The non-target organisms tested were both plants (wheat and two ferns) and animals (the Prussian carp and marsh frog tadpoles). The tested insecticide significantly inhibited the growth of roots in wheat, a result that was also confirmed by a microbiotest application (62% root growth inhibition in sorghum and 100% germination inhibition in white mustard and garden cress). In ferns, even for the lowest concentration, the percentage of germinated spores was inhibited by 40% for Asplenium scolopendrium. The recorded toxicological effects of Actellic 50 EC upon the Prussian carp included a decrease in the respiratory rate and oxygen consumption, an increase in the number of erythrocytes and leukocytes, and an increase in blood glucose levels. The highest concentration (0.1 mg L−1 of Actellic 50 EC) caused a 50% decrease in the survival rate of marsh frog tadpoles after 5 days of exposure, negatively affecting body volume and length. Given the high degree of toxicity of the insecticide Actellic 50 EC, we recommend continuing investigations on non-target species, including both plants and animals, as the sub-chronic effects are quite little known in the scientific literature.This paper contributes to the ecotoxicological risk assessment of the Actellic 50 EC insecticide (with 50% pirimiphos-methyl as the active substance) tested on non-target organisms. The insecticide concentrations tested were the same for all organisms (0.1, 0.01, and 0.001 mg L Among pesticides, organophosphates have been introduced because of their lower persistence in the environment compared to organochlorines . In addihan 0.01 . The toxicity tests for the approval of Actellic 50 EC as a commercial product were carried out mainly on laboratory animals  and were related to the use of the insecticide for phytosanitary protection only in indoor storage spaces . −1 LC50 (96 hours) in rainbow trout; 0.21 µg L−1 LC50 (48 hours) in Daphnia; and 1.0 mg L−1 EC50 in algae. Biodegradation is reported to take between 3.5 and 30 days (DT50-soil), but mutagenic and carcinogenic effects are not known by this producer.In a recent posting, one of the Actellic 50 EC manufacturers provided information [−1 in unbuffered water at 20 °C) and a mean hydrolysis time (DT50) of 75 days at a pH of 9 and 25 °C [−1), because these compounds are acetylcholinesterase inhibitors [The active ingredient of Actellic 50 EC is pirimiphos-methyl . It is snd 25 °C . Even thnd 25 °C . The rephibitors ,12. Therhibitors . Their ehibitors . Due to hibitors .50 48-hour values for the commercial product Actellic 50 in the adults and juveniles of Oreochromis mossambicus at 28–29 °C under static conditions were reported to be 1.3804 and 2.1928 mg L−1, respectively [−1of Actellic 50, as well as excess mucus around the eyes. The LCectively ; the autCarassius gibelio is an invasive species that is becoming dominant in Europe. The Prussian carp was chosen as the bioindicator species in this study, since it is a common species in Romanian rivers, ponds, and lakes; easily available and maintained under laboratory conditions; and highly resistant to adverse environmental effects and the presence of pollutants. Amphibians are more sensitive to the action of xenobiotics in the environment than other vertebrates, because they live in both aquatic and terrestrial environment and have a thin and highly permeable skin through which they exchange gases, water, and electrolytes with the environment . Much ofBesides amphibian vertebrates and fish, the study also followed the effects of the spread of this insecticide on some plants  that exist in areas at risk of contamination by improper use or storage of this insecticide.We wanted to demonstrate the variability of the toxicity of the contaminated soils to plants, as well as the effectiveness and usefulness of the Phytotoxkit microbiotest as a practical and reliable tool  for asse-Lepidium sativum, Sinapis alba, and Sorghum saccharatum;The germination and growth of seedlings, their biomass, and their assimilation of pigments for -Triticum aestivum;The plant growth and biomass of -Athyrium filix-femina and Asplenium scolopendrium;The germination of spores and the differentiation of gametophytes for -Carassius gibelio;The survival rate, behavioral changes, respiratory rate, oxygen consumption, number of figurative elements, and blood glucose of -Pelophylax ridibundus tadpoles, their survival rate, and changes in the morphology of the body.The successful hatching of The purpose of this research was to highlight the effects induced by Actellic 50 EC in exposed non-target organisms by evaluating: The toxicity tests focused on the effects on the germination process and the early development of vegetative and reproductive organs for ferns, wheat, sorghum, white mustard, and garden cress. The last three plants are part of a standardized microbioassay commercially called Phytotoxkit.Lepidium sativum (garden cress) and Sinapis alba (white mustard)) and a monocotyledon species (Sorghum saccharatum (sorghum)) were exposed in triplicate to evaluate the effects of contaminated soil (72 h in darkness at 25 °C ± 2 °C) and control soil (reference or standard soils can be used as negative controls) [The Phytotoxkit is a 3-day bioassay  that is ontrols) ,24. Comm−1, A0.01 = 0.01 mg L−1, and A0.001 = 0.001 mg L−1, with 3–5 days of exposure. After 3 days of exposure, the germination percentage and the degree of root growth inhibition were determined. The soil was contaminated with Actellic 50 EC at the same concentrations/variants of pirimiphos-methyl used for the other plants tested, i.e., A0.1 = 0.1 mg LAfter 5 days of exposure, the inhibition of shoot and leaf growth and the variation in dry biomass and pigments compared to the control group were determined. Plant root, shoot, and leaf length were determined by photographing plant batches with a 13 MP digital camera and analyzing the images using ImageJ software . The plant biomass at the end of the experiment was determined by oven drying each batch of sorghum plants at 105 °C for 5 h and weighing the dry residue. The average dry biomass per plant was determined by considering the number of plants taken for analysis from each batch.a, chlorophyll b, and total carotenoid concentrations were determined using specific equations [The concentration of foliar pigments was determined by grinding 0.1 g of leaf tissue in 10 mL of acetone solution (80%). The extract was filtered and its spectrophotometric absorbance was read at 470, 647, and 663 nm. The chlorophyll quations .Triticum aestivum  were provided from the Pitești Agricultural Research and Development Station, Albota. Seed sterilization (5 min in 75% ethanol) [This test is commonly used to assess the toxicity of pesticides, heavy metals, drugs, nanomaterials, etc. , with whethanol)  was follAthyrium filix-femina and Asplenium scolopendrium. Spores were provided by the Romanian Pteridological Association. The spores were grown on the surface of liquid culture media (Knop solution with/without Actellic 50 EC)  . The culThe state of the culture. For both experiments, culture dishes were kept in a POL EKO 350 growth chamber. The temperature values were 25 °C during daytime and 15 °C at night, and the humidity and light conditions were controlled (photoperiod: 16 h of light and 8 h of darkness).Carassius gibelio) and marsh frogs (Pelophylax ridibundus), captured from the surrounding lakes of Piteşti. The biological materials used were Prussian carp samples  with an average weight of 14.54 ± 1.24 g, caught from the Argeș River, the Mihăilești downstream sector upstream from the Dâmbovița confluence. According to the most recent report—2020 [The experiments were carried out on male and female Prussian carps . Some authors have reported residues of pirimiphos-methyl in surface waters: 8.4–12.3 µg L−1 in runoff water from clay soil plots cultivated with potatoes and of differing soil surfaces [−1 in the river water dissolved phase (WDP), n.d., 1.54 ng L−1 in suspended particulate matter (SPM), and 2.23 ± 0.27 ng L−1 in sediment samples from the Sele River estuary, Southern Italy [After 10 days of adaptation in the lab, where they were fed ad libitum once a day, the fish were divided into three groups of 10 fish each: the control group, without the addition of insecticide, and two experimental groups exposed to the Actellic 50 EC insecticide at concentrations of 0.002 µL Lsurfaces ; 23.3 ppsurfaces ; and 0.3rn Italy .−1, a pH of 7.83 ± 0.65, a total hardness of 100 mg L−1 CaCO3, and a natural light/dark photoperiod. The fish were not fed during the 14 days of the experiment in order to avoid the intervention of this factor [The exposure method chosen was the semi-static test; the immersion solution was changed every 24 h by transferring the fish to another aquarium, and the water was continuously aired. Fish were kept in 100 L glass aquaria with gently aerated tap water at a dissolved oxygen level of 7.53 ± 0.32 mg Ls factor . In the three batches, we evaluated fish respiration by determining the oxygen consumption  and the respiratory rate of the fish at intervals of 24, 48, 72, 96, and 336 hours from the initiation of the experiments .After 14 days of setting up the experiments, the fish were sacrificed, and blood samples were taken from the tail vein to determine the number of figure elements and the blood glucose value. The counting of figure elements was carried out under an Olympus microscope using Bürker counting chambers after prior staining with neutral red and crystal violet . The gluPelophylax ridibundus eggs and tadpoles were carried out with the approval of the Bioethics Commission No. 13925 and in accordance with the provisions of Directive 2010/63/EU of the European Parliament and of the Council on 22 September 2010 on the protection of animals used for scientific purposes [The collection and handling of purposes .The tadpoles came from 3 egg masses collected from Strand Lake, bordering Pitești, in April 2022 and transported in isothermal packaging to avoid thermal and mechanical shocks. The eggs were distributed into 40L aquaria and maintained under controlled climatic conditions .Pelophylax ridibundus. Two experimental variants were set up, in which we followed the action of Actellic 50 EC on the survival, growth, and development of the embryos and tadpoles of Pelophylax ridibundus, randomly selected from the 3 egg masses collected, as follows:-Control I, consisting of 30 eggs kept in dechlorinated, permanently aerated water under controlled climatic conditions for 9 days.-−1 Actellic 50 EC solution for 9 days.Lot I.1, consisting of 30 eggs kept in a 0.1 mg L-−1 Actellic 50 EC solution for 9 days.Lot I.2, consisting of 30 eggs kept in a 0.01 mg L-−1 Actellic 50 EC solution for 9 days.Lot I.3, consisting of 30 eggs held in a 0.001 mg LIn variant I, we used eggs of In all variants, the aquarium water was renewed every other day, and fresh working solutions were added to maintain the quality of the working solutions . The numPelophylax ridibundus, obtained from the hatching of the rest of the eggs collected under the climatic conditions mentioned. After hatching, the tadpoles in Gosner stage 25 [-Control II, consisting of 15 tadpoles kept in dechlorinated, permanently aerated and climate-controlled water for 5 days.-−1 Actellic 50 EC solution for 5 days.Lot II.1, consisting of 15 tadpoles kept in a 0.1 mg L-−1 Actellic 50 EC solution for 5 days.Lot II.2, consisting of 15 tadpoles in a 0.01 mg L-−1 Actellic 50 EC solution for 5 days.Lot II.3, consisting of 15 tadpoles in a 0.001 mg LIn variant II, we used tadpoles of stage 25 , the mosstage 25 , were alThe specimens used in the experiments were healthy, with their health status being determined by morphological appearance and the behavior of the individuals .Throughout the experiment, the tadpoles were fed ad libitum with boiled and minced salad (boiled in boiling water for 4 min) .The Actellic 50 EC concentration was verified by high-performance liquid chromatography (HPLC) using a Varian Pro Star chromatograph . The measurements were performed with a Varian Liberty 110 spectrometer using a five-point calibration curve . For theAt the end of the experiment, biometric assessments of body length and volume were made, avoiding trauma as much as possible . Triticum and fern test results, the data obtained were statistically analyzed using IBM SPSS Statistics 23 software. Means and standard errors were calculated, and the means were compared with Duncan’s test. The results obtained from Duncan’s test are shown in charts with letters  (p = 0.05). For the p < 0.05. For the fish and tadpole tests, a statistical interpretation of the results obtained was performed using the ANOVA “one-way” test for all the physiological parameters investigated, comparing the results obtained in the groups exposed to the insecticide with those of the control group and the dynamic values of respiratory parameters from one determination interval to another. The results were judged to be significantly different using a significance threshold of Lepidium sativum and Sinapis alba for all the Actellic 50 EC concentrations tested. In the case of the monocotyledonous species S. saccharatum, the germination was inhibited by 25% compared to the control group for the higher concentrations of A0.1 and A0.01 of the insecticide. Germination was totally inhibited in the two dicotyledonous species After 3 days of exposure, the root growth inhibition was recorded for sorghum and was found to be proportional to the increase in the Actellic 50 EC concentration in the soil .Lepidium sativum and Sinapis alba) showed a higher sensitivity  than Sorghum saccharatum (monocotyledonous), wherein germination was achieved at all tested concentrations. As for sorghum root growth, a percentage of 62% inhibition for the highest concentration of 0.1 mg L−1 was recorded. Similar results were obtained by applying a Phytotoxkit in the study to assess the risk of infiltration into soil/groundwater of long-term buried/stored insecticides among the three species used, with Lepidium and Sinapis demonstrating a greater sensitivity than sorghum [L. sativum < S. alba < S. saccharatum [The two dicotyledonous species , there were high variations in the chlorophyll content. Variants A0.1 and A0.01 showed a decrease in chlorophyll concentration , and in variant A0.001, an increase was observed in the concentration of both chlorophylls.a and b, either through an enhanced breakdown or a lower synthesis rate). After each spray, the total chlorophyll content can drop by 15–20%. However, while pirimiphos-methyl was found to cause a major initial drop, compared to other similar pesticides , the recovery of foliar pigment levels was also more pronounced with this compound [The organophosphorus insecticides tested in the present study are known to have various physiological effects on plant growth. A study involving several such insecticides sprayed on cabbage plants showed that organophosphates affect the normal synthesis of chlorophylls  was also observed in wheat plants after treatment with organophosphates  .b levels in tomato and chlorophyll a in cucumber plants, while the effects on carotenoids were mixed, with an initial drop followed by a major increase [Tomatoes and cucumbers are other species whose foliar pigment synthesis can be affected by organophosphate spraying . The response depends on the pigment type and species. For instance, profenofos caused a higher decrease in chlorophyll increase .Besides leaves, abnormal pigment levels have also been observed in fruits. In green peppers, Actellic 50 EC treatments induced a 38% increase in the β-carotene concentration [Abelmoschus esculentus) treated with Attack (another insecticide containing pirimiphos-methyl), with some increases in root weight (10%) and shoot weight (16%), plus other parameters [As for plant growth parameters, a field study on onion plants (pirimiphos-methyl in a regular dosage of 0.5%) found significant increases in the average plant height, number of leaves, fresh and dry biomass (up to 100%), bulb yield, etc. However, such effects in field experiments were probably indirect, due to lowering the population of herbivorous insects . Similarrameters .T. aestivum (Actellic 50 EC exposure significantly inhibited the root and stem growth in aestivum . The inhaestivum . For thiaestivum . The 24 Triticum aestivum cultivar Misr 1). They observed that Actellic (375 mL/100 L) significantly decreased the percentage of abnormal seedlings and increased the final germination percentage, germination rate index, germination energy, root and shoot length, seedling dry weight, and seedling vigor index. The effect of Actellic on two-month-old cabbage plants was studied by Shams-El-Din et al. [Actellic (pirimiphos-methyl) induces various physiological and biochemical changes in plants, depending on the species, the dose applied, the ontogenetic stage of the plant, and other factors. Kandil et al.  studied n et al. . The chln et al.  confirmeTriticum aestivum: in variant A1 of both species, the spores did not germinate , we recorded mortality in both groups exposed to Actellic 50 EC (10% in group I and 30% in group II). At the end of the 14-day experiment, the survival percentage in group I was 70%, and in group II, it was 60%. Fish were considered dead when they showed no opercular movements and no reaction to touch with a glass rod.Oreochromis niloticus using the continuous flow test at 21.34 ± 0.39 °C. At the end of the acute test (96 h), the mortality levels recorded were 30, 40, 60, and 85% at the test concentrations of 1.563, 3.125, 6.25, and 12.5 µg L−1 of Actellic 25 EC, respectively. Omoregie and Ufodike  studied Behavioral changes are the most sensitive indicators of potential toxic effects, with most insecticides altering behavioral patterns by interfering with the nervous system and sensory receptors . The inhIf the heptachlor insecticide  is presPoecilia reticulata during the acute test. Forty-eight hours after exposure to the Actellic 50 EC insecticide, the carps of both groups showed a number of behavioral changes: restlessness; hyperactivity followed by increasingly weak swimming; a loss of balance; and, especially at 96 h after exposure, increased mucus secretion. Similar symptoms of Actellic 25 EC intoxication were also observed by Lawal and Samuel [Carassius auratus [Tilapia oreochromis mossambicus [The reversibility of the behavioral effects of organophosphorus pesticides (due to the long-term inhibition of brain acetylcholinesterase) has been reported in  auratus  and Tilasambicus .In a 3-month chronic toxicity study on the sensitivity of Atlantic salmon to the insecticide Actellic 50 EC present in plant-based feeds, Berntssen et al.  reported−1) are shown in Variations in the respiration rate and oxygen consumption of carps exposed to Actellic 50 EC insecticide at two concentrations .The respiratory rate of carps exposed to Actellic 50 EC decreased significantly from the first 24 h after the fish were introduced to the insecticide solutions, with the mean values recorded at the end of the acute test (after 96 hours of exposure) being 8.83% lower than the initial values at 0.002 µL L−1 was also reported by Omoregie and Ufodike [Oreochromis niloticus niloticus, with the authors observing a significant decrease in the frequency of opercular movements after the first 24 h after exposure to the insecticide Actellic 25 EC, followed by a less marked decrease in this physiological index until the end of the acute test. Decreased opercular ventilation in response to exposure to 0.002 mg L Ufodike  in OreocAphanius disparus species), chloral cell degradation, shedding, detachment, epithelial lifting, hypertrophy, and gill fusion [The decrease in oxygen consumption can be linked to the effect of the insecticide at the gill level, as organophosphorus insecticides affect the respiratory organs of fish  at 27 °C. Mgbenka et al.  reported−1.In another study on Actellic, Oluah and Mgbenka  reportedOreochromis niloticus to Kinalux 25 EC (an organophosphorus insecticide). Nannu et al.  reported−1 of insecticide compared to the control group and 28.65% higher in those exposed to 0.002 mg L−1). The mean glycemia values were significantly higher after 14 days of exposure to the Actellic 50 EC insecticide . Increased blood glucose in Dichlorvos intoxication was reported by Gautam et al. [Clarias batrachus and by Srinivas et al. [Catla catla. Significant increases in blood glucose have been reported in different species when exposed to Fenitrothion: Oreochromis niloticus [Sarotherodon mossambicus [Heteropneustes fossilis [Oreochromis mossambicus following exposure to RPR-II (2-butenoic acid-3-(diethoxyphosphinothioyl) methyl ester) [Similar results of an increase in blood glucose as a result of Actellic exposure were reported by Olsvik et al. . The autm et al.  for Clars et al.  for Catliloticus , Sarothesambicus , and Hetfossilis . Glycogefossilis ,80, and l ester) .Changes in hematological parameters can result in alterations to the vital physiological processes of fish: respiration, feeding, reproduction, etc. These parameters could thus serve as sensitive indices to examine the health status and to ascertain the toxic effects on ecosystems under pesticide exposure, as they can provide early warning signals of pesticide exposure, and toxicological effects can be followed by monitoring ,82,83.Pelophylax ridibundus eggs to different concentrations of pirimiphos-methyl showed a negative influence on the hatching percentage . Less toxic was the concentration of 0.001 mg L−1 of Actellic 50 EC, with a hatching percentage of 82.58%. The exposure of rcentage . Thus, aDelayed metamorphosis or a reduced body size may be associated with a lower survival rate, which may have unfavorable consequences for the species . Pelophylax ridibundus tadpoles during the sub-acute test period at all three concentrations investigated is shown in The survival of −1 of Actellic 50 EC, with a survival rate of 100% at the end of the subacute test, while a concentration of 0.1 L−1 of Actellic 50 EC resulted in a survival rate of 50%.An analysis of the data showed that the least toxic concentration was 0.001 mg L−1 in water, while a concentration of 0.001 mg L−1 of Actellic 50 EC did not cause significant changes at the end of the subacute test  tadpoles under glyphosate action, indicating a decrease in body length. Moreover, pendimethalin administered in sublethal doses caused biochemical changes in the antioxidant enzymes of zebrafish larvae [Similar results were observed by Lanctôt et al.  in studih larvae . Exposure to different doses of dinitramine resulted in malformations in zebrafish embryos and the onset of cardiotoxicity . The agrStudies on sub-chronic and chronic toxicity assessments and the impact on reproduction and genotoxicity were conducted in laboratory animals (mice and rats), targeting the risk of chronic exposure of animals or humans in areas of the improper storage and handling of pesticides in the agricultural sector and related activities. Related to the chronic impact on genotoxicity and reproductive function, in a chronic exposure of rats (45 days) to phosalone (PLN), Amniattalab and Razi  noted adRegarding the impact on genotoxicity, Khodabandeh et al.  studied As pointed out by Khodabandeh et al. , the risUnfortunately, there are not enough data on the levels of concentration and types of organophosphorus insecticides existing in the water basins, as well as the quantities applied to the agricultural land, which would be necessary so that the level of risk for the different non-target organisms could be assessed. A monitoring program would be absolutely necessary. These data would be useful precisely to be able to assess the intensity and duration of exposure to these toxic compounds of non-target organisms, in order to initiate studies on the chronic effects on the exposed organisms.A. scolopendrium and A. filix-femina spores to Actellic 50 EC induced significant changes in germination and gametophyte differentiation, suggesting impaired in situ reproduction in contaminated ecosystems. The insecticide Actellic 50 EC adversely affected the germination of the seeds and spores of the non-target species tested, producing either complete inhibition of germination of some of the tested species or a decrease in the percentage of germination. The sub-chronic and chronic phytotoxicity generated by exposure to Actellic 50 EC manifested by affecting the growth process of seedlings, which could ultimately lead to important changes in the life cycle, reproduction, and productivity. The continuous exposure of −1, 0.01 mg L−1, and 0.001 mg L−1 negatively influenced the hatching rate of Pelophylax ridibundus embryos as well as the survival, body length, and volume of tadpoles, which are changes that affect their survival as adults. Intoxication with Actellic 50 EC manifests itself in Prussian carp by causing changes in respiratory function, an increase in the average number of white blood cells and plasma glucose level (as a result of the reduction in hepatic glycolysis), and a series of behavioral changes , which can affect fish feeding and survival. The administration of the Actellic 50 EC insecticide at concentrations of 0.1 mg LAfter highlighting the acute and sub-chronic toxicity of the organophosphorus insecticide Actellic 50 EC on the non-target species tested, we recommend continuing to investigate other parameters in order to complete the profile of changes induced by the exposure of different groups of organisms to this xenobiotic. It is important that these tests also include various plant species, which constitute the first trophic level in most ecosystems. Given the high degree of toxicity of the insecticide Actellic 50 EC, we recommend continuing investigations on non-target species, plants, and animals, as the sub-chronic and chronic effects are quite little known in the scientific literature.Our study can thus contribute to the application of better measures to monitor and protect ecosystems against the uncontrolled spread of the tested pesticide.


This study investigated the effects of annealing on the structural and physicochemical properties of rice starch below the onset temperature (To) by 5 °C and 15 °C. The results revealed that annealing improved the gelatinization temperature of rice starch, decreased the swelling power, solubility, and paste viscosity of rice starch, and had no significant effects on the morphological structure and crystal configuration of rice starch. In one-step annealing, the annealing temperature of 60 °C is more conducive to the rearrangement of starch molecules, so its crystallinity, short-range ordered structure, and gelatinization temperature are higher than at 50 °C; however, its RDS, SDS, and RS contents will be increased. During the two-step annealing treatment, the temperature change is not conducive to the molecular chain rearrangement and to the formation of perfect crystalline structure, which increases the sensitivity of enzymes to starch, so the RDS content of starch increases significantly, while the RS content decreases. Starch is a semi-crystalline polymer composed of glucose, which is deposited in the form of granules in the roots, stems, and leaves of green plants and widely used in foods, medicines, and cosmetics ,2. Howevw/w) or appropriate (40–55% w/w) moisture and at temperatures above glass transition and slightly below gelatinization [Annealing of starch is performed in excessive >65% 5% w/w ornization ,8. Anneanization . It is anization . Comparenization ,12,13. Inization . Liu et nization  found thnization ,17. HoweBased on current researches ,17,18, tThe rice in this study was obtained from Hall 4, Yupei, Nanling, Wuhu, Anhui, China. The rice starch was milled to 100−mesh. A glucose oxidase/peroxidase (GOPOD) was purchased from Megazyme International Ireland . Amyloglucosidase and α-amylase were purchased from Sigma-Aldrich Co. LLC, Santa Clara, CA, USA. All other reagents in the experiments were of analytical grade.The starch isolation from rice flour was based on Bian et al. , with miO) of the starch was determined by DSC at 65° in pre-experiments. The natural rice starch  was dispersed in 0.02% sodium azide and 75 mL of distilled water and divide into 4 equal parts. The first sample was heated at 50 °C (TO −15 °C) for 48 h, which was named ANN−50. The second sample was heated at 60 °C (TO −5 °C) for 48 h, which was named ANN−60; the third sample was first heated at 50 °C for 12 h and then transferred to 60 °C for 12 h to obtain the two-step annealed starch, which was named ANN−50−60. The fourth sample was first heated at 60 °C for 12 h and then transferred to 50 °C for 12 h to obtain the two-step annealed starch, which was named ANN−60−50.The method was based on Xu et al. , with miAfter annealing, the sample was cooled to 25 °C, followed by centrifugation at 4000 r/min for 15 min. The precipitation was collected, dried in an oven (40 °C), and milled to 100−mesh.A drop of starch solution with a mass fraction of 1% was placed onto the slide and covered with the coverslip. The morphologies of the samples were observed using an optical microscope  under crossed-polarized light (magnification 200×). The morphologies of the starch samples were observed using a SEM , prepared for observation as described by Huang et al. . −1 rate. The relative crystallinity (RC) was calculated using the Peakfit software with the following specific formula:XRD analyses of the starch samples were determined by an XRD  equipped with CuKα radiation source . The samples were scanned from 5° to 40° (2θ), at a 5° minw/w), compressed, and scanned at a speed of 4 cm−1 in the range of 4000–400 cm−1; the number of scans was set to 64. After that, the original spectrum was deconvoluted using the Omnic 9.2 software.The FT-IR spectra of the starch samples were based on Ji et al. , with miStarch samples  were mixed with 25 mL of distilled water, and pasting properties were obtained by using a RVA-Super4; the program was set following the procedure of Hu et al. . Peak viw/w) and equilibrated overnight at 25 °C. Then, the samples were scanned at a heating rate of 10 °C min−1 from 20 to 110 °C, and the onset (TO), peak (TP), and conclusion temperatures (TC) and the gelatinization enthalpy change (ΔH) were recorded.Thermal properties of the starch samples were obtained by using a differential scanning calorimeter . Starch samples were mixed with distilled water  and solubility (S) of rice starch samples were obtained according to the method of Xu et al. , with miThe digestibility of the starch samples was obtained according to the method of Englyst et al. , with mip < 0.05) using SPSS 20.0 , by Duncan’s multiple test method. Plots were all performed using Origin 8.5 .Unless otherwise noted, all results in this article were expressed as the mean ± standard deviation of three experiments at 95%  of natural rice starch was 37.09%, and it significantly increased after the annealing modification, corresponding to the findings of Waduge et al. . The inc−1 region were the embodiments of the stretching and bending vibration information of various chemical bonds, which mainly reflected the stretching vibrations of C−C and C−O and the bending vibrations of C−H−O, and they were sensitive to changes in the short-range ordered structure of starch. As shown in The short-range order of rice starch sample molecules was reflected by FT-IR . The ban−1 and 1022 cm−1 were used to characterize the ordered structure of the starch crystalline region and the disordered structure of the amorphous region, respectively, while the absorption strength at 995 cm−1 was used to indicate the amount of intramolecular hydrogen bonds of the hydroxyl group [−1/1022 cm−1 and R1047 cm−1/1022 cm−1 are usually used to characterize the double helix structure and order of starch [−1/1022 cm−1 increased, which was more significant in ANN−60 than that in ANN−50, indicating that annealing at 60°C was more conducive to the increase in the double helix structure. After the two-step annealing, the ratios of R995 cm−1/1022 cm−1 and R1047 cm−1/1022 cm−1 were significantly lower than that of natural starch and one-step annealed starch, indicating a reduced order of starch crystals, which may be due to the fact that the temperature change will disturb the rearrangement of starch molecular chains, thereby reducing the order of starch crystals. In addition, Vela et al. [−1/1022 cm−1 of the rice flour samples remained unchanged at annealing temperatures of 20 °C, 40 °C, and 50 °C, and only the annealing temperature of 60 °C caused a significant difference from the control group, which might be related to partial gelatinization during treatment.Absorption strengths at 1047 cmyl group . Therefof starch . After ta et al.  found thThe gelatinization characteristic curve and corresponding parameters of rice starch before and after annealing are presented in The breakdown is related to the thermostability and shear stability of the starch granules. Compared with NRS, the breakdown of annealed starch was reduced to varying degrees, which showed that annealing was conducive to increasing the thermostability and shear stability of starch. That is mainly because intramolecular imperfect crystallization in rice starch will form a tighter perfect crystal structure through molecular rearrangement under hydrothermal action, resulting in a more stable structure of the starch ,8. The rO, TP, and TC for starch are related to the crystallization degrees and particle sizes of the starch granules. The tighter and more orderly the association between starch molecules is, the more energy is required to destroy its inherent structure in the gelatinization process and the higher the gelatinization temperature is. The effect of annealing treatment on the thermal properties of rice starch is shown in P, decreased in TC−TO, and had no significant change in enthalpy value. It indicates that annealing improves the stability and uniformity of the crystals inside the starch molecules and forms a tighter crystalline structure than the natural starch, resulting in an increase in its gelatinization temperature [O, TP, and Tc of barley starch and decreased the gelatinization temperature range (TC−TO), attributing this change to the formation of a new double helix structure due to the improvement of the crystal structure and the interaction between amylose–amylose or amylose–amylopectin.Thermal properties such as Tperature . This reperature , who repO of starch was increased by annealing treatment in the first step. Combined with the one-step annealed rice starch in which TO, TP, and TC of ANN−60 were significantly higher than ANN−50, it was shown that starch was getting higher and higher in intermolecular association and tighter and tighter in crystallization structure when the annealing temperature was getting closer to the gelatinization temperature of the starch. Significantly, the DSC spectrum began to gradually form a multimodal morphology after annealing, which may be due to the fact that part of the new double helix structure formed in the phase change process, since part of the imperfect crystallization transforms into perfect crystallization in starch during annealing. It gradually generated double peaks under heating and melting because of the difference in stability of that part of the structure and the inherent thermostability of starch.In the two-step annealing, ANN−60−50 had a higher gelatinization temperature than ANN−50−60, which may be because TThe solubility and swellability of starch samples were measured in 55 °C−95 °C, as shown in During the one-step annealing, the sample ANN−50 had a significantly higher solubility and swellability than ANN−60. When annealing at 60 °C, the imperfect crystallization of starch formed perfect crystallization through unwinding, and the crystallinity increased significantly, compared with that annealed at 50 °C, thereby achieving stronger thermostability, as well as relatively lower solubility and swellability. The two-step annealed rice starch had significantly lower solubility than the one-step annealed rice starch, which may be related to the gradual improvement of starch microcrystals and the enhanced intermolecular interaction of amylose during the annealing process. This change formed a more stable configuration and reduced the leaching of amylose. Meanwhile, the decrease in the swelling power of annealed starch was consistent with the decrease in gelatinization viscosity.The digestion curves of rice starch before and after annealing are shown in The increase in ANN-60-50 was most significant in the two-step annealing, with RDS and SDS increasing by 13.2% and 0.12% over NRS and RS content reducing by 12.57%. Combined with SEM analysis, the increase in RDS content and the decrease in RS content may be due to the folds generated on the surface layer of the annealed rice starch, thereby increasing enzyme sensitivity and accelerating the digestion of rapid digestion starch (RDS). The increase in SDS may be due to the annealing process enhancing the strong interaction between the intermolecular chains of starch, which limits the effect of enzymes on the starch molecular chains to some extent . Song etP, which also showed an improvement of the thermostability of rice starch. FTIR showed that no functional groups and new chemical bonds were produced after different annealings of rice starch; the pretreatment prior to two-step annealing increased the TO of starch, and the change of annealing temperature destroyed the natural crystallization integrity, reducing the short-range order of rice starch molecules. Therefore, when starch is modified by annealing, it is important to keep the temperature of the hydrothermal treatment consistent. This is more conducive to the rearrangement of starch molecular chains and the formation of a perfect crystalline structure. The obtained result may be helpful for the development of annealing modified starch with appropriate applications.The results showed that the surface folds of rice starch granules increased, and the Maltese cross was more obvious after annealing. The annealing modification increased the hydration of the amorphous region and crystallization integrity for starch, resulting in decreases in its breakdown, peak viscosity, and retrogradation value and some increase in To and T


Coronary artery bypass graft (CABG) surgery still represents the gold standard for patients with complex multivessel coronary artery disease. However, graft occlusion still occurs in a significant proportion of CABG conduits, and oxidative stress is currently considered to be a potential contributor. Human serum albumin (HSA) represents the main antioxidant in plasma through its reduced amino acid Cys34, which can efficiently scavenge several oxidants. In a nested case–control study including 36 patients with occluded grafts and 38 age- and sex-matched patients without occlusion, we assessed the levels of the native mercaptoalbumin (HSA-SH) and oxidized thiolated form of albumin (Thio-HSA) in relation with graft occlusion within 5 years after CABG. We found that the plasma level of preoperative HSA-SH was significantly lower in patients with occluded graft at 5 years follow-up than in patients with graft patency. Furthermore, low HSA-SH remained independently associated with graft occlusion even after adjusting for preoperative D-dimer, a well-known marker of activated coagulation recently found to be associated with graft occlusion. In conclusion, the preoperative level of HSA-SH is independently associated with graft occlusion in CABG and represents a measurable and potentially druggable predictor. According to the US and European guidelines, coronary artery bypass graft (CABG) surgery represents the gold standard of care for patients with complex multivessel coronary artery disease and/or left main disease, diabetes, or reduced left-ventricular function ,2.Despite significant improvements in the outcome of patients undergoing CABG procedure, graft occlusion is the ‘Achilles’ heel’ of this procedure, occurring in a significant number of CABG conduits. Biological mechanisms, characteristics of the target vessels, surgical procedure, and type of graft used all are critical factors in determining failure ,4. IndeeIndeed, the increased production of reactive oxygen species (ROS) is considered a potential contributor to the incidence of perioperative or postoperative complications occurring after CABG, causing damage and dysfunction of macromolecules, cells, and tissues by overwhelming the local antioxidant defense mechanisms. Several studies have shown an increase of oxidative stress during the intraoperative period and the very early hours after surgery, with increases in several pro-oxidant markers  and a coBeyond the perioperative period, levels of oxidative stress markers at longer periods have not been assessed. To the best of our knowledge, the only study with a follow-up of 1 week after surgery showed that myeloperoxidase levels increased up to the second postoperative day .S-thiolated albumin isoforms. As these oxidized isoforms are not present on albumin after its secretion from hepatocytes but occur after being released into the circulation, they may represent potential biomarkers of oxidative stress in human diseases [Human serum albumin (HSA), beyond its crucial physiological functions  , represediseases ,15.Therefore, in search of biomarkers that may predict graft occlusion and possibly suggest strategies to reduce graft failure, we assessed preoperative and postoperative levels of native and thiolated forms of albumin in relation to graft occlusion evaluated up to 5 years after CABG.In this study, we used samples from an existing plasma biorepository of a cohort of 330 consecutive patients (age 18 to 89 years) enrolled for elective primary surgical myocardial revascularization between November 2006 and February 2010 at Centro Cardiologico Monzino IRCCS   . The CAGFor all patients, postoperative therapy followed guidelines for CAD and surgical myocardial revascularization including always at least single antiplatelet therapy, optimized blood pressure control therapy, and statin. As described in For this work, a nested case–control study was designed. Suitable samples were available from 36 patients with occluded grafts at 5 years follow-up (cases) and were compared with 38 patients without occlusion (control subjects), frequency-matched for age and sex. Analysis was performed on preoperative plasma samples (T0) and postoperative plasma samples collected at patient discharge (T1), usually between postoperative days 6 and 9 after surgery.® M-Class system , as previously described [The relative composition of albumin isoforms in human plasma samples was evaluated by direct infusion of diluted plasma (500-fold dilution) using the Xevo TQ-S micro triple-quadrupole mass spectrometer coupled with the ACQUITY UPLCescribed . After described .t-test and the Wilcoxon rank sum test when appropriate. Multivariable logistic regression was used to assess whether albumin isoforms measured before surgery were independently associated with graft occlusion. Variables included in the model were selected among potential confounders, i.e., baseline variables that were associated with both the dependent variable (graft occlusion) and the variable under investigation (HSA-SH). The ability of mercaptoalbumin to discriminate between cases and controls was assessed by receiver operating characteristic (ROC) curve analysis. Net reclassification improvement (NRI) was used to check the improvement in risk prediction by adding albumin isoforms to the reference model including D-dimer, logistic EuroSCORE, and extracorporeal circulation (ECC) time. Three risk categories were defined as low (0–33%), middle (33–66%), and high (66–100%). SAS statistical software version 9.4  was used for all analyses, and a p-value < 0.05 was considered statistically significant.Quantitative variables were reported as the mean ± SD or median and interquartile range (IQR) according to their distribution. Spearman’s correlation was used to find monotonic associations between variables. Categorical variables were compared between the two groups using the chi-square test or Fisher’s exact test when appropriate, and quantitative variables were compared using Student’s This study was performed on a cohort of 74 sex- and age-matched patients, selected from the CAGE study population divided into two groups according to graft patency at 5 years follow-up. We analyzed a total of 36 patients with graft occlusion (cases) and 38 patients with graft patency (controls), with no significant differences between the two groups in terms of age, sex, risk factors, graft vessel origin, and medications .Albumin isoforms were analyzed in plasma samples collected before surgery (T0) in cases vs. controls by mass spectrometry, to quantify the percentage of HSA-SH and its main modified isoforms: Thio-HSA and Gly-HSA.p = 0.007 in cases and controls, respectively). On the contrary, Thio-HSA showed an inverse behavior , whereas Gly-HSA, another modification often occurring in diabetes as the result of glycation reactions, was not different between the two groups .On the other hand, Thio-HSA did not result significantly different between cases and controls after adjustment for the above variables  of 0.7126 . This anp = 0.007, In addition, to quantify the capacity of HSA-SH to properly classify patients undergoing occlusion, on top of the other variables considered as confounders, we performed a reclassification analysis. On the basis of the NRI, HSA-SH was able to better reclassify 30% of the patients in comparison with a model including D-dimer, logistic EuroSCORE, and duration of ECC (We found a marked increase in the levels of Thio-HSA at patient discharge (T1) compared with preoperative levels, with a concomitant decrease in HSA-SH . Furtherp = 0.007).This study provides evidence, for the first time, that the preoperative plasma level of HSA-SH was significantly lower in patients with occluded graft than in patients with graft patency at 5 years follow-up, and that it was independently and negatively associated with graft occlusion at 5 years after CABG. Furthermore, low HSA-SH remained independently associated with graft occlusion even after adjusting for preoperative D-dimer, a well-known marker of activated coagulation recently found to be associated with graft occlusion . Further2), the most objective functional method to assess the cardiopulmonary exercise capacity [The novelty of these findings is that it is the quality of albumin, rather than its quantity, that is relevant as a disease biomarker in the cardiovascular setting. Until now, the interest in albumin in the cardiovascular field has been mainly limited to its levels in the bloodstream. Hypoalbuminemia has been associated with poor prognosis in many conditions, such as acute coronary syndrome, heart failure (HF), and in patients undergoing CABG ,18,19,20capacity ,21. Furtcapacity . Taken tAfter CABG, there is a marked and prolonged activation of many molecular pathways leading to increased inflammation and oxidative stress, hemostasis activation, and unfavorable endothelial milieu. Thus, several biomarkers reflecting the activation of these metabolic systems have been proposed as predictors of early and late graft occlusion ,16,22,232α excretion was observed in CABG patients during surgery, with levels returning to baseline 24 h after surgery. Similarly, malondialdehyde increased significantly during surgery and declined immediately after [Considering that oxidized isoforms of albumin are not present in the protein immediately after secretion from the liver, they might represent potential biomarkers of oxidative stress in human diseases ,15. Furtly after ,24. Of nIn addition to its role as a potential marker of oxidative stress, it should be emphasized that HSA represents a significant source of antioxidant defense in the plasma, which is constantly exposed to oxidative stress. HSA is involved in scavenging the vast majority of free radicals, thanks to the presence of the free thiol group on Cys34, which constitutes the largest pool of free thiols in circulation and works as a radical scavenger due to its peculiar acidity (pKa = 8.1) and spatial accessibility.Lastly, we recently showed that Thio-HSA can be restored to HSA-SH by a disulfide-breaking agent, which can, thus, preserve the antioxidant potential of the extracellular milieu .Although some limitations should be acknowledged such as the relatively small sample size and lack of validation on a larger cohort of patients, together with the fact that we could not precisely establish the timing of graft failure within the follow-up period, we believe that we identified a measurable and druggable predictor of graft occlusion.


Fritillaria taipaiensis under long-term single planting, the bacterial community structure in the rhizospheric soils of F. taipaiensis with different cultivation years from 1 to 5 were analyzed. The result showed the Chao1 and the ACE indices of the bacterial community had no significant difference among samples while the Shannon and Simpson indices declined with the cutivation year; the intra group beta diversity of the rhizospheric bacteria increased after a initial decline with the cultivation year; in the sample with 1 year of cultivation, the dominant bacterial genera were mainly the species that can improve the soil nutrient status and promote plant growth while with the increase of cultivation year, the dominant genera in samples then gradually reflected the pathogen accumulation and soil nutrient status deterioration; pH was the most significant factor affected by the bacterial community composition. These results indicated long term continuous cropping changed the bacterial community structure and soil nutritional status in the F. taipaiensis rhizospheric soils, which could badly affect its growth.Rhizospheric microorganisms can profoundly influence the nutritional status of soil and the growth of plant. To reveal the change on the bacterial diversity in the rhizosphere of  Fritillaria 1. The Chuan Beimu medicine contain multiple compounds such as alkaloids, steroids, terpenoids, purine and fatty acid, which can cool down body temperature, relieve coughing and asthma, and resolve phlegm4. Fritillaria taipaiensis P. Y. Li, which has been collected in Chinese Pharmacopoeia as one of the medical material provenances of Chuan BeiMu in 20155, has important economic value. In recent years, the production of F. taipaiensis has been popularized in suitable areas8. However, in the actual production, the seedlings of F. taipaiensis may appear the problems such as growth potential reducing9, disease accumulation10, and quality decline11 after 3–4 years’ continuous planting, which badly affected its production and quality. In response to this situation, current researches mainly focus on the fertilization control and the planting technology improvement13.Chuan BeiMu is a kind of the precious traditional Chinese herbal medicine, its source materials are the bulbs of 5 species in genus 15. Meanwhile, the quality of soil can also be reflected by the soil enzyme activities and the microbial community composition16. Researches have demonstrated continuous cropping and long-term single plating pattern could affect the diversity and structure of the microorganism population in the planting soil19, these microorganism and then could directly or indirectly affect the growth of plants by changing the chemical properties of soil20. In addition, the pathogenic microorganisms accumulated during the long-term single planting can also hinder the growth of plants24. The studies on the changes of rhizospheric microbial community structure during the F. taipaiensis cultivation have not been deeply progressed at present.Soil contains different and wide variety of microbial communities, and the interaction between soil microorganism and soil factors plays an important role in the process of soil organic matter degradation and soil nutrient cyclingF. taipaiensis was planted for 1–5 years, were compared and analyzed to reveal the change on the bacterial diversity in the rhizosphere of Fritillaria taipaiensis under long-term single planting. Moreover, we analyzed the relationship between the soil bio-chemical factors and the rhizospheric bacterial community to explore the interaction between soil environment and soil microorganisms. The result of this study could provide a theoretical basis for the soil management in the sustainable cultivation of F. taipaiensis.In this study, the population structure compositions, richness and evenness of the bacteria in the rhizospheric soils, in which 25. The field was 20 × 20 m for the F. taipaiensis with each cultivation year, The plant and row spacing of F. taipaiensis was 15 × 15 cm, and the fields were uncultivated wastelands with the same soil basement condition before F. taipaiensis planting. The fertilization of the field was N-P-K 15:15:15, 225 kg hm−2 for each year.The sampling site is a private planting base located in Lanying Village, Wuxi County, Chongqing, China , with the annual average temperature of 7.2 °C, and the annual average precipitation of 1 100–1 300 mm, the soil in the study site was classified as mountain yellow–brown soil F. taipaiensis rhizospheric soils with different cultivation years from 1 to 5  were sampled. 3 sampling plots (3 × 3 m) were randomly selected in the field with each cultivation year. The regolith, impurities and gravels on the surface of fields were removed to expose the roots, and the soils attached on the roots 6 ~ 10 cm below the ground were collected by shaking root method13. Soils from the rhizosphere of 5 F. taipaiensis plants randomly selected in each sampling plot were collected and then were mixed. Then the soil samples were placed into the aseptic bags in ice box and then were transferred into laboratory after removed the litters and residual cover. The experimental samples were sifted by 2 mm sieve and then were stored in − 80 °C ultra-low temperature freezer after freeze drying to be used in soil total DNA extraction. The bio-chemical properties26 of the F. taipaiensis rhizospheric soils with different cultivation years were shown in Table In May, 2017, with the permission of the planting base owner, F. taipaiensis rhizospheric soil sample was used for DNA extraction by CTAB method27. The genomic DNA after diluted to 1 ng μl-1 was amplified by PCR via high-fidelity DNA polymerase and Phusion® High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs) by using specific primer 515F (5′-GTGCCACCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) for the bacterial 16sRNA gene V4 sequence, the amplification condition was pre-denaturation at 98 °C for 2 min, denaturation at 98 °C for 30 s, annealing at 50 °C for 30 s, extending at 72 °C for 1 min, after 30 cycles, and then extending 72 °C again for 5 min to end the PCR reaction. The PCR products were tested by 2% agarose gel electrophoresis and then were extracted by gel extraction kit (Qiagen). The DNA library was constructed by using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina) and then was sequenced by HiSeq2500 PE250.0.25 g http://ccb.jhu.edu/software/FLASH/) after removed the Barcode sequence and the PCR amplification primer sequence to obtain the Raw date. The Raw date were filtered and spliced to get Clean date refer to the Tags quality control process in QIIME . And then, the chimeras in the Clean date were eliminated by comparing with UCHIME  and database  to obtain the Effective tags. The depth of sequencing was 40 000 reads at least in each original library, the operational taxonomic unit (OTU) was analyzed and clustered by using the UPARSE software (version 7.0.1090) based on 97% similarity. According to the result of OTU clustering, the representative sequence of each OTU was annotated by a species by using QIIME (version 1.9.1). The relative abundance was analyzed via Mothur (version.1.44.0) in the SS rRNA database of SILVA (http://arb-silva.de/).The data of each sample was separated from the off machine data according to the Barcode sequence and PCR amplification primer sequence, and the reads of each sample were assembled by FLASH  was used to calculate the Goods-coverage, Chao1, ACE, Shannon and Simpson indices. The QIIME software (version 1.9.1) was used to calculate the Unifrac distance and plot the unweighted pair-group method using arithmetic average (UPGMA) cluster tree. The R-software (version 2.15.3) was used to plot the Rank abundance curve. Meanwhile, the relative abundance, the Alpha diversity of OTUs was analyzed to get the information such as the unique species abundance in one sample, the common and the unique OTUs among the different samples and groups by Tukey-test. Non-Metric Multi-Dimensional Scaling (NMDS) analysis, distance-based redundancy analysis (db-RDA) and permutational MANOVA analysis (Adonis) were performed by vegan bag on R-software.The data were analyzed by single factor (one-way ANOVA) and Duncan for variance analysis and multiple comparison via SPSS 22.0 and Excel 2003 (α = 0.05). Correlation analysis was conducted on SPSS 22.0 using the Pearson method (α = 0.05).F. taipaiensis rhizospheric soil with 2 years of cultivation had the maximum bacterial species richness; and the curve of sample Y1 had the softest changing trend in ordinate which indicated the F. taipaiensis rhizospheric soil with 1 year of cultivation had the maximum bacterial species evenness; the curve of sample Y4 had the minimum abscissa span and the maxim ordinate changing trend which indicated the F. taipaiensis rhizospheric soil with 4 years of cultivation had the minimum bacterial species richness and evenness.The Rank abundance curves Fig.  which weAs shown in Table F. taipaiensis rhizospheric soils with different cultivation years were Proteobacteria (34.16–54.82%), Acidobacteria (10.19–18.16%), Bacteroidetes (9.49–13.75%), Verrucomicrobia (4.91%-7.21%), Firmicutes (0.18–6.05%), Gemmatimonadetes (2.87–5.93%), Actinobacteria (1.51–4.55%), Planctomycetes (2.05–3.06%), Nitrospirae (1.01–2.48%) and Chloroflexi (0.96–1.83%). The dominant phylum was Proteobacteria which presented a rising relative abundance from Y1 to Y4 with the cultivation year. And the relative abundance of Firmicutes, Actinobacteria and Chloroflexi showed a downward changing trend from Y1 to Y4, in which the relative abundance of Firmicutes had great variation among samples, declined from 6.05% in Y1 to 0.65% in Y3. Meanwhile, the samples were clustered by unweighted pair group method with arithmetic means (UPGMA). Samples Y4 and Y5 could be grouped together but be separated from the cluster of samples Y1, Y2 and Y3, which revealed that the samples with 4 and 5 years of cultivation had the similar bacterial community structures but were different from the samples with 1, 2, and 3 years of cultivation. The samples Y1, Y2, and Y3 could be grouped in a large cluster, in which the samples with 1 and 3 years of cultivation could be further grouped in a smaller cluster.As shown in Fig. F. taipaiensis rhizospheric soil were Methylotenera, Pseudomonas, Lactobacillus, Gemmatimonas, Ralstonia, Bryobacter, Sphingobium, Flavobacterium, Collimonas, Sphingomonas, Candidatus, Rhizomicrobium, Pedobacter, Bacteroides and unculturable bacterium RB41  and Mucilaginibacter; Y4: H16, Methylotenera, Pedobacter, Lysobacter, Polycyclovorans, Massilia and Flavobacterium; Y5: Duganella, Pseudomonas, Pseudarthrobacter, Haliangium, RB41 and Acidibacter. As shown in the Venn diagram, the samples with different cultivation years shared 2 928 common OTUs, the number of the unique OTUs in each sample from Y1 to Y5 was 339, 490, 213, 281 and 332, respectively . The values corresponding to the heat map are Z-values obtained by standardizing the relative abundances of the species in each row. The Z-value of a sample in a certain classification means the difference between the relative abundance of the sample and the average relative abundance of all samples divided by the standard deviation of all samples in the classification.Heat map was created via QIIME   Table .Figure 4p < 0.01), was significantly positively correlated with the activity of invertase (p < 0.01), and was negatively correlated with the activity of alkaline phosphatase (p < 0.05); the activity of acid phosphatase was significantly positively correlated with the content of available potassium (p < 0.01); the activity of alkaline phosphatase was significantly positively correlated with the contents of available phosphorus, available nitrogen, organic matter and the activity of urease (p < 0.01), and was significantly negatively correlated with the activities of protease and invertase (p < 0.01); the urease activity was significantly positively correlated with the contents of available phosphorus, available nitrogen and organic matter (p < 0.01), was significantly negatively correlated with the activity of invertase (p < 0.01), and was negatively correlated with the protease activity (p < 0.05); the activity of invertase was significantly negatively correlated with the contents of available phosphorus, available nitrogen, organic matter, and was significantly positively correlated with the activity of protease (p < 0.01); the activity of protease was significantly negatively correlated with the contents of available phosphorus and available nitrogen (p < 0.01), and was negatively correlated with the content of organic matter (p < 0.05); the activity of catalase was positively correlated with the content of organic matter (p < 0.05); the content of organic matter was significantly positively correlated with the content of available phosphorus (p < 0.01), and was positively correlated with the content of available nitrogen (p < 0.05); the content of available nitrogen was significantly positively correlated with the content of available phosphorus (p < 0.01) , was positively correlated with the abundance of Pedobacter (p < 0.05), was significantly negatively correlated with the abundances of Bryobacter, Candidatus and Rhizomicrobium (p < 0.01), and was negatively correlated with the abundances of Lactobacillus and Gemmatimonas (p < 0.05); the acid phosphatase activity was significantly negatively correlated with the abundance of Flavobacterium (p < 0.01), negatively correlated with the abundances of Methylotenera and Collimonas (p < 0.05), and was positively correlated with the abundances of Gemmatimonas and RB41 (p < 0.05); the alkaline phosphatase activity was significantly positively correlated with the abundances of Lactobacillus, Gemmatimonas and Bryobacter (p < 0.01), was significantly negatively correlated with the abundances of Pseudomonas and Sphingobium (p < 0.01), was positively correlated with the abundances of Candidatus, Rhizomicrobium and Bacteroides (p < 0.05), and was negatively correlated with the abundances of Methylotenera and Ralstonia (p < 0.05); the urease activity was significantly positively correlated with the abundances of Lactobacillus, Bryobacter, Candidatus and Rhizomicrobium (p < 0.01), was positively correlated with the abundance of Gemmatimonas and Bacteroides (p < 0.05), was significantly negatively with the abundances of Methylotenera and Flavobacterium (p < 0.01), and was negatively correlated with the abundance of Ralstonia (p < 0.05); the invertase was significantly positively correlated with the abundances of Ralstonia and RB41 (p < 0.01), was positively correlated with the abundances of Methylotenera and Pseudomonas (p < 0.05), was significantly negatively correlated with the abundances of Bryobacter, Candidatus and Rhizomicrobium (p < 0.01), and was negatively correlated with the abundance of Lactobacillus (p < 0.05); the protease activity was significantly positively correlated with the abundances of Pseudomonas and RB41 (p < 0.01), was positively correlated with the abundance of Ralstonia and Collimonas (p < 0.05), was significantly negatively correlated with the abundance of Bryobacter (p < 0.01), and was negatively correlated with the abundances of Lactobacillus, Candidatus and Rhizomicrobium (p < 0.05); the catalase activity was positively correlated with the abundance of Gemmatimonas and was negatively correlated with the abundance of Sphingomonas (p < 0.05); the available nitrogen content was significantly positively correlated with the abundances of Bryobacter, Candidatus and Rhizomicrobium (p < 0.01), was positively correlated with the abundance of Lactobacillus and Sphingomonas (p < 0.05), and was negatively correlated with the abundances of Methylotenera, Ralstonia and Flavobacterium (p < 0.05); the available phosphorus content was significantly positively correlated with the abundances of Lactobacillus, Gemmatimonas, Bryobacter, Candidatus and Rhizomicrobium (p < 0.01), was positively correlated with the abundance of Bacteroides (p < 0.05), was significantly negatively correlated with the abundances of Methylotenera, Ralstonia and Sphingobium (p < 0.01), and was negatively correlated with the abundances of Pseudomonas and Flavobacterium (p < 0.05); the content of organic matter was significantly positively correlated with the abundances of Lactobacillus and Gemmatimonas (p < 0.01), was positively correlated with the abundances of Bryobacter, Candidatus and Rhizomicrobium (p < 0.05), was significantly negatively correlated with the abundances of Methylotenera, Pseudomonas, Ralstonia and Sphingobium (p < 0.01), and was negatively correlated with the abundance of Flavobacterium (p < 0.05)  Table .Table 6CF. taipaiensis rhizospheric soils with different cultivation years explained 68.36% of the total characteristic value , the urease activity , the available phosphorus content  and the organic matter content  were the dominate factors that affected by the change of the rhizospheric soil bacterial community structure, among which the pH was the most significant factor  is negatively correlated to the content of soil organic carbon38; for Y4 and Y5: Flavobacterium, Duganella and Pseudomonas are pathogen suppressive, and are more abundant in fungal pathogen diseased plant rhizosphere39, the abundance of Acidibacter is positively correlated with the damage of plant cell40.Long-term single planting is considered as a process of pathogen accumulation and soil nutrient status deterioration44. Here, our study proves that pH is also the most significant factor affected by the soil bacterial community structure and its explanation degree exceeds 90%. However, previous studies have shown that, with the increase of continuous cropping years, the pH in the planting soil would gradually decrease, resulting in soil acidification46. Therefore, the increase of the pH in the rhizosphere of F. taipaiensis with the cultivation year in this study provided a special case. Among the genera with the top 15 relative abundances, Flavobacterium and Ralstonia were significantly positively correlated with pH while Bryobacter, Candidatus and Lactobacillus were negatively correlated with pH in this study. Flavobacterium is a kind of important soil saprophytic bacteria, the ammonia produced by the process of its organic matter degradation can alkalize the soil47, the increasing trend of pH from Y1 to Y4 may also associated to the increasing of Flavobacterium. In addition, the ammonia produced by the process of Flavobacterium’s organic matter degradation may be toxic to F. taipaiensis. The suitable soil environment for the growth of Fritillaria plants is weakly acidic (pH = 6.66)48, therefore, in the actual planting, the rising soil pH may cause the growth and quality decline of F. taipaiensis. The metabolic process of Ralstonia can produce alkaline substances that could rise the soil pH49. The relative abundance of Ralstonia in sample Y4 was obviously higher than those in Y1, Y2 and Y3, and The pH of Y4 was 7.47, the weak alkaline environment can also associated to the increasing of Ralstonia, which can badly affect the growth of F. taipaiensis. Bryobacter, Candidatus and RB41 are affiliated to Acidobacteria, the abundances of bacteria in Acidobacteria are generally negatively correlated with soil pH51, the decline on the relative abundance of these 3 genera may also lead the soil pH rising. In addition, the relative abundance of Lactobacillus which can produce lactic acid, gradually declined to 0% in Y4 and Y5, that process hastened the rising of soil pH.Previous studies have confirmed that pH is one of the most important factors affecting the soil bacterial community structure54. Previous studies showed that when the relative abundance of plant pathogenic fungi Fusarium rose, the the growth of Gemmatimonas could be seriously inhibited, and the relative abundance of Pseudomonas would rise55. Therefore, in this study, the decline of the Gemmatimonas’s relative abundance and the increase of the Pseudomonas’s relative abundance may corresponded to the accumulation of pathogenic fungi with the cultivation years. In addition, Gemmatimonas was positively correlated with the activity of phosphatase and the contents of available phosphorus and organic matter in this study. Previous study have found that Gemmatimonas can dissolve insolubilized phosphate and convert it into solubilized phosphate for plant growth56, its decline in relative abundance would hasten the impoverishment of planting soil. Moreover, the bacteria that declined in the relative abundance include various kind of nitrogen fixing bacteria, potash dissolving bacteria and phosphate dissolving bacteria53. Thus, the above conclusion explained the reasons for the decline of the contents of available nitrogen, available phosphorus and available potassium.Some studies found that with the cultivation years’ increase, the abundances of bacteria in the cultivated soil would decline while the abundance of fungi would rise, the soil microbial population type would turn to fungal type from bacterial type, and the fungal plant pathogens would accumulate in soil during this process57. Urease can promote the hydrolysis of urea to ammonia in soil, its activity is often used to characterize the intensity of soil nitrogen transformation58. Protease can promote the hydrolysis of proteins and peptides to amino acids, and its activity is an important characterization of soil nitrogen supply capacity59. The significant higher protease activities in Y4 and Y5 compared with those in Y1, Y2, and Y3 may corresponded to the increase of Collimonas which was positively correlated with protease activities in Y3, for the Collimonas’s metabolites had high protease activity54. The changing trend of urease activity was opposite to that of protease and the urease activity was also negatively correlated with the protease activity in this study, which was consist with the result of Chang et al60. Previous study found that high protease activity in the soil could significantly increase soil total nitrogen content, while the excessive total nitrogen content could inhibit the secretion of urease61. Another study showed that the soil urease activity was positively correlated with the organic matter content and was negatively correlated with the invertase activity62, which was consist with our study. Therefore, in our study, the urease activity showed a changing trend similar with the organic matter content and contrary to the invertase activity.Urease and protease are the key enzymes involved in the transformation of soil organic nitrogen to inorganic nitrogen, which play an extremely important role in the process of soil nitrogen cyclingF. taipaiensis was mainly began as seed sowing, which usually took at least 5 years from germination to harvest63. And because of this long-term single planting pattern, the plant would release large amount of secondary metabolites such as phenolic acids into the soil which would be toxic to the plants and affect the soil microbial structure67. Study have confirmed that phenolic acids could reduce the number of beneficial microbial population under the condition of long-term single planting68. Previous study on F. taipaiensis also found that the number of culturable beneficial bacteria in the rhizospheric soil would significantly decline in the 3rd and 4th cultivation years during the single planting9, this phenomenon could be explained by the accumulation of phenolic acids that also badly affect the growth of F. taipaiensis.Moreover, in the actual production, the cultivation of F. taipaiensis rhizospheric soil, the evenness of the soil bacterial community declined, the relative abundance of the beneficial bacteria decreased and the pathogenic microorganisms accumulated. The decrease of the available nitrogen, available phosphorus and organic matters impoverished the soil. This study can provide a preliminary scientific basis for the soil management in the sustainable cultivation of F. taipaiensis, solving the germplasm decline problem appeared in the production of F. taipaiensis by combining with reasonable rotation, application of organic fertilizer, development of biological fertilizer and control of harmful pathogens. In our future research, we are going to use metagenomic analysis to identify more enzymes and proteins that may be involved in the changes on the bio-chemical properties of F. taipaiensis rhizospheric soil, moreover, we will analyze the relationship between the rhizospheric microorganisms and the growth and the medicinal quality of F. taipaiensis to find the potentially beneficial microorganisms that can be used in the sustainable cultivation of F. taipaiensis.In summary, long-term single planting pattern cause the obvious change of the bacterial community structure in the Supplementary Information.


Atopy is a genetic condition predisposing individuals to develop immunoglobulin E (IgE) against common allergens through T-helper 2 (Th2) polarization mechanisms. The impact of atopy on graft survival in solid organ transplantation is unknown.We analyzed 268 renal allograft recipients from the Swiss Transplant Cohort Study, a prospective multicenter cohort studying patients after solid organ transplantation, with a 9-year median follow-up (IQR 3.0). We used the Phadiatop assay to measure IgE antibodies against a mixture of common inhaled allergens  to identify pre-transplantation atopic patients (>0.35 KU/L).Of 268 kidney transplant recipients, 66 individuals were atopic (24.6%). Atopic patients were significantly younger than non-atopic patients . No significant difference was found for gender, cold/warm ischemia time, preformed donor-specific antibodies (DSA), HLA mismatches, induction and maintenance immunosuppressive therapy, CMV serostatus, or cause of kidney failure. Patient and graft survival at ten years of follow-up were significantly better in the atopic group, 95.2% versus 69.2% patient survival (P < 0.001), and 87.9% versus 60.8% graft survival (P < 0.001), respectively. A multivariate Cox analysis revealed that atopy predicted recipient and graft survival independently of age and living donor donation. Finally, we found similar rates of biopsy-proven acute cellular and antibody-mediated rejections between atopic and non-atopic recipients.Atopy was associated with better long-term patient and graft survival, independently of age and living donor donation after kidney transplantation. Yet, atopy should not be used as a predictor for acute rejection. The T helper 1/T helper 2 (Th1/Th2) balance is a key element involved in regulation of various immunological responses, such as host defense against microorganisms, autoimmune diseases or allergy. Appreciation of its complexity has substantially increased since the discovery of various other T helper cell subsets, including Th17, T follicular helper (Tfh), or regulatory T cells, as well as with the recognition of the interplay with the innate immune system  4)..4).The genetic predisposition for the development of specific IgE against common allergens, such as pollens, dusts, or animals defining atopy may be associated with rhino-conjunctivitis, asthma, atopic dermatitis and food allergies . Atopic The primary objective of this study was to compare kidney allograft and patient survival, between atopic and non-atopic recipients in a Swiss prospective multicenter cohort. We defined atopy by the presence of a positive Phadiatop prior to transplantation, as the latter has a high sensitivity (92%) and specificity 98%) (% (8). WeAll patients were enrolled in the Swiss Transplant Cohort Study (STCS), a prospective multicenter cohort, including all solid organ recipients at the Swiss national level as of May 2008 onwards , 10. 268Data were collected at day 0 before transplantation, 6 and 12 months post transplantation, and then every year, using standardized follow-up forms. Baseline donor and recipient characteristics included ethnicity, age and gender, HLA mismatches, anti-HLA antibodies and pre-formed donor-specific antibodies (DSA), CMV serostatus, as well as recipient diagnosis of end-stage renal disease and pre-existing co-morbidities. Transplantation-related characteristics included type of donor, cold and warm ischemia time, induction and initial maintenance immunosuppressive therapy, and surgery-related complications. Recipient follow-up data included graft loss and rejection status (biopsy-proven acute rejection episodes and corresponding Banff classification) . The Phadiatop is a test quantifying IgE against a mixture of common respiratory allergens including grass, birch, olive, mugwort, parietaria, dog, cat, horse, house dust mite and flour mite, and osporium . Total Icoxph function of the survival package. Multivariate analysis was performed by using stratified multivariable Cox model. Wald test was used to assess significance in Cox proportional hazards regression analysis. Bar plots were generated using the ggpubr package.All statistical analysis were performed using R environment (version 4.0.4). A p-value smaller than 0.05 was considered to be statistically significant. Differences between groups were assessed by a Mann-Whitney test for continuous variables, and a Chi-squared test for proportions. Pearson’s Chi-squared test was used for categorical variables. Survival analyses were performed with the Kaplan–Meier method and the log rank test. Deaths with a functioning graft were not recorded in this study and were considered as graft loss. Multivariate Cox proportional-hazards regression analysis was used to adjust for the recipient’s age, non-living donation, and long cold ischemia time. Univariate Cox regression model was computed for each independent variable, using the Two hundred sixty-eight Caucasian kidney transplant recipients from the STCS were included in this study. Median follow-up was 8.8 years (interquartile range (IQR) 4.1) for the non-atopic and 9.0 years (IQR 2.0) for the atopic group. Their baseline characteristics are described in 66 transplant recipients (24.6%) had a positive Phadiatop. Atopic recipients were significantly younger than non-atopic recipients  and had significantly higher levels of total IgE , whereas graft survival was 98.5% and 93.6.0%, respectively (P = 0.12). Long-term patient survival was significantly better in the atopic group . When excluding individuals with preformed antibodies from the analysis, difference between atopic and non-atopic patient and graft survivals remains statistically significant (P < 0.001). Additionally, we did not find any significant correlation between total IgE and cumulative DSA  and 16 non-atopic (80%). DSA MFI were however not reported limiting the interpretation of the results.Preformed and To understand if the differences in graft survival were related to rejection episodes, we next studied the cumulative events of biopsy-proven acute cellular rejection (ACR) or acute antibody-mediated rejection (AMR) in both groups. One-year ACR cumulative hazard for non-atopic and atopic patients were 32.8% versus 33.8% respectively for ACR (P = 0.89), and 7.0% versus 7.6% (P = 0.86) for AMR  deposits and the microvascular inflammation score, were assessed by combining glomerulitis and peritubular capillaritis scores. No differences were observed between both groups . Unlike mouse models however, atopic patients were not better protected against acute rejection episodes. This suggests that atopy should not be used as an early marker to predict short-term graft outcomes, nor to modulate immunosuppressive protocols.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was reviewed and approved by the ethical committee , as well by the STCS (FUP 098). The patients/participants provided their written informed consent to participate in this study.YM designed the study. RP and YM wrote of the manuscript. RP, RM, and YM analyzed the data. JM and DG identified the sera. VA performed the allergic testing. MP, TH, and DG reviewed and edited the manuscript. All authors have read and approved the final version of the manuscript.


Meanwhile, A-HBS had the highest peak viscosity, indicating the best water absorption (p < 0.05). Moreover, E-HBS had not only higher G′ and G″ values, but also the highest gel hardness value, reflecting its strong gel structure (p < 0.05). These results confirmed that E-HBS provided better pasting stability and rheological properties, while U-HBS provides benefits of reducing starch retrogradation.The purpose of this study was to compare the gelatinization and retrogradation properties of highland barley starch (HBS) using different extraction methods. We obtained HBS by three methods, including alkali extraction (A-HBS), ultrasound extraction (U-HBS) and enzyme extraction (E-HBS). An investigation was carried out using a rapid viscosity analyzer (RVA), texture profile analysis (TPA), differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier-transform infrared spectrometry (FTIR). It is shown that the different extraction methods did not change the crystalline type of HBS. E-HBS had the lowest damaged starch content and highest relative crystallinity value ( Highland barley (HB) is a high-quality raw material for the development of functional foods, among which the most abundant component is starch, accounting for around 65% by dry weight of grains . The regAs is well known, the granular structure of starch is correlated with its physicochemical properties. The pasting behavior of starch is central to many starch-based food matrices and is generally characterized by changes in viscosity during the process of heating, holding and cooling . The degThe scanning electron micrographs of HBS obtained from three extraction methods are shown in As shown in Gelatinization is a vital property for starch-based food processing. The pasting curves for HBS were different  and the As displayed in To further evaluate the gel systems of different HBS, the rheological properties were characterized. As the angular frequency increased, the storage modulus (G′) and the loss modulus (G″) both tended to increase gradually  A,B. FurTo explore the thermal properties of starch, thermograms of all HBS samples treated were revealed in XRD measurement was performed to further investigate the long-range ordered structure of HBS extracted by three methods; the results are shown in −1 to 3300 cm−1, which was mainly attributed to the existence of -OH groups occurring normally in carbohydrates [−1 and 1022 cm−1 represent the crystalline ordered and amorphous regions of starch, respectively. To reveal short-range order, absorbance ratios were calculated at 1047/1022 cm−1 (R1047/1022) [The FTIR spectra of HBS obtained by different extraction methods are shown in hydrates . In addihydrates , which a47/1022) . As seenThe principal component analysis (PCA) was further applied in this study in order to understand the potential mechanism of pasting and retrogradation. The HB kernels (cultivar: Kunlun 15) were purchased from Xinlvkang food Co., Ltd. . Cellulase (≥400 U/mg protein) was purchased from Yuanye Technology Co., Ltd. . Xylanase (≥6500 U/g protein) was purchased from Novozymes (China) Biotechnology Co., Ltd. . All other reagents were analytical grade.HB kernels were milled and sieved through 100 mesh to obtain highland barley flour (HBF). Afterwards, HBS was extracted using three methods based on the conditions of the extraction. The obtained HBS samples were denoted as A-HBS, U-HBS and E-HBS, respectively.g, 20 min) to discard the supernatant and the top gray layer was removed. Then, the precipitates were washed with deionized water and ethanol three times to remove soluble impurities, respectively. The precipitate obtained after washing was collected as starch and dried at room temperature, followed by passing through a 100-mesh sieve. The yield was then calculated as the percentage of the HBF weight.According to Yang et al., with some modifications , HBF (10HBF (100 g) was added to 600 mL distilled water, which was treated by an ultrasonic device  working at a frequency of 20 kHz and input power of 200 W for 20 min. Then, the sample was shaken in a water bath at 50 °C for 5 h. In order to ensure that the purity of HBS was greater than 90%, it was sieved through 300-mesh (0.05 mm) before centrifugation and repeating the above steps.According to Ozturk et al., with some modifications , HBF (10According to the standard methods described by AACC (2010), the contents of moisture, crude protein, crude fat, ash and total dietary fiber were determined. The contents of total starch, amylose and damaged starch were determined by an assay kit . A laser particle size analyzer  was chosen to measure the particle size distribution of HBS samples.The morphology of HBS granules was observed by a scanning electron microscope  under the voltage of 10 kV. All samples were sputter-coated with gold and then observed at magnifications of 500×. The pasting properties of HBS samples were analyzed by a rapid viscosity analyzer , following the method of Qin et al. . A 3.0 gThe retrograded starch samples stored at 4 °C for 1 and 7 d in The viscoelastic and flow behaviors were determined by a stress-controlled rheometer  equipped with parallel plate geometry and a 1 mm gap . The samThe thermal properties were measured by differential scanning calorimetry  according to Chen et al., with some modifications . 3 mg HBa is the total area and Afa is the area of the amorphous fraction.The crystalline structure of HBS samples extracted by different methods was measured by an X-ray diffractometer  equipped with a copper tube operating at 40 kV and 200 mA and producing Cu-Kα radiation at a wavelength of 0.1542 nm. The diffractograms were obtained by scanning from 5° to 45 ° (2θ) at room temperature at a rate of 10°/min and in steps of 0.02°. The crystalline peak and total area of the diffractogram were analyzed using MDI Jade 5.0 software . The relative crystallinity was calculated as the percentage of the area of the crystalline region to the area of total diffraction, as represented in Equation (1):−1 of wavelength, 4 cm−1 of resolution and 64 scans. The HBS samples (2 mg) and the dried KBr powders (100 mg) were mixed and ground thoroughly. The mixture was then pressed into tablets and observed by Fourier-transform infrared spectroscopy . The blank background was completed by the KBr powder alone. The scanning conditions were 400–4000 cmp < 0.05) was determined using the Duncan procedure. PCA was performed to visualize gelatinization and retrogradation properties of three kinds of HBS samples, respectively. PCA was achieved using Origin 2018b software . All experiments were carried out at least in triplicate.Analysis of variance (ANOVA) was used to measure statistical differences by SPSS Version 16.0 software . Significant difference (In conclusion, the gelatinization and retrogradation characteristics of HBS obtained by the different extraction methods were significantly different. It was attributed to the granular structure of the HBS. E-HBS had the highest relative crystallinity value, thus it exhibited better thermal stability, which increased the crispness of foods as cookies. On the other hand, A-HBS showed the weakest hydrogen bonding, which resulted in the highest viscosity and could be used as a food thickener. Moreover, U-HBS had the highest damaged starch content, as well as the lowest gelatinization enthalpy after 1 and 7 days of storage, respectively, providing anti-aging properties, which made it suitable for foods with a short shelf life such as bread or noodles. The findings above provide a theoretical basis for the development of HBS and highland barley food with different expected properties.


Croatian Medical Journal (CMJ); yet, some constants prevail. The Journal is a war child, born among ruined infrastructure, during air raids, amid uncertainty and chaos. It stood for something far greater than a scholarly journal. It was a materialization of a collective striving toward a better future and an act of defiance. The guiding spirit behind its coming to life was the same one that bolstered Croatian sports and cultural achievements and that ultimately catalyzed social and political changes. In other words, the Journal arose from a community wider than the medical one and it disseminated ideas that reverberated beyond the scientific community.Much has changed in the thirty years since the launching of the CMJ – to serve as a gateway between Croatian medical researchers and the global scientific community. This translated into more than 40% entries by Croatian authors out of more than 2700 articles published so far in the CMJ covered by Web of Science and Scopus. Notably, articles from Croatian authors and authors from neighboring and regional countries together account for more than half of the journal contributions . These numbers illustrate a remarkable role of the CMJ in medical publishing in the regional context. Likewise, the Journal has been striving to educate local and regional researchers on the principles of good peer-review by recruiting them into its pool of peer-reviewers, acknowledging their contribution, and helping them grow as reviewers  reflected in the editorial policy aimed at educating local and regional medical professionals on the principles of scientific research and communication ,2. Over In the new millennium, the social environment profoundly changed. Those who occupied the scientific publishing landscape dominated by hard copies, enveloped submissions, and dial-up speeds (if any) could have hardly imagined the today’s almost exclusively virtual niche populated by social networks, alternative metrics, and predatory publishing. To keep pace, the Journal has adapted to the selection pressures on various levels. Trivial and technical amendments, such as the new web design and submission interface, were implemented, but also major policy directives regarding issues such as Journal ownership and autonomy, business and publishing model, and author rights . Again, CMJ. This constant is communication – honest, unbiased, open-access, and widely available communication between researchers and the general public, physicians and patients, mentors and students. This communication should be about not only medical science but about broader topics as well. The field of medicine and its practitioners do not exist in a vacuum  but depend on the entirety of social factors and get increasingly specialized. For this reason, communication is essential for interdisciplinary cohesion and serves to bond and guide us forward. We believe that the CMJ has been a reliable partner and mediator in this communication, and that the products of our labor reach far beyond the journal impact factor.Today, thirty years after the Journal launching, blood has again been spilled on the European soil, and we are facing a future filled with unknowns and uncertainty. As the society grows more interconnected, problems become more global, immediate, and existential – from pandemics to climate change and culture wars. We remain vigilant about these universal societal issues affecting public health and well-being -9. Yet a