COVID-19, which is accompanied by acute respiratory distress, multiple organ failure, and death, has spread worldwide much faster than previously thought. However, at present, it has limited treatments.To overcome this issue, we developed an artificial intelligence (AI) model of COVID-19, named EDRnet , to predict in-hospital mortality using a routine blood sample at the time of hospital admission.We selected 28 blood biomarkers and used the age and gender information of patients as model inputs. To improve the mortality prediction, we adopted an ensemble approach combining deep neural network and random forest models. We trained our model with a database of blood samples from 361 COVID-19 patients in Wuhan, China, and applied it to 106 COVID-19 patients in three Korean medical institutions.In the testing data sets, EDRnet provided high sensitivity (100%), specificity (91%), and accuracy (92%). To extend the number of patient data points, we developed a web application (BeatCOVID19) where anyone can access the model to predict mortality and can register his or her own blood laboratory results.Our new AI model, EDRnet, accurately predicts the mortality rate for COVID-19. It is publicly available and aims to help health care providers fight COVID-19 and improve patients’ outcomes. COVID-19 is a highly contagious infection caused by SARS-CoV2. In severe cases, COVID-19 causes acute respiratory distress, multiple organ failure, and, eventually, death [In a pandemic situation, the most important issue in the management of patients diagnosed with COVID-19 is to select patients at risk of high mortality in the early period of disease and to provide appropriate treatments . ParticuIn this pandemic crisis, the shortage of resources and medical staff causes big problems in the health care system. Accordingly, artificial intelligence (AI) can aid in the management of COVID-19 patients. A recent research study has developed an AI prediction model of mortality based on blood test results . In thisHowever, Yan et al’s study has drawbacks. First, the three biomarkers derived from the XGBoost-based feature selection may not be the best choices. Feature importance provides a score indicating how each feature contributes in the construction of decision trees within the model. However, due to the stochastic nature of machine learning algorithms, each feature’s importance score may vary. Moreover, in decision tree algorithms, such as an XGBoost and a random forest (RF), when multiple features have the same gain during the split, a branch in a tree is made by randomly selecting features among them. Second, numerous studies have shown that the disease progression of COVID-19 is not only associated with LDH ,8-11, lyTherefore, in this study, we aimed to develop an AI model based on a blood test for mortality prediction at the early stage of hospital admission. We deployed the developed AI model on a public website so that all patients and medical staff could predict mortality using individual patient blood test results.This study was approved by Wonkwang University Hospital (WKUH), Chonnam National University Hospital (CNUH), and Samsung Medical Center (SMC) in Korea. Informed consent was waived. For training data, we used the blood test results obtained from 375 COVID-19 patients collected between January 10, 2020, and February 24, 2020, in Tongji Hospital, Wuhan, China . Of thesF test to check for any significant difference between the two groups  according to each blood biomarker. For the feature selection, we also considered the available data rate (ADR), which refers to how much blood biomarker data were available for training the AI model. This is calculated asGiven the total 73 blood biomarkers from the training data, we performed an analysis of variance (ANOVA), which uses an patientsN is the total number of patients (N=361) and biomarkerN is the number of patients having each of the specific biomarker data.where P values less than 10–5. Subsequently, we excluded four biomarkers with ADR values of less than 90%. P values and ADR values. The ANOVA P values and ADR values for all 73 biomarkers in the training data set are summarized in Based on the ANOVA, we first selected the top 32 biomarkers corresponding to Given the selected 28 biomarkers, the mean number of available biomarkers per patient was 27.22 (SD 2.33) for the training data and 16.86 (SD 1.58) for the testing data, as summarized in With the 30 features, we performed data set standardization, which is a common requirement for machine learning estimators. The standardization changes the data distribution of each feature with zero mean and standard deviation of 1 asmean(train) and SD(train) are the mean and standard deviation values, respectively, for each feature from the training data. The standardization was applied to the training and testing data sets.where As illustrated in For the 5-layer DNN, a 10-time-repetition 10-fold stratified cross-validation was performed to confirm the model’s generalization ability. The training data (N=361) were randomly shuffled and partitioned into 10 equal subgroups in a stratified manner. Of the 10 subgroups, a single subgroup was retained as the validation data set for testing the model, and the remaining nine subgroups were used as the training data set. The process was then repeated 10 times, with each of the 10 subgroups used exactly once as the validation data set. By repeating this stratified 10-fold cross-validation process 10 times, a total of 100 models from the 5-layer DNN were derived. Then, we ensembled the models with the weighted average asm(DNN)p is the predicted mortality probability value from the mth model of the DNN, p(DNN) is the ensemble result corresponding to the predicted mortality prediction probability, and m(DNN) is the normalized weight value for the mth model. We obtained the normalized weight value m(DNN) aswhere m(DNN)w was obtained using the validation loss from the mth model, m(DNN)l, aswhere the weight Along with the 5-layer DNN, we separately trained an RF model. For the RF model, 100 decision trees were trained with a maximum depth of 4 and maximum feature number of 5. Similar to the 5-layer DNN, we performed a 10-time-repetition 10-fold stratified cross-validation and ensembled the 100 models with the weighted average asm(RF)p is the predicted mortality probability value from the mth model of the RF, p(RF) is the ensemble result corresponding to the predicted mortality prediction probability, and m(RF) is the normalized weight value for the mth model. We obtained the normalized weight value m aswhere m(RF)w was obtained using the validation loss from the mth model, m(RF)l, aswhere the weight p(DNN) and p(RF), we finally obtained the final predicted mortality probability value using soft voting. Based on the average of the two probability values p(DNN) and p(RF), if the value is greater than or equal to 0.5, then the prediction result represents death; otherwise, it represents survival.Given the two ensemble results We implemented and trained EDRnet using TensorFlow, version 1.13.1 for graphics processing unit (GPU), and Keras, version 2.2.4 for GPU. NumPy, version 1.16.4; Pandas, version: 0.25.3; Matplotlib, version 3.1.2; and scikit-learn, version 0.22.1, were used to build the model and analyze the results. We trained the models with the Adam optimizer and a binary cross-entropy cost function in equation 9 with a learning rate of 0.0001 and a batch size of 64 on the NVIDIA GeForce GTX 1080 Ti GPU asiy is the label  and i)p, each of which was evaluated as a single model and as an ensemble model combined with DNN, resulting in four models: XGBoost, AB, ensemble with DNN and XGBoost (EDX), and ensemble with DNN and AB (EDA). For the training of these models, we searched the optimal hyperparameters providing the highest validation accuracy value, as presented in The cross-validation of RF, DNN, and our ensemble model EDRnet showed that the accuracy on the validation data set is 89% for RF, 92% for DNN, and 93% for EDRnet. Thus, EDRnet provides the highest sensitivity, specificity, accuracy, and balanced accuracy values see .Moreover, we applied EDRnet to 106 Korean patients as an independent testing data set to check the TP, TN, FP, FN, sensitivity, specificity, accuracy, and balanced accuracy. The results show a sensitivity of 100%, specificity of 91%, accuracy of 92%, and balanced accuracy of 96%, indicating that the model trained and validated on Chinese patient data can be applied to Korean patients for mortality prediction see . The comNext, we summarized the performance comparison results between XGBoost, AB, RF, DNN, EDX, EDA, Li et al’s model , and EDROur proposed EDRnet model used 28 blood biomarkers for prediction, but it does not require all 28 blood biomarkers. In our testing data sets, EDRnet training was validated using available biomarkers, ranging from 14 to 24, for each patient see . The resTo further investigate the effect of the number of available biomarkers, we estimated the accuracy values according to the number of available biomarkers see . For theFurthermore, our developed AI model, EDRnet, was successfully deployed on a public website  so that P=.04). All patients from the deceased group required oxygen supply. The deceased group had more frequent altered mentality than the survivor group . There was no significant difference in terms of antiviral drugs  or anti-inflammatory drugs  between the deceased and survivor groups. However, the deceased group received more antibiotics or combination therapy.Regarding clinical characteristics see , there wOur new AI model, EDRnet, was able to predict the mortality of COVID-19 patients using 28 blood biomarkers obtained within 24 hours after hospital admission. In the independent testing data sets, EDRnet showed excellent prediction performance with high sensitivity (100%), specificity (91%), and accuracy (92%). We were able to improve the prediction performance by adopting the ensemble approach combining DNN and RF models. Of note, EDRnet was developed by training with Chinese patients’ data and testing with Korean patients’ data.EDRnet has several advantages. First, EDRnet can predict which patients are at a high risk of mortality in the early stage of hospital admission . This is a substantial improvement compared to the prior AI prediction model reported by Yan et al, which predicted mortality 10 days before the occurrence of survival or death . The morThe hematological changes in lymphocytes, neutrophils, monocytes, eosinophils, and platelets are common, as these changes are related to viral replication and hyperinflammation in COVID-19 infection ,13. In sRegarding blood chemistry, hs-CRP is a major biomarker that represents acute phase inflammation ,10,14-17In this study, no significant differences were observed in the use of pharmacologic agents between the deceased and survivor groups except antibiotics and in the use of antiviral drugs, such as remdesivir. Antibiotics or combination therapy is usually used for suspected bacterial superinfection that represents severe diseases. To date, there has been no successfully effective pharmacologic agent to treat COVID-19. The pharmacologic treatment is not significantly related to survival in this study.EDRnet does not require all 28 blood biomarkers for the prediction of mortality. EDRnet worked well as long as there were at least 19 blood biomarkers at the time of admission. Compared to prior AI prediction models for COVID-19 mortality, which used three biomarkers, there might be concern that EDRnet requires too many biomarkers. However, these blood tests are commonly performed in our daily clinical practice for hospitalized patients with COVID-19. If more data are accumulated, then we can reduce the number of blood biomarkers for mortality prediction.Our study has several limitations. First, the number of patients available for testing might be small. According to Johns Hopkins Coronavirus Resource Center, the mortality rate in South Korea is 1.7%. In the testing data set of 106 Korean patients, the mortality rate was 1.9%, which is almost equivalent to the actual mortality rate. It might be necessary to update EDRnet by training with a large population data set from all over the world. To update EDRnet, we made a web application  so that In conclusion, our new AI model, EDRnet, was developed to predict the mortality of COVID-19 patients at the time of hospital admission using blood biomarkers only. It is now open to the public with the hope that it can help health care providers fight COVID-19 and improve patients’ outcomes.


Novel three-dimensional (3D) nanohydroxyapatite-PLGA scaffolds with high porosity was developed to better mimic mineral component and microstructure of natural bone. To perform a final assessment of this nanomaterial as a potential bone substitute, its toxicological profile was particularly investigated. Therefore, we performed a comet assay on human monocytes for in vitro genotoxicity investigation, and the systemic subchronic toxicity investigation on rats being per oral feed with exactly administrated extract quantities of the nano calcium hydroxyapatite covered with tiny layers of PLGA (ALBO-OS) for 120 days. Histological and stereological parameters of the liver, kidney, and spleen tissue were analyzed. Comet assay revealed low genotoxic potential, while histological analysis and stereological investigation revealed no significant changes in exposed animals when compared to controls, although the volume density of blood sinusoids and connective tissue, as well as numerical density and number of mitosis were slightly increased. Additionally, despite the significantly increased average number of the Ki67 and slightly increased number of CD68 positive cells in the presence of ALBO-OS, immunoreactive cells proliferation was almost neglected. Blood analyses showed that all of the blood parameters in rats fed with extract nanomaterial are comparable with corresponding parameters of no feed rats, taken as blind probe. This study contributes to the toxicological profiling of ALBO-OS scaffold for potential future application in bone tissue engineering. Increased number of skeletal system disorders, due to the increase of the human lifespan, imposes a need for a new and more efficient bone healing procedure, since conventional treatments with auto- and allografts have many drawbacks, such as infections, pain, long-term recovery, and rejection by a host immune system ,2. Tissu10(PO4)6(OH)2] is one of the most widely utilized, since it possesses structural and chemical similarity to natural bone. It is biocompatible, bioresorbable, and bioactive, and it enables excellent cell adhesiveness [Biocompatible porous three-dimensional 3D) scaffolds that mimic the natural bone structure have given the most promising results due to their biostimulative effect on cells proliferation and neovascularization ,2,3,4,5.D scaffolsiveness ,10,11.The main drawbacks of HAP represent its lower rate of degradation in comparison to natural bone, and its mechanical strength, which mostly depends on the microstructure. To overcome it, HAP was modified due to available nanotechnology and widely combined with synthetic polymers. These composite scaffolds exhibited better mechanical properties and bioactivity, in comparison to ceramics or polymer alone ,10,12. BHowever, after being implanted in the tissue, the degradation process of the material in local tissue occurs, and dissolved products obtain to systematic circulation. The chronical presence of scaffolds’ products in circulation could lead to toxicity, and this aspect of nanostructured HAP has been poorly investigated so far. Systemic subchronic toxicity studies, usually conducted in rodents by repeated administration of tested compounds for more than 90 days , are necessary before clinical application of bone substitute, especially at the large scale bone defects. A combined investigation of genotoxicity in vitro and systemic chronic toxicity could provide new insights in safety evaluation and potential long-term adverse effects of investigated materials and its leachable products .The aim of this study was to provide toxicological profile based on in vitro genotoxicity and in vivo systemic subchronic toxicity. A range of endpoints was evaluated, including biochemical analyses, and histological and stereological measurements on liver, kidney, and spleen tissues, after 120 days of oral administration. This study contributes valuable understanding of the novel three-dimensional (3D) nanohydroxyapatite-PLGA scaffold (ALBO-OS) interaction on the systemic level to organism under subchronic exposure scenario. A novel composite scaffold that was based on calcium hydroxyapatite and poly(lactide-co-glycolide) (PLGA), named ALBO-OS, was used for in vivo toxicity study. The detailed procedure of material’s synthesis and characterization is explained in our previous investigations ,14,15, a2) and (NH4)2HPO4 . The obtained hydroxyapatite powder was further used for the production of HAP granules. Further, ceramic slurry was made by mixing HAP granules and starch with water, and then poured over polyurethane foam with the required pore size distribution and dried at room temperature. Finally, it was heated in an oven at 600 °C and sintered at 1200 °C for 4 h. The obtained porous HAP compact was further disintegrated into 300 μm–1 mm granules. Finally, PLGA  thin film was deposited onto the surface of HA granules to obtain final product.Briefly, HAP powder was hydrothermally synthesized from an approximately stoichiometric mixture of calcium hydroxide (Ca(OH)4 cells per well. The next day cells were exposed to ALBO-OS extract. Extract contained maximal concentrations of Ca2+ ions, which exact value, was previously exactly ordered by ICP. The concentration corresponded to concentration of released Ca2+ ions in saturated solution obtained after immersion of 0.05, 5, 10, and 50 mg/mL ALBO OS in 10 mL distilled water with previously adjusted pH at 7.37, during 120 h. For negative control, the cells were not treated with material, while cells exposed to methyl methanesulfonate (MMS) solution (40 µM) were used as a positive control. The cells were exposed to various treatments for 1 h, after which they were centrifuged  and resuspended in Dulbecco’s phosphate-buffered saline . Prior to alkaline comet assay, Trypan blue exclusion assay [THP-1 cells were seeded in 12-well plates in concentration 15 × 10on assay  was usedThe comet assay (single cell gel electrophoresis assay) was used to detect possible single- strand DNA breaks, double-strand DNA breaks, alkali labile site, and incomplete excision repair sites . The micThe Ethical Committee of the Faculty of Veterinary Medicine, University of Belgrade and Ministry of Agriculture and Environment Protection of the Republic of Serbia approved the experimental protocol (decision number 01-831/2), and realized in accordance with recommendations of European Good Laboratory Practice (86/609 EEC) related to protection and use of laboratory animals.Ratus norvegicus), at the age 8–12 weeks, and body weight 240–260 g. Animals were placed in individual cages. During the experiment, the animals were kept under following conditions: temperature 23 ± 3 °C, air humidity 55 ± 5%, 8–12 air changes/h, and artificial lighting at intervals of 12 h day/night. The animals had free access to food and water. The animal health status was monitored daily during the experiment. The investigation included 21 Wistar rats  in distilled water  was used for per oral rats feeding. After seven days of adaptation period, the animals were randomly divided into experimental (n = 12) and control group (n = 9). To the animals in the experimental group, 1 mL of ALBO-OS extract was per oral fed for 120 days, every day at the same time, while, for the control group, distilled water was used. During the experiment, control of the health status, behavior, changes in skin and haircut, food and water consummation, urination, and defecation, was performed and recorded daily. The body weight of animals was checked after the adaptation period, and afterward three times through the experiment.At the end of the experiment, the blood samples were taken from lateral tail vein for hematological  and biochemical analyses , aspartate aminotransaminase (AST), urea, creatinine, bilirubin, serum protein, glucose, and alkaline phosphatase (ALP)).The animals were sacrificed by intravenous administration of thiopentone sodium solution (170 mg/kg). The rats’ livers, kidneys, and spleens were removed, immersed in Buoin’s fixative solution for 24 h, and, for better absorption, further cut into smaller pieces and then immersed into fresh Buoin’s solution. Subsequently, all of the dissected organs were prepared for the light microscopy using the standard tissue preparation procedure. Tissue was embedded in paraffin. Tissue sections of 5 µm thickness were cut using rotary microtome . Every fifth section was used for the analysis in order not to repeat the analyzed structures. Tissue was stained with hematoxylin-eosin (H&E) . The qualitative analysis of the microscopic slides was performed while using the light microscope . For quantitative, stereological analysis, micrographs were acquired in the RGB layout and converted to binary format. Measurements were made using a stereo-universal test system according to Cavalier’s principle, while using 16.0 point-counting system , with P2 spacing grid at the maximum 400× magnification. 0), where Pf represents the number of the desired phase on the test system  and Pt represents the total number of points of the test system [Stained tissue sections of liver, kidney, and spleen were used to measure the number and volume density (Vv) of epithelial cells. Volume density was calculated through the following formula: Vv = Pf/Pt  all cells that have a clearly visible contour and nucleus, (ii) the cells contour do not touch the test system frame, and (iii) the cells contour is clearly visible without a visible nucleus. The numerical density (Nv) of all epithelial cells was calculated based on the cell count (Q) in the volume of analyzed tissues (Vo). The volume of analyzed tissue was evaluated as the product of the number of counted frames (ΣPi), the space of counted frame , and the height factor (h) , as presented in the following formula :(1)Nv=QVMBF software system was used to measure the surface and volume of epithelial cells and nuclei, by the thickness of their diameters. Measuring the surface of epithelial cells and nuclei enabled the measurement of the cells, cytoplasm, and nuclei volumes. The ratio between the cells’ nuclei and cytoplasm volumes was used to determine the nucleocytoplasmic ratio (NCO). The mitotic index was determined as the ratio between the number of cells in mitosis and the total number of cells in 10 visible optical fields per slide, with maximum 200× magnification.2O2 solution in PBS. Tor epitope retrieval, tissue sections were heated in microwave oven for 21 min., at 680 W, in 10 mmol/L citrate buffer, pH 6.0. The tissue sections were incubated with appropriate antibodies  and Ki67 protein ) over night, at +4 °C, in the humid chamber. Streptavidin-biotin technique was used for immunostaining . Immunoreactivity complex was visualized with DAKO Liquid DAB+ Substrate/Chromogen System  and then counterstained with Mayer’s hematoxylin . As negative control served the tissue sections with omitted primary antibody. For positive control, the tissue sections that were known to express CD68 and Ki67 were used. CD68 and Ki67 positive cells in tissues were analyzed by a light microscope  with 40× magnification.For the immunohistochemical analysis, the paraffin tissue sections of rat liver, spleen, and kidney were analyzed cut at 5 µm, and the slides were heated for 60 min. at 56 °C. Prior to the antigen retrieval step, the sections were deparaffinized and rehydrated through the series of xylenes and alcohols. To block endogenous peroxidase activity, tissue sections were treated with 3% HIn all of analyzed sample of tissue, five hot spots were selected for image analysis in ImageJ . Ki67 and CD68 expressions were calculated by determining the positive Ki67/CD68 areas (brown-colored cells) in microscopic fields (40× magnification), based on the threshold . Median t-test) or nonparametric tests , depending on data distribution, with significance set at p < 0.05. Statistical software SPSS 20.0  was used for data processing.All of the values were presented as mean ± standard deviation. Group comparisons were performed while using parametric  was cytotoxic to THP-1 cells after 1 h exposure. In all tested samples, cell viability was over 90%. The results of the comet assay showed that none of the used ALBO-OS concentrations was genotoxic to THP-1 cells . There is no comet tail for any of material’s concentration, which means that there is no damaged DNK (broken fragments) that would migrate from the “head” during the electrophoresis, like in the case of positive control, as it can be seen in During the course of the exposure, no adverse effects that were related to the behavior of the tested animals were observed, as investigated at a daily level. No changes in skin and haircut, as well as changes in food and water consummation, or urinating and defecation, have been reported. Body mass was measured four times during the experiment, and in all animals it was mildly and consistently increased through the observation period . p < 0.001 .In p < 0.05), and surface area of capillary endothelial cells (p < 0.05) increased in the experimental group relative to control  was not significantly different to the control. Hepatocytes and hepatic plaques were normal and neatly placed, without abnormality in the form of a central vein. There were no pathological changes in liver tissue, such as cysts, fibrosis, loss of hepatocytes, necrosis, or lymphocyte aggregates, due to inflammatory reactions, in the experimental or in control group. Further histological and cytological analysis of liver tissue was based on the comparison of fifteen stereological parameters . The num control A. p > 0.05)  . p > 0.05, p > 0.05, p < 0.05), while the surface of Bowman’s space slightly increased . Histological analysis revealed no pathological changes in the spleen tissue, such as change in the morphology of the spleen tissue, loss of lymphocytes, cysts, fibrosis, loss of epithelial and radial cells, necrosis, or large lymphocytes accumulation. Epithelial, endothelial, and connective tissue cells have increased in their number, volume density, and numerical density in the treated group when compared to control A,B, mostp < 0.05, The number and numerical density of lymphocytes in the experimental group were higher in comparison to control . Additionally, the average number of Ki67 positive cells was detected in liver (p < 0.05) and spleen (p < 0.05) experimental groups in comparison to control  during 120 days per oral feeding were particularly important for the determination of the toxicological profile ALBO OS. Accordingly, the liver is chosen due to its vital role in organism being reflected by its physiological functions, synthesis and detoxification of various metabolites. In other words, any lesions caused by drugs, chemicals, and pathological status could interrupt its functions . The ALTThe increased mitotic index and the number of hepatocytes with two nuclei could be explained by the liver adaption to the presence of new material in the organism . It coulIn our previous study, the effect of calcium silicate and calcium aluminate implantation on systematic subchronic toxicity was evaluated . It was p < 0.05)  indicated a statistically significant increased blood flow through the liver in experimental group, when comparing with control group of rats, which is characteristic for processes with more intense blood filtration. Additionally, significant increase the number of endothelial cells (p < 0.05) participating in the formation of new blood sinusoids was observed, which indicated that the process of the necessary adaptation of the rats’ liver to ALBO-OS is occurred. As it is well known, the liver sinusoidal endothelial cells (LSEC) constitute the sinusoidal wall, or endothelial lining. Therefore, they can be observed as unique capillaries that differ from other capillaries in the body, because of the presence of open pores or fenestrae lacking a diaphragm and a basal lamina underneath the endothelium. The increasing quantity of the Ca2+ ions, interacting with lipid soluble ionophores, which transport Ca2+ ions across liver cell membranes, can induce increased transport Ca2+ ions inside of the liver sinusoidal endothelial cells, influencing their contraction and, consequently blood flow diminishing through them. Therefore, the balance establishing in blood flow through sinusoids, the new blood sinusoids should be formed, simultaneously leading to the increased number of the endothelial cells [An increase in the number and density of capillary endothelial cells , while immunostaining revealed significantly higher number of Ki67 positive cells, indicating cell proliferation in experimental group, increased kidney activity, and blood flow. In the study of mineral trioxide aggregate effect on the liver and kidney function, it was reported that the kidney function had subsequently increased after a month, while the increased liver function was observed during the first week and further increased during the observed period [p < 0.05) was statistically significantly reduced during the treatment by ALBO-OS, probably as a consequence of the increase of filtration blood flow into the them. Fray has developed a mathematical model suggesting the stretch-dependent influx of extracellular calcium ions into juxtaglomerular cells to account for the pressure control of renin secretion [The number of epithelial cells in collecting ducts and the connective tissue cells, as well as other stereological and cytological parameters, were not significant  of the number of connective cells in the spleen of rats, during the treatment with ALBO-OS, which is a consequence of the increased production of lymphocytes in the spleen and their increased activity in the blood. Additionally, the increase of the number and numerical density of lymphocytes in the spleen of rats during the treatment by ALBO OS was also statistically significant (p < 0.05). Furthermore, number of CD68 positive immune cells in spleen was similar in the experimental and control group. In previous investigations of systemic toxicity of new endodontic material based on hydroxyapatite and calcium silicates, the slight hypertrophic changes in white pulp were reported, with no adverse effect on the spleen function and its immune response activation . Statist2+ ions and subsequent protein kinase C activation is indispensable for T-lymphocyte proliferation and an increase of their number [2+ ions promotes IL-2 mRNA gene expression and protein synthesis and that IL-2 is a potent T-lymphocyte growth factor [Calcium plays an essential role in the activation and maturation of lymphocytes. As a second messenger, calcium ions are involved in most biochemical processes that transduce the external signals into cell responses. An increase in Car number . Previouh factor ,44,45. Sh factor . Further2+ ions in rat’s blood, the pathological effect expressed over significant changes of morphology of tissue of the liver, kidney, and spleen were not registered, suggesting that the used nanomaterial is biocompatible. An estimate of blood volume in the rat is 4.7 to 8.0 mL per 100 g body volume (BW)  . The BW The tested ALBO OS proved that it could immitate natural bone by its arhitecture and mechanical properties, as well by the rate of resorption, as confirmed by our previous investigations of its structure and physico mechanical characteristics briefly described in the


Background and Objectives: Functional dyspepsia (FD) is one of the most common functional gastrointestinal disorders; it has a great impact on patient quality of life and is difficult to treat satisfactorily. This study evaluates the efficacy and safety of trimebutine maleate (TM) in patients with FD. Materials and Methods: A multicenter, randomized, double-blind, placebo controlled, prospective study was conducted, including 211 patients with FD. Participants were randomized to receive TM 300 mg twice per day  or placebo BID (103 patients) for 4 weeks. The Glasgow Dyspepsia Severity Score (GDSS) was used to evaluate the relief of dyspepsia symptoms. Moreover, as a pilot secondary endpoint, a substudy (eight participants on TM and eight on placebo) was conducted in to evaluate gastric emptying (GE), estimated using a 99mTc-Tin Colloid Semi Solid Meal Scintigraphy test. Results: Of the 211 patients enrolled, 185 (87.7%) (97 (52.4%) in the TM group and 88 (47.6%) in the placebo group) completed the study and were analyzed. The groups did not differ in their demographic and medical history data. Regarding symptom relief, being the primary endpoint, a statistically significant reduction in GDSS for the TM group was revealed between the first (2-week) and final (4-week) visit . The 99 mTc-Tin Colloid Semi Solid Meal Scintigraphy testing showed that TM significantly accelerated GE obtained at 50 min . Adverse effects of low to moderate severity were reported in 12.3% of the patients on TM. Conclusion: TM monotherapy appears to be an effective and safe approach to treating FD, although the findings presented here warrant further confirmation. Functional dyspepsia (FD), sometimes referred as idiopathic or nonulcer dyspepsia, is a bothersome and heterogeneous clinical syndrome affecting 10–40% [The term “dyspepsia”, derived from the Greek words “g 10–40% . FD is cg 10–40% . AlthougFor a better comprehension and definition of functional gastrointestinal syndromes, the Rome Foundation introduced the internationally recognized and applied Rome diagnostic criteria. FD is characterized according to the third Rome revision by four cardinal symptoms: epigastralgia, bothersome postprandial fullness, epigastric burning and early satiety. FD patients are classified into two subgroups: epigastric pain syndrome (EPS) and postprandial distress syndrome (PDS). The last revision of Rome Criteria (IV) slightly updated the aforementioned definitions. Now, not only postprandial fullness, but also EPS symptom, as well as early satiation, should be considered as “bothersome symptoms”. Furthermore, the latest Rome IV classification implicated not only PDS and EPS as separate entities, but also the overlapping PDS and EPS; the latter syndrome is more common in the hospital-based population than among the general public .Helicobacter pylori infection (Hp-I), nervous system dysregulation, low-grade inflammation, lifestyle influences, medication side effects, peripheral immune activation, genetic susceptibility and influences of socio-psychological factors . Nevertheless, the definite mechanism remains unidentified.The pathophysiology FD is poorly defined, leading to much uncertainty regarding therapeutic approaches. The proposed pathophysiologic mechanisms ,7,8,9,10Since its pathophysiology is largely unknown, treatment of FD is difficult, and often yields unsatisfactory results. Camilleri and Tack  proposedHp eradication, prokinetics, neuromodulators, dopamine-2 receptor antagonists, serotonin-4 receptor agonists, nonpharmacological regimens  and acotiamide (a muscarinic receptor antagonist)), relative evaluations concluded that treatments lack efficacy and/or safety, and for most therapeutic options, the precise mechanisms of action and true value in the treatment FD remain uncertain due to conflicting results [Regarding current pharmacological and nonpharmacological therapeutic options for FD  vs. placebo, administered for a period of four weeks in patients diagnosed with FD.A double-blind, randomized, placebo-controlled prospective trial was carried out in tertiary university hospitals at 13 sites in five countries  over a 65-month period (January 2011 to May 2016). Patients with FD as determined by the Rome III criteria, with exacerbated disease experiencing epigastric pain and/or discomfort at least one of the seven days before randomization, were candidates for enrollment. All participants provided written informed consent before enrollment, and the study protocol was approved by the medical ethics committee of the hospitals where the study was conducted .Inclusion criteria were the following: (1) ambulant male and nonpregnant female subjects, (2) age between 18 and 75 years old at the randomization, (3) history of epigastric pain or discomfort at least one of the last seven days before the randomization, (4) absence of macroscopic findings from esophagus, stomach and duodenum during the endoscopy performed 16 ± 4 days before randomization, and (5) willingness to sign the informed consent form prior to entry into the study.Exclusion criteria were the following: (1) alarm symptoms , (2) IBS as defined by the Rome Criteria III, (3) chronic serious constipation, (4) predominantly retrosternal burning pain, (5) serious or uncontrolled diseases, (6) peptic ulcer disease (diagnosed by endoscopy or radiogram) or gastroesophageal reflux disease (diagnosed by endoscopy) and other significant GI disease, (7) history of peptic, gall-bladder or other abdominal surgery, (8) pregnancy or breastfeeding, (9) participation in a clinical trial within the previous month or current participation in any other clinical research study or clinical trial, (10) the detection of abnormal physical, hematology and biochemistry characteristics prior to randomization (at the investigator’s discretion), (11) usage of nonsteroidal anti-inflammatory drugs, (12) usage of oral contraceptives, (13) usage of platelet aggregation inhibitors, (14) usage of drugs that, from the investigators’ point of view, could cause dyspeptic symptoms, (15) alcoholism or psychological illness which could lead to noncompliance with the investigator’s instructions, and (16) known hypersensitivity to TM.Three separate visits were scheduled. In the baseline visit (16 ± 4 days prior to randomization), basic laboratory testing was performed, and the absence of macroscopic endoscopic findings was confirmed. Demographic and medical data were also collected in this visit, and subsequently, the included participants were randomized to receive either TM 300 mg BID or placebo BID for four weeks. Two additional visits occurred at 2 and 4 weeks, according to the following parameters:The primary efficacy endpoint of the study was relief of symptoms, which was evaluated by the Glasgow Dyspepsia Severity Scale (GDSS) , recordeA secondary efficacy endpoint was the effect of treatment on patient quality of life, estimated using the Gastrointestinal Quality of Life Index (GIQLI) , recorde1/2 and Initial Activity score were recorded. Patients were instructed to fast and refrain from smoking overnight before the study. All 16 patients received a radiolabeled meal  and a glass of water (250 mL) within 10 minutes); immediately afterwards, they were positioned at a γ-camera. One-min images were captured at 10-min intervals during the first hour and at 60, 120, 180 and 240 min with the patient standing. A computer-assisted method was used to determine the percentage of intragastric residual contents at 60, 120, 180, 240 min. The lag phase was determined either by a 5% drop in peak counts or by the first appearance of bowel activity. For the analyses, data were recorded as integrals (1–100) representing the percentage of activity retained in the stomach at various times. The duration of the lag phase, as well as the time required for the retained gastric activity to reach the 50% of the initial gastric activity, were estimated.This test was performed and evaluated by an expert investigator (I.T.). The lag phase, TThroughout the study, and for 48 hours after administration of the last drug dose, TM safety evaluation included the recording of any adverse events, as well as their severity, their effect on the study drug and their management. All patients with adverse events were followed until symptoms resolved.t-test, after evaluating for normality, was performed in the analysis, undertaken according to protocol, within groups to test the reported relief from dyspeptic symptoms, graded using the GDSS. Comparisons of mean and median scores for each treatment group and between treatment groups were conducted. Also, patients who showed marked improvement were evaluated to determine whether there was a difference between those receiving TM and those receiving placebo.Descriptive statistics were used for demographic and medical data. Data are presented as mean ± standard deviation (SD) and numbers and/or percentages for continuous and categorical variables, respectively. Improvement vs. nonimprovement for patients receiving TM was also related to a list of variables through a logistic regression model. A paired p < 0.05.A nonparametric Wilcoxon test was used to test median differences in the GDSS and GIQLI scores within each treatment group and between the two treatment groups. A secondary analysis tested whether the outcome measure was different in size between the participating countries using ANOVA, with the outcome measure as the primary response variable, the treatment as the explanatory factor and the country as the adjusting factor. A chi-square test with Yates correction was used to compare GDSS decrease proportion, complete symptom relief and the proportion of patients with adverse events, between the two treatment groups. Logistic regression analysis was used to correlate GDSS decrease with the demographic and medical data collected at the baseline visit, and also to analyze the two response variables for the quality of life results (more than 50% improvement in GIQLI scores and less than 50% improvement in GIQLI scores at the end of the study) where treatment, country and site were used as predictors. Logistic regression analysis was also used to analyze the proportion of recorded adverse events using treatment, severity, frequency, relation to medication and withdrawal due to treatment as predictors. Descriptive statistics analyses were used and confidence intervals were estimated for the reported differences in the estimated time for the retained gastric activity to reach 50% of the initial gastric activity using the 99 mTc-Tin Colloid Semi Solid Meal Scintigraphy test. Statistical significance was set at Two hundred and fifty-nine patients with FD were initially enrolled to participate in the study . A totalp-value = 0.81), height  or weight . With regard to patient medical history, 79% did not report any symptoms apart from dyspepsia, while 21% did. Patient examination proved normal for 98% of the patients. Sixty-five percent of the TM patients ate fried food, 50.5% ate salted food, 86.6% ate vegetables, 53.6% ate spicy food, whereas only 7.2% of patients had any food allergies. More than 25% occasionally consumed alcohol and 65% had never smoked, whereas 19.6% were regular smokers . The statistical difference remained robust, even after excluding patients who had GDSS equal to or less than 2 (complete symptom relief) .Symptom relief compared to the baseline visit was evaluated as the primary endpoint by means of differences in GDSS. A statistically significant reduction of GDSS in the TM group was revealed between the first (2-week) and final (4-week) visit (p = 0.036), and shortened the lag period, albeit at an insignificant level  10.50 and 89.50 min, TM group vs. 12.50 and 90.00 min, placebo group, respectively).Regarding 99mTc-Tin Colloid Semi Solid Meal Scintigraphy testing, as a secondary endpoint of the Greek pilot substudy, the following observations were made; TM significantly accelerated gastric emptying, i.e., occurring at 50 min .Additional results from this analysis include the following: More patients on TM showed improvement of symptoms and complete symptom relief in comparison to placebo, although the differences were not statistically significant. By constructing a logistic regression model with a list of independent variables for the patients on TM (dependent variable was the 3-point GDSS reduction), and separating patients into two groups (improvement vs. non improvement) , only alRegarding the safety of TM, of the 211 patients who were randomized in the study, 26 (12.3%) reported adverse events (AE). Of those 26 patients, 15 (57.7%) were in the placebo group and 11 (42.3%) in the TM group. Of the 108 patients who received TM, 11 presented AE, representing a percentage of 10.2%. Similarly, of the 103 patients who received placebo, 15 presented AE (14.6%). These two percentages did not display a statistically significant difference. AEs were classified as mild, moderate or severe. AE reported in the TM group were mostly of mild intensity . The placebo group included one AE case of severe headache and two cases of nausea. In only one of these cases, the drug was permanently discontinued whereas in remaining five AE cases, drug discontinuation was temporary . Gastric testing was well tolerated in all Greek participants studied.p < 0.0005). Specifically, when compared to pretreatment values, 17 patients exhibited accelerated gastric emptying, 2 showed no change, and 1 exhibited slower gastric emptying. The mean lag period after TM treatment was insignificantly slightly shorter than the lag period of controls (p > 0.05) and no significant difference was observed in the mean symptom score of patients with prolonged gastric emptying in the posttreatment period [In our prospective study, a potential benefit of TM was shown in a rather adequate patient sample following a randomized, placebo-controlled, double-blind design, which ensured the validity of the results. A primary efficacy outcome was achieved, with TM treatment being shown to be more effective in reducing GDSS than a placebo by the completion of the study. One of the two secondary efficacy outcomes, gastric emptying, as part of a smaller pilot substudy, was partly achieved, with TM significantly accelerating gastric emptying, i.e., within 50 min. Another study including patients with nonulcer dyspepsia, and probably with concomitant GERD disease, and healthy controls showed that an oral dose of 100 mg of TM three times daily for three weeks augmented gastric emptying rates with a concomitant decrease in lag period and less retention of food at 100 min  were recruited. The prevalence of FD was 80% for the IBS group. Half of the patients were treated with TM for three weeks and the other half with a placebo. A statistically significant difference in clinical recovery was noted between the groups (94.9% TM group vs. 20.5% placebo group) .Prokinetic agents have shown a beneficial effect in FD, as confirmed in a meta-analysis of all studies involving prokinetic drugs . FourteeIn a further meta-analysis (Bayesian network)  involvinStanghellini et al.  examinedHp-I has been as regarded as contributor in the pathogenesis, at least partly, of these overlapping entities [Hp-I is frequent in patients with GERD-IBS-FD and/or erosive esophagitis, and that Hp eradication, along with PPIs and/or TM regimens, offer improvement of HR-QOL, predominantly in patients treated with TM [Hp-induced mast cells are regarded as important effectors of the gut–brain axis that translate stress signals into the induction of variable neurotransmitters and proinflammatory mediators that might contribute to GI tract pathophysiology. Hp-stimulated chronic perceived stress results in decreased host defense and initiates intestinal inflammation through mast cell-dependent mechanisms, thereby signifying the activation of peripheral corticotropin-releasing factor receptors (CRF-Rs) and mast cells as significant mechanisms involved in stress-linked GI pathophysiology [2 appears to play an essential role in gastric hyperalgesia induced by stress which evokes mast cell degranulation; thus, CRF2 signaling could be a valuable therapeutic target for FD [There is emerging evidence that FD coexists as an overlapping syndrome with IBS and GERD. Such patients are characterized by a worse health-related quality of life (HR-QOL) score. Moreover, entities ,16. Our entities  that Hp- with TM . Mechaniysiology ,16. In tysiology . Specifit for FD . Likewist for FD .Regarding preclinical studies, TM was recently shown to be effective at maintaining upper and lower GI motor function in a peripheral CRF overlapping model with guinea pigs . It is wHp status as a potential contributor to the pathophysiology of the studied patients. The main reason for this was the obvious bias attributable to the high rate of false negative results due to the study’s design, which excluded patients with macroscopic findings from esophagus, stomach and duodenum during the endoscopy; endoscopic abnormal patterns such as “mosaic-like” appearance, “diffuse homogeneous redness” and/or “irregular redness with groove,” predict the presence of Hp-I infection with a sensitivity of 93.3 %, a specificity of 89.1 %, and an overall accuracy of 91.6 % in patients with any of the “abnormal” patterns [Hp-I eradication. Moreover, distinctions were not made among PDS, EPS and PDS–EPS overlap syndromes. Lastly, a meal scintigraphy test was performed as a pilot substudy in one center, involving a small number of participants. Thus, further, large-scale prospective studies are required to confirm our preliminary data regarding its potential objective value in estimating the effect of TM in FD patients.Despite its adequate number of participants, our study was also characterized by several limitations which necessitate further research; we have to acknowledge that the attrition rate was significant for both groups, being over 10%. Moreover, there was no available data in the literature regarding a validated cutoff value of GDSS. We therefore assumed that a decrease of at least three points in the GDSS would clinically reflect a reasonable level of relief of FD symptoms. Furthermore, we did not use the latest Rome IV criteria, since our study was initiated before the introduction of the new criteria. Moreover, the long-term efficacy of TM in FD treatment was not evaluated; as in previous studies, we were constrained to four-week period, noting the early onset of antispasmodic efficacy in this time frame . Our anapatterns . Since Tpatterns ,15, in tWithin this multicenter, randomized, double-blind, placebo-controlled, prospective study, a sufficient number of patients was recruited. It was demonstrated for the first time that TM monotherapy is an effective and safe option for the treatment of FD.Hp) and the relationship between symptoms and pathophysiologic mechanisms [All in all, this study might provide further insights into the treatment of FD, which is, at present, inadequate, despite the amount of knowledge gathered in recent years about the condition, in particular, with regards to its etiologic associations (e.g., the minor role of chanisms .Further large-scale randomized control trials are required to verify the safety and efficacy of TM, which might lead to the inclusion of this promising pharmaceutical agent in future guidelines.


Background: Concomitant chemo-radiation for pelvic cancers remains challenging to be delivered at full doses. We hypothesized that fewer delays in chemotherapy would occur if the sequence of radiotherapy would be reversed, starting with the boost volume followed by the elective nodal volume. We report the result of a Phase II randomized study for high risk prostate cancer.Patients and Method: The study was a double-blinded phase II randomized trial. Patients were eligible if they had non-metastatic high-risk prostate cancer. All patients received 2.5 years of hormonal therapy and 46.5 Gy in 25 fractions to the pelvic lymph nodes. Patients received a radiation boost to the prostate, either before or after whole pelvic irradiation. Concurrent (20 mg/m2) Docetaxel was given on the first day of radiotherapy and weekly thereafter for a total of eight treatments until predefined toxicity stopping rules.Results: Ninety patients were included and randomized. Four were ineligible for the analysis. In total, 42 patients were randomized to the standard sequence, 44 patients to the experimental sequence. There were statistically fewer GI or GU toxicities leading to a docetaxel dose reduction or omission in the experimental sequence compared to the standard sequence, 5 vs. 15 events (p = 0.027). There was no difference in overall survival, cause-specific survival, or biochemical-relapse free survival between the two sequences.Conclusions: This is the first study to test sequence inversion for pelvic radio-chemotherapy in a randomized double-blind trial. Less chemotherapy interruptions or dose reductions occurred by inverting the radiation sequence of the large field and the boost.Clinicaltrials.gov: NCT00452556The trial was registered with  Chemo-radiation is a standard treatment for several pelvic malignancies, including those arising from the cervix, rectum, and anal canal, and has also been tested for high risk prostate cancer –6. When In developed countries, prostate cancer is the most frequent non-cutaneous cancer in men . Twenty We hypothesized that there would be fewer dose reductions or delays in docetaxel chemotherapy if the sequence of radiotherapy would be reversed, starting with the boost volume followed by the large elective nodal volume. This is a proof of principle study of sequence inversion, using prostate cancer as an example. In this manuscript, we report the result of a phase II randomized study of concomitant chemoradiation in high risk prostate cancer patients comparing the standard sequence treating the large volume followed by the boost volume, with an experimental sequence delivering the boost dose before the loco-regional treatment. Outcomes include the number of dose reductions and/or delays in docetaxel, as a result of gastrointestinal or genitourinary toxicity, survival, and health-related quality of life measured by the Expanded Prostate Cancer Index Composite (EPIC) .Patients were eligible if they had a high risk prostate cancer, defined in the current trial either as untreated patients with a 2003 TNM clinical stage T2c, T3a, or T3b, or a Gleason score 8 to 10, or a PSA ≥ 20 μg/L but less than 50 μg/L , a life ClinicalTrials.gov.The study was a single center double-blinded phase II randomized design. Patients were approached for the study during the initial consultation by the study co-ordinator, and after informed consent was obtained, they were referred for medical oncology consultation. When deemed eligible for combined chemo-radiation treatment, patients were randomized in a 1:1 ratio to treatment sequence at the time of registration into the study. Randomization was performed by a computer algorithm, using SAS software version 9.4 , using the permuted block design, using block sizes of four and six patients. To ensure blinded assessment of toxicities, the attending radiation oncologist completed the delineation of the target volumes and organs at risk and approved the final plans for each phase. All subsequent quality assurance, including verification dosimetry and daily image guidance, was reviewed by an independent radiation oncologist involved in the study, but not in the patient's treatment nor the assessment of toxicity. The attending physician was responsible for assessing and scoring toxicity at the time of weekly review within the hospital and was blinded to patient treatment sequence. Prior to study initiation, approval was obtained from the institutional ethics review board, and the trial was registered with Patients underwent radiation simulation following institutional guidelines. In brief, patients were immobilized supine in a Vak-Loc  and CT simulation. A planning MRI was performed in the treatment position using a 1.5 T magnet without an endorectal coil. All patients, including those having had radical prostatectomy, had insertion of gold fiducial markers in the prostate or prostate bed. The clinical target volume (CTV) for the larger volume which included the pelvic nodes included the external and internal iliac vessels plus 7 mm except where vessels were in direct abutment to bone or muscle, where the CTV included only the vessel. The CTV for the larger volume included the nodes and either the prostate plus 3 mm, except at the prostate-rectal interface, or the prostate bed, from the bottom of the anastomosis, superiorly, to the inferior aspect of the proximal vas deferens. The CTV for the smaller boost volume included only the prostate bed, or the prostate plus any extraprostatic extension, if present. The planning target volume (PTV) was equal to the CTV plus 7 mm. The dose fractionation was intended to be biologically equivalent to 70 Gy in 35 fractions, with the elective nodal dose equivalent to 46 Gy in 23 fractions. All patients received 46.5 Gy in 25 fractions to the pelvic lymph nodes. Patients who had not had previous radical prostatectomy received a boost, either before, or after whole pelvic irradiation, to the prostate and to the seminal vesicles for T3b tumors to a dose of 26.78 Gy in 13 fractions. The boost dose was reduced to 20.6 Gy in 10 fractions, if patients had previous radical prostatectomy. There were no specified dose constraints to organs at risk. Plans were approved if 95% of the dose covered 98% of the PTV volume. All patients were treated with static port intensity-modulated radiotherapy (IMRT), using either five or seven ports, with six MV photons. All radiation plans were verified by Medical Physics prior to treatment as per institutional policy. Daily image guidance was performed with on-board kV imaging, matching to the fiducial markers.2 over 30 min. Chemotherapy was withheld for grade 3 or greater: diarrhea, thrombocytopenia, absolute neutrophil count (ANC) < 500× 109/L, febrile neutropenia with ANC < 1.0 × 109/L, nausea, and vomiting, stomatitis, grade 2 peripheral neuropathy, or abnormal liver function tests. When acceptable toxicity was reached, docetaxel was restarted at 16 mg/m2. If grade 3 toxicity recurred, or if docetaxel was delayed more than 2 weeks, docetaxel was discontinued.All patients received hormonal therapy with leuprolide acetate 45 mg subcutaneously every 6 months. This was given 4 months prior to starting concurrent chemoradiation and continued for two years post-treatment. In addition, patients received 4 weeks of daily bicalutamide 50 mg at the time of the first leuprolide acetate administration. Concurrent Docetaxel was given on the first day of IMRT (week 16 of protocol therapy) and weekly thereafter for a total of eight treatments at a dose of 20 mg/mThe primary endpoint of the study was the comparison between sequences of the patients' proportion experiencing Docetaxel dose reductions or omissions due to gastrointestinal or genitourinary toxicity. The number of dose reductions and/or delays was compared using Poisson regression. Secondary endpoints included the time to selected grade 2 and 3 NCI CTCAE version 3.0 toxicity, the difference in incidence of grade 2 and 3 toxicity, as well as the difference in overall bowel domain score of the EPIC at weeks 16, 20, and 24. Differences in rates of toxicity were compared using Poisson regression, when the modeling fit the data, and when the assumptions of Poisson regression were not met, the proportions of toxicity between sequences were calculated using the Chi-squared test. Differences in Overall survival and time to selected grades of toxicity were calculated using the Log Rank test, and differences in biochemical relapse-free survival and prostate cancer related mortality were calculated using competing risk proportional hazards modeling. The date of biochemical failure in the patients with no prior surgery occurred at the time the PSA reached a value of the PSA nadir +2, and the date of biochemical failure in the patients who had had a prior radical prostatectomy occurred at a PSA of 0.2. Statistical analyses were conducted using SAS software version 9.4 .2 weekly in all subsequently treated patients, and if more than 30% of patients subsequently experienced grade 3 toxicity in the next 10 patients treated after the dose reduction, the study would have been discontinued.Based on the Kumar et al.  study, wp = 0.027), using Poisson regression. Secondary study endpoints included the total amount of Docetaxel that can be delivered. In the standard sequence 78.6% of patients received 8 weeks of chemotherapy compared to 81.8% in the experimental sequence (p = 0.88), and 76.2% of patients did not require a docetaxel dose reduction compared to 77.2% in the experimental sequence (p = 0.88). Goodness of fit testing indicated that the Poisson regression fit the data well (p = 0.44) for the primary endpoint of the study.All patients received radiotherapy, as per protocol, without breaks or dose reductions and the vast majority were able to receive the full dose of chemotherapy, There were significantly fewer GI or GU toxicities leading to a docetaxel dose reduction or omission in the experimental sequence compared to the standard sequence, 5 vs. 15 events (p = 0.0109). The rates of Grade 2 or higher diarrhea corresponding to 3 or more bowel movements per day above baseline were similar with the standard sequence, 50%, compared to 52.2% in the experimental sequence (p = 0.83). Conversely, there were more combined grade 2 and 3 gastrointestinal toxicities in the experimental sequence, with a non-statistically significant trend to more proctitis in the experiment sequence, 22.7 vs. 11.9% (p = 0.186). Constipation was the only statistically significant individual item (p = 0.045). There was a statistically significant delay in time to grade 1 diarrhea in the experimental sequence (p = 0.04). There was also a non-statistically significant trend to more combined grade 2 and 3 urinary frequency corresponding to 2 times increase of the normal voiding frequency, 70.5 vs. 50% (p = 0.0525).Secondary endpoints also included individual GI and GU toxicities, selected ones of which are listed in It is important to note that bone marrow toxicity was infrequent in both sequences, with no patients experiencing febrile neutropenia see . One patp = 0.66), cause-specific survival (Gray's Test p = 0.47), or biochemical recurrence . For all the other domains, there was no difference between sequences in terms of score decline for the bowel, urinary, sexual, hormonal domains, nor in the AUA symptom scores, or Physical Component score.During the period of concurrent chemo-radiation (week 16–24 of protocol therapy) there was statistically significant declines in all domains of the EPIC, except on the Mental Component score of the SF12. For this domain there was a statistically decline in the standard sequence only using repeated measures analysis of variance  vs. 2.38% (one event in one patient) grade 3 urinary frequency, p = 0.18), but this excess was not sufficient to fit the pre-specified criteria to warrant chemotherapy dose reductions or delays, and resulted in 15 events resulting in dose reductions or delays in the standard sequence vs. five events in the experimental sequence, due to GU or GI toxicity. In essence, slightly more toxicity was seen in the experimental sequence, as a percentage of patient affected, but it didn't translate into more events that required reduction or omission of the systemic therapy, in the experiment arm. In fact, they had less events, and perhaps more bother, although this was not detected by bother as measured by the EPIC.This is the first proof of principle study, to test the inversion of the sequence of loco-regional radiation followed by the boost as the standard sequence or the reverse as the experimental sequence, in a randomized, double-blind trial. The study found that there were fewer dose-reductions or delays due to gastrointestinal or genitourinary toxicity when the radiotherapy started with the boost phase followed by the large loco-regional phase that includes the nodal volume. This could potentially enable more radio-sensitization of prostate cancer cells by docetaxel, and our study demonstrates that by inverting the order of the radiation sequences there was no detriment in cancer control to addressing only the grossly apparent tumor first. There was more grade 2 toxicity, mostly grade 2 urinary frequency, in the experimental sequence arm (70.4% vs. 50% combined grade 2 and 3 urinary frequency, Clearly, however, docetaxel is out of favor to be combined with radiotherapy in a concurrent fashion, and is not recommended.Similar sequence inversion of the boost and whole pelvic irradiation for concomitant chemo-radiation of prostate cancer with paclitaxel had been reported by Sanfilippo et al. , but thiThe strength in the present study is its randomized nature, blinding of participants and investigators, which limits potential bias. One other important aspect is that the incidence of grade 2 or higher bone marrow toxicity was less than 10%, without febrile neutropenia, likely owing to the weekly docetaxel regimen compared to docetaxel given every 3 weeks, when marrow suppression is more pronounced. Ideally, the optimal systemic agent would be radio-sensitizing only to tumor cells, would have a high degree of independent anti-tumor effect, and itself would not cause treatment-related diarrhea, or urinary toxicity.What is the meaning for other cancer sites? The same sequence inversion strategy could be tested for anal canal, cervix, or rectal cancer or used as option when patients present with significant co-morbidities presenting a challenge to protocol completion.In conclusion, our study demonstrates the proof of principle that sequence inversion of the large and boost volumes results in fewer dose deductions or delays in systemic therapy when there is overlapping normal tissue toxicity between the two. There was no detriment in cancer control to addressing only the grossly apparent tumor first. While the study will not turns the heels of radiotherapy on its head, it does provide scientific proof that in special circumstances it may offer an approach of how to optimize combined modality therapy, with radiotherapy and a systemic agent, when there is overlapping toxicity.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Nova Scotia Health Authority, Research Ethics Board. The patients/participants provided their written informed consent to participate in this study.DW contributed to conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing—original draft, and writing—review and editing. LW contributed to conceptualization, data curation, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization, and writing—review and editing. SC contributed to data curation, investigation, project administration, and writing—review and editing. RR contributed to conceptualization, data curation, investigation, methodology, project administration, visualization, and writing—review and editing. HH contributed to conceptualization, data curation, investigation, methodology, project administration, and writing—review and editing. DB contributed to data curation, investigation, project administration, and writing—review and editing. NP contributed to data curation, investigation, project administration, and writing—review and editing. CA contributed to conceptualization, data curation, funding acquisition, investigation, methodology, project administration, resources, supervision, and writing—review and editing. J-PP contributed to formal analysis, investigation, methodology, project administration, resources, supervision, visualization, writing—original draft, and writing—review and editing. All authors contributed to the article and approved the submitted version.DW reports grants and personal fees from Allergan, grants, and personal fees from AstraZeneca, grants, and personal fees from AbbVie, personal fees from Amgen, personal fees from Astellas, personal fees from Jansen, grants and personal fees from Paladin Labs, grants and personal fees from Sanofi, outside the submitted work. LW participates in advisory boards for BMS, Merck, Astellas, Pfizer, Novartis with no personal financial compensation. LW participates in research with Merck, AZ, BMS, Roche, Pfizer with money going to her institution. NP reports personal fees from Ferring Pharmaceuticals, personal fees from AbbVie Pharmaceuticals, outside the submitted work. CA reports personal fees from Allergan, grants and personal fees from AstraZeneca, grants and personal fees from AbbVie, grants and personal fees from Paladin Labs, grants and personal fees from Sanofi, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


One of the assets, assigned to the phytoplankton resting stages, is that of serving as the “memory” of the aquatic ecosystems and preserved biodiversity in the course of time. However, an accurate cyst identification proves to be a more difficult and extremely challenging process, even today. In order to gain a better taxonomic coverage of cyst assemblages in the Black Sea, an integrated approach of the classical morphological identification with metabarcoding methods (MySeq sequencing of V7-V9 regions of the 18S rDNA) was applied on thirteen surface sediment samples collected from different sites. A total number of 112 dinoflagellate taxa was detected at the species level and ascribed to 51 genera. In general, it is the molecular analysis that yields a higher number of taxa as compared to those obtained through the morphological taxonomy . Besides, it should be pointed out that the integrated dataset includes 14 potentially toxic dinoflagellate species. Discerned, subsequently, was a good dataset consistency for ten species, followed by some discrepancies as to a number of taxa, identified with one of the methods only, due to specific methodological biases. On the whole, it could be concluded that the combination of morphological and molecular methods is likely to increase the potential for a more reliable taxonomic assessment of phytoplankton diversity in marine sediments which, in turn, proves conclusively the utmost importance of the integrated approach. Biodiversity of phytoplankton as key primary producers is of utmost importance for the state and activity of the marine ecosystems  and the Gonyaulacales, Gymnodiniales or Peridiniales species share the same “simple” subspherical shape with no evidence of any processes and are characteried by the following common distinctive features - the size, the presence of mucous or the outline of the archeopyle, when visible. This is precisely the reason why the recent molecular methods have appeared to be gaining in popularity when studying microbial diversity in sediment samples, unlocking their potential for more sensitive detection and exact taxonomical identification  or Van-Veen Grab sampler, by a 10 x 10 cm frame at 13 stations located in different areas across the Black Sea during the period from May to June 2016  was selected from each subsample for the cyst analyses while another one (≈ 10 cm3) was concurrently oven-dried for one night at 70°C to calculate the water content. The wet aliquots were weighed and screened through a 10 µm mesh  using filtered natural (0.45 µm) seawater (Lingulodinium and Polykrikos) interspersed with zooplankton resting eggs. The material retained on to the 200 µm mesh was discarded. No chemicals were used to dissolve the sediment particles in order to preserve the calcareous cyst walls.An aliquot of homogenied sediment (from 2.00 to 2.20 cmseawater . The mathttps://www.marum.de/en/Modern_Dinocyst_Key.html). The identification was performed at the species level whenever possible and, as a rule, the modern taxonomic appellation was applied. For those taxa whose active stages are not determined, the paleontological nomenclature was adopted. Quantitative data are reported as cysts x g-1 of dry sediment (cysts g-1).A quali-quantitative analysis for cysts taxonomic identification was conducted under an inverted microscope (Zeiss Axiovert 200M), equipped with a Leica MC17o HD digital camera at 320-400x magnification. Next determined was the number of both the full (i.e. with cytoplasmic content) and the empty  cysts. As for the 10-20 µm and 20-75 µm fractions, at least 200 full cysts were counted in an attempt to obtain such values of abundance that are as homogeneous as possible and as a means to evaluate the rare species as well. From this perspective, a detailed and thorough analysis was performed on the > 75 µm fractions, while all the resting stage morphotypes were identified on the basis of published descriptions and the Modern Dinocyst Key Website  using the ISOIL DNA extraction kit . All DNA samples were used as templates for PCR amplification of the V7–9 hypervariable regions of the 18S-rRNA gene (amplicon length ~484 bp) using universal primers SSR-F1289-sn and F: TGGAGYGATHTGTCTGGTTDATTCCG; SSR-R1772-sn, R: TCACCTACGGAWACCTTGTTACG  and, next, only species that were present at all sites were selected and viewed as “shared”. The shared species datasets comprised between 17 and 57% of the species numbers per station identified by the morphological method and between 12 and 32% of those specified by the molecular approach. The percentage fraction of each “shared” species in the total cyst abundance was calculated and expressed in numerical value per habitat.SimScoreTreshold = 0.75)  .-1 for the full dinoflagellate cysts and between 66 (st. 9) and 5296 (st. 12) cysts g-1 for the empty dinocysts. On the whole, most species were unevenly spread. Scrippsiellaacuminata (Ehrenberg) Kretschmann, Elbrächter, Zinssmeister, S.Soehner, Kirsch, Kusber & Gottschling, 2015 and Scrippsiella sp. dominated by frequency  and abundance . Amongst the identified resting stages, five were assigned to potentially toxic dinoflagellates  Diesing, 1866, Lingulodiniumpolyedra (F.Stein) J.D.Dodge, 1989, Polykrikoshartmannii W.M.Zimmermann, 1930 and Protoceratiumreticulatum (Claparède & Lachmann) Bütschli, 1885). Nonetheless, the cysts of these harmful microalgae were too sporadic and occasional to be found in great abundance with the exception of L.polyedra, identified at 11 out of 13 stations and measured in high densities (up to 1722 full cysts g-1).From the total amount of 56 taxa, 51 were morphologically differentiated as full cysts and 33 as empty cysts, out of which 39  were identified at the species level. Considerable spatial variability in cysts abundance has been observed, ranging between 269 (st. 9) and 6963 (st. 1) cysts gCalciodinellumoperosum Deflandre, 1947 †, Calciperidiniumasymmetricum Versteegh, 1993 †, Follisdinellumsplendidum Versteegh, 1993 †, Melodomunculaberlinensis Versteegh, 1993 †, Posoniellatricarinelloides (G.Versteegh) Streng, Banasová, D.Reháková & H.Willems).The majority of cysts belong to the predominant Black Sea-specific phytoplankton species, except for those originally known as fossils only  Gert Hansen, 2000). It is worth noting that, amongst the most abundant OTUs found at all the stations, some HAB species  J.Larsen, 2000) were also preserved in the records. The selected OTUs, closely affiliated with potentially toxic dinoflagellates , were represented with just a few sequences and a single record, whereas the others, such as Gonyaulaxspinifera, Prorocentrumcordatum (Ostenfeld) J.D.Dodge, 1975 and Protoceratiumreticulatum, appeared more regularly with higher sequence numbers.The total number of dinoflagellate sequences in the dataset was 904,816 representing 36.4% of all sequences obtained by massively parallel sequencing (MPS). The sequences were clustered into 348 18S operational taxonomic units (OTUs) accounting for 15.5% of all the obtained OTUs. More than half of the dinoflagellate sequences (60.4%) could be assigned to references with high value of similarity varying from 100 to 98%. The number of OTUs that satisfied the taxonomic assignment criteria (> 0.980 BLAST top hit similarity) was 99 ranging by samples between 29 (st. 3) and 64 (st. 11). The number of sequences also fluctuated between samples with a minimum value of 18,871 (st. 8) and maximum approaching 87,693 (st. 7). More than 71% of OTUs (66 taxa) were determined at the genus level and 58% were identified at the species level (56 species)  were overlooked by the classical method, just as others  that were subject to morphological identification only . Most of them were represented by a single species, each accounting for less than 3% of the total species number and only 13 yielding a higher proportion. Species-level taxonomic compositions revealed a total number of 112 dinoflagellates  with 85 species that could be clearly distinguished and the remaining ones identified as sp.  were not present in the morphological results (K.veneficum is not known as a cyst producer). The potentially-toxic species detected by LM  were reflected in NGS data, but with a low degree of congruence.In total, 14 HAB dinoflagellate species were identified in the combined dataset, out of which five were accurately identified by the two methods. Apparently, the dominant species recorded with NGS  Montresor, Zingone & Marino, 1993, Polykrikoshartmannii and Protoceratiumreticulatum) have a similarity score above the threshold (SimScoreTreshold = 0.75); for example, 70% overall similarity between datasets for the coastal habitat , were slightly different Alexandriumminutum  Siano, 2012)), Prorocentrum , Scrippsiella/Peridinium (Scrippsiellahangoei (J.Schiller) J.Larsen, 1995 and Peridiniumaciculiferum Lemmermann, 1900) and Scrippsiella/Theleodinium  species, it could not be established in view of the inadequate set of recognisable identifiers in the target region. The 18S rRNA region is not useful for differentiating some species because of a low variability . A practical solution to such an issue might be achieved through filling the taxonomic gaps with integrated registration for both morphological and genetic information. The development of reliable databases with data from different regions will further facilitate the precise identification of OTUs. In the current study, the morphological dataset covered 13 Protoperidinium species (nine identified at the species level and four at the genus level), whereas the filtered NGS dataset, including only sequence with similarity ≥ 98% with the reference was related to only one species. Actually, there were more Protoperidinium species determined in the molecular data, but were excluded from any further analyses due to their low similarity (below the set threshold).Taking into consideration the advantages and drawbacks of the morphological and molecular methods and their appropriateness for biodiversity assessment, suggests that the two methods should be cross-checked if they are to ensure the accuracy of the dinocyst identification. To that effect, the integration and comparison of data derived by different approaches seem imperative, regardless of the subtle interpretation of the combined dataset. A critical issue in the comparative analyses is to use the same taxonomic concept in all attempted approaches as biodiversity can be measured at different taxonomic levels . Thus thfication . The resiability . Irrefuiability . Indeed,Alexandrium, though, discerned under a light microscopy, there were two species and seven noted as sp. On the other hand, the traditional identification technique via LM proved to be even more indispensable for the correct validation of the data obtained by metabarcoding. Yet, some identified morphospecies exist that could not be detected using the NGS analyses .Given the fact that the application of different methods increases the potential for substantial growth in biodiversity studies, both approaches can benefit from the integration of the information contained in the datasets generated by metabarcoding and morphology methods . Our resThe combined results broaden our knowledge on the dinoflagellate diversity in Black Sea sediments and highlight the importance of carrying out integrated investigations. Moreover, the presence of species, both typical and exceptional for the present-day plankton community, defines the role of sediments as seed banks where resting stages accumulate over time. Some cysts are ubiquitously distributed in Black Sea sediments, whereas others are irregularly scattered in patchy dispersion. Most notably, however, is that, unlike other studies , a separ12D7AE79-559E-5FAF-BA07-DD987511883610.3897/BDJ.8.e55172.suppl1Supplementary material 1Minimum and maximum abundance of the dinoflagellate species identified in the Black Sea sediment samples by morphological and molecular approachData typeTableFile: oo_433752.DOCXhttps://binary.pensoft.net/file/433752Dzhembekova, N., Rubino, F., Nagai, S., Zlateva, I., Slabakova, N., Ivanova, P., Slabakova, V., Moncheva, S.


The polymers of wheat glutenins are studied here using asymmetric flow field flow fractionation (A4F). Molecular mass (Mw), gyration radius (Rw), and the polydispersity index (PI) of polymers were measured over a four-year, multi-local wheat trial in France. The experiment, involving 11 locations and 192 cultivars, offered the opportunity to approach the genetic and environmental factors associated with the phenotypic values of the polymer characteristics. These characteristics, which were all highly influenced by environmental factors, exhibited low broad-sense heritability coefficients and were not influenced by grain protein content and grain hardness. The 31 alleles encoding the glutenin subunits explained only 17.1, 25.4, and 16.8% of the phenotypic values of Mw, Rw, and PI, respectively. The climatic data revealed that a 3.5 °C increase between locations of the daily average temperature, during the last month of the grain development, caused an increase of more than 189%, 242%, and 434% of the Mw, Rw, and PI, respectively. These findings have to be considered in regard to possible consequences of global warming and health concerns assigned to gluten. It is suggested that the molecular characteristics of glutenins be measured today, especially for research addressing non-celiac gluten sensitivity (NCGS). Triticum aestivum L.) and durum wheat (Triticum durum L.) are known to be largely influenced by the grain storage proteins (GSPs): gliadins and glutenins. Dough properties are influenced by the quantity of gliadins, which are monomeric and confer extensibility, whereas glutenins are polymeric and associated with dough strength and elasticity [The end-use quality of bread wheat  using size exclusion (SE) for molecular profiling has rapidly become the preferred tool for gluten sub-fraction analysis and quality characterization ,13. The for GPPs ,20. The for GPPs , the inffor GPPs , or the for GPPs . Using Afor GPPs . This alfor GPPs . This prfor GPPs . So far,for GPPs ). When gThe main A4F-MALLS’ monomeric and polymeric protein profiles are specifically investigated here for the three major glutenin polymer characteristics: average molecular mass (Mw), gyration radius (Rw), and polydispersity index (PI). The aim of the present publication is to estimate the effect of allelic diversity of HMW-GS and LMW-GS and of environmental factors, like temperature and rainfall in particular, during GSP accumulation on specifically the polymer characteristics using a large set of cultivars grown in experimental fields. Climatic records over four years for 11 experimental locations in France, where 192 cultivars have been grown, provided the data, allowing for estimation of the genetic and environmental influences on the variation of the three major polymer characteristics. Consequences of the findings, including some current questions raised regarding non-celiac gluten sensitivity (NCGS), will also be discussed.https://www.fsov.org). Of the 12 locations, the Clermont Ferrand location was used three times, in 2004 and 2005, for “Indice of Quality” and in 2009 for “Tenacity–Extensibility Bread Loaf Volume.” To avoid overinfluence of the central area of France, one trial of the Clermont Ferrand location was removed. The trial harvested in 2005 was withdrawn since unusual climatic conditions (very high temperature and severe drought) occurred in that location for the three last months of wheat crop, causing shrivelled grains for the majority of cultivars. For the present study, a total of 885 samples harvested on 11 locations were used. The 11 locations are given in 2) with two replicates and using conventional agronomic practices, with mineral fertilization and fungicide protection, to achieve high grain yields without any water irrigation. The average nitrogen supply was around 155 Kg ha−1 and varied according the residual soil nitrogen available in the trials locations to ensure optimum grain production. The daily climatic records over the three last months of the wheat cycle  provided by the closest climatic station of the experimental fields and have been registered. The sum of the temperatures and water precipitations averaged for May, June, and July each year are given in In the frame of collaborative research between INRA  and the majority of European private breeding companies operating in France, multi-local trials were carried out over four years: 2004–2006  and 2008–2010 (Program “Tenacity–Extensibility Bread Loaf Volume”). The institutes Unilasalle and Arvalis were also associated with this research, led by INRA and financed by the FSOV . Many biochemical, genetic, and technological parameters were measured on the collected samples: 780 samples (130 cultivars in six locations) and 240 samples , respectively, in these two programs. Main results and presentations given can be consulted (w/w), and then, the resulting sample (approx. 1000 g) was maintained for at least three weeks at 15 °C and 65% relative hygrometry for moisture homogenization between samples. Moisture and test weight (TW) of the grain were measured in 400 g of sample using a Multigrain TR-Dj apparatus . The thousand kernel weight (TKW) was determined according to the protocol AFNOR NF V03-702, now available under AFNOR NF EN ISO 520 [TM1093  with a 0.75-mm sieve. The whole meal was used for PC and grain hardness (GH) determination by near-infrared spectroscopy (NIRS), as described by the AACC methods 39-11 and 39-70, respectively [For each cultivar, the harvested grains of the two plots after cleaning were blended  data were collected from proteomics studies carried out by Tasleem-Tahir et al.  on develw/v) SDS and was filtered through a 0.1-µm membrane . The fractionation method used a constant detector flow of 1 mL/min and a gradient cross-flow. The method started with a focus time of 0.5 min at a flow rate of 2 mL/min. This step was followed by a focus/injection time of 1.0 min at 0.2 mL/min and a relaxation/focusing time of 0.5 min. Elution then followed at an outflow rate of 1.0 mL/min and with a cross-flow rate decreasing exponentially from 3.0 to 0.0 mL/min for 14 min. Finally, elution at a cross-flow rate of 0.0 mL/min was maintained for 9 min. The recorded MALLS and UV data were processed using Astra software . The Mw and radius were obtained by fitting the MALLS data using the Berry method, performing a second order fit to the data collected at scattering detectors 5–17.The principle of the separation technique and the wheat protein extraction protocol were previously described ,22. The i for slice i of the fractionated sample. iM is the weight-average molar mass for the solute. r2> is derived. Molar mass and radius can be obtained by MALLS combined with concentration measurements by applying the Rayleigh-Gans-Debye approximation  . The number-average mean square radius is determined as Equation (6). Lastly, the z-average root mean square radius Rz is derived from the mean square radii (Equation (7)).The average weight-average molar mass (Equation (2)) is derived by measuring ® software. The broad-sense heritability, 2H, values of the technological parameters and polymer characteristics were computed using the GLM of variance analysis, considering cultivars grown on several locations in a given year as the random variate. The heritability was computed as described in Equation (8), where 2H values reported in All statistical analyses , and partial least square regression (PLS)) were performed using Statgraphics−1 to 86.3 kg hL−1 for TW, evidencing that genetic and growing environmental factors had impacted the grain filling (p < 0.05) with Mw, Rw, and PI, respectively. The three characteristics were not associated to GH, TKW, and TW as well.The phenotypic values measured on the 885 samples  exhibited a wide range of variation for all traits measured. The grain PC was between 8.7% and 15.1% of dry matter (dm) and the GH was from very soft to very hard types of wheat. The range of variation was from 29.0 g to 64.9 g for TKW and from 64.0 kg hL filling . The higR2 percentages were found for the locations or for the years of experimental trial  could be obtained for each polymer characteristic (data not shown). Being of low heritability, the polymer characteristics can be employed, however, for phenotypic evaluation and cultivar ranking in the breeding program. The analysis of the alleles at the six glutenin loci, encoding HMW-GS and LMW-GS, offered the opportunity to better approach their genetic influence on the phenotypic variation of polymer characteristics.ANOVA, when computed with genotypes, locations, or years, revealed the maximum variance explained by each of these factors used as unique explanatory variate. As expected, genetic differences were prominent in GH variations, but the percentage for TW, although significant, was low. Of particular interest for the polymer characteristics, the part of phenotypic variance explained by genotypes are relatively high: 55.0%, 79.6%, and 61.5% for Mw, Rw, and PI, respectively. The phenotypic variance of each polymer characteristic was better explained by environmental factors; greater al trial . This hiThe PLS regression first evidenced that PC and GH had very low, but significant, influences on Mw and PI characteristics . AlthougThe standardized coefficient, described by the PLS regression, for the 14 HMW-GS alleles are shown in The standardized coefficient attributed to each of the explanatory variates clearly evidenced the low impact of the different glutenin alleles compared to the monthly averaged temperature of the last three months of wheat crop in the 11 locations for Mw, Rw, and PI  of the phenotypic variations of Rw, Mw, and PI, respectively. These regressions clearly evidence the influence of temperature in the experimental field trial during the last month of the wheat crop, which is highly associated to increasing the polymer characteristics. Other regressions performed using either the average temperature of May or of June or the average temperatures cumulated during the 50 days usually acquired from grain formation to maturity did not explain as much of the polymer characteristics as did the average temperature of the month of July. The vertical dots, corresponding to phenotypic values measured in each location, on As shown in 6 Da to 17.4 × 106 Da) by 189%, and the PI of the polymers (from 5.71 to 30.49) by 434%.The highly significant increasing polymer characteristics, associated with different average temperatures from the month of July, were computed from hourly (night and day) daily temperatures recorded in locations where the multilocal trials were implanted: three locations in 2004, two in 2005, three in 2009, and three in 2010. The difference between lowest sum of the daily average temperatures in July (546 °C) registered at La Chapelle d’Armentière (North of France) in 2004, and the highest average temperature (656.6 °C) registered at Clermont Ferrand  in 2010 was 110.6 °C. It corresponds to a difference of only 3.5 °C of the daily average temperature between the two experimental sites (17.6 °C versus 21.1 °C). When introduced in the fitted equations , this 3.Some approaches may provide possible explanations of the observed phenomenon. Many technological studies have reported the influence of experimentally monitored temperatures on wheat flour proteins—for example on albumins and globulins on gluten and on flour protein during extrusion cooking , like hydrogen peroxide, dehydroascorbate, and oxidized glutathione. Proteomics analyses of developing wheat endosperm at 21 stages led to the identification of eight PDIs, specifically present in the endoplasmic reticulum, where polymerization occurs: one PDI-2 precursor, three PDIs, and four PDI-likes . ExpressDuring the 15 last days of grain maturation, the average abundance of PDIs dropped to 55.8%. This decline was recorded for unstressed wheat grown in a controlled environment where air temperature close to the ears did not exceed 24.2 °C , far belProtein disulfide isomerase (PDI) is a multifunctional redox chaperone of the endoplasmic reticulum (ER) . PDI is 6 Da)—that is, more than 50 times the classic Mw, were recently measured on wheat grown in France where both the high temperature and drought were prevalent [In wheat stress-related proteins, characteristics of the UPR were found in both soft and hard near-isogenic lines, these stress-related proteins being more abundant in the hard ones . Althougrevalent . MoleculWhen wheat varieties are studied for human health, the amount of insoluble polymers is usually measured by SE-HPLC. This amount, often closely related to the grain PC, gives no measure of polymer Mw. These glutenin polymers are hydrophobic and heat-resistant. Their size is often further increased by oxidation resulting from intense kneading, and they are only partially hydrolyzed during the short fermentations generally practiced today for many baked products. Are these gluten proteins easily hydrolyzed if they are packed in a huge polymer mass?Numerous approaches were developed to reduce the gluten exposure for celiac and sensitive persons ,39, likeGlobal warming is observed on all continents where wheat is produced, from varieties adapted to each country and originating from different pedigrees. The high temperatures registered in France during our four experimental years are encountered in many other countries, as well. They cause an increase in the molecular weights of glutenin polymers, which appear to be a likely hypothesis behind non-celiac gluten sensitivity (NCGS). No glutenin epitope specific to this sensitivity has been revealed to date. Consequently, several alternative hypotheses are currently being investigated to explain NCGSs ,27. Howe