N = 172) who were diagnosed with functional constipation per Rome III criteria were randomized to four treatment groups: 10 g lactitol, 3.5 g psyllium, a combination of 10 g lactitol and 3.5 g psyllium, or placebo. The primary endpoint was change in BMF from Day 0 to 28 as compared to placebo. Secondary endpoints were assessed by inventories, including stool consistency, patient assessment of constipation symptoms and quality of life, relief of constipation, 24-h food recall, physical activity, product satisfaction and adverse events (AE). BMF increased by 3.0 BMs with lactitol, by 2.9 with psyllium, and by 3.1 with the combination, but was not different from placebo (3.7 BMs). Other clinical endpoints were similar between treatments. No serious AEs were reported. In conclusion, this study showed a similar effect on relief of constipation in all treatment groups. The treatments that were administered to the volunteers were well tolerated.Psyllium and lactitol have been reported to increase fecal volume, moisture content and bowel movement frequency (BMF). However, the benefits of their combined use on constipation has not been examined. The aim of this study was to evaluate the effects of a 4-week intervention with lactitol and/or psyllium on bowel function in constipated volunteers. Adults ( Constipation, functional constipation, and chronic constipation are often used interchangeably, although the latter includes conditions that are caused by diseases, such as neurogenic and drug-induced diseases . In genePer the Rome III diagnostic criteria for functional constipation in adults, the complaint is defined as unsatisfactory defecation that is characterized by infrequent stools (fewer than 3 defecations per week) and difficult stool passage, with the presence of at least 2 of the following symptoms for at least 25% of defecations: straining, a sense of difficulty passing stool, incomplete evacuation, hard/lumpy stools, prolonged time to stool, and the need for manual maneuvers to pass stool . FurtherThe multisymptomatic, heterogeneous nature of constipation is a major hurdle in the management of subjects with this condition . TreatmeBulking agents are generally considered the first-line therapy for constipation. These agents expand with water to increase stool bulk and enhance bowel movements. Adequate fluid intake is necessary for bulk-forming agents—a lack of water increases bloating and, paradoxically, can increase the incidence of bowel obstruction . Among bIn subjects who are unresponsive or intolerant to bulking agents, osmotic laxatives may be recommended . OsmoticLactitol, a synthetic disaccharide osmotic laxative, is produced by the hydrogenation of lactose. It increases osmotic pressure in the intestinal lumen, resulting in greater fecal volume and moisture content and the stimulation of peristalsis . The effThis prospective, randomized, 4-arm, double-blind, placebo-controlled, phase III intervention study was performed in 6 clinics throughout India . DuPont,The study volunteers were healthy adults (aged 25–65 years) with self-reported constipation (≤3 stools per week for the last 3 months and in the run-in period), attending local practitioners for bowel function related disorders. Other inclusion criteria were body mass index (BMI) between 18.5 (inclusive) and 29.9 (inclusive), at least 80% compliance during the run-in period, willingness to maintain a stable diet throughout the study, and the ability to comprehend the study, including the possible risks and side effects that are associated with both investigational products (IPs) and constipation-associated medications . Blood and urine tests for safety were performed at the screening visit, and volunteers without clinically significant findings were considered eligible.Volunteers were excluded if they had consumed probiotics, prebiotics, laxatives, fiber supplements, or other medications for constipation that could have affected the study outcome within 2 weeks prior to the screen and during the run-in period. Other exclusion criteria were diagnosed or suspected organic gastrointestinal disease  or severely impaired general health, history of alcohol or drug abuse, and participation in another clinical trial within 3 months prior to the screen. Volunteers who were planning major lifestyle changes , were pregnant, were planning a pregnancy or breastfeeding, or had given birth in the last 3 months were also excluded.Medications that excluded participation from the study included antibiotics and oral steroids (1 month prior to the start of the study) and current use of anticholinergic medications, pain medications that contained opiates or morphine, weight loss medications, misoprostol, 5-HT3 receptor antagonists, antacids with magnesium or aluminum, diarrhea medication, medication that accelerated emptying of the stomach, sulfasalazine, cholestyramine, cytostats, and biological medications or probiotics, because they could have affected the study outcomes. Iron, antidepressants, statins, thyroxine, coxibs, acid medications, inhaled steroids, and other non-excluding medications that would not be expected to affect the study outcomes in the clinician’s opinion were allowed if they had been consumed at least 30 days prior to the study on a stable dose; otherwise, volunteers who failed to meet the criteria were excluded. Volunteers who were unsuitable for the study for any other reason, as judged by the clinician, or had known allergies to any substance in the study product, were excluded.Eligible volunteers were enrolled in a 6-week intervention study, comprising a 2-week run-in period and a 4-week treatment period . A windoAt the end-of-study (EOS) visit on Day 28, volunteers returned empty and unused sachets of IPs for accountability. Rescue medications, including suppositories (bisacodyl or glycerin) and enemas, were permitted at the discretion of the investigator. Concomitant treatment for all groups—including iron, antidepressants, statins, thyroxine, coxibs, acid medications, inhaled steroids, and other non-excluding medications that did not affect the study outcomes in the clinicians’ opinions—was allowed if it had been consumed for at least 30 days on a stable dose. Diagnoses were made by the investigators, based on ROME III criteria for functional constipation in adults, during Visits 1 and 2 at the clinics.The primary outcome was the change in weekly BMF from baseline to the end of the 4-week treatment. BMF was recorded daily by the volunteers in a diary throughout the study.The secondary outcome of Stool Consistency (SC) was assessed using the Bristol Stool Form Scale , in whicThe IPs were powder sachets that contained 10 g lactitol, 3.5 g psyllium, a combination of 10 g lactitol and 3.5 g psyllium, and placebo. Sucrose, instant waxy maize starch, citric acid, trisodium citrate, carotene powder, and orange flavor were used as excipients and as placebo. The IPs were produced by Danisco USA Inc. The products complied with the microbial quality standards per the European Pharmacopoeia. All 4 study treatments were as similar in smell, taste, and appearance as possible. The formulations of the IPs are listed in At randomization , all ranNew Delhi, ECR/20/Inst/DL/2013/RR-16Lucknow, ECR/262/Inst/UP/2013/RR-16Jaipur, ECR/26/Inst/RJ/2013/RR-16Kolkata, ECR/373/Inst/WB/2013/RR-16New Delhi, ECR/329/Inst/DL/2013/RR-16Pune, ECR/54/Inst/Maha/2013/RR-16This study was conducted in full accordance with the Sixth Revision (2008) of the Declaration of Helsinki; the EMA Note for Guidance on Good Clinical Practice , also known as the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines for GCP; and the laws and regulations on clinical research in the participating countries. A notification was submitted to the national authority; i.e., the Drug Controller General of India (DCGI), before commencement of the trial on the 26 June 2014, as applicable according to local regulations. Written informed consent was obtained from each volunteer before any study-specific procedure or assessment was performed. The study was reviewed and approved by six local ethical committees of the research sites in India. The study was registered in the Clinical Trials Registry for India (WHO approved Registry) with the registration identification number CTRI/2015/02/005580. The EC approval numbers per research site are listed below:The trial hypothesis was to obtain a significant difference between the 3 treatments in parallel against placebo. Thus, the significance level for each comparison was assumed to be 0.05/3 = 0.0167. The sample size was based on the comparison of change in bowel movements per week from baseline to EOS between the 3 treatment groups and placebo. Based on the literatures, a standard deviation of 3–5 for bowel movements per week and an effect size of 2–4 more bowel movements per week after treatment were assumed ,40,41,42The intention-to-treat (ITT) population was defined as all randomized subjects. The per-protocol (PP) population was defined as those subjects in the ITT population who had completed the EOS visit and for whom there were no serious protocol deviations . Moreover, the PP population should have less than three Bowel Movements (BM) per week during the run-in period, based on a self-reporting participant’s diary, as defined during the Blind Data Review process.® for Windows version 9.3/Proc PLAN . The sealed envelopes containing randomization codes were sent directly to JSS Medical Research India Private Limited. Mechanism of concealment was to use 1280 random numbers between 0001-9999 for kits with prefix “K” . These numbers were randomly assigned to the subject numbers used over the study and merged with the output of the randomization for the subjects. This merged output of randomization was automatically written in an output text file which was read in to MS Word to construct a Randomization List. The subject number, associated treatment sequence and kit number were provided within each stratum in the Randomization List. The kit (number) linked to each subject and subject’s randomized treatment sequence contained the treatment randomized for the subject. All subjects and investigators were masked to the treatment assigned until completion of the trial to minimize any subject- or investigator-induced bias in the outcome measures. Blocked randomization was stratified in order to include both vegetarian and non-vegetarian subjects at equal ratio. Subjects were randomized 1:1:1:1 to one of the four treatment groups with the block size for 4. The randomization lists were prepared by Oy 4Pharma Ltd.  using a validated computer program, SASThe efficacy was analyzed in the ITT population. Missing values were imputed by the last-observation-carried-forward approach.n) of non-missing observations, mean, standard deviation, median, and minimum and maximum (range). For categorical data, the descriptive statistics were presented as the number of exposed patients and number (n) with percentage of observations in various categories of the variable, for which the percentage was based on the exposed patients. Unless otherwise stated, all statistical tests were two-sided hypothesis tests, performed at a 5% level of significance for the main effects, and all confidence intervals were two-sided 95% confidence intervals. Descriptive analyses also included graphical presentations of the data, wherever appropriate. The descriptive statistics for continuous variables was presented as number  was performed with treatment, population strata, and research center as factors and baseline as the covariate for treatment comparisons for continuous variables. Dunnett’s test was used for group-wise comparisons (treatments versus placebo). All tests were two-tailed, and the significance level was 0.05.® Version 9.4 package .All statistical analyses were performed at JSS Medical Research India Private Limited using SASAll randomized volunteers were defined as the safety population. Adverse events (AEs) were coded by system organ class (SOC) and their preferred terms using the Medical Dictionary for Drug Regulatory Affairs (MedDRA) (v. 19.0 or later). All AEs were listed and categorized as AEs before dosing and treatment-emergent adverse events (TEAEs), i.e., AEs with onset on the date of or after dosing. TEAEs were tabulated by indicating the number and percentage of volunteers and the number of events. The number of volunteers who experienced any adverse drug reaction, including any adverse drug reaction, was summarized for each treatment group. AEs were collected, evaluated, and tabulated in relation to study drug, seriousness, severity, action taken, outcome, SOC, and preferred term for each treatment group. Serious adverse events (SAEs) were summarized by SOC and their preferred term. The AEs that were related to severity were presented as number of occurrences and % for the respective group. The AEs and their relationship with the study product were presented as above or by severity. The SAEs that were related to seriousness were expressed as the number of occurrences and % for the respective group. Clinical laboratory parameters , vital signs , and physical examination findings were measured descriptively. AEs were recorded in a daily diary throughout the intervention.® Version 9.4 package .All statistical analyses were performed at JSS Medical Research India Private Limited using SASN = 32): self-reported constipation >3 stools per week , noncompliance with the study product and methods , deviations from the protocol , failure in the safety tests , and less than 80% compliance with the treatment during the run-in period . All volunteers attended all visits; the flowchart is presented in From April 20, 2015 to April 28, 2016, 204 volunteers were screened, out of which 172 were enrolled. Their disposition is shown in N = 80, 46.5%) and females . Although significant differences were observed in gender, height, and BMI compared with placebo, they were not considered to have had any influence on the clinical endpoints in the efficacy analysis. Overall, the demographics were comparable across the 4 treatment groups. Of 172 randomized volunteers, 39 reported a clinically relevant medical history but were evenly distributed across all treatment arms . Over 1%No statistically significant difference was observed in total consumed sachets between active treatment groups versus placebo . Overallp < 0.01, The primary endpoint was the mean change in BMF between pre-treatment and post-treatment compared with placebo. There was a significant increase in BMF from baseline to EOS in all 4 treatment groups, with all treatments effecting an improvement in constipation over the 4-week intervention (p = 0.02) rise in total physical activity was observed in the placebo group . This tendency was seen in the placebo group for walking activity .There was no significant increase in walking or moderate, vigorous, or total physical activity in the 3 active treatment groups from baseline to EOS. Notably, from baseline to EOS, a significant (Adequate relief of constipation (ARC) was measured at 2 site visits: baseline and EOS, with respect to a response of “ARC since last visit.” The proportion of volunteers with ARC was comparable between all treatment groups at baseline and EOS. Most volunteers (≥80%) without ARC during the run-in period reported ARC at EOS in all treatment groups.A higher proportion of volunteers in all 4 treatment groups were ‘satisfied’ with the product (47.6% to 67.5%), followed by ‘very satisfied’ (7.5% to 23.8%), ‘neutral’ (12.5% to 17%), ‘dissatisfied’ (8.5% to 12.5%), and ‘very dissatisfied’ (0% to 4.7%). On all subscales, the proportion of those who experienced product satisfaction was comparable between all active treatments and between active groups versus placebo. p = 0.046; liquid intake: 1061.5 ± 827.0 mL vs. 921.3 ± 666.5 mL, p = 0.04) (p = 0.02). Fiber intake did not change significantly in the treatment groups but trended toward a decline in the lactitol and combination groups . No significant change in total calories , carbohydrate (g), fat (g), protein (g), fiber (g), or liquid intake (mL) was observed from baseline to EOS in any treatment groups, except for a significant increase in fat intake and a decline in liquid intake from baseline to EOS in the placebo group  . In addip = 0.06) from baseline to EOS in the lactitol versus placebo group.The changes in total calories, macronutrients, and liquid intake were comparable between active treatments versus placebo. A trend was observed towards a decrease in fiber intake  volunteers : 6 AEs iThe most common TEAEs were gastrointestinal disorders; 4 TEAEs in 3 (1.7%) volunteers, which were abdominal pain; 2 TEAEs in 2 (1.2%) volunteers, and pain in the extremities; 2 TEAEs in 1 (0.6%) volunteer. Seven TEAEs in 5 (2.9%) volunteers were unrelated to the study drug, and 1 TEAE in 1 (2.3%) volunteer in the lactitol 10 g + psyllium 3.5 g group was possibly related. No specific action was deemed to have been needed against any AE. In this prospective, randomized, double-blind, placebo-controlled, parallel-group study, a statistically significant increase in BMF was noted in all 4 treatment groups from baseline to Days 7, 14, and 21 and from baseline to EOS (Day 28) . Fibers To rule out the possibility of effects from the ingredients in the placebo, we assessed BMF during the run-in phase. As seen in The Patient Assessment of Constipation Symptoms (PAC-SYM) and assessment of constipation Quality of Life (PAC-QoL) questionnaires are the best validated and most specific tools for measuring the quality of life of patients with constipation. These questionnaires were developed, based on the literature, clinical expert interviews, and patient interviews, to measure health-related quality of life in constipated patients . In our Additionally, no significant difference was observed in product satisfaction in any active treatment group compared with placebo in the ITT population, indicating no superior satisfaction with the active products versus placebo. A significant increase in fat intake and a decline in liquid intake were seen in the placebo group from baseline to Day 28 in the ITT population.Our study products were well tolerated by the study patients. It is reported that both lactitol and psyllium are accompanied by uncommon adverse reactions. Lactitol, like most sugar alcohols, causes cramping, flatulence, and diarrhea, because humans lack a suitable beta-galactosidase in the upper gastrointestinal (GI) tract, and most ingested lactitol reaches the large intestine . PsylliuA possible limitation of the study is that we failed to record the potential use of the traditional ayurvedic remedies for constipation relief. Another potential shortcoming is that rescue medication use was not defined, and therefore, this aspect was left to the judgement of the principal investigators. Overall, BMF increased similarly in all 4 treatment groups. The treatments that were administered to the subjects were well tolerated. All 4 treatments were safe, and no adverse reactions required further action. No serious AEs or TEAEs were reported. All laboratory findings and vital signs were comparable across all treatment groups.


The opioid epidemic in the United States is averaging over 100 deaths per day due to overdose. The effectiveness of opioids as pain treatments, and the drug-seeking behavior of opioid addicts, leads physicians in the United States to issue over 200 million opioid prescriptions every year. To better understand the biomedical profile of opioid-dependent patients, we analyzed information from electronic health records (EHR) including lab tests, vital signs, medical procedures, prescriptions, and other data from millions of patients to predict opioid substance dependence.We trained a machine learning model to classify patients by likelihood of having a diagnosis of substance dependence using EHR data from patients diagnosed with substance dependence, along with control patients with no history of substance-related conditions, matched by age, gender, and status of HIV, hepatitis C, and sickle cell disease. The top machine learning classifier using all features achieved a mean area under the receiver operating characteristic (AUROC) curve of ~ 92%, and analysis of the model uncovered associations between basic clinical factors and substance dependence. Additionally, diagnoses, prescriptions, and procedures prior to the diagnoses of substance dependence were analyzed to elucidate the clinical profile of substance-dependent patients, relative to controls.The predictive model may hold utility for identifying patients at risk of developing dependence, risk of overdose, and opioid-seeking patients that report other symptoms in their visits to the emergency room.The online version of this article (10.1186/s13040-019-0193-0) contains supplementary material, which is available to authorized users. In a highly visible report it was described how drug overdose deaths have substantially increased in the United States from 2010 to 2015 , examiniPrevious studies of biomedical variables predictive of opioid misuse and abuse have unraveled several salient factors, including chronic opioid prescriptions, history of psychiatric illness, non-opioid substance disorders, having a family member diagnosed with an opioid use disorder, the use of multiple pharmacies to fill prescriptions, having hepatitis C, and tobacco addiction –10. ThesFinding a clinically objective signature of opioid abuse would assist physicians in offering the proper treatment to those patients who attempt to hide their addiction for other clinical conditions. Such a signature will be a composite biomarker that can be detected by machine learning methods. EHR systems have proliferated in the past decade, and are increasingly used to perform predictive diagnosis with non-linear machine learning methods –14. EHR The MSMC EHR  data were organized into a de-identified collection. 42,825,357 diagnoses from the EHR were queried to find all patients with diagnoses belonging to the 304. * family of the International Classification of Diseases (ICD-9) codes , referring to various forms of substance dependence . PatientModified z-scores  were calThe distribution of lab tests and vital signs per case contains a large portion of cases with fewer than 20 lab tests and vital signs, while the rest of the lab tests and vital signs per case distribution forms an approximate bell curve Fig. a. To retUsing the lab tests and vital signs from the cases and matched controls, median effect sizes were calculated for all 95 lab tests and vital signs. The value of the lab test or vital sign from each case is used to calculate an effect size, then for each lab test or vital sign, the median of these effect sizes is taken. Medians were calculated because they are more robust to outliers. Mean effect sizes were also calculated to check for consistency of their direction. Additionally, we examined the values of the lab tests and vital signs in both cases and controls during the 100 days prior to the diagnosis of substance dependence. For comparing these profiles to the controls, we examined the 100 days prior to the mean day of their lab tests and vital signs. Finally, diagnoses, prescriptions, and procedures in the five years preceding the first diagnosis of substance dependence were compared to those found in the age matched controls using odds ratios.To examine opioid prescriptions in the MSMC-EHR, the percentages of patients with at least one or more opioid prescriptions were calculated along with the percentage of total opioid prescriptions. Additionally, the distribution of opioid prescriptions by patient was examined, and a Wilcoxon rank-sum test was applied to quantify the difference between the number of opioid prescriptions given to patients with an opioid dependence prior to the substance abuse diagnosis, and the number of opioid prescriptions given to patients who have at least one opioid prescription, but no history of opioid dependence.A Random Forest classifier was implemented with Scikit-learn  with 100In the experiments using imputation by the mean or the median, classification performance was measured for including all patients, only those with no less than 17 lab tests and vital signs, and only those with less than 17 lab tests and vital signs. Additionally, we ran a test case with patients that had ICD-9 code families in the range of 291–293 but did not have ICD-9 codes in the 304.* family. These 291–293 ICD codes denote alcohol- and drug-induced mental disorders and withdrawal. Because the data in these analyses had higher dimensionality, i.e., more lab tests and vital signs due to the retention of all patients, only 10 bootstraps of equal size were sampled from the matched controls, and 10-fold cross-validation was conducted on each bootstrap.The lab tests and vital signs during the 20 days prior to the first diagnosis of substance dependence, as well as 10 days before and 10 days after the first diagnosis, were used as the features to train the main set of classifiers. However, other classifiers were developed using only the diagnoses, prescriptions, and procedures during the 5 years prior to the first diagnosis of substance dependence. Furthermore, rather than predicting substance dependence status, we also constructed models to predict non-medical opioid poisoning events, i.e. overdose, denoted by ICD-9 codes 965.0, 965.00, 965.01, 965.02, 965.09, E850.0, E850.1, E850.2, using lab tests and vital signs during the 6 months prior to the event.From the 12,112 patient records within the MSMC-EHR that had at least one substance dependence diagnosis, 64.12% were males (7745) and 35.84% were females (4329). Out of these we retained 11,573 cases whose first diagnosis of substance dependence was made at 20 years of age or later. The mean age of these patients at their first substance dependence diagnosis was 45.6 years, with the youngest patient being 20 years old, and the oldest 89.4 . In terms of cases and their matched control populations, similar percentages had a prior non-methadone opioid prescription . However, for the patients that had a prior opioid prescription, the case patients had a mean of 13.07 prescriptions, and the controls had a mean of 4.25 prescriptions.Out of the 880,605 patients that have at least one prescription in the MSMC-EHR, 356,734 patients (40.51%) had at least one opioid prescription. Out of 45,392,334 total prescriptions, 2,029,008 prescriptions (4.47%) were for opioids. While 356,734 patients had at least one opioid prescription, 214,757 had at least two, 155,120 had at least three, 118,208 had at least four, 42,515 had at least five, and 5312 had at least fifty  and white blood cell (WBC) markers  Table . This can = 7797 case patients was achieved by the filtering steps described in the methods. Stratified 10-fold cross-validation was implemented to evaluate the performance of the classifiers.Lab tests and vital signs from the cases and matched controls were used to train various Random Forest classifiers. A bootstrapping method was used to match different sets of controls to equal size of the case population. The initial set of p-value. The mean AUROC was 0.619, with the confusion matrix showing correct identification of 11.6% of controls and 95.4% of cases.The initial Random Forest classifier achieved, across 100 bootstraps, each with 10 folds, AUROCs ranging from 0.813–0.875, with a mean of 0.846 , the results showed more variability. Without imputation, AUROCs ranged from 0.694–0.922, with a mean of 0.822 , backache , abdominal pain , chronic pain , chest pain , and others. Diagnoses underrepresented in the cases include those related with pregnancy, such as “supervision of other normal pregnancy,” “outcome of delivery, single liveborn,” and “post term pregnancy, delivered, with or without mention of antepartum condition.” This is related to the suggestion that pregnant patients are among the least likely to seek care from multiple institutions, while HIV and chronic pain patients are among the most likely to seek care from multiple institutions [Medical non-adherence, a condition where patients do not follow therapeutic recommendations, is overrepresented in the cases. This finding may support a socioeconomic difficulty in adhering to medical advice, or general apathy to medical treatment, or a refusal to take alternative medications that are not opioids, or a refusal for any psychiatric treatment. Lumbago, an older term for low back pain, is also overrepresented in the cases. Patients with lumbago are often treated with opioids, and may become addicted; or conversely, opioid users with correspondingly lower thresholds for pain may present to clinics or emergency departments with lumbago. Other pain-related diagnoses are also overrepresented in the cases. These include limb pain . Compared to the substance-dependent cases, our control cohort showed the same pattern as prior studies of trauma patients. In addition to classification using clinical measures, we attempted to classify patients with, as well as examined the prevalence of, diagnoses, prescriptions, and procedures in the case and control populations during the five years before diagnosis of substance dependence. The diagnoses most overrepresented were psychiatric, supporting the close association between substance abuse and psychiatric comorbidities as reported before . AgreeinThrough analyzing of the health records of hundreds of thousands individuals in the MSMC-EHR with a machine learning framework, we furthered characterized opioid dependent patients using physiological measurements. We found that opioid dependent patients have significantly higher WBC and respiratory disturbances. Opioid dependent patients are also commonly malnourished which is characterized by low RCDW and blood albumin compared to controls. Clinical phenotyping analysis discovered that opioid dependent patients are more likely to suffer from psychiatric disorders and manifest pain-related symptoms. The predictive model may hold utility for identifying patients at risk of developing dependence, risk of overdose, and opioid-seeking patients that report other symptoms in their visits to the emergency room. It should be noted that marking a patient with an opioid dependency ICD code, which is commonly used for insurance purposes, is often inaccurate and inconclusive. Hence, we recommend that the results from our study should be considered preliminary, and the quality of the real cases disputable. The study should be validated by other independent EHR systems and different computational approaches. Regardless, the multi-variate non-linear characteristic of the classifiers developed here, combine unique mixture of the values of many measured variables together to produce predictions not possible by looking at a single biomarker. The complex relationships between measured variables would be difficult to detect via an in-person clinical assessment alone. Hence, the predictive machine learning classifiers we developed can alert physicians about the potential of patients to have opioid dependency from routine lab tests and vital signs. However, there are still technical, administrative, and bureaucratic barriers for real implementation.Additional file 1:Figure S1. Flowchart illustrating the steps of the creating the case and control populations. (PDF 131 kb)Additional file 2:Figure S2. Pain score ratings for cases (purple) and controls (gray) during the 100 days prior to diagnosis of substance dependence. For the controls, the 100 days are from prior to their mean day of analyzed lab tests and vital signs. The lines represent a moving average. (PDF 104 kb)Additional file 3:Figure S3. Receiver operating characteristic curves, normalized, and non-normalized confusion matrices for classifiers using all features , the top 10 by Gini importance , and the top 10 by p-value from the Wilcoxon rank-sum test . (PDF 419 kb)Additional file 4:Figure S4. Receiver operating characteristic curves, normalized, and non-normalized confusion matrices for a classifier using 10 sets of 10 random features (1/10 are shown) , and a dummy classifier outputting random predictions . (PDF 44 kb)Additional file 5:Figure S5. Raw values for cases and matched controls of the top 9 lab tests and vital signs by mean Gini importance. (PDF 174 kb)Additional file 6:Figure S6. Receiver operating characteristic curves, normalized, and non-normalized confusion matrices for classifiers using no imputation , imputation by the mean , and imputation by the median . (PDF 602 kb)Additional file 7:Figure S7. Receiver operating characteristic curves, normalized, and non-normalized confusion matrices for labs and vitals from the 20 days prior to substance dependence diagnosis, classified using no imputation , imputation by the mean , and imputation by the median . (PDF 1335 kb)Additional file 8:Figure S8. Precision-recall curves for labs and vitals from the 20 days prior to substance dependence diagnosis, using classifiers with no imputation (A), imputation by the mean (B), imputation by the median (C). (PDF 199 kb)Additional file 9:Figure S9. Receiver operating characteristic curves, normalized, and non-normalized confusion matrices for diagnoses, prescriptions, and procedures from the 5 years prior to substance dependence diagnosis, classified using no imputation , imputation by the mean , and imputation by the median . (PDF 596 kb)Additional file 10:Figure S10. Precision-recall curves for classifiers using diagnoses, prescriptions, and procedures from the 5 years to substance dependence diagnosis, with no imputation (A), imputation by the mean (B), imputation by the median (C). (PDF 212 kb)Additional file 11:Figure S11. Receiver operating characteristic curves, normalized, and non-normalized confusion matrices for lab tests and vital signs from the 6 months prior to opioid poisoning diagnosis, classified using no imputation , imputation by the mean , and imputation by the median . (PDF 597 kb)


The ability to accurately measure multiple proteins simultaneously in a single assay has the potential to markedly improve the efficiency of clinical tests composed of multiple biomarkers. We investigated the diagnostic accuracy of the two multiplex protein array platforms for detecting a bladder-cancer-associated diagnostic signature in samples from a cohort of 80 subjects (40 with bladder cancer). Banked urine samples collected from Kyoto and Nara Universities were compared to histologically determined bladder cancer. The concentrations of the 10 proteins  were monitored using two prototype multiplex array platforms and an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s technical specifications. The range for detecting each biomarker was improved in the multiplex assays, even though the lower limit of quantification (LLOQ) was typically lower in the commercial ELISA kits. The area under the receiver operating characteristics (AUROC) of the prototype multiplex assays was reported to be 0.97 for the multiplex bead-based immunoassay (MBA) and 0.86 for the multiplex electrochemoluminescent assay (MEA). The sensitivities and specificities for MBA were 0.93 and 0.95, respectively, and for MEA were 0.85 and 0.80, respectively. Accuracy, positive predictive values (PPV), and negative predictive values (NPV) for MBA were 0.94, 0.95, and 0.93, respectively, and for MEA were 0.83, 0.81, and 0.84, respectively. Based on these encouraging preliminary data, we believe that a multiplex protein array is a viable platform that can be utilized as an efficient and highly accurate tool to quantitate multiple proteins within biologic specimens. While there continues to be increasing reports of biomarker discovery and early validation studies being published, few new biomarkers have entered clinical practice over the past 30 years. There are many reasons for this, but one of the overwhelming reasons has been the reliance on single biomarkers for the evaluation of cancers that we know can have a broad range of molecular changes. When coupling the tumor’s heterogeneity with the observed variation, even between individuals with similar tumors, it is no wonder very few accurate biomarkers have progressed to clinical relevance. Thus, a shift has occurred to molecular signatures comprised of multiple biomarkers being favored over single biomarkers. The advent of advanced molecular profiling techniques has enabled the derivation of molecular signatures that can more accurately diagnose cancer and make individualized patient evaluation and care feasible. Several molecular signature assays  are now being incorporated into clinical practice ,2,3. HowThe objective of the current pilot study was to test the ability of two customized prototype multiplex array platforms to accurately and simultaneously monitor 10 urinary protein biomarkers that comprise our bladder-cancer-associated diagnostic signature; A1AT, APOE, ANG, CA9, IL8, MMP9, MMP10, PAI1, SDC1, and VEGF . The levp = 0.004) and CA9 . Furthermore, the combination of all ten biomarkers using optimal cutoff values defined by Youden index calculations resulted in an AUROC (p = 0.003). The sensitivities and specificities for MBA were 0.93 and 0.95, respectively, and for MEA were 0.85 and 0.80. Accuracy, positive predictive values (PPV), and negative predictive values (NPV) for MBA were 0.94, 0.95, and 0.93, respectively, and for MEA were 0.83, 0.81, and 0.84, respectively.In order to test the diagnostic accuracy of the two multiplex array platforms, 80 urine samples (cohort #2) were monitored for the 10 biomarkers, using both MEA and MBA. The subjects’ demographics and disease characteristics are summarized in an AUROC  of 97% fThere is a growing demand to incorporate molecular signatures into single assays in order to obtain favorable assay properties, such as reduced sample volume, decreased processing time, lower cost of analysis, and lower reagent consumption. Several multiplex protein measurement services are already available , and several studies have reported that multiplex ELISA procedures appear suitable and reliable for tissue lysate and serum analyses. The overall approach and goals of protein multiplexing are straightforward—a rapid, cost effective, and reliable detection assay.n = 127; 64 with bladder cancer) [Previously, in order to identify a novel, accurate bladder-cancer-associated diagnostic signature, we applied two complementary techniques: gene expression array of shed urothelia in voided urine ,10 and g cancer)  and subs cancer) ,15. Fina cancer) . TherefoIn the current study, we reported the lower and upper limits of quantification and coefficient of variation between commercial ELISA kits and two multiplex array platforms (MEA and MBA), in addition to reporting the diagnostic performance of the two multiplex array platforms in detecting bladder cancer diagnostic signatures. Firstly, we must stress that all assays are for research use only and have not been optimized for urine sampling or for clinical purposes. The multiplex assays could quantify biomarkers over a wider range than commercial ELISA kits, but commercial ELISA kits typically had a lower level of quantification. Variability, as evidenced by CV, was present in all assays. Briefly, the variability of the ELISA and other methods of chemical assays that produce continuous-type values is summarized by CV, which is defined as the SD divided by the mean, with the result often reported as a percentage . The maiThe most encouraging aspect of the current study is the diagnostic accuracy of the multiplex array platforms. Urinary PAI1 was the most accurate single biomarker in both MEA and MBA, with an AUROC of 0.67 in MEA and 0.85 in MBA (95% CI 0.06–0.30). Next, in the MBA, CA9 was the most accurate, with an AUROC of 0.80, while IL8 with an AUROC of 0.60 was the next most accurate biomarker in the MEA. Thus, the difference between the MBA and MEA assays were due to the ability of the platforms to more accurately detect concentration of these analtyes. As we previously reported, when all 10 biomarkers are combined we see the best accuracy, with an AUROC of 0.86 in MEA and 0.97 in MBA. Thus, the bladder cancer signature comprised of the 10 biomarkers achieved greater diagnostic power than any biomarker alone and has the potential to consider the heterogeneity of bladder tumors.Though the sensitivity and specificity achieved by the described multiplex array platforms far exceed those achieved by voided urine cytology (32% sensitivity and 94% specificity) or single biomarker tests for bladder cancer detection , we feelClinically, accurate non-invasive bladder cancer tests would have a clear impact on the clinical management of patients with bladder cancer. Therefore, our goal is to be able to detect bladder cancer in a swifter and more expedient manner , so that the patient can expect an improved survival and perhaps improved quality of life. Therefore, to successfully translate a diagnostic test from the basic science laboratory to the clinic, the diagnostic needs to be cost-effective, as well as accurate, especially if it is to be used over a long period of follow-up, as in the case for bladder cancer. The detection of urinary proteins through multiplex array platforms has the potential to be relatively simple to perform, interpret, and is affordable enough to meet this requirement. With that said, the multiplex bead-based immunoassay (MBA) is currently being applied to quantitatively detect and monitor a bladder-cancer-associated diagnostic signature in three large multicenter, international prospective clinical trials .n = 34; 6 with bladder cancer) from University of Hawaii Cancer Center (UHCC) to assess the lower level of quantification (LLOQ), upper level of quantification (ULOQ), and coefficient of variation (CV); and cohort #2  from Kyoto University and Nara Medical University to assess diagnostic accuracy of the multiplex array platforms. For the bladder cancer cases, histological confirmation of urothelial carcinoma, including grade and stage, was defined from excised tissue.Ethical review of the study was performed by Western Institutional Review Board. Banked voided urine samples were made available for analysis. Urine samples were collected prior to any instrumentation, centrifuged to remove cellular pellets, and stored frozen until analysis, as previously reported ,13,14,15Urine samples were monitored for protein content by using a Pierce 660-nm protein assay kit , while the concentration of urinary creatinine was measured using an enzymatic assay  according to the products’ manufacturer instructions. Laboratory personnel were blinded to final diagnoses.Levels of A1AT , apolipoprotein E , angiogenin , carbonic anhydrase 9 , interleukin 8 , matrix metalloproteinase 9 , matrix metalloproteinase 10 , plasminogen activator inhibitor 1 , syndecan , and vascular endothelial growth factor  were monitored in voided urine samples using commercial enzyme-linked immunosorbent assays (ELISA), as previously reported [® SQ 120 (MSD) instrument, while the MBA plates were read on the Luminex 200. As per above, calibration curves were generated along with optimal fit in conjunction with Akaike’s information criteria (AIC) values [The concentrations of the 10 proteins  were monitored using two prototype multiplex array platforms; multiplex electrochemoluminescent assay (MEA) from Meso Scale Discovery  and multiplex bead-based assay (MBA) from R&D Systems Inc.  for Luminex 200. Final monoclonal antibody pairs (capture and detection) for the multiplex assays were selected based on sensitivity, specificity, physical properties, and recognition of native protein, as described previously . In orde) values ,20.p < 0.05 and all reported p values were 2-sided. All analyses were performed using SAS software version 9.3 .Descriptive statistics were used to report the LLOQ and ULOQ, as well as CV in cohort #1. In cohort #2, Wilcoxon rank sum tests were used to determine the association between each biomarker and bladder cancer. We investigated the diagnostic performance of the protein biomarkers for bladder cancer (BCa) detection using the logistic regression analysis with BCa status (yes vs. no) as the response variable and 10 biomarkers as the explanatory variables. The individual biomarkers were combined into a linear combination, with the regression coefficients obtained in logistic regression as the weights, and the linear combination used as a combined score for the detection of BCa. Using cutoff thresholds previously reported ,15,19,20Based on these encouraging early results, we believe that the multiplex array platform is a feasible avenue to exploit as a simple, yet accurate tool to quantitate the amount of 10 diagnostic proteins in voided urine samples. At this stage in assay development, the detection range, variability, and accuracy are acceptable, and thus the method warrants further development. Furthermore, the prescribed platform is already in many clinical laboratories, thus facilitating clinical uptake of a new robust assay.


Re-irradiation after radiotherapy is a common treatment for locally recurrent esophageal cancer. However, the side effects of re-irradiation are serious. The most serious adverse reactions of re-irradiation include esophageal perforation and hemorrhage caused by esophageal perforation. Studies have shown that pulsed low-dose rate radiotherapy (PLDR) induces a hypersensitivity effect on tumor tissue and a hyper-repair effect on normal tissue, which can simultaneously reduce damage on the normal tissue and increase the therapeutic effect on the tumor. The objective of this study is to explore whether PLDR can reduce rate of esophageal perforation and improve efficacy in patients with recurrent esophageal squamous cell carcinoma (ESCC) after radiotherapy.This study is a prospective, multi-center, open, single-arm clinical trial designed to enroll 27 patients with locally recurrent ESCC after radiotherapy with or without chemotherapy. Re-irradiation will be performed using intensity modulated radiation therapy in 50 Gy/25 fractions. The strategy of PLDR includes dividing 2 Gy into 10 fractions, and administering each irradiating dose of 20 cGy at an interval of 3 minutes before the next low-dose irradiation. The actual dose rate of administration each time will be 16.67 cGy /minute. The primary endpoint in this study is the rate of esophageal perforation. The secondary endpoints are the objective remission rate, the palliative effect on quality of life and pain, and the time of disease progression. The observation time is 2 years after the end of the study.Clinical trial number: ChiCTR1900020609. In China, EC ranks third in incidence and mortality, and more than 90% of the EC cases are reported with ESCC.–4 Concurrent chemoradiotherapy is the standard treatment for locally advanced ESCC in patients who are resistant to or unsuitable for surgical treatment. About 50% of the ESCC cases are reported to recur locally within 3 years after chemoradiotherapy, and most recurrences occur in the target area of gross tumor volume (GTV).,6 Most patients with recurrence die within 1 year without treatment. At present, there is no standard treatment for local recurrence after radiotherapy. Common treatment methods include surgery, re-irradiation, chemotherapy, and supportive treatment. The proportion of patients receiving esophagectomy after recurrence is very small. According to data from Anderson Cancer Center, definitive chemoradiation failed in 184 of the 276 patients treated, while only 23 (8%) underwent esophagectomy. In general, re-irradiation is superior to other treatments in patients who cannot be treated with esophagectomy.–11 Re-irradiation is limited by the tolerance dose of normal tissues in the lungs, spinal cord, and esophagus and hence, increasing the target dosage is difficult. Re-irradiation can increase the risk of adverse reactions, such as esophageal perforation, hemorrhage, and radiation pneumonia. The most serious adverse reaction is esophageal perforation with an incidence of approximately 30%.,12Esophageal cancer (EC) is a common malignant tumor, and ranks seventh in incidence and sixth in mortality worldwide. According to 2018 statistics data, there have been 572,000 new cases of esophageal cancer and 509,000 deaths. hypersensitivity (HRS) is observed in tumor cells exposed to <0.2 Gy low dose rate irradiation with a steeper slope of survival curve (αs) than that of higher dose irradiation(αr). When the dose reaches 0.2 to 0.8 Gy, radiosensitivity began to shift from sensitivity to irradiation resistance (IRR). An interval between each pulse exists during pulsed low-dose rate radiotherapy (PLDR). Sufficient interval time can promote repair of normal tissues and reduce side effects on normal tissues thereby increasing the therapeutic effect on tumors and reducing damage on normal tissues.Low-dose rate radiation is considered effective to alleviate the side effects of radiotherapy. According to the classical cell survival curve of hypersensitivity Fig. ,[13] hyp The main reason for this is that PLDR causes less vascular damage, and retaining vascular network can improve tumor oxygenation. Normal tissue can be repaired during the treatment interval. Clinical studies of recurrent breast cancer, head and neck cancer, and glioma after radiotherapy have further demonstrated the effectiveness and safety of PLDR re-radiotherapy, and PLDR shows an increase in the ability to protect normal tissues.–18 However, clinical studies of PLDR in patients with ESCC do not exist. Based on the effects of low dose radiosensitivity and low dose hyperrepair, we designed this study to explore whether intensity modulated radiation therapy (IMRT)-PLDR can reduce rate of esophageal perforation and improve efficacy in patients with recurrent esophageal squamous cell carcinoma (ESCC) after radiotherapy.Researchers found that PLDR was more effective and exhibits lower toxicity to normal tissues than conventional radiotherapy in mouse models.22.1This study is a prospective, multi-center, open, single-arm phase II clinical trial designed to enroll 27 patients with locally recurrent ESCC after radiotherapy with or without chemotherapy •Palliative effect on quality of life and pain•Time of disease progression (TTP)2.31.Age, 18 to 75 years;2.Pathological confirmation of ESCC;3.Local recurrence and/or lymph node metastasis in the irradiation field of first radiotherapy and life expectancy of >3 months;4.No distant metastasis;5.Eastern Cooperative Oncology Group (ECOG) score of 0 to 2, which measures the patient's physical condition;6.Patients who cannot tolerate surgery or the tumor cannot be removed surgically;7.The patient is conscious and capable of coordination with positioning and treatment;8.Availability of previous radiotherapy information, including total dose in the target area, segmentation mode, dose distribution, irradiation dose to normal tissue and isodose curve, can be obtained;9.The patient has a measurable solid tumor ; Women of childbearing age must be in a non-pregnancy state (pregnancy test negative), menopausal women with menopause for >12 months and considered infertile, women of childbearing age who have taken effective contraceptive measures to prevent pregnancy during the study period and 3 months after the end of the study;10.9/L; Platelet (PLT) count – 75 × 109 or higher/L;Absolute neutrophil count (ANC) > 1.0 × 1011.Patients must sign a written informed consent and be willing to participate in and complete the study and follow up.2.41.patients with other malignant tumors;2.patients who received radiotherapy for the target area within 4 weeks before entering this study;3.patients with previous history of ataxia due to telangiectasia or other radiosensitivity reactions;4.patients with scleroderma or active connective tissue disease;5.patients with serious and uncontrollable infectious diseases;6.Patients with severe mental or psychological disorders, central nervous system disorders, cardiovascular and cerebrovascular complications, epilepsy and other diseases which may limit their understanding, implementation, and treatment compliance of informed consent.2.5Criteria that will lead to the drop-out of a patient before completion of this study may be treatments for medical reasons or impairment that will result in an interruption or early completion of treatment, including:1.Patient safety events;2.Any other physical condition that researchers suspect to increase the risk of radiation-related toxicity;3.Subjects voluntarily withdraw informed consent and request withdrawal from the clinical trial;4.Disease progression with medical imaging evidence  or clinical progress judged by researchers (the cause of progress needs to be recorded in detail);5.Pregnancy events during the study;6.Other researchers consider it necessary to withdraw from the study ;7.Patient participated in other clinical studies during this study;8.Inadequate patient compliance.2.6Radiotherapy will be performed using IMRT in 50 Gy /25 fractions. The implementation strategy of PLDR include dividing 2 Gy into 10 fractions, and administering each irradiating dose of 20 cGy at an interval of 3 minutes before the next low-dose irradiation. The actual dose rate of administration each time will be 16.67 cGy/minutes  found in known generally tumor. GTV includes primary tumor (GTV-t) and enlarged metastatic lymph nodes (GTV-n). The definition of GTV-n is any of the following: the measured value of short diameter >1 cm during the diagnosis of CT scan or less than 1 cm, but met one of the following criteria: PET scan showed high metabolic absorption; lymph nodes fused or clustered; short diameter of tracheoesophageal groove lymph nodes >0.5 cm. In this study, all the enrolled patients were not exposed to preventive irradiation, and there was no need to delineate the clinical tumor volume (CTV). The planned tumor volume (PTV) includes the positioning error due to tissue and organ movement, and PTV= GTV + 0.5 cm. Actual output data can be determined from the quality control data of each center.2.7A case report by researchers will accompany each case. Mianyang Central Hospital will be responsible for establishing the database and data entry, and will use a data acquisition system and an electronic data management system to manage data. Main researchers will audit the data and will lock it. The final version of the statistical plan will be completed and will be used to analyze the locked data. When the month and the year of treatment are clearly available, but the day is unknown, the date will be recorded as the fifteenth day of the month. When the year is clear, but the day and the month are unknown, the date will be recorded as the intermediate point between the end of the year and the last known date.2.8Rate of esophageal perforation is the primary endpoint in this study, which will be observed from the initiation of re-irradiation to 60 days after the end of the study. Esophageal perforation should be diagnosed by esophageoscope. ORR is one of the secondary endpoints in our study. Ninety days after the end of treatment, we will evaluate the efficacy by using the RESICT 1.1 criterion through CT scan and esophageoscope. Quality of life scale QLQ-C30 (V3.0) according to the European Organization for Cancer Research and Treatment (EORTC) and digital pain scale will be assessed to determine the palliative effect on quality of life and pain in 2 years after the end of the study. Kaplan Meier curves will be performed to evaluate TTP, 2 years after the end of the study.2.9 The proportion of patients with clinical response and the 95% confidence intervals will be calculated and reported. The Chi-Squared test and Kaplan–Meier method will be used to analyze the rates and severity of disease progression according to NCI-CTCAE (version 4), and Fisher exact probability test will be used to analyze the correlations between clinical outcomes and toxicity.The power of the test is 80% and α = 0.05. According to the literature, the incidence of esophageal cancer perforation after re-irradiation is 30%. It is expected that after re-irradiation with PLDR technology, the rate of esophageal perforation will decrease to 10%. The optimal sample size for re-irradiation is 27 cases by checking the table .2.10IMRT-PLDR QA group will be established with 2 radiation oncologists and 1 physicist.2.11This trial was approved by the Ethics Committee of Mianyang Central Hospital, Sichuan Province, China with trial number: S2018071. The trial is subject to the supervision and management of the ethics committee. Written informed consent is obtained from all participants.2.12The clinical registration was approved on January 10, 2019, This study opened to recruitment in February 2019, with a planned recruitment period of 1 year.3Currently, there is no standard treatment available for local recurrence after radiotherapy. Numerous basic research studies reveal low-dose rate radiation to be effective to alleviate the side effects of radiotherapy. Based on the effects of low dose radiosensitivity and low dose hyperrepair, we designed IMRT-PLDR radiotherapy for local recurrence of ESCC after radiotherapy.This study is novel in testing the safety and efficacy of PLDR for the treatment of locally recurrent ESCC. Our expected results are that PLDR causes fewer side effects and offers effective treatment, when compared with the historical data of conventional radiotherapy. The study results will indicate more treatment options for locally recurrent ESCC.Study conception LL and XBD. Initial Study design: XBD, LL and JL.Revision of study design and protocol: XBD, LL,TZD and JL. Study coordination:MX, YXW, LDG, XYY, GF, and YZ. Participating centers: Department of Oncology, Affiliated Hospital of North Sichuan Medical College, Nan Chong and Department of Oncology, Lang Zhong People‘s Hospital, Lang Zhong, China. Provide subjects for this study: ZHZ, GBD, XHZ, HWC, DBL. Drafting the manuscript:XBD, JL and JB. All authors read and approved the final manuscript.Conceptualization: Jie Li, Lei Liu, Xiaobo Du.Data curation: Zhenhua Zhao, Guobo Du, Tangzhi Dai, Xuhai Zhen, Hongwei Cai, Dongbiao Liao.Investigation: Jie Li, Zhenhua Zhao, Guobo Du, Tangzhi Dai, Hongwei Cai, Dongbiao Liao, Miao Xiang, Yixue Wen, Lidan Geng, Xiyue Yang, Gang Feng, Yu Zhang.Methodology: Jie Li, Zhenhua Zhao, Guobo Du, Tangzhi Dai, Xuhai Zhen, Dongbiao Liao, Miao Xiang, Yixue Wen, Lidan Geng, Xiyue Yang, Gang Feng, Yu Zhang.Project administration: Xiaobo Du.Resources: Xiaobo Du.Writing – original draft: Jie Li, Xiaobo Du.Writing – review & editing: Jie Bai, Lei Liu, Xiaobo Du.


Allium cepa L. Aggregatum group). Selenocystine showed the best effect on the growth and yield of mycorrhized plants, whereas sodium selenate was the most effective on the non-inoculated plants. The soluble solids, total sugars, monosaccharides, titratable acidity, and proteins attained higher values upon AMF inoculation. Sodium selenate resulted in higher soluble solids, total sugars and monosaccharide content, and titratable acidity than the non-treated control, and it also resulted in higher monosaccharides when compared to selenocystine; the latter showed higher protein content than the control. Calcium, Na, S, and Cl bulb concentrations were higher in the plants inoculated with the beneficial microorganisms. Calcium and sodium concentrations were higher in the bulbs of plants treated with both the selenium forms than in the control. Selenocystine-treated plants showed enhanced accumulation of sulfur and chlorine compared to the untreated plants. The AMF inoculation increased the bulb selenium content by 530%, and the Se biofortification with selenocystine and sodium selenate increased this value by 36% and 21%, respectively, compared to control plants. The AMF-based formulate led to increases in ascorbic acid and antioxidant activity when compared to the non-inoculated control. The bulb ascorbic acid was increased by fortification with both selenium forms when compared to the non-treated control. The results of our investigation showed that both AMF and selenium application represent environmentally friendly strategies to enhance the overall yield and quality performances of shallot bulbs, as well as their selenium content.Plant biofortification with selenium in interaction with the application of an arbuscular mycorrhizal fungi (AMF)-based formulate, with the goal of enhancing Se bioavailability, is beneficial for the development of the environmentally friendly production of functional food with a high content of this microelement. Research was carried out in order to assess the effects of an AMF-based formulate and a non-inoculated control in factorial combination with two selenium treatments with an organic (selenocystine) or inorganic form (sodium selenate) and a non-treated control on the yield, quality, antioxidant properties, and elemental composition of shallot ( Allium cepa L. Aggregatum group) is one of the most widespread Allium species across the world, and its bulbs are characterized by high levels of biological activity, including antioxidant [The shallot  with a pronounced anticarcinogenic effect [Allium species are particularly sensitive to the presence of arbuscular mycorrhizal fungi in the soil, which increase the root absorption surface through their hyphae, thus encouraging soil nutrient accumulation and water exploitation. AMF inoculation in Allium crops can also enhance plant antioxidant properties [In this respect, the c effect ,16. Moreoperties .+6, Se+4, Se−2, and Seo [The presence of Se bioavailable forms in soil depends to a large extent on the presence of microorganisms in the rhizosphere which take part in redox reactions between Se and Seo , resultiAllium cepa L. Aggregatum group and to the controversial results relevant to the effects of AMF on plant selenium biofortification, further investigations regarding the prospects of this approach are needed. The present research aimed to assess the interactions between inoculation with AMF and the supply of organic or inorganic Se forms to shallot plants in terms of the root mycorrhizal colonization, bulb yield, quality, antioxidant properties, and elemental composition.Due to the scant data available about the effects of AMF and selenium application on the Se biofortification of Allium cepa L. Aggregatum group) cultivar Alba at the experimental fields of the Federal Scientific Center of Vegetable Production, Moscow region, Russia  in 2017 and 2018. The trial was carried out on a clay-loam soil with pH 6.8, 2.1% organic matter, 108 mg·kg−1 N, 450 mg·kg−1 P2O5, 357 mg·kg−1 K2O, and an exchangeable base sum as high as 95.2%. The average air temperature values, recorded at plant level, were 13.5 °C in May, 17.1 °C in June, and 20.1 °C in July.Research was conducted on shallot (−2 soil) and a non-inoculated control, two selenium treatments , and a non-treated control.The experimental protocol was based on the factorial combination ofan arbuscular mycorrhizal fungi (AMF)-based formulate  is a plant-growth-stimulating preparation which predominantly contains the endomycorrhizal fungus 2 (5 × 3 m) surface area. A split plot design with three replicates was used for the treatment distribution in the field, with the AMF inoculation assigned to the main plot and each elementary plot covering a 15 m2).Shallot bulbs were planted on 5 and 4 May in 2017 and 2018, respectively, and bulbs were spaced 7 cm apart along single rows 35 cm apart  were determined at 20 °C using a digital refractometer by Bellingham and Stanley, model RFM 81, on the supernatant obtained by centrifuging the raw homogenate.The monosaccharides were determined using the ferricyanide colorimetric method based on the reaction of monosaccharides with potassium ferricyanide . The totThe Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, Hg, I, K, Li, Mg, Mn, Na, Ni, P, Pb, Si, Sn, Sr, V, and Zn contents of the shallot bulbs were assessed using an ICP-MS on a quadruple mass spectrometer Nexion 300D  equipped with the 7-port FAST valve and ESI SC DX4 autosampler  in the Biotic Medicine Center . Rhodium 103 Rh was used as an internal standard to eliminate instability during measurements. Quantitation was performed using an external standard , potassium iodide for iodine calibration, and Perkin-Elmer standard solutions for P, Si, and V. All the standard curves were obtained at five different concentrations.3 diluted 1:150 with distilled deionized water  in the Berghof SW-4 DAP-40 microwave system . For quality checking purposes, internal controls and reference materials were tested together with the samples on a daily basis. Microwave digestion of samples was achieved with sub-boiled HNO−1, respectively; aux argon flow, 1.6 L min−1; nebulizer argon flow, 0.98 L min−1; sample introduction system, ESI ST PFA concentric nebulizer and ESI PFA cyclonic spray chamber ; sampler and slimmer cone material, platinum; injector, ESI Quartz 2.0 mm I.D.; sample flow, 637 L min−1; internal standard flow, 84 L min−1; dwell time and acquisition mode, 10–100 ms and peak hopping for all analytes; sweeps per reading, 1; reading per replicate, 10; replicate number, 3; DRC mode, 0.55 L min−1 ammonia  for Ca, K, Na, Fe, Cr, V, optimized individually for RPa and RPq; STD mode, for the rest of analytes at RPa = 0 and RPq = 0.25.The instrument conditions and acquisition parameters were as follows: plasma power and argon flow, 1500 and 18 L minNitrates were assessed using an ion selective electrode by ionomer Expert-001 . Five grams of fresh shallot bulb homogenate were homogenized with 50 ml of distilled water. A quantity of 45 ml of the resulting extract wasmixed with 5 ml of 0.5 M potassium sulfate background solution (necessary for regulating the ionic strength) and analyzed through the ionomer for nitrate determination.+6) to selenite (Se+4) with a solution of 6 N HCl, and formation of a complex between Se+4 and 2,3-diaminonaphtalene. Calculation of the Se concentration was achieved by recording the piazoselenol fluorescence value in hexane at 519 nm λ emission and 376 nm λ excitation. Each determination was performed in triplicate. The precision of the results was verified using a reference standard of lyophilized cabbage in each determination with a Se concentration of 150 μg·Kg−1.Se was analyzed using the fluorimetric method previously described for tissues and biological fluids . Dried hThe ascorbic acid contentwas determined by visual titration of plant extracts in 6% trichloracetic acid with Tillmans reagent . Three g2CO3 solution and 0.25 ml of diluted (1:1) Folin–Ciocalteu reagent were added. The volume was brought to 25 ml with distilled water. One hour later the solutions were analyzed through aspectrophotometer , and the concentration of polyphenols was calculated according to the absorption of the reaction mixture at 730 nm. As an external standard, 0.02% gallic acid was used.Polyphenols were determined in water extract using the Folin–Ciocalteu colorimetric method as previously described . One gra3) complexation [3, 0.5 mL of 1 M sodium acetate solution, and 1 mL of distilled water were added. The mixture was incubated for 30 min at room temperature and the absorption at 415 nm was measured. The total flavonoid content was determined from astandard curve built using five different concentrations of quercetin–AlCl3 complex. Quercetin was purchased from Fluka (Switzerland).The total flavonoids content was determined by a spectrophotometric method based on flavonoid–aluminum chloride ·100 g−1d.w. The use of KMnO4 acidic solution has been successfully used for the determination of the Ocimum basilicum antioxidant potential [The antioxidant activity of the shallot bulbs was assessed using a redox titration method  via titrotential  and the otential .Data were processed by two-way analysis of variance, and mean separations were performed through Duncan’s multiple range test, with reference to a 0.05 probability level, using SPSS software version 21. Data expressed as percentage were subjected to angular transformation before processing. All the variables analyzed in this research were not significantly affected by the year of research; therefore, only the mean data of the two years areshown. The mycorrhizal index did not significantly change between the two determinations performed one month after the transplant and at the end of crop cycle; therefore, the mean values of the two determinations are reported in As reported in Glomus and Scutellospora, with Glomus predominating [Allium cepa, AMF application also increased bulb yield by 2–18 timesdepending on the amount of phosphorus in the soil [The shallot root system isbeneficially colonized by the two AMF genera minating ; this exminating . In Allithe soil .As for the Se supply, the decrease inshallot bulb number in the plants treated with selenocystine suggests that the organic form of this microelement promoted the formation of fewer but heavier bulbs, whereas the bulbs were not affected by the Se inorganic compound in terms of either weight or number . Moreove+6 applicationalso led toa higher monosaccharide concentration compared to Se−2, but these did not differ with respect to titratable acidity. A higher content of total proteinsresulted in the treatment with the Se organic form than in the control l plants .2 led to a higher concentration of Cu or Zn, respectively, compared to the non-Se-treated control; however, the beneficial microorganism inoculation resulted in higher Cu content not only in non-Se-treated plants but also under the Se treatments for Zn content. Se treatments enhanced Mn accumulation in non-inoculated plants, whereas the Se organic form caused a reduction of this element in mycorrhized plants; the latter showed higher Mn content than non-inoculated plants only in the non-Se-treated control. The Si content was reduced by sodium selenate application in non-inoculated plants and by both Se forms in mycorrhized plants; the latter always showed higher Si content than non-inoculated plants.The interaction between microorganism inoculation and Se treatment was statistically significant on the content of B, Cu, Fe, Mn, Si, and Zn in shallot bulbs . B and FThe application of AMF formulates to shallot plants resulted in the following average increases in microelements: 64.7% B, 47.4% Cu, 141% Fe, 77.9% Mn, 44.7% Zn, and 78.4% Si. These elements are essential for plant growth and development as they are involved in physiological processes. Indeed, Fe and Mn participate in redox reactions promoting photosynthesis; Cu, Mn, Zn, and Fe are important components of many enzymes; and Si encourages plant growth and development, increases B accumulation, and decreases the toxicity of heavy metals. The high levels of Si in AMF-inoculated plants detected in our research are consistent with the effect of AMF on increased Si content which is known to reduce Al accumulation , the redAllium species to AMF [+4 < Se+6 < SeMet [Allium species. In a previousinvestigation [The effect of AMF on Se accumulation is of remarkable interest. Previous investigations ,12 showes to AMF . This phs to AMF . Neverths to AMF . Literat < SeMet . The restigation , the Se In our research, the amount of Se ingested from 50 g of shallot bulbs treated with AMF formulates and selenocystine meets the optimal Se requirement, whereas 50 g of bulbs from plants sprayed with sodium selenate provides 63% of the daily Se requirement.3; K with Na and NO3; and Mg with P and Al.The correlation coefficients between the mineral elements analyzed are reported in As reported in A. cepa, and leek [At present, the mechanisms concerning AMF, Se, and antioxidant interactions in plants are not clear. Several papers suggest that root AMF colonization can induce the accumulation of secondary metabolites, polyphenols, flavonoids, and vitamins in lettuce , potato and leek . Other rand leek ,53. Thisand leek , or relaAscorbic acid affects mitosis and cell growth in plants, and it is an important cofactor for several enzymes involved in the synthesis of a number of secondary metabolites including phytohormones and in protection against environmental stresses . In our Shallot is characterized by high antioxidant activity due to the presence of sulfur compounds , flavonoids (glucosides of quercetin), polyphenols, ascorbic acid, and saponins ,56. The A. cepa [Notably, the application of an AMF formulate provided a significant increase in plant antioxidant activity in our research, in accordance with previous reports related to A. cepa .One of the possible factors contributing to these properties is the close relationship between AMF, Se, and phytohormones. It is known that hormones participate in AMF–host plant symbiosis and in antioxidant biosynthesis. In particular, gibberellins and auxins are intensive regulators promoting the synchronization of AMF spore formation and host plant growth ,59. Furthermore, plant biofortification with inorganic Se is also strictly connected with the plant hormonal status; this is reflected in the increase of female forms of hemp as a result of sodium selenite application  and highThe results from research carried out in northern Europe suggest interesting application prospects of the use of formulates based onarbuscular mycorrhizal fungi (AMF) in interaction with selenium biofortification by organic or inorganic compounds in shallot crops. Indeed, AMF inoculation resulted in higher production, whereas the selenium treatment effect depended on the compound form: sodium selenate positively affected bulb yield only when applied to mycorrhized plants, whereas selenocystine was only effective on non-inoculated plants. The values of bulb quality indicators, macro- and microelements, ascorbic acid, and antioxidant activity increased upon AMF inoculation; the two selenium forms positively affected most of the quality attributes and macroelements, as well as selenium and ascorbic acid, but their effect on the remaining microelements was unclear. The use of AMF-based formulates has proved to be a cost-effective and ecologically friendly method for enhancing the yield, nutritional properties, and selenium content of shallot bulbs.


Coptis chinensis, which has been listed in the Chinese Fisheries Pharmacopeia as a common drug for the control of bacterial fish diseases. However, BBR is poorly absorbed into the systemic circulation but is significantly accumulated in the intestine. It is difficult to explain the mechanism of clinical effects of BBR based on systemic genes and pathways; it has been proved that the function of BBR in mammals is associated with the host metabolic phenotypes mediated by the structural modulation of gut microbiota. The mechanism of pharmacological effects of BBR in fish remains unclear. Here, we fed grass carp (Ctenopharyngodon idellus) a diet supplemented with BBR at a dose of 30 mg/Kg body weight daily and compared them with grass carp fed a regular fish feed diet. Biochemical analysis revealed that fish fed BBR had significantly reduced serum glucose, total cholesterol (TC), and triglyceride (TG) levels, and increased TC (p < 0.05) and TG (p < 0.01) levels in the liver. Deep amplicon sequencing of the V4 region of 16S rRNA genes of the gut microbiota revealed: (i) the composition of gut microbiota after BBR feeding was more diverse than that in the control group; (ii)before fish were fed BBR, the enriched operational taxonomic units (OTUs) mainly belonged to Firmicutes while most enriched OTUs came from Proteobacteria, Planctomycetes, Bacteroidetes, and Firmicutes during BBR feeding and after BBR feeding stopped; (iii) the ratio of Firmicutes to Bacteroidetes was significantly decreased in fish fed BBR. Spearman’s rank correlation showed that 32 berberine-OTUs were significantly negative correlated with glucose (p < 0.05). It indicates that BBR may affect the levels of serum glucose by the structural modulation of gut microbiota. Our results provide insight into the effect of BBR on fish metabolism and gut microbiomes, which would be beneficial for the fish welfare.Berberine (BBR), an isoquinoline alkaloid, is a major pharmacological component of the Chinese herb  The grass carp is a herbivorous freshwater fish and one of the most important economic farmed fish in China; it has been introduced into more than 100 countries . GeneralCoptis chinensis . As a botanical drug, BBR or BBR-containing herbs have been used to treat intestinal infections, particularly bacterial diarrhea, for at least 2000 years in China  (Berberine (BBR), an isoquinoline alkaloid, is a major pharmacological component of the Chinese herb in China . BBR hasin China . Recentlin China . BBR alscephala) . HoweverOreochromis niloticus) is 2.95 ng/mL after oral administration of 30 mg/kg BBR  was purchased from Sigma-Aldrich (United States). BBR was suspended in distilled water before being sprayed evenly onto grass carp feeds. The formulated grass carp diet included the following: fish meal, 5 g⋅kg0 g⋅Kg-1 .-1, and <0.35 mg⋅L-1, and <0.01 mg⋅L-1, respectively. For 56 days, the fish received a daily feeding rate of approximately 2% body weight. The weight of fish in each group was determined 0, 7, 14, 28, 42, and 56 and the daily ration adjusted accordingly.Juvenile grass carp with mean body weight 34.0 g (standard deviation (SD) 0.73) and mean total length 14.8 cm (SD 0.26) were collected from the same spawn and kindly provided by the farm of PRFRI. The fish were maintained in tanks with filtered water in a flow-through system. All experimental protocols were approved by the animal Ethics Committee of the Guangdong Provincial Zoological Society, China (permit number: GSZ-AW003). To remove parasites or pathogens from the fish and feed, fish skin and gills were checked using a microscope, and bacterial and viral contamination was checked using bacterial isolation and reverse transcription quantitative real-time PCR (RT-PCR), respectively . The fis-1 MS-222 . Then the body length and weight of each fish was measured. Random samples of blood, liver, and the hindgut were taken from the BBR group and control group on days 0, 1, 3, 7, 9, 14, 21, and 28 under strictly sterile conditions. Fish bled from the caudal vein using non-heparinized sterile syringes. To isolate serum, blood samples were left at room temperature in sterile centrifuge tubes for 30 min to allow clotting, and then centrifuged at 3000 ×g for 10 min at 4°C , and triglyceride (TG) in the serum, and TC and TG in the liver were measured using commercial kits  and a fully automatic biochemical analyzer  following the manufacturer instructions. Briefly, the glucose level was measured using the GOD-PAP method . TC and ® stool DNA Kit  following the manufacturer instructions under required aseptic conditions  quality filters . The higt-test for two sample groups and Kruskal-Wallis rank sum test for multiple sample groups was used to identify the significant differences in alpha and beta diversity between and among different groups  were calculated to estimate the species diversity and richness for each sample using QIIME software . The rart groups . We alsot groups . The P-vp < 0.05, p < 0.05 and P < 0.05, respectively) lower than those in the control group from days 3 to 9; however, these significant differences disappeared after day 14 when BBR-supplemented feeding stopped  in the BBR group from day 7 to 21, but there were no significant differences days 1 to 3 before BBR-supplemented feeding, or on day 28 after BBR-supplemented feeding had stopped for 3 weeks (Although the body weight and relative fatness (condition factor) were not significantly different between the BBR-fed and control group , the lev stopped . Compare 3 weeks .Cetobacterium, Bacteroides, Bacillus, Lactococcus, Enterococcus, Erysipelatoclostridium, and Proteocatella were dominant at the genus level , while the Firmicutes, including Bacillus, Lactococcus, and Enterococcus significantly decreased (from 55 to 10%) by day 28 in the control group. In addition, the relative abundance of Bacteroides was obviously increased from the beginning to the mid-sampling days, and significantly decreased at the end of the sampling days in the control group . At the end of the sampling days, the relative abundance of potential pathogenic bacteria such as Vibrio was also significantly increased in the control group , the composition of gut microbiota was more diverse and stable after the BBR feeding was stopped than in the control group. For example, compared to the control group, the relative abundance of Firmicutes, Bacteroidetes, and Proteobacteria were significantly increased at day 28, while the abundant bacteria Fusobacteria were significantly decreased in the BBR group.The strong antimicrobial activity of BBR on both gram-positive and negative bacteria changed the pattern of gut microbiota, and the composition was significantly different in the BBR-fed group  comparedEven though most gut microbes were found in both groups, some species were enriched in different groups . In totap < 0.0001, p < 0.05, t-test, p < 0.05).All the rarefaction curves indicated that the sequencing depth was sufficient for each sample ,3. Compap < 0.005, p < 0.00001, Compared with the control group, the gut microbial community distances were significant decreased from days 0 to 28  is 2.95 ng/mL after oral administration of 30 mg/kg BBR (in vitro assays cannot be achieved as a result of low bioavailability of BBR (p < 0.05). These results broaden our view that BBR lowers blood glucose level in teleost fishes as well as mammals. In addition, BBR or BBR-derived products may be useful in the regulation of blood glucose levels and gut microbiota in grass carp culture.Berberine can significantly reduce glucose levels in mammals . The orag/kg BBR . Effectiy of BBR . Therefoy of BBR . BBR diry of BBR . It has y of BBR . The dosy of BBR . The preVibrio. Furthermore, most of the 32 negatively corelated OTUs were more abundant in the BBR group than in the control, indicating that these OTUs may have been affected by BBR and further affected the metabolism of grass carp.Besides the effects on fish metabolism, BBR also dramatically affected the structure of host gut microbiota. Consistent with previous findings in mice , we founIn summary, the levels of glucose, TC, and TG in blood were significantly decreased, and the diversity and structure of intestinal microbial bacteria in grass carp were affected by BBR-supplemented feed. BBR may have directly affected fish metabolism by controlling and modifying the structure of the gut microbiota, such as adjusting the ratio of Firmicutes to Bacteroidetes, increasing diversity, and recruiting more beneficial microbes. These findings indicate that BBR lowers blood glucose levels in teleost fishes via the gut microbiota, and that BBR or BBR-derived products may be used to maintain the growth and health of grass carp culture.The experiments protocols were approved by the animal Ethics Committee of the Guangdong Provincial Zoological Society, China (permit number GSZ-AW003).HP, JX, and DL conceived and designed the experiments. HP, ZL, and CS performed the experiments with help from JX and DL. HP and HW collected the data. JX, HP, ZL, DL, and HW analyzed the data. HP, JX, DL, ZL, ZH, QZ, and DY co-wrote the manuscript. All authors discussed the results and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this regard, dental thread structures were machined on fully sintered zirconia (ZrO2), alumina toughened zirconia (ATZ), and zirconia toughened alumina (ZTA) bioceramics. The ground workpieces were analysed through a scanning electron microscope (SEM), X-ray diffraction (XRD), and white light interferometry (WLI) to evaluate the microstructure, residual stresses, and surface roughness, respectively. Moreover, the grinding processes were monitored through forces measurement. Based on the machining parameters tested, the results showed that low peripheral speed (vc) and low depth of cut (ae) were the main conditions investigated to achieve the optimum surface integrity and the desired low grinding forces. Finally, the methodology proposed to investigate the surface integrity of the ground workpieces was helpful to understand the zirconia-based ceramics response under micro-grinding processes, as well as to set further machining parameters for dental implant threads.Metallic implants were the only option for both medical and dental applications for decades. However, it has been reported that patients with metal implants can show allergic reactions. Consequently, technical ceramics have become an accessible material alternative due to their combination of biocompatibility and mechanical properties. Despite the recent developments in ductile mode machining, the micro-grinding of bioceramics can cause insufficient surface and subsurface integrity due to the inherent hardness and brittleness of these materials. This work aims to determine the influence on the surface and subsurface damage (SSD) of zirconia-based ceramics ground with diamond wheels of 10 mm diameter with a diamond grain size (d Dental implants aim to replace a partially or totally, damaged or diseased tooth structure, i.e., restoring the function and also the aesthetics ,2. In th2O3) and zirconium dioxide (zirconia—ZrO2) have become the better alternatives due to their combination of biocompatibility, mechanical properties, like high flexural strength and wear resistance, as well as minimum thermal and electrical conductivity [Among the bioceramics in the market, aluminium oxide , feed rate (vf), and depth of cut (ae), using the design of experiments (DOE) method as the statistical approach for planning, conducting, analyzing and interpreting data from grinding experiments. The results evaluate the bioceramics with regard to their machinability and, thus, their suitability as materials for dental ceramics. The structuring of bioceramics using micro-grinding is still an area under investigation, and the surface integrity characterisation of ground ceramics is considered to be a key aspect of their further applications as dental implants on the market ,2,3. In The success of ceramic dental implants is correlated with effective osseointegration, such as the formation of direct contact between the implant and the surrounding bone ,12. AccoIn order to avoid defect parts during manufacturing and failures during its use, enhanced micro-grinding processes are still necessary and, consequently, have been subject to several research investigations ,17,18,192-TZP),—also known as zirconium dioxide (ZrO2) and commonly called ‘‘zirconia’’—alumina toughened zirconia (ATZ), and zirconia toughened alumina (ZTA). These materials are zirconium-based ceramics commonly used for dental applications and have intrinsic toughening mechanisms, but differ in their mechanical properties [The ceramic workpieces (5.0 mm × 7.0 mm × 33.0 mm) are fully sintered and commercially-available tetragonal polycrystalline zirconia  along the x-direction, while the tool spindle rotated (np). In this case, diamond galvanic-bonded wheels of 10 mm in diameter, commercialised by the company SCHOTT Diamantwerkzeuge GmbH, with a specific width (bw) of 0.9 mm, and an average diamond grain size of 75 μm (D75) were used [w) of 0.2 mm, as well as the overall design of a square thread profile of a dental implant. In order to machine dental threads, as illustrated in ere used ,25. Morec), feed speed (vf), and depth of cut (ae), which were selected after a screening campaign and a literature review concerning the critical depth of cut, as well as the equivalent chip thickness, to achieve ductile grinding mode machining [The machining conditions performed are summarised in achining ,18,19,26In the present work, the Taguchi method was used as the design of experiment (DOE) approach to examine the influence of the grinding process parameters on the surface integrity and grinding forces of the three bioceramics materials ,28,29,30c of 0.08 mm and an objective of 50×.In order to evaluate the surface and subsurface damage (SSD), the microstructure and surface topography were studied by means of a scanning electron tabletop microscope TM3030  under magnifications of 60× and 2500×. An X-ray diffraction (XRD) machine  was used to measure the residual stresses along the diagonal stress axis . Finally, the surface roughness was measured by using a white light interferometer Talysurf CCI HD  within an air-conditioned laboratory. Herein, the roughness data was acquired using a Gaussian filter with a specified cut off λ2 dental threads, with a 75-grit diamond wheel, at peripherical speed (vc) 10.00 m/s, feed rate (vf) 100 mm/min, and a depth of cut (ae) 50 μm, i.e., process condition P8, showed ductile streaks and a smooth surface as indicated in c 18.33 m/s, vf 300 mm/min, and an ae 250 μm, i.e., process condition P1, as shown in 2 ground workpieces showed a greater amount of ductile areas than the ATZ and ZTA specimens, where the material was removed in a more partial ductile grinding and brittle mode. The surface of the ground ZrO2 workpieces predominantly increased the compressive stresses, i.e., −141 MPa for P1 and −52 MPa for P8. Both machined ATZ samples exhibited tensile stresses after grinding, for instance, 178 MPa and 133 MPa for P1 and P8, respectively. For ZTA specimens, the P1 tended to generate a slightly tensile stress of 6 MPa and substantial compressive stress for P8, herein −92 MPa. This phenomenon was due to the toughening mechanism, which also involves the phase transformation already mentioned. In general, compressive values are, likewise, desired in the specimen surface for biomedical application. For example, the compressive residual stress tends to increase the fatigue strength and the fatigue life of ceramic dental implants [According to the XRD analysis, shown in implants ,19.2, ATZ, and ZTA specimens. Although the same process conditions were applied for all the three bioceramics, different surface roughness were achieved. The values for the ATZ dental threads were all considered to be the optimum results for the successful osseointegration of biomedical implants. For example, dental implants are suggested to exhibit a surface roughness, Sa, of between 500 and 1000 nm [2 ceramics, this was only observed for P3, P4, P7, and P8. 1000 nm ,22. Ther2 workpieces, i.e., from ca. 715 nm up to 2100 nm. In comparison, ATZ and ZTA ceramics ranged between ca. 510 nm and 960 nm, and between ca. 820 nm and 1200 nm, respectively. In general, the highest variation in Sa values, with respect to the eight machining conditions, was observed on ground ZrOe of 250 μm), higher mean forces (Fy) were also measured in comparison to the machining conditions that followed . Specifically, for the machining of ATZ, higher forces were reported in regard to the grinding of ZrO2 and ZTA, which obtained similar values. In general, lower forces are beneficial for the tool wear and tool life [ool life ,31.In this section, further analysis of the surface and subsurface damage (SSD) of ground bioceramics threads are discussed. Moreover, the Taguchi method is used for understanding the mean Sa and F responses, based on the eight designed machining parameters ,28,29,302 and ATZ ceramics. This showed that brittle materials led to different surface topographies although grinding conditions did not vary.In The grinding direction is clearly discernible in both figures. The surfaces consist mostly of a series of parallel grinding marks, and the width of these forms are better visible in 2 ground ceramics have a tendency to have a higher osseointegration response once a better-machined surface is obtained than the ATZ and ZTA bioceramics [An important characteristic of a ceramic dental implant is the ability to create correct interaction between the ceramic implant and the bone tissue through the ground threads . Since mceramics ,21,22,32c = 18.33 m/s, vf = 300 mm/min, and ae = 250 μm) were more beneficial for ZrO2 workpieces in comparison to the less demanding grinding conditions investigated  once more compressive stress was measured. However, in the case of the ATZ specimens, condition P1 resulted in higher tensile stresses than P8. No significant stress differences were seen in ZTA ceramics ground with condition P1, but the machining parameter P8 generated considerable compressive stresses.According to the XRD results, the highest machining parameters , feed speed (vf), and depth of cut (ae), and their response on the surface roughness (Sa) and grinding forces (Fy) on the three bioceramics were analysed [The Taguchi method was used as a statistical tool for the optimisation of the grinding process by analysing the machining parameters’ influence on surface roughness and grinding forces. Therefore, an analysis of variance (ANOVA) was performed and evaluated using the statistical software Minitab 17 . ANOVA ranalysed ,29,30,35To examine the differences between the most and the least demanding machining parameters conditions (P1 and P8), the main effect plots were generated with Minitab 17 support . Basicalc), feed speed (vf), and depth of cut (ae), on the surface roughness of ZrO2, ATZ and ZTA specimens are presented in c), which had the highest influence on surface roughness. Feed speed (vf) and depth of cut (ae) had the lowest contribution factor, but different inclinations according to the tested material, i.e., the slope rose or fell with the increase of the respective machining parameter level tested.The individual grinding parameters effects, i.e., peripheral speed . Therefore, increasing the rotation speed also led to an increase in surface roughness. The highest vc response on surface roughness indicated that the setting of 10.00 m/s peripherical speed was beneficial to achieve an optimal Sa range, independent of the vf and ae designed in this study. This was the reason that the machining conditions P3, P4, P7, and P8 were indicated for grinding all three zirconia-based ceramics investigated once the Sa achieved between 500 and 1000 nm are an optimum range for further dental applications as also mentioned in For all three bioceramic materials, v2 and ATZ ceramics and increased for ZTA. Although the ae main effect line tended to be horizontal for the machined bioceramics and, consequently, no significant Sa response was observed, the increase in the depth of cut for grinding ZrO2 and ZTA seemed to have a slightly positive influence on the material surface, while the opposite was observed for ATZ specimens.Additionally, once the feed rate increased, the surface roughness decreased for the ZrOThe quality of the dental part produced by the micro-grinding process is influenced by the grinding tool and the conditions, which are linked in particular by the induced mechanical forces ,10,29,332 and ZTA ceramics. Both materials showed a significant lower process force response than the ATZ materials. Moreover, the highest forces measured in the process conditions with the higher depth of cut, i.e., ae = 250 μm, were not desired because of expected higher tool wear during machining.Similar force values were monitored during the grinding of the ZrO2 and ZTA materials.The forces measured while machining of the ATZ workpieces were essentially two- to three-times higher than the other two bioceramics. Basically, the long term machining of the ATZ ceramics tended to introduce more damage to the grinding tool life and to the surface integrity of the implant in comparison to the ZrOc = 10 m/s, vf = 300 mm/min, and ae = 250 μm) exhibited the highest forces monitored, while the lowest forces were seen with condition P6 . Therefore, in this study, high rotation, low feed speed, and low cutting depth tended to be beneficial to keep process forces low. This machining configuration is explained in The parameter setting P3  had the highest contribution, and peripheral speed (vc) and feed speed (vf) had the lowest impact factor on the ceramics. The Figure indicates the process forces response during the grinding. In general, the ZrOe had a very strong line inclination, which points out the highest influence on the grinding forces (Fy) response once the cutting depth was increased. Furthermore, the same tendency with a lower slope was seen for the vf main effect, where the force response also increased when the feed speed set was higher. The opposite configuration was seen once the peripheral speed rose. Herein, the forces applied to the ceramics decreased.For all bioceramics, a2), alumina-toughened zirconia (ATZ), and zirconia-toughened alumina (ZTA) were structured by micro-grinding process. In order to replicate dental threads with a square profile, a grinding wheel with a diameter of 10 mm and a specific width (bw) of 0.9 (grain size: D75) was used. Eight machining conditions were designed and the process forces (F) were monitored. The microstructure of the ground bioceramics was analysed via SEM, the XRD technique accessed the residual stresses, and surface roughness (Sa) was measured with WLI.Three fully sintered types of zirconia-based ceramics, zirconium dioxide  was beneficial to achieve an optimal Sa for dental uses. Regarding the process monitoring, the depth of cut (ae) had the highest influence on the grinding forces (Fy) response when it was larger—herein, the machining process with an ae of 50 μm are indicated for less tool wear and best implant integrity.The different surface roughness (Sa) and force (F) responses due to the different grinding parameters were directly correlated to the intrinsic physical properties and chemical composition of the bioceramics investigated. Basically, the machining conditions P3, P4, P7, and P8 generated the optimal surface roughness suggested for dental implants, i.e., between 500 and 1000 nm, on all machined bioceramic materials. This was due to the highest peripheral speed, v2 ceramics machined with the grinding conditions P7  and P8  are suggested for further dental applications due to their optimal Sa range, smoother microstructures, compressive residual stress, as well as low forces generated during machining.ZrOThe following conclusions were drawn from the investigation:2O3). This will extend the validity of this approach and will allow a fundamental material and process analysis in the micro-grinding of bioceramics. Finally, the next steps are the machining of ceramic dental parts based on the optimum grinding parameters investigated for further mechanical and biological evaluation.Based on the results of this work, a future investigation should include a similar approach to machine aluminium oxide (alumina—Al