaluate the antihyperglycemic  effect in vivo . Normal S. lavandulifolia aqueous extract on pancreatic-amylase enzymatic activity. Increasing volumes of S. lavandulifolia extract/acarbose were added to test tubes, which were then filled to a capacity of 200 µL with distilled water.The Nageswara Rao Thalapaneni et al.  method wThe tubes were then filled with 200 µL of pancreatic α-amylase solution (61.33 U/mL) and 200 µL of phosphate buffer (PB) solution , with the exception of the correction series, where the PB solution was used in its place. A time of 10 min at 37 °C were spent pre-incubating the tubes.The tubes were then re-incubated for 15 min at 37 °C with 200 µL of a 0.5% starch solution. After adding 600 µL of DNSA (2.5%) to halt the enzymatic reaction, the tubes were heated in a boiling water bath for 8 min. The tubes were placed in an ice bath to stop the reaction, and then 10 mL of diluted water was added to each tube. In order to compare the absorbance at 540 nm to the correction series, a spectrophotometer was used. The percentage inhibition was calculated using the equation shown below:Control: absorption of enzyme activity without inhibitor; AbTest: Absorption of enzymatic activity in the presence of the extract or acarbose; AbBlank/Blank of the corresponding tube: a series of corrections is carried out in parallel with each series where the enzyme is replaced by the PB.AbS. lavandulifolia aqueous extract in vivo in normal rats by taking into account the effect of intestinal lumen on the extract’s inhibitory effect. Normal fasting rats  were divided into three groups : the control group was given distilled water (10 mL/kg), while the treated groups were given the extract (400 mg/kg) or acarbose (10 mg/kg). After measuring blood glucose, an adequate volume of the extract/distilled water/acarbose was administered (t0) to begin the oral starch tolerance test. A second measurement of glycemia was taken 30 min later (t1), and the rats were then overloaded with starch (3 g/kg). The variation in glycemia was measured at t2 = 60 min, t3 = 90, and t4 = 120 min.The purpose of this test is to confirm the enzymatic activity of pancreatic-amylase-inhibitory effect of p-values of p < 0.05, p < 0.01, and p < 0.00.The results were statistically analyzed using ANOVA , and they are shown as means and standard deviation. Statistics were considered to be significant at Salvia lavandulifolia Vahl is a widespread plant in Morocco; its leaves are often used for their high content of essential oil whose virtues are well known. The extracts, on the other hand, are very little studied by researchers, and their properties are found most of the time in articles—ethnopharmacological surveys or some very old ones. The aqueous extract of the leaves of S. lavandulifolia harvested in Ouled Ali-Boulemane-Morocco is very rich in polyphenols; it recorded a remarkable total antioxidant capacity and antiradical power. It is also considered bactericidal against Proteus mirabilis, fungicidal against Aspergillus niger, Candida albicans, Candida tropicalis, and Saccharomyces cerevisiae, and fungistatic against Candida krusei. Administered 30 min before an acute oral administration of glucose, our extract significantly lowered hyperglycemia in vivo in rats. Thanks to its content of rosmarinic acid, apigenin, and luteolin, it essentially could also inhibit the activity of the enzyme α-amylase in vitro and in vivo.Aromatic and medicinal plants are a precious gift of nature for human beings; they have been used since antiquity in food, beverages, traditional medicine, and cosmetics.


The purpose of this retrospective study was to introduce an alternative technique for the treatment of type II symptomatic ulnar styloid nonunion by the reinsertion of the triangular fibrocartilage complex and the ulnar collateral ligament.Between March 2009 and May 2017, 45 patients  suffering from the nonunion of type II ulnar styloid fractures all underwent the subperiosteal resection of the avulsed fragments and the reinsertion of the TFCC and ulnar collateral ligament. Outcome assessments included the ranges of motion of the wrist, grip strength, pain, and Mayo wrist score. The preoperative and postoperative parameters were compared. A P-value less than 0.05 was considered to be statistically significant.The mean follow-up period was 21.66 ± 7.93 months . At the final follow-up, the mean preoperative flexion and extension were 79.32 ± 4.52° and 74.40 ± 4.36° respectively. The mean preoperative pain score, grip strength, and Mayo wrist score were 32.48 ± 4.00; 23.88 ± 8.38 kg, and 77.72 ± 8.31 respectively. The mean postoperative flexion and extension of the wrist were 80.56 ± 6.32° and 75.43 ± 3.12° respectively. The mean postoperative pain score, grip strength, and Mayo wrist score were 12.41 ± 3.27, 26.31 ± 8.30 kg, and 90.71 ± 7.97 respectively. There were significant differences in pain, grip strength, and Mayo wrist score (P < 0.05), but no significant differences concerning the range of motion of the wrist.In the treatment of the nonunion of type II ulnar styloid fractures, the resection of the avulsed fragments followed by the reinsertion of the TFCC and the ulnar collateral ligament with an anchor was a reliable alternative technique, bringing the satisfactory function of the wrist. Ulnar styloid fractures occur as isolated injuries or in association with distal radius and other wrist fractures . Ulnar sThe triangular fibrocartilage complex (TFCC) is located at the base of the ulnar styloid or fovea. The structures consist of the articular disc, the dorsal and volar radioulnar ligaments, the meniscus homologue, the ulnar collateral ligament, and the sheath of the extensor carpi ulnaris . Hauck eSimple excision of the ulnar styloid fragment may relieve wrist pain, but wrist instability is a major concern . Many suThe purpose of this retrospective study was to introduce an alternative technique for the treatment of type II symptomatic ulnar styloid nonunions by reinserting the TFCC and ulnar collateral ligament. We also reported the results using the technique.This study was approved by the institutional review boards of the hospitals involved. Informed consent was obtained from each patient.From March 2009 to May 2017, 63 patients with painful styloid nonunion were treated consecutively in our hospitals .The mean age of the 45 patients  undergoing the surgery was 45.48 ± 11.50 years  old. All patients had sustained injuries. The average time from injury to surgery was 17.57 ± 7.89 months . The mean preoperative wrist pain score, grip strength, and Mayo wrist scores were 32.48 ± 4.00 mm, 23.88 ± 8.38 kg, and 65.72 ± 8.31 respectively . At the final follow-up, the DRUJ instability was achieved in all patients. The mean postoperative pain score of the wrist, grip strength, and Mayo wrist scores were 12.41 ± 3.27; 26.31 ± 8.30 kg, and 90.71 ± 7.97 respectively (Table There were significant differences in the pain score (P = 0.0001), grip strength (P = 0.0012), and Mayo wrist scores (P = 0.0001). There were no significant differences regarding wrist flexion (P = 0.223), and wrist extension (P = 0.238). There were 29 excellent, 14 good, and 2 fair results.Ulnar styloid fractures are often accompanied by radius fractures. The most common cause of an ulnar styloid fracture is a fall on an outstretched arm. Most of these are small avulsion fractures (type I) involving the tip of the ulnar styloid. The ulnar styloid is an important structure for the TFCC. Some clinical findings help establish the diagnosis of symptomatic ulnar styloid nonunion, including wrist pain on resisted ulnar deviation and dorsiflexion, a clicking sensation on wrist movement, reduced grip strength, and tenderness and swelling over the dorsal ulnar aspect of the ulna , 15. SurMany surgical treatments are available for the treatment of symptomatic type II ulnar styloid nonunion, such as fixation with K-wires, tension banding wiring, compression screws, and mini-fragment plates , 17. HauOur technique avoids the prominence of the implant and tendon irritation. The loss of anatomical structure and the disability of bony union are the main disadvantages. The indication of our technique is failed conservative treatments of type II ulnar styloid nonunion with persistent ulnar-sided wrist pain, especially due to small fragments.The study has limitations. There was neither a control group nor randomization because of the small sample size.In the treatment of the nonunion of type II ulnar styloid fractures, the resection of the avulsed fragments followed by the reinsertion of the TFCC and ulnar collateral ligament with an anchor was a reliable alternative technique, bringing the satisfactory function of the wrist.


Therefore, the utilization of ultrasound for the extraction of pepsin soluble collagen is a good alternative technology to expand the application at industrial level.The objective of this study was to evaluate the effect of ultrasound pre-treatment on the characterization from Bactrian camel skin. It was possible to produce and characterize collagen extracted from Bactrian camel skin. The results showed that the yield of collagen was higher in ultrasound pre-treatment (UPSC) (41.99%) than the pepsin-soluble collagen extraction (PSC) (26.08%). All extracts were identified as type I collagens using sodium dodecyl sulfate polyacrylamide gel electrophoresis and retained their helical structure, as confirmed through Fourier transform infrared spectroscopy. The scanning electron microscopy analysis of UPSC revealed that some physical changes were caused by sonication. UPSC had smaller particle size than PSC. The viscosity of UPSC always plays a leading role in the range of 0–10 Hz. However, the contribution of elasticity to the solution system of PSC increased in the range of 1–10 Hz. Moreover, ultrasound-treated collagen had superior solubility property at pH 1–4 and at <3% ( Animal tissues contain abundant levels of collagen, which mainly exists in skin, bone, cartilage, tendon, and other connective tissues, accounting for about 30% of total animal protein . Up to 2According to statistics, 89% of camels are dromedaries located in North Africa, West Asia, and Australia, and the other 11% are Bactrian camels, mainly distributed in China and Mongolia . China iGenerally, collagen fiber-rich tissues, such as skin or tendons, are treated with neutral salts, acids, alkalis, and proteases to extract collagen . UnfortuBactrian camel was purchased from local producers in Alxa League, Inner Mongolia, China. The skin was dissected into 3 × 3 cm2 pieces and frozen at −20 °C for 2 weeks. Neutral protease , pepsin from porcine gastric mucosa , and SDS-PAGE gel preparation kit, protein maker, and sodium dodecyl sulfate (SDS) were purchased from solaibao Technology Co., Ltd. . Other reagents were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. .Fresh skin of a three-year-old Bactrian camel skin was thawed at 4 °C and cut into 3 cm × 3 cm blocks, added with 0.5% Na2CO3 with a skin/solution ratio of 1:10 (g/mL), soaked for 12 h, and then washed with distilled water. To remove the hair on the Bactrian camel hides, 0.6% neutral protease with a skin/solution ratio of was added to the soaked raw hides and incubated at 25 °C for 4 h. After depilation, the Bactrian camel skin was cut into 0.5 cm × 0.5 cm pieces, soaked in 5% Na2CO3 solution at 1:10 (g/mL), and incubated with continuous stirring at 20 °C for 18 h to remove fat and pigment. The fat-removed raw materials were soaked in 2% NaCl solution at 1:10 (g/mL) and incubated with continuous stirring at 20 °C for 18 h to remove salt-soluble non-collagen components, and then washed with distilled water. Finally, the pretreated Bactrian camel skin was placed at −20 °C until use.Before use, the w/v)), together with 4% (w/v) pepsin and incubated for 48 h. Following centrifugation  at 10,000 g/min at 4 °C for 20 min, NaCl in 0.05 M Tris buffer was added to the supernatant to a final concentration of 0.9 M. The mixture was centrifuged for 10 min at 10,000 g/min and the pellet was dissolved in 5 volumes of 0.5 M acetic acid. Following sequential dialysis against 0.1 M acetic acid followed by distilled water for 24 h each , the sample was lyophilized (PSC from Bactrian camel skin).All PSC extractions were performed at 4 °C. 0.5 M acetic acid and pretreated skin tissues were mixed at a ratio of 1:20 (skin: acid (Bactrian camel skin was suspended in 0.5 M acetic acid containing 4% (w/v) pepsin at 1:20 (w/v) ratio and incubated for 24 h at 4 °C. An ultrasonic cell pulverizer  was used to extract PSC ultrasonically. The reaction was carried out at a single frequency of 24 kHz and a power of 200 W. The samples were sonicated for 20 min and then centrifuged for 10 min at 10,000 g/min for 10 min. Subsequent steps were the same as those detailed in The pretreated Bactrian camel skin Hyp content (mg/g); B1 represents the collagen dry weight (g); and B2 represents the Bactrian camel skin dry weight (g).The hydroxyproline content in collagen was measured according to the method of Li et al. . The yieThe amino acid composition of PSC and UPSC was analyzed using an amino acid analyzer  using a previously published method .The electrophoretic mobility patterns of collagens were assessed in accordance with Song et al. . BrieflyA UV-visible spectrophotometer  was used to determine the collagens’ UV spectra according to a previously published method with slight modifications . Dry col−1 spectral range with a 2 cm−1 resolution [An FTIR spectrometer  was used to measure the collagen samples’ FITR spectra. A 2 mg collagen sample was mixed with 100 mg of potassium bromide (KBr) and formed into a tablet, which was mounted in the sample holder of the FTIR spectrometer. The samples were scanned and measured using a 500–4000 cmsolution .The method of Indriani et al.  was usedA scanning electron microscope  was used to observe the morphology of lyophilized collagen. Freeze-dried collagen was cut into small pieces and fixed after spraying gold. SEM images were captured under an accelerating voltage of 10 kV at ×200 magnification .Rheological characterization was carried out in a rotational rheometer (Thermo RS6000) using a 49 mm diameter parallel stainless steel plate with a 1 mm gap. Collagen cross-linking behavior was determined using dynamic frequency sweeps. In the linear viscoelastic region, the sample was selected with a strain setting of 0.5%, a scanning frequency of 0.01–10 Hz, and a temperature of 25 °C. Dynamic temperature sweeps were performed from 10 to 70 °C over a linear range of 0.5% constant strain, and an accuracy of 1.0 °C/min. We then analyzed changes in the elastic modulus (G′), viscous modulus (G″), and loss factor (tanδ = G″/G′) of collagen under the two modes.Freeze-dried samples of PSCs and UPSCs were dissolved in 0.5 M acetic acid and stirred slowly for 6 h at 4 °C to achieve a concentration of 2.5 mg/mL.g and 4 °C, and the biuret method was used to measure the protein content in the supernatant [pH is the protein content in mg/g at different pH values. WpH.max is the maximum dissolved amount of protein when the pH value changes, mg/g.The collagen solutions were adjusted to achieve a pH range of 1–10. The samples were stirred evenly, centrifuged for 20 min at 10,000 ernatant . The relw/v) containing final NaCl concentrations of 1–6% (w/v), after centrifugation at 10,000 g/min for 30 min at 4 °C. Using the above method, the protein content in the supernatant was determined and the relative solubility of collagen was calculated using Equation (3) [NaCl is the protein content under different NaCl concentrations, mg/g. WNaCl.max is the maximum dissolved amount of protein when the NaCl concentration changes, mg/g.The collagen solutions (5 mL) were mixed with 5 mL of acetic acid (0.5 M) with different concentrations of NaCl 1–6% (tion (3) .(3)Relap < 0.05. In the figures, data are displayed as the mean ± SD.IBM SPSS software  was used to carry out all the statistical analyses. The least significant difference (LSD) test was used to compare the means. Statistically significant differences were accepted at An appropriate ultrasound treatment time was beneficial to the extraction of collagen. In this experiment, the yields of PSC and UPSC were 26.08 ± 2.26% and 41.99 ± 1.57%, respectively. Ultrasonication increased the collagen content of UPSC by 16% compared with that of PSC. This increase was possibly caused by the cavitation and mechanical effects of ultrasound .The amino acid composition is an important factor that determines collagen’s physicochemical properties, including thermal stability, cross-linking ability, and solubility. PSC and UPSC had similar amino acid profiles. As shown in Bactrian camel skin belonged to type I collagen. The identification of the γ-chain suggested abundant intermolecular and intramolecular collagen cross-links [PSC and UPSC exhibited similar protein electrophoretic mobility patterns, with α1 and α2 chains and their dimers (β chains) and trimers (γ chains), respectively . Previouss-links . In collss-links . Moreovess-links .2 groups are believed to be mainly responsible for these observations [The PSC and UPSC absorption peaks were observed at approximately 218 nm A. These rvations . Additiorvations . A previrvations .−1, respectively. The absorption characteristic of amide A mainly occurs in the wavenumber range of 3400~3450 cm−1, and is usually associated with the stretching vibration of N–H; however, it might shift to a lower frequency if the N-H group forms a hydrogen bond [−1, respectively, reflecting CH2’s asymmetrical stretch and CH3’s symmetrical stretch [The main absorption peaks of PSC and UPSC were amide A, B, and amide I, II, III B. The amgen bond . In PSC  stretch .−1 usually encompasses the amide I band, which is mainly associated with the stretching vibration of the C = O bond that forms H-bonds between adjacent chains [−1, representing N–H bending vibrations and C–N stretching vibrations [−1, ~1543 cm−1, and ~1236 cm−1, respectively , and a sample’s viscosity is reflected by the G″ modulus . Over a Bactrian camel skin collagen are presented in Changes in G′ and G″ observed during temperature sweeps of w/v) NaCl (w/v) NaCl, followed by a gradual decline in higher salt concentrations. At high concentrations, salt ions disturb the hydration shell on the collagen’s surface to expose hydrophobic sites. This exposure increases the likelihood of hydrophobic interactions, resulting in aggregation and precipitation of collagen [UPSC and PSC showed high solubility up to 3% (/v) NaCl B. Collagcollagen .Bactrian camel skin could be used as a novel source and will be beneficial in promoting the commercialization of Bactrian camel skin.PSC and UPSC were successfully extracted and characterized from the Bactrian camel. Ultrasound facilitated the pepsin-mediated extraction of collagen and promoted collagen yield. The FTIR spectra, UV absorption spectra, electrophoretic mobility pattern, and amino acid composition demonstrated that the extracted collagen is type I collagen and retains its structure. UPSC from


Ruxolitinib (RUX) is a Janus kinase 1/2 inhibitor (JAKi) approved in the EU for treating disease‑related splenomegaly or symptoms in adults patients with myelofibrosis (MF). This is an interim analysis of JAKoMo, a prospective, non‑interventional, phase IV study in MF. Between 2012–2019 (cutoff March 2021), 928 patients (JAKi-naïve and -pretreated) enrolled from 122 German centers. This analysis focuses on JAKi-naïve patients. RUX was administered according to the Summary of Product Characteristics. Compared to the COMFORT-I, -II, and JUMP trials, patients in JAKoMo were older (median 73 years), had poorer Eastern Cooperative Oncology Group (ECOG) performance statuses (16.5% had ECOG ≥ 2), and were more transfusion dependent (48.5%). JAKoMo represents the more challenging patients with MF encountered outside of interventional studies. However, patients with low-risk International Prognostic Scoring System (IPSS) scores or without palpable splenomegaly were also included. Following RUX treatment, 82.5% of patients experienced rapid (≤ 1 month), significant decreases in palpable spleen size, which remained durable for 24 months (60% patients). Symptom assessment scores improved significantly in Month 1 (median –5.2) up to Month 12 (–6.2). Common adverse events (AEs) were anemia (31.2%) and thrombocytopenia (28.6%). At cutoff, 54.3% of patients had terminated the study due to, death, AEs, or deterioration of health. No new safety signals were observed. Interim analysis of the JAKoMo study confirms RUX safety and efficacy in a representative cohort of real-world, elderly, JAKi-naïve patients with MF. Risk scores were used in less than half of the patients to initiate RUX treatment.Trial registration: NCT05044026; September 14, 2021. BCR::ABL–negative myeloproliferative neoplasm (MPN) that is characterized by changes in blood counts, splenomegaly, disease-associated symptoms, and bone marrow (BM) fibrosis. It is caused by constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway  count < 4 × 109/L). Median hemoglobin level at baseline was 13.3 g/dL. Transfusion dependency was present in 48.5% of patients  patients having a platelet count > 200 × 109/L, 83 (20.1%) patients with a count between 100 and 200 × 109/L, 51 (12.4%) patients with a count between 50 and 100 × 109/L, and five (1.5%) patients with a count < 50 × 109/L.Baseline blood counts can be found in Table Splenomegaly was reported by physicians in 83% of patients; however, spleen palpation at baseline was only documented in 62.7% of patients. Median spleen length at baseline was 13.9 cm below the costal margin (BCM), with a median size of 17 cm BCM for PPV-MF patients. Median spleen length by computerized tomography (CT) or magnetic resonance imaging (MRI) scan was 19.5 cm.n = 406) of the patients, a bone marrow biopsy was performed at initial diagnosis. As per investigator assessment, 68:5% of patients (n = 317) had a diagnosis according to the WHO criteria from 2008. It was then up to the treating physician to report the biopsies in detail on a data sheet in the eCRF. For 343 patients, a detailed description was provided, and 314 patients of those were reported to have evaluable biopsies. Furthermore, 59.7% of patients (n = 277) had not undergone BM examination during the year before enrollment into the study; 187 patients were reported to have evaluable BM biopsies. Intriguingly, and perhaps counterintuitively, dry taps (punctio sicca) were not frequent, and physicians reported evaluable BM aspirations in as many as 86.7% of cases. In total, 67.5% of BM biopsies were reported to be hypercellular or strongly hypercellular, 14.1% were normocellular, and 17.2% were hypocellular. Histologic staining was reported in only 136 cases, with Grade 0–1, Grade 2, or Grade 3 MF  diagnosed in 28.0%, 45.6%, and 26.5% of cases, respectively; 43.9% of patients had 0% blasts in their BM biopsies, and only six cases showed blast counts above 5% (up to 14%).In 87.5% (n = 385) of the patients. It was then at the discretion of the treating physician to report the results of the mutational analysis in detail on a data sheet in the eCRF. For 295 patients, a detailed description on their molecular status was provided.At study entry of patients, physicians were asked if they had performed a molecular analysis of the patients before study start. A molecular analysis, which was not mandatory, was done in 83%  had cytogenetic results available from the last year before baseline. The low numbers of patients with results and with CALR mutations reflect the low prevalence of these tests at the time of trial initiation in 2012.As stated above, the study protocol did not provide mandatory examinations, so this may reflect the real-world behavior of German physicians treating MF patients. Only 151 (32.5%) patients had at least one molecular examination performed as a possible examination in the study (up to three in some patients), and results were reported from 140 patients (30.2%), with 110 of these patients harboring the p = 0.0549; 0.7 years vs 3.0 years, p ≤ 0.0001).The median RUX dose administered was 23.77 mg/day. Patients were started on a median dose of 30 mg/day , not differing between the three subentities. 39.5% and 25.4% of patients received 30 mg/day or 40 mg/day, respectively; however, 35.1% of patients were dosed with 20 mg/day or less, and 20.8% of patients received 10 mg/day or less. There were no relevant differences between PMF, PPV-MF, or PET-MF patients regarding average, start, or last doses of RUX. Median treatment duration was 1.5 years, with a slightly shorter duration in patients with PMF and PET-MF than in PPV-MF patients. As expected, patients who dropped out showed a lower median average dose and a lower median treatment duration than patients who had completed the trial ; however, median study duration was comparable between the IPSS risk groups: .Figure The extent of spleen length reduction among the three disease subentities  was similar, with the largest reduction observed in patients with PPV-MF and the smallest in patients with PET-MF, but the effect was dose dependent, with a more pronounced reduction observed with 40 mg/day RUX compared with 20 mg/day or 10 mg/day.Spleen assessment by abdominal ultrasound was performed in 75.7% of cases at baseline. While spleen volume was only rarely reported, spleen length by ultrasound was frequently reported, with a median length of 18.5 cm. Spleen length by ultrasound after 2 months of RUX therapy was reported as 16.2 cm (median change of –2.0 cm vs baseline). The extent of spleen length reduction by ultrasound was again highest in the PPV-MF group and lowest in the PET-MF group, and dose dependent, with the most pronounced dose reduction seen with 40 mg/day RUX.Interestingly, correlation between spleen size measured by palpation  vs spleen length by ultrasound was accurate Fig. , Fig. 4,p = 0.0623, sign test). Furthermore, there was a significant improvement in symptom burden during RUX treatment, as reported by both physicians and patients.ECOG performance status of patients showed a trend towards improvement after RUX treatment (16.5% of patients with ECOG 2–4 at baseline vs 12.7% at Month 36), with the proportion of patients with no restrictions (ECOG 0) increasing over time (29.9% at baseline vs 43.7% at Month 36). In 29.0% of patients, the ECOG performance status decreased, while it tended to be increased in 18.8% of patients at Month 36, as compared with baseline (p < 0.0001) at Month 1, 31.3% (p < 0.0001) at Month 2, and 20.3% (p < 0.0001) at Month12, remaining stable afterwards until the end of follow-up at Month 36. There were no major differences between the three disease subentities. Similarly, weakness , sweating , fever , pruritus , bone pain (9.1% vs 4.5% p < 0.0001), and weight loss  decreased during RUX treatment.When assessed by physicians, overall constitutional symptoms were present in 66.2% of patients at baseline, but only in 37.2% patients (p = 0.0308). At the end of the study, 41.2% of patients reported an at least five-point improvement in TSS, and 26.8% of patients reported an at least 50% improvement in TSS vs baseline . Figure 80 (17.2%) patients died during the study, 25 (5.4%) patients terminated the study due to progression of primary disease, and seven (1.5%) patients terminated the study because of transformation to acute myeloid leukemia (AML).86.9% of all patients experienced at least one AE Table , the mosSAEs were reported in 51.3% of patients. SAEs that occurred in at least 5% of patients included anemia (11.9%), thrombocytopenia (5.0%), general physical health deterioration 5.6%), and pneumonia (5.4%). For SAEs occurring in more than 1% of patients, please see Table %, and pnn = 7) and 1.9% (n = 9) of patients, respectively, discontinued treatment, indicating that these AEs were manageable in most patients.The most common adverse hematologic events were anemia  and thrombocytopenia . However, as expected, baseline hemoglobin levels differed between the three MF subentities, and, while median hemoglobin eventually recovered in patients with PMF, it remained significantly decreased in patients with PET-MF and PPV-MF at the end of the study.Median hemoglobin levels decreased initially upon RUX treatment but subsequently stabilized or even returned to their initial values . While the WBC count was initially highest in patients with PPV-MF, this decrease was seen across all three MF subentities and, at the end of the study, the median WBC count was around 8 × 109/L in all MF subentities.Median WBC count decreased during RUX treatment from 13.3 × 109/L at baseline to 181 × 109/L at 2 months (p < 0.0001) but increased afterwards to 209 × 109/L at the end of the study.During RUX treatment, the median number of platelets initially decreased from 287 × 10The most common non-hematologic AEs (occurring in ≥ 5% of patients) included nausea, diarrhea, pyrexia, fatigue, and asthenia. Atrial fibrillation (AF) occurred in 3.9% of patients .JAK2V617F induces leukocyte AP; therefore, we investigated whether RUX treatment changed AP levels in our cohort overall. Indeed, median AP levels decreased from 99.0 U/L at baseline to 67.0 U/L at the end of the study (p = 0.0068).Elevated alkaline phosphatase (AP) levels have been described in Philadelphia chromosome–negative MPNs, and p < 0.0001).There were no relevant changes in bilirubin, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, glutamate dehydrogenase, serum protein, or lipase levels during RUX treatment. Also, urea, sodium, potassium, and chloride levels were not elevated at baseline and did not change relevantly during RUX treatment. Median creatinine slightly increased over time, with the increase being most severe in patients with PET-MF and PPV-MF  or tuberculosis reactivation. One single case of progressive multifocal leukoencephalopathy was reported.Here, we report on the largest German cohort of patients with MF treated according to the approved label with RUX almost exclusively by hematologists/oncologists in the outpatient office setting and, thus, reflective of the true, real-world scenario of RUX treatment as per the SmPC. This interim analysis focusses on 464 patients with PMF, PPV-MF, or PET-MF not previously treated with RUX or any other JAKi (Arm A).There were no further restrictions regarding comorbidities, dynamic (D)IPSS risk, or spleen size. Patients with low-risk disease were included independent of their blood counts. Despite the liberal inclusion criteria, median spleen length at baseline (14 cm BCM) was comparable to the JUMP 13.3 cm) and COMFORT-II (14 cm) trials , 13. Des.3 cm andOne rather surprising finding was the high percentage (58%) of missing data for IPSS (or DIPSS) scoring, suggesting that physicians involved in this trial did not employ (D)IPSS scoring when deciding to commence RUX treatment. This contrasts current guidelines by the German Society of Hematology and Medical Oncology , which rInterestingly, the JAKoMo study cohort showed signs of both earlier disease  and, concomitantly, more advanced disease, with around half of the patients in the JAKoMo trial (48.5%) having been transfusion dependent at baseline, almost twice as much as in the JUMP trial (25.9%) . The JAKMedian treatment duration in patients participating in the JAKoMo trial was 1.5 years, a little longer than in the JUMP trial. Primary reasons for RUX discontinuation were also similar in both trials, particularly thrombocytopenia and disease progression. Importantly, kidney and liver dysfunction were almost never stated as reasons for dose adjustments in our trial, suggesting that this was not deemed a significant clinical problem.Median spleen length was markedly and rapidly reduced from 13.9 cm BCM at baseline to 8.0 cm after 2 months of therapy (reduction of 42%); this was comparable to efficacy reported in the JUMP trial (40%) and slightly lower than that reported in the COMFORT-II (56%) trial. Comparison of spleen length by palpation and by ultrasound suggested that spleen length by palpation may be overestimated; however, despite the overall overestimation by palpation, intraindividual correlation between both measures in our patients was very accurate, leading us to conclude that overestimation by palpation does not vary between several visits. As spleen sizes via palpation correlated well with ultrasound length measurements, this suggests that palpation is a cost-effective and simple alternative in the routine setting, but it might overestimate actual spleen size.Since the beginning of RUX clinical development, clinical trials in MF have included spleen size and symptom reductions as clinical endpoints. Although these endpoints translate into benefits for patients by providing better quality of life, a landmark trial  demonstrIn the real-world setting of our study, RUX was fairly well tolerated, revealing no new safety signals. As described, the most frequent AEs included anemia in 31% and thrombocytopenia in 27% of patients. Anemia-related SAEs occurred in 13% of patients in this population (in comparison to 4.5% of anemia SAEs in the JUMP trial  and 5% oIn addition to the points addressed above, limitations to this analysis include the observatory character of the trial with limited on-site monitoring of the data, and this may also explain the missing values, particularly regarding IPSS scoring and molecular genetics assessments.In conclusion, this large analysis of RUX treatment in therapy-naïve MF patients confirms feasibility in the real-world setting and demonstrates efficacy and tolerability comparable to those observed in the more selected JUMP and COMFORT-II drug approval trial populations.


Camellia sinensis L.) is promoted by the presence of aluminum (Al), a beneficial element under acidic conditions, but the influence of rhizosphere pH on this interaction is not known. To understand the mechanisms underlying the adaptation to acidic rhizosphere conditions, we evaluated ionome profiles and the effect of pH on tea growth in hydroponic culture. The optimum pH for tea growth was around pH 4.2, and growth was inferior under a pH less than 3.8 or higher than 5.0. Under the optimum pH growth and Al accumulation were markedly stimulated by Al treatment. Al content and accumulation in new and mature leaves and new roots  gradually declined with decrease in pH, especially in new roots. Ionome profiles drastically altered Al treatment, but changes were more pronounced in new roots than in new or mature leaves and did not depend on pH. Although the uptake of most cationic minerals in new roots was decreased by Al treatment, cationic mineral contents in new and mature leaves were not decreased by Al. In contrast to other plant species, the content and accumulation of manganese, despite it being a cationic nutrient, were significantly increased by Al treatment. These results indicated that one role of Al as a beneficial element was to maintain the shoot nutrient status by effectively utilizing Al‐limited elements in the roots.The growth of tea plants ( A slight modification of the culture method described by Konishi et al. (2(SO4)3‧14‐18H2O, at pH 4.2 3‧14‐18H2O. Each experiment was conducted using three biological replicates. The solutions were replaced at 2‐day intervals to maintain the pH. After 5 weeks, tea plants were harvested following the methods described by Morita et al. (2SO4) for 3 min to remove Al absorbed on the root surface. After washing with deionized water, the plants were divided into leaves, stems, and roots, and each part was further separated into new and mature parts: new parts were those that had emerged during treatment, and mature parts were those that were present at the start of treatment. Thus, the growth of new shoots, comprising new leaves and stems, and new roots were evaluated as the growing parts. Each sample was weighed fresh, then freeze‐dried and re‐weighed to determine the dry weight (DW).An initial hydroponic experiment (in spring of 2017) was performed to evaluate broadly the effects of pH and the presence of Al on plant growth. Plants were transferred to nutrient solutions adjusted using Ha et al. . At harvThe second hydroponic experiment (in spring of 2018) was performed to evaluate in detail the effects of pH and Al on the growth and ionome profile of new roots and leaves. Plants were precultivated under aforementioned standard hydroponic conditions at pH 4.2 with 400 μM Al until the first new shoots had developed five leaves, and then the plants were grown for approximately 2 months to investigate the effects of different treatments on new roots. The first new shoots with five leaves were excised leaving the lowest two leaves, and the plants were transferred to hydroponic solutions adjusted to various pH values from 3.0 to 5.8 in pH 0.2 increments with or without 400 μM Al. The plants were sampled using the same methods as described for the first growth experiment. The freeze‐dried samples were ground into a fine powder then stored in a desiccator at room temperature until analysis. The subsequent mineral analyses were conducted using the samples grown at pH 3.0–5.2 for new leaves and roots, and pH 3.0–5.8 for mature leaves, as the predominant tissues that accumulate minerals in tea plants. New leaves and roots of plants cultured under pH 5.4–5.8 could not be analyzed because the plants did not grow under these extreme conditions.2.23 at 110°C in DigiTUBE® tubes  for approximately 2 hr. Once the samples had cooled, 2 ml of 60% HClO was added and the samples were heated at 110°C for a further approximately 2 hr. Once digestion was completed, the samples were cooled and made up to a volume of 10 ml with ultra‐pure water. The total concentration of the following 13 elements was measured, based on selected specific wavelengths using an ICP‐OES : Al 396.152 nm, Fe 259.940 nm, sodium (Na) 589.592 nm, boron (B) 249.773 nm, P 213.618 nm, sulfur (S) 180.731 nm, silicon (Si) 251.611 nm, Ca 315.887 nm, copper (Cu) 324.754 nm, K 766.490 nm, Mg 285.213 nm, Mn 257.610 nm, and zinc (Zn) 206.200 nm.Fine powder (50 mg) of freeze‐dried samples was digested in 2 ml of 60% HNOTotal carbon (C) and N were measured by dry combustion using a Vario MAX cube  with aspartic acid as a standard.2.3s and among pH values were determined using two‐way analysis of variance (ANOVA) and simple linear regression, respectively. Significance of correlations between Al and other minerals was determined using Pearson correlation analysis, while correcting for multiple comparisons. The q‐values were calculated for multiple testing using the Benjamini–Hochberg false discovery rate  and mineral accumulation (mg/plant). Mineral accumulation (mg/plant) was calculated from the mineral content (mg/g DW) and the plant tissues dry weight (g DW). Significant differences in growth, mineral contents, and mineral accumulation between pH and Al treatmentRevelle, . The q‐vz‐score values for each mineral in principal component analysis (PCA). The PCA was performed using the R function “prcomp,” and the principal component scores and biplots were plotted using the R package “ggplot2” ver. 3.1.0 . Root growth stimulation by Al was observed at pH 3.2–5.2 . The development of new roots in the –Al and +Al treatments was observed at pH 3.0–5.2 and 3.0–5.4, respectively . Root growth stimulation by Al was observed especially at pH 4.2 and 5.0 , and especially under the optimum hydroponic growth conditions at around pH 4.2. The Al accumulation in mature leaves and new roots was increased by Al treatment . The Al content and accumulation were higher in new roots, mature leaves, and new leaves  and lowered with decrease in pH . The highest Al content at pH 4.4 in new roots was approximately threefold that observed at pH 3.0.We analyzed the Al content and accumulation in new leaves, mature leaves, and new roots, as the predominant tissues for mineral accumulation in tea plants, under different pH conditions. The Al content in the three tissues was increased by Al treatment , especially around the optimum pH 4.2 for the growth of tea plants. An Al‐induced increase in Mn accumulation in new leaves and new roots was also observed .To determine the effect of pH and Al on the distribution of a range of elements in the predominant mineral‐accumulating tissues, we used ICP‐OES and a CN analyzer to analyze the following 14 elements, in addition to Al, constituting the ionome: Fe, Na, B, P, S, Si, Ca, Cu, K, Mg, Mn, Zn, N, and C Table . PERMANO3.4Correlation analyses of the ionome dataset revealed positive and negative correlations among the 15 elements in each of the three mineral‐accumulating tissues Figure . To unde4+ and Al3+, which causes inhibition of root growth and nutrient uptake (Kochian et al., The major rhizotoxicity factor in acid soils is the excess of H+ competitively inhibited Al uptake in the roots of tea plants.The Al content and accumulation were increased by Al treatment, and this was observed especially under the optimal conditions for the growth of tea plants around pH 4.2 Figure . These r3+ (Kinraide et al., Ionomics is a useful tool to understand changes in physiological processes in response to nutrient status (Baxter et al., Interestingly, the only element significantly increased in content by Al treatment was Mn, despite it being a cationic nutrient; Mn content was not affected by pH conditions, and an Al‐induced increase in Mn accumulation in the leaves was also observed Figure . In riceCorrelation analysis confirmed the results revealed by PCA Figure . B and MC. sinensis var. sinensis (Wei et al., assamica (Xia et al., In conclusion, we revealed that the growth of tea plants was stimulated by both Al and acidic pH, with optimum growth observed in a narrow range around pH 4.2 and inferior growth at pH less than 3.8 or higher than 5.0. Under the optimum pH conditions, Al markedly stimulated growth and Al accumulation at the whole‐plant scale. Furthermore, we showed that the alteration to ionome profiles caused by Al in tea plants did not depend on pH Figure . Our finH.Y. conducted the hydroponic growth tests and harvested the samples. H.Y., Y.F., and S.Y. performed the mineral analysis. H.Y. and T.I. performed the ionome data analysis. A.M. and T.I. acquired the funding. H.Y., A.M., and T.I. designed the experiments and wrote the manuscript. All the authors read and approved the manuscript.The authors declare no conflict of interest associated with this manuscript.Figure S1–S3Click here for additional data file.Table S1Click here for additional data file.


Chlorella sorokiniana is a microalgae which flourishes under conditions of high light intensity and temperature, thus its prevalence in algal blooms is expected to increase with climate change. However, Chlorella sorokiniana AOM has not been previously investigated as a DBP precursor. In this context, this study evaluated the effect of AOM concentration, humic acid (HA), and pH on DBP formation from chlor(am)ination of AOM Chlorella sorokiniana. DBP yields determined by linear regression for trichloromethane (TCM) and chloral hydrate (CH) were 57.9 and 46.0 µg·mg DOC−1 in chlorination, while the TCM, CH, dichloroacetonitrile (DCAN), 1,1,1-trichloropropanone , and chloropicrin (CPN) concentrations were 33.6, 29.8, 16.7, 2.1, and 1.2 µg·mg DOC−1 in chloramination. Chloramination reduced the formation of TCM and CH but increased CPN, DCAN, and 1,1,1-TCP yields. AOM Chlorella sorokiniana showed a higher DBP formation than 9 of 11 algae species previously investigated in the literature. At basic pH, the concentration of TCM increased while the concentration of other DBP classes decreased. Bromide was effectively incorporated into the AOM structure and high values of bromine incorporation factor were found for THM (1.81–1.89) and HAN (1.32) at 1.5 mg Br·L−1. Empirical models predicted successfully the formation of THM and HAN (R2 > 0.86). The bromide concentration had more impact in the model on the DBP formation than AOM and HA. These results provide the first insights into the DBP formation from AOM chlor(am)ination of Chlorella sorokiniana. Eutrophication in water reservoirs releases algal organic matter (AOM), which is an important precursor of disinfection by-products (DBPs) formed during water treatment.  The phenomenon has been becoming increasingly frequent and severe over time as a result of climate change ,2. Algalam)ination has been prioritized since AOM is recognized as one of the major precursors of DBP formation. Together algae and AOM can contribute from 20 to 50% of the DBP formed during algal blooms [−1 in Europe [−1, respectively [AOM is a well-known precursor of DBPs, such as chloral hydrate (CH), haloacetonitriles (HANs), and trihalomethanes (THMs) ,11,12. Rl blooms . Carbonal blooms ,15,16, cl blooms ,17. THMsn Europe . Investin Europe . They aram)ination of different algae species, such as Anabaena flos-aquae, Aphanizomenon flos-aquae, Asterionella Formosa, Chlorella vulgaris, Chlorella sp., Dolichospermum circinale; Microcystis aeruginosa, Chaetoceros mulleri, Melosira sp., Oscillatoria prolifera, Scenedesmus quadricauda, and Scenedesmus subspicatus [am)ination of drinking water source  (main species Microcystus sp. and Anabaena sp.) [Synecococcus sp.) [Microcystis aeruginosa is the most analyzed specie due to its abundance in eutrophication events [Chlorella genus other than Chlorella vulgaris, have been overlooked even though they can be the prevalent species in algal blooms [Chlorella sorokiniana is found worldwide [Chlorella species, can flourish under conditions of high light intensity (2100 µmol·m−2·s−1) [am)ination of AOM of Chlorella sorokiniana. To our best knowledge, it is the first the Chlorella sorokiniana AOM has been investigated regarding DBP formation.Research has been conducted to examine the DBP formation from AOM chlor  and Red n events . Despitel blooms ,32. The orldwide ,34,35 anm−2·s−1)  and tempm−2·s−1) . These cChlorella sorokiniana 211-8k was obtained from the Culture Collection of Algae and Protozoa, Argyll, Scotland. Chlorella sorokiniana was cultivated according to Leite et al. (2021) [g, 10 min), washed twice with ultrapure water, and frozen at −20 °C.. (2021) . The celChlorella sorokiniana cells was extracted using the protocol suggested by Leite et al. (2019) [g, 10 min) and the supernatant was filtered by a 0.45 µm membrane. Then, the extracted AOM was kept at −20 °C until use. The dissolved organic carbon (DOC) content of the AOM was quantified using a TOC-L analyser .The AOM from . (2019) . In brieam)ination protocol was adapted from method 5710 [3·L−1 of alkalinity), a mass ratio of 5:1 mg Cl2∙mg DOC−1, the contact time in the dark of 7 days, and temperature of 20 °C. The alkalinity and pH values were selected based on the water quality found in eutrophicated reservoirs in São Paulo State, Brazil [2:NH3-N [The chlor colourimetric method using powder pillows . The concentration of residual disinfectant at the end of tests was always above 2.0 mg Clam)ination experiments was assessed in this study, including THMs (trichloromethane (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM)), HANs (bromochloroacetonitrile (BCAN), dibromochloroacetonitrile (DBCAN), and DCAN, trichloroacetonitrile (TCAN)), HKs  and 1,1,1-trichloropropanone ), chloropicrin (CPN), and CH. Their concentrations were quantified by a gas chromatograph- electron capture detector  with polysiloxane column  following USEPA 551 method [The presence of target DBPs in the chlor were normalized to DOC dividing the DBP concentration (µg·L−1) by the AOM concentration (mg DOC·L−1).A mixed standard of EPA 551B Halogenated Volatiles Mix  and EPA 501/601 Trihalomethanes Calibration Mix , and CH  were used to construct the calibration curves. The calculated limits of detection (LD) were ≤ 0.7 µg·L−) concentrations, and pH values. The selected values represent a typical range of pH [In total, the impact of 25 different experimental conditions of a single factor on DBP formation was evaluated , encompage of pH ,44, orgage of pH ,46, and ge of pH ,47,48 fo−1).The pH of the water test was modified using 1 N NaOH or 1 N HCl . HA solution was prepared by diluting 0.5 g of HA  in 1 L ultrapure water, mixing for 2 h and filtered using a 0.45 μm membrane. HA concentration was also expressed as DOC value. Required bromide concentration was added from a standard solution of KBr  was also assessed in four levels  .am)ination, since brominated DBPs are more genotoxic and cytotoxic than chlorinated DBPs [The BIF quantifies the amount of bromine incorporated into a DBP class as a proportion of the total formation of chlorinated and brominated DBPs. It is a useful parameter to compare the degree of bromination of the DBP classes in chlor(ted DBPs . The BIFn = 7–8). Experimental data of THM and HAN were modelled using empirical models , as summarised by Chowdhury et al. (2009) [R2). Modelling was undertaken using Microsoft Excel Solver.Correlations between DBPs yield and relevant water quality parameters were assessed by linear regression. The DBP concentrations per mg of AOM/HA were reported based on the slope of the equations generated by a linear equation of each data set (. (2009)  using boR2 > 0.98 for all DBPs. Based on the regression lines, yields of TCM and CH were 57.9 and 46.0 µg·mg DOC−1 following chlorination, while the TCM, CH, DCAN, 1,1,1-TCP, and CPN concentrations were respectively 33.6, 29.8, 16.7, 2.1, and 1.2 µg·mg DOC−1 following chloramination. TCM and CH were the DBPs detected following chlorination in the absence of bromide, while TCM, CH, DCAN, CPN, and 1,1,1-TCP were quantified following chloramination . Thus, Tam)ination of different algae species and testing conditions (−1) > CH (0.2–32.5 µg·mg DOC−1) > HAN (0–62.5 µg·mg DOC−1) > HK (0.0–77.5 µg·mg DOC−1) > CPN (0–27.5 µg·mg DOC−1) [Chlorella sorokiniana showed a higher DBP formation than 9 of 11 algae species previously investigated in the literature (−1) following chlorination experiments in the current work is higher than literature values  ,26,27,28terature . The CH e values . This isChloramination reduced the formation of some DBP species (42.0% of TCM and 35.2% of CH) but promoted the formation of N-DBPs (HAN and CPN) and 1,1,1-TCP. The lower concentration of these species in chloramination than in chlorination is consistent with previous studies, though the magnitude of the reduction varies between studies and experimental conditions ,26,30. The absence of N-DBPs during chlorination indicates that the inorganic nitrogen present in chloramine was responsible for the formation of nitrogenous species. Previous studies also reported chloramine acts as a precursor for some N-DBPs  during AOM chloramination, even if the precise formation pathway are sometimes unclear ,26. −1 at pH 10 for chloramination and chlorination, respectively . Similar trends in the DBP formation were also reported by a previous study of AOM chlorination of Microcystis aeruginosa. Fang et al. (2010a) [TCM concentration increased at high pH, reaching the highest concentration of 50.0 and 144.8 µg·mg DOCectively . This reectively . In gene (2010a)  observedam)ination reactivity with the AOM and the stability of the DBPs formed in the solution. Hydrolysis decomposition of unstable DBPs  has higher rates at basic pH [The pH affects the chlor while CH remained essentially constant. Following chloramination, all the DBP species increased with the increasing HA concentration. For instance, 194.2 µg·mg DOC−1 of TCM, 53.4 µg·mg DOC−1 of CH, 111.0 µg·mg DOC−1 of HAN, 12.9 µg·mg DOC−1 of 1,1,1-TCP, and 5.0 µg·mg DOC−1 of CPN were the concentrations per mg HA generated in the presence of 0.1–1.5 mg DOC L−1 of HA , contact time (3 to 7 d), pH (6–7.5), and organic matter present , and a potential synergistic effect when both HA and AOM are present in the solution. In general, the chloramination reduced the TCM and CH yield by 35.9–47.8% and 2.8–29.3%, respectively, compared to the chlorination. These results indicate that although AOM may not be the biggest contributor to the formation of DBP compared to NOM, it still has a significant contribution to the DBP yield.The NOM is the major precursor of DBP formation and is primarily comprised of HA and fulvic acid. The effect of HA, a widely used surrogate for NOM in studies on AOM ,56, on D−1 of HA . These v sources ,57,58. T−1 at 0·mg Br L−1 to 0 µg·mg DOC−1 at 1.5·mg Br L−1. THM and HAN formation following chloramination reached concentrations of 97 and 72 µg·mg DOC−1 for THMs and HANs at 1.5 mg Br L−1, respectively. Chloramination in the presence of bromide reduced the THMs and CH formation by 34.4–56.3% and 21.5–100% compared to chlorination. These findings align with the results of other studies, which have also demonstrated that using chloramination can decrease the formation of DBPs even in the presence of bromide [Bromide addition increased the concentration of brominated types of HANs and THMs, as well as the overall yields of these groups . Yields  bromide ,59.−1 for chlorination and chloramination, respectively, while the BIF of HAN was 1.32 at the same bromide dose. These values are indicative of high bromine incorporation (−1·mg−1) as observed in other studies [BIF results are shown in poration . This su studies .Chlorella vulgaris (BIF = 1.4) and Microcystis aeruginosa (BIF = 1.4) with 1.0 mg Br·L−1 [Microcystis aeruginosa of 0.51–0.68 for chlorination and 0.38–0.87 for chloramination with 0.5 mg Br·L−1. The incorporation of bromide in the structure of AOM depends on the algae species and the Br/DOC ratios [Similar results were observed in THMs formed from AOM chlorination of g Br·L−1 . While Cg Br·L−1  found BIC ratios ,61, whic−1 for THMs and CH after chlorination, respectively, and 167.8, 146.8, 95.9, 10.3, and 5.8 µg·L−1 for THMs, CH, HANs, 1,1,1-TCP, and CPN after chloramination. Meanwhile, the worst case scenario  formed 2430.7 and 289.6 µg·L−1 for THMs and CH following chlorination, respectively, and 1106.2, 765.9, and 6.9 µg·L−1 for THMs, HANs, and 1,1,1-TCP following chloramination. Considering all conditions, chloramination reduced THM and CH formation by 29.5–54.5% and 38.0–100% compared with chlorination.The effect of the multiple parameters on DBP formation previously tested individually (HA + Br + AOM) was also assessed simultaneously to simulate the complexity of real water matrices . The res−1 for THM4 in the EU [−1 for TCM, 100 µg·L−1 for TBM and DBCM, 100 µg·L−1 for BDCM, 20 µg·L−1 for DCAN, and 70 µg·L−1 for DBAN) [Chlorella sorokiniana were higher) this does nonetheless highlight the potential for this species of algae to generate significant concentrations of DBPs. In turn, the study indicates that Chlorella sorokiniana blooms can threaten drinking water quality and this species’ presence should be therefore monitored and mitigated. It emphasises the importance of removing AOM before the addition of chemical disinfectants during drinking water treatment.These conditions show the potential for DBPs to exceed regulatory limits . The most appropriate model to predict the DBP yield (µg·L−1) had the following general form:a, b, and c are the model constants.Modeling of DBP yields is a helpful tool to quantify and understand the influence of key water quality parameters on DBP formation. Various empirical models have been reported in the literature to predict the impacts of water quality parameters on DBP yields as summarised by Chowdhury et al. (2009) . For exaR2 = 0.95) had a greater fit (Equation (4)) to the chlorination data, meanwhile, THM ) and HAN ) had a greater fit to the chloramination data.The data were modelled by linear regression. THM model ination to compare with ours. Ersan et al. (2021) [Chlorella sp. using AOM properties  and obtained an exponential equation. No previous study reported models with the same equation form and variables for AOM chlor(. (2021)  develope. (2021)  modelledChlorella sorokiniana is a more potent DBP precursor than 80% of algae previously investigated in literature. Considering its prevalence in algal blooms is expected to increase due to climate change, it is important to develop control strategies for reactive algae such as this one. The modeling results suggest bromide removal would perhaps be the most effective approach, but currently, there are no efficient/economic treatment options for bromide removal [am)ination. Oxidation processes are inherently a high-risk approach as they have the potential to increase downstream DBP formation. In particular, ozonation (22–781%) [am)ination. Therefore, physical removal processes, for example, ultrafiltration and/or activated carbon adsorption, are expected to be the most pragmatic and effective methods.This study has shown  removal . Convent removal ,9. Thus,22–781%) ,26,65 an22–781%) ,66 of AOam)ination of AOM Chlorella sorokiniana. DBP yields determined by linear regression for TCM and CH were 57.9 and 46.0 µg·mg DOC−1 following chlorination, while mean concentrations of TCM, CH, DCAN, 1,1,1-TCP, and CPN concentrations were 33.6, 29.8, 16.7, 2.1, and 1.2 µg·mg DOC−1, respectively during chloramination. Chloramination reduced the formation of TCM and CH but increased CPN, DCAN, and 1,1,1-TCP yields. At basic pH, the concentration of TCM increased while the concentration of other DBP classes decreased. Bromide was effectively incorporated into the AOM structure and high BIF values were found for THM (1.81–1.89) and HAN (1.32) at a bromide dose of 1.5 mg·L−1. Empirical models were generated using the data obtained and effectively predicted the formation of THMs and HANs (R2 > 0.86). Changes in bromide concentration had more impact on the DBP formation than AOM and HA in the model. These results provide the first insights into the DBP formation from AOM chlor(am)ination of Chlorella sorokiniana, which was found to be more reactive as a DBP precursor than 80% of algae previously investigated in literature. Nonetheless, removing AOM and natural organic matter before disinfection is anticipated to be an effective control strategy for this species of algae. This study investigated the effect of AOM concentration, HA, and pH on the DBP formation from chlor(


At the cellular level, acute temperature changes alter ionic conductances, ion channel kinetics, and the activity of entire neuronal circuits. This can result in severe consequences for neural function, animal behavior and survival. In poikilothermic animals, and particularly in aquatic species whose core temperature equals the surrounding water temperature, neurons experience rather rapid and wide-ranging temperature fluctuations. Recent work on pattern generating neural circuits in the crustacean stomatogastric nervous system have demonstrated that neuronal circuits can exhibit an intrinsic robustness to temperature fluctuations. However, considering the increased warming of the oceans and recurring heatwaves due to climate change, the question arises whether this intrinsic robustness can acclimate to changing environmental conditions, and whether it differs between species and ocean habitats.Hemigrapsus sanguineus and Carcinus maenas that have seen a worldwide expansion in recent decades.We address these questions using the pyloric pattern generating circuits in the stomatogastric nervous system of two crab species, H. sanguineus, but not in C. maenas. Comparing the temperature responses of C. maenas from a cold latitude (the North Sea) to those from a warm latitude (Spain) demonstrated that similar shifts in robustness occurred in natural environments. Our results thus demonstrate that neuronal temperature robustness correlates with, and responds to, environmental temperature conditions, potentially preparing animals for changing ecological conditions and shifting habitats.Consistent with their history as invasive species, we find that pyloric activity showed a broad temperature robustness (>30°C). Moreover, the temperature-robust range was dependent on habitat temperature in both species. Warm-acclimating animals shifted the critical temperature at which circuit activity breaks down to higher temperatures. This came at the cost of robustness against cold stimuli in  Cancer borealis and Cancer pagurus) that live in the epipelagic and mesopelagic zones (up to a depth of 750 m). With ongoing global ocean warming and recurring heatwaves with rapidly changing temperatures affecting intertidal and pelagic habitats of marine crustaceans, it now becomes crucial to explore the limits of temperature robustness, and to investigate potential regulatory mechanisms, species-specific differences, and adaptation to altered environmental conditions.Temperature is a critical environmental factor that profoundly influences the physiological processes and behaviors of organisms . The nerHemigrapsus sanguineus and Carcinus maenas. These species live in the intertidal zone  twice a week. In Greifswald, crabs were kept in air-bubbled artificial seawater  that was exchanged every 10 days. Animals were fed frozen shrimp (Litopenaeus vannamei) once a week. Habitat temperatures were 8°C (C. maenas from Helgoland), 15°C (C. maenas from Vigo), and 15°C (H. sanguineus from Helgoland). A subset of the C. maenas crabs collected in Helgoland was acclimated to 15°C for at least 2 months. A subset of the H. sanguineus crabs was acclimated to 25°C for at least 2 months. The research presented in this paper complies with national (Germany) and international laws (guidelines from the directives 2010/63/EU of the European parliament and of the Council of 22nd September 2010) on the protection of animals used for scientific purposes.Adult Carcinus maenas saline solution was composed of 410 mM NaCl, 11 mM KCl, 11 mM CaCl2, 21 mM MgCl2, 30 mM NaHCO3, 11 mM Tris, 5.1 mM maleic acid; pH 7.4–7.6 , pyloric dilator nerve (pdn), pyloric constrictor nerve (pyn), medial ventricular nerve (mvn), and lateral gastric nerve (lgn) were electrically isolated from the bath with petroleum jelly wells . Files were analyzed in Spike 2  using original Spike 2 scripts, after conversion to Spike 2 format. All neurons could be individually identified on the respective nerve recordings. Cycle periods and cycle frequencies (1/cycle period) were calculated from the beginning of one burst of the pyloric dilator neuron (PD) to the beginning of the next burst. Duty cycles and phase relationships were calculated by dividing burst parameters  by cycle period.F value, p value, post hoc test. “N” denotes the number of preparations. Post hoc tests are at a significance level of 0.05. Data was prepared in Excel and finalized in Coreldraw . In figures, data is presented as mean ± SD or mean ± SEM unless otherwise noted.We used parametric tests (ANOVA and t-test) for normally distributed data. For non-normally distributed data, we used Friedman Analysis of Variance on Ranks. In cases where multiple variables were measured in the same animals, repeated measures tests were considered. The factors and specific designs of each test are given in We define robustness as the tolerance of the pyloric network against acute temperature perturbations. Specifically, we classified pyloric rhythms as “regular” when they were triphasic and continuously active, i.e., when the three main pyloric neurons showed continuous regular and rhythmic burst activity see , bottom.Preparations were continuously superfused with physiological saline , the single lateral pyloric neuron (LP) and the pyloric constrictor neurons (PY) – and recorded their spontaneous activities at different temperatures  based on its anatomical features, the muscles it innervates, and its similarity with previously studied pdn of other species , an incomplete separation of the pyn from the pdn, or because in this species, no clearly separatable pyn exists . Nevertheless, the different action potential amplitudes and firing patterns allowed a straight-forward separation of the three pyloric neuron types. As evident from the recordings of the three animals in H. sanguineus. Accordingly, the phase plot . The pyloric rhythm was recognizable, showed the familiar PD, LP, and PY phases, and was stable over time (C. maenas), recorded on the lvn and pdn, demonstrating that all C. maenas generate the canonical pyloric rhythms observed in other decapod crustaceans. Unlike in H. sanguineus, however, the pdn never contained PY activity, suggesting that there is a complete separation between pdn and pyn in this species. In fact, in a few preparations, we recorded the pyn instead or in addition to the pdn, and it contained only the PY neuron activity, but never the PDs. The phase plot  to 0°C, then heated it to 39°C, and then cooled it back down to 21°C. Three temperatures are shown . At temperatures close to 0°C, the rhythm became slow, and eventually disorganized. Individual neuron types failed to produce action potentials, with LP typically being the first neuron to be missing, while PD and PY continued to generate alternating rhythmic activity, albeit at a very slow rate. Eventually, rhythmicity seized entirely . entirely , rangingentirely . The heaC. maenas  to roughly 2.5 Hz before rhythmicity stopped and the rhythm crashed. The rhythm resumed after cooling the preparation.We found similar pyloric responses for . maenas . When coactivity . The speactivity  demonstrH. sanguineus , cycle frequencies in C. maenas were significantly higher at cold and warm temperatures , even though phase relationships and cycle frequency changed and how they changed was distinct between species. The most dramatic change to pyloric activity occurred when the rhythm stopped at the low and high ends of our temperature ramps. To further analyze the temperature robustness of the pyloric rhythm, we thus classified pyloric activity  during cold ramps, and finally failed entirely (crashed) at 1.64 ± 0.8°C (N = 7). Irregular or crashed conditions could not be achieved in all preparations: In two preparations we could not cool the preparation enough to reach intermittent activity, and in one additional preparation we did not reach the cold crash point. These data were thus excluded from the calculation of the average temperatures. During hot ramps, the pyloric rhythm remained regular up to 30.46 ± 2.9°C (N = 10), after which it entered the intermittent activity condition. It crashed at 31.71 ± 3.0°C (N = 10). There was substantial variability of the crash temperatures between individual animals, much more so than during cold ramps, as indicated by the approximately three-fold larger standard deviation during the hot ramp.H. sanguineus experiences temperatures between 0 and 23°C throughout the year  during cold ramps and crashed at 3.97 ± 1.1°C (N = 8). Both temperatures were significantly higher than in the 15°C group (N = 8), after which it entered the intermittent activity condition. It crashed at 37.35 ± 1.3°C (N = 8). There was variability between individual animals: For cold ramps, inter-animal variability appeared similar to the 15°C group, but variability was reduced for hot crashes compared to the 15°C group. In fact, in the 25°C group, the variability for the hot crashes now seemed similar to that for the cold crashes. In summary for H. sanguineus, all crash temperatures were significantly shifted after warm acclimation . It crashed at 1.16 ± 1.4°C (N = 10). During hot ramps, it entered the intermittent activity condition at 30.07 ± 1.9°C (N = 10), and crashed at 33.38 ± 2.2°C (N = 10). There was substantial variability in crash temperatures between individual animals, during both cold and hot ramps.For N = 9) was reached. It crashed at 1.81 ± 0.5°C (N = 9). During hot ramps, the rhythm remained in the teal, regular condition up to 33.29 ± 0.9°C (N = 9). The intermittent activity condition was observed up to 37.37 ± 1.1°C (N = 9) above which the rhythm crashed. The variability of the crash temperatures between individual animals was lower than in cold-acclimated animals, with the standard deviation being about half of that of the cold-acclimated group. The hot crash temperature thus shifted significantly after acclimation , where the average habitat temperature was 15°C, and kept them at this temperature for at least 2 months. The pyloric rhythm in these animals was also regular over a wide range of temperatures (N = 12) was reached, after which it showed intermittent activity. The rhythm crashed at 1.39 ± 0.7°C (N = 9). In 3 animals, the final crash temperature could not be reached (see Methods). They were thus excluded from the calculation. During hot ramps, the rhythm remained regular up to 32.49 ± 2.1°C (N = 11). The intermittent activity condition was observed up to 37.37 ± 1.1°C (N = 11) where the rhythm crashed. In one of the recordings  were not significantly different from each other, but they differed significantly from the 8°C cold-acclimated animals . In these species, hot crashes occur well below 30°C, and while the cold crash points have never been measured, animals inhabiting waters between 5°C and 14°C show behavioral responses at temperatures close to freezing  and for C. pagurus .C. maenas and H. sanguineus as eurythermic. C. maenas survives temperatures from freezing to over 35°C, with intact cardiac function up to 37°C  to Taiwan . The hoo Taiwan , and alto Taiwan , it is pH. sanguineus and C. maenas, suggesting that a precise coordination of the rhythmic pyloric output also contributes to the rhythm’s functional integrity in these species. However, in contrast to previous studies  is already close to the critical thermal maximum, suggesting that additional warm acclimatization cannot elevate the hot crash temperature any further.Looking at the robustness of the pyloric rhythm, we made an interesting observation: When acclimated to 15°C, the temperature robustness of the C. maenas, the CTmax of the righting response shifts from 31.8°C to 35.4°C when the animals are acclimating from 8°C to 22°C  between individuals in each species , 4. ThesWe noticed that cold crash variability was consistently smaller than hot crash variability . Most coC. maenas, the total range of regular activity increased after acclimation (H. sanguineus. This predicts that inter-animal variability should be reduced more strongly in C. maenas. Our data are consistent with this idea, showing that cold crash temperature variability diminished in C. maenas, but not in H. sanguineus.There is further support for this idea in limation . A largeDegeneracy is a key characteristic of complex biological systems and has been shown to exist in several other neuronal systems . In the The original contributions presented in the study are included in the article/WS: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. GT: Methodology, Resources, Writing – review & editing. LG: Methodology, Resources, Writing – review & editing. NE-N: Methodology, Resources, Writing – review & editing. JPG: Methodology, Resources, Writing – review & editing. AV-G: Conceptualization, Validation, Writing – review & editing. SH: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing – review & editing.