While testing the antimicrobial activity, Ch-NPs showed the least minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values exerting eightfold higher bactericidal activity than O3-oil against both Enterococcus faecalis and Streptococcus mutans as well as fourfold higher fungicidal activity against Candida albicans. Antimicrobial synergy test revealed synergism between O3-oil and Ch-NPs against the test pathogens (FIC index ≤ 0.5). Ch-NPs was superior at inhibiting immature single and mixed-species biofilm formations by 97 and 94%, respectively. Both of O3-oil and Ch-NPs had a complete anti-fibroblast adherent effect. The safety pattern results showed that O3-oil was the safest compound, followed by Ch-NPs. The double combination of Ch-NPs and O3-oil reduced the mature viable biofilm on premolars ex vivo model by 6-log reductions, with a fast kill rate, indicating potential use in treating root canals. Therefore, the double combination has the potential to eradicate mature mixed-species biofilms and hence it is potent, novel and safe.This study was conducted to investigate the antimicrobial-biofilm activity of chitosan (Ch-NPs), silver nanoparticles (Ag-NPs), ozonated olive oil (O The primary goal of endodontic therapy is to eradicate microbial infection and promote periapical tissue healing. Endodontic infections are polymicrobial and made up of various microorganisms that differ between failed endodontic cases and primary endodontic infections .Enterococcus faecalis and Streptococcus mutans, and Candida albicans are more common in failed or persistent endodontic infections  containing active oxygen in the form of peroxide ranged between 560–590 mmol-equiv/kg, prepared by introducing olive oil through a device generating ozone  were prepared.Stock solutions (100 mg/ml) of Ag-NPs in sterile distilled water, Ch-NPs in 5% glacial acetic acid (HAC) and OS. mutans ATCC 2419, E. faecalis OG1RF, and C. albicans MTCC 227, were used, which were obtained from the Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Tanta University, Egypt.Freeze-dried reference microorganisms, ® PCS-201-012TM) were obtained from the laboratory of the Tissue Culture Department of the Holding Company for Biological Products and Vaccines (VACSERA), Cairo, Egypt.Human Gingival Fibroblast cells . MIC was defined as the lowest concentration of each medicament that inhibited the microbial growth compared to the untreated control culture. MBC was determined by performing viable count (CFU/mL) following 24 h of incubation at 37°C and was defined as the lowest medicament concentration that reduced the viable cells by more than 3 log10 steps (>99.9%) compared to the untreated control cultures. Independent assays were performed in triplicates  along the columns. The same concentrations of Ch-NPs are present in the columns which was diluted twofold (from 2.5 to 0.0391 mg/ml) along the rows. The wells were inoculated by the test pathogen at 106 CFU/ml and the plates were incubated at 37°C for 24 h. For detecting the combined synergy effect, fractional inhibitory concentration (FIC) index was calculated as follows:The antimicrobial activity of the tested root canal disinfectants was examined by determining the minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and minimum fungicidal concentrations (MFCs) against endodontic pathogens using broth microdilution assay. The turbidity of each microbial suspension was adjusted to that of a 0.5 MacFarland standard then diluted in RPMI 1640 to give a final inoculum of 10plicates . The antWhere FIC A = MIC of drug A in the combination/MIC of drug A alone.FIC B = MIC of drug B in combination/MIC of drug B alone.3-oil/Ag-NPs was assessed by the same procedure.The results were interpreted as synergism if the FIC index ≤0.5; additive effect if the FIC index of >0.5 and ≤4; and antagonism if the FIC index >4. The procedure was replicated three times. Similarly, the antimicrobial synergy effect of Ch-NPs/Ag-NPs and O4 cell/ml) on 96-well plates. After 48 h, the cellular cytotoxic effects were quantified using the neutral red assay protocol (50) was calculated for each test medicament.The safety of the test compounds was evaluated on normal human fibroblasts. Briefly, approximately 100 μl of each of serially diluted compound was incubated with pre-cultured  were considered and inoculated medium without treatment was used as a positive control. After incubation at 37°C for 24 h, the planktonic cells were removed by aspiration of the spent media. All wells were washed twice using phosphate buffered saline solution (PBS) (pH 7.4) to remove loosely bound microbial cells and media components. Next, 125 μl of 0.1% crystal violet solution was added to each well to stain biofilm forming cells, incubated for 10 min at room temperature then rinsed three times with distilled water. For the quantitative assay, crystal violet was solubilized by adding 125 μl of 30% acetic acid to each well and incubated for 10 min at room temperature. The absorbance was measured for both the control and test groups by a plate reader at 550 nm with a reference filter of 570 nm.The effect of the test medicaments on the biofilm biomass was evaluated by crystal violet binding assay . Overnig5 cells/ml then incubated at 37°C in a 5% CO2 atmosphere until they formed semi-confluent monolayers. Twenty-four hours post-incubation, the exhausted media were replaced with fresh DMEM. Overnight cultures of the test microbes were standardized to a final inoculum of 107 CFU/ml using 0.5 MacFarland standard and confirmed by viable count. Approximately 50 μl of each standardized microbial cell cultures, pretreated with non-toxic doses of the medicaments, were incubated at 37°C for 3 h. After incubation, the medium was discarded and the wells were washed three times with PBS to remove the unbound microbial cells. One milliliter of methanol was added to each well for 1 min, then replaced with 4 ml of freshly diluted Giemsa stain (1:10) and incubated for 30 min at room temperature. The stain was discarded, and the fibroblast monolayers were washed sequentially with tap water, acidified water (1 ml concentrated H2SO4 in 1 L of tap water) and tap water again. The wells were examined at 20× magnification under a light microscope . The microbial strains were considered adherent if they formed characteristic microcolonies on >40% of the fibroblast cell surface. The data represented at least three trials (Fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  and 1% streptomycin/penicillin in 6-well plates  at approximately 1 × 10e trials .3-oil were selected due to antibiofilm activity at their IC50. The effect of the later medicaments, either separate or combined together, was evaluated on the mature multi-species biofilm ex vivo using extracted premolar teeth. The effect of DMSO or 5% HAC, as vehicles, on the microbial biofilm was also assessed.Among the tested medicaments, Ch-NPs and OA total of 168 intact single rooted human premolars with single straight root canals extracted for periodontal or orthodontic reasons were collected after obtaining the patients’ written consent. This was based on the protocol approved by The Research Ethics Committee at the Faculty of Dentistry, Tanta University, Egypt. The teeth were collected in 2.5% sodium hypochlorite solution then mechanically cleaned and decoronated under water coolant using a diamond disk . The step-back technique was used to chemomechanically debride the canals using stainless steel k-files  and 2.5% sodium hypochlorite as an irrigant with a master apical file size 40.The apical 3 mm as well as crown (2–3 mm) of each root specimen was cut using the diamond disk to obtain 12 mm-long standardized root specimens. The canal lumen for each specimen was prepared to a standardized size by passing rotating peeso drills, sizes 1 and 2, from the coronal end until just visible at the apical end. A 2 mm-deep cavity was prepared on the apical ends of each canal using #4 slow speed round bur (Dentsply/Maillefer) and sealed with acrylic resin to prevent micro leakage. The prepared root canals were conditioned with 10 mL of 17% EDTA  for 60 s to remove the smear layer, washed with 10 mL of 5.25% NaOCl, flushed with 10 mL of saline solution and dried with paper points . Teeth were subjected to sterilization by steam autoclave for 15 m at 121°C .6 CFU/ml using the 0.5 McFarland standard. Equal portions of each microbial suspension were mixed together in a Falcon tube to be used for generating multiple species biofilm. Premolar teeth were aseptically transferred to the tube containing the mixed microbial suspension, then incubated under anaerobic conditions at 37°C for 1 week to obtain a mature multi-species biofilm. The culture broth was refreshed every 2nd day to ensure bacterial viability and remove dead cells. At the end of the incubation period, specimens were aseptically removed from the tubes and washed with sterile PBS to remove unbound microbes and culture media. Another Falcon tube contained teeth in a medium without inoculation was used as negative control  to test the three different intracanal medicaments including; O3-oil, Ch-NPs and a combination of both. The control group  was inoculated and kept untreated in the incubator for the remainder of the test. The negative control group  in which the medium was free from inoculum and treatment to exclude contamination throughout the experiment. Vehicles (DMSO or 5% HAC) were also tested to exclude their effect on mature biofilm . The positive control group (G. 8) which test calcium hydroxide suspension. Test samples were irrigated with sterile saline for 2 m and dried with paper points. The test medicaments were injected into the corresponding root using syringe and canal orifices, then aseptically sealed with modeling wax . The specimens were transferred into sterile petri dishes and covered with humid sterile gauze. The plates were incubated at 37°C for 1 week and evaluated daily for viability of microbial cells  were calculated . This procedure was repeated three times. The CFU values were converted to loglculated .50 from the plotted curves of the cytotoxicity assay.The data were analyzed using IBM SPSS statistics for Windows, version 21.0 . One-way ANOVA, and Tukey’s test at α = 0.05 were used to determine significant differences between the groups. Graph Pad Prism was used to calculate the ICTable 1. Ch-NPs exerted 4- and 8-fold increases in the microbicidal activity compared to O3-oil against C. albicans, and both of E. faecalis and S. mutans, respectively. Synergism (FIC index ≤ 0.5) was recorded for O3-oil/Ch-NPs combination when it was assessed for the antimicrobial synergy in vitro against the test endodontic pathogens. Moreover, an additive effect (FIC index of >0.5 and ≤4) was noticed when O3-oil/Ag-NPs and Ch-NPs/Ag-NPs combinations were tested as recorded in Table 2.Broth microdilution assay revealed that all of the test medicaments had high MIC and MBC values as recorded in Figure 1). The IC50 for the three test disinfectants was calculated from the plotted curves and ranged from 0.039–10 mg/ml. One-way ANOVA revealed a significant difference (p < 0.001) between the test groups and the untreated control group. The IC50 of O3-oil was the safest, followed by the Ch-NPs (1.25 mg/ml).The neutral red assay results for testing cytotoxicity on human fibroblasts are shown in . Ch-NPs reduced single and mixed species biofilm by 97 and 94%, respectively. Furthermore, O3-oil exerted 86 and 79% reduction in biofilm, respectively. However, Ag-NPs showed non-significant reduction. Notably, the control group was significantly different from the other groups.Effect of the test medicaments on the formation of single or mixed microbial species biofilms was evaluated by a crystal violet assay that measures only biomass by binding of positively charged CV to all negatively charged residues in the biofilm. The Ch-NP solutions had the lowest mean value of absorbance, followed by OFigure 3). It revealed that the Ch-NPs and O3-oil were the most effective at reducing the fibroblast-microbial cell adhesion by reducing the number of fibroblast-associated microbes to almost zero (p < 0.05) compared to the untreated microbial cells (control), which showed high adhesion indices. Non-significant reduction in fibroblast-bacterial cell adhesion was also noted with Ag-NPs. Moreover, it had less effect on C. albicans-fibroblast adhesion (Table 3). The time-kill kinetics for the different test treatments showed that killing was time-dependent throughout the 7-day incubation with the mature biofilm. ANOVA revealed a significant difference between groups (p < 0.05). The fastest kill rate was recorded for the double combination (O3-oil/Ch-NPs), as it significantly reduced (6-log reduction) the number of survivors by the 2nd day of treatment. The separate medicaments had slower kill rates than the combined one . There were no significant effect for DMSO or 5% HAC on the microbial biofilm as compared to the untreated control group .Antiadherent test was assessed using Giemsa staining and microscopy  secretion helps other pathogens, such as S. mutans, adhere and form microcolonies that enable growth in the presence of C. albicans. The acidity of the biofilm increases as it matures in the presence of acidogenic pathogens such as S. mutans. The coexistence of E. faecalis with these pathogens stimulates production of superoxides that trigger hyphae formation in C. albicans. This results in a more complex biofilm due to the complexity of the bacterial-fungal association  reduced the total biofilm mass compared to the control groups, specifically the Ch-NPs, which showed 97 and 94% reductions for single and mixed species biofilms, respectively. This agrees with the results of C. albicans and S. mutans up to 92.5 and 93.4%, respectively. This supports the finding that our medications acted against the biofilm. The slightly lesser effect of the treatments on the mixed species biofilm in this study can be explained by the findings of C. albicans was resistant in the presence of the encapsulated E. faecalis ATCC 29212 biofilm at the intermediate stage (24-h). E. faecalis produces peptidoglycan that induces hyphal growth in C. albicans. Consequently, this stimulatory effect at the intermediate stage of biofilm development indicates the ability of E. faecalis and C. albicans to cohabitate. Additionally, E. faecalis produces extracellular superoxides that can trigger reactive oxidative stress, inducing filamentation in C. albicans. However, this relationship can become antagonistic with unencapsulated E. faecalis isolates in mature biofilm. Similarly, S. mutans can promote C. albicans biofilm due to their mutualistic relationship . It worked faster than the other treatments and the traditional medications that need at least 1 week to provide acceptable results. Calcium hydroxide was used as a positive control in our study. It was less effective than O3-oil, Ch-NPs, and their combination. This result was supported by the findings of E. faecalis biofilm. E. faecalis cells at 200 and 400 micron depths compared to the control; however, this did not differ significantly from the triple antibiotic paste (In our study, mixed species mature biofilm was developed in premolars to simulate clinical cases. The effect of the medicaments on mature biofilm in premolars was evaluated using viable counts of CFU/ml to test their viability in biofilm within the dentinal tubules. The Oic paste .in vitro study results, further in vivo investigations are recommended to determine the best concentration, and application time for each medicament, as well as the optimum ratio of the double combination, to obtain the best antimicrobial effect with the least adverse reactions.In view of the current Based on the current study, ozonated olive oil had the least cytotoxic effects, while chitosan nanoparticles showed better antimicrobial activity against the tested endodontic pathogens when used at its MIC and MBC/MFC compared to both silver nanoparticles and ozonated olive oil. Our tested double combination has the potential to prevent microbial biofilm formation and eradicate mature mixed-species biofilms. Incorporating the tested medicaments, either separately or in the novel double combination, into intracanal treatments, root canal sealers and irrigating solutions can prevent biofilm formation and eradicate resistant endodontic pathogens from the root canal, thereby increasing the success rate of endodontic treatment. These medicines might also be valuable if added to toothpastes and/or mouth rinses to combat harmful oral microbial biofilms.LA-M and NE-D conceived the experiments. LA-M, NE-D, and WG conducted the experiments. LA-M and NE-D analyzed the results. All authors wrote and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recent evidences suggest the involvement of DYRK1A  in Alzheimer’s disease (AD). Here we showed that DYRK1A undergoes a proteolytic processing in AD patients hippocampus without consequences on its kinase activity. Resulting truncated forms accumulate in astrocytes and exhibit increased affinity towards STAT3ɑ, a regulator of inflammatory process. These findings were confirmed in APP/PS1 mice, an amyloid model of AD, suggesting that this DYRK1A cleavage is a consequence of the amyloid pathology. We identified in vitro the Leucettine L41 as a compound able to prevent DYRK1A proteolysis in both human and mouse protein extracts. We then showed that intraperitoneal injections of L41 in aged APP/PS1 mice inhibit STAT3ɑ phosphorylation and reduce pro-inflammatory cytokines levels  associated to an increased microglial recruitment around amyloid plaques and decreased amyloid-β plaque burden. Importantly, L41 treatment improved synaptic plasticity and rescued memory functions in APP/PS1 mice. Collectively, our results suggest that DYRK1A may contribute to AD pathology through its proteolytic process, reducing its kinase specificity. Further evaluation of inhibitors of DYRK1A truncation promises a new therapeutic approach for AD.The online version of this article (10.1186/s40478-019-0678-6) contains supplementary material, which is available to authorized users. AD neuropathology is mostly defined by the accumulation of amyloid-beta (Aβ) plaques and neurofibrillary tangles (NFTs) in patient brains. Extracellular Aβ plaques are mainly formed by the aggregation of amyloid peptides , encoded by a gene localized in the Down syndrome critical region of chromosome 21, is a serine/threonine protein kinase which contributes to various biological processes in the embryonic and adult central nervous systems . In AD, T) is associated with a decrease of full-length form of DYRK1A (DYRK1AFL) thus confirming previous report by Jin and colleagues [FL, DYRK1AT exhibit stronger affinity toward STAT3ɑ. We identified Leucettine L41, derived from the marine sponge alkaloid Leucettamine B [T in AD pathology and demonstrate the relevance of inhibitors of DYRK1A cleavage as a potentially relevant therapeutic strategy.Here, we show that DYRK1A is truncated in the AD context. This increase of truncated forms of DYRK1A  and 12 age-matched littermate control mice were used for behavioral (Morris Water Maze), pathology, and biochemistry studies. A second cohort composed of 14 APP/PS1 and nine littermates were used for behavioral (Y-maze) and electrophysiological analysis. APP/PS1 mice express the human APP gene carrying the Swedish double mutation (K595 N/M596 L). In addition, they express the human PS1∆E9 variant lacking exon 9 [Fourteen APPg exon 9 . Only maMice were anesthetized with ketamine/xylazine (100 and 10 mg/kg respectively) and decapitated. One hemisphere was post-fixed by incubation for 72 h in 4% PFA, cryoprotected in 30% sucrose in PBS, and cut into 40 μm sections with a freezing microtome (Leica) for histological analyses. The contralateral hemisphere was dissected for hippocampus isolation. Samples were homogenized in a lysis buffer  containing phosphatase (Pierce) and protease (Roche) inhibitors and centrifuged for 20 min at 15000 x g. The same procedure was applied to human samples.The pre-weighed compound was dissolved in DMSO/PEG300/water (5/35/60) to a final concentration of 2 mg/mL for a dose of 20 mg/kg. The formulation was prepared on the day of the in vivo experiment. The mice received five intraperitoneal injections per week for four weeks.2+ with or without Harmine, Leucettine LeuI or Leucettine L41 at various concentrations  for 10 min at 30 °C. The reactions were terminated by the addition of 4-fold concentrated SDS-PAGE sample buffer, followed by heating in boiling water for 5 min. The products of proteolysis were analyzed by Western blots developed with antibody to DYRK1A.Human hippocampus tissue and 4 months-aged mouse (C57Bl6) hippocampus were homogenized in 9 volumes of buffer consisting of 50 mM Tris-HCl (pH 7.4), 8.5% sucrose, 10 mM β-mercaptoethanol, 2.0 mM EDTA, followed by centrifugation at 16,000×g at 4 °C for 10 min. The supernatants were incubated in the presence or absence of various concentrations of Caα-DYRK1A-Nter antibody (DYRK1A D1694) overnight at 4 °C. The proteins interacting with DYRK1A were revealed by Western blots developed with STAT3 , NFATc1 , APP, Tau and PS1.Homogenized total proteins from mouse hippocampus tissue were incubated with 2 mM EDTA, 0 or 2 mM of Ca2+ and 0 or 1 μM of Leucettine L41 during 10 min at 30 °C. 200 μg of total proteins were incubated with 2 μg of Equal amounts of protein (30 μg) were separated by electrophoresis in NuPAGE Bis-Tris Gels (Life Technologies) and transferred to nitrocellulose membranes. The membranes were hybridized with various primary antibodies , DYRK1A D1694 , GFAP , Vimentin , pSTAT3(Y705) , STAT3 , IBA1 , CD68 , IDE , TREM2 , GAPDH ). Various secondary antibodies were also used . Membranes were developed using enhanced chemiluminescence (Thermo Fisher Scientific). Signals were detected with Fusion FX7 (Vilber Lourmat) and analyzed and quantified using ImageJ (NIH).Inflammatory cytokines and interleukin concentrations were measured using the MSD Mouse V-PLEX Plus Proinflammatory Panel 1 kit . Extracted soluble Aβ forms were quantified with the MSD Human and rodents V-Plex Plus Aβ Peptide Panel 1 (4G8) . All ELISA were performed according to each kit manufacturer’s instructions.2+ or 10 mM EGTA to determine calcium-independent activity, thus excluding cathepsin activity.Calpain activity was measured using the fluorogenic peptide N-Succinyl-Leu-Tyr-7-Amido-4-Methylcoumarin as described by Kohli et al. . BrieflyCatalytic activity of total and endogenous DYRK1A was assessed using high-performance liquid chromatography (HPLC), based on the separation and quantification of specific fluorescent peptides (substrate and phosphorylated product), as previously described . Both ne2O2 for 5 min, followed by amplification using the ABC system . Horseradish peroxidase conjugate and 3,3′-diaminobenzidine were then used according to the manufacturer’s manual . The sections were mounted, dehydrated by passing twice through ethanol and toluol solutions, and cover-slipped with Eukitt (O. Kindler). Images were captured with a Leica DM6000 microscope and analyzed using ImageJ software (NIH). For fluorescent immunostainings, slices were incubated with secondary Alexa Fluor conjugated antibodies (Invitrogen). Slices were stained with DAPI , mounted in Vectashield fluorescent mounting media (Vector laboratories) and conserved at 4 °C. Images were captured with a Leica SP8 confocal microscope (Leica) and analyzed using ImageJ software (NIH). Laser power, numeric gain, and magnification were kept constant between animals to avoid potential technical artefacts. Images were first converted to 8-bit gray scale and binary thresholded to highlight positive staining. At least three sections per mouse  were quantified. The average value per structure was calculated for each mouse. For quantification of Iba1 and GFAP immunoreactivity around plaques, a region of interest (ROI) was drawn around the center of the plaque. The diameter of the circular ROI was set to two times the diameter of the plaque. Mean fluorescence intensity values were measured for either DYRK1A (7D10) (α-DYRK1A-Cter), DYRK1A (D1694) (α-DYRK1A-Nter) Iba1, or GFAP immunoreactivity and were processed using Icy software . Experiments and data analysis were performed blind with respect to treatments and genotypes.Cryosections were washed with 0.25% Triton in 0.1 M PBS, incubated in an 88% formic acid solution for 15 min , and saturated by incubation in 0.25% Triton in 0.1 M PBS/5% goat serum. They were then incubated with primary antibodies , DYRK1A D1694 , GFAP , APP 4G8-Biotin, , IBA1 ). For non-fluorescent immunostaining, endogenous peroxidase was quenched with PBS containing 3% HY-maze. Experiments were performed in a maze consisting of three transparent plastic arms, 46 × 11 × 25 cm each, set at a 120° angle relative to each other. During the first trial, mice could freely explore two arms, called familiar arms (FA), for 3 min, whereas the third arm was blocked by an opaque door. Assignment of arms was counterbalanced randomly within each experimental group to avoid any preference-related bias. Mice were then returned to their home cage for 5 min. Finally, mice were returned to the maze and allowed to explore all three arms, including the novel arm (NA), for 3 min. The maze was carefully cleaned with a 70% ethanol solution between each exploration phase to remove any olfactory cues. EthoVision software was used for recording and analysing each exploration trial.Experiments were performed in a tank 180 cm in diameter and 50 cm deep, filled with opaque water maintained at 21 °C, equipped with a platform of 18 cm in diameter, submerged 1 cm below the surface of the water. Visual clues were positioned around the pool and luminosity was maintained at 350 lx. The mice were initially exposed to a learning phase, which consisted of daily sessions  on five consecutive days. The starting position varied pseudo-randomly, between the four cardinal points. There was a mean interval of 20 min between trials. The trial was considered to have ended when the animal reached the platform. Long-term spatial memory was assessed 72 and 120 h after the last training trial in a probe trial in which the platform was no longer available. Animals were monitored with EthoVision software.Slice preparation. Mice were anesthetized with halothane and decapitated. The brain was rapidly removed from the skull and placed in chilled (0–3 °C) artificial cerebrospinal fluid (ACSF) containing 124 mM NaCl, 3.5 mM KCl, 1.5 mM MgSO4, 2.5 mM CaCl2, 26.2 mM NaHCO3, 1.2 mM NaH2PO4, and 11 mM glucose. Transverse slices (300–400 μm thick) were cut with a vibratome and placed in ACSF in a holding chamber, at 27 °C, for at least one hour before recording. Each slice was individually transferred to a submersion-type recording chamber and submerged in ACSF continuously superfused and equilibrated with 95% O2, 5% CO2.Electrically induced long-term potentiation (LTP) was studied. Theta-burst stimulation (TBS), mimicking the natural stimulation at the theta frequency from the medial septum to the hippocampus, consisting of five trains of four 100 Hz pulses each, separated by 200 ms, was applied at the test intensity. The sequence was repeated three times, with an interburst interval of 10s. Testing with a single pulse was then performed for 60 min (TBS) or 75 min (3 × 100 Hz), to determine the level of LTP.P-value < 0.05 for all tests.Data are expressed as the mean ± SEM. Statistical analyses were performed with GraphPad Prism  software. One-way ANOVA followed Tukey’s post-hoc tests were used to determine the significance of differences between groups. Student’s t test was used when only two groups were analyzed. All values are given as mean ± SEM. Statistical significance was set to a n = 4) and age-matched controls (n = 4). Using the anti-DYRK1A antibody 7D10 (named after α-DYRK1A-Cter) targeting the C-terminal region of DYRK1A, we observed decreased levels of DYRK1A in AD patients compared to controls (p < 0.05)    associated to an increase of the truncated form  in the hippocampus of AD patients  n =  and age-2+ dose-dependent decrease of DYRK1AFL and increase DYRK1AT were observed while the Ca2+ chelating agent EGTA was used as a control  and L41. We then performed western-blots using STAT3α and IkBα antibodies        Fig. a, b. The05) Fig. c, leadinFL forms, showed only marginal co-localization between GFAP and DYRK1AFL in all mice groups, as revealed by the level of DYRK1AFL in GFAP-positive cells, which was the same for all three groups   showed lower DYRK1A staining in the hippocampi of vehicle-treated APP/PS1 mice compared to littermates for both antibodies, confirming biochemical analysis. Strikingly, treatment of APP/PS1 mice with L41 restored DYRK1A staining levels in the hippocampus to those of wild-type mice. Most pyramidal neurons in the CA1 region and interneurons in the Stratum Radiatum (StrR) exhibited DYRK1A staining in littermates and APP/PS1 mice treated or not with L41 Fig. g. This wups Fig. h. The α-th) Fig. i. These T toward STAT3ɑ  . Interestingly, L41-treated APP/PS1 exhibited lower phospho-STAT3α levels compared to vehicle-treated APP/PS1 mice (p < 0.0005), thereby leading to a restored ratio pSTAT3ɑ/STAT3ɑ similar to littermates . These levels were reversed by L41 treatment      Fig. a-b. Westly) Fig. c-d. TREMly) Fig. e. Becausn = 13 slices/N = 5 mice) exhibited impaired LTP relative to littermates treated with the vehicle solution (n = 11 slices/N = 4 mice) (p < 0.0005)  relative to APP/PS1 mice that received vehicle injections. LTP generated in L41-treated APP/PS1 mice was still significantly lower compared to littermates (p < 0.0005), suggesting that synaptic plasticity measured by the LTP was partially rescued by the L41 treatment. To evaluate whether improved synaptic plasticity was also reflected at the behavioral level, we tested the mice by the Morris water-maze and the Y-maze tasks , whereas L41-treated APP/PS1 mice were statistically undistinguishable from littermate controls (ns)   but not different compared to littermates (ns). We also used the Y-maze paradigm to evaluate the spatial working memory which is mediated by hippocampus but also prefrontal cortex [p < 0.05) whereas L41-treated APP/PS1 mice tended to exhibit similar behavior (p = 0.06)  of synaptic transmission associated with impaired learning and memory . It has l cortex Fig. 5f)fn = 13 sFL) and producing truncated forms (DYRK1AT). The increase of DYRK1A kinase activity was suspected to contribute to AD. However, we demonstrate that this proteolysis is not associated with modification of the global DYRK1A kinase activity but affect its specificity. DYRK1AT forms exhibit increased affinity toward STAT3α, an activator of neuroinflammation. We then show that inhibiting DYRK1A proteolysis through the peripheral administration of Leucettine L41 in APP/PS1 mice is associated with increased number of phagocytic-microglia around amyloid-β deposits and reduction of the plaque load. This is associated to improved synaptic plasticity and reduced cognitive and memory deficits in APP/PS1 mice. Specific inhibitors of DYRK1A proteolysis could be therapeutic interest for AD.The present study provides compelling evidence for DYRK1A involvement in AD and describes a new mechanism through which DYRK1A modulation contributes to AD pathology. We first describe a proteolytic processing of DYRK1A in the hippocampus of AD patients and APP/PS1 mice reducing level of full-length form of DYRK1A (DYRK1ADYRK1A gene is located on chromosome 21 (21q22.2), a region known as the Down- Syndrome Critical Region (DSCR) [APP gene on chromosome 21 [FL and increased DYRK1AT forms have been reported in the frontal and temporal cortex of AD patients (Braak V-VI/Tangles score = 14) through upregulated calpain I activity, the major calpain isoform in brain [FL forms in APP/PS1 mice, together with increased DYRK1AT forms and increased calpain activity.The n (DSCR) . People osome 21 . In addiosome 21 , 35. Howin brain . We confDYRK1A has a long kinase domain, followed by a PEST region, and histidine-repeat and serine/threonine-rich domains . PEST seSeveral molecules able to reduce DYRK1A kinase activity have been developed . One of α and IL-12, (ii) increased microglial cells recruitment around amyloid plaques, and (iii) decrease of the amyloid-β burden. The immune system, driven by inflammatory mediators, influences AD progression [FL levels and DYRK1AT in astrocytes or no significant decrease of pro-inflammatory cytokines were observed in LeuI treated animals  in APP/PS1 mice. STAT3ɑ is a transcription factor and a major regulator of cytokine production . The tyrgression . In partgression  and elevgression . In addigression , 40. Emegression . Activatgression . In APP/gression . Therefogression , 48. A pgression . We showIn conclusion, we provide several evidences that DYRK1A is proteolyzed in both AD patients and APP/PS1 mice. We show that truncated forms of DYRK1A accumulate in astrocytes without consequences on its kinase activity. However, kinase specificity of truncated DYRK1A is reduced leading to increase its affinity toward STAT3α, a major regulator of inflammation We demonstrate that decreasing production of DYRK1A truncated forms by Leucettine L41 in an AD-like mouse model, reduces levels of inflammatory cytokines, improves clearance of amyloid-β plaques through microglia recruitment and activation, and consequently improves synaptic plasticity and memory. These data confirm the interest of L41 and eventually other inhibitors of DYRK1A proteolysis for AD.Additional file 1:Supplementary data figure 1. (JPG 27 kb)Additional file 2:Supplementary data figure 2. (JPG 137 kb)Additional file 3:Supplementary data figure 3. (JPG 415 kb)Additional file 4:Supplementary data figure 4. (JPG 647 kb)Additional file 5:Supplementary data figure 5. (JPG 186 kb)Additional file 6:Supplementray Data text. (DOCX 230 kb)


Caulerpa cupressoides modified the morphology, size and surface charge of CaOx crystals. Thus, these crystals became similar to those found in healthy persons. In the presence of SPs, dihydrate CaOx crystals showed rounded or dumbbell morphology. Infrared analysis, fluorescence microscopy, flow cytometry (FITC-conjugated SPs) and atomic composition analysis (EDS) allowed us to propose the mode of action between the Caulerpa’s SPs and the CaOx crystals. This study is the first step in understanding the interactions between SPs, which are promising molecules for the treatment of urolithiasis, and CaOx crystals, which are the main cause of kidney stones.Urolithiasis affects approximately 10% of the world population and is strongly associated with calcium oxalate (CaOx) crystals. Currently, there is no efficient compound that can be used to prevent this disease. However, seaweeds’ sulfated polysaccharides (SPs) can change the CaOx crystals surface’s charge and thus modify the crystallization dynamics, due to the interaction of the negative charges of these polymers with the crystal surface during their synthesis. We observed that the SPs of  Urinary lithiasis or urolithiasis is a pathophysiological condition resulting from the formation of kidney stones. The incidence of urinary lithiasis in the world is increasing rapidly. In the last decade, prevalence doubled from approximately 6% to 11% among men and 4% to 7% among women . This diUrolithiasis can be influenced directly by the types of formed crystals, as each crystal has its own different ability to bind to the renal epithelium and to initiate the formation of the stones. Calcium oxalate (CaOx) crystals make up about 70% of urinary stones , and theThe formation of these CaOx crystals is derived from a physicochemical process divided into three phases: nucleation, aggregation and crystal growth. The preponderant condition for crystal formation (nucleation) is urinary supersaturation due the superfluous ions that cannot be stored in the buffer system; the condensation of these salts occurs forming tiny crystals, which are called nuclei. The aggregation occurs by the union of one or more growing nuclei, which form crystals of larger dimension and mass that can precipitate in the renal epithelium, being able to adhere to or internalized by cells . The groThe changes in the CaOx crystal’s face are caused by the other molecules present in the urinary tract, considering the fact that the concentration of ions in healthy persons and lithogenic patients is practically the same. These molecules, such as citrate, pyrophosphate, glycosaminoglycans and glycoproteins, present in the renal and urinary ducts, have the function of stabilizing CaOx crystals, mainly in the COD morphology ,12.Recently, seaweeds sulfated polysaccharides (SPs) have begun to be evaluated for their anti-lithic capacity. The studies are done in vitro as well as with cells under culture conditions and in vivo. The data are well reviewed in a recently published paper , which vCaulerpa cupressoides var. flabellata have been studied by our research group for almost a decade. The Brazilian coast has a wide diversity of marine seaweed, especially on the northeast coast; however, the SPs of few species are characterized and evaluated for their therapeutic potential. The SPs  from C. cupressoides exhibited several biological activities, including anticoagulant, antiproliferative and antioxidant activities  rather than growth in the direction [100]. This gave COM edges and tips with less sharp angles, as they were elliptical . Thus, the morphological characteristic assumed by COM crystals after incubation with C. cupressoides SPs is advantageous, as, by leaving the edges of their faces with a slight angle (rounded), the possible interaction of these crystals in renal epithelium could be decreased, which would consequently favor their passage in the urinary tract, thus reducing the formation of kidney stones. In agreement with our hypothesis, studies analyzing CaOx crystals in the urine of lithogenic patients verified that the COM crystals had edges and tips with sharp angles and concluded that these are important factors for the anchorage of these crystals in the renal epithelium [Regarding COM crystals, when formed after treatment with ithelium ,33.−1. While, in COM crystal spectra, we observed at least five prominent bands  of crystals formed in the presence of The ζ mean value of untreated CaOx crystals was +8.85 ± 3.30 mV. This positive profile of crystal charge surface can be mainly related with the presence of calcium ions in the crystal structure. All SPs decreased the zeta potential of CaOx crystals, ranging from −25.82 ± 6.36 mV in the presence of CCB-F0.3 to −68.70 ± 12.01 mV in the presence of CCB-F2.0.An interesting fact is that the increase of crystals ζ did not correspond to the sulfate/sugar ratio , that isC. cupressoides, which explains part of the formation of many small crystals. The elevated negative charge on the crystal surface led to repulsion of other crystals and blocked crystal aggregation and growth. This interference in crystal aggregation/growth has also been observed by other authors while working with other seaweeds SPs [The ζ increased with the presence of SPs of eeds SPs ,16. In tZeta potential analysis provides great indications that SPs interact with CaOx crystals structure. However, there are no studies that prove direct binding between SPs from seaweed and CaOx crystals. However, this connection is well described for other molecules that also have negative charges in their structure, such as polyacrylate , osteopoIn another set of experiments, the SPs were covalently conjugated to the FITC, as described in As expected, crystals formed in the presence of inflorescent SPs and FITC were not detected by flow cytometry, indicating that FITC alone did not bind to CaOx crystal. However, using this technique, we verified that, in all condition evaluated, more than 90% of crystals formed in the presence of fluorescent SPs showed positive FITC signal A.When those crystals were analyzed by fluorescence microscopy, we observed that FITC alone did not label the crystals and did not change the crystal morphology. On the other hand, we noticed that the crystals appeared marked by FITC (green) in almost totality B when foC. cupressoides induced higher amount of COD crystals in comparison to the positive control. In addition, SPs gave COD crystals’ higher stability. This behavior was already observed by Escobar and colleagues [The data presented thus far show that, except for CCB-F2.0, all other SPs of lleagues , in the lleagues .C. cupressoides SPs may take different forms, which seems to indicate that SPs can stabilize COD in different ways, perhaps because they associate with crystals in different faces.However, not every sulfated polysaccharide promotes stabilization of COD crystals, as shown by Melo and colleagues  and by o42-) groups of SPs, since CaOx is composed only of calcium, carbon and oxygen, thus some considerations were made.To confirm this hypothesis, the surfaces of the COD crystals obtained after the incubation with the SPs studied here had their atomic composition characterized through the spectroscopic chemical microanalysis of X-rays by energy dispersion (EDS). The sulfur atoms were quantified at different points of the crystals: apex, face and base A. The suThe results from this analysis are summarized in the table of We found no other articles that report this type of analysis; therefore, it was not possible to compare our results with those of other authors. However, some considerations were made.Is it necessary for the SPs to be distributed differently throughout the crystal so that there is a greater stabilization of the COD crystals? It does not seem so, since CCB-F1.0 had this type of distribution, but the COD:COM ratio obtained with this sample was only 2:1 .The other SPs were more concentrated on the base. However, there is a subtle relationship between the amount of sulfur that should be at the base, apex and face. If there was a lot of sulfur in the base (about ten times more) than in the other points, as occurred with the presence of CCB-F0.5, there was stabilization of COD, but with only a COD:COM ratio of 3:1 . The ideCaulerpa cupressoides var. flabellata, was collected from Nísia Floresta, on the southern coast of the state of Rio Grande do Norte, Brazil (S 6°1′819′′ and W 35°6′33.40′′) and then transported to the Laboratory of Natural Polymer Biotechnology, Department of Biochemistry, Federal University of Rio Grande do Norte, UFRN. The seaweed was identified according to its morphology [The seaweed, rphology . The matC. cupressoides var. flabellata Børgesen was cleaned with running water and oven dehydrated at 45 °C. It was sprayed and then treated four times with two volumes of ethanol for depigmentation and delipidation of the material. Two volumes of 0.25 M NaCl were added to the powder obtained, with the pH being adjusted to 8.0. The proteolytic enzyme maxatase  was added to this material. The suspension was then centrifuged at 10,000× g for 20 min. The precipitate was discarded, and the volume of the supernatant was measured fractionated with increasing volumes of acetone, obtaining SPs according to a method established by Costa and collaborators [After collection, the green seaweed borators .2SO4 reaction, as described previously [The total sugars were determined according to Dubois and collaborators (1956) by the phenol–Heviously . The suleviously . Proteineviously . C. cupressoides SPs was evaluated by gel electrophoresis in PDA buffer, according to the reported method [m/v) agarose in PDA buffer . Subsequently, aliquots of the polysaccharides (about 50 μg) were applied to the gel and subjected to electrophoresis  for 60 min. After the electrophoretic run, the polysaccharides were precipitated with 0.1% cetyltrimethylammonium bromide  for 2 h at room temperature and the gels were dried using warm air stream. To visualize the SPs, gels were stained with a solution of 0.1% toluidine blue in 1% acetic acid and 50% ethanol. The gel was then de-stained with the same solution without the dye. Three independent analyzes were performed.The electrophoretic mobility of d method . InitialThe molecular weight and homogeneity of samples were determined by gel permeation chromatography (GPC). Each sample was dissolved to a final concentration of 10 mg/mL and 200 µg were applied to a column containing Sephadex G-100 . GPC was performed using an isocratic elution mode. The molecular weight was estimated by reference to a calibration curve made by dextran sulfate standards  . Homogeneity of samples was evaluated by chromatographic profile. The total sugars were determined according to Dubois and collaborators (1956) by the phenol–H2SO4 reaction, and the sulfate was determined by metachromasia, as described previously .C. cupressoides were obtained using infrared spectroscopy via Fourier transform  equipped with the IRsolution 1.20 software. SPs and crystals samples (5 mg) were completely mixed with dried potassium bromide powder (KBr) and then compressed. The sweep frequency range was 4000–400 cm−1. Thirty-two scans at a resolution of 4 cm−1 were evaluated and referenced against air. The Infrared Spectroscopy Analysis was carried out at the Department of Chemistry of the Federal University of Rio Grande do Norte.The infrared spectra of the CaOx crystals controls formed after incubation with the SPs of 2+ and oxalate to a mixture of calcium chloride (8 mM/L), sodium oxalate, sodium chloride (200 mM/L) and sodium acetate (10 mM/L) as final concentrations of this solution from physiological concentrations of urine. The formation of the CaOx crystals was evaluated in the presence or absence (control) of Caulerpa’s polysaccharides (0.1 mg/mL) [g and the supernatant was discarded. The precipitate was composed mainly of crystals of CaOx, and was resuspended in 0.5 mL of water, and a solution of 0.1 mL was placed on a histological slide and observed under an optical microscope (600×). Images of 10 different fields were obtained for each slide, and then the crystal diameters and sizes were analyzed using the NIS Elements AR Ver4.30.01 DU1 64-bit software, 2014 . The conclusions about the measurements of the CaOx analyzes were obtained after a trial of distinct experiments, being repeated four times.The CaOx crystals can be formed in vitro from Ca1 mg/mL) . When neC. cupressoides, scanning electron microscopy  and dispersive energy spectroscopy  images were generated with 1280 × 960 pixels resolution. The scanning electron microscopy was carried out at the Department of Materials Engineering of the Federal University of Rio Grande do Norte. The images showed in the results are representative of three independent experiments.To observe the superstructure and composition of generated crystals in the presence of the SPs of g). The crystal concentrate was then suspended in 1.5 mL of ultrapure water (pH ~7.0), and the zeta potential of the ζ samples was obtained using a Zeta Plus® analyzer  . Since pH value and ionic strength are factors that affect zeta potential results, the values of these two parameters, in all solutions evaluated, were always the same.After 30 min of crystal formation in the presence and absence of the polysaccharides , the solTo better understand the morphology and arrangement of the SPs in the CaOx crystals, polysaccharide was labeled with fluorescein isothiocyanate (FITC). Five milligrams of each polysaccharide were added to 0.1 mL of phosphate buffer (PBS) at pH 7.0 containing 0.1 mg FITC. The solution was kept in an environment with reduced brightness, at room temperature for 1 h. The material was then labeled with deionized water in membranes with pores of 6 kDa, and then lyophilized. Samples without SPs as well as samples of FITC-labeled polysaccharides were not subjected to new production of CaOx crystals and slides, assembled according to n = 3). The ANOVA test was performed to check the difference between results. The Student–Newman–Keuls test (p < 0.05) was used to solve similarities found by ANOVA. All tests were performed in GraphPad Prism 5 .All data are presented as the mean ± standard deviation (Caulerpa cupressoides and were named CCB-F0.3; CCB-F0.5; CCB-F1.0; and CCB-F2.0. These SPs interacted in vitro with calcium oxalate crystals, making their surface more negative. They also induced a decrease in the size and an increase in the number of formed crystals. This effect did not depend on the amount of sulfate groups present in SPs. Except for CCB-F2.0, SPs induced the formation of a greater amount of COD crystals compared to the control group, with CCB-F0.3 being the most efficient. This occurred because CCB-F0.3 was distributed in the base:apex:face of the crystal in a ratio of 2:1:1. We believe that this balance/proportion between SPs at the interaction points was crucial to avoid dehydration of the COD crystal to COM, which enhanced their stability.Four sulfated polysaccharide populations were extracted from the seaweed


Ecklonia cava within alginate microparticles for the first time. Microparticles with immobilized spores were generated by printing alginate-spore suspensions into a calcium chloride solution. We demonstrate that the inkjet technique can control the number of spores in an ejected droplet in the range of 0.23 to 1.87 by varying spore densities in bioink. After the printing-based spore encapsulation, we observe initial sprouting and continuous growth of thallus until 45 days of culture. Our study suggest that inkjet printing has a great potential to immobilize algae, and that the ability to control the number of encapsulated spores and their microenvironments can facilitate research into microscopic interactions of encapsulated spores.The algal cell immobilization is a commonly used technique for treatment of waste water, production of useful metabolites and management of stock culture. However, control over the size of immobilized droplets, the population of microbes, and production rate in current techniques need to be improved. Here, we use drop-on-demand inkjet printing to immobilize spores of the alga  Entrapped algal cells can also be used to generate metabolite production4, measure toxicity6, preserve cells by freezing7, and to manage stock cultures8. The technique has also enabled improvement of metabolism, function, and growth of immobilized algal cells10. Methods to entrap microorganisms in hydrogel particles include conventional dripping of cell suspension into a receptacle that contains hardening solution11; extrusion dripping12; gravity-driven dripping13; and suspension spraying14. All of these methods are either slow or not allow sufficient control over the size of droplets, their content of microbes or production rate. A practical method would overcome these drawbacks.Immobilization of algal cells in polymeric hydrogels has a range of applications. Immobilized algal cells can be used for effluent treatment to remove nutrients, metals and industrial pollutantsEcklonia cava is an edible, perennial marine brown alga  that grows in the ocean around South Korea and Japan15. E. cava is a major source of alginate, which is used for foods, pharmaceuticals, therapeutics and tissue engineering17. An extract of E. cava, phlorotannin, has been evaluated for ability to suppress tumor growth18 and as an antioxidation19. Therefore, E. cava has wide applications in science and industry. However, populations of E. cava are decreasing around east Asia20.22, printed electronics25 and 3D fabrication27. The DOD piezoelectric inkjet printing uses a piezoelectric actuator in the channel of the nozzle of piezoelectric inkjet printer. A voltage pulse reduces the volume of a chamber that contains ink, so some is ejected as a droplet28. Piezoelectric inkjet printing can generate droplets of size 1–100 pL at >10 kHz. The size of the ejected droplet can be controlled by adjusting the input voltage pulse or by selecting an appropriate nozzle and be smaller than the diffusion limits of nutrients and metabolites in hydrogels (100–200 µm)12. The small size of microparticles can minimize the inhibitory effect during the growth of the entrapped algal cells29. Owing to the ability to eject small volume of ink, inkjet printing has been used to encapsulate macromolecules30, drugs17 and mammalian cells32.Drop-on-demand (DOD) inkjet printing is extensively used in various fields such as bioprinting2+) or magnesium (Mg2+) ions. Especially G blocks in alginate contributes to form strong and reversible crosslinking networks so called “egg-box structure”  and α-L-guluronic acid (G block). Alginate can form a hydrogel in the presence of calcium  sodium alginate solution from brown algae  was autoclaved for 20 min at 120 °C, cooled at room temperature and filtered using 0.45-µm syringe filter. The solution was mixed with the spore suspension to prepare a final 0.5% (w/v) sodium alginate solution with concentrations of 0.125 × 106, 0.500 × 106 or 2.00 × 106 cells ml−1.Sodium alginate was purchased from MP Biomedical LLC, USA. Reproductive thalli of −1 and 1,500 s−1 were applied for 60 s; the viscosities were derived by averaging data of the final 10 s (n = 3).Shear viscosities of the suspensions were measured using a cone-plate rotational viscometer  at room temperature. The viscometer has a rotating cone with a radius of 2.4 cm and a stationary plate. Sodium alginate-spore suspension (0.5 ml) was placed between the plate and the cone. Shear rates between 50 sA DOD inkjet printing system  was used to print the sodium alginate-spore suspensions. A piezoelectric inkjet nozzle with a diameter of 80 µm was used and voltage pulse was applied at ±80 V in bipolar mode. The wave pulse had a rise time of 6.0 µs, dwell time of 13 µs, fall time of 9.0 µs, and echo time of 22 µs. Images of jet formation were acquired every 30 µs by a stroboscopic CCD camera equipped in the printing system, and a short-duration LED light.−1. Printed arrays were observed under an optical microscope and the average number of spores per drop was calculated (n = 30).Arrays of printed sodium alginate-spore suspensions were generated on glass slides. The drop spacing of the arrays was 400 µm and stage speed was 50 mm s2 in seawater. The seawater had been autoclaved for 20 min at 120 °C, cooled at room temperature and passed through a 0.45-µm filter. Printing was conducted at an ejection frequency of 1,000 Hz for 30 min. The solution was strirred using a 1-cm long magnetic stirring bar to prevent aggregation of the hydrogel particles. After printing, 70 µl of generated microparticle suspension was mixed with 200 µl of PESI medium37 supplemented with GeO2 and delivered into each well of 96-well plates. The samples were incubated at 15 °C, light intensity of 30 μmol m−2 s−1, and diel cycle of 10 h light: 14 h dark. Thallus length of gametophytes of E. cava was measured under a microscope on days 3, 8, 14 and 21 of culture. The number of measured gametophytes of E. cava was different according to the used sodium alginate solutions with concentrations of 0.125 × 106, 0.500 × 106 or 2.00 × 106 cells ml−1. .Sodium alginate-spore suspensions were printed using the inkjet printing system, the the printed drops were collected in a 35-mm Petri dish containing 1% (w/v) dissolved CaClp-values < 0.05 were considered to be statistically significant.All quantitative data are presented as the mean ± standard deviation. A one-way analysis of variance (ANOVA) with a Bonferroni post-hoc test was performed using Origin 2016 . 2 solution in a receptacle. As soon as a droplet impacts on the surface of the solution, Ca2+ enters guluronate blocks of alginate molecule, thus forming junctions with adjacent guluronate blocks38. As a result, alginate-based droplets are crosslinked and transformed into hydrogel microparticles that encapsulate spores. The solution in the receptacle was magnetically stirred to prevent aggregation of hydrogel particles .Figure 1 and 103 s−1 to examine their printability. All of the suspensions had viscosity <20 mPa∙s, which is suitably low for stable printing . Then, we observed their jetting behaviors for 2 hours. No clogging of the nozzle was observed during the experiments. Their representative high-speed images of jet formation are shown in Fig. E. cava spores inside the suspensions might slightly increase the elasticity of bioink.Viscosities of sodium alginate-spore suspensions was measured in the range of shear rate between 5 × 10ing Fig. . The spo6 ml−1, 0.67 at 0.500 × 106 ml−1, and 1.87 at 2.00 × 106 ml−1 (Fig. 39. This ability to control the number of encapsulated spores and their microenvironments demonstrates that the piezoelectric inkjet printing technique has possible uses as a method to study microscopic interactions of encapsulated spores.In order to quantify the controllability of delivery of spores, we investigated the number of spores in a droplet by printing dot arrays of sodium alginate-spore suspensions onto the glass substrate without crosslinking. Then, the number of spores in a drop was counted using microscope Fig. . Examinal−1 Fig. , and decl−1 Fig. . However2 crosslinking solution as shown in Fig. E. cava were immobilized inside alginate microparticles (Fig. Microparticles were generated using inkjet printing system with a receptacle containing CaClE. cava showed a bush-like morphology. The growing E. cava showed the gametophyte phenotype because the spores had been collected from sporophytes10. The results show that inkjet printing has applications for immobilization of algal cells. Furthermore, the growth trend was not significantly affected by spore density. This result indicates that E. cava spores at low to moderate density of within hydrogel microparticles could not significantly influence each other’s thallus morphology. The range of the spore density at which such influence occurs should be determined in future studies.Sprouting and growth of the entrapped spores were monitored for 45 d to prove a compatibility of the immobilization technique of algal spores. Spores immobilized in microparticles remained dormant until day 8 Fig. , then a E. cava in sodium alginate suspensions. Viscosities and drop-formation behaviors demonstrated that they were suitable inks for the inkjet printing. The spatial density of spores may affect the viscosity and the elasticity of sodium alginate-spore suspension, and thereby affect jetting formation and lower-than-expected average number of spores per droplet. The printing technique allowed fabrication of spore-immobilized alginate microparticles. Culture of the microparticles yielded healthy gametophytes of E. cava without significant dependence on the spore density. These results show that inkjet printing has applications for immobilization of algal cells and single cell encapsulations.In this study, piezoelectric inkjet printing technology was used to immobilize spores of Supplementary Figure 1


Differences were observed in the tumor vasculature and metabolism in tumors based on ascites volumes that provide new insights into the development of this condition.Epithelial ovarian cancer is the leading cause of death from gynecologic malignancy among women in developed countries. Epithelial ovarian cancer has a poor prognosis, due to the aggressive characteristics of the disease combined with the lack of effective therapies. Options for late-stage ovarian cancer are limited and invasive, especially once malignant ascites develops. Malignant ascites, a complication observed in terminal ovarian cancer, significantly contributes to poor quality of life and to mortality. Excess accumulation of fluid in the peritoneal cavity occurs due to a combination of impaired fluid drainage and increased net filtration, mostly due to increasing intraperitoneal vascular permeability. Here we applied non-invasive magnetic resonance imaging (MRI) and spectroscopic imaging (MRSI) of syngeneic mouse tumors  Epithelial ovarian cancer is the leading cause of death from gynecologic malignancy among women in developed countries with an estimated incidence of 205,000 cases worldwide per year resulting in ~125,000 deaths . TherapeMalignant ascites is a complication observed in terminal ovarian cancer that significantly contributes to poor quality of life and to mortality. The excess accumulation of fluid in the peritoneal cavity arises from a combination of impaired fluid drainage and increased net filtration. Malignant ascites formation is thought to occur due to increasing intraperitoneal vascular permeability . Local sin vivo MRI and 1H MRSI, and 1H MRS of tumor extracts, to better understand the relationship between tumor vasculature, metabolism, and ascites build-up in an experimental model of ovarian cancer. The ID8 cell line is an ovarian epithelial papillary serous adenocarcinoma cell line, originating from mouse ovarian surface epithelial cells transformed after multiple passages in vitro , while others had no or low-volume ascites (< 50 μl). We applied non-invasive MRI and MRSI to better characterize differences in tumor vasculature and metabolism between tumors that produced low and high-volume ascites.Non-invasive magnetic resonance imaging (MRI) and magnetic resonance spectroscopic imaging (MRSI) can be used to characterize the tumor microenvironment and understand its role in ascites formation. Here we applied in vitro . The celin vitro . These Iin vitro . When inin vitro . These ain vitro . For mosin vitro . Tumors 2. Cells were orthotopically implanted in C57BL/6J mice using a two-step process as previously described . All surgical procedures and animal handling were performed in accordance with protocols approved by the Johns Hopkins University Institutional Animal Care and Use Committee, and conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH.ID8-Defb29 Vegf cells were grown in RPMI 1640 medium with 10% fetal bovine serum, and cultured under standard cell culture incubator conditions at 37°C in a humidified atmosphere containing 5% COescribed . First, 1-weighted images were acquired to localize the orthotopic tumors. To characterize tumor vascular volume and permeability, we used a previously published protocol . The albumin-GdDTPA was synthesized as previously described (1) maps were obtained by a saturation recovery method combined with fast T1 SNAPSHOT-FLASH imaging. First, an M0 map with a recovery delay of 10 s was acquired. Then, images of 4 slices (1 mm thick), acquired with an in-plane spatial resolution of 250 μm  were obtained for three relaxation delays . These T1 recovery maps were obtained before i.v. injection of albumin-GdDTPA and repeated over a 23-min period, starting 3 min after i.v. injection of the contrast agent. At the end of the imaging studies, the T1 of blood was measured. Relaxation maps were reconstructed from data sets for three different relaxation times and the M0 dataset on a pixel-by-pixel basis. Vascular volume (VV) and permeability surface area product (PSP) maps were generated from the ratio of (1/T1) values in the images to that of blood.Imaging studies were performed on a 9.4T Bruker spectrometer  using a 25 mm diameter volume coil placed around the torso of the mouse. Mice were anesthetized with a mixture of ketamine and acepromazine. Anatomic Tprotocol , 13. Briescribed . The taiMetabolic maps of tCho were obtained from a 4 mm thick slice using two dimensional-chemical shift imaging (2D-CSI)  [echo tiMice were sacrificed, and the ascitic fluid volume was measured. Lungs, liver, and lymph nodes were excised and fixed in formalin to quantify metastatic spread. Tumors were cut in half with one half freeze clamped for MR extracts and protein analysis, and the other half fixed in formalin.2O) containing 2.4 × 10−7 mol of 3-(trimethylsilyl)propionic 2,2,3,3-d4 acid  as an internal standard. Lipid-soluble extracts were resuspended in 0.4 mL of chloroform-D and 0.2 mL of methanol-D4 with 0.05 v/v% tetramethylsilane (TMS)  as an internal standard  using a 5-mm HX inverse probe, and the following acquisition parameters: 30° flip angle, 6,000 Hz sweep width, 9.5 s repetition time, time-domain data points of 32K, and 128 transients  , 19. Briansients . SpectraProteins were extracted from freeze-clamped tumors using radioimmunoprecipitation lysis buffer fortified with a protease inhibitor cocktail, dithiothreitol, phenylmethylsulfonyl fluoride, sodium orthovanadate, and sodium fluoride . Protein concentration was estimated using the Bradford Bio-Rad protein assay kit . About 60 μg of total protein was resolved on 7.5% SDS-PAGE gels from Bio-Rad, transferred onto nitrocellulose membranes, and probed with antibodies directed against mouse FAS (A-5) , cPLA2 , ApoE (M-20) . GAPDH was used as a loading control and detected with a monoclonal antibody . Immunoblots were developed using SuperSignal West Pico chemiluminescent substrate kit .3. As shown in Figures ex vivo. Metastases were more frequent in mice with ascites, especially in organs in the peritoneal cavity, including the diaphragm (67 vs. 0%), liver (100 vs. 20%) and intestine (17 vs. 0%), as shown in Figures Mice were imaged when tumors were ~200–300 mm1H MRSI in all the orthotopic tumors imaged  was detected with  Figures . The tChs Figure . There ws Figure  suggestiWe measured vascular volume (VV) and permeability surface area product (PSP) in the ID8-Defb29 Vegf tumors using MRI of the macromolecular contrast agent albumin-gadolinium-DTPA. Representative maps are shown in Figure 1H MRS. Analysis of the lipid phase obtained after dual phase extraction revealed higher concentrations of cholesterol, phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdE), and lower CH2/CH3 ratio in tumors from mice with high-volume ascites . No significant differences were observed in the water phase tumor extracts (data not shown).To further examine the metabolic differences between both tumor types, we extracted the tumors and performed high resolution s Figure . Figure To better understand the differences observed in tumor lipid patterns, we investigated the expression levels of some of the proteins involved in lipid and cholesterol metabolism Figure . While nin vivo between the groups do not appear to be directly related to VEGF expression in the tumors.We also performed qRT-PCR to analyze tumor Chk and VEGF mRNA expression levels and found no significant differences between the no or low-volume and high-volume ascites groups (data not shown). The differences in ascites volume, and in VV and PSP measured via the lymphatic system, resulting in a high rate of pelvic and para-aortic lymph node involvement. Hematogenous spread is less predominant but can also occur in ovarian cancer. In a retrospective clinical study, 372 ovarian cancer patients were divided into 2 groups, depending of the presence or absence of ascites  plays a critical role in ovarian tumor progression and ascites formation (2 activity is higher in epithelial ovarian cancer tissues compared to benign or normal tissues (Phospholipase Aormation . This en tissues , and hig tissues , 29. Her tissues .Higher levels of cholesterol were measured in the tumors that presented with high-volume ascites. High levels of cholesterol have been previously detected in aggressive mouse ovarian surface epithelial cells . The eleIn summary, despite similar genetic backgrounds and VEGF expression levels, the ID8-Defb29 Vegf tumors implanted orthotopically induced very different ascites volumes, highlighting the importance of tumor microenvironmental factors in the accumulation of ascites. Our data suggest that large volumes of ascites may act to occlude vessels affecting delivery of therapeutic agents. These studies provide new insights into vascular and metabolic differences in no or low-volume ascites and high-volume ascites that merit expanded future investigation.M-FP and ZB contributed to the conception and design of the study. M-FP, BK, YM, FW acquired the data. M-FP performed the data analysis and wrote the first draft of the manuscript. BK, YM, FW, TW, C-FH, and ZB revised the manuscript critically. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tertiary oral health services  represent high-cost and ineffective ways to improve a child’s oral health. We measured the impact of increased Texas Medicaid reimbursements for preventive dental care on use of tertiary oral health services.We used difference-in-differences models to compare the effect of a policy change among children (≤9 y) enrolled in Medicaid in Texas and Florida. Linear regression models estimated 4 outcomes: preventive care dental visit, dental sedation, emergency department use, and surgical event.P < .001) and decreased emergency department visits  for children aged 9 years or younger. We saw no significant change in dental surgical rates associated with increased preventive dental care reimbursements.Increased preventive care visits led to increased sedation visits (1.7 percentage points, Increased access to preventive dentistry was not associated with improved long-term oral health of Medicaid-enrolled children. Policies that aim to improve the oral health of children may increase the effectiveness of preventive dentistry by also targeting other social determinants of oral health. Increasing dental provider reimbursements for preventive care visits is an effective policy intervention to increase preventive dental visit use among Medicaid-enrolled children.Increased preventive care dental visits did not translate into significant changes in pediatric dental surgeries after a reimbursement-focused policy intervention.Interventions to reduce pediatric dental surgery should reflect the social determinants of oral health, including access to regular dental care and household oral health behaviors.Dental caries is the most common childhood chronic disease in the United States and worldwide. It disproportionately affects vulnerable children such as those who are poor, receiving Medicaid, of a racial/ethnic minority group, or residents of an underserved area ,2. The iClinical care alone does not address the multifactorial causes of caries. Oral health behaviors, caregiver psychosocial state and parenting style, dietary choices, health literacy, and fluoride exposure are only a few factors that influence a child’s oral health –10. HoweAlthough increasing access to preventive dental care is important, preventive care alone does not reduce the likelihood of needing tertiary oral health services  . PreviouWe studied children aged 9 years or younger who were enrolled in the Texas and Florida Medicaid programs. Texas children were the treatment group, and Florida children were the control. Florida was selected as the control because its trends in reimbursement for preventive dental care were stable during the study period as a ratio of private insurance to Medicaid rates .The estimated prevalence of DGA in the Medicaid population during our study period was ~0.5% to 1% ,19. A dePatient demographics and outcomes were derived from Medicaid enrollment and claims files, which we obtained from the Centers for Medicare and Medicaid Services Research Data Assistance Center. Because of budget constraints, we limited our requests for data files for treatment and control states to the pre-reform (2007) and post-reform years 2011 and 2012). This period was selected to reflect prior work that evaluated the impact of these natural experiments on use of preventive dental care . To address this, we measured the effect of increased reimbursements for preventive dental care on appendectomies as a falsification test. If dental surgeries and appendectomies were associated with increased reimbursement for dental care in similar fashion, we would interpret this to signify confounding factors that influence both dental surgeries and preventive dental care reimbursement levels.Data management. Outcomes were identified on the basis of American Dental Association Code on Dental Procedures and Nomenclature (CDT)    or the Are (CDT)  and InteCD-9-CM) . Surgicare claim , or a coThis observational study used difference-in-differences models to establish causal inference between the intervention (policy change) and outcomes. We assumed that any changes in use of dental general anesthesia would not be immediate and theorized that the largest changes in surgery would overlap with children at risk for early childhood caries, because most dental surgeries among Medicaid enrollees occur in children aged 1 to 5 years .We used a linear regression model to estimate 4 outcomes: receipt of a preventive care dental visit, caries-related dental sedation visits, DGA, and an emergency department dental visit. State fixed effects accounted for time-invariant aspects of each state’s environment related to outcomes. To control for the possibility that other changes between 2007 and 2012 in the treatment state (Texas) could confound the effect of the Medicaid reforms on use, we used Medicaid-enrolled children from Florida, where no known policy changes occurred during that same period, as a comparison group. The econometric model used for each outcome was as follows:2 represents the change in DGA in Texas after the Medicaid policy change took effect. The coefficient vector (βS) represents state fixed effects, which adjusts for varying outcome rates across states.The vector X includes control variables . Although the control variables are known to be important correlates to receipt of DGA and emergency department visits, the main variables of interest are the year by state interaction effects. For example, βA total of 7,748,850 children met study inclusion criteria. Demographic differences between Texas and Florida were primarily based on race/ethnicity, because approximately 60% of Medicaid enrollees in Texas were Latino, compared with approximately 30% Latino in Florida . Use of Reimbursements for preventive care, sedation, general anesthesia, and other caries-related treatment services increased over the study period in Texas . ReimburP < .001, standard error [SE], 0.0008) from a baseline of 50.5%. The policy-associated increase in preventive care dental visits accounted for 93% of the overall increase in such visits. Dental surgery use increased by 0.01 percentage points  in association with the policy intervention , without significance. However, surgery for a medical indication (appendicitis) increased significantly by 0.01 percentage points . Sedation visits increased by 1.7 percentage points  from a baseline of 3.2%. Emergency department visits for caries decreased in the postpolicy period by 0.3 percentage points  from a baseline of 0.41%.To isolate changes in use in the policy intervention, we employed a difference-in-differences study design. We found that use changed significantly for all difference-in-differences outcomes  when we We found that increasing provider reimbursements was an effective way to increase access to preventive care dental visits. Although tertiary services were not the intended target of our study, use of those services provides useful outcomes to assess long-term effects of increased preventive care visits. We hypothesized that increased preventive care dental visits would improve oral health to the degree that need for tertiary oral health services would be decreased. Our results partially supported this hypothesis by showing decreased dental emergency department visits. Increased reimbursements for preventive dental care were associated with increased sedation visits. Rather than an outcome for severe disease, sedation visits may indicate a population’s access to dental providers who diagnose and treat caries.Although the overall frequency of emergency department visits increased over time in Texas, our model attributed a decline in these visits to a policy intervention to increase preventive care dental visits. Our findings support the idea that the use of emergency department visits is a sensitive indicator of a population’s lack of access to preventive dental care. Our findings provide a counterbalance to prior work on the association between declining reimbursements and increased ED visits for caries . Such viWe were concerned about the potential for omitted variable bias, which would lead us to incorrectly attribute changes in use of dental service to a policy intervention that increased reimbursements for preventive dental care. To address this concern, we employed a difference-in-differences model with appendectomies as an outcome. We assumed that appendectomies were unlikely to be directly influenced by preventive dental care reimbursements. Although both dental and medical surgeries increased in association with increased dental reimbursements, only changes in appendectomies were found to be significant. We interpret the difference in significant change between medical versus dental surgical outcomes to further strengthen the validity of our findings that associate changes in use of dental care with increased reimbursements for preventive dental care. Had both dental and medical surgical outcomes changed in similar fashion, we would have concluded that our findings represented more general trends in health care use. Furthermore, a nonsignificant increase in dental surgeries of 0.01 percentage points with a baseline prevalence of 1.43% does not appear to be clinically meaningful at a population level. We interpret this to signify the limitations of an isolated policy intervention to increase access to preventive dental care on the oral health status of the dental surgery population.https://publichealth.nc.gov/oralhealth/partners/IMB.htm) have facilitated preventive dental care by nondental providers. However, because we defined preventive dental care as a claim for a service rather than specific to type of provider, our results reflect any preventive dental care, including that of primary care providers, reimbursed by Medicaid. Finally, we were unable to specify the mechanisms between increased provider reimbursements for prevention and use of tertiary oral health services. It has been demonstrated that reimbursement affects use of preventive services by expanding dental provider capacity, either by increasing the total number of participating providers or increasing the volume of patients seen by participating providers  with a gap in data between the prepolicy and postpolicy period 2011–2012). Second, the ability to detect changes in disease burden was limited by use of the CDT coding system. The CDT coding system is an accurate system to track dental procedures, but it is an inadequate measure for the extent and severity of caries. Third, other possible sources of preventive dental care extend beyond dentists. State programs, such as North Carolina’s “Into the Mouths of Babes” (. Second,Our findings suggest that a focus on other social determinants of oral health may be particularly influential in young children. The contribution of oral health behaviors, such as regular toothbrushing, restricted sugar intake, and exposure to fluoride may have greater impact than preventive care dental visits in families with young children who require dental surgery, particularly if these families do not seek care until after caries have developed. Future interventions may build on our findings by investigating the impact of multilevel interventions that address access to dental care as well as household oral health behaviors to change a population’s oral health status.


The objective of the present study was to explore the long-term postpartum glucose metabolism in women with previous GDM, and study the mechanism of hyperglycemia from gestation to postpartum by investigating the postpartum insulin resistance and insulin secretion. β were used to assess insulin resistance and insulin secretion levels with different glucose statuses.  A total of 321 females with previous GDM were followed up once during 1- to 6-years postpartum. Characteristics during pregnancy, perinatal period, and postpartum were compared between postpartum NGT and hyperglycemic women. HOMA-IR and HOMA-P=0.006). After ROC analysis, the best equilibrium between sensitivity (70.3%) and specificity (60.4%) for 2 hPG was 9.03 mmol/L. HOMA-IR was increased in postpartum normal glucose tolerance (NGT), prediabetes, and T2DM . After ROC analysis, the best equilibrium between sensitivity (70.3%) and specificity (60.4%) for 2 hPG was 9.03 mmol/L. HOMA-IR was increased in postpartum normal glucose tolerance (NGT), prediabetes, and T2DM . After ROC analysis, the best equilibrium between sensitivity (70.3%) and specificity (60.4%) for 2 hPG was 9.03 mmol/L. HOMA-IR was increased in postpartum normal glucose tolerance (NGT), prediabetes, and T2DM  (β-cell function contributes more to T2DM development.β were used to assess insulin resistance and insulin secretion levels with different glucose statuses.  75 g OGTT 2h PG during pregnancy higher than 9.03 mmol/L is regarded as an independent risk factor of postpartum hyperglycemia. Insulin resistance with insufficient insulin secretion compensation is still common phenomenon during long-term postpartum. Women with heavier insulin resistance in the postpartum period are more likely develop prediabetes, while decreased  Maternal age, family history of DM, weight in early pregnancy, weight at delivery, weight change during pregnancy, FPG, 1 h postprandial glucose (1 hPG), 2 hPG, and insulin use during pregnancy were collected. Neonatal birth weight and feeding patterns were recorded. During follow-up, FPG, 2 hPG, fasting insulin (FINS), and 2-hour postprandial insulin (PINS) were measured. Follow-up age, weight, postpartum weight change, waist, hip, and waist hip ratio (WHR) were recorded. HOMA-IR = FPG (mmol/L) Plasma glucose levels were determined by glucose oxidase method. Insulin levels were measured by chemiluminescence.t-test, one-way ANOVA, nonparametric test, or chi-square test as appropriate. Multiple logistic regression (forward: likelihood ratio) was used to analyze the predictors of postpartum hyperglycemia. Receiver operating characteristic (ROC) analysis was used to evaluate the predictive power of identified predictors. P values < 0.05 were considered as statistically significant in most of the analyses. Bonferroni method was used to adjust the α level for multiple comparisons.SPSS 21.0 was used for statistical analyses. Values were expressed as mean ± standard deviation (SD) for normally distributed measurement data, median  for abnormally distributed measurement data, and number (percentage) for categorical variables. Comparisons were performed by independent-samples P=0.051, 0.283, ). Compared to the included group, the women in excluded group had lower weight  and BMI  during early pregnancy period, while the weight and BMI at delivery were similar . 75 g OGTT FPG, 1 hPG, and 2 hPG showed no significant differences between groups , as well as the proportion of women using insulin to control blood glucose levels during pregnancy (P=0.138). As to offspring, sex and born weight of the excluded group were also similar to the included group .After followed up by telephone, 321 (28.3%) of the total population came to the hospital and underwent 75 g OGTT once at different postpartum years, while the rest 813 (71.7%) women refused and were excluded from our postpartum study. Characteristics during pregnancy and perinatal period were compared between the included group and excluded group . Age at There were 54, 127, 34, 20, 41, and 45 women for 1-, 2-, 3-, 4-, 5- and 6-year postpartum study, respectively. 116 women diagnosed by NDDG criteria were followed up at 5 -year postpartum, and 205 women diagnosed by IADPSG criteria were followed up at 2 -year postpartum. During the follow-up period, 92 (28.7%) women developed hyperglycemia, which included 16 (5.0%) T2DM women and 76 (23.7%) prediabetes women. From 1- to 6-year postpartum, there were, respectively, 14 (25.9%), 24 (18.9%), 9 (26.5%), 6 (30.0%), 22 (53.7%), 17 (37.8%) women diagnosed with hyperglycemia .P=0.065, vs 15.3%, P=0.001), and demonstrated to have a significantly higher weight  and BMI during early pregnancy period . However, there were no significant differences in weight or BMI between the two groups at delivery , and similarly in the neonatal birth weight (P=0.748). In the hyperglycemic group, more women used insulin to control blood glucose levels during pregnancy , and their 75 g OGTT 1 hPG and 2 hPG during pregnancy were significantly higher than those in the NGT group (P < 0.001), but FPG showed no significant differences (P=0.073). A greater proportion of women in the hyperglycemia group used formula to feed their offspring . At the time of follow-up, the maternal age, weight, BMI, waist, hip circumference, FPG, 2 hPG, FINS, and PINS were all higher in the hyperglycemic group (P=0.06). Weight change was smaller in the hyperglycemic group both during pregnancy  and in the postpartum period . 8 (3.5%) women in the NGT group and 4 (4.3%) women in the hyperglycemic group had intervening pregnancies (P=0.748). Of all the intervening pregnancies, one woman completed delivery without GDM during the following pregnancy and developed NGT, another woman developed hyperglycemia at the time of follow-up with similar situation in the following pregnancy, and the rest of the women had abortion during early pregnancy.Compared to the NGT group, the hyperglycemic group included a slightly older women at the time of pregnancy, but the difference was not statistically significant P=0.065, . The womic group . But, WHP=0.006). ROC analysis was used to identify the optimal cut-off level of 2 hPG for predicting hyperglycemia postpartum   (< 0.001) . The besβ. HOMA-IR was 1.80 , and HOMA-β was 1.13  in the overall population. HOMA-IR in the hyperglycemic group was significantly higher than that in the NGT group  vs 1.64 , P < 0.001). HOMA-IR in the NGT, prediabetes, and T2DM groups was increased successively . The differences between NGT and prediabetes, NGT, and T2DM showed significant differences , while the difference was not statistically significant when comparing the pre-diabetes and T2DM groups after adjusting the α level (P=0.025). HOMA-β in the NGT group was higher than that in the hyperglycemic group  vs. 1.04 ), but the difference was not significant (P=0.115). However, when the HOMA-β of NGT, prediabetes, and T2DM groups were calculated separately, the results were different . Both HOMA-β of NGT and prediabetes groups were significantly higher than that of the T2DM group , while the HOMA-β of the NGT group was not significantly higher than that of the prediabetes group (P=0.583).FPG and FINS during follow-up were used to calculate HOMA-IR and HOMA-Due to increasing prevalence and adverse outcomes, especially during the high risk of metabolic disorders postpartum, GDM caused a huge economic burden to the society, and gradually became a serious public health problem. The risk of progression from GDM to T2DM varies with ethnicity, length of follow-up period, and cohort retention. Through systematic review, Kim C et al.  found thβ-cell dysfunction. When various hormones and cytokines change with increasing gestation period, insulin resistance is progressively increased to the level of T2DM [β-cell function should be increased to compensate insulin resistance [β-cell function as well as systematic insulin resistance. It is known that both increased insulin resistance and inadequate insulin secretion contribute to hyperglycemia in nonpregnant women in varying degrees, and their roles in GDM are also different. According to a study [GDM is obviously considered as a risk factor for T2DM, but this phenomenon is not fully explained. Similar to T2DM, more and more studies , 13–15 s of T2DM . To remasistance , 16. GDM a study , almost β were both still higher than 100% overall after delivery. This meant that they still had insulin resistance and insulin compensatory secretion, and some of their glucose levels were even recovered to normal. Within 6-year postpartum, the women who developed hyperglycemia had significantly higher levels of HOMA-IR than those with normal glucose levels, while the difference between prediabetes and T2DM was not significant. According to the HOMA-β levels, the normal glucose, prediabetes, and diabetes were decreased successively. In contrast to HOMA-IR, only the diabetes group demonstrated a significant decrease of HOMA-β than normal glucose and prediabetes. These results were in accordance with the study conducted by Ekelund M et al. [β-cell function, insulin secretion ultimately decreased, with consequent development of T2DM.As insulin resistance and insulin secretion defects contribute to both GDM and T2DM, the changes after delivery in the short term and long term are necessary to explain the transition from GDM to T2DM. In short-term studies , 17, insM et al. . This stSince not all GDM women would develop hyperglycemia postpartum, the women who developed may have some characteristics during pregnancy and after delivery. In the previous studies , 20, preβ could reflect the levels of insulin resistance and insulin secretion during the course. As serum insulin might be influenced by many factors, HOMA2-IR and HOMA2-β [In our study, 28.3% of the total population accepted the postpartum 75 g OGTT. Women who refused postpartum tests had lower weight and BMI during early pregnancy period than the included women, which could cause some selection bias. However, other characteristics during pregnancy and perinatal period showed no significant differences, indicating that the included women had representativeness to a certain extent. Our study was retrospective in nature, experiencing the change of diagnostic criteria of GDM, women were followed up only once at different postpartum years, and lack of data between perinatal and follow-up time may lead to the difficulty of acquiring the exact incidence of hyperglycemia in different postpartum years. We could only get the prevalence, but the small sample size in some postpartum years might influence the accuracy. HOMA-IR and HOMA- HOMA2-β , 22 baseβ-cell function contributes more to T2DM development.In conclusion, women with previous GDM are still at a greater risk of developing prediabetes and even T2DM in long-term postpartum. 75 g OGTT 2 hPG during pregnancy higher than 9.03 mmol/L is regarded as an independent risk factor of postpartum hyperglycemia. Entering postpartum stage, insulin resistance with insufficient insulin secretion compensation is still common phenomenon during long-term postpartum. Women with severe insulin resistance during postpartum are more likely to develop prediabetes, but decreased