Collectively, we demonstrate the therapeutic potential of the human TNFR1 antagonist Atrosimab for treatment of chronic inflammatory diseases.Therapeutics that block tumor necrosis factor (TNF), and thus activation of TNF receptor 1 (TNFR1) and TNFR2, are clinically used to treat inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. However, TNFR1 and TNFR2 work antithetically to balance immune responses involved in inflammatory diseases. In particular, TNFR1 promotes inflammation and tissue degeneration, whereas TNFR2 contributes to immune modulation and tissue regeneration. We, therefore, have developed the monovalent antagonistic anti-TNFR1 antibody derivative Atrosimab to selectively block TNFR1 signaling, while leaving TNFR2 signaling unaffected. Here, we describe that Atrosimab is highly stable at different storage temperatures and demonstrate its therapeutic efficacy in mouse models of acute and chronic inflammation, including experimental arthritis, non-alcoholic steatohepatitis (NASH) and experimental autoimmune encephalomyelitis (EAE). Our data support the hypothesis that it is sufficient to block TNFR1 signaling, while leaving immune modulatory and regenerative responses  Tumor necrosis factor (TNF) is recognized as a master pro-inflammatory cytokine that plays a dominant role in the initiation and perpetuation of chronic inflammation , 2. Elevin vitro due to bivalent TNFR1 binding of the full IgG molecule  and TNFR2. It was shown that expression levels of TNFR1 and TNFR2 are regulated during inflammatory conditions and that cellular fate may be dependent on cytokine-mediated TNFR expression . Whereasmolecule . Thus, wmolecule . In vitr Atrosab . In this® NH2-750F, flow-through mode) and CHT multi modal  chromatography. Virus inactivation was performed by incubation of Amsphere load with Triton X-100 and virus removal by Nanofiltration (Virosart®) of CHT eluate.Atrosab, Atrosimab, FcΔab and human TNFR1-Fc were provided by Baliopharm . Mouse, rat, rhesus and cynomolgus TNFR1-Fc and human and mouse TNFR2-Fc were obtained from Sino Biologicals . Atrosimab was produced by Catalent  in chinese hamster ovary (CHO) cells after lentiviral transfection and purified by depth filtration (Zeta Plus™), anion exchange hybrid purifier (Emphaze AEX filter), protein A affinity , anion exchange . Standard proteins  included Thyroglobulin , Apoferritin , beta Amylase , bovine serum albumin , and Carbonic anhydrase .Atrosimab was analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) using 4 μg protein under reducing and non-reducing conditions. Proteins were stained using Coomassie-Brilliant Blue G-250 and acrylamide gels were de-stained with water. Correct assembly under native conditions was visualized by size exclusion chromatography (SEC) using a Waters 2695 HPLC and a TSKgel SuperSW mAb HR column  reached at least a factor 2.0. Melting points were further determined by differential scanning calorimetry in the laboratory of Prof. Franz Hagn .Temperature-dependent aggregation of Atrosimab was analyzed by dynamic light scattering using the ZetaSizer Nano ZS . Atrosimab (100 µg in PBS) was heated stepwise from 35°C to 80°C with intervals of 1°C and equilibration times of 2 min prior to each measurement and signal of the particle size (kcps) was determined. The mean of two measured kcps values at each temperature was calculated and plotted over temperature. The aggregation temperature was defined as the temperature T where the quotient kcpsR2  and twice with PBS.The indicated fusion proteins were diluted to 1 μg/ml in PBS, transferred to a 96-well microtiter plate  and incubated overnight at 4°C. Skim milk in PBS (2% MPBS) was used to block residual binding sites [200 μl/well for 2 h at room temperature (RT)]. Atrosimab or EHD2-sc-mTNF2 overnight. Then, the medium was removed, Atrosimab samples were diluted in steps of 1 to 3.16 (square root of 10) in culture medium and applied to the cells. In the case of inhibition experiments (antagonist assay), addition of Atrosimab samples was followed by addition of TNF (0.01 nM). For agonist assays, cells were incubated with Atrosimab dilution, but without addition of TNF. After 16 - 20 h incubation at 37°C, 5% CO2, the supernatants were harvested subsequent to 5 min centrifugation at 500 g and analyzed for IL-8 concentration by ELISA according to the manufacturer’s instructions .7,500 HT1080 cells were plated in 100 μl RPMI 1640 supplemented with 5% FCS in 96-well plates and incubated at 37°C, 5% CO2. The data generated were fitted to a 1:1 binding model.The affinity measurements were performed by SPR technology using a Biacore T200. Cynomolgus and human TNFR1 was immobilized on a CM5 (carboxymethylated Dextran matrix) chip surface (one flow cell each with remaining flow cells being blanks) using standard amine coupling chemistry (10 mM Sodium Acetate pH 4.0). Subsequently Atrosimab was serially diluted in assay buffer  from 500 nM to 2 nM (2-fold dilution) and injected over each flow cell pair in separate assay sections (multi cycle kinetic), the regeneration was performed with 3M MgClhCMEC/D3 cells  were culAll animal studies were performed according to the EU guidelines and have been approved by University authorities and local governmental institutions . The huTNFR1-k/i mice were described previously . Brieflyg, 4°C, 10 min) and stored at −20°C prior to analysis. The concentration of Atrosimab in serum samples was determined by an ELISA specific for Atrosimab as described for the binding studies. Results were analyzed using the PKSolver Excel add-in (i.v. studies) or GraphPad Prism One-Phase decay non-linear fitting algorithm (s.c.).Atrosimab and Atrosab were injected at doses of 1.0 mg/kg or 30.0 mg/kg body weight intravenously (i.v.) or subcutaneously (s.c.) in C57BL/6J wild type mice or C57BL/6J huTNFR1-k/i mice. In transgenic huTNFR1-k/i mice the endogenous mouse TNFR1 is exchanged with a chimeric TNFR1, composed of the extracellular domain of human TNFR1 fused to the mouse transmembrane and intracellular region . Blood sg, 4°C, 10 min). Serum samples were stored at -20°C subsequently. At the end of the study, serum samples were analyzed for IL-6  and CRP levels  according to the instructions of the manufacturer. Individual values were excluded using the ROUT method of outlier identification (Q=1%).Mice received i.v. injections of 100-150 µl Atrosimab (30.0 mg/kg), FcΔab (30.0 mg/kg) or TNF (0.3 mg/kg), or were left untreated, respectively. Body weight was recorded at the indicated time points. Blood samples were collected at the indicated time points, incubated on ice for 20 min and serum was separated by centrifugation . Serum samples were stored at -20°C subsequently. At the end of the study, serum samples were analyzed for IL-6  according to the manufacturer’s instructions. Individual values were excluded using the ROUT method of outlier identification (Q=1%).Mice first received an i.v. injection (100-150 µl) of Atrosimab (30.0 mg/kg), FcΔab (30.0 mg/kg), or PBS, respectively, followed 30 minutes later by i.v. injection of TNF (0.3 mg/kg). Body weight was recorded at the indicated time points. Blood samples were collected at the indicated time points, incubated on ice for 20 min and serum was separated by centrifugation  was induced in male huTNFR1-k/i mice by feeding a high-fat diet (HFD) consisting of 60% kcal of fat , supplemented with 42 g/L sugar  in the drinking water  for 21 w35−55 in PBS emulsified in an equal volume of complete Freund’s adjuvant  supplemented with Mycobacterium tuberculosis H37RA  to a final concentration of 10 mg/ml. Immediately afterwards and again 2 days later 300 ng pertussis toxin was injected intraperitoneally (i.p.). Mice were weighed and scored on a daily basis, with disease severity assessed using a scale ranging from 0 to 5: 0, no clinical disease; 0.5, distal paresis of the tail; 1.0, complete paralysis of the tail; 1.5, paresis of tail and mildly impaired righting reflex; 2.0, gait ataxia and severely reduced righting reflex; 2.5, bilateral severe hind limb paresis; 3.0, complete bilateral hind limb paralysis; 3.5, complete bilateral hind limb paralysis and forelimb paresis; 4, hind and fore limb paralysis; 5, moribund state or death. Mice received i.p. injections with Atrosab or Atrosimab on the days of EAE as stated. Control mice were injected with the appropriate species-specific control protein Fc∆ab.EAE was induced as previously described , 17. BriThe ankle joints were dissected, the skin was removed, and tissue was subsequently fixed in 4% aqueous formaldehyde solution overnight at RT, demineralized in EDTA decalcification solution (13% EDTA in 0.1M sodium phosphate buffer) at RT for 30 days and further placed in PBS at 4°C until further processing. Decalcified ankle joints were paraffin embedded in the sagittal plane. Paraffin blocks were sectioned using a microtome into 4 μm thick sections. The sections were then stained with H&E and evaluated by light microscopy for histopathological hallmarks of arthritis. The evaluation process was performed in a blinded fashion. Histopathological score (H score) was assessed based on the following scoring system: 0, no detectable pathology; 1, hyperplasia of the synovial membrane and presence of polymorphonuclear infiltrates; 2, pannus and fibrous tissue formation and focal subchondrial bone erosion; 3, cartilage destruction and bone erosion; 4, extensive cartilage destruction and bone erosion, bone outline structure is lost. Group means were calculated from the individual highest scores per joint in each mouse in the specific group.For immunohistological analysis of liver tissue, the organ was embedded in paraffin and sections were prepared and analyzed as described . BrieflySpinal cord histopathology was performed as described previously , 17. Bripost-hoc Dunn’s analysis for multiple group comparisons. A p value of <0.05 was considered to be statistically significant.Data was assessed for normality using the Shapiro-Wilk Test, followed by either a Mann-Whitney or a two-tailed student’s t-test for comparing two experimental groups, or the one-way analysis of variance (ANOVA) with post-hoc Bonferroni, Dunnett’s or Tukey’s test, or Kruskal-Wallis test with 50 values ranging between 21.9 nM and 34.9 nM without statistically significant differences. No major differences were observed between batches stored up to 9 months at 5°C or 25°C/60%RH  and SDS-PAGE analysis using Coomassie staining revealed correct assembly and integrity of the heterodimeric protein comprising a VL1C chain (38 kDa) and a VHκC chain (43 kDa)  . No binding of Atrosimab to mouse and rat TNFR1 was observed  or subcutaneous (s.c.) injections using doses of 1.0 mg/kg and 30.0 mg/kg body weight, respectively (vs 150 kDa).Pharmacokinetic analyses of Atrosimab in comparison to Atrosab were performed in C57BL/6 wild-type and C57BL/6 huTNFR1-k/i mice after intravenous  injection after 30 minutes to induce a TNF-mediated inflammatory response. PBS- and FcΔab-treated groups showed a TNF-induced reduction of body weight, lasting for 48 hours post injection  in a prophylactic setting by starting injections at 4 weeks of age, before onset of arthritic disease. All Atrosimab doses and the anti-TNF therapeutics Etanercept and Infliximab prevented development of arthritic disease  was delayed until week 9 of age, when mice had developed a profound arthritic score. Atrosimab treatment was compared to the anti-TNF therapeutics Infliximab and Certolizumab pegol. All therapeutics alleviated arthritic disease  or NASH in patients with immune-related diseases . HoweverWhereas anti-TNF drugs failed in a clinical MS trial, selective blocking of TNFR1 was therapeutic in the EAE model of MS , 17. TheOur published data indicate that anti-TNFR1 therapy restricts immune cell infiltration across the blood-brain barrier through the downregulation of TNF-induced adhesion molecules resulting in ameliorated neuronal injury and EAE motor disease . We therBivalent binding of TNFR1 antagonists harbors the risk for dose-dependent activation of TNFR1 and therefore renders bivalent TNFR1 antagonists, such as Atrosab, unsuitable for clinical use , 19, 26.We previously demonstrated that the epitope of Atrosab is located at the ligand-binding site of the receptor . Epitopevia TNFR1 while leaving regenerative and immunomodulatory TNFR2 signaling intact . This can be explained by the shorter half-life of the antibody derivative Atrosimab compared to the full antibody Infliximab or the PEGylated, and thus half-life extended, Fab Certolizumab pegol. This limitation can be circumvented by either increasing the dose or the application frequency of Atrosimab. In particular, Atrosimab therefore may be a suitable treatment for patients that lose their therapeutic response to conventional anti-TNF drugs, e.g. due to formation of anti-drug antibodies. Altogether, our data obtained in the arthritis and EAE model demonstrate that Atrosimab is a suitable drug candidate for the treatment of chronic inflammatory diseases.via TNFR1, but also inhibits lymphotoxin α (LTα) signaling via TNFR1 (in vitro studies suggest that LTα induces proliferation of RA fibroblast-like synoviocytes (In contrast to conventional anti-TNF drugs, Atrosimab not only blocks TNF signaling ia TNFR1 . This maia TNFR1 –45. In pia TNFR1  and, likia TNFR1  and lesiia TNFR1  of MS paia TNFR1 , 50 and viocytes . Altogetviocytes , 52. Notviocytes . TherefoIn summary, we have demonstrated that Atrosimab is therapeutic in different models of acute and inflammatory diseases and provide necessary pre-clinical data to support further clinical development of Atrosimab as a novel drug candidate. Altogether our data confirm the high potential of the TNFR1 antagonists Atrosimab as a safe and effective novel drug to treat acute and chronic inflammatory diseases.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Regierungspräsidium Stuttgart, Regierungspräsidium Heidelberg and Regierungspräsidium Hannover. Written informed consent was obtained from the owners for the participation of their animals in this study.FR, SW, KJ, CH, CV, HZ, RiF, KPi, SM, and AV conducted experiments. M-AD, SH, AH, KPf, HB, and RD designed parts of the experimental work, analyzed results, and contributed to writing the manuscript. RK and RoF designed and supervised the project, analyzed the results, and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a research grant from Baliopharm, Basel, CH.Author M-AD, SH and AH were employed by company Baliopharm. FR, AH, KP and REK are named inventors on patents and patent applications covering the Atrosimab technology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The authors declare that this study received funding from Baliopharm. The funder was involved in the design of the experimental arthritis study.


Lateral S’ was 36% higher at T3 than at T1 (p < 0.05). Changes in diastolic function were demonstrated by 10% lower E wave and 15% A wave at T1, compared to T4 (p < 0.05). E’ and A’ were 23% and 42% higher at T3 compared to T4 (p < 0.01). Both lateral E/E’ and E’/A’ were 6% and 19% lower at T3 compared to T1 . At T3, MPI was 51% and 34% lower when compared to T1 or T2 (p < 0.05). The echocardiographic evaluation of maternal cardiac function is important, as structural, and functional changes occur throughout pregnancy.This study aimed to evaluate maternal left ventricular (LV) systo-diastolic function using conventional and TDI echocardiography and included 10 healthy Saint-Bernard pregnant bitches. M-mode, peak transmitral flow velocities during early diastole (E) and atrial contraction (A), aortic and pulmonic flow, myocardial performance index (MPI), TDI studies , atrial contraction (A’) and peak systole (S’)), and blood pressure were measured at 21 to 28 (T1), 40 (T2) and 60 (T3) days of gestation and four to eight weeks postpartum (T4). Cardiac output and heart rate were 20% and 9% higher at T3, respectively, compared to T4 ( Appropriate development of the foetuses during pregnancy induces several hemodynamic alterations, with consequent cardiac changes. Echocardiographic and Doppler studies have been widely used to assess heart function during pregnancy in humans ,2,3 and Pulsed wave Doppler transmitral inflow velocity may not truly reflect the left ventricle (LV) diastolic function during pregnancy since it is highly influenced by ventricular loading conditions, which are naturally altered by the pregnancy volume overload state . Tissue Myocardial performance index  is a valuable tool in evaluating systo-diastolic function. This index correlates with the New York Heart Association (NYHA) class and some echocardiographic indices , serving as a prognostic measure . In addiThe cardiac changes in human pregnancy have been widely studied. A primary fall in vascular tone in early pregnancy leads to a rapid decrease in preload and afterload with compensatory increase in heart rate and activation of the volume-restoring mechanisms, with resulting increase in cardiac output . This coIn the bitch, significant changes have been observed by late pregnancy, characterized by an increase in heart rate ,19, stroUnderstanding the physiological adaptations of the heart to the pregnancy is essential for monitoring maternal cardiac function and detecting early signs of cardiac failure in high-risk pregnancies  or obste®, with a 3.5-MHz linear transducer), at the first visit, and the gestational age was calculated from the date of mating. The bitches were evaluated three times during pregnancy at early , mid  and late pregnancy , as well as 4 to 8 weeks postpartum (PP). In each visit, the dogs were evaluated by clinical examination, obstetric ultrasound for monitoring foetal development  × 100 [The aortic (Ao) and left atrium (LA) diameters and the LA/Ao ratio were evaluated by the right parasternal short-axis view at the level of the aortic valve. The fractional shortening (FS) and the ejection fraction (EF) of LV were calculated according to the formulas: FS (%) = [(LVIDd − LVIDs) /LVIDd] × 100 and EF = [ and during atrial contraction (A-wave) were measured.3]/(2.4 + LVDd/10) and ESV is the end systolic volume, calculated by ESV = [7 × (LVDs/10)3]/(2.4 + LVDs/10). Cardiac output (CO) was calculated as the product of SV and HR, and the LV mass was calculated by the formula: 0.8 × [1.04 × (((ISVd/10 + LVDd/10 + LVPWd/10)3) − (LVDd/10)3)] + 0.6  + 0.6 .Left ventricular myocardial performance index (MPI), also known as Tei Index, was calculated from pulsed-wave Doppler, according to the formula: (IVCT+IVRT)/ET, where IVCT, IVRT and ET represent, respectively, isovolumetric contraction time, isovolumetric relaxation time, and left ventricular ejection time . Value fSpectral TDI recordings were made from the apical four-chamber, left parasternal view. Ventricular myocardial velocities were assessed during both contraction and relaxation. The sample volume was placed at the lateral margin  and at the common septal margin  of the mitral annulus, as previously described . From th®) was used to collect five consecutive measurements, after discarding the first one, from which the means for systolic , diastolic  and mean  arterial pressures were calculated.Blood pressures measurement was performed as previously described . The bitp < 0.05 was considered statistically significant.Data were graphed and analysed using the Prism 9.0 software . Data were expressed as mean and standard deviation (SD). Comparisons between groups were analysed by one-way analysis of variance (ANOVA), that compared echocardiographic variables obtained at early, mid and late pregnancy as well as at postpartum. Post hoc evaluation was performed using the Tukey comparison test. Selected echocardiographic variables are listed in p < 0.01). Blood pressure  was higher at T1, T2 or T3 than at postpartum (p < 0.01) and reached maximal values at T1 that progressively decreased until the end of pregnancy (T3).Post hoc testing revealed several statistically significant differences. Body weight was significantly higher on late pregnancy (T3) than at T1, T2 or postpartum (p < 0.05), and the cardiac output was 20% higher at T3 than at postpartum (p = 0.02). The LA/Ao ratio was 7% higher at T2 than at T3 (p = 0.03). Transmitral early diastolic flow (E) was 9% lower at T1 than at T2 or T3 (p < 0.05). At T1, transmitral late diastolic flow (A) was 13%, 16% and 15% lower than at T2, T3 or postpartum, respectively (p < 0.05). Pulmonic flow was 26% and 21% greater at T3 than at T1 or T2, respectively (p < 0.05). Similarly, aortic flow was also 13% higher at T3 than at T1 (p < 0.01).Heart rate was 10% and 9% higher at T3 when compared to T1 or postpartum, respectively (p < 0.05).Even though ejection time did not change throughout pregnancy, both IVRT+IVCT and MPI decreased, with the latter being 56% and 34% lower at T3 when compared to T1 or T2 (p < 0.01). The lateral S’ and A’ were also higher at T3 than at T1 or T2 (p < 0.01). Lateral S’ was 35% and 30% higher at T3, and lateral A’ was 66% and 34% higher at T3 when compared to T1 and T2, respectively. The E’/A’ ratio decreased with gestational age, being 19% lower in T3 when compared to T1 (p < 0.05). The E/E’ ratio of the lateral mitral annulus decreased throughout pregnancy, being 6% lower at T3 compared to T1 (p < 0.01) and was always within normal limits as described for healthy dogs [Changes in TDI variables throughout pregnancy are shown in thy dogs .Blood pressure values were elevated throughout the entire pregnancy (>160 mmHg), opposing a previous study carried out in small pregnant bitches, where systolic blood pressure did not change ; observeOur data suggest that early systemic hypertension possibly triggers the following cardiac events: a pressure overload in canine early pregnancy and consequent increased left ventricular filling pressure  induces an increase in left atrium size by day 40. As the placental-foetal circulation develops , the decIn human gestation, it is well established that an early endocrine mediated fall in vascular tone triggers volume-restoring mechanisms, responsible for the increasing cardiac output seen through pregnancy, primarily caused by an increased stroke volume, but later on also due to increased heart rate . This naIn the current study, no cardiac hypertrophy was observed, despite the tendency of some variables to increase at the end of gestation . Previous studies reported contradictory results, with some reporting no increase of LV end-diastolic dimension or volume ,8, whilsIn bitches, several cardiac indices of systolic function increase in late pregnancy ,5. In thThere are few studies that evaluated diastolic function in pregnant bitches. In human pregnancy, studies on mitral inflow velocities showed an increase in E wave on the first trimester of pregnancy ,18, follThe compensatory increase in preload (increased plasma volume) observed in pregnancy  induces The MPI is a valuable tool in evaluating LV systo-diastolic function. MPI seems to be independent of ventricular geometry, preload and heart rate ,15. In tOne potential limitation of the current study would be the sample size, since serial evaluation of cardiac function during pregnancy was often not feasible; however, we may assume that it is minimized as we have only included Saint Bernard bitches. As previously referred, we intended to characterize the cardiac adaptations during pregnancy in a large breed dog prone to develop cardiac diseases. However, the obtained results may not be representative of the large breed dog population.The present work demonstrated an elevation of blood pressure throughout pregnancy, with the highest values observed in early pregnancy. Cardiac output increased during pregnancy, probably due to increased heart rate. Pregnancy, as a chronic, natural volume-overload state, also caused alterations on TDI parameters, where S’, E’ and A’ values increased with advanced gestation. On the other hand, the values obtained for MPI, in conjunction with other systolic function parameters, suggested an improvement of cardiac function throughout gestation in a large breed dog such as the Saint Bernard. In the future, increasing the sample size, or even including different breeds of the same size, would help to better describe these cardiac adaptations. It would also be interesting to study animals with different obstetric complications, to deepen the knowledge already published ; also, t


The aim of the study was to explore the relationship between vitamin D status and islet function in patients with type 2 diabetes mellitus.The participants were recruited from Hebei General Hospital. Basic characteristics and blood indicators were collected after fasting overnight. The data were analyzed statistically using SPSS 22.0. Analysis of variance, a nonparametric test, or a trend Chi-square test was used for the comparisons. The association between 25-hydroxy vitamin D and modified homeostasis model assessment-β was assessed using multivariate ordinal logistic regression.One hundred seventy-four patients aged 26 to 79 years with type 2 diabetes mellitus were included in this study. Patients with vitamin D deficiency had a lower modified homeostasis model assessment-β level compared with those without vitamin D deficiency. There were differences in body mass index, diabetes course, glycosylated hemoglobin, fasting blood glucose, fasting blood C-peptide, triglyceride, and 25-hydroxy vitamin D among different modified homeostasis model assessment-β groups based upon the tertiles. 25-hydroxy vitamin D, as continuous or categorical variables, was positively related to modified homeostasis model assessment-β whether or not cofounding factors were adjusted.There is an association between increased 25-hydroxy vitamin D levels and improvement in modified homeostasis model assessment-β function in patients with type 2 diabetes mellitus.ChiCTR2000029391, Registration Date: 29/01/2020.Cross-sectional trails The online version contains supplementary material available at 10.1186/s12902-021-00862-y. Widespread type 2 diabetes mellitus (T2DM) has caused a burden worldwide. Evaluation of islet function is important for patients with T2DM because it is usually used to assess the progress of T2DM and to guide drug usage. Traditionally, homeostasis model assessment (HOMA)-β has been used to evaluate islet function using fasting blood glucose (FBG) and insulin levels , but thiThe relationship between vitamin D and numerous diseases including metabolic abnormalities such as insulin resistance (IR) and T2DM has been identified over time . It is gPrevious studies explored the relationship between vitamin D status and islet function using traditional HOMA-β. The aim of the present study was to explore the association between 25OHD and the modified HOMA-β in patients with T2DM.This was a cross-sectional study. This study was registered at Clinical Trial  and approved by the Hebei General Hospital Ethics Committee. It was performed in accordance with the Declaration of Helsinki.n = 69) and vitamin D deficiency group (n = 105). According to tertiles, modified HOMA-β was grouped three groups and 58 participants were included in each group.The population was from the Department of Endocrinology of Hebei General Hospital, China. All participants were hospitalized between June 2018 and December 2019. We included adult patients (≥18 years) with T2DM. A diagnosis of diabetes mellitus (DM) was made on the basis of the World Health Organization definition, as follows: FPG level ≥ 7.0 mmol/L or 2-h post-load value ≥11.1 mmol/L. Patients who had incomplete data or were diagnosed with severe diseases  or infectious diseases  within 3 months were excluded. Two hundred and forty-eight patients were enrolled and 174 patients were included in this cross-sectional study. On the basis of vitamin D deficiency, the participants were grouped into control group (2), as follows: weight (kg) / height2 (m2). Height and weight were rounded up to the nearest 0.1 and the mean value was obtained by measuring twice and taking the average of the two measurements.Basic data were collected by questionnaire, which included sex, age, DM course, diabetes history, smoking, alcohol drinking, hypertension, and insulin use. Height in cm and weight in kg were measured without shoes and with light clothes, respectively, and the body mass index (BMI) was calculated using the body mass index , and bone turnover biomarkers . The modified HOMA-β was calculated as follows: 270 × FCP / (FBG − 3.5) × 0.333 (FCP in ng/mL and FBG in mmol/L).All data were inputted by one researcher and checked by a second researcher. If errors were observed, data were checked and revised under two researchers’ supervision.P-value < 0.1. We used multivariate ordinal logistic regression analysis to explore the relationship of vitamin D and modified HOMA-β. Results were shown as the odds ratio (OR) and 95% confidence interval (95%CI). In Model 1, no cofounding factors were adjusted. In Model 2, the DM course, age, and BMI were adjusted. In Model 3, the DM course, age, BMI, HbA1C, TG, β-CTX, and PINP were adjusted. A P-value < 0.05 was considered to be statistically significant.The data were analyzed statistically using SPSS 22.0 . The modified HOMA-β results were grouped into tertiles. Differences in the measurement data were compared using an analysis of variance (ANOVA) if the data had a normal distribution, and these data were expressed as the mean ± standard deviation (SD). A nonparametric test was used if the data had a skewed distribution, and these data were expressed as the median (interquartile range). A trend Chi-square test was used for counting data, and these data were expressed as the number (percentage). The association between 25OHD and modified HOMA-β was assessed using linear correlation analysis. After comparisons among different groups, cofounding factors were screened based on a A flow diagram for patient recruitment is shown in Fig. p = 0.027; Fig. Vitamin D deficiency was defined as 25OHD < 20 ng/mL. On the basis of the presence or absence of vitamin D deficiency, the modified HOMA-β level decreased significantly in the vitamin D deficiency group compared with the control group (no vitamin D) . There were differences in the DM course among different modified HOMA-β groups . Compared with T1 group, the HbA1c level was decreased in the other two groups . The FBG level decreased from the T1 group to the T3 group , and FCP showed the opposite trend . TG and 25OHD levels were higher in the T3 group than in the T1 and T2 groups . We screened BMI, DM course, age, HbA1c, TG, 25OHD, β-CTX, and P1NP as confounding factors on the basis of P < 0.1. Because FBG and FCP were needed to calculate the modified HOMA-β, they were not seen as confounding factors.Table r = 0.199, P = 0.009; Fig. Linear correlation analysis showed that as the 25OHD level increased, the modified HOMA-β level also increased   levels are essential for insulin action. Vitamin D is involved in regulating Ca2+ flux in pancreatic cells [2D3 improves glucose metabolism via the SIRT1/IRS1/GLUT4 signaling cascade in C2C12 myotubes [The mechanisms of vitamin D that regulate islet function may be involved in several aspects. First, the vitamin D receptor (VDR) was expressed in pancreatic β cells, and vitamin D can exert its effect by directly binding to the VDR . Mice laic cells . Fourth,ic cells . For examyotubes . In addimyotubes , sub-infmyotubes , and epimyotubes .The hyperinsulinemic–euglycemic clamp is the gold standard that is currently used to quantitatively evaluate insulin secretion and IR. Its accuracy and stability are widely recognized. However, because of the invasiveness of surgery and technical difficulties, its use has been limited. In clinical studies, HOMA-IR and HOMA-β are an evaluation method that uses fasting glucose and insulin levels. Patients with T2DM, especially patients with poor islet function, usually inject insulin subcutaneously to have good glycemic control. Therefore, the modified HOMA-β is need for these patients.The current study has some limitations. First, for cross-sectional studies, our sample size was small. The results are more convincing when there is a large sample size in a cross-sectional observational study. Second, exposure to sunshine, physical exercise, vitamin D, calcium intake, or drugs that treat osteoporosis may be confounding factors, but we did not take these factors into account. Third, because of this study type, we could not obtain a causal relationship between vitamin D and islet function. Fourth, patients with or without insulin therapy were all included in this study. It is more appropriate to recruit patients without insulin therapy to compare the traditional HOMA-β and modified HOMA-β.In conclusion, taking all the potential confounding factors into account, the vitamin D status and modified HOMA-β showed a positive relationship. Thus, an increase in or supplementation of vitamin D may be related to improved modified HOMA-β function and delayed pancreatic β cell functional decline.Additional file 1. Questionnaire.


In vivo pharmacokinetic and pharmacodynamic evaluation of ODTs showed better behavior compared to the raw drug suspension and the commercial tablet (p > 0.05). Orally disintegrating tablets revealed a promising potential to improve Pioglitazone poor aqueous solubility, dissolution profile and bioavailability.Pioglitazone Hydrochloride (PGZ) suffers from poor aqueous solubility. The aim of this research was to design orally disintegrating tablets with self-nanoemulsifying properties (T-SNEDDS) to improve the Pioglitazone solubility and dissolution rate. Three liquid self-nanoemulsifying systems (L-SNEDDS) were formulated and evaluated for transmittance percentage, emulsification time, particle size, Poly dispersity index (PDI), percentage of content, solubility and stability. The optimum L-SNEDDS formula was converted to a solidified self-nanoemulsifying drug delivery system (S-SNEDDS) by adsorption on Syloid (SYL). Powder characterization tests, such as flowability tests, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM), were performed for the selected S-SNEDDS formulation. Orally disintegrating tablets (ODT) were formulated by blending S-SNEDDS with tableting excipients. The ODT tablet batch composed of Prosolv was selected for tablet quality control tests, such as hardness, friability, disintegration time, content uniformity, weight variation, in vitro release, in vivo studies and accelerated stability studies. ODT tablets showed accepted mechanical properties and rapid disintegration time (<30 s). No drug degradation was observed at 3 months into the accelerated stability study. The optimized L-SNEDDS, S-SNEDDS and ODT (T-SNEDDS), showed significant enhancement of PGZ in vitro dissolution profiles compared to the pure drug ( Pioglitazone HCl (PGZ) is an antidiabetic drug with an insulin-sensitizing effect. It binds to peroxisome proliferator-activated receptor gamma (PPAR-γ) that increases the transcription of insulin-responsive genes, thus improves tissue sensitivity towards insulin. PGZ is classified as class II under the biopharmaceutics classification system (BCS), meaning it is poorly soluble and highly permeable. Its poor aqueous solubility 0.015 mg/mL) has been a major constrain that hinders its dissolution rate, resulting in therapeutic level failure [5 mg/mL ho/w) emulsion once agitated with the aqueous phase at a low energy requirement. Upon oral administration, the stomach’s digestive motility provides the necessary agitation for self-emulsification, resulting in a nanosized emulsion (20–200 nm) with a large interfacial area for drug absorption [Liquid self-emulsifying drug delivery systems (L-SNEDDS) have recently gained importance in the field of pharmaceutical technology for enhancement of solubility and bioavailability of orally administered poorly water-soluble drugs (PWSDs). They are isotropic mixtures of an oil, a surfactant and a cosurfactant . Their psorption . Howeversorption . Additiosorption .®244 FP. Last, S-SNEDDS was further formulated into orally disintegrating tablets (T-SNEDDS) that have a self-nanoemulsifying nature in aqueous gastrointestinal media. The optimized tablets were evaluated by tablet quality control tests, in vitro dissolution, in vivo studies and stability studies.Solidification of L-SNEDDS into S-SNEDDS is a remarkable approach to solve such problems and provides formula stabilization benefits . Porous TM 90, TranscutolTM HP, Labrafil®M1944CS and Labrasol® were purchased from Gattefossé . Capmul MCM was obtained by Abitec . Olive oil and Soybean oil were gifted from Croda . Tween® 80 and Oleic acid were acquired from Sigma–Aldrich . Propylene glycol (PG) was obtained from Winlab . Cremophor® EL and Cremophor® RH 40 were supplied from BASF . Pure gelatin capsules (size 000) were supplied by the Center of Applied Research and Advanced Studies, Faculty of Pharmacy, Cairo University (CARAS). Silica powders, Aerosil 200®, Syloid® 244 FP (SYL), Neusilin® grades US2 and UFL2 were donated from Fuji Chemical Industries . Prosolv® ODT G2, mannitol, fructose and crospovidone were gifted by GRS pharma . All other chemicals were of analytical grade.Pioglitazone HCl (PGZ) (purity > 99.5%) was obtained by Riyadh Pharma manufacturer . Capryol® RH40, Labrafil® M1944CS, Labrasol®, Tween®80 and Cremophore® EL), and cosurfactants (Transcutol HP and Propylene glycol (PG)). According to the shake flask method described by Higuchi and Connors [The saturated equilibrium solubility of PGZ was examined in different oils , surfactants  as cosurfactants. For each selected SNEDDS system, oil/surfactant/cosurfactant were prepared at 36 predetermined ratios with a 10% interval for a total concentration of 100% w/w [Components utilized to construct the pseudo-ternary phase diagrams were chosen based on solubility study results. Accordingly, three phase diagrams were constructed using Caproyl 90 and Campul MCM as oily phases, Cremophore100% w/w . At each100% w/w ,15. ThesTwelve formulations were selected from each ternary phase plot, four from each system . For each 1 mL formula, 15 mg PGZ was dissolved in the accurately weighed amount of oil and cosurfactant, vortexed, then heated at 50 °C until the mixture became transparent, the mixture was left to cool down, then, the weighed amount of surfactant was further added and vortexed to obtain the final L-SNEDDS formulations. The compositions of PGZ loaded selected liquid self-nanoemulsifying formulations (L-SNEDDS) are shown in Selected SNEDDS preconcentrates were visually evaluated for their clarity after dilution and the percentage of transmittance was assessed spectrophotometrically at 638 nm using deionized water as a blank. Then, the time required for nano emulsion formation was recorded at 200 times dilution with 0.3 M HCl buffer solution under constant stirring at 100 rpm at 37 ± 0.5 °C . Later, Droplet size and polydispersity index (PDI) analysis were performed using Malvern Zetasizer  at 25 °C. Droplet size is crucial for self-emulsification, it affects the rate and extent of drug release, and is a good indication for an emulsion’s physical stability. PDI is an indication for uniformity of size distribution within the formula. The higher PDI values, the lower uniformity of the formulation, while closer to zero values indicate more homogenous emulsions. All samples were sonicated prior to droplet size and PDI determination.PGZ loaded SNEDDS formulations equivalent to 15 mg PGZ were tested for their content. 0.1 gm of each preparation was diluted to 25 mL methanol, vortexed, then analyzed spectrophotometrically at 269 nm using methanol as a blank.In vitro release studies were performed according to FDA specifications using the United States Pharmacopeia (USP) dissolution apparatus type II  coupled with a paddle stirrer at 75 rpm in 0.3 M HCl at 37 ± 0.5 °C (pH = 2) . One graIn order to assess the maximum PGZ loading ability of selected L-SNEDDS in systems I, II and III, a solubility experiment was performed. An excess amount of PGZ (100–500 mg) was added to one gram of each L-SNEDDS formulation. Mixtures were vortexed then placed in a shaker water bath adjusted at 37 ± 0.5 °C for 72 h. After equilibrium, mixtures were centrifuged at 4000 rpm for 10 min. The supernatant was then collected and measured spectrophotometrically after appropriate dilutions with methanol at 269 nm; results are shown in A three-phase thermodynamic stability test was conducted for L-SNEDDS formulations. The phase 1 test included three hearting-cooling cycles carried between 4 °C (refrigerator) and 50 °C (Oven) with storage at each temperature for 48 h. Passed formulations were then subjected to a phase 2 test, where centrifugation of formulations was carried out at 3500 rpm, 25 °C for 30 min. Formulations that did not show phase separation were subjected to a phase 3 test that included the storage of formulations at three freeze/thaw cycles between −21 °C (freeze) and +25 °C (thaw) for not less than 48 h . Results® US2, Neusilin® UFL2, Syloid® 244FP, Aerosil® PH200) with a ratio of 1:0.5 (w/w) (L-SNEDDS: adsorbent) as shown in One gram of the optimized L-SNEDDS formula was added in a drop-wise manner over four types of adsorbent powders (Neusilinw/w) with the selected adsorbent Syloid (SYL). Then, powder flowability tests were performed for determination of powder micrometric properties, such as compressibility index (CI), Hausner ratio (HR) and angle of repose (θ)  . For thermotropic properties assessment, thermal analysis was carried out using Shimadzu differential scanning calorimeter . Samples of 3–5 mg were heated in aluminum pans at a scanning rate of 10 °C/min under dry nitrogen flow (20 mL/min) and over a temperature range of 0–230 °C. Empty aluminum pans were used as references. Differential scanning calorimetry (DSC) thermograms were recorded for pure PGZ, pure SYL and the S-SNEDDS formulation.−1 and the average of 16 scans was recorded.Fourier transform infrared (FTIR) spectroscopy was recorded for the characterization of crystallinity and polymorphic changes using a Shimadzu FTIR- 8400 spectrophotometer, Japan. FTIR Spectrum of pure PGZ was compared to the S-SNEDDS formulation. Samples were blended with potassium bromide powder. The test was conducted over a frequency range of 4000–400 with a resolution of 4 cmUsing an Ultima IV X-ray diffractometer , powder X-ray diffraction (PXRD) patterns of pure PGZ, pure SYL and the S-SNEDDS formulation were obtained for additional detection of crystallinity and polymorphic changes. Samples were analyzed using cobalt radiation (CuKa) at a voltage of 40 kV and a current of 40 mA. The scan step was 0.02° with a 2θ range of 5–70° at angular increment of 4 degrees/minute.Surface morphology was visualized using a Philips’s scanning electron microscope . Samples were fixed on an aluminum stub by a double-sided sticky disc of conductive carbon, then coated with a thin gold pallidum layer. The electron beam was scanned over the specimen to produce a digital image for pure SYL and S-SNEDDS formulation.S-SNEDDS powder containing SYL was blended with different tableting excipients; all ingredients were sieved and blended, then, compressed at a compression force of 6 kN (Kilonewton) using Shimadzu IR hydraulic press machine . Hardness of produced tablets was measured at each compression process. The composition of three prepared ODT batches is shown in The force needed to break a tablet was measured using the Copley Hardness Tester, Switzerland. For friability testing, 20 tablets were weighed accurately (Wi) and rotated using Erweka friabilator  for 4 min at a 25 rpm rotation speed. Then, tablets were weighed after performing the test (Wa). The friability percentage was calculated according to Equation (1). Another six tablets were placed individually in the baskets of the disintegration tester  containing the dissolution medium at 37 ± 0.5 °C. The time required for a tablet to pass totally through the basket mesh was recorded.In order to measure content uniformity, 10 tablets were crushed individually, dissolved in methanol, centrifuged, and filtered using a 0.45 µm syringe filter, then analyzed spectrophotometrically at 269 nm. Tablet weight variation was determined by weighing 10 tablets individually; the percentage of weight variation was calculated by dividing each tablet weight over the average weight of the 10 tablets.In vitro release for the selected ODT batch was tested in triplicate using the United States (USP) dissolution apparatus type II  at 37 ± 0.5 °C. Each ODT was placed in 900 mL 0.3 M HCl buffer (pH = 2). The paddle rotation speed was adjusted to 75 rpm. 5 mL samples were withdrawn at predetermined time intervals of 5, 10, 15, 30, 45, 60 and 120 min, collected, filtered using 0.45 µm syringe filter, replaced by fresh buffer and analyzed spectrophotometrically at 269 nm. n = 6); Group I , Group II (ODT), Group III (control) and Group IV (raw PGZ). Orally administered dose was equivalent to 30 mg/kg dissolved in 10% DMSO for the three groups  calculated upon a rat’s individual weight to avoid any possible toxicity. Approximately 0.5 mL blood samples were collected from rat tail veins into lithium heparinized test tubes at 0, 1, 2, 3, 4, 5, 6, 12 and 24 h post dose. Plasma was obtained by centrifugation at 14,000 rpm for 15 min. Then samples were stored in deep freezer at −80 °C. Sample analysis was performed according to the validated UPLC-MS/MS analytical method reported by Imran et, al [Twenty-four Wistar Albino male rats (200 ± 10 gm) were divided into four groups ; Group I , Amman, Jordan), Group II (ODT), Group III (+ve control) and Group IV (-ve control). Three days before conducting the experiment, diabetes was induced in groups I, II and III by a single intraperitoneal injection of aqueous Streptozocin (STZ) (50 mg/kg) solubilized in phosphate buffer pH 4.5. Blood glucose level was determined by a blood drop from the tail veins of the rats using the Accu-check blood glucose meter. Rats showing serum glucose levels above 200 mg/dL were considered diabetic. Diabetic rats received the calculated dose of the corresponding formula equivalent to 30 mg/kg through an oral gavage needle. Blood glucose level was measured at 1, 2, 4, 6, 12 and 24 h.Twenty-four Wistar Albino male rats (200 ± 10 gm) were divided into four groups  followed by post-hoc (LSD) tests were performed using SPSS 25 software to interpret significant difference between data obtained by in vitro, in vivo and stability tests; a Among oil materials, the highest PGZ solubility was attained by Caproyl 90 with a value of 44.69 mg/mL and Capmul MCM with a value of 32.93 mg/mL. Crempohore EL showed the best solubilizing potential among all the tested surfactants with a solubility of 48.38 mg/mL, while Propylene glycol (249.99 mg/mL) followed by Transcutol HP (109.38 mg/mL) showed the highest PGZ solubility as cosurfactants .w/w respectively (w/w), the same as to higher cosurfactant content (40–60% w/w) that resulted in mixtures with lower viscosities. However, all formulations were robust to 200 times dilution with 0.3 M HCl buffer; no signs of phase separation and precipitation after 24 h storage were detected.All selected formulations were clear (graded as A), except, formulations S18-i and S24-ii were bluish and translucent (graded as B) with oil amount of 30% and 40% ectively , while tThe mean droplet size of the formulations was shown in The maximum and minimum assays for drug content in all prepared L-SNEDDS formulations were 110.13% and 94.28%, respectively, which complies with the USP guidelines acceptance criteria (±15%).PGZ solubility in L-SNEDDS reflects the drug loading capacity. In that, a remarkable variability was observed owing to the formulation composition, specifically the type of and the amount of cosurfactant added rather than the type of oil . S14-i , high drug loading capacity, accepted stability and enhanced release profile . Accordi®244FP (SYL) was preferred over the other formulations to be utilized in further ODT tablet formulation.According to the in vitro release results obtained by S-SNEDDS formulations containing four different types of adsorbents a shown in As shown in Pure PGZ exhibited a sharp endothermic peak detected at 197.3 °C, corresponding to its melting point and its crystalline state, while, SYL had no characteristic endothermic peak due to its amorphous state . DSC the−1 for N–H stretching, whereas peaks at 2964.59 cm−1 and 2742.66 cm−1 represented the stretching of aliphatic C–H structure. A sharp absorption peak was observed at 1741.72 cm−1 that may be attributed to carbonyl C=O stretching structure vibration. A peak at 1510.26 cm−1 indicated the presence of C=N group and a peak at 693.5 cm−1 was attributed to the C–S group. The absence of PGZ characteristic peaks in the S-SNEDDS pattern confirmed that the drug was transformed into an amorphous state, and proves the complete adsorption of L-SNEDDS on the surface of the adsorbent molecules  contributing to its crystalline state. In contrast, XRPD patterns of pure SYL and S-SNEDDS were clearly distinct from that of the pure drug. Characteristic peaks of PGZ were not detectable in PGZ loaded S-SNEDDS, suggesting the conversion to an amorphous state .SEM study indicated that pure SYL showed spherical shaped rough particles with a small particle at the top of its surface a, while Three ODT tablet batches were formulated: ODT1, ODT2 and ODT3 . ODT1 coThe optimized ODT tablets showed weight variation, hardness, friability, percentage of content and disintegration time within the official limits . All tabp < 0.05). S14-iii formulation achieved 100% release after 60 min, as the behavior of the dissolution profile is directly proportional to the droplet size, and also to the solubility of the drug in the formulation, S14-iii showed nanosized droplets with the highest solubilizing capacity that would increase the drug absorption and thus bioavailability [Raw PGZ showed poor dissolution with 44% maximum release within 60 min run time, while all prepared liquid SNEDDS formulations had shown similar immediate release patterns of >80% after 10 min a–c. Accolability .® UFL2 and Aerosil® PH200, respectively among the other solidified formulations. However, the release was 92.7% attained by S-SNEDDS-1 composed of Neusilin US2® and 93.2% obtained by S-SNEDDS-3 composed of Syloid® 244FP, over 120 min run time (p > 0.05).The percentage of drug release from S-SNEDDS-2 and S-SNEDDS-4 with a release of 77.8% and 76.2% over 120 min run time, those formulations composed of Neusilinrun time . Hence, p < 0.05) (p > 0.05). However, the ODT tablet obtained a release of 88% of PGZ after 15 min and reached 100% after 30 min, which proved the solubility enhancement of PGZ by self-nanoemulsifying properties by a 2.5-fold increase in the release of raw PGZ (p < 0.05) (ODT tablets showed a significant higher release than the solidified S-SNEDDS formulation ( < 0.05) a. In add < 0.05) b (p > 0. < 0.05) c. p < 0.05) than in those treated not only with the raw drug suspension, but also the commercial product (max) after about 3 h for ODT formulation was 27.77 ± 2.55 µg/mL as compared to 17.40 ± 3.38 µg/mL and 12.91 ± 2.24 µg/mL for the commercial product and raw drug suspension, respectively. Corresponding AUC values of ODT formulation showed 1.69- and 2.06-fold increase in the bioavailability as compared to the commercial product and raw drug suspension, respectively (max) values among the three formulations with values of 7 h and 3 h, respectively (p > 0.05).Drug plasma concentration in rats receiving ODT (T-SNEDDS) formulation was shown to be significantly higher ( product . Maximumectively . There wp < 0.05). Similarly, the commercial product (ACTOS®) attained a significant reduction (p < 0.05) in blood glucose level when it was compared to the diabetic control group (p > 0.05).A significant reduction in blood glucose level was observed with ODT formulation as compared to the STZ diabetic control group (ol group . The maxp > 0.05). All tested tablets showed no changes in their physical appearance.It was shown that T-SNEDDS were able to keep >95% of intact PGZ after 3 months storage at 25 ± 2 °C, 60 ± 5% RH and 40 ± 2 °C, 75 ± 5% conditions . There wp = 2.3) favors solubilization in small or medium molecular structures of fatty acids and/or mono- or diglycerides with their high ester content [Self-nanoemulsifying formulations should be liquid monophasic systems of an oil-surfactant-cosurfactant mixture to present the drug in its solubilized form . Therefo content , amphiphw/w [During the development of L-SNEDDS, psuedoternary diagram data revealed that wider phase regions in system I > system II > system III indicated a better self-emulsification ability , this waw/w . Moreovew/w ,39. Howew/w . As repow/w .p value > 0.05), where Capryol 90 was utilized as an oil phase. There was a marginal difference  in the droplet sizes in system III; Capmul MCM was utilized as the oil phase, the composition of the monoglycerides and free fatty acids contributed to the higher droplet size within the formulation [Emulsion droplet size (PS) is an important index that reflects the rate and extent of drug release and consequently its absorption . A good mulation . In rega® 244FP (380 m2/g) > Neusilin US2® and Neusilin® UFL2 (300 m2/g) > Aerosil® PH200 (200 m2/g) [2) is associated with more hydrophobic surface properties, which resulted in a decreased moisture uptake, as compared to the mesoporous structure of Mg–Al-silicates i.e., Syloid that have higher adsorption capacities and improved moisture uptake, that in turn improves the dissolution rate of the corresponding formulation [For solidified SNEDDS, the slower release of PGZ from S-SNEDDS as compared to L-SNEDDS counterparts was attributed to the physical interaction of L-SNEDDS carrying the drug with the hydrophobic structures of silica particles within the adsorbent powder ,47. Upon00 m2/g) . Furthermulation ,46. AccoODT formulation using Prosolv showed excellent tableting characteristics and enhancement of the PGZ release profile; the tablets disintegrated rapidly to release the self-nanoemulsifying formula where PGZ is solubilized upon exposure to GI fluids.max and AUC values for ODT formulation as compared to the raw drug suspension (p < 0.05). Moreover, the pharmacokinetic parameters obtained in the prepared ODT tablet were compared to the relevant commercially available film coated tablet (Actos®) and it was found that the prepared ODT formulation attained significant superior bioavailability (p < 0.05). Similarly, the correlated pharmacodynamic data showed a significant decrease in blood glucose levels with ODT formulation as compared to the commercially available tablet and raw drug suspension (p < 0.05). However, stability studies for ODT formulation showed no drug degradation or change in tablet characteristics.In vivo data revealed a significant improvement in PGZ pharmacokinetic profile in terms of Cmax and AUC values than those of the commercially available tablet and raw drug suspension. The collective mechanism of self-nanoemulsifying drug delivery systems and orally disintegrating tablets led to improved solubility, stability, absorption and bioavailability of Pioglitazone Hydrochloride.Twelve PGZ liquid SNEDDS formulations were developed in this study. In that, the best formulation was selected with 20% Campul MCM, 20% Cremophore EL and 60% Propylene glycol, as an oil, a surfactant and a cosurfactant, respectively. The corresponding droplet size (121 nm) was less than 200 nm and a PDI value of 0.379 indicated the formation of emulsion with nanosized droplets and a uniform distribution. Moreover, it was stable against all phases of tested stability conditions and provided the maximum drug loading capacity. Solidification of the optimized L-SNEDDS into S-SNEDDS using SYL was successful in producing a freely flowing powder, free from drug interactions. However, the incorporation of S-SNEDDS into an ODT tablet was carried out using various tableting excipients. The selected ODT batch formulated with Prosolv was able to pass tablet quality control tests and stability tests. The ODT release profile was comparable to that of the marketed product and 2.5 folds higher than the raw drug. In vivo presentation of the optimized ODT batch showed evidence of improved bioavailability by higher C


Halomicrobium sp. IBSBa (HmcIsc). The HmcIsc enzyme was recombinantly expressed and purified in Escherichia coli and shown to synthesize inulin as proven by nuclear magnetic resonance (NMR) analysis. In accordance with the halophilic lifestyle of its native host, the enzyme showed maximum activity at very high NaCl concentrations (3.5 M), with specific adaptations for that purpose. Phylogenetic analyses suggested that Archaeal inulosucrases have been acquired from halophilic bacilli through horizontal gene transfer, with a HX(H/F)T motif evolving further into a HXHT motif, together with a unique D residue creating the onset of a specific alternative acceptor binding groove. This work uncovers a novel area in fructan research, highlighting unexplored aspects of life in hypersaline habitats, and raising questions about the general physiological relevance of inulosucrases and their products in nature.Fructans are fructose-based (poly)saccharides with inulin and levan being the best-known ones. Thanks to their health-related benefits, inulin-type fructans have been under the focus of scientific and industrial communities, though mostly represented by plant-based inulins, and rarely by microbial ones. Recently, it was discovered that some extremely halophilic Archaea are also able to synthesize fructans. Here, we describe the first in-depth functional and molecular characterization of an Archaeal inulosucrase from  Pyrolobus fumarii holding the upper temperature limit for life , like organism  and Picrund pH 0 . Howeverund pH 0 . HoweverHitherto, there had not been much hard evidence that the Archaea superkingdom harbours genuine pathogenic organisms according to Koch’s disease postulates . On the Lactobacillus johnsonii EPS on bacteria-host interaction in the context of the gastrointestinal tract [Microbes can build different types of extracellular carbohydrate polymers in their outer layers as part of their ExoPolymeric Substances (EPS), for purposes of overall protection and/or as a temporal energy reserve , althougal tract .One of the most intriguing classes of carbohydrate-type EPS are the fructans, which are fructose (Fru) polymers built from sucrose (Suc)  and inulins  that are produced by single enzymes termed levansucrases and inulosucrases, respectively ,30. Typifructans . Howeverfructans . Levansufructans . While mfructans . All thefructans ,37,38,39fructans .Halomonas smyrnensis  or HmcIsc (protein) from this point onwards.Following the characterization of the first halophilic levansucrase from  , and as . We fulHmc. sp. IBSBa was originally isolated from crude salt samples of Tuz Lake, Turkey [4.7H2O, 2 g/L KCl, 3 g/L Na3 citrate, 5 g/L casamino acid, 5 g/L yeast extract, 1 g/L glutamic acid, 36 mg/L FeCl2.4H2O and 0.36 mg/L MnCl2.4H2O. pH was adjusted to 7.0 and the medium was autoclaved at 121 °C for 15 min. Fifty mL of the medium in a 250 mL Erlenmeyer flask was inoculated with a frozen culture stock at 1% (v/v) ratio and left to grow at 45 °C and 180 rpm for 4–5 days., Turkey . DSMZ MeEscherichia coli cultures, Luria-Bertani (LB) medium was prepared with 10 g/L NaCl, 10 g/L peptone and 5 g/L yeast extract. Medium was autoclaved at 121 °C for 15 min. Antibiotics (kanamycin or chloramphenicol) were added after sterilization for the selection of transformants. Cultures were incubated at 37 °C and 180 rpm.For Hmc. sp IBSBa was extracted using DNeasy® UltraClean® Microbial Kit  following the manufacturer’s instructions and used as a template for PCR amplification of the inulosucrase HmcIsc gene. The Whole Genome Shotgun project was deposited at DDBJ/ENA/GenBank under the accession JADPQC000000000. The version described in this paper is version JADPQC010000000. Primers were designed for directional cloning  and 17 µL nuclease-free water. The thermocycling conditions were 30 s initial denaturation at 98 °C in a VWR® PCR Thermal Cycler XT96 , followed by 30 cycles with 10 s denaturation at 98 °C, 30 s annealing at 69 °C for C-terminal/66 °C for N-terminal, and 35 s extension at 72 °C, with a final extension of 2 min at 72 °C. PCR products at expected size and quantity were purified by the Wizard® SV Gel and PCR Clean-up System  and then digested with respective restriction enzymes. After digestion, samples were incubated at 80 °C for 20 min to inactivate the enzymes then put on ice, run on a 0.8% agarose gel, and digested bands were cut and subjected to clean-up.The genomic DNA of  cloning . Both C-Vector DNA pET-28a(+) and insert DNA  were ligated in a 20 µL reaction mixture that contained T4 DNA Ligase Buffer (1X), 50 ng vector DNA, 36.79 ng insert DNA and 0.02 U/µL New England BioLabs T4 DNA ligase at final concentrations. Ligation samples were mixed gently and incubated at room temperature for 30 min. Samples were incubated at 16 °C overnight. Heat inactivation was carried out at 65 °C for 10 min, and samples were stored at 4 °C until transformation.HmcIsc was used to transform Invitrogen One Shot E. coli TOP10 Chemically Competent Cells . Mixtures were incubated on ice for 30 min, followed by a heat shock at 42 °C for 45 s, and placed back on ice for 2 min. LB medium (250 µL) was added to the cell mixture and incubated at 37 °C . 100 µL of each transformation was plated onto LB agar plates with kanamycin (50 ng/µL). Plates were incubated overnight at 37 °C. Plasmids were isolated from transformed E. coli TOP10 cells using QIAprep Spin Miniprep Kit  following manufacturer’s instructions.To multiply and store the vector, 5 µL of pET-28a(+) plasmid containing ® Buffer (Promega), 0.3 µM GH68 forward primer, 0.3 µM GH68 reverse primer, 0.3 mM dNTPs and 0.02 U/µL Promega GoTaq® DNA Polymerase. The thermocycling conditions were 3 min initial denaturation at 95 °C in a VWR® PCR Thermal Cycler XT96, followed by 30 cycles with 50 s denaturation at 95 °C, 50 s annealing at 63 °C, and 1 min 30 s extension at 72 °C, with a final extension of 10 min at 72 °C. PCR samples were run on 0.8% agarose gel. Successful results were sent to sequencing. Three µL of isolated pET-GH68C and pET-GH68N were used to transform to E. coli Rosetta(DE3) competent cells and transformants were selected on LB plates with kanamycin (50 ng/µL) and chloramphenicol (34 ng/µL).The GH68 family gene sequence with C-terminal and N-terminal 6xHis-Tag sequences were amplified by PCR on 0.17 µg of DNA of isolated pET-GH68C and pET-GH68N in a reaction mixture of 50 µL also containing 1X GoTaqHmcIsc inulosucrase, a preculture was prepared by picking a pET-GH68C transformant colony from LB selection agar and transferring it to 5 mL LB medium with kanamycin (50 ng/µL) and chloramphenicol (34 ng/µL), which was incubated at 37 °C and 180 rpm overnight. Two milliliters from the preculture were then transferred to two 200 mL of LB media with kanamycin (50 ng/µL) in 1 L Erlenmeyer flasks. Flasks were incubated at 37 °C and 180 rpm. Gene expression was induced by adding 1 mM of isopropyl β-D-l-thiogalactopyranoside (IPTG) when OD600 reached ~0.6. Cells were incubated at 20 °C with 180 rpm shaking overnight. An uninduced culture was used as negative control.For the production of the g and 4 °C for 20 min. Pellets were suspended in 5–6 mL of 20 mM potassium phosphate buffer with 300 mM NaCl (pH 6.6). Suspended cells were disrupted by ultrasonication (Bandelin Sonopuls UW 2200) with 28–31% of nominal power, 30 s on, 30 s off, 30 cycles on ice. Cell debris was removed by centrifugation at 8000× g for 10 min. Supernatant containing the HmcIsc inulosucrase enzyme was subjected to purification via immobilized metal affinity chromatography (IMAC) using nickel-nitrilotriacetic acid (Ni-NTA) agarose resin according to manufacturer’s instructions (QIAGEN). Briefly, equilibration, wash and elution buffers for IMAC contained 10, 20 and 300 mM imidazole, respectively, in 20 mM potassium phosphate buffer with 300 mM NaCl (pH 7.4). Fractions of 1 mL were collected and checked for protein content and HmcIsc enzyme activity.For the purification, cells were centrifuged at 10,000× ® Tetra Cell . Acrylamide concentrations of the separating and stacking gels were 10% and 5%, respectively. Coomassie Brilliant Blue R-250 was used to visualize the protein bands. PeqGOLD ProteinMarker I  was used as the protein ladder.To check the purity and molecular weight of protein fractions, a modified version of the standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protocol  was foll280 value of 1 g/L of HmcIsc is 1.7 . Protein concentrations were calculated by using this molar extinction coefficient. To determine HmcIsc enzyme activity, one volume of enzyme fraction was incubated in nine volumes of 50 mM potassium phosphate buffer (pH 7.0) with 0.15 M Suc and 3.5 M NaCl at 45 °C for 24 h. Activity of the enzyme was determined by measuring the concentration of released reducing sugars by the 3,5-dinitrosalicylic acid (DNS) method [Protein concentrations of the collected fractions were determined by measuring their absorbance at 280 nm via a BIO-RAD SmartSpec Plus spectrophotometer. According to ExPASy ProtParam, an A2 or MgSO4 to assess the effects of different salts at 3.5 M concentration each. To optimize salt concentration, enzyme activities of reactions with seven different NaCl concentrations between 0 M and 4.5 M were determined in the presence of 50 mM potassium phosphate buffer with 0.15 M Suc (pH 7.0). All reactions were incubated at 45 °C. Enzyme activities were determined via the DNS method at 24 h.Enzymatic reactions were carried out as described above with NaCl, KCl, KI, NaBr, MgClEnzymatic reactions were carried out at nine different pH values ranging from pH 4.0 to pH 9.0 in the presence of 0.15 M Suc and 3.5 M NaCl. 50 mM sodium acetate buffer for pH 4 and pH 5, 50 mM potassium phosphate buffer for pH 6 to pH 7.5, 50 mM Tris buffer for pH 8.0 and pH 8.5 and 50 mM carbonate-bicarbonate buffer for pH 9.0 were used. Reactions were carried out at 45 °C for 24 h. To analyze the effect of temperature, standard reactions were incubated at different temperatures . Enzyme activities were determined with DNS method at 24 h.2+, Fe2+, Ca2+, Cu2+, Co2+, Ni2+, Mg2+, Mn2+), detergents (SDS and Triton X-100) and EDTA on enzyme activity at 1 mM and 5 mM final concentrations each were investigated. Divalent cations were added to reactions as BaCl2, FeSO4, CaCl2, CuSO4, CoSO4, NiCl2, MgSO4, MnCl2, respectively, to give final metal ion concentrations of 1 mM and 5 mM. Standard enzymatic reactions were run to assess activity.Effects of various divalent cations  at 45 °C for 24 h. The DNS method was used to determine enzyme activities. GraphPad Prism5 software  was used to obtain a kinetic model.HmcIsc enzyme and cooled on ice. Two volumes of ice-cold ethanol were added to the mixture and mixed immediately for precipitation overnight at −20 °C, followed by centrifugation at 8000× g for 10 min. The pellet still contained some small sugars and precipitated salts, which were progressively removed by five freeze/thaw cycles in 20% ethanol. The pellet was dissolved in 100 µL water and ethanol was mostly removed through Speedvac.A standard enzymatic reaction in a total volume of 8 mL was carried out in 50 mM potassium phosphate buffer with 3.5 M NaCl and 0.15 M Suc (pH 7.0) at 45 °C for 4–5 days. The mixture was boiled at 90 °C for 5 min to denature the Nuclear Magnetic Resonance (NMR) spectroscopy was applied as described  to deterHmcIsc inulosucrase was created through the multitemplate protein structure prediction tool I-TASSER [www.glycam.org, accessed date: 1 February 2021; [The homology model of the I-TASSER ,45,46 usry 2021; ) and modry 2021;  using ChThe model with the glycosylation chain was created through GOLD covalent docking, minimized and analyzed with MOE. The top five solutions were analyzed to find all the amino acids interacting with the glycosyl residues. All figures were prepared using PYMOL 2.4.Nucleotide sequences from fourteen species of systematically important Haloarchaea, Gram-positive and Gram-negative bacteria were selected from NCBI for the family GH68 and the well-known molecular marker 16S rRNA. The sequences were downloaded manually, converted to FASTA files and aligned in Clustal W . The aliHalomicrobium strains [www.cazy.org/GH68_archaea.htmL, Access Date: 01/02/2021), although there are exceptions. It should be noted that not all Haloarchaean species are fructanogenic though, suggesting that other types of EPS may be able to take over the function of fructans [www.cazy.org/GH70.htmL, Access Date: 01/02/2021). This indicates that fructans, rather than glucans, play important roles in salt-loving organisms, as suggested before [After isolation of different fructan producing  strains , the genfructans ,23. Howefructans ) could nd before .HmcIsc (1305 bp including the stop codon) encodes the HmcIsc inulosucrase enzyme consisting of 434 amino acids with a predicted MW of 48.2 kDa and a isoelectric point (pI) of 4.58. Two putative N-glycosylation sites (NGS and NDS) were identified . HmcIsc is a highly negatively charged/hydrophilic enzyme, in line with other extracellular haloenzymes [HmcIsc (1.94) is similar to the one observed for the halophilic levansucrase for the bacterium H. smyrnensis (2.13) but much higher than the one recorded for Bacillus subtilis levansucrase (1.10). Strikingly, the K/R ratio differed strongly between HmcIsc (0.06) and the bacterial levansucrases from H. smyrnensis (1.14) and B. subtilis (4.78), respectively. Although no signal peptide could be detected with SignalP 5.0 [Sulfolobus acidocaldarius [HmcIsc is in full accordance with the previously reported purification of inulin-type fructans from the Suc supplemented growth medium of Hmc. sp. IBSBa [The predicted ORF of oenzymes . The numnalP 5.0 , the PREnalP 5.0  predictealdarius . This prp. IBSBa .Halomicrobium spp. classify within the domain of the Archaea, kingdom Euryarchaeota, Stenosarchaea group, class Halobacteria, order Halobacteriales, family Haloarculaceae . Overalry 2021;  combinedry 2021;  leads tory 2021; , and notry 2021;  strengththanogen , possiblthanogen , has nevthanogen  and not thanogen . Here, wHmcIsc ORF. Besides a list of expected (putative) Haloarchaean GH68 sucrases such as those from Haloarcula sp. CBA1115, Halorubrum saccharovorum [Natronococcus amylolyticus [Halalkalicoccus jeotgali [Pontibacillus chungwhensis and Salipalidubacillus agaradhaerens), showed surprisingly high homology. Together with a series of selected structurally or functionally characterized sucrases from Gram-positive and Gram-negative origin, a phylogenetic tree was produced  strain, as advised [Halalkalicoccus jeotgali B3T inulosucrase.PCR amplification was performed from genomic DNA, followed by sticky end cloning into the pET-28a(+) vector, with N- (pET-GH68N) and C-terminal (pET-GH68C) His-tagged versions. Heterologous expression in  advised , for theE. coli of pET-GH68N and pET-GH68C showed that crude extracts of the former showed no enzymatic activity (Glc production from Suc). Therefore, for upscaling and purification, GH68C was selected. Growth and expression in E. coli occurred as described [HmcIsc right above the 45 kDa ladder on SDS-PAGE . Preliminary expression in escribed , compariSDS-PAGE . AlthougSDS-PAGE . For com .HmcIsc was incubated together with 150 mM Suc at pH 7.0 at 45 °C and Glc production was followed. The produced fructan after four days was ethanol precipitated, further purified and dissolved in water prior to NMR analysis. The 1D proton spectra of the fructans produced by the recombinant HmcIsc enzyme were compared to previously-reported spectra on inulin  and H. smyrnensis levan reference samples [HmcIsc produced inulin-type fructans from Suc, as expected, since Halomicrobium sp. IBSBa cultures produced inulin [From preliminary IBSBa growth experiments we learned that neutral to mild alkaline conditions and temperatures between 37 and 45 °C are suitable growth conditions (data not shown). We reasoned that such conditions would also be suitable for the extracellular sucrase under study. Therefore, the purified recombinant  samples . Slow tud inulin .HmcIsc enzyme was characterized comprehensively in terms of the effects of pH, temperature, salt and heavy metals on the enzyme activity as well as its kinetics by following Glc formation from Suc.While the recently reported Haloarchaean inulosucrase was not a subject of enzymatic characterization , here, tHmcIsc (7.0–7.5) was situated in this region, with rapid activity loss in the acidic region  and broad (between 40 °C and 60 °C)  b. In gen+ (114.25 g/L) and Cl− (201.67 g/L), being much higher than SO42− (19.85 g/L), Mg2+ (11.44 g/L), K+ (6.55 g/L) and Ca2+ (0.32 g/L) [HmcIsc activity  . When coactivity a, it is t extent a. Curiou+ 114. g/L and ntration b, reachiin vitro b. It sho2+, Co2+ and Ni2+ showed detrimental effects, and Ca2+, though an important cofactor for several GH sucrases [HmcIsc. Fe2+ progressively inhibited activity at enhanced concentrations. Remarkably, HmcIsc was not inhibited by Ba2+, in contrast to HsLsc which was completely inhibited by 5 mM Ba2+.sucrases , inhibitHmcIsc enzyme activity was followed as a function of time starting with 150 mM Suc, and reducing sugars were analyzed through the DNS method , and this will be considered in the future. However, the enzyme proved stable, and the obtained profiles fitted well with Michaelis-Menten kinetics  with a Vmax  and a Km of 38 ± 3 mM Suc, respectively. Contrastingly, Hill kinetics and much higher Vmax (296 U/mg protein) K0.5 (104.79 mM) values were observed for HsLsc [Leuconostoc citreum CW28 . Both ial cells . Taken tal cells  and muciK0.5 104. mM valueHmcIsc, could that be of physiological relevance for Halomicrobium spp. in Tuz Lake? These microorganisms are believed to occur in a free-living state in the upper layers of the lake. The only possible access to carbon (Suc) for growth and fructan formation would be through association with a photosynthetic Suc-producing partner [Halomicrobium spp. to adhere to such photosynthetic partners  and establishing such  relationships, which usually depends on a series of signal exchanges between the two partners over time. At extreme salt concentrations (3.5–5.0 M NaCl), biomolecular interactions between the two microorganisms may be much less dependent on specific protein:protein interactions, since this is a rather costly option in terms of ATP requirements. We speculate that Suc-dependent carbohydrate:carbohydrate interactions may become more interesting to serve as the glue between the two organisms. From the perspective of Halomicrobium, directly using the energy released from the extracellular splitting of symbiotic Suc to drive fructan synthesis is the energetically cheaper option. Indeed, ATP can be saved by limiting the amount of Suc importers, and especially by avoiding the synthesis of nucleoside diphosphate (NDP)-sugars and lipid carriers that are typically required for the synthesis of other exopolysaccharide types [Halomicrobium and used for ATP production, serving growth. Perhaps fructans are relatively more important compared to other exopolysaccharides in Haloarchaea, which remains an interesting topic for future research.Assuming that such low reaction rates would also occur for the native  partner ,23. Therde types . This maHmcIsc is still enzymatically active at 4.5 M NaCl, the free water at such concentrations becomes extremely scarce. Indeed, although the water binding capacities of fructans and NaCl are very similar [Halomicrobium spp. themselves or with their photosynthetic partners, potentially forming symbiotic microbial networks. Accordingly, it was observed before that the flocculation rate and exopolysaccharides both increased at increasing salt concentrations in halotolerant bacteria from the plant root context [Although extracellular  similar ,82, extr context . The ste context ,65 and r context . TherefoHmcIsc with its C-terminal His tag and further minimized according to the optimal working conditions for the enzyme (pH 7.0 and 3.5 M NaCl) and compared to the inulosucrase from Lactobacillus johnsonii , D200 (transition state stabilizer: RDP motif) and E272  resides within the central deep pocket of the five-bladed propeller  revealed no major changes in its overall structure (not shown). However, no less than 14 salt bridges were observed (A multitemplate model (ITASSER) was produced for i , Bacills , and Laosucrase , are parobserved  potentiaHmcIsc amino acid sequence, two classical NX(S/T) glycosylation sites (N239GS and N320DS) were detected 2-Methylated hexuronic acid-Mannose; [HmcIsc N320 residue and its mobility was studied  was ins studied . The sugble 2 in ). It is,L. johnsonii inulosucrase [2+ binding sites  and alpha helix secondary structures  .Contrary to the 3D structures of osucrase  and SacBructures a, HmcIscmotif)  were performed with Suc as donor substrate and DP6 inulin (GF5) as acceptor substrate. Donor substrate docking revealed that the binding of the Fru part of Suc at the -1 subsite was similar to all previously published poses. HmcIsc (a) and the SacB E342A mutant (b), while an overlay of the two poses is presented in C. The Fru at the -1 donor subsite (-1d) interacts with the nucleophile (D53/D86), the transition state stabilizer (D200/D247), and with a S (S338/S412) residue. When S345, the homologue of S338 in HmcIsc, was mutated to an A residue in Zymomonas mobilis levansucrase, an increase in the 1-kestotriose/6-kestotriose product ratio from Suc as only substrate was observed, while the opposite occurred for an A343S mutant in a GH32 member residing in the same operon of that organism [Considering the substrate specificity of organism .HmcIsc and HsLsc to SacB. S126 from SacB was replaced by A121/A131 in HmcIsc/HsLsc. Considering a multiple alignment of approved and potential Haloarchean inulosucrases and GH68 members of Gram-positive organisms, their ancestors serving as donors through HGT, the A121 homologue is strictly conserved  subsite followed by the +2a till +5a Fru subsites and terminating with the +6a Glc subsite , but thEIER motif, and R199 from the RDP motif, all involved in interactions with the Suc donor, also interacted with several Fru residues at different acceptor subsites  inulosucrases and their closest homologues in Gram-positive bacilli, tentatively termed as the “inulosucrase subfamily” for this purpose . E270 ansubsites . In partsubsites , conservsubsites . H290 anrmediate . When N8organism . This obstriking . Althougstriking .Bacillus sp. SB49 [S. agaradhaerens inulosucrase  inulosucrases carrying typical signatures. Its low substrate conversion rate raised questions on sucrose availability and the physiological function of the generated inulin-type fructans in symbiotic events in salt lakes. By extension, thought provoking questions were raised on the potential roles of inulosucrases and their products in the human body  and in food fermentation processes. This work suggests that inulosucrases, as compared to levansucrases, are more widespread than originally thought, and have been largely underexplored. Our combined enzymatic and structural analyses provide tools to better discriminate between putative inulosucrases and levansucrases in the future. The discovery of a putative alternative acceptor binding groove will boost further research into product linkage type and degree of polymerization within GH68 and, by extension, in GH32, also comprising plant fructosyltransferases. These fundamental insights will allow, through rational design, the development of novel enzymes that can provide us with novel types of fructans for specific industrial or agronomical applications.


After stagnation, total cell counts were not significantly different among seasons; however, the composition of the microbial community varied seasonally. The relative abundance of Pseudomonas spp. was dominant in winter, whereas Sphingomonas spp. were dominant in most faucets after stagnation in other seasons. Opportunistic pathogens, such as Legionella pneumophila, Mycobacterium avium, Pseudomonas aeruginosa, and Acanthamoeba spp., were below the quantification limit for real-time quantitative PCR in all samples. However, sequences related to other opportunistic pathogens, including L. feeleii, L. maceachernii, L. micdadei, M. paragordonae, M. gordonae, and M. haemophilum, were detected. These results indicate that health risks may increase after stagnation due to the regrowth of opportunistic pathogens.In premise plumbing, microbial water quality may deteriorate under certain conditions, such as stagnation. Stagnation results in a loss of disinfectant residual, which may lead to the regrowth of microorganisms, including opportunistic pathogens. In the present study, microbial regrowth was investigated at eight faucets in a building over four seasons in one year. Water samples were obtained before and after 24 h of stagnation. In the first 100 mL after stagnation, total cell counts measured by flow cytometry increased 14- to 220-fold with a simultaneous decrease in free chlorine from 0.17–0.36 mg L However, stagnation, which is defined as intermittent water use at individual outlets for an extended period, may occur in premise plumbing, causing longer incubation times  to water-borne infections, including Legionnaires’ disease, Pontiac fever, and nontuberculous pulmonary disease , which is distributed to the faucets on each floor. Water is supplied from a water treatment plant that treats surface water by coagulation followed by sedimentation, primary rapid sand filtration, ozonation, biological activated carbon filtration, and secondary rapid sand filtration. The typical free chlorine concentration in treated water at the plant is 0.5–0.7 mg L–1 to maintain a free chlorine residual at faucets of >0.1 mg L–1, which is regulated by the Japanese drinking water quality guidelines.The present study was conducted at an 11-story building at the University of Tokyo, Tokyo, Japan. Drinking water samples were collected from eight cold water faucets (F1–F8) at the handwashing basins in laboratory rooms. The target faucets were located on the 3–1. The pre-stagnation sample represents drinking water samples with the minimized influence of stagnation. Afterwards, the faucets were closed for 24 h. Post-stagnation samples were collected from the same faucet after approximately 24 h of stagnation at a flow rate of approximately 5 L min–1. The first 2 L of the flushed post-stagnation sample was collected in incremental samples of 100 mL.To evaluate seasonal variations in microbial regrowth, pre- and post-stagnation samples were collected in four seasons. Samples were collected from eight faucets (F1–F8) in summer (June–July 2018) and from six faucets  in autumn (November 2018), winter (January 2019), and spring (April 2019) (52 samples) . Prior tDaily variations in stagnation-induced microbial growth were evaluated by repeating the above sample collection procedure at F1 on six consecutive days in April 2019.N,N-diethyl-p-phenylenediamine method (Hach), respectively. The limit of quantification (LOQ) of the free chlorine measurement was 0.02 mg L–1.Temperature and free chlorine were measured with a digital thermometer  and HACH chlorine pocket colorimetric according to the ® Green I (Invitrogen) diluted 100-fold with dimethylsulfoxide  was used as a working dye solution. Then, 490 μL of the sample was stained with 5 μL of the working dye solution and 5 μL of 0.5 mol L–1 EDTA (Invitrogen). In the analysis, 0.2-μm filtered samples were used as a blank. This mixture was vortexed and incubated at 37°C for 10 min under dark conditions and analyzed using a BD Accuri C6® flow cytometer (BD Biosciences). Electronic gating for bacterial cell quantification was selected on density plots of green fluorescence in the FL1 channel (535±15 nm) and red fluorescence in the FL3 channel (>670 nm)  . Constan–1, pH 7.2–7.4) . One hundred-microliter aliquots of non-diluted or diluted samples were spread on R2A agar medium  and incubated at 20°C for 7 days.HPC were enumerated using the spread plate method following Standard Method 9215 C . Water s2 , following a previously described method  (Sigma-Aldrich). The mixture of concentrates and elution buffer was circulated for 2 min to further recover cells from the HFUF cartridge. The final concentrated sample was filtered through a 0.2-μm polycarbonate membrane filter. Filters were stored at –20°C until DNA extraction.To collect the biomass from samples, the first 1 L of post-stagnation samples was filtered through 0.2-μm polycarbonate filters . Regarding pre-stagnation samples, 10 L of samples were concentrated using a hollow fiber ultrafiltration (HFUF) unit with a surface area of 25 m–1 for 40 s. The DNA extraction efficiency of this method evaluated using E. coli was 93.8±4.9% (n=3). A negative control experiment using milli-Q water revealed that DNA contamination was negligible.DNA was extracted from the filter using the FastDNA SPIN Kit for Soil  following the manufacturer’s instructions. Briefly, the filter was placed in a lysing matrix tube, and dissolved in phenol–chloroform–isoamyl alcohol (25:24:1) (Nippon Gene). Bead beating was performed with Fast-Prep  at a speed of 6.0 m sLegionella spp. (23S-5S rRNA spacer region), L. pneumophila (mip gene), Mycobacterium spp. (16S rRNA gene), M. avium (16S rRNA gene), Pseudomonas aeruginosa (regA gene), and Acanthamoeba spp. (18S rRNA gene) were quantified using TaqMan-based qPCR (® Probes Master solution (Roche Life Science), 0.1 μL of each primer (100 pmol μL–1), 0.1 μL of the probe (40 pmol μL–1), and 2 μL of the DNA template. Standards were prepared using a 10-fold serial dilution (5.0×100–5.0×106 copies μL–1) of an artificially synthesized plasmid containing the target genes (® LC480 system (Roche Life Science). PCR reaction efficiencies were in the range of 90.15–99.50% (The number of gene copies of et genes . LOQ was5–99.50% . PCR inhACACTCTTTCCCTACACGACGCTCTTCCGATCT-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-GGACTACHVGGGTWTCTAAT-3′) (http://hannonlab.cshl.edu/fastx_toolkit/) was applied to filter reads whose sequences had a perfect match with the primer sequences. The deletion of primer sequences, paired-end reads under 250 bases, and low qualified bases and the merging of paired-end reads were processed using Quantitative Insights into Microbial Ecology 2 (QIIME 2) : 515F . Primers with adapter sequences (underlined) were used to amplify full-length 16S rRNA genes: 16S-F (5′-ATCGCCTACCGTGAC-Barcode-AGAGTTTGATCMTGGCTCAG-3′) and 16S-R (5′-ATCGCCTACCGTGAC-Barcode-CGGTTACCTTGTTACGACTT-3′). The thermal conditions of the first PCR consisted of 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 65°C for 80 s, with a final extension at 65°C for 5 min. LongAmp Taq 2× Master Mix (New England BioLabs) was used for the PCR reaction. The library was loaded onto R9.4 flow cells on the GridION platform (Oxford Nanopore Technologies) at the Bioengineering Lab, Japan. Base-calling and de-barcoding were performed using GUPPY software (ver. 3.2.6) (Oxford Nanopore Technologies). Adapter sequences were trimmed using Porechop (ver. 0.2.3). The SeqIO module in Biopython was used to extract reads with 1,400–1,600 bp. After subsampling by Filtlong software (ver 0.2.0), 221,978–678,877 sequences per sample were obtained. The taxonomic assignment was performed with RDP_16S_V16_sp using Usearch (ver. 11.0.667).In addition to the Illumina MiSeq platform, DNA from 12 post-stagnation samples, which showed that the relative abundance of All sequence data in the present study were submitted to the DNA Data Bank Japan (DDBJ) Sequence Read Archive and are available under accession number DRA011523.t-test and an analysis of variance (ANOVA) followed by Tukey’s honestly significant difference test (HSD) were performed using R statistical software (ver. 3.4.1), and P<0.05 was considered to be significant. The relevant statistical test results, including P values, F values, and 95% CI, are shown in Statistical analyses of TCC, HPC, and qPCR data were performed after log transformation. The paired Student’s t-test, an ANOVA, and HSD test were performed. To identify any significant differences (P<0.05) in the microbial community between sample groups, an analysis of similarity (ANOSIM) was performed based on the Bray–Curtis dissimilarity index at the OTU level using the vegan package in R statistical software with 999 permutations. The value generated from random permutations  ranged between 0 (complete similarity) and 1 (complete dissimilarity). Additionally, hierarchical clustering at the OTU level was created with the vegan package (ver. 2.5-7) in R statistical software based on the Bray–Curtis dissimilarity index.Alpha diversity indices (observed richness and the Shannon diversity index) were calculated with the vegan package (ver. 2.5-7) in R statistical software. To evaluate the significance of differences in alpha-diversity indices between sample groups, the paired Student’s –1 free chlorine and 2.1×103 cells mL–1 TCC. After 24 h of stagnation, free chlorine in the first 100 mL decreased to <0.02 mg L–1, while TCC increased to 5.5×104 cells mL–1. The temperature pre-stagnation was 18.3°C and increased to 20.7°C in the first 100 mL after 24 h of stagnation   a. Microbon, n=6) b. These t-test, P=3.059×10–5), with the highest increase being 8.3°C for an F1 sample collected during winter (–1) was not significantly different among seasons, as confirmed with ANOVA test (P=0.365), the significant depletion of free chlorine to <0.02 mg L–1 in the first 100 mL sampled after stagnation  was consistently observed after stagnation for all samples . Several samples, such as F1  and F3 , exceeded the target levels for drinking water quality in Japan   . Average TCC levels post-stagnation were the highest in summer; however, differences were not significant . TCC levels peaked in the first 100 mL of water collected after stagnation in all samples. The first 100 mL contributed to 10–47% of all cells discharged in the first 2 L of post-stagnation samples.Culturable bacteria were not detected in pre-stagnation samples c. HPC inFU mL–1) . TCC in P=0.521) d. A signP=0.0299; Tukey’s HSD, P=0.039). OTU richness in post-stagnation samples in winter was significantly lower than in summer and spring . In winter, microbial communities pre- and post-stagnation were also less diverse; the Shannon index was significantly lower in winter than in other seasons . Average OTU richness decreased after stagnation in all seasons; however, the decrease was not significant in spring .Microbial community structures in pre- and post-stagnation samples were analyzed. An amplicon sequencing analysis revealed that 1,591 and 872 OTUs were recovered from pre- and post-stagnation samples, respectively, ranging between 110 and 339 OTUs per sample. Alpha diversity measured by OTU richness and the Shannon diversity index showed seasonal trends in pre- and post-stagnation samples . Winter P<0.05)  .Phreatobacter spp. were predominant with a relative abundance of 15–46%. The abundance of this genus was significantly lower in winter (1–3%). The shift in the microbial community after stagnation in F1, F2, F4, F5, and F6 in summer, autumn, and spring was characterized by a predominance of Sphingomonas spp. The microbial community after stagnation from these samples was clustered in post-stagnation cluster 1. Phreatobacter spp. decreased in abundance from 27 to 8%, while Sphingomonas spp. increased from 5 to 22%. On the other hand, the shift in dominant bacteria in F3, F7, and F8 after stagnation in summer, autumn, and spring (post-stagnation cluster 2) was generally characterized by genera in Comamonadaceae, Dechloromonas spp., Cyanobacteria, and Porphyrobacter spp. Differences in the microbial communities of post-stagnation samples between these two groups of samples in these seasons were supported by ANOSIM .The pre-stagnation cluster consisted of the most pre-stagnation samples in summer, autumn, and spring, in which Pseudomonas spp. were dominant in these samples, with a relative abundance of 37–59%. After stagnation, Pseudomonas spp. were still dominant at several faucets . F7 and F8 were grouped in post-stagnation cluster 2 after stagnation in winter, and the relative abundance of Pseudomonas spp. was higher (16–27%) than that of the other samples.Most pre-stagnation samples in winter were clustered in the winter cluster, and M. avium, L. pneumophila, P. aeruginosa, and Acanthamoeba spp., were below LOQ in all faucets before and after stagnation . On the other hand, Legionella spp. and Mycobacterium spp. significantly increased after stagnation in all seasons (Legionella spp. were detected in 62% (16/26) of samples collected from several faucets before stagnation. After stagnation, the detection frequency increased to 81% (21/26), and their abundance significantly increased from 9.6±5.0×10–1 to 9.7±4.1×100 gene copies mL–1 . Mycobacterium spp. were positive for all samples before and after stagnation (52/52), and their abundance significantly increased from 2.5±0.6×100 to 8.8±3.0×10 gene copies mL–1 . The abundance of Legionella spp. and Mycobacterium spp. post-stagnation was lower in summer; however, these differences were not significant .The abundance of opportunistic pathogens was quantified by real-time PCR. Clinically important opportunistic pathogens, including  seasons . qPCR reLegionella spp. and Mycobacterium spp. were subjected to nanopore sequencing targeting full-length 16S rRNA genes in order to obtain detailed phylogenetic information. On average, 60.7% of Legionella-related sequences were assigned to species in Legionella spp.  and F8 (winter) was less than that of other detected species, such as L. drozanskii. Other Legionella sequences, which were closely affiliated with pathogenic L. maceachernii and L. micdadei, were also detected with an average relative abundance of 0.9 and 0.02% of all Legionella-related sequences, respectively. On the other hand, only 18.7% of Mycobacterium-related sequences were assigned to species in Mycobacterium spp.  and HPC (8.7×102 CFU mL–1) were observed after overnight stagnation  has been suggested to affect the regrowth of bacteria in drinking water  from which samples were collected in four different seasons. TCC levels after stagnation were not significantly different between seasons. Higher TCC in the first 100 mL were consistently found in all samples, which indicated that the last meter of premise plumbing was vulnerable to microbial regrowth.Seasonal differences in microbial richness and diversity were observed in the present study, in which richness (observed OTUs) and the Shannon diversity index were significantly lower in winter. Previous studies also observed lower bacterial richness in winter and variations in the microbial community structure over time  than in the cold period (water temperature <15°C) in the drinking water distribution system in Paris   Distinct changes in community structures were observed before and after stagnation because the dominant species changed from Phreatobacter spp. to Sphingomonas spp., Comamonadaceae, Dechloromonas spp., Cyanobacteria, and Porphyrobacter spp. (3) Seasonal variations appeared to play a role in shaping the composition of the microbial community in drinking water, particularly in winter when Pseudomonas spp. were dominant. (4) Although clinically important opportunistic pathogens were not detected, several sequences, which were closely related to pathogenic Legionella spp. and Mycobacterium spp., were identified. Further guidelines regarding water usage practices in premise plumbing are essential for maintaining the good microbial water quality of drinking water prior to consumption.Microbial water quality was monitored for one year before and after stagnation in different faucets of the premise plumbing in a building. The main conclusions of the present study are as follows: (1) The depletion of free chlorine was observed in all faucets after stagnation, which resulted in a significant increase in TCC from 10Microbes Environ 37: ME21065.Rahmatika, I., Kurisu, F., Furumai, H., and Kasuga, I. (2022) Dynamics of the Microbial Community and Opportunistic Pathogens after Water Stagnation in the Premise Plumbing of a Building. https://doi.org/10.1264/jsme2.ME21065Supplementary Material